HK40060356A

HK40060356A - Fusion of an antibody binding cea and 4-1bbl

Info

Publication number: HK40060356A
Application number: HK62022050385.3A
Authority: HK
Inventors: C·费拉拉·科勒; T·霍弗; C·克雷恩; E·莫斯纳; C·克劳斯; R·霍斯; B·舍雷尔; P·尤马纳
Original assignee: 豪夫迈·罗氏有限公司
Priority date: 2019-06-26
Filing date: 2020-06-24
Publication date: 2022-05-13

Description

Fusion of CEA-binding antibodies to 4-1BBL

Technical Field

The present invention relates to novel antigen binding molecules, particularly humanized CEA antibodies, that bind to carcinoembryonic antigen (CEA), and to CEA-targeting antigen binding molecules containing a 4-1BBL trimer comprising these CEA antibodies, and their use in the treatment of cancer. The invention further relates to methods of producing these molecules and methods of using them.

Background

Carcinoembryonic antigen (CEA), also known as carcinoembryonic antigen-associated cell adhesion molecule 5(CEACAM-5 or CD66e), is a glycoprotein having a molecular weight of about 180 kDa. CEA is a member of the immunoglobulin gene superfamily and comprises seven domains linked to the cell membrane by a Glycosylphosphatidylinositol (GPI) anchor (Thompson J.A., J Clin Lab anal.5: 344-366, 1991). These seven domains include a single N-terminal variable domain-like region and three sets of constant domain-like regions (A1B1, A2B2, and A3B 3; the A domain is 92 amino acids and the B domain is 86 amino acids, Hefta L J et al, Cancer Res.52:5647-5655, 1992).

The human CEA family contains 29 genes, 18 of which are expressed: 7 belong to the CEA subgroup and 11 belong to the pregnancy specific glycoprotein subgroup. Some CEA subgroup members are believed to have cell adhesion properties. CEA is thought to play a role in innate immunity. Due to the presence of proteins that are closely related to CEA, it can be challenging to generate anti-CEA antibodies that are specific for CEA and have minimal cross-reactivity with other closely related proteins.

CEA has long been identified as a tumor-associated antigen (Gold and Freedman, J Exp Med.1965, 121,439-462 CEA plays a key role in cell adhesion, invasion and metastasis of cancer cells endogenous expression of CEA affects expression of various cancer-associated genes, especially cell cycle and apoptotic genes, protecting colon tumor cells from various apoptotic stimuli, such as treatment with 5-fluorouracil (Soeth et al, Clin. cancer Res.2001,7(7),2022-2030) CEA inhibits a process known as anoikis in which cells deprived of anchorage to the extracellular matrix subsequently undergo apoptosis (a process in which cells deprived of anchorage to extracellular matrix subsequently undergo apoptosis: (B) (CEA)Et al, Cancer Research 2000,60(13), 3419-. Thus, CEA expression may be one way by which cancer cells gain survival benefits and overcome apoptosis-inducing therapies.

High expression of CEA was found in various tumor types (Thompson et al, J.Clin.Lab.anal.1991,5, 344-. High incidence thereof is observed in colorectal cancer (CRC), pancreatic cancer, gastric cancer, non-small cell lung cancer (NSCLC), breast cancer, and the like; low expression was found in small cell lung cancer and glioblastoma. Both epithelial-derived tumors and their metastases comprise CEA as a tumor-associated antigen. The presence of CEA itself does not indicate that it has been transformed into cancer cells, but the distribution of CEA is indicative. In normal tissues, CEA is normally expressed on the apical surface of cells (S) ((R))S. Semin Cancer biol.1999,9(2),67-81), rendering it unabsorbable by antibodies in the bloodstream. In contrast to normal tissue, CEA tends to be expressed on the entire surface of cancer cells. This change in expression pattern allows CEA to readily bind to antibodies in cancer cells. Furthermore, expression of CEA in cancer cells is increased. Furthermore, an increase in CEA expression promotes an increase in intercellular adhesion, which may lead to metastasis (Marshall J., Semin. Oncol.2003,30(suppl.8):30-36)。

CEA is readily cleaved from the cell surface and flows from the tumor into the bloodstream, either directly or through lymphatic vessels. Because of this property, serum CEA levels have been used as clinical markers for diagnosing cancer and screening for cancer recurrence (especially colorectal cancer). This property is also one of the challenges of using CEA as a target, as serum CEA binds to most of the currently available anti-CEA antibodies, blocking them from reaching cell surface targets and limiting potential clinical efficacy.

Accordingly, various monoclonal antibodies have been generated against CEA for research purposes, as well as diagnostic tools and for therapeutic purposes. One is the murine antibody T84.66(Wagener et al, J Immunol 1983,130,2308; Neumaier et al, 1985, J Immunol 135,3604), which has also been chimeric (WO 1991/01990) and humanized (WO 2005/086875). Another CEA antibody is the mouse monoclonal antibody PR1A3(Richman et al, int.J. cancer 1987,59, 317-. Humanized variants of which affinity matures are described in WO 2011/023787. Humanized antibodies derived from the murine antibody A5B7 are disclosed in WO 92/01059 and WO 2007/071422. However, there is a continuing need to provide new CEA antibodies with advantageous properties, particularly for use in targeting therapeutic molecules to tumor cells.

4-1BB (CD137) is a member of the TNF receptor superfamily, which was first identified as an inducible molecule expressed by T cell activation (Kwon and Weissman,1989, Proc Natl Acad Sci USA 86, 1963-. Subsequent studies have shown that many other immune cells also express 4-1BB, including NK cells, B cells, NKT cells, monocytes, neutrophils, mast cells, Dendritic Cells (DCs) and cells of non-hematopoietic origin, such as endothelial cells and smooth muscle cells (Vinay and Kwon,2011, Cell Mol Immunol 8, 281-. Expression of 4-1BB in different cell types is mostly inducible and driven by various stimulatory signals such as T Cell Receptor (TCR) or B cell receptor triggering and receptor-induced signaling by co-stimulatory molecules or pro-inflammatory cytokines (Diehl et al, 2002, J Immunol 168, 3755-2762; Zhang et al, 2010, Clin Cancer Res 13, 2758-2767).

4-1BB ligand (4-1BBL or CD137L) was identified in 1993 (Goodwin et al, 1993, Eur J Immunol 23, 2631-2641). Expression of 4-1BBL has been shown to be limited to professional Antigen Presenting Cells (APC) such as B cells, DCs and macrophages. Inducible expression of 4-1BBL is characteristic of T cells (including both. alpha. beta. and. gamma. delta. T cell subsets) and endothelial cells (Shao and Schwarz,2011, J Leukoc Biol 89, 21-29).

Co-stimulation via the 4-1BB receptor (e.g., via 4-1BBL ligation) activates T cells (CD 4)⁺And CD8⁺Two subsets) strongly enhances the activation of T cells (Bartkowiak and Curran, 2015). In combination with TCR triggering, agonistic 4-1 BB-specific antibodies enhance T cell proliferation, stimulate lymphokine secretion and reduce T lymphocyte sensitivity to activation-induced cell death (Snell et al, 2011, Immunol Rev 244, 197-217). This mechanism is further advanced as the first demonstration of cancer immunotherapy concept. Potent anti-tumor effects have been produced in preclinical models in which agonistic antibodies against 4-1BB are administered to tumor-bearing mice (Melero et al, 1997, Nat Med 3, 682-685). Later, there is increasing evidence that 4-1BB generally only shows its efficacy as an anti-tumor agent when administered in combination with other immunomodulatory compounds, chemotherapeutic agents, tumor-specific vaccination or radiotherapy (Bartkowiak and Curran,2015, Front Oncol 5,117).

The signaling of the TNFR superfamily requires cross-linking of trimeric ligands to engage with receptors, as does the 4-1BB agonistic antibodies that require wild-type Fc binding (Li and Ravetch,2011, Science 333, 1030-1034). However, systemic administration of 4-1 BB-specific agonistic antibodies with functionally active Fc domains resulted in CD8 associated with hepatotoxicity⁺Influx of T cells (dublot et al, 2010, Cancer Immunol Immunother 59,1223-1233), which were attenuated or significantly improved in the absence of functional Fc receptors in mice. In the clinic, Fc competent 4-1BB agonistic Ab (BMS-663513) (NCT00612664) caused grade 4 hepatitis, leading to termination of the experiment (Simeone and Ascieto, 2012, J immunotoxin 9, 241-. Therefore, there is a need forA potent and safer 4-1BB agonist.

For example, in WO 2016/075278 a novel antigen binding molecule is described which binds a moiety capable of forming a trimer of costimulatory 4-1BBL to an antigen binding domain capable of binding to a tumor-associated target, such that cross-linking occurs only in the presence of the tumor-associated target. However, there is still a need to optimize tumor targeting by introducing new CEA antigen binding domains with advantageous properties.

Disclosure of Invention

Antigen binding molecules containing 4-1BBL trimers targeting CEA have increased activity at CEA-expressing tumor sites, contain natural human 4-1BB ligand, and therefore should have fewer safety issues than traditional fusion proteins with higher 4-1BB agonistic antibodies or artificial components. Novel antigen binding molecules combine an anti-CEA antigen binding domain with a moiety that is capable of forming a costimulatory 4-1BBL trimer and is sufficiently stable to be pharmaceutically useful. Surprisingly, the antigen binding molecules of the present invention provide trimeric and thus biologically active human 4-1BB ligands, although one of the trimerized 4-1BBL ectodomains is located on another polypeptide than the other two 4-1BBL ectodomains of the molecule.

In one aspect, the invention provides an antigen binding molecule comprising a trimer of 4-1BBL, comprising

An antigen binding domain capable of specifically binding to CEA,

a first polypeptide and a second polypeptide, said first polypeptide and said second polypeptide being linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises two 4-1BBL extracellular domains or fragments thereof linked to each other by a peptide linker and in that the second polypeptide comprises one 4-1BBL extracellular domain or fragment thereof, and

an Fc domain comprising a first subunit and a second subunit capable of stable association,

wherein the antigen binding domain capable of specifically binding to CEA comprises

(a) A variable heavy chain domain (VH) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:17, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:18, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19; and a variable light chain domain (VL) comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:20, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:21, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22; or

(b) A VH domain comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:25, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:26, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and a VL domain comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:28, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30; or

(c) A VH domain comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:65, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:66 or SEQ ID NO:67, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 68; and a VL domain comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:69 or SEQ ID NO:70 or SEQ ID NO:313, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:71 or SEQ ID NO:72 or SEQ ID NO:73, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 74.

In another aspect, there is provided an antigen binding molecule comprising a trimer of 4-1BBL as described hereinbefore, wherein the extracellular domain of 4-1BBL or a fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO 87, SEQ ID NO 88, SEQ ID NO 89, SEQ ID NO 90, SEQ ID NO 91, SEQ ID NO 92, SEQ ID NO 93 and SEQ ID NO 94, in particular the amino acid sequence of SEQ ID NO 91.

In a further aspect, there is provided an antigen binding molecule comprising a trimer of 4-1BBL as described herein, comprising

An antigen binding domain capable of specifically binding to CEA,

Wherein the antigen binding molecule is characterized in that the first polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO 95, SEQ ID NO 96, SEQ ID NO 97 and SEQ ID NO 98 and in that the second polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO 87, SEQ ID NO 91, SEQ ID NO 89 and SEQ ID NO 94 and

In one aspect, the Fc domain is an IgG, in particular an IgG1 Fc domain or an IgG4 Fc domain. More specifically, the Fc domain is an IgG1 Fc domain. In a particular aspect, the Fc domain comprises a modification that facilitates association of the first subunit and the second subunit of the Fc domain. In a particular aspect, the invention provides an antigen binding molecule comprising a trimer of 4-1BBL, wherein the Fc domain comprises a knob-and-hole (knob-hole) modification that facilitates association of a first subunit and a second subunit of the Fc domain. In a particular aspect, the invention provides an antigen binding molecule comprising a trimer of 4-1BBL, wherein the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (numbering according to the Kabat EU index) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to the Kabat EU index).

In another aspect, the present invention relates to an antigen binding molecule comprising a trimer of 4-1BBL as defined above, comprising (c) an Fc domain comprising a first subunit and a second subunit capable of stable association, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, in particular to an fey receptor. In particular, the Fc domain comprises amino acid substitutions at positions 234 and 235 (EU numbering according to Kabat) and/or 329 (EU numbering according to Kabat) of the IgG heavy chain. In particular, an antigen binding molecule comprising a trimer of 4-1BBL is provided, wherein the Fc domain is a human IgG1 Fc domain comprising the amino acid substitutions L234A, L235A and P329G (numbered according to the Kabat EU index).

In one aspect, the antigen binding molecule comprising a trimer of 4-1BBL is an antigen binding molecule wherein the antigen binding domain capable of specifically binding to CEA is a Fab molecule capable of specifically binding to CEA. In another aspect, the antigen binding domain capable of specifically binding to CEA is a cross-Fab molecule or scFV molecule capable of specifically binding to CEA.

In one aspect, an antigen binding molecule comprising a trimer of 4-1BBL is provided, wherein the antigen binding domain capable of specifically binding to CEA comprises

(b) A VH domain comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:25, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:26, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and a VL domain comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:28, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

In one aspect, the antigen binding domain capable of specifically binding to CEA comprises a variable heavy chain domain (VH) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:17, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:18, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19; and a variable light chain domain (VL) comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:20, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:21, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22.

In another aspect, the antigen binding domain capable of specifically binding to CEA comprises a VH domain comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:25, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:26, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and the VL domain comprises: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:28, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

In a particular aspect, there is provided an antigen binding molecule comprising a trimer of 4-1BBL, wherein the antigen binding domain capable of specifically binding to CEA comprises

(a) A VH domain comprising the amino acid sequence of SEQ ID NO. 23 and a VL domain comprising the amino acid sequence of SEQ ID NO. 24; or

(b) A VH domain comprising the amino acid sequence of SEQ ID NO. 31 and a VL domain comprising the amino acid sequence of SEQ ID NO. 32; or

(c) A VH domain comprising the amino acid sequence of SEQ ID NO 33 and a VL domain comprising the amino acid sequence of SEQ ID NO 34; or

(d) A VH domain comprising the amino acid sequence of SEQ ID NO 35 and a VL domain comprising the amino acid sequence of SEQ ID NO 36; or

(e) A VH domain comprising the amino acid sequence of SEQ ID NO 37 and a VL domain comprising the amino acid sequence of SEQ ID NO 38; or

(f) A VH domain comprising the amino acid sequence of SEQ ID NO 39 and a VL domain comprising the amino acid sequence of SEQ ID NO 40; or

(g) A VH domain comprising the amino acid sequence of SEQ ID NO 41 and a VL domain comprising the amino acid sequence of SEQ ID NO 42; or

(h) A VH domain comprising the amino acid sequence of SEQ ID NO 43 and a VL domain comprising the amino acid sequence of SEQ ID NO 44; or

(i) A VH domain comprising the amino acid sequence of SEQ ID NO 45 and a VL domain comprising the amino acid sequence of SEQ ID NO 46; or

(j) A VH domain comprising the amino acid sequence of SEQ ID NO 47 and a VL domain comprising the amino acid sequence of SEQ ID NO 48; or

(k) A VH domain comprising the amino acid sequence of SEQ ID NO. 49 and a VL domain comprising the amino acid sequence of SEQ ID NO. 50;

(l) A VH domain comprising the amino acid sequence of SEQ ID NO 51 and a VL domain comprising the amino acid sequence of SEQ ID NO 52; or

(m) a VH domain comprising the amino acid sequence of SEQ ID NO:53 and a VL domain comprising the amino acid sequence of SEQ ID NO: 54.

In a further aspect, the present invention provides an antigen-binding molecule VH domain comprising a trimer of 4-1BBL as described herein, wherein the antigen-binding domain capable of specifically binding to CEA comprises: a VH domain comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:65, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:66 or SEQ ID NO:67, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 68; and a VL domain comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:69 or SEQ ID NO:70 or SEQ ID NO:313, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:71 or SEQ ID NO:72 or SEQ ID NO:73, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 74. In one aspect, the antigen binding domain capable of specifically binding to CEA comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO 75, 76, 77, 78, 79 or 80; and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO 81, 82, 83, 84, 85 or 86.

(a) A VH domain comprising the amino acid sequence of SEQ ID NO 75 and a VL domain comprising the amino acid sequence of SEQ ID NO 85; or

(b) A VH domain comprising the amino acid sequence of SEQ ID NO. 79 and a VL domain comprising the amino acid sequence of SEQ ID NO. 85; or

(c) A VH domain comprising the amino acid sequence of SEQ ID NO:76 and a VL domain comprising the amino acid sequence of SEQ ID NO: 85; or

(d) A VH domain comprising the amino acid sequence of SEQ ID NO:80 and a VL domain comprising the amino acid sequence of SEQ ID NO: 84; or

(e) A VH domain comprising the amino acid sequence of SEQ ID NO. 79 and a VL domain comprising the amino acid sequence of SEQ ID NO. 84; or

(f) A VH domain comprising the amino acid sequence of SEQ ID NO 77 and a VL domain comprising the amino acid sequence of SEQ ID NO 84; or

(g) A VH domain comprising the amino acid sequence of SEQ ID NO 75 and a VL domain comprising the amino acid sequence of SEQ ID NO 84.

In another aspect, an antigen binding molecule comprising a trimer of 4-1BBL is provided, wherein a first peptide comprising two extracellular domains of 4-1BBL or fragments thereof connected to each other by a first peptide linker is fused at its C-terminus to a CL domain which is part of a heavy chain by a second peptide linker, and a second peptide comprising one of said extracellular domains of 4-1BBL or fragments thereof is fused at its C-terminus to a CH1 domain which is part of a light chain by a third peptide linker. In another aspect, an antigen binding molecule comprising a trimer of 4-1BBL is provided, wherein a first peptide fusion comprising two extracellular domains of 4-1BBL or fragments thereof connected to each other by a first peptide linker is connected at its C-terminus to a CH domain which is part of a heavy chain by a second peptide linker, and a second peptide comprising one of said extracellular domains of 4-1BBL or fragments thereof is fused at its C-terminus to a CL domain which is part of a light chain by a third peptide linker.

In a further aspect, there is provided an antigen binding molecule comprising a trimer of 4-1BBL as described herein, wherein the antigen binding molecule comprises

(i) A first heavy chain and a first light chain, both comprising a Fab molecule capable of specific binding to CEA;

(ii) a second heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO 99, SEQ ID NO 101, SEQ ID NO 103 and SEQ ID NO 105; and

(iii) a second light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO 100, SEQ ID NO 102, SEQ ID NO 104 and SEQ ID NO 106.

In particular, antigen binding molecules comprising a trimer of 4-1BBL as described herein are provided, wherein the antigen binding molecule comprises

(a) A first heavy chain comprising the amino acid sequence of SEQ ID NO 99, a first light chain comprising the amino acid sequence of SEQ ID NO 100, a second heavy chain comprising the amino acid sequence of SEQ ID NO 238 and a second light chain comprising the amino acid sequence of SEQ ID NO 239; or

(b) A first heavy chain comprising the amino acid sequence of SEQ ID NO 99, a first light chain comprising the amino acid sequence of SEQ ID NO 100, a second heavy chain comprising the amino acid sequence of SEQ ID NO 240, and a second light chain comprising the amino acid sequence of SEQ ID NO 241; or

(c) A first heavy chain comprising the amino acid sequence of SEQ ID NO 99, a first light chain comprising the amino acid sequence of SEQ ID NO 100, a second heavy chain comprising the amino acid sequence of SEQ ID NO 242 and a second light chain comprising the amino acid sequence of SEQ ID NO 243; or

(d) A first heavy chain comprising the amino acid sequence of SEQ ID NO 99, a first light chain comprising the amino acid sequence of SEQ ID NO 100, a second heavy chain comprising the amino acid sequence of SEQ ID NO 244 and a second light chain comprising the amino acid sequence of SEQ ID NO 245; or

(e) A first heavy chain comprising the amino acid sequence of SEQ ID NO 99, a first light chain comprising the amino acid sequence of SEQ ID NO 100, a second heavy chain comprising the amino acid sequence of SEQ ID NO 246 and a second light chain comprising the amino acid sequence of SEQ ID NO 247

A chain; or

(f) A first heavy chain comprising the amino acid sequence of SEQ ID NO 99, a first light chain comprising the amino acid sequence of SEQ ID NO 100, a second heavy chain comprising the amino acid sequence of SEQ ID NO 248 and a second light chain comprising the amino acid sequence of SEQ ID NO 249; or

(g) A first heavy chain comprising the amino acid sequence of SEQ ID NO 99, a first light chain comprising the amino acid sequence of SEQ ID NO 100, a second heavy chain comprising the amino acid sequence of SEQ ID NO 250, and a second light chain comprising the amino acid sequence of SEQ ID NO 251; or

(h) A first heavy chain comprising the amino acid sequence of SEQ ID NO 99, a first light chain comprising the amino acid sequence of SEQ ID NO 100, a second heavy chain comprising the amino acid sequence of SEQ ID NO 252, and a second light chain comprising the amino acid sequence of SEQ ID NO 253; or

(i) A first heavy chain comprising the amino acid sequence of SEQ ID NO 99, a first light chain comprising the amino acid sequence of SEQ ID NO 100, a second heavy chain comprising the amino acid sequence of SEQ ID NO 254, and a second light chain comprising the amino acid sequence of SEQ ID NO 255; or

(j) A first heavy chain comprising the amino acid sequence of SEQ ID NO 99, a first light chain comprising the amino acid sequence of SEQ ID NO 100, a second heavy chain comprising the amino acid sequence of SEQ ID NO 256, and a second light chain comprising the amino acid sequence of SEQ ID NO 257; or

(k) A first heavy chain comprising the amino acid sequence of SEQ ID NO 99, a first light chain comprising the amino acid sequence of SEQ ID NO 100, a second heavy chain comprising the amino acid sequence of SEQ ID NO 258 and a second light chain comprising the amino acid sequence of SEQ ID NO 259; or

(l) A first heavy chain comprising the amino acid sequence of SEQ ID NO 99, a first light chain comprising the amino acid sequence of SEQ ID NO 100, a second heavy chain comprising the amino acid sequence of SEQ ID NO 260 and a second light chain comprising the amino acid sequence of SEQ ID NO 261; or

(m) a first heavy chain comprising the amino acid sequence of SEQ ID NO:49, a first light chain comprising the amino acid sequence of SEQ ID NO:50, a second heavy chain comprising the amino acid sequence of SEQ ID NO:262 and a second light chain comprising the amino acid sequence of SEQ ID NO: 263.

In another particular aspect, there is provided an antigen binding molecule comprising a trimer of 4-1BBL, wherein the antigen binding molecule comprises

(a) A first heavy chain comprising the amino acid sequence of SEQ ID NO 99, a first light chain comprising the amino acid sequence of SEQ ID NO 100, a second heavy chain comprising the amino acid sequence of SEQ ID NO 266, and a second light chain comprising the amino acid sequence of SEQ ID NO 267; or

(b) A first heavy chain comprising the amino acid sequence of SEQ ID NO 99, a first light chain comprising the amino acid sequence of SEQ ID NO 100, a second heavy chain comprising the amino acid sequence of SEQ ID NO 268, and a second light chain comprising the amino acid sequence of SEQ ID NO 267; or

(c) A first heavy chain comprising the amino acid sequence of SEQ ID NO 99, a first light chain comprising the amino acid sequence of SEQ ID NO 100, a second heavy chain comprising the amino acid sequence of SEQ ID NO 269, and a second light chain comprising the amino acid sequence of SEQ ID NO 267; or

(d) A first heavy chain comprising the amino acid sequence of SEQ ID NO 99, a first light chain comprising the amino acid sequence of SEQ ID NO 100, a second heavy chain comprising the amino acid sequence of SEQ ID NO 270, and a second light chain comprising the amino acid sequence of SEQ ID NO 271; or

(e) A first heavy chain comprising the amino acid sequence of SEQ ID NO 99, a first light chain comprising the amino acid sequence of SEQ ID NO 100, a second heavy chain comprising the amino acid sequence of SEQ ID NO 272 and a second light chain comprising the amino acid sequence of SEQ ID NO 271; or

(f) A first heavy chain comprising the amino acid sequence of SEQ ID NO 99, a first light chain comprising the amino acid sequence of SEQ ID NO 100, a second heavy chain comprising the amino acid sequence of SEQ ID NO 273 and a second light chain comprising the amino acid sequence of SEQ ID NO 271; or

(g) A first heavy chain comprising the amino acid sequence of SEQ ID NO 99, a first light chain comprising the amino acid sequence of SEQ ID NO 100, a second heavy chain comprising the amino acid sequence of SEQ ID NO 274 and a second light chain comprising the amino acid sequence of SEQ ID NO 271.

In another aspect, the present invention provides a novel humanized antibody that binds carcinoembryonic antigen (CEA) (hu CEACAM5) comprising

In one aspect, the present invention provides a novel humanized antibody that binds to the A2 domain of carcinoembryonic antigen (CEA) (hu CEACAM5), i.e., to the domain comprising the amino acid sequence of SEQ ID NO: 311. Accordingly, the present invention provides a novel humanized antibody that binds to the a2 domain of carcinoembryonic antigen (CEA) (hu CEACAM5) comprising: (a) a variable heavy chain domain (VH) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:17, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:18, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19; and a variable light chain domain (VL) comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:20, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:21, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22; or (b) a VH domain comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:25, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:26, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and a VL domain comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:28, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

In one aspect, a humanized antibody that binds to the a2 domain of carcinoembryonic antigen (CEA) (huCEACAM5) comprises

In another aspect, there is provided a novel humanized antibody that binds carcinoembryonic antigen (CEA), the humanized antibody comprising: a VH domain comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:65, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:66 or SEQ ID NO:67, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 68; and a VL domain comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:69 or SEQ ID NO:70 or SEQ ID NO:313, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:71 or SEQ ID NO:72 or SEQ ID NO:73, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 74. In one aspect, the invention provides a humanized antibody that binds to the A1 domain of carcinoembryonic antigen (CEA) (hu CEACAM5), i.e., to the domain comprising the amino acid sequence of SEQ ID NO: 312. Accordingly, the present invention provides a humanized antibody that binds to the a1 domain of carcinoembryonic antigen (CEA) (hu CEACAM5), the humanized antibody comprising: a VH domain comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:65, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:66 or SEQ ID NO:67, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 68; and a VL domain comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:69 or SEQ ID NO:70 or SEQ ID NO:313, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:71 or SEQ ID NO:72 or SEQ ID NO:73, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 74.

In particular, a humanized antibody that binds to the a1 domain of carcinoembryonic antigen (CEA) (hu CEACAM5) comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO 75, 76, 77, 78, 79 or 80 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO 81, 82, 83, 84, 85 or 86. In a particular aspect, the antigen binding domain capable of specifically binding to CEA comprises

In another aspect, humanized antibodies that bind to the a2 or a1 domain of carcinoembryonic antigen (CEA), respectively, are antibody fragments, particularly Fab molecules, that specifically bind to CEA. In one aspect, the antibody is a full length IgG1 antibody.

According to another aspect of the present invention there is provided an isolated nucleic acid encoding an antigen binding molecule or antibody comprising a trimer of 4-1BBL as defined above. The present invention further provides a vector, in particular an expression vector, comprising the isolated nucleic acid molecule of the present invention; and provides a host cell comprising the isolated nucleic acid or vector of the invention. In some embodiments, the host cell is a eukaryotic cell, particularly a mammalian cell.

In another aspect, a method for producing an antigen binding molecule of the invention comprising a trimer of 4-1BBL is provided, the method comprising culturing a host cell of the invention under conditions suitable for expression of an antigen binding molecule comprising a trimer of 4-1BBL, and isolating the antigen binding molecule comprising a trimer of 4-1 BBL. The invention also encompasses an antigen binding molecule comprising a trimer of 4-1BBL produced by the method of the invention.

The invention further provides a pharmaceutical composition comprising an antigen binding molecule comprising a trimer of 4-1BBL of the invention or an antibody of the invention and at least one pharmaceutically acceptable excipient. In another aspect, a pharmaceutical composition is provided comprising an antigen binding molecule comprising a trimer of 4-1BBL of the invention and at least one pharmaceutically acceptable excipient, further comprising an additional therapeutic agent, such as a chemotherapeutic agent and/or other agent for use in cancer immunotherapy. In a further aspect, a pharmaceutical composition is provided, further comprising a T cell activating anti-CD 3 bispecific antibody, in particular an anti-CEA anti-CD 3 bispecific antibody.

The invention also encompasses the antigen binding molecule of the invention comprising a trimer of 4-1BBL, or the antibody or pharmaceutical composition of the invention, for use as a medicament. In one aspect, an antigen binding molecule of the invention comprising a trimer of 4-1BBL or a pharmaceutical composition of the invention is provided for use in the treatment of a disease in an individual in need thereof. In a particular aspect, there is provided an antigen binding molecule of the invention comprising a trimer of 4-1BBL, or an antibody or pharmaceutical composition of the invention, for use in the treatment of cancer. In another aspect, an antigen binding molecule of the invention comprising a trimer of 4-1BBL or a pharmaceutical composition of the invention is provided for use in up-regulating or prolonging cytotoxic T cell activity. In another aspect, the antigen binding molecule comprising a trimer of 4-1BBL of the present invention or the pharmaceutical composition of the present invention is provided for use in the treatment of cancer, wherein the antigen binding molecule comprising a trimer of 4-1BBL is used in combination with another therapeutic agent, in particular a T cell activating anti-CD 3 bispecific antibody. In one aspect, the T cell activating anti-CD 3 bispecific antibody is administered simultaneously, prior to, or after the antigen binding molecule comprising a trimer of 4-1 BBL.

Furthermore, there is provided the use of an antigen binding molecule comprising a 4-1BBL trimer according to the invention or an antibody according to the invention for the manufacture of a medicament for the treatment of a disease in an individual in need thereof, in particular for the manufacture of a medicament for the treatment of cancer, and a method of treating a disease in an individual comprising administering to said individual a therapeutically effective amount of a composition comprising an antigen binding molecule comprising a 4-1BBL trimer as disclosed herein, said composition being in a pharmaceutically acceptable form. In a particular aspect, the disease is cancer. Further provided is the use of an antigen binding molecule comprising a trimer of 4-1BBL according to the invention for the preparation of a medicament for the treatment of cancer, wherein the antigen binding molecule comprising a trimer of 4-1BBL is used in combination with a T-cell activating anti-CD 3 bispecific antibody, in particular an anti-CEA/anti-CD 3 antibody. Furthermore, a method of treating an individual having cancer is provided, comprising administering to the subject an effective amount of the antigen binding molecule comprising a trimer of 4-1BBL of the invention or a pharmaceutical composition thereof, and an effective amount of a T cell activating anti-CD 3 bispecific antibody, in particular an anti-Her 2/anti-CD 3 antibody. Also provided is a method of up-regulating or prolonging cytotoxic T cell activity in an individual having cancer comprising administering to the individual an effective amount of the antigen binding molecule comprising a trimer of 4-1BBL of the invention or the pharmaceutical composition of the invention. In any of the above embodiments, the individual is preferably a mammal, in particular a human.

Drawings

Figures 1A and 1B show the components used to assemble a monovalent CEA-targeted split trimeric 4-1BB ligand Fc fusion antigen binding molecule. FIG. 1A shows dimeric 4-1BB ligand fused at C-terminus to the human IgG1-CL domain with mutations E123R and Q124K (charged variant), while FIG. 1B shows monomeric 4-1BB ligand fused at C-terminus to the human IgG1-CH1 domain with mutations K147E and K213E (charged variant). Figure 1C schematically illustrates the structure of a monovalent CEA-targeting split trimeric 4-1BB ligand fc (kih) P329G LALA fusion antigen-binding molecule comprising CH-CL crossed by charged residues. The thick black dots represent knob and hole structure modifications. Represents amino acid modifications in the CH1 and CL domains (so-called charged variants).

Figure 2 shows the binding of the humanized A5B7 huIgG 1P 329G LALA variant to MKN-45 compared to the binding of the parent murine A5B7 antibody. The antibody was detected with a fluorescently labeled secondary antibody and fluorescence was measured by flow cytometry.

FIGS. 3A to 3C are schematic representations of recombinant proteins displaying different domains of CEACAM5 protein, which are used as antigens in phage display activities. Figure 3A shows the construct NABA-avi-His consisting of 4 Ig-like domains N, A1, B, and a 2. FIG. 3B shows construct N (A2B2) A-avi-His, and FIG. 3C schematically illustrates construct NA (B2) A-avi-His.

FIGS. 4A and 4B show the VH and VL sequences, respectively, of humanized CEA antibody A5H1EL1D, with random positions labeled with X.

Schematic representations of phage vectors for affinity maturation libraries are shown in FIG. 5A (CDRH1/H2 affinity maturation library), FIG. 5B (CDRL1/H2 affinity maturation library) and FIG. 5C (CDRH3/CDRL3 amplification library).

Figures 6A and 6B show an alignment of the VH amino acid sequence (figure 6A) and VL amino acid sequence (figure 6B) of humanized CEA (A5H1EL1D) antibody variants.

Fig. 7A and 7B show an alignment of VH amino acid sequence (fig. 7A) and VL amino acid sequence (fig. 7B) of humanized MFE23 antibody variants.

Figures 8A, 8B and 8C show binding of the humanized MFE23 huIgG 1P 329G LALA variant to MKN-45 compared to the binding of the parental murine MFE23 antibody. The antibody was detected with a fluorescently labeled secondary antibody and fluorescence was measured by flow cytometry. The three panels show low binding, moderate binding and similar binding to the parent MFE23 clone, respectively.

FIGS. 9A to 9H relate to the simultaneous binding of CEA-targeted trimer cleaving 4-1BBL molecules to hu4-1BB and hu (A2-B2) A or hu (NA1) BA. Fig. 9A shows the assay setup. FIG. 9B shows simultaneous binding of CEA (A5B7) -4-1BBL to huN (A2-B2) A and hu4-1BB-Fc (kih). FIG. 9C shows simultaneous binding of CEA (A5H1EL1D) -4-1BBL to huN (A2-B2) A and hu4-1BB-Fc (kih). FIG. 9D shows simultaneous binding of CEA (MFE23) -4-1BBL to hu (NA1) BA and hu4-1BB-Fc (kih). FIG. 9E shows simultaneous binding of CEA (MFE23-L28-H24) -4-1BBL to hu (NA1) BA and hu4-1BB-Fc (kih). FIG. 9F shows simultaneous binding of CEA (MFE23-L28-H28) -4-1BBL to hu (NA1) BA and hu4-1BB-Fc (kih). FIG. 9G shows simultaneous binding of CEA (P001.177) -4-1BBL to huN (A2-B2) A and hu4-1BB-Fc (kih). FIG. 9H shows simultaneous binding of CEA (P005.102) -4-1BBL to huN (A2-B2) A and hu4-1BB-Fc (kih). Duplicate or triplicate are shown.

The cell surface CEACAM5 expression levels of different clones expressing CEACAM5 used in the binding assay are shown in fig. 10. Chinese hamster ovary cell line CHO-k1(ATCC CRL-9618) was transfected with either cynomolgus monkey CEACAM5(CHO-k1-cyno CEACAM5 clone 8) or human CEACAM5(CHO-k1-huCEACAM5 clone 11, clone 12, clone 13 and clone 17). Expression levels were determined by titration of APC-labeled anti-CD 66 specific detection antibody (clone cd66ab.1.1) using flow cytometry. The median of the fluorescence intensity is shown relative to the concentration of the detection antibody, whereby the median of the fluorescence intensity is positively correlated with the amount of detection antibody bound and therefore with the level of expression of CEACAM5 molecules on the cell surface. CHO-k1-cynoCEACAM5 clone 8 and CHO-k1-huCEACAM5 clone 11 showed similar cell surface CEACAM5 expression, while CHO-k1-huCEACAM5 clones 12, 13 and 17 showed high cell surface CEACAM5 expression levels.

FIGS. 11A to 11E show binding to CHO-k1 cells expressing either Macacam fascicularis CEACAM5 or human CEACAM 5. The concentrations of different bispecific CEA-4-1BBL molecules containing MFE23 (parental) or humanized MFE23(humFE23-L28-H24 or humFE23-L28-H28) or T84.66-LCHA (reference) or control molecules as CEA binders were plotted against the median fluorescence intensity of PE-conjugated secondary detection antibodies. Baseline correction was performed for all values by subtracting baseline values for blank controls (e.g., no primary detection antibody, only secondary detection antibody included). All constructs containing CEACAM5 antigen binding domain were effectively bound to cells expressing human CEACAM5 (fig. 11B, 11C, 11D and 11E). In contrast, none of the CEA binding agents detected binding to cynomolgus monkey CEACAM5 (fig. 11A).

Binding to CHO-k1 cells expressing either Macaca fascicularis CEACAM5 or human CEACAM5 is also shown in FIGS. 12A to 12E. The concentrations of different bispecific CEA-4-1BBL molecules containing A5B7 (parental) or humanized A5B7(A5H1EL1D or MEDI-565) or T84.66-LCHA (reference) or control molecules as CEA binding agents were plotted against the median fluorescence intensity of PE conjugated secondary detection antibodies. Baseline correction was performed for all values by subtracting baseline values for blank controls (e.g., no primary detection antibody, only secondary detection antibody included). All constructs comprising CEACAM5 antigen binding domains bind effectively to cells expressing human CEACAM5 (fig. 12B, 12C, 12D and 12E) and cynomolgus monkey CEACAM5 (fig. 12A), for example the indicated binders are cynomolgus/human cross-reactive.

FIGS. 13A to 13C show binding to CHO-k1 cells expressing either Macacam fascicularis CEACAM5 or human CEACAM 5. The concentration of different bispecific CEA-4-1BBL molecules containing A5B7 (parental) or humanized A5B7(A5H1EL1D or MEDI-565) or affinity matured A5H1EL1D (aff. mat. A5H1el1d P001.177 and aff. mat. A5H1el1d P005.102) or T84.66-LCHA (reference) or control molecules as CEA binding agents was plotted against the median fluorescence intensity of PE-conjugated secondary detection antibodies. Baseline correction was performed for all values by subtracting baseline values for blank controls (e.g., no primary detection antibody, only secondary detection antibody included). All constructs containing CEACAM5 antigen binding domain bound efficiently to human CEACAM5 expressing cells (fig. 13B and 13C) and cynomolgus CEACAM5 (fig. 13A), e.g. the indicated binders were cynomolgus/human cross-reactive, whereas binder A5H1EL1D showed limited affinity. Affinity maturation increased the affinity of A5H1EL1D for human CEACAM5, and in the case of affinity maturation, A5H1EL1D P005.102 also increased the affinity for cynomolgus monkey CEACAM 5.

FIGS. 14A to 14D show NF-. kappa.B-mediated luciferase expression activity in 4-1 BB-expressing reporter cell line Jurkat-hu4-1 BB-NF-. kappa.B-luc 2. To test the functionality of the bispecific CEA-4-1BBL antigen binding molecule compared to a control, the molecule was incubated with the reporter cell line Jurkat-hu4-1BB-NF κ B-luc2 at a ratio of 1:5 in the absence or presence of CHO-k1 cell line expressing cynomolgus monkey or human CEACAM5 for 5 h. The concentration of CEA-4-1BBL molecule or control thereof is plotted against the units of emitted light (RLU) measured 5h after incubation and addition of luciferase assay solution. Baseline correction was performed for all values by subtracting the baseline values of the blank control (e.g., no antibody added). In relation to binding assay molecules containing anti-human CEACAM5 specific clone MFE23 (parental) or humanized versions (huMFE23-L28-H24, huMFE23-L28-H28, sm9B) or reference clone T84.66-LCHA, the molecules bound and cross-linked by human CEACAM5 and thus induced 4-1 BB-mediated activation of the reporter cell line Jurkat-hu4-1BB-nfkb-luc2 (fig. 14C and 14D). In contrast, in the absence of human CEACAM5 (fig. 14A) or in the presence of macaca fascicularis CEACAM5, no activation was induced (fig. 14B).

FIGS. 15A to 15D also show the NF-. kappa.B-mediated luciferase expression activity in the 4-1BB expressing reporter cell line Jurkat-hu4-1 BB-NF-. kappa.B-luc 2. To test the functionality of the bispecific CEA-4-1BBL antigen binding molecule compared to a control, the molecule was incubated with the reporter cell line Jurkat-hu4-1BB-NF κ B-luc2 at a ratio of 1:5 in the absence or presence of CHO-k1 cell line expressing cynomolgus monkey or human CEACAM5 for 5 h. The concentration of CEA-4-1BBL molecule or control thereof is plotted against the units of emitted light (RLU) measured 5h after incubation and addition of luciferase assay solution. Baseline correction was performed for all values by subtracting the baseline values of the blank control (e.g., no antibody added). In association with binding assay molecules containing either the anti-human CEACAM5 specific clone A5B7 (parental) or humanized version (A5H1EL1D or MEDI-565) or affinity matured version (P005.102 and P001.177) or reference clone T84.66-LCHA, the molecules bound and crosslinked by human CEACAM5, thus inducing 4-1 BB-mediated activation of the reporter cell line Jurkat-hu4-1BB-NFkB-luc2 (fig. 15C and 15D). In contrast, there was no molecular induced activation in the absence of human CEACAM5 (fig. 15A). In the presence of Macacam fascicularis CEACAM5, only the molecule containing the A5B 7-derived clone induced activity, whereas CEA (T84.77-LCHA) -4-1BBL did not (FIG. 15B).

Detailed Description

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purpose of interpreting the specification, the following definitions will apply and, where appropriate, terms used in the singular will also include the plural and vice versa.

As used herein, the term "antigen binding molecule" refers in its broadest sense to a molecule that specifically binds to an antigenic determinant. Examples of antigen binding molecules are antibodies, antibody fragments and scaffold antigen binding proteins.

The term "antigen binding domain" refers to a portion of an antigen binding molecule that comprises a region that specifically binds to and is complementary to a portion or all of an antigen. In the case of large antigens, the antigen binding molecule may bind only a specific part of the antigen, which part is called an epitope. The antigen binding domain may be provided by, for example, one or more variable domains (also referred to as variable regions). Preferably, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

As used herein, the term "antigen binding domain capable of specifically binding to CEA" or "moiety capable of specifically binding to CEA" refers to a polypeptide molecule that specifically binds to CEA. In one aspect, the antigen binding domain is capable of activating or inhibiting signaling by CEA. In a particular aspect, the antigen binding domain is capable of directing the entity (e.g., 4-1BBL trimer) to which it is attached to a target site, e.g., to a particular type of tumor cell that carries CEA on its surface. Antigen binding domains capable of specifically binding to CEA include antibodies and fragments thereof as further defined herein. With respect to antibodies or fragments thereof, the term "portion capable of specifically binding to a target cell antigen" refers to a portion of a molecule that comprises a region that specifically binds to and is complementary to part or all of an antigen. The moiety capable of specific antigen binding may be provided, for example, by one or more antibody variable domains (also referred to as antibody variable regions). In particular, the moiety capable of specific antigen binding includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). By "specifically binds," it is meant that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions.

The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen binding activity.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of a monoclonal antibody preparation, such variants typically being present in minor amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen.

As used herein, the term "monospecific" antibody refers to an antibody having one or more binding sites, each binding site binding to the same epitope of the same antigen. The term "bispecific" means that the antigen binding molecule is capable of specifically binding to at least two distinct antigenic determinants. Typically, bispecific antigen binding molecules comprise two antigen binding sites, each of which is specific for a different antigenic determinant. In certain embodiments, the bispecific antigen binding molecule is capable of binding two antigenic determinants simultaneously, particularly two antigenic determinants expressed on two distinct cells. The term "multispecific" means a unique antigenic determinant with which an antigen-binding molecule is capable of binding at least two specificities. The multispecific antigen-binding molecule may be, for example, a bispecific antigen-binding molecule.

The term "valency" as used in this application denotes the presence of a specified number of binding sites in an antigen binding molecule. Thus, the terms "monovalent", "divalent", "tetravalent" and "hexavalent" indicate the presence of one binding site, two binding sites, four binding sites and six binding sites, respectively, in an antigen binding molecule.

The terms "full-length antibody" and "intact antibody" are used interchangeably herein to refer to an antibody having a structure that is substantially similar to the structure of a native antibody. "native antibody" refers to a native immunoglobulin molecule having a different structure. For example, a natural IgG class antibody is a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two light and two heavy chains that are disulfide-bonded. From N-terminus to C-terminus, each heavy chain has a variable region (VH) (also known as the variable heavy chain domain or heavy chain variable domain) followed by three constant domains (CH1, CH2, and CH3) (also known as heavy chain constant regions). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL) (also known as a variable light chain domain or light chain variable domain) followed by a light chain constant domain (CL) (also known as a light chain constant region). The heavy chain of an antibody may be assigned to one of five types, referred to as α (IgA), δ (IgD), epsilon (IgE), γ (IgG), or μ (IgM), some of which may be further divided into subtypes such as γ 1(IgG1), γ 2(IgG2), γ 3(IgG3), γ 4(IgG4), α 1(IgA1), and α 2(IgA 2). The light chain of an antibody can be assigned to one of two types, called kappa (. kappa.) and lambda (. lamda.), based on the amino acid sequence of its constant domain.

An "antibody fragment" refers to a molecule other than a whole antibody that comprises a portion of a whole antibody that binds to an antigen to which the whole antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab '-SH, F (ab')₂(ii) a Diabodies, triabodies, tetrabodies, cross-Fab molecules; a linear antibody; single chain antibody molecules (e.g., scFv); and single domain antibodies. For a review of certain antibody fragments, see Hudson et al, Nat Med 9, 129-. For reviews on scFv fragments see, for example, Plouckthun, published on The Pharmacology of Monoclonal Antibodies, Vol.113, Rosenburg and Moore eds, Springer-Verlag, New York, pp.269 to 315 (1994); see also WO 93/16185; and U.S. patent nos. 5,571,894 and 5,587,458. For a discussion of Fab fragments and F (ab')2 fragments that contain salvage receptor binding epitope residues and have extended half-lives in vivo, see U.S. Pat. No. 5,869,046. Diabodies, which can be bivalent or bispecific, are antibody fragments with two antigen binding sites, see, e.g., EP 404,097; WO 1993/01161; hudson et al, Nat Med 9, 129-; and Hollinger et al, Proc Natl Acad Sci USA 90, 6444-. Tri-and tetrad antibodies are also described in Hudson et al, Nat Med 9,129-134 (2003). A single domain antibody is an antibody fragment comprising all or part of a heavy chain variable domain of an antibody or all or part of a light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516B 1). Antibody fragments can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies, and production from recombinant host cells (e.g., e.

Papain digestion of whole antibodies produces two identical antigen-binding fragments, called "Fab" fragments, eachThe "Fab" fragment contains the heavy and light chain variable domains and the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Thus, as used herein, the term "Fab fragment" or "Fab molecule" refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a light chain constant domain (CL), and a VH domain of a heavy chain and a first constant domain (CH 1). Fab 'fragments differ from Fab fragments in that the Fab' fragment has added to the carboxy terminus of the heavy chain CH1 domain residues that include one or more cysteines from the antibody hinge region. Fab '-SH is a Fab' fragment in which the cysteine residues of the constant domains carry a free thiol group. Pepsin treatment to yield F (ab')₂A fragment having two antigen binding sites (two Fab fragments) and a portion of an Fc region.

The term "crossover Fab molecule" or "crossover Fab fragment" or "xFab fragment" or "crossover Fab molecule" refers to a Fab fragment in which the variable or constant regions of the heavy and light chains are exchanged. Two different chain compositions of the crossover Fab molecule are possible and are comprised in the bispecific antibody of the invention: in one aspect, the variable regions of the Fab heavy and light chains are exchanged, i.e., the exchanged Fab molecule comprises a peptide chain consisting of the light chain variable region (VL) and the heavy chain constant region (CH1), and a peptide chain consisting of the heavy chain variable region (VH) and the light chain constant region (CL). This exchanged Fab molecule is also called CrossFab _(VLVH). On the other hand, when the constant regions of the Fab heavy and light chains are exchanged, the exchanged Fab molecule comprises a peptide chain consisting of the heavy chain variable region (VH) and the light chain constant region (CL), and a peptide chain consisting of the light chain variable region (VL) and the heavy chain constant region (CH 1). This exchanged Fab molecule is also called CrossFab_(CLCH1)。

A "single chain Fab fragment" or "scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1(CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein the antibody domain and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH 1-linker-VL-CL, b) VL-CL-linker-VH-CH 1, c) VH-CL-linker-VL-CH 1, or d) VL-CH 1-linker-VH-CL; and wherein the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single chain Fab fragment is stabilized via the native disulfide bond between the CL domain and the CH1 domain. Furthermore, these single chain Fab molecules may be further stabilized by creating interchain disulfide bonds via insertion of cysteine residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

An "exchange-type single chain Fab fragment" or "x-scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1(CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein the antibody domain and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CL-linker-VL-CH 1 and b) VL-CH 1-linker-VH-CL; wherein VH and VL together form an antigen binding site that specifically binds to an antigen, and wherein the linker is a polypeptide of at least 30 amino acids. In addition, these x-scFab molecules can be further stabilized by creating interchain disulfide bonds via insertion of cysteine residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain, according to Kabat numbering).

"Single chain variable fragment (scFv)" is the variable region of the heavy chain (V) of an antibody_H) And light chain variable region (V)_L) The fusion proteins of (a), linked by a short connecting peptide of 10 to about 25 amino acids. The linker is generally glycine rich for flexibility and serine or threonine rich for solubility, and V may be substituted_HN-terminal of (5) and V_LOr vice versa. Despite the removal of the constant region and the introduction of the linker, the specificity of the original antibody is retained by the protein. scFv antibodies are described, for example, in Houston, J.S., Methods in enzymol.203(1991) 46-96). In addition, antibody fragments comprise single chain polypeptides characterized by a VH domain, i.e., capable of assembly with a VL domain to a functional antigen binding site; or a VL domain, i.e.capable of assembling with a VH domain to a functional antigen binding site, thereby providing an antigen junction of a full length antibody And (4) synthesizing the characteristics.

"scaffold antigen binding proteins" are known in the art, e.g., fibronectin and designed ankyrin repeat proteins (DARPins) have been used as alternative scaffolds for antigen binding domains, see, e.g., Gebauer and Skerra, Engineered protein scaffold as next-generation antibody therapy. curr Opin Chem Biol 13: 245-. In one aspect of the invention, the scaffold antigen binding protein is selected from the group consisting of: CTLA-4(Evibody), lipocalin (Anticalin), protein a-derived molecules such as the Z-domain of protein a (affibody), a-domain (Avimer/macroantibody), serum transferrin (trans-body); designed ankyrin repeat proteins (darpins), variable domains of antibody light or heavy chains (single domain antibodies, sdabs), variable domains of antibody heavy chains (nanobodies, aVH), V_NARFragments, fibronectin (AdNectin), C-type lectin domains (tetranectin); variable domain (V) of the neoantigen receptor beta-lactamase_NARFragments), human gamma-crystallin or ubiquitin protein (Affilin molecules); the kunitz-type domain of human protease inhibitors, minibodies (such as proteins from the knottin family), peptide aptamers, and fibronectin (adnectins). CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) is predominantly CD4 ⁺The CD28 family of receptors expressed on T cells. Its extracellular domain has a variable domain-like Ig fold. The loops corresponding to the CDRs of the antibody can be substituted with heterologous sequences to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also known as evibods (e.g., US7166697B 1). Evibody is about the same size as the isolated variable region of an antibody (e.g., a domain antibody). For further details, see Journal of Immunological Methods 248(1-2),31-45 (2001). Lipocalins are a family of extracellular proteins that transport small hydrophobic molecules, such as steroids, cholesterol, retinoids, and lipids. They have a rigid β -sheet secondary structure with many loops at the open ends of the cone structure, and can be engineered to bind different target antigens.Anticalin is between 160-180 amino acids in size and is derived from lipocalin. For further details, see Biochim Biophys Acta 1482:337-350(2000), US7250297B1 and US 20070224633. Affibodies are scaffolds of protein a derived from Staphylococcus aureus (Staphylococcus aureus), which can be engineered to bind antigen. This domain consists of a triple helix bundle of about 58 amino acids. Libraries have been formed by randomization of surface residues. For further details, see Protein Eng.Des.Sel.2004,17,455-462 and EP 1641818A 1. Avimer is a multidomain protein derived from the a domain scaffold family. The native domain of about 35 amino acids adopts a defined disulfide bonding structure. Diversity is created by natural variation exhibited by the recombinant a domain family. For further details, see Nature Biotechnology 23(12), 1556-. Transferrin is a monomeric serum transport glycoprotein. Transferrin can be engineered by inserting peptide sequences in permissive surface loops to bind different target antigens. Examples of engineered transferrin scaffolds include the trans body. For further details, see J.biol.chem 274,24066-24073 (1999). The designed ankyrin repeat protein (DARPin) is derived from ankyrin, a family of proteins that mediate the attachment of integral membrane proteins to cell scaffolds. The single ankyrin repeat is a 33 residue motif consisting of two alpha helices and one beta turn. They can be engineered to bind different target antigens by randomizing residues in the first alpha-helix and beta-turn in each repeat sequence. Their binding interface can be increased by increasing the number of modules (affinity maturation method). For further details, see J.mol.biol.332,489-503(2003), PNAS 100(4),1700-1705(2003) and J.mol.biol.369,1015-1028(2007) and US20040132028A 1. Single domain antibodies are antibody fragments consisting of a single monomeric variable antibody domain. The first single domain is derived from the variable domain of the heavy chain of an antibody of the camelid family (nanobody or V) _HH fragment). Furthermore, the term single domain antibody comprises autologous human heavy chain variableDomain (aVH) or shark derived V_NARAnd (3) fragment. Fibronectin can be engineered to bind to a scaffold of an antigen. Adnectin consists of a backbone of the native amino acid sequence of domain 10 of the 15 repeat unit of human fibronectin type III (FN 3). The three loops at one end of the β -sandwich can be engineered to enable the Adnectin to specifically recognize the target therapeutic target of interest. For further details, see Protein eng.des.sel.18,435-444(2005), US20080139791, WO2005056764, and US6818418B 1. Peptide aptamers are combinatorial recognition molecules consisting of a constant scaffold protein, usually thioredoxin (TrxA), containing a constrained variable peptide loop inserted at the active site. For further details, see Expert opin. biol. ther.5,783-797 (2005). The minibodies are derived from naturally occurring miniproteins of 25-50 amino acids in length containing 3-4 cysteine bridges, examples of which include KalataBI and conotoxins, and knottin. The micro-proteins have loops that can be engineered to include up to 25 amino acids without affecting the overall folding of the micro-protein. For further details on engineered knottin domains see WO 2008098796.

Lipocalins are a family of extracellular proteins that transport small hydrophobic molecules, such as steroids, cholesterol, retinoids, and lipids. They have a rigid β -sheet secondary structure with many loops at the open ends of the cone structure, and can be engineered to bind different target antigens. Anticalin is between 160-180 amino acids in size and is derived from lipocalin. For further details, see Biochim Biophys Acta 1482:337-350(2000), Biodrugs 19(5),279-288(2005), US7250297B1 and US 20070224633.

"antigen-binding molecule that binds to the same epitope" as a reference molecule refers to an antigen-binding molecule that blocks binding of the reference molecule to its antigen by 50% or more in a competition assay, and conversely, blocks binding of the antigen-binding molecule to its antigen by 50% or more in a competition assay.

As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site (e.g., a contiguous stretch of amino acids or a conformational configuration composed of different regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen-binding portion binds, thereby forming an antigen-binding portion-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, in serum free and/or in extracellular matrix (ECM). Unless otherwise indicated, a protein used herein as an antigen can be any native form of the protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In a particular embodiment, the antigen is a human protein. When referring to a particular protein herein, the term encompasses "full-length," unprocessed protein, as well as any form of protein that results from intracellular processing. The term also encompasses naturally occurring protein variants, such as splice variants or allelic variants.

The term "carcinoembryonic antigen (CEA)" also referred to as carcinoembryonic antigen-associated cell adhesion molecule 5(CEACAM5) refers to any native 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 indicated. The amino acid sequence of human CEA is shown in UniProt accession number P06731 (version 151, SEQ ID NO: 108). CEA has long been identified as a tumor associated antigen (Gold and Freedman, J Exp Med., 121: 439-. CEA was originally classified as a protein expressed only in fetal tissues and has now been identified in a variety of normal adult tissues. These tissues are mainly of epithelial origin, including cells of the gastrointestinal, respiratory and genitourinary tracts as well as cells of the colon, cervix, sweat glands and prostate (Nap et al, Tumour biol., 9(2-3):145-53, 1988; Nap et al, Cancer Res.,52(8):2329-23339, 1992). Both epithelial-derived tumors and their metastases comprise CEA as a tumor-associated antigen. The presence of CEA itself does not indicate that it has been transformed into cancer cells, but the distribution of CEA is indicative. In normal tissues, CEA is normally expressed on the apical surface of cells (S) ((R)) S. Semin Cancer biol.9(2):67-81(1999)), rendering it unabsorbable by antibodies in the bloodstream. CEA tends to be expressed on the entire surface of cancer cells compared to normal tissues: (S., Semin Cancer biol.9(2):67-81 (1999)). This change in expression pattern allows CEA to readily bind to antibodies in cancer cells. Furthermore, expression of CEA in cancer cells is increased. Furthermore, an increase in CEA expression promotes an increase in intercellular adhesion, which may lead to metastasis (Marshall J., Semin Oncol.,30(a suppl.8):30-6,2003). Expression of CEA in various tumor entities is generally very high. Based on published data, the high incidence of CEA has been demonstrated in its own analyses in tissue samples, with an incidence of about 95% in large bowel cancer (CRC), 90% in pancreatic cancer, 80% in gastric cancer, 60% in non-small cell lung cancer (NSCLC, co-expressed with HER 3) and 40% in breast cancer; and found to be low in expression levels in small cell lung cancer and glioblastoma.

CEA is readily cleaved from the cell surface and flows from the tumor into the bloodstream, either directly or through lymphatic vessels. Because of this property, serum CEA levels have been used as a clinical index for diagnosing Cancer and screening for recurrence of Cancer, particularly colorectal Cancer (Goldenberg D M., The International Journal of Biological Markers, 7:183-188, 1992; Chau I et al, J Clin Oncol., 22:1420-1429, 2004; Flarni et al, Clin Cancer Res; 12(23): 6985-6986, 2006).

The term "anti-CEA antigen binding molecule" or "antigen binding molecule capable of specifically binding to CEA" refers to an antigen binding moiety capable of binding with sufficient affinity to CEAAnd (b) a synthetic molecule, such that the antigen binding molecule is useful as a diagnostic and/or therapeutic agent targeting CEA. Antigen binding molecules include, but are not limited to, antibodies, Fab molecules, exchange Fab molecules, single chain Fab molecules, Fv molecules, scFv molecules, single domain antibodies, and VH and scaffold antigen binding proteins. In one aspect, the extent of binding of the anti-CEA antigen binding molecule to an unrelated, non-CEA protein is less than about 10% of the binding of the antigen binding molecule to CEA, as measured, for example, by Surface Plasmon Resonance (SPR). In particular, antigen binding molecules capable of specifically binding to CEA have the following dissociation constant (K)_d): less than or equal to 1 μ M, less than or equal to 500nM, less than or equal to 200nM, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, less than or equal to 0.1nM, less than or equal to 0.01nM or less than 0.001nM (e.g., 10 nM)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M). In certain aspects, the anti-CEA antigen binding molecule binds to CEA from a different species. In particular, the anti-CEA antigen binding molecules bind to human and cynomolgus monkey CEA.

The term "epitope" refers to the site on the antigen of a protein or non-protein to which an anti-CEA antibody binds. Epitopes can be formed from contiguous stretches of amino acids (linear epitopes) or comprise non-contiguous amino acids (conformational epitopes), for example due to spatial proximity by antigen folding, i.e. by tertiary folding of the protein antigen. Linear epitopes are typically still bound by antibodies after exposure of the protein antigen to a denaturant, whereas conformational epitopes are typically destroyed after treatment with the denaturant. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique conformation.

By "specific binding" is meant that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen-binding molecule to bind to a particular antigen can be measured by enzyme-linked immunosorbent assays (ELISAs) or other techniques familiar to those skilled in the art (e.g., Surface Plasmon Resonance (SPR) techniques (analyzed on a BIAcore instrument) (Liljeblad et al, Glyco J17, 323-329(2000)) as well as conventional binding assays (Heeley, Endocr Res28,217-229(2002))The extent of binding of the antigen binding molecule to an unrelated protein is less than about 10% of the extent of binding of the antigen binding molecule to an antigen as measured by SPR. In certain embodiments, the dissociation constant (Kd) of the molecule that binds to the antigen is less than or equal to 1 μ M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, less than or equal to 0.1nM, less than or equal to 0.01nM or less than or equal to 0.001nM (e.g., 10 nM)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M)。

"affinity" or "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise specified, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (Kd), which is the dissociation and association rate constants (k, respectively) _offAnd k_on) The ratio of. Thus, equivalent affinities may comprise different rate constants, as long as the ratio of rate constants remains the same. Affinity can be measured by conventional methods known in the art, including those described herein. A particular method for measuring affinity is Surface Plasmon Resonance (SPR).

As used herein, the term "affinity matured" in the context of an antigen binding molecule (e.g., an antibody) refers to an antigen binding molecule derived from a reference antigen binding molecule, e.g., by mutation, binds to the same antigen as the reference antibody, preferably binds to the same epitope; and has a higher affinity for the antigen than the reference antigen binding molecule. Affinity maturation typically involves modification of one or more amino acid residues in one or more CDRs of an antigen binding molecule. Typically, affinity matured antigen binding molecules bind to the same epitope as the original reference antigen binding molecule.

As used herein, "target cell antigen" refers to an antigenic determinant presented on the surface of a target cell, e.g., a T cell, a B cell, a cell in a tumor such as a cancer cell, or a tumor stroma. In certain aspects, the target cell antigen is an antigen on the surface of a cancer cell. In one aspect, the target cell antigen is CEA.

The term "variable domain" or "variable region" refers to a domain of an antibody heavy or light chain that is involved in the binding of an antigen binding molecule to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved Framework Regions (FR) and three hypervariable regions (HVRs). See, e.g., Kindt et al, Kuby Immunology, 6 th edition, w.h.freeman and co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity.

As used herein, the term "hypervariable region" or "HVR" refers to the various regions of an antigen-binding variable domain which are hypervariable in sequence and determine antigen-binding specificity, e.g., "complementarity determining regions" ("CDRs"). Typically, the antigen binding domain comprises six CDRs; three in VH (CDR-H1, CDR-H2, CDR-H3) and three in VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include:

(a) highly variable loops occurring at the following amino acid residues: 26 to 32(L1), 50 to 52(L2), 91 to 96(L3), 26 to 32(H1), 53 to 55(H2) and 96 to 101(H3) (Chothia and Lesk, J.mol.biol.196:901-917 (1987));

(b) CDRs appearing at the following amino acid residues: 24 to 34(L1), 50 to 56(L2), 89 to 97(L3), 31 to 35b (H1), 50 to 65(H2) and 95 to 102(H3) (Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and

(c) Antigen contacts that occur at the following amino acid residues: 27c to 36(L1), 46 to 55(L2), 89 to 96(L3), 30 to 35b (H1), 47 to 58(H2), and 93 to 101(H3) (MacCallum et al, J.mol.biol.262:732-745 (1996)).

Unless otherwise indicated, the CDRs are determined according to the methods described by Kabat et al, supra. Those skilled in the art will appreciate that the CDR names may also be determined according to the method described by Chothia supra, McCallum supra, or any other scientifically accepted nomenclature. Kabat et al also define a numbering system for the variable region sequences applicable to any antibody. One of ordinary skill in the art can unambiguously assign this "Kabat numbering" system to any variable region sequence, without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al, U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise indicated, reference to the numbering of specific amino acid residue positions in the variable region of an antibody is according to the Kabat numbering system.

"framework" or "FR" refers to variable domain residues other than the Complementarity Determining Regions (CDRs). The FRs of a variable domain typically consist of the following four FR domains: FR1, FR2, FR3 and FR 4. Thus, CDR and FR sequences typically occur in VH (or VL) as follows: FR1-CDR-H1(CDR-L1) -FR2-CDR-H2(CDR-L2) -FR3-CDR-H3(CDR-L3) -FR 4.

For purposes herein, an "acceptor human framework" is a framework comprising an amino acid sequence derived from a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework of a human immunoglobulin framework or a human consensus framework as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or human consensus framework may comprise the same amino acid sequence as the human immunoglobulin framework or human consensus framework, or it may comprise amino acid sequence variations. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The "class" of antibodies refers to the type of constant domain or constant region that the heavy chain of an antibody has. There are five major classes of antibodies: IgA, IgD, IgE, IgG and IgM, and several of these classes may be further divided into subclasses (isotypes), e.g. IgG₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively.

The term "constant region derived from human origin" or "human constant region" denotes the constant heavy chain region and/or constant light chain kappa or lambda region of a human antibody of subclass IgG1, IgG2, IgG3 or IgG 4. Such constant regions are well known in the art and are described, for example, by: kabat, E.A., et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also, e.g., Johnson, G., and Wu, T.T., Nucleic Acids Res.28(2000) 214-. Unless otherwise specified herein, the numbering of amino acid residues in the constant region is according to the EU numbering system, also known as the EU index of Kabat, as described in Kabat, E.A. et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

The term "CH 1 domain" denotes the portion of an antibody heavy chain polypeptide extending substantially from EU 118 to EU 215 (EU numbering system according to Kabat). In one aspect, the CH1 domain has the amino acid sequence ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKV (SEQ ID NO: 301). Typically, a fragment having the amino acid sequence of EPKSC (SEQ ID NO:302) follows to connect the CH1 domain with the hinge region.

The term "hinge region" denotes the portion of an antibody heavy chain polypeptide that connects the CH1 domain and the CH2 domain in a wild-type antibody heavy chain, e.g., from position 216 to position 230 according to the EU numbering system of Kabat, or from position 226 to position 230 according to the EU numbering system of Kabat. The hinge region of the other IgG subclasses can be determined by alignment with the hinge region cysteine residues of the IgG1 subclass sequence. The hinge region is typically a dimeric molecule consisting of two polypeptides with identical amino acid sequences. The hinge region typically comprises up to 25 amino acid residues and is flexible, allowing independent movement of the associated target binding site. The hinge region can be subdivided into three domains: an upper hinge region, a middle hinge region, and a lower hinge region (see, e.g., Roux, et al, j. immunol.161(1998) 4083).

In one aspect, the hinge region has the amino acid sequence DKTHTCPXCP (SEQ ID NO:303), wherein X is S or P. In one aspect, the hinge region has the amino acid sequence HTCPXCP (SEQ ID NO:304), where X is S or P. In one aspect, the hinge region has the amino acid sequence CPXCP (SEQ ID NO:305), where X is S or P.

The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which contains at least part of a constant region. The term includes native sequence Fc domains and variant Fc domains. In one aspect, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxy terminus of the heavy chain. However, the antibody produced by the host cell may undergo post-translational cleavage of one or more (in particular one or two) amino acids from the C-terminus of the heavy chain. Thus, an antibody produced by a host cell by expression of a particular nucleic acid molecule encoding a full-length heavy chain may comprise the full-length heavy chain, or the antibody may comprise a cleaved variant of the full-length heavy chain. This may be the case for the last two C-terminal amino acids of the heavy chain, glycine (G446) and lysine (K447, according to the EU index). Thus, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. The amino acid sequence of the heavy chain comprising the Fc region is represented herein as without the C-terminal glycine-lysine dipeptide if not otherwise indicated. In one aspect, a heavy chain comprising an Fc region as specified herein comprising an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the EU index) is comprised in an antigen binding molecule according to the present invention. In one aspect, a heavy chain comprising an Fc region as specified herein is comprised in an antigen binding molecule according to the invention, the heavy chain comprising an additional C-terminal glycine residue (G446, numbering according to the EU index). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

The IgG Fc region comprises an IgG CH2 domain and an IgG CH3 domain. The "CH 2 domain" of the human IgG Fc region typically extends from amino acid residue at approximately position 231 to amino acid residue at approximately position 340. In one aspect, the carbohydrate chain is linked to a CH2 domain. The "CH 2 domain" of the human IgG Fc region typically extends from amino acid residue at about EU 231 to amino acid residue at about EU 340 (EU numbering system according to Kabat). In one aspect, the CH2 domain has the amino acid sequence APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQESTYRW SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAK (SEQ ID NO: 299). The CH2 domain is unique in that it is not closely paired with another domain. In contrast, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of the intact native Fc region. It is speculated that carbohydrates may provide alternatives to the domain-domain pair and help stabilize the CH2 domain. Burton, mol.Immunol.22(1985) 161-206. In one embodiment, the carbohydrate chain is attached to a CH2 domain. The CH2 domain herein may be the native sequence CH2 domain or a variant CH2 domain.

The "CH 3 domain" comprises a stretch of residues from the C-terminus to the CH2 domain in the Fc region (i.e., from the amino acid residue at about position 341 to the amino acid residue at about position 445 of an IgG, according to the EU numbering system of Kabat). In one aspect, the CH3 domain has an amino acid sequence of GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSP (SEQ ID NO: 300). The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain with an introduced "knob" in one strand and a corresponding introduced "cavity" in the other strand (a "mortar"); see U.S. Pat. No. 5,821,333, expressly incorporated herein by reference). Such variant CH3 domains may be used to promote heterodimerization of two non-identical antibody heavy chains as described herein.

"knob-into-hole" techniques are described in, for example, US 5,731,168; US 7,695,936; ridgway et al, Prot Eng 9,617- & 621(1996) and Carter, J Immunol Meth 248,7-15 (2001). Generally, the method involves introducing a bulge ("knob") at the interface of the first polypeptide and a corresponding cavity ("hole") in the interface of the second polypeptide such that the bulge can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. The bulge is constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities having the same or similar size as the projections are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). The projections and cavities can be prepared by altering the nucleic acid encoding the polypeptide (e.g., by site-specific mutagenesis or by peptide synthesis). In a particular embodiment, the knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, while the hole modification comprises the amino acid substitutions T366S, L368A and Y407V in the other of the two subunits of the Fc domain. In another specific embodiment, the subunit comprising a knob modified Fc domain further comprises the amino acid substitution S354C, and the subunit comprising a hole modified Fc domain further comprises the amino acid substitution Y349C. The introduction of these two cysteine residues results in the formation of disulfide bridges between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J immunological Methods 248,7-15 (2001)). Numbering is the EU index according to Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

"region equivalent to the Fc region of an immunoglobulin" is intended to include naturally occurring allelic variants of the Fc region of an immunoglobulin, as well as modified variants having the ability to make substitutions, additions or deletions without substantially reducing the ability of the immunoglobulin to mediate effector functions, such as antibody-dependent cellular cytotoxicity. For example, one or more amino acids may be deleted from the N-terminus or C-terminus of an Fc region of an immunoglobulin without substantial loss of biological function. Such variants may be selected according to general rules known in the art so as to have minimal effect on activity (see, e.g., Bowie, J.U. et al, Science 247:1306-10 (1990)).

The term "wild-type Fc domain" denotes an amino acid sequence identical to the amino acid sequence of an Fc domain found in nature. Wild-type human Fc domains include native human IgG1 Fc region (non-a and a allotypes), native human IgG2 Fc region, native human IgG3 Fc region, and native human IgG4 Fc region, as well as naturally occurring variants thereof. The wild-type Fc domain is contained in SEQ ID NO 306(IgG1, Caucasian allotype), SEQ ID NO 307(IgG1, African American allotype), SEQ ID NO 308(IgG2), SEQ ID NO 309(IgG3), and SEQ ID NO 310(IgG 4).

The term "variant (human) Fc domain" denotes an amino acid sequence that differs from the amino acid sequence of a "wild-type" (human) Fc domain by at least one "amino acid mutation". In one aspect, the variant Fc region has at least one amino acid mutation as compared to the native Fc region, such as from about 1 to about 10 amino acid mutations in the native Fc region, and in one aspect, from about 1 to about 5 amino acid mutations. In one aspect, the (variant) Fc region has at least about 95% homology to a wild-type Fc region.

A "humanized" antibody is a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise 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. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. An antibody, e.g., a non-human antibody, in "humanized form" refers to an antibody that has been subjected to humanization. Other forms of "humanized antibodies" encompassed by the present invention are antibodies in which the constant regions have been otherwise modified or altered relative to the original antibody to produce properties according to the present invention, particularly with respect to C1q binding and/or Fc receptor (FcR) binding.

The term "effector function" refers to those biological activities that can be attributed to the Fc region of an antibody that vary with the isotype of the antibody. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC), Fc receptor binding, antibody dependent cell mediated cytotoxicity (ADCC), Antibody Dependent Cellular Phagocytosis (ADCP), cytokine secretion, immune complex mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

An "activating Fc receptor" is an Fc receptor that, upon engagement of the Fc region of an antibody, causes a signaling event that stimulates receptor-bearing cells to perform effector functions. Activating Fc receptors include Fc γ RIIIa (CD16a), Fc γ RI (CD64), Fc γ RIIa (CD32), and Fc α RI (CD 89). A particular activating Fc receptor is human Fc γ RIIIa (see UniProt accession No. P08637, version 141).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that results in the lysis of antibody-coated target cells by immune effector cells. The target cell is a cell to which an antibody or derivative thereof comprising an Fc region specifically binds, typically via a protein moiety at the N-terminus of the Fc region. The term "reduced ADCC" as used herein is defined as a reduction in the number of target cells lysed in a given time by the ADCC mechanism as defined above at a given concentration of antibody in the medium surrounding the target cells and/or an increase in the concentration of antibody in the medium surrounding the target cells required to achieve lysis of a given number of target cells in a given time by the ADCC mechanism. The reduction in ADCC is relative to ADCC mediated by the same antibody produced by the same type of host cell using the same standard production, purification, formulation and storage methods (known to those skilled in the art) but not engineered. For example, the reduction in ADCC mediated by an antibody comprising an amino acid substitution in its Fc domain that reduces ADCC is relative to ADCC mediated by the same antibody without such amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, e.g., PCT publication No. WO 2006/082515 or PCT publication No. WO 2012/130831)

The term "TNF ligand family member" or "TNF family ligand" refers to a proinflammatory cytokine. In general, cytokines, and in particular members of the TNF ligand family, play a crucial role in the stimulation and coordination of the immune system. Currently, nineteen 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, called the TNF Homeodomain (THD), has 20-30% amino acid identity between superfamily members and is responsible for receptor binding. The TNF ectodomain is also responsible for the formation of TNF ligands into trimeric complexes recognized by their specific receptors. The member of the TNF ligand family is selected from the group consisting of: lymphotoxin alpha (also referred to as LTA or TNFSF1), TNF (also referred to as TNFSF2), LT beta (also referred to as TNFSF3), OX40L (also referred to as TNFSF4), CD40L (also referred to as CD154 or TNFSF5), FasL (also referred to as CD95L, CD178 or TNFSF6), CD27L (also referred to as CD70 or TNFSF7), CD30L (also referred to as CD153 or TNFSF L), 4-1BBL (also referred to as TNFSF L), TRAIL (also referred to as APO2L, CD253 or TNFSF L), RANKL (also referred to as CD254 or TNFSF L), TWEAK (also referred to as TNFSF L), APRIL (also referred to as CD256 or TNFSF L), BAFF (also referred to as CD257 or TNFSF 13L), LIGHT (also referred to as CD258 or TNFSF L), twtl 1 or TNFSF L), ecto gi (also referred to as TNFSF L), and ectopic protein (also referred to as TNFSF L). Unless otherwise indicated, the term refers to any 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). The term "co-stimulatory TNF ligand family members" or "co-stimulatory TNF family ligands" refers to a subset of TNF ligand family members that are capable of synergistically stimulating T cell proliferation and cytokine production. These TNF family ligands can collectively stimulate TCR signaling upon interaction with their corresponding TNF receptors, and interaction with their receptors results in the recruitment of TNFR-related factors (TRAFs), thereby initiating a signaling cascade leading to T cell activation. The co-stimulatory TNF family ligand is selected from the group consisting of 4-1BBL, OX40L, GITRL, CD70, CD30L, and LIGHT, more particularly, the co-stimulatory TNF family ligand member is 4-1 BBL.

As previously described, 4-1BBL is a type II transmembrane protein and is a member of the TNF ligand family. Complete or full-length 4-1BBL having the amino acid sequence of SEQ ID NO:297 has been described to form trimers on the cell surface. Trimer formation is facilitated by specific motifs in the extracellular domain of 4-1 BBL. The motif is designated herein as a "trimerization region". Amino acids 50-254 of the human 4-1BBL sequence (SEQ ID NO:298) form the extracellular domain of 4-1BBL, but even fragments thereof are capable of forming trimers. In particular embodiments of the invention, the term "4-1 BBL ectodomain or fragment thereof" refers to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:90 (amino acids 52-254 of human 4-1 BBL), SEQ ID NO:87 (amino acids 71-254 of human 4-1 BBL), SEQ ID NO:89 (amino acids 80-254 of human 4-1 BBL), and SEQ ID NO:88 (amino acids 85-254 of human 4-1 BBL), or a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:91 (amino acids 71-248 of human 4-1 BBL), SEQ ID NO:94 (amino acids 52-248 of human 4-1 BBL), SEQ ID NO:93 (amino acids 80-248 of human 4-1 BBL), and SEQ ID NO:92 (amino acids 85-248 of human 4-1 BBL), but other fragments of the extracellular domain capable of trimerization are also included herein.

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

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, (G)₄S)_n、(SG₄)_nOr G₄(SG₄)_nA peptide linker, wherein "n" is typically a number between 1 and 10, typically between 1 and 4, in particular 2, i.e. a peptide selected from the group consisting of: GGGGS (SEQ ID NO:112), GGGGSGGGGS (SEQ ID NO:113), SGGGGSGGGG (SEQ ID NO:114), (G) ₄S)₃Or GGGGSGGGGSGGGGS (SEQ ID NO:117), GGGGSGGGGSGGGG or G4(SG4)₂(SEQ ID NO:115), and (G)₄S)₄Or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:118), but further comprises the sequence GSPGSSSSGS (SEQ ID NO:116), GSGSGSGSGS (SEQ ID NO:119), GSGSGNGS (SEQ ID NO:120), GGSGSGSGSG (SEQ ID NO:121), GGSGSG (SEQ ID NO:122), GGSG (SEQ ID NO:123), GGSGNGSG (SEQ ID NO:124), GGNGSGSG (SEQ ID NO:125) and GGNGSG (SEQ ID NO: 126). A specific target peptide linker is (G4S)₁Or GGGGS (SEQ ID NO:112), (G)₄S)₂Or GGGGSGGGGS (SEQ ID NO:113) and (G)₄S)₃(SEQ ID NO:117)。

The term "amino acid" as used in this application denotes the group of naturally occurring carboxy alpha-amino acids comprising: alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain containing an Fc region as defined herein.

As used herein, "fusion polypeptide" or "fusion protein" refers to a single chain polypeptide that includes antibody fragments and peptides not derived from antibodies. In one aspect, the fusion polypeptide consists of one or two 4-1BBL extracellular domains or fragments thereof fused to a portion of an antigen binding domain or an Fc portion. Fusion can occur by linking the N-or C-terminal amino acid of the antigen-binding portion directly to the C-or N-terminal amino acid of the extracellular domain of 4-1BBL or a fragment thereof via a peptide linker.

By "fusion" or "linked" is meant that the components (e.g., the extracellular domains of the polypeptide and the TNF ligand family members) are linked by peptide bonds, directly or via one or more peptide linkers.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide (protein) sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after aligning the amino acid residues in the candidate sequence with the amino acid residues in the reference polypeptide sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignments to determine percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, align. sawi, or megalign (dnastar) software. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, the sequence comparison computer program ALIGN-2 is used to generate values for% amino acid sequence identity. The ALIGN-2 sequence comparison computer program was written by Genentech, inc and the source code has been submitted with the user document to u.s.copy Office, Washington d.c.,20559 where it was registered with us copyright registration number TXU 510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, which includes the digital UNIX V4.0D. All sequence comparison parameters were set by the ALIGN-2 program and were unchanged. In the case of amino acid sequence comparisons using ALIGN-2, the% amino acid sequence identity (which may alternatively be expressed as a percentage of the amino acid sequence identity of a given amino acid sequence A with or including a given amino acid sequence B) of a given amino acid sequence A to a given amino acid sequence B is calculated as follows:

100 times a fraction X/Y

Wherein X is the number of amino acid residues scored as identical matches in the alignment of program A and B by the sequence alignment program ALIGN-2, and wherein Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not be equal to the% amino acid sequence identity of B to A. Unless otherwise specifically indicated, all values of% amino acid sequence identity as used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

In certain embodiments, amino acid sequence variants of the antigen binding molecules comprising TNF ligand trimers provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antigen binding molecule comprising a trimer of TNF ligands. Amino acid sequence variants of the antigen-binding molecules comprising TNF ligand trimers can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecule or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into, and/or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding. The target sites for substitution mutagenesis include HVRs and Frameworks (FRs). Conservative substitutions are provided below the head "preferred substitutions" in table a and are described further below with reference to amino acid side chain classes (1) to (6). Amino acid substitutions may be introduced into the molecule of interest and the product screened for a desired activity (e.g., retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

TABLE A

Amino acids can be grouped according to common side chain properties:

(1) hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilicity: cys, Ser, Thr, Asn, Gln;

(3) acidity: asp and Glu;

(4) alkalinity: his, Lys, Arg;

(5) residues that influence chain orientation: gly, Pro;

(6) aromatic: trp, Tyr, Phe.

Non-conservative substitutions will require the exchange of a member of one of these classes for another.

The term "amino acid sequence variant" includes substantial variants in which there is an amino acid substitution in one or more hypervariable region residues of a parent antigen-binding molecule (e.g., a humanized or human antibody). Typically, one or more of the resulting variants selected for further study will be altered (e.g., improved) in certain biological properties (e.g., increased affinity, decreased immunogenicity) and/or will substantially retain certain biological properties of the parent antigen-binding molecule relative to the parent antigen-binding molecule. Exemplary substitution variants are affinity matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and variant antigen binding molecules are displayed on phage and screened for a particular biological activity (e.g., binding affinity). In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs so long as such changes do not substantially reduce the antigen-binding molecule's ability to bind antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in the CDRs. A method that can be used to identify antibody residues or regions that can be targeted for mutagenesis is referred to as "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science,244: 1081-1085. In this method, a residue or set of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether antibody interaction with an antigen is affected. Additional substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antigen binding molecule complex is used to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues that are candidates for substitution may be targeted or eliminated. Variants can be screened to determine if they possess the desired properties.

Amino acid sequence insertions include amino and/or carboxyl terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of one or more amino acid residues. Examples of terminal insertions include antigen binding molecules containing a trimer of 4-1BBL with an N-terminal methionyl residue. Other insertional variants of the molecule include fusions to the N-or C-terminus of a polypeptide that increases the serum half-life of an antigen-binding molecule comprising a trimer of 4-1 BBL.

In certain aspects, the CEA antibodies or antigen binding molecules comprising a trimer of 4-1BBL provided herein are altered to increase or decrease the degree of antibody glycosylation. Glycosylated variants of the molecule may conveniently be obtained by altering the amino acid sequence such that one or more glycosylation sites are created or removed. When an antigen binding molecule containing a trimer of 4-1BBL comprises an Fc region, the carbohydrate to which it is attached may be altered. Natural antibodies produced by mammalian cells typically comprise bi-antennary oligosaccharides with a branched chain, typically attached through an N-linkage to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "backbone" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in an antigen binding molecule containing a trimer of a 4-1BBL ligand can be modified to produce variants with certain improved properties. In one aspect, variants of an antigen binding molecule comprising a trimer of 4-1BBL are provided, which have a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. Such fucosylated variants may have improved ADCC function, see, e.g., U.S. patent publication No. US 2003/0157108(Presta, L.) or US 2004/0093621(Kyowa Hakko Kogyo co., Ltd.). Other variants of the 4-1BBL trimer containing antigen binding molecules of the invention include variants having bisected oligosaccharides, for example where the biantennary oligosaccharides attached to the Fc region are bisected by GlcNAc. Such variants may have reduced fucosylation and/or improved ADCC function, see for example WO 2003/011878(Jean-Mairet et al); U.S. Pat. No. 6,602,684(Umana et al); and US 2005/0123546(Umana et al). Also provided are variants having at least one galactose residue in an oligosaccharide attached to an Fc region. Such antibody variants may have improved CDC function and are described, for example, in WO 1997/30087(Patel et al); WO 1998/58964(Raju, S.); and WO 1999/22764(Raju, S.).

In certain embodiments, it may be desirable to produce cysteine engineered variants of the CEA antibodies of the invention or antigen binding molecules containing 4-1BBL trimers, such as "thioMAbs", in which one or more residues of the molecule are substituted with cysteine residues. In particular embodiments, the substituted residue is present at an accessible site on the molecule. By replacing those residues with cysteine, the reactive thiol group is thereby localized to an accessible site of the antibody and can be used to conjugate the antibody to other moieties, such as a drug moiety or linker-drug moiety, to produce an immunoconjugate. In certain embodiments, any one or more of the following residues may be substituted with cysteine: v205 of the light chain (Kabat numbering); a118 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region (EU numbering). Cysteine engineered antigen binding molecules can be formed as described, for example, in U.S. patent No. 7,521,541.

In certain aspects, the CEA antibody or antigen binding molecule comprising a trimer of 4-1BBL provided herein can be further modified to comprise additional non-protein moieties known and readily available in the art. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (homopolymers or random copolymers), and dextran or poly (N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may have any molecular weight and may or may not have branches. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the bispecific antibody derivative will be used in therapy under defined conditions, and the like. In another aspect, a conjugate of an antibody and a non-proteinaceous moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam, N.W. et al, Proc. Natl. Acad. Sci. USA 102(2005) 11600-. The radiation can be of any wavelength and includes, but is not limited to, wavelengths that are not harmful to normal cells, but heat the non-proteinaceous part to a temperature at which cells in the vicinity of the antibody-non-proteinaceous part are killed.

In another aspect, the CEA antibody or antigen binding molecule immunoconjugates comprising trimers of 4-1BBL provided herein can be obtained. An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

The term "nucleic acid molecule" or "polynucleotide" includes any compound and/or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, in particular a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (a), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose) and a phosphate group. Generally, nucleic acid molecules are described by the sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is usually expressed from 5 'to 3'. In this context, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) (including, for example, complementary DNA (cdna) and genomic DNA), ribonucleic acid (RNA) (particularly messenger RNA (mrna)), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. The nucleic acid molecule may be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-and double-stranded forms. In addition, the nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the invention in vitro and/or in vivo (e.g., in a host or patient). Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or expression of the encoding molecule so that the mRNA can be injected into a subject to produce in vivo antibodies (see, e.g., Stadler et al, Nature Medicine 2017, published on 2017 at 12.6.7, doi:10.1038/nm.4356 or EP 2101823B 1).

By "isolated" nucleic acid molecule or polynucleotide, it is meant a nucleic acid molecule, DNA or RNA, that has been removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the purposes of the present invention. Additional embodiments of the isolated polynucleotide include a recombinant polynucleotide maintained in a heterologous host cell or a purified (partially or substantially purified) polynucleotide in solution. An isolated polynucleotide includes a polynucleotide molecule that is contained in a cell that normally contains the polynucleotide molecule, but which is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. Isolated RNA molecules include RNA transcripts of the invention, either in vivo or in vitro, as well as both positive and negative stranded forms and double stranded forms. Isolated polynucleotides or nucleic acids according to the invention also include such molecules produced synthetically. In addition, the polynucleotide or nucleic acid may be or include regulatory elements such as a promoter, ribosome binding site or transcription terminator.

With respect to a nucleic acid or polynucleotide having a nucleotide sequence that is at least, e.g., 95% "identical" to a reference nucleotide sequence of the present invention, it is meant that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may include up to five point mutations every 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with additional nucleotides, or up to 5% of the number of nucleotides of the total nucleotides in the reference sequence may be inserted into the reference sequence. These changes to the reference sequence can occur at the 5 'or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, or interspersed either individually among residues of the reference sequence, or in one or more contiguous groups within the reference sequence. As a practical matter, it can be routinely determined whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention using known computer programs, such as those discussed above for polypeptides (e.g., ALIGN-2).

The term "expression cassette" refers to a polynucleotide, generated recombinantly or synthetically, with a series of specific nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plasmid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of the expression vector includes, among other sequences, the nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassettes of the invention comprise a polynucleotide sequence encoding a bispecific antigen binding molecule of the invention or a fragment thereof.

The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule for introducing a particular gene into a target cell with which it is operably associated and directing the expression of the gene. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. The expression vector of the present invention comprises an expression cassette. Expression vectors allow for the transcription of a large number of stable mrnas. Once the expression vector is inside the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by cellular transcription and/or translation machinery. In one embodiment, the expression vector of the invention comprises an expression cassette comprising a polynucleotide sequence encoding the bispecific antigen binding molecule of the invention or a fragment thereof.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include a primary transformed cell and progeny derived from the primary transformed cell, regardless of the number of passages. Progeny may not be completely identical to the nucleic acid content of the parent cell, 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 herein. The host cell is any type of cellular system that can be used to produce the bispecific antigen binding molecules of the invention. Host cells include cultured cells, for example, cultured mammalian cells such as CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, per.c6 cells or hybridoma cells, yeast cells, insect cells and plant cells, as well as cells included in transgenic animals, transgenic plants or cultured plant or animal tissues, to name a few.

An "effective amount" of an agent is that amount necessary to produce a physiological change in the cell or tissue to which it is administered.

A "therapeutically effective amount" of an agent (e.g., a pharmaceutical composition) is an amount effective to achieve the desired therapeutic or prophylactic result at the necessary dosage and for the period of time. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the adverse effects of a disease.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

The term "pharmaceutical composition" refers to a formulation that is in a form that allows the biological activity of the active ingredient contained therein to be effective, and that is free of additional components that have unacceptable toxicity to the subject to which the formulation is to be administered.

By "pharmaceutically acceptable excipient" is meant an ingredient of the pharmaceutical composition other than the active ingredient that is not toxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, or preservatives.

The term "package insert" is used to refer to instructions typically included in commercial packaging for therapeutic products that contain information regarding the indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings concerning the use of such therapeutic products.

As used herein, "treatment" (and grammatical variations thereof, such as "treatment" or "treating") refers to a clinical intervention that attempts to alter the natural course of the treated individual, and may be for the purpose of prevention or in the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis. In some embodiments, the molecules of the invention are used to delay the progression of a disease or to slow the progression of a disease.

The term "cancer" as used herein refers to a proliferative disease, such as various lymphomas, carcinomas, lymphomas, blastomas, sarcomas, leukemias, lymphocytic leukemias, lung cancer, non-small cell lung (NSCL) cancer, bronchoalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer (stomach cancer), stomach cancer (gastrotic cancer), colorectal cancer (CRC), pancreatic cancer, breast cancer, negative breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, hodgkin's disease, esophageal cancer, small bowel cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, sarcoma soft tissue, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer or renal cell cancer, renal cell carcinoma, cervical cancer, bladder, Renal pelvis cancer, mesothelioma, hepatocellular carcinoma, cholangiocarcinoma, a Central Nervous System (CNS) tumor, a vertebral axis tumor, a brain stem glioma, glioblastoma multiforme, an astrocytoma, a schwannoma, an ependymoma, a medulloblastoma, a meningioma, a squamous cell carcinoma, a pituitary adenoma, and ewing's sarcoma, melanoma, multiple myeloma, a B-cell cancer (lymphoma), Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, refractory forms including any of the above cancers, or a combination of one or more of the above cancers.

The term "metastatic cancer" refers to a cancer state in which cancer cells are transported from an original site to one or more sites elsewhere in the body via blood or lymphatic vessels, thereby forming one or more secondary tumors in one or more organs other than the breast.

"advanced" cancer refers to cancer that spreads beyond the primary site or organ due to local invasion or metastasis. Thus, the term "advanced" cancer includes locally advanced and metastatic disease.

"recurrent" cancer refers to cancer that recurs at an initial site or beyond in response to an initial therapy (e.g., surgery). "locally recurrent" cancer refers to cancer that recurs at the same location after treatment as previously treated cancer. A "operable" or "resectable" cancer is a cancer that is confined to a major organ and is amenable to surgery (resection). A "non-resectable" or "unresectable" cancer cannot be removed (resected) by surgery.

Antigen binding molecules binding to CEA according to the invention

The present invention provides novel antigen binding molecules that bind to carcinoembryonic antigen (CEA) with particularly advantageous properties, such as the epitope to which they bind, cynomolgus/human cross-reactivity, producibility, stability, binding affinity, biological activity, targeting efficiency, reduced toxicity, and the ability to bind to other CEA-targeting antigen binding molecules, wherein the CEA antigen binding domain binds to a different epitope.

In a first aspect, the present invention provides novel humanized antibodies that bind to the a2 domain of carcinoembryonic antigen (CEACAM 5). Thus, in one aspect, novel humanized antibodies are provided that bind to a domain comprising the amino acid sequence of SEQ ID NO 311. In one aspect, an antibody that competes for binding with an antibody comprising: a variable heavy chain domain (VH) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a variable light chain domain (VL) comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In particular, these antibodies compete for binding with antibodies comprising: a VH domain comprising the amino acid sequence of SEQ ID NO. 7 and a VL domain comprising the amino acid sequence of SEQ ID NO. 8. Thus, antibodies are provided which specifically bind to CEA, which do not compete for binding with antibodies comprising the amino acid sequence: a VH domain comprising the amino acid sequence of SEQ ID NO 63 and a VL domain comprising the amino acid sequence of SEQ ID NO 64.

In one aspect, there is provided a humanized antibody capable of specifically binding to CEA, wherein the antibody comprises: a variable heavy chain domain (VH) comprising (i) CDR-H1 comprising SEQ ID NO:17, (ii) CDR-H2, which comprises the amino acid sequence of SEQ ID NO:18, and (iii) CDR-H3, which comprises the amino acid sequence of SEQ ID NO:19, and a variable light chain domain (VL), it comprises (iv) CDR-L1 comprising SEQ ID NO:20, (v) CDR-L2, which comprises the amino acid sequence of SEQ ID NO:21, and (vi) CDR-L3, which comprises the amino acid sequence of SEQ ID NO:22, and wherein the framework of the VH domain is based on a polypeptide comprising SEQ ID NO:127(IGHV3-23-02), and wherein the framework of the VL domain is based on a polypeptide comprising SEQ ID NO:139(IGKV 3-11). In a particular aspect, an antibody capable of specifically binding to CEA comprises a VH domain comprising the amino acid sequence of SEQ ID NO. 23 and a VL domain comprising the amino acid sequence of SEQ ID NO. 24. Thus, in one aspect, there is provided a humanized antibody that competes for binding with an antibody comprising: a variable heavy chain domain (VH) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a variable light chain domain (VL) comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID No. 4, (v) CDR-L2 comprising the amino acid sequence of SEQ ID No. 5, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID No. 6, wherein the antibody comprises a VH domain comprising the amino acid sequence of SEQ ID No. 23 and a VL domain comprising the amino acid sequence of SEQ ID No. 24.

In a further aspect, humanized and affinity matured antibodies are provided. In one aspect, the humanized and affinity matured antibody comprises: a VH domain comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:25, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:26, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and a VL domain comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:28, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

In a particular aspect, a humanized and affinity matured antibody capable of specifically binding to CEA comprises

In one aspect, a humanized and affinity matured antibody capable of specifically binding to CEA comprises a VH domain comprising the amino acid sequence of SEQ ID NO 39 and a VL domain comprising the amino acid sequence of SEQ ID NO 40, or a VH domain comprising the amino acid sequence of SEQ ID NO 41 and a VL domain comprising the amino acid sequence of SEQ ID NO 42.

In a second aspect, the present invention provides novel humanized antibodies that bind to the a1 domain of carcinoembryonic antigen (CEA). Thus, in one aspect, novel humanized antibodies are provided that bind to a domain comprising the amino acid sequence of SEQ ID NO: 312. In one aspect, an antibody that competes for binding with an antibody comprising: a variable heavy chain domain (VH) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:55, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:56, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 57; and a variable light chain domain (VL) comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:58, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:59, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 60. In particular, these antibodies compete for binding with antibodies comprising: a VH domain comprising the amino acid sequence of SEQ ID NO 61 and a VL domain comprising the amino acid sequence of SEQ ID NO 62. Thus, antibodies are provided which specifically bind to CEA, which do not compete for binding with antibodies comprising the amino acid sequence: a VH domain comprising the amino acid sequence of SEQ ID NO 63 and a VL domain comprising the amino acid sequence of SEQ ID NO 64.

In one aspect, there is provided a humanized antibody capable of specific binding to CEA, the humanized antibody comprising: a variable heavy chain domain (VH) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:65, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:66 or SEQ ID NO:67, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 68; and a VL domain comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:69 or SEQ ID NO:70 or SEQ ID NO:313, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:71 or SEQ ID NO:72 or SEQ ID NO:73, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 74. In particular, the framework of the VH domain is based on the human acceptor framework comprising the amino acid sequence of SEQ ID NO:232(IGHV1-2-02) and the framework of the VL domain is based on the human acceptor framework comprising the amino acid sequence of SEQ ID NO:235(IGKV 1-39-01). In one aspect, a humanized antibody capable of specific binding to CEA is provided, wherein the antigen binding domain capable of specific binding to CEA comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79 or SEQ ID NO:80 and a light chain variable region (Vl) comprising the amino acid sequence of SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85 or SEQ ID NO: 86.

In a particular aspect, there is provided a humanized antibody capable of specifically binding to CEA, wherein the antigen-binding domain capable of specifically binding to CEA comprises

In one aspect, there is provided a humanized antibody capable of specific binding to CEA, wherein the antibody comprises a variable heavy chain domain (VH) and a VL domain, said VH comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:65, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:66, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 68; and the VL domain comprises: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:313, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:71, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 74. In a particular aspect, there is provided a humanized antibody capable of specifically binding to CEA comprising a variable heavy chain domain (VH) comprising the amino acid sequence of SEQ ID NO:75 and a variable light chain domain (VL) comprising the amino acid sequence of SEQ ID NO: 85.

Antigen binding molecules of the invention comprising a trimer of 4-1BBL

The present invention provides novel antigen binding molecules comprising trimers of 4-1BBL with particularly advantageous properties, such as binding to different epitopes, producibility, stability, binding affinity, biological activity, targeting efficiency and reduced toxicity. The antigen binding molecules of the invention comprising a trimer of 4-1BBL are particularly suitable for (co-) stimulating cytotoxic T cells, e.g.in combination with a T cell activator such as a T cell bispecific antibody (TCB). The antibodies of the invention can also effectively activate other 4-1BB expressing immune cells without the simultaneous stimulation by activating Fc receptors such as Fc γ RIIIa (CD16 a). By avoiding the need for Fc receptor binding and activation of its function, the antigen binding molecules of the invention containing the 4-1BBL trimer can achieve efficient immune cell activation with less risk of systemic side effects compared to 4-1BB antibodies that require Fc receptor binding and activation of their function. By binding to CEA, antigen binding molecules containing 4-1BBL trimers achieve activation of tumor-specific immune cells and reduce the risk of systemic side effects.

In a first aspect, the invention provides an antigen binding molecule comprising a trimer of 4-1BBL, comprising

An antigen binding domain capable of specifically binding to CEA,

(b) A VH domain comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:25, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:26, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and a VL domain comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:28, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30; or

An antigen binding domain capable of specifically binding to CEA,

(e) A first heavy chain comprising the amino acid sequence of SEQ ID NO 99, a first light chain comprising the amino acid sequence of SEQ ID NO 100, a second heavy chain comprising the amino acid sequence of SEQ ID NO 246 and a second light chain comprising the amino acid sequence of SEQ ID NO 247; or

An antigen binding molecule comprising a trimer of 4-1BBL as defined hereinbefore comprises an Fc domain consisting of a first subunit and a second subunit capable of stable association. In one aspect, the antigen binding molecules of the invention that contain a trimer of 4-1BBL comprise: (a) a Fab molecule capable of specific binding to CEA, wherein the Fab heavy chain is fused at the C-terminus to the N-terminus of the CH2 domain in the Fc domain, and (C) an Fc domain consisting of a first and a second subunit capable of stable association.

In particular, the Fc domain is an IgG, in particular an IgG1Fc domain or an IgG4 Fc domain. More specifically, the Fc domain is an IgG1Fc domain.

Fc domain modifications that reduce Fc receptor binding and/or effector function

The Fc domain of the 4-1 BBL-containing trimeric antigen binding molecules of the invention consists of a pair of polypeptide chains comprising the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin g (IgG) molecule is a dimer, each subunit of which comprises a CH2 and CH3 IgG heavy chain constant domain. The two subunits of the Fc domain are capable of stably associating with each other.

The Fc domain confers advantageous pharmacokinetic properties to the antigen binding molecules of the invention, including a long serum half-life and a favorable tissue-to-blood partition ratio that contribute to good accumulation in the target tissue. At the same time, however, it may result in the bispecific antibodies of the invention undesirably targeting Fc receptor expressing cells rather than the preferred antigen carrying cells. Thus, in particular aspects, the Fc domain of the antigen binding molecules of the invention comprising a trimer of 4-1BBL exhibits reduced binding affinity to Fc receptors and/or reduced effector function compared to the native IgG1Fc domain. In one aspect, the Fc does not substantially bind to Fc receptors and/or does not induce effector function. In a particular aspect, the Fc receptor is an fey receptor. In one aspect, the Fc receptor is a human Fc receptor. In a particular aspect, the Fc receptor is an activated human Fc γ receptor, more specifically human Fc γ RIIIa, Fc γ RI, or Fc γ RIIa, most specifically human Fc γ RIIIa. In one aspect, the Fc domain does not induce effector function. Reduced effector function may include, but is not limited to, one or more of the following: reduced Complement Dependent Cytotoxicity (CDC), reduced antibody dependent cell mediated cytotoxicity (ADCC), reduced Antibody Dependent Cellular Phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex mediated antigen uptake by antigen presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling induced apoptosis, reduced dendritic cell maturation, or reduced T cell priming.

In certain aspects, one or more amino acid modifications can be introduced into the Fc region of an antigen binding molecule comprising a trimer of 4-1BBL provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising amino acid modifications (e.g., substitutions) at one or more amino acid positions.

In a particular aspect, the invention provides an antigen binding molecule comprising a trimer of 4-1BBL, comprising

(a) An antigen binding domain capable of specifically binding to CEA,

(b) a first polypeptide and a second polypeptide, said first polypeptide and said second polypeptide being linked to each other by a disulfide bond,

(c) an Fc domain consisting of a first subunit and a second subunit capable of stable association, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, particularly to an Fcyreceptor,

In one aspect, the Fc domain of the antigen binding molecules of the invention that contain a trimer of 4-1BBL comprises one or more amino acid mutations that reduce the binding affinity of the Fc domain to an Fc receptor and/or effector function. Typically, the same amino acid mutation or mutations are present in each of the two subunits of the Fc domain. In particular, the Fc domain comprises amino acid substitutions at positions E233, L234, L235, N297, P331 and P329 (EU numbering). In particular, the Fc domain comprises amino acid substitutions at positions 234 and 235 (EU numbering) and/or 329 (EU numbering) of the IgG heavy chain. More specifically, antigen binding molecules comprising a trimeric TNF family ligand according to the invention are provided, comprising an Fc domain having the amino acid substitutions L234A, L235A and P329G ("P329G LALA", EU numbering) in the IgG heavy chain. The amino acid substitutions L234A and L235A refer to the so-called LALA mutations. The "P329G LALA" combination of amino acid substitutions almost completely abolished Fc γ receptor binding of the human IgG1 Fc domain as described in international patent application publication No. WO2012/130831 a1, which also describes methods of making such mutant Fc domains and methods for determining properties thereof, such as Fc receptor binding or effector function. "EU numbering" refers to numbering according to the EU index of Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

Fc domains with reduced Fc receptor binding and/or effector function also include those with substitution of one or more of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acids 265, 269, 270, 297 and 327, including so-called "DANA" Fc mutants in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581).

In another aspect, the Fc domain is an IgG4 Fc domain. Compared to the IgG1 antibody, the IgG4 antibody exhibits reduced binding affinity to Fc receptors and reduced effector function. In a more specific aspect, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat numbering), in particular the amino acid substitution S228P. In a more specific aspect, the Fc domain is an IgG4 Fc domain comprising the amino acid substitutions L235E and S228P and P329G (EU numbering). Such IgG4 Fc domain mutants and their Fc γ receptor binding properties are also described in WO 2012/130831.

The mutant Fc domain may be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide change can be verified, for example, by sequencing.

Binding to Fc receptors can be readily determined, for example, by ELISA or by Surface Plasmon Resonance (SPR) using standard instruments such as BIAcore instruments (GE Healthcare), and Fc receptors can be obtained, for example, by recombinant expression. Suitable such binding assays are described herein. Alternatively, cell lines known to express specific Fc receptors (such as human NK cells expressing Fc γ IIIa receptors) can be used to assess the binding affinity of the Fc domain or Fc domain containing cell activating bispecific antigen binding molecule to the Fc receptor.

The effector function of an Fc domain, or a bispecific antibody comprising an Fc domain of the invention, can be measured by methods known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Pat. nos. 5,500,362; hellstrom et al, Proc Natl Acad Sci USA 83, 7059-; U.S. Pat. nos. 5,821,337; bruggemann et al, J Exp Med 166, 1351-. Alternatively, non-radioactive assay methods may be employed (see, e.g., for flow cytometry Non-radioactive cytotoxicity assay (CellTechnology, inc. mountain View, CA); and CytotoxNon-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, the ADCC activity of the target molecule may be assessed in vivo, for example in an animal model such as disclosed in Clynes et al, Proc Natl Acad Sci USA 95, 652-.

In some embodiments, Fc domain binding to complement components, particularly C1q, is reduced. Thus, in some embodiments, wherein the Fc domain is engineered to have reduced effector function, said reduced effector function comprises reduced CDC. A C1q binding assay may be performed to determine whether a bispecific antibody of the invention is capable of binding C1q and thus has CDC activity. See, e.g., WO 2006/029879 and WO 2005/100402 for C1q and C3C binding ELISA. To assess complement activation, CDC assays may be performed (see, e.g., Gazzano-Santoro et al, J Immunol Methods 202,163 (1996); Cragg et al, Blood 101, 1045-.

In a particular aspect, the Fc domain comprises a modification that facilitates association of the first subunit and the second subunit of the Fc domain.

Fc domain modification to promote heterodimerization

In one aspect, the antigen binding molecules of the invention comprising a trimer of 4-1BBL comprise (a) an antigen binding domain capable of specifically binding to CEA,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises two 4-1BBL extracellular domains or fragments thereof linked to each other by a peptide linker, and in that the second polypeptide comprises one 4-1BBL extracellular domain or fragment thereof, and (c) an Fc domain consisting of a first subunit and a second subunit capable of stable association. Thus, they comprise different portions, fused to one or the other of the two subunits of the Fc domain, usually comprised in two different polypeptide chains ("heavy chains"). Recombinant co-expression and subsequent dimerization of these polypeptides results in several possible combinations of the two polypeptides. In order to increase the yield and purity of the antigen-binding molecule comprising a trimer of 4-1BBL in recombinant production, it would therefore be advantageous to introduce a modification in the Fc domain of the antigen-binding molecule of the invention comprising a trimer of 4-1BBL that promotes the association of the desired polypeptide.

Thus, the Fc domain of the antigen binding molecules of the invention that contain a trimer of 4-1BBL comprises modifications that facilitate association of the first and second subunits of the Fc domain. The most extensive site of protein-protein interaction between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, the modification is particularly in the CH3 domain of the Fc domain.

In a particular aspect, the modification is a so-called "knob" modification, which includes a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain. Thus, in one particular aspect, the invention relates to an antigen binding molecule comprising a trimer of 4-1BBL as described herein above, which comprises an IgG molecule, wherein the Fc portion of the first heavy chain comprises a first dimerization module and the Fc portion of the second heavy chain comprises a second dimerization module that allows heterodimerization of the two heavy chains of the IgG molecule, and the first dimerization module comprises a knob, and the second dimerization module comprises a hole according to the knob-and-hole structure technique.

The technique of mortar and pestle construction is described, for example, in the following documents: US 5,731,168; US 7,695,936; ridgway et al, Prot Eng 9, 617-. Generally, the method involves introducing a bulge ("knob") at the interface of the first polypeptide and a corresponding cavity ("hole") in the interface of the second polypeptide such that the bulge can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. The bulge is constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities having the same or similar size as the projections are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine).

Thus, in a particular aspect, in the CH3 domain of the first subunit of the Fc domain of the 4-1BBL trimer-containing antigen binding molecule of the invention, the amino acid residue is substituted with an amino acid residue having a larger side chain volume, thereby generating a bulge within the CH3 domain of the first subunit, which bulge is locatable in a cavity within the CH3 domain of the second subunit; whereas in the CH3 domain of the second subunit of the Fc domain, the amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit can be positioned.

The projections and cavities can be prepared by altering the nucleic acid encoding the polypeptide (e.g., by site-specific mutagenesis or by peptide synthesis).

In a particular aspect, in the CH3 domain of the first subunit of the Fc domain, the threonine residue at position 366 is substituted with a tryptophan residue (T366W), while in the CH3 domain of the second subunit of the Fc domain, the tyrosine residue at position 407 is substituted with a valine residue (Y407V). More specifically, in the second subunit of the Fc domain, the threonine residue at position 366 is additionally replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A). More specifically, in the first subunit of the Fc domain, the serine residue at position 354 was additionally replaced with a cysteine residue (S354C), and in the second subunit of the Fc domain, the tyrosine residue at position 349 was additionally replaced with a cysteine residue (Y349C). The introduction of these two cysteine residues results in the formation of a disulfide bridge between the two subunits of the Fc domain. Disulfide bridges further stabilize the dimer (Carter, J Immunol Methods 248,7-15 (2001)).

In another alternative aspect, the modification that facilitates association of the first and second subunits of the Fc domain comprises a modification that mediates electrostatic steering effects, for example, as described in PCT publication WO 2009/089004. Typically, the method involves substituting one or more amino acid residues at the interface of two Fc domain subunits with charged amino acid residues such that homodimer formation becomes electrostatically unfavorable, but heterodimerization is electrostatically favorable.

In a particular aspect, the invention provides an antigen binding molecule comprising a trimer of 4-1BBL, wherein the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (numbering according to the Kabat EU index) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to the Kabat EU index).

Modification of the CH1/CL Domain

To further improve correct pairing, antigen binding molecules containing trimers of 4-1BBL may contain amino acid substitutions (so-called "charged residues") with different charges. These modifications were introduced into the crossover or non-crossover CH1 and CL domains. In a particular aspect, the invention relates to an antigen binding molecule comprising a trimer of 4-1BBL, wherein in one of the CL domains the amino acid at position 123(EU numbering) has been substituted with arginine (R) and the amino acid at position 124(EU numbering) has been substituted with lysine (K); and wherein in one of the CH1 domains, the amino acids at position 147(EU numbering) and position 213(EU numbering) have been substituted with glutamic acid (E).

More particularly, the invention relates to an antigen binding molecule comprising a trimer of 4-1BBL, wherein in the CL domain adjacent to members of the TNF ligand family the amino acid at position 123(EU numbering) has been substituted with arginine (R) and the amino acid at position 124(EU numbering) has been substituted with lysine (K); and wherein in the domain of CH1 adjacent to a member of the TNF ligand family, the amino acids at position 147(EU numbering) and position 213(EU numbering) have been substituted with glutamic acid (E).

Thus, in a particular aspect, there is provided an antigen binding molecule comprising a trimer of 4-1BBL, comprising

(a) An antigen binding domain capable of specifically binding to CEA,

(b) a first polypeptide comprising a CL domain comprising the amino acid mutations E123R and Q124K, and a second polypeptide comprising a CH1 domain comprising the amino acid mutations K147E and K213E, wherein the second polypeptide is linked to the first polypeptide by a disulfide bond located between the CH1 domain and the CL domain,

and wherein the antigen binding molecule is characterized in that the first polypeptide comprises two 4-1BBL extracellular domains or fragments thereof linked to each other and to a CL domain by a peptide linker, and in that the second polypeptide comprises one 4-1BBL or fragment thereof linked to a CH1 domain of said polypeptide via a peptide linker; and

(c) An Fc domain consisting of a first subunit and a second subunit capable of stable association.

In one aspect, the invention provides an antigen binding molecule comprising a trimer of 4-1BBL, wherein in the CL domain adjacent to a member of the TNF ligand family the amino acid at position 123(EU numbering) has been substituted with arginine (R) and the amino acid at position 124(EU numbering) has been substituted with lysine (K); and wherein in the domain of CH1 adjacent to a member of the TNF ligand family, the amino acids at position 147(EU numbering) and position 213(EU numbering) have been substituted with glutamic acid (E). These modifications result in so-called charged residues, which have the advantageous property of avoiding undesirable effects such as mismatches.

In particular, the CL domain comprises the amino acid mutations E123R and Q124K, and the CH1 domain comprises the amino acid mutations K147E and K213E.

Polynucleotide

The invention further provides isolated nucleic acid molecules encoding an antigen binding molecule or antibody or fragment thereof comprising a trimer of 4-1BBL as described herein.

An isolated polynucleotide encoding an antigen binding molecule comprising a trimer of 4-1BBL of the invention may be expressed as a single polynucleotide encoding the entire antigen binding molecule, or as multiple (e.g., two or more) polynucleotides that are co-expressed. Polypeptides encoded by the co-expressed polynucleotides may associate via, for example, disulfide bonds or other means to form a functional antigen binding molecule. For example, the light chain portion of an immunoglobulin may be encoded by a separate polynucleotide from the heavy chain portion of an immunoglobulin. When co-expressed, the heavy chain polypeptide will associate with the light chain polypeptide to form an immunoglobulin.

In some aspects, the isolated nucleic acid encodes a complete antigen binding molecule according to the invention comprising a trimer of 4-1BBL as described herein. In particular, the isolated polynucleotide encodes a polypeptide comprised in an antigen binding molecule according to the invention comprising a trimer of 4-1BBL as described herein.

In one aspect, the invention relates to an isolated nucleic acid molecule encoding an antigen binding molecule comprising a trimer of 4-1BBL, wherein the nucleic acid molecule comprises (a) a sequence encoding an antigen binding domain capable of specifically binding to CEA, (b) a sequence encoding a polypeptide comprising two extracellular domains of 4-1BBL or fragments thereof linked to each other by a peptide linker and (c) a sequence encoding a polypeptide comprising one of said extracellular domains of 4-1BBL or fragments thereof.

In another aspect, an isolated nucleic acid encoding an antigen binding molecule comprising a trimer of a 4-1BB ligand is provided, wherein the polynucleotide comprises (a) a sequence encoding a moiety capable of specifically binding to CEA, (b) a sequence encoding a polypeptide comprising two 4-1BBL ectodomains or two fragments thereof linked to each other by a peptide linker, and (c) a sequence encoding a polypeptide comprising one 4-1BBL ectodomain or fragment thereof.

In another aspect, isolated nucleic acids encoding the antibodies described herein that are capable of specifically binding to CEA are provided.

In certain aspects, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the invention is RNA, for example in the form of messenger RNA (mrna). The RNA of the present invention may be single-stranded or double-stranded.

Recombination method

The antigen binding molecules of the invention comprising a trimer of 4-1BBL can be obtained, for example, by solid state peptide synthesis (e.g., Merrifield solid phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides, e.g., as described above, encoding an antigen binding molecule comprising a trimer of 4-1BBL, or a polypeptide fragment thereof, are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such polynucleotides can be readily isolated and sequenced using conventional methods. In one aspect of the invention, there is provided a vector, preferably an expression vector, comprising one or more of the polynucleotides of the invention. Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of the antigen binding molecule (fragment) containing the 4-1BBL trimer, together with appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination/genetic recombination. See, for example, the techniques described in: maniatis et al, Molecula clone, A Laboratory Manual, Cold Spring Harbor LABORATORY, N.Y. (1989); and Ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, N.Y. (1989). The expression vector may be part of a plasmid, virus, or may be a nucleic acid fragment. The expression vector includes an expression cassette into which a polynucleotide encoding an antigen-binding molecule comprising a trimer of 4-1BBL, or a polypeptide fragment thereof (i.e., the coding region) is cloned in operable association with a promoter and/or other transcriptional or translational control elements. As used herein, a "coding region" is a portion of a nucleic acid that consists of codons that are translated into amino acids. Although the "stop codon" (TAG, TGA or TAA) is not translated into an amino acid, it (if present) can be considered part of the coding region, whereas any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, 5 'and 3' untranslated regions, etc., are not part of the coding region. The two or more coding regions may be present in a single polynucleotide construct (e.g., on a single vector), or in separate polynucleotide constructs (e.g., on separate (different) vectors). In addition, any vector may contain a single coding region, or may contain two or more coding regions, e.g., a vector of the invention may encode one or more polypeptides that are separated into the final protein by proteolytic cleavage post-or post-translationally. In addition, the vectors, polynucleotides or nucleic acids of the invention can encode a heterologous coding region that is fused or not fused to a polynucleotide encoding an antigen-binding molecule comprising a trimer of 4-1BBL or a polypeptide fragment thereof of the invention, or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretion signal peptides or heterologous functional domains. Operable association is when the coding region of a gene product (e.g., a polypeptide) is associated with one or more regulatory sequences in a manner such that expression of the gene product is under the influence or control of the regulatory sequences. Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in transcription of mRNA encoding the desired gene product, and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression control sequences to direct expression of the gene product or with the ability of the gene template to be transcribed. Thus, if a promoter is capable of affecting transcription of the nucleic acid, the promoter region will be operably associated with the nucleic acid encoding the polypeptide. The promoter may be a cell-specific promoter that directs substantial transcription of DNA only in predetermined cells. In addition to promoters, other transcriptional control elements, such as enhancers, operators, repressors, and transcriptional termination signals, may be operably associated with a polynucleotide to direct cell-specific transcription.

Suitable promoters and other transcriptional control regions are disclosed herein. Various transcriptional control regions are known to those skilled in the art. These transcriptional control regions include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (e.g., immediate early promoter-binding intron-a), simian virus 40 (e.g., early promoter), and retroviruses (such as, for example, rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes (such as actin, heat shock proteins, bovine growth hormone, and rabbit a-globin), as well as other sequences capable of controlling gene expression in eukaryotic cells. Other suitable transcriptional control regions include tissue-specific promoters and enhancers and inducible promoters (e.g., tetracycline-inducible promoters). Similarly, various translational control elements are known to those of ordinary skill in the art. These translation control elements include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly internal ribosome entry sites, or IRES, also known as CITE sequences). The expression cassette may also include other features, such as an origin of replication, and/or chromosomal integration elements, such as retroviral Long Terminal Repeats (LTRs), or adeno-associated virus (AAV) Inverted Terminal Repeats (ITRs).

The polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions encoding a secretion peptide or signal peptide which direct secretion of the polypeptide encoded by the polynucleotide of the present invention. For example, if secretion of an antigen binding molecule comprising a trimer of 4-1BBL or a polypeptide fragment thereof is desired, a DNA encoding a signal sequence can be placed upstream of a nucleic acid encoding an antigen binding molecule comprising a trimer of 4-1BBL or a polypeptide fragment thereof of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once the protein chain has been initiated to grow across the rough endoplasmic reticulum export. One of ordinary skill in the art will recognize that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to yield a secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide (e.g., an immunoglobulin heavy or light chain signal peptide) is used, or a functional derivative of that sequence that retains the ability to direct secretion of a polypeptide with which it is operably associated. Alternatively, a heterologous mammalian signal peptide or functional derivative thereof may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human Tissue Plasminogen Activator (TPA) or mouse β -glucuronidase.

DNA encoding short protein sequences (e.g., histidine tags) or to aid in labeling fusion proteins that can be used to facilitate subsequent purification can be included within or at the ends of polynucleotides encoding the 4-1BBL trimer-containing antigen-binding molecules of the invention or polypeptide fragments thereof.

In another aspect of the invention, host cells comprising one or more polynucleotides of the invention are provided. In certain embodiments, host cells comprising one or more vectors of the invention are provided. The polynucleotide and vector may be introgressed, individually or in combination, with any of the features described herein with respect to the polynucleotide and vector, respectively. In one aspect, the host cell comprises a vector (e.g., has been transformed or transfected with a vector) comprising a polynucleotide encoding (part of) the antigen binding molecule comprising a trimer of 4-1BBL according to the invention. As used herein, the term "host cell" refers to any kind of cellular system that can be engineered to produce a fusion protein of the invention or a fragment thereof. Host cells suitable for replicating and supporting the expression of antigen binding molecules are well known in the art. Such cells can be appropriately transfected or transduced with a particular expression vector, and large numbers of vector-containing cells can be grown for seeding large-scale fermentors to obtain sufficient quantities of antigen binding molecules for clinical use. Suitable host cells include prokaryotic microorganisms such as E.coli, or various eukaryotic cells such as Chinese hamster ovary Cells (CHO), insect cells, and the like. For example, the polypeptide may be produced in bacteria, particularly when glycosylation is not required. The polypeptide can be isolated from the bacterial cell paste after expression in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding polypeptides, including fungi and yeast strains whose glycosylation pathways have been "humanized" resulting in the production of polypeptides having a partially or fully human glycosylation pattern. See Gerngross, Nat Biotech 22, 1409-.

Suitable host cells for the expression of (glycosylated) polypeptides also originate fromMulticellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. A number of baculovirus strains have been identified which can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda (Spodoptera frugiperda) cells. Plant cell cultures may also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIIES for antibody production in transgenic plants^TMA technique). Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lines (293 or 293T cells, as described for example in Graham et al, J Gen Virol 36,59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells, as described for example in Mather, Biol Reprod 23, 243-. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including dhfr-CHO cells (Urlaub et al, Proc Natl Acad Sci USA 77,4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63, and Sp 2/0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol.248 (B.K.C.Lo eds., Humana Press, Totowa, NJ), pp.255-268 (2003). Host cells include cultured cells such as mammalian cultured cells, yeast cells, insect cells, bacterial cells and plant cells, to name a few, and also include cells contained in transgenic animals, transgenic plants or cultured plant or animal tissues. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, Human Embryonic Kidney (HEK) Cells or lymphocytes (e.g., Y0, NS0, Sp20 cells). Standard techniques for expressing foreign genes in these systems are known in the art. Cells expressing a polypeptide comprising the heavy or light chain of an immunoglobulin can be engineered to also express another immunoglobulin chain, such that the expressed product is an immunoglobulin with a heavy and light chain.

In one aspect, a method of producing an antigen-binding molecule comprising a 4-1BBL trimer according to the invention or a polypeptide fragment thereof is provided, wherein the method comprises culturing a host cell comprising a polynucleotide encoding an antigen-binding molecule comprising a 4-1BBL trimer according to the invention or a polypeptide fragment thereof as provided herein under conditions suitable for expression of an antigen-binding molecule comprising a 4-1BBL trimer according to the invention or a polypeptide fragment thereof, and recovering the antigen-binding molecule comprising a 4-1BBL trimer according to the invention or a polypeptide fragment thereof from the host cell (or host cell culture medium).

In the 4-1BBL trimer-containing antigen-binding molecules of the present invention, the components (at least one portion capable of specifically binding to a target cell antigen, one polypeptide comprising two extracellular domains of TNF ligand family members or fragments thereof, and one polypeptide comprising one of said extracellular domains of 4-1BBL family members or fragments thereof) are not genetically fused to each other. The polypeptides are designed such that their components (two TNF ligand family member extracellular domains or fragments thereof and other components such as CH or CL) are fused to each other either directly or through a linking sequence. The composition and length of the linker can be determined according to methods well known in the art, and the efficacy of the linker can be tested. Examples of linker sequences between the different components of the antigen binding molecules of the invention are found in the sequences provided herein. Additional sequences (e.g., endopeptidase recognition sequences) may also be included to incorporate cleavage sites to separate the individual components of the fusion protein, if desired.

In certain embodiments, the portion (e.g., Fab fragment) that is capable of specifically binding to a target cell antigen that forms part of the antigen binding molecule comprises at least one immunoglobulin variable region that is capable of binding to an antigen. The variable regions may form part of and be derived from naturally or non-naturally occurring antibodies and fragments thereof. Methods for producing polyclonal and monoclonal Antibodies are well known in the art (see, e.g., Harlow and Lane, "Antibodies, a Laboratory", Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be recombinantly produced (e.g., as described in U.S. patent No. 4,186,567), or can be obtained, for example, by screening combinatorial libraries comprising variable heavy and variable light chains (see, e.g., U.S. patent No. 5,969,108 to McCafferty).

Immunoglobulins of any animal species may be used in the present invention. Non-limiting immunoglobulins for use in the present invention may be of murine, primate or human origin. If the fusion protein is intended for human use, a chimeric form of an immunoglobulin may be used, wherein the constant region of the immunoglobulin is from a human. Immunoglobulins may also be prepared in humanized or fully human form according to methods well known in the art (see, e.g., U.S. Pat. No. 5,565,332 to Winter). Humanization can be achieved by a variety of methods including, but not limited to, (a) grafting non-human (e.g., donor antibody) CDRs onto human (e.g., acceptor antibody) frameworks and constant regions with or without retaining critical framework residues (e.g., critical framework residues important for maintaining good antigen binding affinity or antibody function), (b) grafting only non-human specificity determining regions (SDRs or a-CDRs; residues critical for antibody-antigen interaction) onto human frameworks and constant regions, or (c) grafting entire non-human variable domains but "hiding" them with human-like regions by replacing surface residues. Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, Front Biosci 13,1619-1633(2008), and further described, for example, in Riechmann et al, Nature 332,323-329 (1988); queen et al, Proc Natl Acad Sci USA 86, 10029-; U.S. Pat. nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; jones et al, Nature 321,522-525 (1986); morrison et al, Proc Natl Acad Sci 81,6851-6855 (1984); morrison and Oi, Adv Immunol 44,65-92 (1988); verhoeyen et al, Science 239,1534-1536 (1988); padlan, Molec Immun 31(3),169-217 (1994); kashmiri et al, Methods 36,25-34(2005) (SDR (a-CDR) grafting is described); padlan, Mol Immunol 28,489-498(1991) (described as "surface remodeling"); dall' Acqua et al, Methods 36,43-60(2005) (describes "FR shuffling"); and Osbourn et al, Methods 36,61-68(2005) and Klimka et al, Br J Cancer 83, 252-. A particular immunoglobulin according to the invention is a human immunoglobulin. Various techniques known in the art can be used to generate human antibodies and human variable regions. Human antibodies are generally described in van Dijk and van de Winkel, Curr Opin Pharmacol 5,368-74(2001) and Lonberg, Curr Opin Immunol 20, 450-. The human variable region may form part of and be derived from a human Monoclonal Antibody produced by the hybridoma method (see, e.g., Monoclonal Antibody Production Techniques and Applications, pp.51-63(Marcel Dekker, Inc., New York, 1987)). Human antibodies and human variable regions can also be prepared by: the immunogen is administered to a transgenic animal that has been modified to produce a fully human or intact antibody having human variable regions that respond to antigen challenge (see, e.g., Lonberg, Nat Biotech23,1117-1125 (2005)). Human antibodies and human variable regions can also be generated by: fv clone variable region sequences selected from a Human-derived phage display library were isolated (see, e.g., Hoogenboom et al Methods in Molecular Biology 178,1-37 (O' Brien et al ed., Human Press, Totowa, NJ, 2001); and McCafferty et al, Nature 348, 552-554; Clackson et al, Nature 352,624-628 (1991)). Phage typically display antibody fragments as single chain fv (scfv) fragments or Fab fragments.

In certain aspects, the portion (e.g., Fab fragment) capable of specific binding to a target cell antigen included in the antigen binding molecules of the invention is engineered to have enhanced binding affinity, for example, according to the methods disclosed in PCT publication WO 2012/020006 (see examples relating to affinity maturation) or U.S. patent application publication No. 2004/0132066. The ability of the antigen binding molecules of the invention to bind to a particular Epitope can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art (e.g., surface plasmon resonance (Liljeblad, et al, Glyco J17, 323-329(2000)) as well as conventional binding assays (Heeley, Endocr Res 28,217-229(2002)) that can be used to identify antigen binding molecules that compete with a reference antibody for binding to a particular antigen. NJ). In an exemplary competition assay, an immobilized antigen is incubated in a solution comprising a first labeled antigen binding molecule that binds to the antigen and a second unlabeled antigen binding molecule that is being tested for its ability to compete with the first antigen binding molecule for binding to the antigen. The second antigen binding molecule may be present in a hybridoma supernatant. As a control, the immobilized antigen is incubated in a solution comprising the first labeled antigen binding molecule but not the second unlabeled antigen binding molecule. After incubation under conditions that allow the first antibody to bind to the antigen, excess unbound antibody is removed and the amount of label associated with the immobilized antigen is measured. If the amount of label associated with the immobilized antigen is substantially reduced in the test sample relative to the control sample, it is indicative that the second antigen binding molecule competes with the first antigen binding molecule for binding to the antigen. See Harlow and Lane (1988) Antibodies, Chapter 14 of A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

The 4-1BBL trimer containing antigen binding molecules of the invention prepared as described herein can be purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors or antigens bound by antigen binding molecules containing trimers of 4-1BBL can be used. For example, for affinity chromatography purification of the fusion protein of the invention, a matrix with protein a or protein G may be used. The antigen binding molecules can be separated using sequential protein a or G affinity chromatography and size exclusion chromatography, essentially as described in the examples. The purity of the antigen binding molecule or fragment thereof comprising the trimer of 4-1BBL can be determined by any of a variety of well known analytical methods, including gel electrophoresis, high pressure liquid chromatography, and the like. For example, antigen binding molecules containing a trimer of 4-1BBL expressed as described in the examples are shown to be intact and properly assembled, as shown by reducing and non-reducing SDS-PAGE.

Measurement of

The physical/chemical properties and/or biological activities of the antigen binding molecules provided herein can be identified, screened, or characterized by various assays known in the art. Biological activity may include, for example, the ability to enhance the activation and/or proliferation of various immune cells, particularly T cells. For example, they enhance the secretion of immunomodulatory cytokines. Other immunomodulatory cytokines that may be enhanced or may be enhanced are, for example, IL2, granzyme B, and the like. Biological activity may also include cynomolgus binding cross-reactivity and binding to different cell types. Antigen binding molecules having such biological activity in vivo and/or in vitro are also provided.

1. Affinity assay

The affinity of the 4-1BBL trimer-containing antigen binding molecules provided herein for 4-1BB (CD137) can be determined by Surface Plasmon Resonance (SPR) according to the methods set forth in the examples using standard instruments such as BIAcore instruments (GE Healthcare) and receptors or target proteins such as can be obtained by recombinant expression. The affinity of an antigen binding molecule containing 4-1BBL trimer for CEA or an antibody capable of specifically binding CEA can also be determined by Surface Plasmon Resonance (SPR) using standard instruments such as BIAcore instruments (GE Healthcare) and receptors or target proteins obtainable by recombinant expression. Specific illustrative and exemplary embodiments for measuring binding affinity are described in example 1.1.5 or example 2.2. According to In one aspect, use at 25 ℃The T100 instrument (GE Healthcare) measures K by surface plasmon resonance_D。

2. Binding assays and other assays

Binding of the 4-1BBL trimer-containing antigen binding molecules provided herein to cells expressing the corresponding receptor can be assessed using cell lines expressing the particular receptor or target antigen, for example, by flow cytometry (FACS). In one aspect, fresh Peripheral Blood Mononuclear Cells (PBMCs) expressing 4-1BB can be used in a binding assay. These cells are used directly after isolation (initial PMBC) or stimulation (activated PMBC). In another aspect, activated mouse splenocytes (expressing 4-1BB) can be used to demonstrate binding of the 4-1BBL trimer-containing antigen binding molecules of the invention to cells expressing 4-1 BB.

In a further aspect, the binding of the antigen binding molecule to the target cell antigen is demonstrated using a CEA expressing cell line. The binding assay with CEACAM5 is described in more detail in example 3.1.

In another aspect, a competition assay can be used to identify antigen binding molecules that compete with a particular antibody or antigen binding molecule for binding to CEA or 4-1BB, respectively. In certain embodiments, such competing antigen binding molecules bind to the same epitope (e.g., a linear or conformational epitope) bound by a specific anti-CEA antibody or a specific 4-1BB antibody. Detailed exemplary methods for locating an epitope to which an antibody binds are provided in: morris (1996), "Epitope Mapping Protocols", from Methods in Molecular Biology Vol.66 (Humana Press, Totowa, NJ).

3. Activity assay

In one aspect, an assay method is provided for identifying antigen binding molecules containing a trimer of 4-1BBL that bind to CEA and to biologically active 4-1 BB. Biological activity can include, such as, through 4-1BB on CEA expressing cancer cells on the excitability of signal transduction. Antigen binding molecules comprising trimers of 4-1BBL identified by assays having such in vitro biological activities are also provided.

In certain aspects, the antigen binding molecules of the invention comprising a trimer of 4-1BBL are tested for such biological activity. Assays for detecting biological activity of the molecules of the invention are described in example 3.2. In addition, methods for detecting cell lysis (e.g., by measuring LDH release), induced apoptosis kinetics (e.g., by measuring caspase 3/7 activity), or apoptosis (e.g., using TUNEL assays) are well known in the art. In addition, the biological activity of such complexes can be assessed by assessing the effect of such complexes on survival, proliferation and lymphokine secretion of various lymphocyte subpopulations such as NK cells, NKT cells or γ δ T cells, or by assessing their ability to modulate the phenotype and function of antigen presenting cells such as dendritic cells, monocytes/macrophages or B cells.

Pharmaceutical compositions, formulations and routes of administration

In a further aspect, the invention provides a pharmaceutical composition comprising any of the 4-1BBL trimer-containing antigen binding molecules provided herein, for example for use in any of the following methods of treatment. In one embodiment, the pharmaceutical composition comprises any of the antigen binding molecules comprising a trimer of 4-1BBL provided herein and at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition comprises any of the antigen binding molecules comprising a trimer of 4-1BBL provided herein and at least one additional therapeutic agent as described below. In a further aspect, a pharmaceutical composition comprising any antibody capable of specifically binding to CEA is also provided.

The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of one or more antigen binding molecules comprising a trimer of 4-1BBL or CEA antibodies dissolved or dispersed in a pharmaceutically acceptable excipient. The term "pharmaceutically or pharmacologically acceptable" means that the molecular entities and compositions are generally non-toxic to recipients at the dosages and concentrations employed, i.e., do not produce adverse, allergic, or other untoward reactions when administered to an animal (e.g., a human) as appropriate. The preparation of a Pharmaceutical composition containing at least one antigen binding molecule containing a trimer of 4-1BBL or an antibody capable of specifically binding to CEA, and optionally further active ingredients, will be known to the person skilled in the art in view of this disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18 th edition, Mack Printing Company,1990, which is incorporated herein by reference. Specifically, the composition is a lyophilized formulation or an aqueous solution. As used herein, "pharmaceutically acceptable excipient" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, salts, stabilizers, and combinations thereof, as known to one of ordinary skill in the art.

Parenteral compositions include those designed for injection (e.g., subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal, or intraperitoneal injection). For injection, the antigen binding molecules of the invention comprising a trimer of 4-1BBL may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks 'solution, ringer's solution or physiological saline. The solution may contain formulating agents (formulations), such as suspending, stabilizing and/or dispersing agents. Alternatively, the fusion protein may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use. Sterile injectable solutions are prepared by incorporating the fusion protein of the invention in the required amount in the appropriate solvent with various other ingredients enumerated below, as required. For example, sterility can be readily achieved by filtration through sterile filtration membranes. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains a basic dispersion medium and/or other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsions, the preferred methods of preparation are vacuum drying or lyophilization techniques that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium. The liquid medium should be suitably buffered if necessary, and sufficient saline or glucose should first be used to render the liquid diluent isotonic prior to injection. The composition must be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept to a minimum at a safe level, for example below 0.5ng/mg protein. Suitable pharmaceutical excipients include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, and the like. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil; or synthetic fatty acid esters such as ethyl oleate or triglycerides; or liposomes.

The active ingredient may be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively); embedded in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules); or embedded in the crude emulsion. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18 th edition, Mack Printing Company, 1990). Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g., films, or microcapsules. In certain embodiments, prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate, gelatin or combinations thereof.

Exemplary pharmaceutical excipients herein also include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (r: (r))Baxter International, Inc.). Certain exemplary shasegps and methods of use (including rHuPH20) are described in U.S. patent publication nos. 2005/0260186 and 2006/0104968. In one aspect, the sHASEGP is admixed with one or more additional glycosaminoglycanases (such as chondroitinase).

Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulations comprising histidine-acetate buffer.

In addition to the previously described compositions, the fusion proteins may also be formulated as long acting preparations. Such long acting formulations may be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the fusion protein may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).

Pharmaceutical compositions comprising the fusion proteins of the invention may be manufactured by conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes. The pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations which can be used pharmaceutically. Suitable formulations depend on the route of administration chosen.

Antigen binding molecules or antibodies containing a trimer of 4-1BBL capable of specifically binding to CEA can be formulated as compositions in free acid or base, neutral or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or free base. Such pharmaceutically acceptable salts include acid addition salts, for example, formed with the free amino groups of the proteinaceous composition, or formed with inorganic acids such as hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, or mandelic acid. Salts formed with free carboxyl groups may also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron hydroxide; or an organic base such as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutically acceptable salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms.

The compositions herein may also contain more than one active ingredient necessary for the particular indication being treated, preferably active ingredients having complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in an amount effective for the intended purpose.

In one aspect, a pharmaceutical composition can comprise any of the 4-1BBL trimer-containing antigen binding molecules provided herein and at least one additional therapeutic agent. In one aspect, the pharmaceutical composition can comprise any of the 4-1BBL trimer-containing antigen binding molecules and T cell activating anti-CD 3 bispecific antibodies provided herein.

The formulations to be used for in vivo administration are generally sterile. For example, sterility can be readily achieved by filtration through sterile filtration membranes.

Therapeutic methods and compositions

Any antigen binding molecule or antibody containing a trimer of 4-1BBL provided herein that is capable of specifically binding to CEA can be used in the method of treatment.

For use in a method of treatment, the antigen binding molecules containing a trimer of 4-1BBL or CEA antibodies of the invention can be formulated, administered and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular condition being treated, the particular mammal being treated, the clinical symptoms of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to the practitioner.

In one aspect, there is provided an antigen binding molecule or antibody comprising a trimer of 4-1BBL of the present invention capable of specifically binding to CEA for use as a medicament. In a further aspect, there is provided an antigen binding molecule comprising a trimer of 4-1BBL or a CEA antibody of the invention for use in the treatment of disease, in particular for use in the treatment of cancer. In certain aspects, the antigen binding molecules of the invention that contain a trimer of 4-1BBL are provided for use in a method of treatment. In one aspect, the invention provides an antigen binding molecule comprising a trimer of 4-1BBL as described herein for use in the treatment of a disease in an individual in need thereof. In certain aspects, the invention provides an antigen binding molecule comprising a trimer of 4-1BBL for use in a method of treating an individual having a disease, the method comprising administering to the individual a therapeutically effective amount of the antigen binding molecule. In certain aspects, the disease to be treated is a CEA positive cancer. Examples of CEA-positive cancers include colon, pancreatic, gastric, non-small cell lung cancer (NSCLC), breast, ovarian, bladder, esophageal, cervical or endometrial adenocarcinoma, salivary gland, endometrial, and head and neck small cell carcinoma. In one aspect, the CEA-positive cancer is selected from the group consisting of colon adenocarcinoma, pancreatic adenocarcinoma, gastric adenocarcinoma, non-small cell lung cancer (NSCLC), breast cancer, cervical cancer, and esophageal adenocarcinoma. In particular, the CEA-positive cancer is colon cancer or non-small cell lung cancer (NSCLC). Accordingly, antigen binding molecules containing a trimer of 4-1BBL as described herein are provided for the treatment of these cancers. The subject, patient or "individual" in need of treatment is typically a mammal, more particularly a human.

In another aspect, there is provided an antigen binding molecule comprising a trimer of 4-1BBL as described herein for use in the treatment of infectious diseases, in particular for use in the treatment of viral infections. In a further aspect, there is provided an antigen binding molecule comprising a trimer of 4-1BBL as described herein for use in the treatment of an autoimmune disease, such as lupus disease.

In a further aspect, the invention relates to the use of an antigen binding molecule comprising a trimer of 4-1BBL in the manufacture or preparation of a medicament for the treatment of a disease in an individual in need thereof. In one aspect, the medicament is for use in a method of treating a disease, the method comprising administering to an individual having the disease a therapeutically effective amount of the medicament. In certain embodiments, the disease to be treated is a proliferative disorder, particularly cancer. Thus, in one aspect, the invention relates to the use of an antigen binding molecule of the invention comprising a trimer of 4-1BBL in the manufacture or preparation of a medicament for the treatment of cancer, in particular CEA positive cancer. Examples of CEA-positive cancers include colon, pancreatic, gastric, non-small cell lung cancer (NSCLC), breast, ovarian, bladder, esophageal, cervical or endometrial adenocarcinoma, salivary gland, endometrial, and head and neck small cell carcinoma. In one aspect, the CEA-positive cancer is selected from the group consisting of colon adenocarcinoma, pancreatic adenocarcinoma, gastric adenocarcinoma, non-small cell lung cancer (NSCLC), breast cancer, cervical cancer, and esophageal adenocarcinoma. In particular, the CEA-positive cancer is colon cancer or non-small cell lung cancer (NSCLC). One skilled in the art will recognize that in some cases, antigen binding molecules containing 4-1BBL trimers may not provide a cure, but may only provide a partial benefit. In some aspects, physiological changes with certain benefits are also considered to have therapeutic benefits. Thus, in some aspects, the amount of the antigen binding molecule comprising a trimer of 4-1BBL that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount".

In another aspect, the invention provides a method for treating a disease in an individual comprising administering to the individual a therapeutically effective amount of an antigen binding molecule comprising a trimer of 4-1BBL according to the invention. In one aspect, a composition comprising a fusion protein of the invention in a pharmaceutically acceptable form is administered to the individual. In certain aspects, the disease to be treated is a proliferative disorder. In a particular aspect, the disease is cancer. In certain aspects, if the disease to be treated is cancer, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent. An "individual" according to any of the above embodiments may be a mammal, preferably a human.

For the prevention or treatment of disease, the appropriate dosage of the 4-1BBL trimer-containing antigen binding molecule of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the patient's body weight, the type of antigen binding molecule, the severity and course of the disease, whether the fusion protein is administered for prophylactic or therapeutic purposes, previous or concurrent therapeutic intervention, the patient's clinical history and response to the fusion protein, and the discretion of the attending physician. In any case, the practitioner responsible for administration will determine the concentration and appropriate dosage of the active ingredient in the composition for the individual subject. Various dosing schedules are contemplated herein, including but not limited to single or multiple administrations at various time points, bolus administrations, and pulsed infusions.

The antigen binding molecule comprising a trimer of 4-1BBL is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1. mu.g/kg to 15mg/kg (e.g., 0.1mg/kg-10mg/kg) of the antigen binding molecule containing trimer of 4-1BBL may be an initial candidate dose administered to the patient, e.g., by one or more separate administrations, or by continuous infusion. Depending on the factors mentioned above, a typical daily dose may range from about 1. mu.g/kg to 100mg/kg or more. For repeated administrations over several days or longer, depending on the condition, the treatment will generally continue until the desired suppression of disease symptoms occurs. An exemplary dose of the fusion protein ranges from about 0.005mg/kg to about 10 mg/kg. In other examples, the dose may further include about 1 μ g/kg body weight, about 5 μ g/kg body weight, about 10 μ g/kg body weight, about 50 μ g/kg body weight, about 100 μ g/kg body weight, about 200 μ g/kg body weight, about 350 μ g/kg body weight, about 500 μ g/kg body weight, about 1mg/kg body weight, about 5mg/kg body weight, about 10mg/kg body weight, about 50mg/kg body weight, about 100mg/kg body weight, about 200mg/kg body weight, about 350mg/kg body weight, about 500mg/kg body weight to about 1000mg/kg body weight or more per administration, and any range derivable therein. In examples of ranges derivable from the numbers listed herein, ranges of about 5mg/kg body weight to about 100mg/kg body weight, about 5 μ g/kg body weight to about 500mg/kg body weight, etc., can be administered based on the numbers above. Thus, one or more doses of about 0.5mg/kg, 2.0mg/kg, 5.0mg/kg, or 10mg/kg (or any combination thereof) may be administered to a patient. Such doses may be administered intermittently, such as weekly or every three weeks (e.g., such that the patient receives from about 2 to about 20 or, for example, about 6 doses of the fusion protein). An initial higher loading dose may be administered followed by one or more lower doses. However, other dosage regimens may be useful. The progress of this therapy is readily monitored by conventional techniques and assays.

The antigen binding molecules of the invention containing 4-1BBL trimers will generally be used in amounts effective to achieve the intended purpose. For use in treating or preventing a disorder, the 4-1 BBL-containing trimeric antigen binding molecules of the invention or pharmaceutical compositions thereof are administered or applied in a therapeutically effective amount. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, particularly in light of the detailed disclosure provided herein.

For systemic administration, the therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. Doses can then be formulated in animal models to achieve IC including as determined in cell culture₅₀Circulating concentration range. Such information can be used to more accurately determine useful doses for humans.

Initial dosages can also be estimated from in vivo data (e.g., animal models) using techniques well known in the art. Administration to humans can be readily optimized by one of ordinary skill in the art based on animal data.

The dose and interval can be adjusted individually to provide plasma levels of the antigen binding molecule containing 4-1BBL trimer sufficient to maintain therapeutic efficacy. The typical patient dose for administration by injection ranges from about 0.1 to 50 mg/kg/day, usually about 0.5 to 1 mg/kg/day. Therapeutically effective plasma levels can be achieved by administering multiple doses per day. Levels in plasma can be measured, for example, by HPLC.

In the case of topical administration or selective ingestion, the effective local concentration of the antigen binding molecule containing the trimer of 4-1BBL may be independent of plasma concentrations. One skilled in the art will be able to optimize therapeutically effective local dosages without undue experimentation.

A therapeutically effective dose of the 4-1BBL trimer-containing antigen binding molecules described herein will generally provide therapeutic benefit without causing significant toxicity. Toxicity and therapeutic efficacy of the fusion proteins can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Cell culture assays and animal studies can be used to determine LD₅₀(dose of 50% of lethal population) and ED₅₀(a therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD₅₀/ED₅₀. Antigen binding molecules containing 4-1BBL trimers that exhibit large therapeutic indices are preferred. In one embodiment, the antigen binding molecules according to the invention that contain a trimer of 4-1BBL exhibit a high therapeutic index. Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages suitable for use in humans. The dosage is preferably selected to include ED with little or no toxicity ₅₀In the circulating concentration range of (c). The dosage may vary within this range depending upon a variety of factors, such as the dosage form employed, the route of administration utilized, the condition of the subject, and the like. The exact formulation, route of administration and dosage may be selected by The individual physician according to The condition of The patient (see, e.g., Fingl et al, 1975, in: The pharmaceutical Basis of Therapeutics, Chapter 1, page 1, The entire contents of which are incorporated herein by reference).

The attending physician of a patient treated with a fusion protein of the invention will know how and when to terminate, discontinue or regulate administration due to toxicity, organ dysfunction, etc. Conversely, if the clinical response is inadequate (excluding toxicity), the attending physician will also know to adjust the treatment to higher levels. The size of the dose administered in the management of the condition of interest will vary with the severity of the condition to be treated, the route of administration, and the like. For example, the severity of a condition can be assessed, in part, by standard prognostic assessment methods. In addition, the dose and possibly the frequency of dosing will also vary according to the age, weight and response of the individual patient.

Other Agents and treatments

The antigen binding molecules of the invention comprising a trimer of 4-1BBL can be administered in combination with one or more other agents in therapy. For example, the fusion protein of the invention can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" includes any agent that can be administered for the treatment of a symptom or disease in an individual in need of such treatment. Such additional therapeutic agents may comprise any active ingredient suitable for the particular indication being treated, preferably active ingredients having complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is another anti-cancer agent.

Such other agents are suitably present in combination in an amount effective for the intended purpose. The effective amount of such other agents will depend on the amount of antigen binding molecule containing the 4-1BBL trimer used, the type of disorder or treatment, and other factors discussed above. Antigen binding molecules containing 4-1BBL trimers are typically used at the same dosages and routes of administration as described herein, or about 1% to 99% of the dosages described herein, or at any dosage and by an empirically/clinically determined appropriate route.

Such combination therapies described above encompass combined administration (where two or more therapeutic agents are contained in the same composition or separate compositions), as well as separate administration, in which case administration of the 4-1BBL trimer-containing antigen binding molecule of the invention can occur prior to, concurrently with, and/or after administration of additional therapeutic agents and/or adjuvants.

Thus, in one aspect, there is provided an antigen binding molecule comprising a trimer of 4-1BBL as described herein for use in the treatment of cancer, in particular CEA-positive cancer, wherein the antigen binding molecule comprising a trimer of 4-1BBL is used in combination with a T cell activating anti-CD 3 bispecific antibody, in particular an anti-CEA/anti-CD 3 bispecific antibody.

In one aspect, the anti-CEA/anti-CD 3 antibody comprises a first antigen-binding domain that specifically binds to CD3 and a second antigen-binding domain that specifically binds to CEA. In a particular aspect, the second binding domain that binds to CEA binds to an epitope on CEA that is different from the epitope to which the antigen binding molecule comprising a trimer of 4-1BBL binds.

In one aspect, the anti-CEA/anti-CD 3 bispecific antibody as used herein comprises a first antigen binding domain comprising a heavy chain variable region (V)_HCD3) comprising the CDR-H1 sequence of SEQ ID NO:275, the CDR-H2 sequence of SEQ ID NO:276 and the CDR-H3 sequence of SEQ ID NO: 277; and/or light chain variable region (V)_LCD3) comprising the CDR-L1 sequence of SEQ ID NO:278, the CDR-L2 sequence of SEQ ID NO:279, and the CDR-L3 sequence of SEQ ID NO: 280. More specifically, the anti-CEA/anti-CD 3 bispecific antibody comprises a first antigen binding domain comprising a heavy chain variable region (V) which is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:281 _HCD3) and/or a light chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:282_LCD 3). In a further aspect, an anti-CEA/anti-CD 3 bispecific antibody comprises: heavy chain variable region (V)_HCD3) comprising the amino acid sequence of SEQ ID NO 281; and/or light chain variable region (V)_LCD3) comprising the amino acid sequence of SEQ ID No. 282.

In another aspect, the anti-CEA/anti-CD 3 bispecific antibody comprises a second antigen binding domain comprising

(a) Heavy chain variable region (V)_HCEA) comprising the CDR-H1 sequence of SEQ ID NO:283, the CDR-H2 sequence of SEQ ID NO:284 and the CDR-H3 sequence of SEQ ID NO: 285; and/or light chain variable region (V)_LCEA) comprising the CDR-L1 sequence of SEQ ID NO. 286, the CDR-L2 sequence of SEQ ID NO. 287 and the CDR-L3 sequence of SEQ ID NO. 288; or

(b) Heavy chain variable region (V)_HCEA) comprising the CDR-H1 sequence of SEQ ID NO:291, the CDR-H2 sequence of SEQ ID NO:292 and the CDR-H3 sequence of SEQ ID NO: 293;and/or light chain variable region (V)_LCEA) comprising the CDR-L1 sequence of SEQ ID NO:294, the CDR-L2 sequence of SEQ ID NO:295 and the CDR-L3 sequence of SEQ ID NO: 296.

In one aspect, the anti-CEA/anti-CD 3 bispecific comprises a second antigen-binding domain comprising a heavy chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:289 _HCEA) and/or a light chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:290_LCEA). In a further aspect, the anti-CEA/anti-CD 3 bispecific comprises a second antigen-binding domain comprising: heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO 63; and/or light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO 64.

In one aspect, an anti-CEA/anti-CD 3 bispecific antibody comprises a first antigen-binding domain comprising: heavy chain variable region (V)_HCD3) comprising the amino acid sequence of SEQ ID NO:281, and/or a light chain variable region (V)_LCD3) comprising the amino acid sequence of SEQ ID NO: 282; the second antigen-binding domain comprises: heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO:289, and/or a light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO 290.

In a further aspect, the antigen binding molecule comprising a trimer of 4-1BBL is used in combination with a T cell activating anti-CD 3 bispecific antibody, and the T cell activating anti-CD 3 bispecific antibody is administered simultaneously with or before or after the antigen binding molecule comprising a trimer of 4-1 BBL.

In a further aspect, there is provided the use of an antigen binding molecule comprising a trimer of 4-1BBL in the manufacture of a medicament for the treatment of cancer, wherein the antigen binding molecule comprising a trimer of 4-1BBL is used in combination with a T cell activating anti-CD 3 bispecific antibody, in particular an anti-CEA/anti-CD 3 bispecific antibody. In certain aspects, the disease to be treated is a CEA positive cancer. Examples of CEA-positive cancers include colon, pancreatic, gastric, non-small cell lung cancer (NSCLC), breast, ovarian, bladder, esophageal, cervical or endometrial adenocarcinoma, salivary gland, endometrial, and head and neck small cell carcinomas. In one aspect, the CEA-positive cancer is selected from the group consisting of colon adenocarcinoma, pancreatic adenocarcinoma, gastric adenocarcinoma, non-small cell lung cancer (NSCLC), breast cancer, cervical cancer, and esophageal adenocarcinoma. In particular, the CEA-positive cancer is colon cancer or non-small cell lung cancer (NSCLC).

In a further aspect, the present invention provides a method of treating cancer in an individual, comprising administering to said individual a therapeutically effective amount of an antigen binding molecule comprising a 4-1BBL trimer according to the invention and an effective amount of a T cell activating anti-CD 3 bispecific antibody, in particular an anti-CEA/anti-CD 3 bispecific antibody as defined above. In some aspects, the method is for a CEA-positive cancer. Examples of CEA-positive cancers include breast cancer, ovarian cancer, gastric cancer, bladder cancer, salivary gland cancer, endometrial cancer, pancreatic cancer, and non-small cell lung cancer (NSCLC). In one aspect, the method is for treating CEA positive metastatic breast cancer.

In a further aspect, the 4-1BBL trimer-containing antigen binding molecules of the present invention are used in combination with an agent that blocks the PD-L1/PD-1 interaction, and the agent that blocks the PD-L1/PD-1 interaction is administered simultaneously with or before or after the 4-1BBL trimer-containing antigen binding molecule. In this aspect, the agent that blocks the PD-L1/PD-1 interaction is a PD-L1 binding antagonist or a PD-1 binding antagonist. In particular, the agent that blocks the PD-L1/PD-1 interaction is an anti-PD-L1 antibody or an anti-PD-1 antibody. In a particular aspect, the agent that blocks the PD-L1/PD-1 interaction is an anti-PD-L1 antibody. In a particular aspect, the anti-PD-L1 antibody is selected from the group consisting of atuzumab (MPDL32 3280A, RG7446), de waguchumab (MEDI4736), avizumab (MSB0010718C), and MDX-1105. More particularly, the anti-PD-L1 antibody is atelizumab. In another aspect, the agent that blocks the PD-L1/PD-1 interaction is an anti-PD-1 antibody. In a particular aspect, the anti-PD-1 antibody is selected from the group consisting of MDX 1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680(AMP-514), PDR001, REGN2810, and BGB-108, particularly selected from pembrolizumab and nivolumab.

Article of manufacture

In another aspect of the invention, an article of manufacture is provided that contains materials useful for the treatment, prevention and/or diagnosis of the above-mentioned conditions. The article of manufacture comprises a container and a label or package insert (package insert) on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container contains the composition, alone or in combination with another composition effective for treating, preventing and/or diagnosing the condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antigen binding molecule of the invention comprising a trimer of 4-1 BBL.

The label or package insert indicates that the composition is for use in treating the selected condition. Further, the article may comprise: (a) a first container having a composition contained therein, wherein the composition comprises an antigen binding molecule of the invention comprising a 4-1BBL trimer; and (b) a second container having the composition contained therein, wherein the composition comprises an additional cytotoxic or other therapeutic agent. The article of manufacture of this embodiment of the invention may further comprise a package insert indicating that the composition is useful for treating a particular condition.

Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. The kit may also include other materials as desired from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

Table B (sequence):

general information on the nucleotide sequences of human immunoglobulin light and heavy chains is given in: kabat, E.A. et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Amino acids of the antibody chain are numbered and referenced according to the EU numbering system according to Kabat (Kabat, e.a. et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)) as defined above.

***

Examples of the invention

The following are examples of the methods and compositions of the present invention. It is to be understood that various other embodiments may be practiced given the general description provided above.

Recombinant DNA technology

DNA is manipulated using standard methods, such as those described in Sambrook et al, Molecular cloning: A laboratory Manual; cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions. General information on the nucleotide sequences of human immunoglobulin light and heavy chains is given in the following references: kabat, E.A. et al, (1991) Sequences of Proteins of Immunological Interest, fifth edition, NIH Publication No 91-3242.

DNA sequencing

The DNA sequence was determined by double-strand sequencing.

Gene synthesis

The desired gene segments were generated by PCR using appropriate templates, or were synthesized from synthetic oligonucleotides and PCR products by automated gene synthesis from Geneart AG (Regensburg, Germany). In the case where the exact gene sequence is not available, oligonucleotide primers are designed based on the sequence of the closest homolog and the gene is isolated by RT-PCR from RNA derived from the appropriate tissue. Gene segments flanked by single restriction enzyme cleavage sites were cloned into standard cloning/sequencing vectors. Plasmid DNA was purified from the transformed bacteria and the concentration was determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. Gene segments with appropriate restriction sites were designed to allow subcloning into the corresponding expression vectors. All constructs were designed with a 5' DNA sequence encoding a leader peptide that targets a protein secreted by eukaryotic cells.

Cell culture technique

Standard Cell culture techniques are used as described in Current Protocols in Cell Biology (2000), Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott-Schwartz, J.and Yamada, K.M (eds.), John Wiley & Sons, Inc.

Protein purification

The protein was purified from the filtered cell culture supernatant according to standard protocols. Briefly, the Fc-containing protein was purified from cell culture supernatants by applying the antigen-binding molecule to protein A affinity chromatography (equilibration buffer: 20mM sodium citrate, 20mM sodium phosphate, pH 7.5; elution buffer: 20mM sodium citrate, pH 3.0). Elution was achieved at pH 3.0, followed by immediate neutralization of the pH of the sample. Aggregated proteins were separated from monomeric antibodies by size exclusion chromatography (Superdex 200, GE Healthcare) in PBS or in 20mM histidine, 140mM NaCl (pH 6.0). The monomeric antigen-binding molecule fractions may be combined, concentrated (if desired) using, for example, a MILLIPORE Amicon Ultra (30MWCO) centrifugal concentrator, frozen and stored at-20 ℃ or-80 ℃. Portions of the sample can be provided for subsequent protein analysis and analytical characterization, for example, by SDS-PAGE, Size Exclusion Chromatography (SEC), or mass spectrometry.

Example 1

Production and production of anti-CEA antibodies

1.1 formation of humanized variants of anti-CEA antibody A5B7

1.1.1 methods

anti-CEA antibody A5B7 is disclosed by, for example, M.J.Banfield et al (Proteins 1997,29(2),161-171), and its structure can be found in PDB ID:1CLO in Protein Structure database (PDB) (www.rcsb.org, H.M.Berman et al, The Protein Data Bank, Nucleic Acids Research, 2000, 28, 235-242). The entries include heavy and light chain variable domain sequences. To identify a suitable human acceptor framework in the humanization process of the anti-CEA binding agent A5B7, a classical approach was taken, namely to find an acceptor framework with high sequence homology, graft the CDRs onto this framework, and evaluate the conceivable back-mutations. More specifically, the effect of each amino acid difference of the identified framework and the parent antibody on the structural integrity of the binding agent is judged, and back mutations towards the parent sequence are introduced as appropriate. Structural assessment Fv region homology models based on the parental antibody and its humanized versions were created by an internal antibody structural homology modeling tool, which was implemented using the Biovia Discovery Studio Environment version 4.5.

1.1.2 selection of acceptor frameworks and Adaptation thereof

The acceptor framework was selected as shown in table 1 below:

table 1: acceptor framework

The CDR3 post-framework region was adapted from the human J element germline IGJH6 for the heavy chain and the sequence was similar to the kappa J element IGKJ2 for the light chain.

Based on structural considerations, back mutations were introduced at positions 93 and 94 of the heavy chain from the human acceptor framework to the amino acids in the parent binding agent.

1.1.3 VH and VL regions of the resulting humanized CEA antibody

The VH domain of the resulting humanized CEA antibody can be seen in table 2 below, and the VL domain of the resulting humanized CEA antibody can be seen in table 3 below.

Table 2: amino acid sequence of VH Domain of humanized CEA antibody (based on human acceptor framework IGHV3-23 or IGHV3-15)

For the heavy chain, the initial variant 3-23A5-1 was found to be suitable for use in a binding assay (but exhibited slightly less binding capacity than the parent murine antibody) and was selected as the starting point for further modification. Variants based on IGHV3-15 showed lower binding activity compared to the humanized variant 3-23A 5-1.

To restore the full binding activity of the parent chimeric antibody, variants 3-23A5-1A, 3-23A5-1C, and 3-23A5-1D were created. The length of the variant 3-23A5-1, CDR-H2, was also tested for adaptation to the human receptor sequence, but the construct lost binding activity altogether. Due to the putative deamidation hot spot in CDR-H2(Asn53-Gly54), we changed the motif to Asn53-Ala 54. Another possible hotspot Asn73-Ser74 was back mutated to Lys73-Ser 74. Thus, variant 3-23A5-1E was created.

Table 3: amino acid sequence of VL domain of humanized CEA antibody (based on human receptor framework IGKV 3-11).

The light chain is humanized based on the human IGKV3-11 acceptor framework. In the series a5-L1 to a5-L4, it was understood that the variant a5-L1 showed good binding activity (but slightly lower than the parent antibody). Partial humanization of CDR-L1 (variant A5-L2; Kabat positions 30 and 31) completely abolished binding. Likewise, humanization of CDR-H2 (variant A5-L3; Kabat positions 50 to 56) also completely abolished binding. Position 90 (variant A5-L4) has a significant contribution to the binding properties. Histidine at this position is important for binding. Thus, variant A5-L1 was selected for further modification.

The series a5-L1A through a5-L1D solved the problem of back mutations required to restore the full binding potential of the parental chimeric antibody. Variant a5-L1A showed that the Kabat positions 1, 2, whole frame 2 and the back-mutations at Kabat position 71 did not increase any more binding activity. Variants A5-L1B and A5-L1C mapped a subset of those positions and confirmed that they did not alter binding properties. The variant A5-L1D, which had back mutations at Kabat positions 46 and 47, showed the best binding activity.

1.1.4 selection of humanized A5B7 antibodies

Based on novel humanized variants of VH and VL, the novel CEA antibody was expressed as a huIgG1 antibody with effector-silenced Fc (P329G; L234A, L235A) to eliminate binding to Fc γ receptors according to the method described in WO 2012/130831 a 1; they were tested for binding to CEA expressed on MKN45 cells and compared to the corresponding parent murine A5B7 antibody.

Table 4: VH/VL combinations expressed as huIgG1_ LALA _ PG antibodies

	A5-L1A	A5-L1B	A5-L1C	A5-L1D
					3-23A5-1A	P1AE2164	P1AE2165	P1AE2166	P1AE2167
3-23A5-1C	-	-	P1AE2176	P1AE2177
					3-23A5-1D	P1AE2179	-	P1AE2181	P1AE2182

MKN45(DSMZ ACC 409) is a human gastric adenocarcinoma cell line expressing CEA. Cells were cultured in high grade RPMI + 2% FCS + 1% Glutamax. The viability of MKN-45 cells was examined and the cells were resuspended and adjusted to a density of 1Mio cells/ml. 100 μ l of this cell suspension (containing 0.1Mio cells) was seeded into a 96-well round bottom plate. The well plates were centrifuged at 400Xg for 4min and the supernatant was removed. Then 40. mu.l of diluted antibody or FACS buffer was added to the cells and incubated at 4 ℃ for 30 minutes. After incubation, cells were washed twice with FACS buffer (150 μ l per well). Then 20. mu.l of diluted secondary PE anti-human Fc specific secondary antibody (109-116-170, Jackson ImmunoResearch) was added to the cells. The cells were incubated at 4 ℃ for a further 30 minutes. To remove unbound antibody, cells were washed twice more with FACS buffer (150 μ Ι per well). To immobilize the cells, 100. mu.l of FACS buffer (containing 1% PFA) was added to the wells. Before measurement, cells were resuspended in 150. mu.l FACS buffer. Fluorescence was measured using a BD flow cytometer.

Figure 2 shows the binding curve for the humanized A5B7 variant. All tested binding agents were able to bind MKN45 cells, but the binding capacity was slightly reduced compared to the parent A5B7 antibody. Among all variants tested, clone P1AE2167 has the best binding capacity and was therefore selected for further development.

1.1.5 determination of the affinity of Fab fragments of the humanized variant of the murine CEA-antibody A5B7 for human CEA Using surface plasmon resonance (BIACORE)

The affinity of Fab fragments of the humanized variant of murine CEA antibody A5B7 for human CEA was assessed by surface plasmon resonance using a BIACORE T200 instrument. Human CEA (hu N (A2-B2) A-avi-His B) was immobilized onto flow cell 2 at a concentration of 40nM for 30s to approximately 100RU by standard amine coupling on a CM5 chip. The Fab fragment of the humanized variant of murine CEA antibody A5B7 was then injected as analyte (3-fold dilution in a concentration range of 500-0.656nM) for a contact time of 120s, a dissociation time of 250s or 1000s, and a flow rate of 30. mu.l/min. Regeneration of human CEA (hu N (A2-B2) A-avi-His B) levels was achieved by 2 pulse injections of 10mM glycine/HCl (pH 2.0) over 60 s. The data were double referenced against the non-immobilized flow cell 1 and zero concentration analyte. Sensorgrams of the analytes were fitted to a simple 1:1Langmuir interaction model. Affinity constant [ K ] for human CEA (A2 Domain) _D]Summarized in table 5 below.

Table 5: fab fragments representing different humanized variants of murine CEA antibody A5B7 have affinity constants for human CEA (a2 domain).

The humanized variant of murine CEA antibody A5B7 has lower affinity than the parent murine antibody. The Fab fragment P1AE4138 derived from P1AE2167 (heavy chain comprising the VH variant 3-23A5-1A and C.kappa.light chain comprising the VL variant A5-L1D) was selected as the final humanized variant. Furthermore, to remove the deamidation site, a glycine to alanine mutation at Kabat position 54(G54A) was introduced in the VH domain, resulting in VL variant 3-23a 5-1E. The final humanized antibody (heavy chain comprising the VH variant 3-23A5-1E and Ckappa light chain comprising the VL variant A5-L1D) was designated A5H1EL1D or huA5B 7.

1.2 production of A5H1EL 1D-derived affinity mature anti-CEA antibodies

1.2.1 preparation, purification and characterization of antigens for phage display campaigns

Murine antibody A5B7 and its humanized derivative A5H1EL1D bound the A2 domain of CEACAM5(CEA) with an affinity of about 0.8 and about 2.5nM, respectively. To generate affinity matured A5H1EL1D variants by phage display, 3 different recombinant soluble antigens were generated. Each protein contained one C-terminal avi-tag for site-specific biotinylation and one His-tag for purification: the first protein consisted of the extracellular portion of CEACAM1, which consisted of 4 Ig-like domains N, A1, B, A2(NABA-avi-His, SEQ ID NO:147, table 6). The second protein is a chimeric protein consisting of 2 CEACAM5 and 2 CEACAM1 Ig domains. Based on the sequence of the four domains of CEACAM1, the DNA encoding the second and third domains (a1 and B domains) of CEACAM1 was replaced with DNA encoding the A2 and B2 domains of CEACAM5 (N (A2B2) a-avi-His, SEQ ID NO:148, table 6). The third protein is a chimeric protein consisting of 1 CEACAM5 and 3 CEACAM1 Ig domains. Based on the sequence of the four domains of CEACAM1, the DNA encoding the third domain (B domain) of CEACAM1 was replaced with the DNA encoding the B2 domain of CEACAM5 (NA (B2) a-avi-His, SEQ ID NO:149, table 6). Schematic representations of the three constructs are shown in fig. 3A, 3B and 3C.

Table 6: amino acid sequence of CEA antigen used

Each plasmid was transiently transfected into HEK 293 cells to stably express EBV-derived protein ebna (HEK ebna). Simultaneously co-transfected plasmids encoding biotin ligase BirA allowed avi tag specific biotinylation in vivo. Proteins were purified from the filtered cell culture supernatant using Immobilized Metal Affinity Chromatography (IMAC) followed by gel filtration according to standard protocols. The monomeric protein fractions were combined, concentrated (if necessary), frozen and stored at-80 ℃. Portions of the sample are provided for subsequent protein analysis and analytical characterization, for example by SDS-PAGE, Size Exclusion Chromatography (SEC), or mass spectrometry.

1.2.2 selection of affinity matured A5H1EL 1D-derived antibodies

Humanization of antibody A5B7 resulted in an approximately 3 to 4 fold decrease in affinity for CEA as measured by SPR. While the affinity for A5B7 was about 0.8nM, the affinity for A5H1EL1D was about 2.5 nM. FACS experiments using cell lines with different CEA expression levels confirmed this finding. To increase the affinity of the humanized clone A5H1EL1D, 3 different affinity maturation libraries were made for selecting clones with increased affinity by phage display.

1.2.2.1 Generation of A5H1EL1D affinity maturation library

The generation of affinity matured A5H1EL 1D-derived antibodies was performed by phage display using standard protocols (Silacci et al, 2005). In a first step, the DNA sequences encoding the VH and VL of the humanized parent clone A5H1EL1D (amino acid sequences SEQ ID No:23 and SEQ ID No:24) were cloned into a phagemid and then used as a template for randomization. In the next step, three libraries were generated for selection of favorable clones by phage display. Maturation libraries 1 and 2 were randomized in either the CDR1 and CDR2 of the heavy chain or the CDR1 and CDR2 of the light chain. The third maturation library was randomized in the CDR3 regions of the heavy and light chains. The random positions in the various CDR regions are shown in fig. 4A and 4B. To generate the mature library 1 randomized in CDR1 and 2 of the heavy chain, two fragments were assembled by "overlap extension splicing" (SOE) PCR and cloned into phage vectors (fig. 5A). The following primer combinations were used to generate library fragments: fragment 1(LMB3(SEQ ID NO:152, Table 7) and A5H1EL 1D-H1-rev-TN (SEQ ID NO:150, Table 7) and fragment 2(A5H1EL 1D-H2-for-TN (SEQ ID NO:151, Table 7) and HCDR 3-rev-invariant (SEQ ID NO:153, Table 22) (Table 7).

Table 7: primers for A5H1EL1D affinity maturation library H1/H2

To generate the mature library 2 randomized in CDR1 and 2 of the light chain, the two fragments were assembled by "overlap extension splicing" (SOE) PCR and cloned into a phage vector (fig. 5B). The following primer combinations were used to generate library fragments: fragment 1(LMB3(SEQ ID NO:152, Table 8) and A5H1EL1D _ L1_ rev _ TN (SEQ ID NO:154, Table 8) and fragment 2(A5H1EL1D _ L2_ for _ TN (SEQ ID NO:155, Table 8) and HCDR 3-rev-invariant (SEQ ID NO:153, Table 8).

Table 8: primer of A5H1EL1D affinity maturation library L1/L2

To generate the mature library 3 randomized in CDR3 of the light and heavy chains, the two fragments were assembled by "overlap extension splicing" (SOE) PCR and cloned into a phage vector (fig. 5C). The following primer combinations were used to generate library fragments: fragment 1(LMB3(SEQ ID NO:152, Table 9) and LCDR 3-rev-invariant (SEQ ID NO:158, Table 9) and fragment 2(A5H1EL 1D-L3-for-TN (SEQ ID NO:156, Table 9) and A5H1EL 1D-H3-rev-TN (SEQ ID NO:157, Table 9).

Table 9: primers for A5H1EL1D affinity maturation library L3/H3

To assemble the fragments for each library, equimolar amounts of each fragment were used and amplified with the respective outer primers. For assembly of fragments of the third library randomized in HCDR3 and LCDR3, primer LMB3(SEQ ID NO:148, Table 7) was used in combination with primer "HCDR 3 amplification" (SEQ ID NO:155, Table 9). This primer was used to extend the C-terminus of VH with a sequence containing a KpnI site. After assembling sufficient quantities of full-length randomized fragments of all libraries, they were digested with NcoI/KpnI together with the identically treated acceptor phagemid vector. A3-fold molar excess of library inserts was ligated to 20. mu.g of phagemid vector. The purified linker was used for 20 transformations, yielding approximately 0.7x 10 ⁹To 2x 10⁹And (4) a transformant. Phagemid particles displaying the A5H1EL1D affinity maturation library were rescued and purified by PEG/NaCl purification for selection.

1.2.2.2 selection of affinity matured A5H1EL1D derived clones

To select affinity matured clones, phage display selection was performed on all 3 libraries using recombinant soluble antigens. Multiple panning rounds were performed in solution according to the following pattern: 1. pre-clearance of non-specific phagemid particles by incubation with 200nM biotinylated NA (B2) A-avi-His and NABA-avi-His for 0.5 h; 2. capture of biotinylated NA (B2) A-avi-His, NABA-avi-His, and addition of 5.4X 10⁷The phagemid particles were bound for 10 minutes by streptavidin-coated magnetic beads; 3. isolating unbound phagemid particles from the supernatant for further selection; 4. phagemid particles were conjugated with 20nM biotinylated N (A2B2) A-avi-His for 0.5 h in a total volume of 1 ml; 5. capture biotinylated N (A2B2) A-avi-His protein and add 5.4X 10⁷Phage particles were labeled with streptavidin-coated magnetic beadsHeterosexual binding for 10 minutes; 6. washing the magnetic beads with 5x 1ml PBS/Tween20 and 5x 1ml PBS; 7. the phage particles were eluted for 10 minutes by adding 1ml 100mM TEA and neutralized by adding 500. mu.l 1M Tris/HCl pH 7.4; 8. infection with exponentially growing E.coli TG1 bacteria; 9. infection with helper phage VCSM 13; and 10, subsequent to the phagemid particle PEG/NaCl precipitation, for subsequent selection rounds. Use of decreasing antigen concentration (20X 10) ^-9M、10x 10^-9M and 2x 10^-9M) for 3 rounds of selection. In round 3, streptavidin beads were washed with 20X 1ml PBS/Tween20 and 5X 1ml PBS.

Specific binders were identified by ELISA as follows: mu.l of 10nM biotinylated N (A2B2) A-avi-His protein or 40nM biotinylated NA (B2) A-avi-His protein per well were coated on a neutral avidin plate. Fab-containing bacterial supernatants were added and bound Fab was detected via their Flag tag by using anti-Flag/HRP secondary antibodies. Clones that were positive for the recombinant N (A2B2) A-avi-His protein ELISA but not for the NA (B2) A-avi-His protein were further tested by SPR.

1.2.2.3 identification of affinity matured A5H1EL1D derived variants by SPR

To further characterize ELISA positive clones, dissociation rates were measured by surface plasmon resonance using the Proteon XPR36 machine and the results were compared to the parental humanized clone A5H1EL 1D.

In this experiment, approximately 2000, 1000 and 500RU of biotinylated N (A2B2) A-avi-His were immobilized in the vertical direction on 3 channels using streptavidin-coated NLC chips. As a control for non-specific binding, 2000RU of biotinylated NA (B2) A-avi-His protein was immobilized on channel 4. For the analysis of the dissociation rate of the identified ELISA positive clones, the injection direction was changed to the horizontal direction. Before injection, each Fab-containing bacterial supernatant was filtered and diluted 3-fold with PBS. The association time was 100 seconds, the flow rate was 100. mu.l/min, and the dissociation time was 600 or 1200 seconds. Bacterial supernatants without Fab fragments were used for reference. Regeneration was carried out using 10mM glycine pH 1.5 at 50. mu.l/min (vertical) for 35 seconds.

The dissociation rate constant (koff) was calculated by simultaneous fitting of sensorgrams using a simple one-to-one Langmuir binding model in the ProteOn Manager v3.1 software. Clones expressing fabs with the slowest off-rate constants were identified and screened. The selected clones were re-evaluated in additional SPR experiments under the same conditions. This time, 4 affinity matured clones were compared directly in parallel to the parental clone A5H1EL1D during each injection. Bacterial supernatants without Fab fragments were used for reference. Clones showing a slower off-rate for N (A2B2) A-avi-His than A5H1EL1D and not binding to NA (B2) A-avi-His were selected and the variable domains of the corresponding phagemids were sequenced. The measured off-rates of the best clones are shown in table 10 and the sequences of the individual variable domains are listed in table 11.

Table 10: kinetic dissociation constant (koff) of selected clones obtained in screening assays with bacterial supernatants

Table 11: amino acid sequences of parental clone A5H1EL1D and selected affinity matured clones

1.2.2.4 Fab purification of affinity matured A5H1EL1D clone

To further characterize the affinity matured clones, individual Fab fragments were purified for accurate analysis of kinetic parameters. For each clone, 500ml of culture was inoculated with bacteria with the corresponding phagemid and induced with 1mM IPTG having an optical density (OD600) of 0.9 measured at 600 nm. Thereafter, the culture was incubated at 25 ℃ overnight and harvested by centrifugation. After incubating the resuspended pellet in 25ml of PPB buffer (30mM Tris-HCl pH8, 1mM EDTA, 20% sucrose) for 20min, the bacteria were centrifuged again and the supernatant harvested. This incubation step was repeated once with 25ml of 5mM MgSO4 solution. The supernatants from both incubation steps were combined, filtered and applied to an IMAC column (His gravitrap, GE Healthcare). Subsequently, 40ml of washing buffer (500mM NaCl, 20mM imidazole, 20mM NaH) was used ₂PO₄pH 7.4) the column was washed. In elution (500mM NaCl, 500mM imidazole, 20mM NaH)₂PO₄pH 7.4), the eluate is re-buffered using a PD10 column (GE Healthcare). The yield of purified protein ranged from 300 to 500. mu.g/l.

1.2.2.5 SPR analysis of purified affinity matured A5H1EL1D Fab fragments

The affinity (KD) of the purified Fab fragments was measured by surface plasmon resonance using the Proteon XPR36 machine, using the same setup as described previously.

Approximately 2000, 1000, 500 and 250RU of biotinylated N (A2B2) A-avi-His were immobilized in the vertical orientation on 4 channels of a streptavidin-coated NLC chip. As a control for non-specific binding, 2000RU of biotinylated NA (B2) A-avi-His protein was immobilized on channel 5. To determine the affinity (KD) of the purified clones, the injection direction was changed to the horizontal direction. Two-fold dilution series (varying concentrations between 100 and 3 nM) of purified Fab fragments were injected simultaneously along separate channels 1-5 at a rate of 100 μ l/min with an association time of 100 seconds and a dissociation time of 1200 seconds. Buffer (PBST) was injected along the sixth channel to provide an "in-line" blank for reference. Regeneration was carried out using 10mM glycine pH 1.5 at 50. mu.l/min (vertical) for 35 seconds.

Association rate constants (kon) and dissociation rate constants (koff) were calculated in the ProteOn Manager v3.1 software using a simple one-to-one Langmuir binding model by simultaneous fitting of the association sensorgram and dissociation sensorgram. The equilibrium dissociation constant (KD) is calculated as the ratio koff/ko. The kinetic and thermodynamic data are listed in table 12.

Table 12: determination of kinetic and thermodynamic parameters of purified Fab fragments by SPR

Clone ID	k on(1/Ms)	k off(1/s)	K_D(nM)
				A5H1EL1D	1.08E+5	2.48E-4	2.3
P006.038	2.25E+5	5.78E-5	0.25
				P005.097	0.94E+5	8.54E-5	0.91
P005.103	1.00E+5	4.99E-5	0.5
				P002.139	1.05E+5	6.53E-5	0.63
P001.177	2.67E+5	7.85E-4	0.29
				P005.102	1.34E+5	3.92E-4	0.29

1.2.2.6 CDR position combinations of affinity matured clones

To further increase the affinity to CEA, the CDR positions of several previously identified affinity matured binding agents were combined with each other. This includes not only specific positions within the CDRs, but also combinations of CDRs from different binding bodies. An alignment of all clones, phage display derived and combined clones is shown in fig. 6A and 6B. The CDRs for all heavy and light chains are listed in tables 13 and 14, respectively. The VH and VL domains are summarized in table 11.

Table 13: CDR sequences of affinity-matured heavy chains

Table 14: CDR sequences of affinity-matured light chains

1.3 formation of humanized variants of anti-CEA antibody MFE23

1.3.1 methods

anti-CEA antibody MFE23 is disclosed by, for example, M.K.Boehm et al (biochem.J.2000,346,519-528), and its structure can be found in PDB ID:11QOK in Protein Structure database (PDB) (www.rcsb.org, H.M.Berman et al, The Protein Data Bank, Nucleic Acids Research, 2000, 28, 235-242). The entries include heavy and light chain variable domain sequences. To identify a suitable human acceptor framework in the humanization process of the anti-CEA binding agent MFE23, a classical approach was taken, i.e., to find an acceptor framework with high sequence homology, to graft CDRs onto this framework, and to evaluate conceivable back-mutations. More specifically, the effect of each amino acid difference of the identified framework and the parent antibody on the structural integrity of the binding agent is judged, and back mutations towards the parent sequence are introduced as appropriate. Structural assessment Fv region homology models based on the parental antibody and its humanized versions were created by an internal antibody structural homology modeling tool, which was implemented using the Biovia Discovery Studio Environment version 4.5.

To improve the confidence of the back-mutation selection, we identified the closest murine homologous sequence from which the antibody was likely derived. We sought therefrom the location where extensive somatic hypermutation occurred during murine B-cell maturation in this antibody. These mutations may be important for introgression into humanized constructs.

1.3.2 selection of acceptor frameworks and Adaptation thereof

The acceptor framework was selected as shown in table 15 below:

table 15: acceptor framework

The CDR3 post-framework region was adapted from the human J element germline IGHJ4-01 for the heavy chain and the sequence was similar to the kappa J element IGKJ4-01 for the light chain. Based on structural considerations, back mutations were introduced at Kabat positions 71 and 93 of the heavy chain from the human acceptor framework to the amino acids in the parent binding agent. Based on the importance of framework mutations in the murine germline (resulting in the final mature MFE23 sequence), the Kabat residue 94 of the VH was changed back to the murine sequence.

To evaluate further improvement in affinity and/or stability of MFE23 sequences, we introgressed the following mutations in the light chain sequence: phe26Leu, Ser30Pro or Tyr, Leu78Val, as described in C.P. Graff et al (Protein Engineering, Design & Selection 2004,17(4), 293-304).

1.3.3 VH and VL domains of the resulting humanized CEA antibody

The VH domains of the resulting humanized CEA antibodies can be seen in table 16 below, and the VL domains of the resulting humanized CEA antibodies can be seen in table 17 below.

Table 16: amino acid sequence of VH Domain of humanized CEA antibody (based on human acceptor framework IGHV1-2-02)

Table 17: amino acid sequence of VL domain of humanized CEA antibody (based on human receptor framework IGKV1-39-01)

FIG. 7 shows an alignment of the sequences listed in tables 16 and 17, respectively.

The variable regions encoding the 6 heavy and 6 light chain DNA sequences of the humanized CEA binding agent were subcloned into frame with the constant heavy or constant light chain of human IgG1 containing the P239G, L234A and L235A mutations to eliminate binding to Fc γ receptors (WO 2012/130831 a 1). Antibodies were produced as follows. The resulting 36 variants (table 18) were tested for binding on MKN45 cells; and 7 variants were selected for further development.

Table 18: nomenclature of VH/VL combinations expressed as huIgG1_ LALA _ PG antibodies

MFE-L24

MFE-L25

MFE-L26

MFE-L27

MFE-L28

MFE-L29

MFE-H24

P1AE3125

P1AE3119

P1AE3113

P1AE3107

P1AE3101

P1AE3095

MFE-H25

P1AE3124

P1AE3118

P1AE3112

P1AE3106

P1AE3100

P1AE3094

MFE-H26

P1AE3123

P1AE3117

P1AE3111

P1AE3105

P1AE3099

P1AE3093

MFE-H27

P1AE3122

P1AE3116

P1AE3110

P1AE3104

P1AE3098

P1AE3092

MFE-H28

P1AE3121

P1AE3115

P1AE3109

P1AE3103

P1AE3097

P1AE3091

MFE-H29

P1AE3120

P1AE3114

P1AE3108

P1AE3102

P1AE3096

P1AE3090

1.3.4 selection of humanized MFE23 antibodies

Binding of 36 humanized MFE23 huIgG 1P 329G LALA variants to CEA expressed on MKN45 cells was compared to the corresponding parental murine MFE23 huIgG 1P 329G LALA antibody. Seventeen clones lost their binding ability to MKN45 cells expressing human CEACAM5 (fig. 8A). Binding was reduced for 8 clones compared to parental clone MFE23 (fig. 8B). The binding capacity of 11 clones was similar compared to the parental clone MFE23 (fig. 8C). Fitting of these binding curves EC ₅₀The values and the area under the curve (AUC) are shown in table 19.

Table 19: EC for binding curves of different humanized MFE23 huIgG 1P 329G LALA antibodies₅₀The values and area under the curve (AUC) are shown in FIGS. 8A, 8B and 8C

Example 2

Generation and production of CEA-targeted 4-1BB agonistic antigen binding molecules

2.1 preparation of CEA-targeted Fc fusion antigen-binding molecules containing 4-1BB ligand trimer

Asymmetric human IgG1 molecules with knob and hole structural mutations were prepared as follows: the variable regions of the heavy and light chain DNA sequences encoding binding agents specific for CEA were subcloned in frame with the constant heavy chain of the socket or the constant light chain of human IgG 1. A DNA sequence encoding a portion of the ectodomain of human 4-1BB ligand (amino acids 71-248) was synthesized based on the P41273 sequence of the Uniprot database.

The polypeptide containing two extracellular domains of 4-1BB ligand separated by a (G4S)2 linker and fused to a human IgG1-CL domain was cloned as described in FIG. 1A: human 4-1BB ligand, (G4S)2 linker, human 4-1BB ligand, (G4S)2 linker, human CL. The polypeptide containing one extracellular domain of the 4-1BB ligand fused to the human IgG1-CH domain was cloned as described in FIG. 1B: human 4-1BB ligand, (G4S)2 linker, human CH.

To improve correct pairing, the following mutations were introduced in the crossed CH-CL. Mutations E123R and Q124K were introduced in dimeric 4-1BB ligand fused to human CL. In monomeric 4-1BB ligand fused to human CH1, mutations K147E and K213E were cloned into the human CH1 domain as described in international patent application publication No. WO 2015/150447.

According to the method described in international patent application publication No. WO 2012/130831, P329G, L234A and L235A mutations were introduced into the constant regions of the knob and hole chains in the Fc domain to eliminate binding to Fc γ receptors. The combination of dimeric ligand-Fc pestle chain containing S354C/T366W mutations, monomeric CH1 fusions, targeted anti-CEA-Fc mortar chain containing Y349C/T366S/L368A/Y407V mutations, and anti-CEA light chain allows for the production of heterodimers, including assembled trimeric 4-1BB ligand and CEA binding Fab (fig. 1C).

Tables 20 and 21 show the amino acid sequences of monovalent CEA-targeting split trimeric 4-1BB ligand fc (kih) fusion antigen binding molecules containing CH1-CL crossover and charged residues based on the CEA binding agent A5B7 and humanized versions thereof.

Table 20: amino acid sequence of a monovalent CEA (murine A5B7) -targeting split trimeric 4-1BB ligand antigen binding molecule (CEA (A5B7) -4-1BBL) targeting, with CH-CL crossover and charged residues

Represents charged residue

Table 21: amino acid sequence of a monovalent CEA (A5H1EL1D) -targeting split trimeric 4-1BB ligand antigen binding molecule (CEA (A5H1EL1D) -4-1BBL) targeting, with CH-CL crossover and charged residues

Tables 22 to 33 show the amino acid sequences of monovalent CEA-targeting split trimeric 4-1BB ligand fc (kih) fusion antigen binding molecules containing CH1-CL crossover and charged residues based on affinity matured variants of the CEA binding agent A5H1EL 1D.

Table 22: amino acid sequence of a monovalent CEA (P006.038) -targeting split trimeric 4-1BB ligand antigen binding molecule (CEA (P006.038) -4-1BBL) having CH-CL crossover and charged residues

Table 23: amino acid sequence of a monovalent CEA (P005.097) -targeting split trimeric 4-1BB ligand antigen binding molecule (CEA (P005.097) -4-1BBL) having CH-CL crossover and charged residues

Table 24: amino acid sequence of a monovalent CEA (P005.103) -targeting split trimeric 4-1BB ligand antigen binding molecule (CEA (P005.103) -4-1BBL) having CH-CL crossover and charged residues

Table 25: amino acid sequence of a monovalent CEA (P002.139) -targeting split trimeric 4-1BB ligand antigen binding molecule (CEA (P002.139) -4-1BBL) having CH-CL crossover and charged residues

Table 26: amino acid sequence of a monovalent CEA (P001.177) -targeting split trimeric 4-1BB ligand antigen binding molecule (CEA (P001.177) -4-1BBL) having CH-CL crossover and charged residues

Table 27: amino acid sequence of a monovalent CEA (P005.102) -targeting split trimeric 4-1BB ligand antigen binding molecule (CEA (P005.102) -4-1BBL) having CH-CL crossover and charged residues

Table 28: amino acid sequence of a monovalent CEA (P005.103-combo1) -targeting split trimeric 4-1BB ligand antigen binding molecule (CEA (P005.103-combo1) -4-1BBL) targeting, with CH-CL crossover and charged residues

Table 29: amino acid sequence of a monovalent CEA (P005.103-combo2) -targeting split trimeric 4-1BB ligand antigen binding molecule (CEA (P005.103-combo2) -4-1BBL) targeting, with CH-CL crossover and charged residues

Table 30: amino acid sequence of a monovalent CEA (P005.102-combo1) -targeting split trimeric 4-1BB ligand antigen binding molecule (CEA (P005.102-combo1) -4-1BBL) targeting, with CH-CL crossover and charged residues

Table 31: amino acid sequence of a monovalent CEA (P005.102-combo2) -targeting split trimeric 4-1BB ligand antigen binding molecule (CEA (P005.102-combo2) -4-1BBL) targeting, with CH-CL crossover and charged residues

Table 32: amino acid sequence of a monovalent CEA (P006.038-combo1) -targeting split trimeric 4-1BB ligand antigen binding molecule (CEA (P006.038-combo1) -4-1BBL) targeting, with CH-CL crossover and charged residues

Table 33: amino acid sequence of a monovalent CEA (P006.038-combo2) -targeting split trimeric 4-1BB ligand antigen binding molecule (CEA (P006.038.combo2) -4-1BBL) with CH-CL crossover and charged residues

Table 34 shows the amino acid sequences of monovalent CEA-targeting split trimeric 4-1BB ligand fc (kih) fusion antigen binding molecules based on CEA binding agent MFE23 containing CH1-CL crossover and charged residues, and tables 35 to 41 show the amino acid sequences of monovalent CEA-targeting split trimeric 4-1BB ligand fc (kih) fusion antigen binding molecules based on humanized versions thereof.

Table 34: amino acid sequence of a monovalent CEA (MFE23) -targeting split trimeric 4-1BB ligand antigen binding molecule (CEA (MFE23) -4-1BBL) with CH-CL crossover and charged residues

Table 35: amino acid sequence of monovalent CEA-targeting (humFE23-L28-H24) -targeting split trimeric 4-1BB ligand antigen binding molecule (CEA (MFE23-L28-H24) -4-1BBL) with CH-CL crossover and charged residues

The CEA binding agent humFE23-L28-H24 corresponds to the clone used in P1AE 3101.

Table 36: amino acid sequence of monovalent CEA-targeting (humFE23-L28-H28) -targeting split trimeric 4-1BB ligand antigen binding molecule (CEA (MFE23-L28-H28) -4-1BBL) with CH-CL crossover and charged residues

The CEA binder humFE23-L28-H28 corresponds to the clone used in P1AE 3097.

Table 37: amino acid sequence of monovalent CEA-targeting (humFE23-L28-H25) -targeting split trimeric 4-1BB ligand antigen binding molecule (CEA (MFE23-L28-H25) -4-1BBL) with CH-CL crossover and charged residues

The CEA binding agent humFE23-L28-H25 corresponds to the clone used in P1AE 3100.

Table 38: amino acid sequence of monovalent CEA-targeting (humFE23-L27-H29) -targeting split trimeric 4-1BB ligand antigen binding molecule (CEA (MFE23-L27-H29) -4-1BBL) with CH-CL crossover and charged residues

The CEA binding agent humFE23-L27-H29 corresponds to the clone used in P1AE 3102.

Table 39: amino acid sequence of monovalent CEA-targeting (humFE23-L27-H28) -targeting split trimeric 4-1BB ligand antigen binding molecule (CEA (MFE23-L27-H28) -4-1BBL) with CH-CL crossover and charged residues

The CEA binding agent humFE23-L27-H28 corresponds to the clone used in P1AE 3103.

Table 40: amino acid sequence of monovalent CEA-targeting (humFE23-L27-H26) -targeting split trimeric 4-1BB ligand antigen binding molecule (CEA (MFE23-L27-H26) -4-1BBL) with CH-CL crossover and charged residues

The CEA binding agent humFE23-L27-H26 corresponds to the clone used in P1AE 3105.

Table 41: amino acid sequence of monovalent CEA-targeting (humFE23-L27-H24) -targeting split trimeric 4-1BB ligand antigen binding molecule (CEA (MFE23-L27-H24) -4-1BBL) with CH-CL crossover and charged residues

The CEA binding agent humFE23-L27-H24 corresponds to the clone used in P1AE 3107.

Bispecific constructs were prepared by transfecting mammalian cells with the corresponding expression vectors in a ratio of 1:1:1 ("vector 4-1BBL Fc-knob chain": vector 4-1BBL light chain ": vector Fc hole chain": vector light chain ") to transfect cells.

Production of IgG-like proteins in HEK293 EBNA or CHO EBNA cells

Antibodies and bispecific antibodies were formed by transient transfection of HEK293 EBNA cells or CHO EBNA cells. The cells were centrifuged and then the pre-warmed CD CHO medium (Thermo Fisher, Cat. No. 10743029) was used in place of the original medium. The expression vectors were mixed in CD CHO medium, PEI (polyethyleneimine, Polysciences, Inc, cat # 23966-1) was added, the solution was vortexed, and incubated at room temperature for 10 minutes. Then, the cells (2Mio/ml) were mixed with the carrier/PEI solution, transferred to flasks, and placed in a shaking incubator at 5% CO₂Was incubated at 37 ℃ for 3 hours under the atmosphere of (2). After incubation, Excell cultures containing supplements (80% of the total volume) were addedNutrient (W.Zhou and A.Kantardjieff, Mammalian Cell Cultures for Biologics Manufacturing, DOI: 10.1007/978-3-642-54050-9; 2014). 1 day after transfection, supplement (feed, 12% of total volume) was added. After 7 days, the cell supernatant was harvested by centrifugation and subsequent filtration (0.2 μm filter), and the protein was purified from the harvested supernatant using standard methods as shown below.

Production of IgG-like proteins in CHO K1 cells

Alternatively, the antibodies and bispecific antibodies described herein were prepared by Evitria using its proprietary vector system by conventional (non-PCR-based) cloning techniques and using suspension-adapted CHO K1 cells (originally received from ATCC and adapted for serum-free growth in suspension culture of Evitria). During the production process, Evtria used its proprietary animal component-free and serum-free medium (eviGrow and eviMake2) and its proprietary transfection reagent (eviFect). The cell supernatant was harvested by centrifugation and subsequent filtration (0.2 μm filter) and the protein was purified from the harvested supernatant using standard methods.

Purification of IgG-like proteins

The protein was purified from the filtered cell culture supernatant according to standard protocols. Briefly, Fc-containing proteins were purified from cell culture supernatants using Protein A affinity chromatography (equilibration buffer: 20mM sodium citrate, 20mM sodium phosphate, pH 7.5; elution buffer: 20mM sodium citrate, pH 3.0). Elution was achieved at pH 3.0, followed by immediate neutralization of the pH of the sample. By centrifugation (Millipore)ULTRA-15, product number: UFC903096) and then size exclusion chromatography was used to separate aggregated from monomeric proteins in 20mM histidine, 140mM sodium chloride (pH 6.0).

Analysis of IgG-like proteins

The concentration of the purified Protein was determined by measuring the absorbance at 280nm using the method described by Pace et al (Protein Science,1995,4,2411-1423) based on amino acidsAnd (5) calculating the mass extinction coefficient by sequence. The purity and molecular weight of the proteins were analyzed by CE-SDS in the presence and absence of reducing agents using LabChipGXII (Perkin Elmer). Aggregate content was determined by HPLC chromatography at 25 ℃ using an analytical size exclusion column (TSKgel G3000 SW XL or UP-SW3000) in running buffer (25 mM K, respectively)₂HPO₄125mM NaCl, 200mM L-arginine hydrochloride (pH 6.7) or 200mM KH₂PO₄250mM KCl (pH 6.2)).

Table 42: biochemical analysis of exemplary CEA-targeting Fc (kih) fusion antigen-binding molecules containing 4-1BB ligand trimers

2.2 functional characterization of Fc fusion antigen-binding molecules containing 4-1BB ligand trimer targeting CEA by surface plasmon resonance

The ability to bind both human 4-1BB Fc (kih) and human CEA in the form of NABA constructs was assessed by Surface Plasmon Resonance (SPR). All SPR experiments used Biacore T200 using HBS-EP as running buffer (0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% surfactant P20, Biacore, Freiburg/Germany) at 25 ℃. Human N (A2B2) a protein was coupled directly to the flow cell of the CM5 chip by amine coupling. The fixed level used was about 600 RU.

Trimeric split 4-1BBL constructs targeting CEA were passed through the flow cell at a concentration range of 200 or 500nM and a flow rate of 30 μ Ι/min for 90 or 240 seconds, and dissociation was set to zero seconds. Human 4-1BB Fc (kih) was injected as a second analyte, at a concentration of 500nM and a flow rate of 30 μ L/min for 90 or 200 seconds through the flow cell (FIG. 9A). Dissociation was monitored for 120 or 600 seconds. Bulk refractive index differences were corrected for by subtracting the responses obtained in the reference flow cell where the protein was not immobilized.

As can be seen in FIGS. 9B to 9H, CEA-targeting 4-1BBL molecules can bind both human CEA (in the form of N (A2B2) A or hu (NA1) BA constructs) and human 4-1 BB.

Example 3

Functional characterization of CEA-targeted split trimeric 4-1BB ligand Fc fusion antigen binding molecules

3.1 binding assay to cells expressing CEACAM5

First, cell lines expressing macaca fascicularis CEACAM5 or human CEACAM5 were generated. The full-length cDNA encoding human and cynomolgus CEACAM5 was subcloned into mammalian expression vectors. The plasmid was transfected into CHO-K1(ATCC CRL-9618) cells using Lipofectamine LTX reagent (Invitrogen, #15338100) according to the manufacturer's protocol. Stably transfected CEACAM5 positive CHO cells were maintained in DMEM/F-12 medium (GIBCO supplied by Life Technologies, #11320033) supplemented with 10% fetal bovine serum (FBS, GIBCO supplied by Life Technologies, Cat. No. 16000-044, batch No. 941273, gamma-irradiated, Mycoplasma-free, heat inactivated) and 2mM L-alanyl-L-glutamine dipeptide (Gluta-MAX-I, GIBCO supplied by Life Technologies, Cat. No. 35050-038). Two days after transfection, puromycin (Invivogen; # ant-pr-1) was added to a concentration of 6. mu.g/mL, and the cells were cultured for several generations. After primary screening, cells with high cell surface expression of human and cynomolgus monkey CEACAM5 were sorted using a BD FACSAria II cell sorter (BD Biosciences) (detection antibody anti-CD 66 clone cd66ab.1.1) and cultured to determine stable cell clones. Expression levels and stability over a period of 4 weeks were confirmed by flow cytometry analysis (see figure 10).

In the binding assay, CHO-k1-cynoCEACAM5 clone 8, CHO-k1-huCEACAM5 clone 11, CHO-k1-huCEACAM5 clone 12, CHO-k1-huCEACAM5 clone 13 or CHO-k1-huCEACAM5 clone 17 were harvested and washed with DPBS (GIBCO supplied by Life Technologies, # 14190-. The cells were washed and incubated at 3X 10⁴Cell/well Density seeding into 384 well plates (Cornin)g # 3830). The cells were centrifuged (350Xg,5min), the supernatant removed, and the cells resuspended in 10. mu.L/well of FACS buffer (DPBS, supplemented with 2% FBS, 5nM EDTA, 7.5mM sodium azide) containing a titrating concentration of CEA-4-1BBL antibody or control (starting concentration 300 nM). Cells were incubated at 4 ℃ for 30 minutes and then washed twice with 80. mu.L/well DPBS. Cells were resuspended in 10. mu.L/well of FACS buffer containing 2.5. mu.g/mL of PE-conjugated AffiniPure anti-human IgG Fc γ -fragment specific goat F (ab')2 fragment (Jackson ImmunoResearch, Cat. No. 109-. Cells were washed twice with 80 μ L/well DPBS and then fixed in 30 μ L/well DPBS containing 1% formaldehyde for at least 15 minutes. Cells from the same or the next day were resuspended in 50 μ L wells of FACS buffer and harvested using macSQurant Analyzer X (Miltenyi Biotec).

As shown in FIGS. 11A-11E and FIGS. 12A-12E and FIGS. 13A-13C, the CEA-4-1BBL antibody binds effectively to CHO-k1 clones 11, 12, 13 and 17 expressing human CEACAM 5. In contrast, only CEA (A5B7) -4-1BBL, CEA (MEDI-565) -4-1BBL and CEA (A5H1EL1D) -4-1BBL and CEA (aff. mat. A5H1EL1D P001.117) -4-1BBL and CEA (aff. mat. A5H1EL1D P005.102) -4-1BBL bound also the CHO-k1-cynoCEACAM5 clone 8 cell line expressing cynomolgus monkey CEACAM5 (FIGS. 11A and 12A and 13A). Thus, MFE23, humFE23-L28-H24, humFE23-L28-H28, T84.66-LCHA do not have cross-reactivity, while A5B7, MEDI565, A5H1EL1D and aff.mat.A5H1EL1D based on clone P001.117 or P005.102 have cynomolgus/human cross-reactivity. However, A5H1EL1D and A5H1EL1D based on clone P001.117 showed lower cynomolgus monkey cross-reactivity than the parent clone A5B 7.

Fitting EC₅₀The values and area under the curve values are listed in tables 43, 44, 45 and 46.

Table 43: EC for binding curves for different humanized CEA-4-1BBL molecules shown in FIGS. 11A-11E and FIGS. 12A-12E₅₀Value of

Table 44: area under the Curve (AUC) of the binding curves for the different humanized CEA-4-1BBL molecules shown in FIGS. 11A-11E and FIGS. 12A-12E

Table 45: EC of binding curves for different humanized CEA-4-1BBL molecules shown in FIGS. 13A-13C ₅₀Value of

Table 46: area under the Curve (AUC) of the binding curves for the different humanized CEA-4-1BBL molecules shown in FIGS. 13A-13C

3.2 activation of NF-. kappa.B in the reporter cell line Jurkat-hu4-1 BB-NF-. kappa.B-luc 2 expressing human 4-1BB and NF-. kappa.B-luciferase reporter genes

Agonistic binding of the 4-1BB (CD137) receptor to its ligand (4-1BBL) induces 4-1BB downstream signaling by activating nuclear factor kappa B (NF kappa B) and promotes survival and activity of CD 8T cells (Lee HW, Park SJ, Choi BK, Kim HH, Nam KO, Kwon BS, J Immunol 2002; 169, 4882-phase 4888). To monitor NF κ B activation mediated by CEA-4-1BBL antigen binding molecules, the Jurkat-hu4-1BB-NF κ B-luc2 reporter cell line was purchased from Promega (Germany). The cell culture method is as described above. For the assay, cells were harvested and resuspended in assay medium RPMI 1640 medium supplemented with 10% (v/v) FBS and 1% (v/v) GlutaMAX-I. 10 μ L of the extract containing 2X 10³A resuspension of Jurkat-hu4-1 BB-NF-. kappa.B-luc 2 reporter cells was transferred to individual wells of a capped sterile white 384-well flat-bottomed tissue culture plate (Corning, Cat. No. 3826). Add 10. mu.L of assay medium containing a titrated concentration of CEA-4-1BBL antibody or control molecule. Finally, 10. mu.L of assay medium alone or containing 1X 10 was provided ⁴Media of individual CHO-k1 cells (transfected with cynomolgus monkey or human CEACAM 5) and the plates were placed in a cell incubator at 37 ℃ and 5% CO₂Incubate for 6 hours. To each well was added 6 μ l of freshly thawed One-Glo luciferase assay detection solution (Promega, catalog No. E6110) and the luminescence intensity was measured immediately using a Tecan plate reader (integration time 500ms, no filter, signal acquisition at all wavelengths).

As shown in FIGS. 14A-14D and FIGS. 15A-15D, in the absence of CEACAM 5-expressing cells, none of the molecules was able to induce strong human 4-1BB receptor activation in Jurkat-hu4-1 BB-NF-. kappa.B-luc 2 reporter cell line, leading to NF-. kappa.B activation and thus luciferase expression. In the presence of human CEACAM5 expressing cells such as CHO-k 1-human CEACAM5 clone 11 and CHO-k 1-human CEACAM5 clone 12, cross-linking of CEA-4-1BBL antibody resulted in a strong increase in the luciferase activity of NF κ B activation in Jurkat-hu4-1BB-NF κ B-luc2 reporter cell line, above that mediated by the non-targeted control DP47-4-1 BBL. In the presence of CHO-k1-cyno CEACAM5 clone 8, the induction of activation was dependent on the CEACAM5 binding clone used. Clone MFE23 (parental) or humanized MFE23 clone huMFE23-L28-H24 or huMFE23-L28-H28 or humanized MFE23 clone sm9B (disclosed in US 2005/0147614) containing CEA-4-1BBL antibody or reference clone T84.66-LCHA did not bind to cynomolgus monkey CEACAM5 and therefore did not induce Jurkat-hu4-1 BB-nfkb-luc 2 reporter cell activation (fig. 14B). On the other hand, cynomolgus monkey/human cross-reactive clone A5B7 (parental), humanized A5B7 clone MEDI-565 or A5H1EL1D or affinity maturation-based clone P005-102 or P001-177 of A5H1EL1D containing CEA-4-1BBL antibody also induced Jurkat-hu4-1BB-NF κ B-luc2 reporter cell activation in the presence of CHO-k1-cyno CEACAM5 clone 8 (FIG. 15B).

EC of activation Curve₅₀The values and area under the curve (AUC) are listed in tables 47 and 48, respectively.

Table 47: EC of activation curves for different humanized CEA-4-1BBL molecules shown in FIGS. 14A-14D and FIGS. 15A-15D₅₀Value of

Table 48: the area under the curve (AUC) of the activation curves for the different humanized CEA-4-1BBL molecules shown in FIGS. 14A-14D and FIGS. 15A-15D

Sequence listing

<110> Haofmai Roche Ltd

<120> novel antigen binding molecules binding to CEA

<130> P35612-WO

<150> 19182505.8

<151> 2019-06-26

<160> 313

<170> PatentIn edition 3.5

<210> 1

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5B7)- CDR-H1

<400> 1

Asp Tyr Tyr Met Asn

1 5

<210> 2

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5B7)- CDR-H2

<400> 2

Phe Ile Gly Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 3

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5B7)- CDR-H3

<400> 3

Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr

1 5 10

<210> 4

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5B7)- CDR-L1

<400> 4

Arg Ala Ser Ser Ser Val Thr Tyr Ile His

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5B7)- CDR-L2

<400> 5

Ala Thr Ser Asn Leu Ala Ser

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5B7)- CDR-L3

<400> 6

Gln His Trp Ser Ser Lys Pro Pro Thr

1 5

<210> 7

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5B7) VH (parent)

<400> 7

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Gln Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Ser Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 8

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5B7) VL (parent)

<400> 8

Gln Thr Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 9

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> CEA CDR-H1

<400> 9

Ser Tyr Trp Met His

1 5

<210> 10

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> CEA CDR-H2

<400> 10

Phe Ile Arg Asn Lys Ala Asn Gly Gly Thr Thr Glu Tyr Ala Ala Ser

1 5 10 15

Val Lys Gly

<210> 11

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA CDR-H3

<400> 11

Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr

1 5 10

<210> 12

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> CEA CDR-L1

<400> 12

Thr Leu Arg Arg Gly Ile Asn Val Gly Ala Tyr Ser Ile Tyr

1 5 10

<210> 13

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> CEA CDR-L2

<400> 13

Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly Ser Gly Val

1 5 10

<210> 14

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA CDR-L3

<400> 14

Met Ile Trp His Ser Gly Ala Ser Ala Val

1 5 10

<210> 15

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> CEA VH

<400> 15

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Arg Asn Lys Ala Asn Gly Gly Thr Thr Glu Tyr Ala Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 16

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> CEA VL

<400> 16

Gln Ala Val Leu Thr Gln Pro Ala Ser Leu Ser Ala Ser Pro Gly Ala

1 5 10 15

Ser Ala Ser Leu Thr Cys Thr Leu Arg Arg Gly Ile Asn Val Gly Ala

20 25 30

Tyr Ser Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Tyr

35 40 45

Leu Leu Arg Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly Ser Gly Val

50 55 60

Ser Ser Arg Phe Ser Ala Ser Lys Asp Ala Ser Ala Asn Ala Gly Ile

65 70 75 80

Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys

85 90 95

Met Ile Trp His Ser Gly Ala Ser Ala Val Phe Gly Gly Gly Thr Lys

100 105 110

Leu Thr Val Leu

115

<210> 17

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5H1EL1D)- CDR-H1

<400> 17

Gly Phe Thr Phe Thr Asp Tyr Tyr Met Asn

1 5 10

<210> 18

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5H1EL1D)- CDR-H2

<400> 18

Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 19

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5H1EL1D)- CDR-H3

<400> 19

Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr

1 5 10

<210> 20

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5H1EL1D)- CDR-L1

<400> 20

Arg Ala Ser Ser Ser Val Thr Tyr Ile His

1 5 10

<210> 21

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5H1EL1D)- CDR-L2

<400> 21

Ala Thr Ser Asn Leu Ala Ser

1 5

<210> 22

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5H1EL1D)- CDR-L3

<400> 22

Gln His Trp Ser Ser Lys Pro Pro Thr

1 5

<210> 23

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5H1EL1D) VH (3-23A5-1E)

<400> 23

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 24

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5H1EL1D) VL (A5-L1D)

<400> 24

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 25

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5H1EL1D af. mat.) CDR-H1 consensus

<220>

<221> variants

<222> (3)..(3)

<223> X is Thr or Tyr

<220>

<221> variants

<222> (5)..(5)

<223> X is Thr or Ser

<220>

<221> variants

<222> (8)..(8)

<223> X is Tyr or Ala or Glu

<400> 25

Gly Phe Xaa Phe Xaa Asp Tyr Xaa Met Asn

1 5 10

<210> 26

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5H1EL1D af. mat.) CDR-H2 consensus

<220>

<221> variants

<222> (1)..(1)

<223> X is Phe or Val

<220>

<221> variants

<222> (3)..(3)

<223> X is Gly or Ser

<400> 26

Xaa Ile Xaa Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 27

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5H1EL1D af. mat.) CDR-H3 consensus

<220>

<221> variants

<222> (4)..(4)

<223> X is Leu or Ile

<220>

<221> variants

<222> (7)..(7)

<223> X is Tyr or Gly or Gln or Ser

<400> 27

Asp Arg Gly Xaa Arg Phe Xaa Phe Asp Tyr

1 5 10

<210> 28

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5H1EL1D af. mat.) CDR-L1 consensus

<220>

<221> variants

<222> (1)..(1)

<223> X is Arg or His

<400> 28

Xaa Ala Ser Ser Ser Val Thr Tyr Ile His

1 5 10

<210> 29

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5H1EL1D af. mat.) CDR-L2 consensus

<400> 29

Ala Thr Ser Asn Leu Ala Ser

1 5

<210> 30

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5H1EL1D af. mat.) CDR-L3 consensus

<220>

<221> variants

<222> (6)..(6)

<223> X is Lys or Val or Gln or Ile

<220>

<221> variants

<222> (7)..(7)

<223> X is Pro or Ser

<400> 30

Gln His Trp Ser Ser Xaa Xaa Pro Thr

1 5

<210> 31

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038) VH

<400> 31

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Ile Arg Phe Gly Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 32

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038) VL

<400> 32

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Val Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 33

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.097) VH

<400> 33

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Ser Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 34

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.097) VL

<400> 34

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Gln Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 35

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103) VH

<400> 35

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Ile Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 36

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103) VL

<400> 36

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Ile Ser Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 37

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P002.139) VH

<400> 37

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Tyr Phe Thr Asp Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Ser Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 38

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P002.139) VL

<400> 38

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys His Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 39

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P001.177) VH

<400> 39

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Tyr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Ser Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 40

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P001.177) VL

<400> 40

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 41

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102) VH

<400> 41

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Ile Arg Phe Gln Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 42

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102) VL

<400> 42

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Ser Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 43

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102 combo1) VH

<400> 43

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Tyr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Ser Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Ile Arg Phe Gln Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 44

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102 combo1) VL

<400> 44

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Ser Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 45

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102 combo2) VH

<400> 45

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Tyr Phe Ser Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Ser Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Ile Arg Phe Gln Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 46

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102 combo2) VL

<400> 46

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Ser Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 47

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103 combo1) VH

<400> 47

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Ile Arg Phe Ser Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 48

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103 combo1) VL

<400> 48

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Ile Ser Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 49

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103 combo2) VH

<400> 49

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Tyr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Ser Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Ile Arg Phe Ser Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 50

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103 combo2) VL

<400> 50

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Ile Ser Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 51

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038 combo1) VH

<400> 51

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Tyr Phe Thr Asp Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Ser Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Ile Arg Phe Gly Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 52

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038 combo1) VL

<400> 52

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Val Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 53

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038 combo2) VH

<400> 53

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Glu Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Ser Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Ile Arg Phe Gly Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 54

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038 combo2) VL

<400> 54

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Val Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 55

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE23)- CDR-H1

<400> 55

Asp Ser Tyr Met His

1 5

<210> 56

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE23)- CDR-H2

<400> 56

Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe Gln

1 5 10 15

Gly

<210> 57

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE23)- CDR-H3

<400> 57

Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr

1 5 10

<210> 58

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE23)- CDR-L1

<400> 58

Ser Ala Ser Ser Ser Val Ser Tyr Met His

1 5 10

<210> 59

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE23)- CDR-L2

<400> 59

Ser Thr Ser Asn Leu Ala Ser

1 5

<210> 60

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE23)- CDR-L3

<400> 60

Gln Gln Arg Ser Ser Tyr Pro Leu Thr

1 5

<210> 61

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE23) VH

<400> 61

Gln Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Thr

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Leu Arg Gln Gly Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Lys Ala Thr Phe Thr Thr Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 62

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE23) VL

<400> 62

Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 63

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (T84.66-LCHA) VH

<400> 63

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 64

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (T84.66-LCHA) VL

<400> 64

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Gly Glu Ser Val Asp Ile Phe

20 25 30

Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 65

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE-H24 to H29)- CDR-H1

<400> 65

Asp Ser Tyr Met His

1 5

<210> 66

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE-H24, H25, H27, H28, H29)- CDR-H2

<400> 66

Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe Gln

1 5 10 15

Gly

<210> 67

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE-H26)- CDR-H2

<400> 67

Trp Ile Asp Pro Glu Asn Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 68

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE-H24 to H29)- CDR-H3

<400> 68

Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr

1 5 10

<210> 69

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE-L24, L25)- CDR-L1

<400> 69

Arg Ala Ser Ser Ser Val Ser Tyr Met His

1 5 10

<210> 70

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE-H26)- CDR-L1

<400> 70

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Met

1 5 10

<210> 71

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE-L24, L25, L27, L28)- CDR-L2

<400> 71

Ser Thr Ser Asn Leu Ala Ser

1 5

<210> 72

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE-L26)- CDR-L2

<400> 72

Tyr Thr Ser Asn Leu Ala Ser

1 5

<210> 73

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE-L29)- CDR-L2

<400> 73

Ser Thr Ser Ser Leu Gln Ser

1 5

<210> 74

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE-L24, L25, L27, L26, L28, L29)- CDR-L3

<400> 74

Gln Gln Arg Ser Ser Tyr Pro Leu Thr

1 5

<210> 75

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-H24

<400> 75

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 76

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-H25

<400> 76

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 77

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-H26

<400> 77

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 78

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-H27

<400> 78

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 79

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-H28

<400> 79

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 80

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-H29

<400> 80

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Thr Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 81

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-L24

<400> 81

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 82

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-L25

<400> 82

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 83

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-L26

<400> 83

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 84

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-L27

<400> 84

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 85

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-L28

<400> 85

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Leu Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 86

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-L29

<400> 86

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Leu Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 87

<211> 184

<212> PRT

<213> Intelligent people

<400> 87

Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp

1 5 10 15

Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu

20 25 30

Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val

35 40 45

Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val

50 55 60

Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg

65 70 75 80

Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His

85 90 95

Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr

100 105 110

Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly

115 120 125

Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val

130 135 140

His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln

145 150 155 160

Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala

165 170 175

Gly Leu Pro Ser Pro Arg Ser Glu

180

<210> 88

<211> 170

<212> PRT

<213> Intelligent people

<400> 88

Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val

1 5 10 15

Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala

20 25 30

Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu

35 40 45

Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu

50 55 60

Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala

65 70 75 80

Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala

85 90 95

Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala

100 105 110

Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu

115 120 125

Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu

130 135 140

Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile

145 150 155 160

Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu

165 170

<210> 89

<211> 175

<212> PRT

<213> Intelligent people

<400> 89

Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu

1 5 10 15

Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser

20 25 30

Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys

35 40 45

Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val

50 55 60

Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly

65 70 75 80

Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly

85 90 95

Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu

100 105 110

Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser

115 120 125

Ala Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg

130 135 140

His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg

145 150 155 160

Val Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu

165 170 175

<210> 90

<211> 203

<212> PRT

<213> Intelligent people

<400> 90

Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala Ala Ser

1 5 10 15

Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly

20 25 30

Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn

35 40 45

Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu

50 55 60

Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys

65 70 75 80

Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu

85 90 95

Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu

100 105 110

Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu

115 120 125

Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser

130 135 140

Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg

145 150 155 160

Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln

165 170 175

Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu

180 185 190

Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu

195 200

<210> 91

<211> 178

<212> PRT

<213> Intelligent people

<400> 91

Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp

1 5 10 15

Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu

20 25 30

Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val

35 40 45

Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val

50 55 60

Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg

65 70 75 80

Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His

85 90 95

Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr

100 105 110

Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly

115 120 125

Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val

130 135 140

His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln

145 150 155 160

Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala

165 170 175

Gly Leu

<210> 92

<211> 164

<212> PRT

<213> Intelligent people

<400> 92

Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val

1 5 10 15

Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala

20 25 30

Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu

35 40 45

Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu

50 55 60

Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala

65 70 75 80

Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala

85 90 95

Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala

100 105 110

Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu

115 120 125

Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu

130 135 140

Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile

145 150 155 160

Pro Ala Gly Leu

<210> 93

<211> 169

<212> PRT

<213> Intelligent people

<400> 93

Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu

1 5 10 15

Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser

20 25 30

Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys

35 40 45

Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val

50 55 60

Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly

65 70 75 80

Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly

85 90 95

Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu

100 105 110

Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser

115 120 125

Ala Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg

130 135 140

His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg

145 150 155 160

Val Thr Pro Glu Ile Pro Ala Gly Leu

165

<210> 94

<211> 197

<212> PRT

<213> Intelligent people

<400> 94

Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala Ala Ser

1 5 10 15

Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly

20 25 30

Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn

35 40 45

Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu

50 55 60

Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys

65 70 75 80

Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu

85 90 95

Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu

100 105 110

Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu

115 120 125

Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser

130 135 140

Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg

145 150 155 160

Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln

165 170 175

Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu

180 185 190

Ile Pro Ala Gly Leu

195

<210> 95

<211> 378

<212> PRT

<213> Artificial sequence

<220>

<223> dimeric hu 4-1BBL (71-254), linked by a (G4S)2 linker

<400> 95

Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp

1 5 10 15

Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu

20 25 30

Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val

35 40 45

Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val

50 55 60

Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg

65 70 75 80

Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His

85 90 95

Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr

100 105 110

Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly

115 120 125

Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val

130 135 140

His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln

145 150 155 160

Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala

165 170 175

Gly Leu Pro Ser Pro Arg Ser Glu Gly Gly Gly Gly Ser Gly Gly Gly

180 185 190

Gly Ser Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu

195 200 205

Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val

210 215 220

Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala

225 230 235 240

Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu

245 250 255

Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu

260 265 270

Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala

275 280 285

Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala

290 295 300

Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala

305 310 315 320

Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu

325 330 335

Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu

340 345 350

Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile

355 360 365

Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu

370 375

<210> 96

<211> 366

<212> PRT

<213> Artificial sequence

<220>

<223> dimeric hu 4-1BBL (71-248) linked by a (G4S)2 linker

<400> 96

Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp

1 5 10 15

Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu

20 25 30

Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val

35 40 45

Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val

50 55 60

Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg

65 70 75 80

Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His

85 90 95

Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr

100 105 110

Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly

115 120 125

Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val

130 135 140

His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln

145 150 155 160

Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala

165 170 175

Gly Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Glu Gly Pro

180 185 190

Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly

195 200 205

Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro

210 215 220

Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly

225 230 235 240

Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala

245 250 255

Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala

260 265 270

Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu

275 280 285

Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro

290 295 300

Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg

305 310 315 320

Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu His Thr

325 330 335

Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val

340 345 350

Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala Gly Leu

355 360 365

<210> 97

<211> 360

<212> PRT

<213> Artificial sequence

<220>

<223> dimeric hu 4-1BBL (80-254), linked by a (G4S)2 linker

<400> 97

Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu

1 5 10 15

Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser

20 25 30

Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys

35 40 45

Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val

50 55 60

Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly

65 70 75 80

Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly

85 90 95

Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu

100 105 110

Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser

115 120 125

Ala Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg

130 135 140

His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg

145 150 155 160

Val Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu Gly

165 170 175

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Pro Ala Gly Leu Leu Asp

180 185 190

Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu

195 200 205

Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val

210 215 220

Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val

225 230 235 240

Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg

245 250 255

Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His

260 265 270

Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr

275 280 285

Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly

290 295 300

Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val

305 310 315 320

His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln

325 330 335

Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala

340 345 350

Gly Leu Pro Ser Pro Arg Ser Glu

355 360

<210> 98

<211> 416

<212> PRT

<213> Artificial sequence

<220>

<223> dimeric hu 4-1BBL (52-254), linked by a (G4S)2 linker

<400> 98

Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala Ala Ser

1 5 10 15

Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly

20 25 30

Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn

35 40 45

Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu

50 55 60

Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys

65 70 75 80

Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu

85 90 95

Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu

100 105 110

Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu

115 120 125

Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser

130 135 140

Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg

145 150 155 160

Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln

165 170 175

Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu

180 185 190

Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu Gly Gly Gly Gly Ser

195 200 205

Gly Gly Gly Gly Ser Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro

210 215 220

Gly Ser Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro

225 230 235 240

Asp Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln

245 250 255

Leu Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr

260 265 270

Ser Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr

275 280 285

Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr

290 295 300

Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser

305 310 315 320

Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala

325 330 335

Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser

340 345 350

Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu

355 360 365

Ser Ala Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala

370 375 380

Arg His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe

385 390 395 400

Arg Val Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu

405 410 415

<210> 99

<211> 708

<212> PRT

<213> Artificial sequence

<220>

<223> dimeric 4-1BBL-CL Fc pestle chain

<400> 99

Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp

1 5 10 15

Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu

20 25 30

Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val

35 40 45

Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val

50 55 60

Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg

65 70 75 80

Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His

85 90 95

Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr

100 105 110

Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly

115 120 125

Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val

130 135 140

His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln

145 150 155 160

Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala

165 170 175

Gly Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Glu Gly Pro

180 185 190

Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly

195 200 205

Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro

210 215 220

Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly

225 230 235 240

Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala

245 250 255

Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala

260 265 270

Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu

275 280 285

Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro

290 295 300

Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg

305 310 315 320

Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu His Thr

325 330 335

Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val

340 345 350

Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala Gly Leu Gly Gly

355 360 365

Gly Gly Ser Gly Gly Gly Gly Ser Arg Thr Val Ala Ala Pro Ser Val

370 375 380

Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly Thr Ala Ser

385 390 395 400

Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln

405 410 415

Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val

420 425 430

Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu

435 440 445

Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu

450 455 460

Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg

465 470 475 480

Gly Glu Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

485 490 495

Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

500 505 510

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

515 520 525

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

530 535 540

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

545 550 555 560

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

565 570 575

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly

580 585 590

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

595 600 605

Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn

610 615 620

Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

625 630 635 640

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

645 650 655

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

660 665 670

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

675 680 685

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

690 695 700

Ser Leu Ser Pro

705

<210> 100

<211> 291

<212> PRT

<213> Artificial sequence

<220>

<223> monomer 4-1 BBL-CH 1 >

<400> 100

Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp

1 5 10 15

Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu

20 25 30

Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val

35 40 45

Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val

50 55 60

Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg

65 70 75 80

Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His

85 90 95

Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr

100 105 110

Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly

115 120 125

Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val

130 135 140

His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln

145 150 155 160

Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala

165 170 175

Gly Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Thr Lys

180 185 190

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

195 200 205

Gly Thr Ala Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro

210 215 220

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

225 230 235 240

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

245 250 255

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

260 265 270

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro

275 280 285

Lys Ser Cys

290

<210> 101

<211> 708

<212> PRT

<213> Artificial sequence

<220>

<223> dimeric 4-1BB ligand (71-248) -CL Fc pestle chain

<400> 101

Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp

1 5 10 15

Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu

20 25 30

Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val

35 40 45

Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val

50 55 60

Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg

65 70 75 80

Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His

85 90 95

Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr

100 105 110

Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly

115 120 125

Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val

130 135 140

His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln

145 150 155 160

Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala

165 170 175

Gly Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Glu Gly Pro

180 185 190

Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly

195 200 205

Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro

210 215 220

Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly

225 230 235 240

Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala

245 250 255

Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala

260 265 270

Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu

275 280 285

Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro

290 295 300

Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg

305 310 315 320

Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu His Thr

325 330 335

Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val

340 345 350

Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala Gly Leu Gly Gly

355 360 365

Gly Gly Ser Gly Gly Gly Gly Ser Arg Thr Val Ala Ala Pro Ser Val

370 375 380

Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser

385 390 395 400

Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln

405 410 415

Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val

420 425 430

Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu

435 440 445

Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu

450 455 460

Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg

465 470 475 480

Gly Glu Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

485 490 495

Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

500 505 510

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

515 520 525

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

530 535 540

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

545 550 555 560

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

565 570 575

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly

580 585 590

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

595 600 605

Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn

610 615 620

Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

625 630 635 640

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

645 650 655

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

660 665 670

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

675 680 685

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

690 695 700

Ser Leu Ser Pro

705

<210> 102

<211> 291

<212> PRT

<213> Artificial sequence

<220>

<223> monomeric 4-1BB ligand (71-248) -CH1

<400> 102

Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp

1 5 10 15

Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu

20 25 30

Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val

35 40 45

Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val

50 55 60

Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg

65 70 75 80

Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His

85 90 95

Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr

100 105 110

Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly

115 120 125

Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val

130 135 140

His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln

145 150 155 160

Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala

165 170 175

Gly Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Thr Lys

180 185 190

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

195 200 205

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

210 215 220

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

225 230 235 240

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

245 250 255

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

260 265 270

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

275 280 285

Lys Ser Cys

290

<210> 103

<211> 720

<212> PRT

<213> Artificial sequence

<220>

<223> dimeric 4-1BB ligand (71-254) -CL _ Fc pestle chain

<400> 103

Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp

1 5 10 15

Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu

20 25 30

Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val

35 40 45

Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val

50 55 60

Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg

65 70 75 80

Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His

85 90 95

Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr

100 105 110

Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly

115 120 125

Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val

130 135 140

His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln

145 150 155 160

Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala

165 170 175

Gly Leu Pro Ser Pro Arg Ser Glu Gly Gly Gly Gly Ser Gly Gly Gly

180 185 190

Gly Ser Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu

195 200 205

Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val

210 215 220

Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala

225 230 235 240

Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu

245 250 255

Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu

260 265 270

Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala

275 280 285

Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala

290 295 300

Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala

305 310 315 320

Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu

325 330 335

Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu

340 345 350

Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile

355 360 365

Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu Gly Gly Gly Gly Ser Gly

370 375 380

Gly Gly Gly Ser Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

385 390 395 400

Pro Ser Asp Arg Lys Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

405 410 415

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

420 425 430

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

435 440 445

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

450 455 460

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

465 470 475 480

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Asp

485 490 495

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

500 505 510

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

515 520 525

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

530 535 540

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

545 550 555 560

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

565 570 575

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

580 585 590

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

595 600 605

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

610 615 620

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

625 630 635 640

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

645 650 655

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

660 665 670

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

675 680 685

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

690 695 700

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

705 710 715 720

<210> 104

<211> 297

<212> PRT

<213> Artificial sequence

<220>

<223> monomeric 4-1BB ligand (71-254) -CH1

<400> 104

Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp

1 5 10 15

Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu

20 25 30

Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val

35 40 45

Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val

50 55 60

Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg

65 70 75 80

Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His

85 90 95

Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr

100 105 110

Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly

115 120 125

Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val

130 135 140

His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln

145 150 155 160

Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala

165 170 175

Gly Leu Pro Ser Pro Arg Ser Glu Gly Gly Gly Gly Ser Gly Gly Gly

180 185 190

Gly Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

195 200 205

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Glu

210 215 220

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

225 230 235 240

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

245 250 255

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

260 265 270

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

275 280 285

Asp Glu Lys Val Glu Pro Lys Ser Cys

290 295

<210> 105

<211> 720

<212> PRT

<213> Artificial sequence

<220>

<223> dimeric 4-1BB ligand (71-254) -CL Fc pestle chain

<400> 105

Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp

1 5 10 15

Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu

20 25 30

Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val

35 40 45

Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val

50 55 60

Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg

65 70 75 80

Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His

85 90 95

Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr

100 105 110

Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly

115 120 125

Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val

130 135 140

His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln

145 150 155 160

Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala

165 170 175

Gly Leu Pro Ser Pro Arg Ser Glu Gly Gly Gly Gly Ser Gly Gly Gly

180 185 190

Gly Ser Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu

195 200 205

Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val

210 215 220

Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala

225 230 235 240

Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu

245 250 255

Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu

260 265 270

Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala

275 280 285

Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala

290 295 300

Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala

305 310 315 320

Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu

325 330 335

Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu

340 345 350

Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile

355 360 365

Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu Gly Gly Gly Gly Ser Gly

370 375 380

Gly Gly Gly Ser Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

385 390 395 400

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

405 410 415

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

420 425 430

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

435 440 445

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

450 455 460

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

465 470 475 480

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Asp

485 490 495

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

500 505 510

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

515 520 525

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

530 535 540

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

545 550 555 560

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

565 570 575

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

580 585 590

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

595 600 605

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

610 615 620

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

625 630 635 640

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

645 650 655

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

660 665 670

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

675 680 685

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

690 695 700

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

705 710 715 720

<210> 106

<211> 297

<212> PRT

<213> Artificial sequence

<220>

<223> monomeric 4-1BB ligand (71-254) -CH1

<400> 106

Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp

1 5 10 15

Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu

20 25 30

Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val

35 40 45

Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val

50 55 60

Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg

65 70 75 80

Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His

85 90 95

Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr

100 105 110

Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly

115 120 125

Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val

130 135 140

His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln

145 150 155 160

Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala

165 170 175

Gly Leu Pro Ser Pro Arg Ser Glu Gly Gly Gly Gly Ser Gly Gly Gly

180 185 190

Gly Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

195 200 205

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

210 215 220

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

225 230 235 240

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

245 250 255

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

260 265 270

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

275 280 285

Asp Lys Lys Val Glu Pro Lys Ser Cys

290 295

<210> 107

<211> 328

<212> PRT

<213> Artificial sequence

<220>

<223> Fc mortar chain

<400> 107

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 108

<211> 702

<212> PRT

<213> Intelligent people

<400> 108

Met Glu Ser Pro Ser Ala Pro Pro His Arg Trp Cys Ile Pro Trp Gln

1 5 10 15

Arg Leu Leu Leu Thr Ala Ser Leu Leu Thr Phe Trp Asn Pro Pro Thr

20 25 30

Thr Ala Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly

35 40 45

Lys Glu Val Leu Leu Leu Val His Asn Leu Pro Gln His Leu Phe Gly

50 55 60

Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Ile

65 70 75 80

Gly Tyr Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser

85 90 95

Gly Arg Glu Ile Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Ile

100 105 110

Ile Gln Asn Asp Thr Gly Phe Tyr Thr Leu His Val Ile Lys Ser Asp

115 120 125

Leu Val Asn Glu Glu Ala Thr Gly Gln Phe Arg Val Tyr Pro Glu Leu

130 135 140

Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro Val Glu Asp Lys

145 150 155 160

Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Ala Thr Tyr

165 170 175

Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln

180 185 190

Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn Val Thr Arg Asn

195 200 205

Asp Thr Ala Ser Tyr Lys Cys Glu Thr Gln Asn Pro Val Ser Ala Arg

210 215 220

Arg Ser Asp Ser Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Ala Pro

225 230 235 240

Thr Ile Ser Pro Leu Asn Thr Ser Tyr Arg Ser Gly Glu Asn Leu Asn

245 250 255

Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Phe

260 265 270

Val Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn

275 280 285

Ile Thr Val Asn Asn Ser Gly Ser Tyr Thr Cys Gln Ala His Asn Ser

290 295 300

Asp Thr Gly Leu Asn Arg Thr Thr Val Thr Thr Ile Thr Val Tyr Ala

305 310 315 320

Glu Pro Pro Lys Pro Phe Ile Thr Ser Asn Asn Ser Asn Pro Val Glu

325 330 335

Asp Glu Asp Ala Val Ala Leu Thr Cys Glu Pro Glu Ile Gln Asn Thr

340 345 350

Thr Tyr Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg

355 360 365

Leu Gln Leu Ser Asn Asp Asn Arg Thr Leu Thr Leu Leu Ser Val Thr

370 375 380

Arg Asn Asp Val Gly Pro Tyr Glu Cys Gly Ile Gln Asn Lys Leu Ser

385 390 395 400

Val Asp His Ser Asp Pro Val Ile Leu Asn Val Leu Tyr Gly Pro Asp

405 410 415

Asp Pro Thr Ile Ser Pro Ser Tyr Thr Tyr Tyr Arg Pro Gly Val Asn

420 425 430

Leu Ser Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser

435 440 445

Trp Leu Ile Asp Gly Asn Ile Gln Gln His Thr Gln Glu Leu Phe Ile

450 455 460

Ser Asn Ile Thr Glu Lys Asn Ser Gly Leu Tyr Thr Cys Gln Ala Asn

465 470 475 480

Asn Ser Ala Ser Gly His Ser Arg Thr Thr Val Lys Thr Ile Thr Val

485 490 495

Ser Ala Glu Leu Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro

500 505 510

Val Glu Asp Lys Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Ala Gln

515 520 525

Asn Thr Thr Tyr Leu Trp Trp Val Asn Gly Gln Ser Leu Pro Val Ser

530 535 540

Pro Arg Leu Gln Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn

545 550 555 560

Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Cys Gly Ile Gln Asn Ser

565 570 575

Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu Tyr Gly

580 585 590

Pro Asp Thr Pro Ile Ile Ser Pro Pro Asp Ser Ser Tyr Leu Ser Gly

595 600 605

Ala Asn Leu Asn Leu Ser Cys His Ser Ala Ser Asn Pro Ser Pro Gln

610 615 620

Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu

625 630 635 640

Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe

645 650 655

Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile

660 665 670

Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu Ser Ala Gly Ala Thr

675 680 685

Val Gly Ile Met Ile Gly Val Leu Val Gly Val Ala Leu Ile

690 695 700

<210> 109

<211> 255

<212> PRT

<213> Intelligent people

<400> 109

Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu

1 5 10 15

Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro

20 25 30

Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys

35 40 45

Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile

50 55 60

Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser

65 70 75 80

Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly

85 90 95

Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu

100 105 110

Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln

115 120 125

Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys

130 135 140

Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro

145 150 155 160

Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala

165 170 175

Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu

180 185 190

Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu

195 200 205

Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

210 215 220

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

225 230 235 240

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

245 250 255

<210> 110

<211> 256

<212> PRT

<213> mice

<400> 110

Met Gly Asn Asn Cys Tyr Asn Val Val Val Ile Val Leu Leu Leu Val

1 5 10 15

Gly Cys Glu Lys Val Gly Ala Val Gln Asn Ser Cys Asp Asn Cys Gln

20 25 30

Pro Gly Thr Phe Cys Arg Lys Tyr Asn Pro Val Cys Lys Ser Cys Pro

35 40 45

Pro Ser Thr Phe Ser Ser Ile Gly Gly Gln Pro Asn Cys Asn Ile Cys

50 55 60

Arg Val Cys Ala Gly Tyr Phe Arg Phe Lys Lys Phe Cys Ser Ser Thr

65 70 75 80

His Asn Ala Glu Cys Glu Cys Ile Glu Gly Phe His Cys Leu Gly Pro

85 90 95

Gln Cys Thr Arg Cys Glu Lys Asp Cys Arg Pro Gly Gln Glu Leu Thr

100 105 110

Lys Gln Gly Cys Lys Thr Cys Ser Leu Gly Thr Phe Asn Asp Gln Asn

115 120 125

Gly Thr Gly Val Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Arg

130 135 140

Ser Val Leu Lys Thr Gly Thr Thr Glu Lys Asp Val Val Cys Gly Pro

145 150 155 160

Pro Val Val Ser Phe Ser Pro Ser Thr Thr Ile Ser Val Thr Pro Glu

165 170 175

Gly Gly Pro Gly Gly His Ser Leu Gln Val Leu Thr Leu Phe Leu Ala

180 185 190

Leu Thr Ser Ala Leu Leu Leu Ala Leu Ile Phe Ile Thr Leu Leu Phe

195 200 205

Ser Val Leu Lys Trp Ile Arg Lys Lys Phe Pro His Ile Phe Lys Gln

210 215 220

Pro Phe Lys Lys Thr Thr Gly Ala Ala Gln Glu Glu Asp Ala Cys Ser

225 230 235 240

Cys Arg Cys Pro Gln Glu Glu Glu Gly Gly Gly Gly Gly Tyr Glu Leu

245 250 255

<210> 111

<211> 254

<212> PRT

<213> crab eating macaque

<400> 111

Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu

1 5 10 15

Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Leu Cys Ser Asn Cys Pro

20 25 30

Ala Gly Thr Phe Cys Asp Asn Asn Arg Ser Gln Ile Cys Ser Pro Cys

35 40 45

Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile

50 55 60

Cys Arg Gln Cys Lys Gly Val Phe Lys Thr Arg Lys Glu Cys Ser Ser

65 70 75 80

Thr Ser Asn Ala Glu Cys Asp Cys Ile Ser Gly Tyr His Cys Leu Gly

85 90 95

Ala Glu Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu

100 105 110

Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln

115 120 125

Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys

130 135 140

Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro

145 150 155 160

Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Ala Thr Pro Pro Ala

165 170 175

Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Phe Phe Leu Ala

180 185 190

Leu Thr Ser Thr Val Val Leu Phe Leu Leu Phe Phe Leu Val Leu Arg

195 200 205

Phe Ser Val Val Lys Arg Ser Arg Lys Lys Leu Leu Tyr Ile Phe Lys

210 215 220

Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys

225 230 235 240

Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

245 250

<210> 112

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker G4S

<400> 112

Gly Gly Gly Gly Ser

1 5

<210> 113

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker (G4S)2

<400> 113

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 114

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker (SG4)2

<400> 114

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

1 5 10

<210> 115

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker G4(SG4)2

<400> 115

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

1 5 10

<210> 116

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker GSPGSSSSGS

<400> 116

Gly Ser Pro Gly Ser Ser Ser Ser Gly Ser

1 5 10

<210> 117

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker (G4S)3

<400> 117

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 118

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker (G4S)4

<400> 118

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 119

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker GSGSGSGS

<400> 119

Gly Ser Gly Ser Gly Ser Gly Ser

1 5

<210> 120

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker GSGSGNGS

<400> 120

Gly Ser Gly Ser Gly Asn Gly Ser

1 5

<210> 121

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker GGSGSGSGSG

<400> 121

Gly Gly Ser Gly Ser Gly Ser Gly

1 5

<210> 122

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker GGSGSG

<400> 122

Gly Gly Ser Gly Ser Gly

1 5

<210> 123

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker GGSG

<400> 123

Gly Gly Ser Gly

1

<210> 124

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> GGSGNGSG

<400> 124

Gly Gly Ser Gly Asn Gly Ser Gly

1 5

<210> 125

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker GGNGSGSG

<400> 125

Gly Gly Asn Gly Ser Gly Ser Gly

1 5

<210> 126

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker GGNGSG

<400> 126

Gly Gly Asn Gly Ser Gly

1 5

<210> 127

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<223> IGHV3-23-02

<400> 127

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Gly Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys

<210> 128

<211> 100

<212> PRT

<213> Artificial sequence

<220>

<223> IGHV3-15*01

<400> 128

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Thr

100

<210> 129

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> 3-23A5-1

<400> 129

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 130

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> 3-23A5-2

<400> 130

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Gly Tyr Thr Thr Tyr Tyr Gly Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 131

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> 3-23A5-3

<400> 131

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Gly Asn Lys Gly Tyr Thr Thr Glu Tyr Ser Ala Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 132

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> 3-23A5-4

<400> 132

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 133

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> 3-23A5-1A (all back mutations)

<400> 133

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 134

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> 3-23A5-1C (A93T)

<400> 134

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 135

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> 3-23A5-1D (K73)

<400> 135

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 136

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> 3-15A5-1

<400> 136

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 137

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> 3-15A5-2

<400> 137

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 138

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> 3-15A5-3

<400> 138

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Gly Gly Thr Thr Asp Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 139

<211> 95

<212> PRT

<213> Artificial sequence

<220>

<223> IGKV3-11

<400> 139

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro

85 90 95

<210> 140

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> A5-L1

<400> 140

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 141

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> A5-L2

<400> 141

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 142

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> A5-L3

<400> 142

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

35 40 45

Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 143

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> A5-L4

<400> 143

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 144

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> A5-L1A (all back mutations)

<400> 144

Gln Thr Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 145

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> A5-L1B (Q1T2)

<400> 145

Gln Thr Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 146

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> A5-L1C (FR2)

<400> 146

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 147

<211> 428

<212> PRT

<213> Artificial sequence

<220>

<223> NABA-avi-His

<400> 147

Gln Leu Thr Thr Glu Ser Met Pro Phe Asn Val Ala Glu Gly Lys Glu

1 5 10 15

Val Leu Leu Leu Val His Asn Leu Pro Gln Gln Leu Phe Gly Tyr Ser

20 25 30

Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Val Gly Tyr

35 40 45

Ala Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Asn Ser Gly Arg

50 55 60

Glu Thr Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Val Thr Gln

65 70 75 80

Asn Asp Thr Gly Phe Tyr Thr Leu Gln Val Ile Lys Ser Asp Leu Val

85 90 95

Asn Glu Glu Ala Thr Gly Gln Phe His Val Tyr Pro Glu Leu Pro Lys

100 105 110

Pro Ser Ile Ser Ser Asn Asn Ser Asn Pro Val Glu Asp Lys Asp Ala

115 120 125

Met Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Thr Thr Tyr Leu Trp

130 135 140

Trp Ile Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln Leu Ser

145 150 155 160

Asn Gly Asn Arg Thr Leu Thr Leu Leu Ser Val Thr Arg Asn Asp Thr

165 170 175

Gly Pro Tyr Glu Cys Glu Ile Gln Asn Pro Val Ser Ala Asn Arg Ser

180 185 190

Asp Pro Val Thr Leu Asn Val Thr Tyr Gly Pro Asp Thr Pro Thr Ile

195 200 205

Ser Pro Ser Asp Thr Tyr Tyr Arg Pro Gly Ala Asn Leu Ser Leu Ser

210 215 220

Cys Tyr Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Leu Ile Asn

225 230 235 240

Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn Ile Thr

245 250 255

Val Asn Asn Ser Gly Ser Tyr Thr Cys His Ala Asn Asn Ser Val Thr

260 265 270

Gly Cys Asn Arg Thr Thr Val Lys Thr Ile Ile Val Thr Glu Leu Ser

275 280 285

Pro Val Val Ala Lys Pro Gln Ile Lys Ala Ser Lys Thr Thr Val Thr

290 295 300

Gly Asp Lys Asp Ser Val Asn Leu Thr Cys Ser Thr Asn Asp Thr Gly

305 310 315 320

Ile Ser Ile Arg Trp Phe Phe Lys Asn Gln Ser Leu Pro Ser Ser Glu

325 330 335

Arg Met Lys Leu Ser Gln Gly Asn Ile Thr Leu Ser Ile Asn Pro Val

340 345 350

Lys Arg Glu Asp Ala Gly Thr Tyr Trp Cys Glu Val Phe Asn Pro Ile

355 360 365

Ser Lys Asn Gln Ser Asp Pro Ile Met Leu Asn Val Asn Tyr Asn Ala

370 375 380

Leu Pro Gln Glu Asn Leu Ile Asn Val Asp Leu Glu Val Leu Phe Gln

385 390 395 400

Gly Pro Gly Ser Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu

405 410 415

Trp His Glu Ala Arg Ala His His His His His His

420 425

<210> 148

<211> 420

<212> PRT

<213> Artificial sequence

<220>

<223> N(A2B2)A-avi-His

<400> 148

Gln Leu Thr Thr Glu Ser Met Pro Phe Asn Val Ala Glu Gly Lys Glu

1 5 10 15

Val Leu Leu Leu Val His Asn Leu Pro Gln Gln Leu Phe Gly Tyr Ser

20 25 30

Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Val Gly Tyr

35 40 45

Ala Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Asn Ser Gly Arg

50 55 60

Glu Thr Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Val Thr Gln

65 70 75 80

Asn Asp Thr Gly Phe Tyr Thr Leu Gln Val Ile Lys Ser Asp Leu Val

85 90 95

Asn Glu Glu Ala Thr Gly Gln Phe His Val Tyr Pro Glu Leu Pro Lys

100 105 110

Pro Phe Ile Thr Ser Asn Asn Ser Asn Pro Val Glu Asp Glu Asp Ala

115 120 125

Val Ala Leu Thr Cys Glu Pro Glu Ile Gln Asn Thr Thr Tyr Leu Trp

130 135 140

Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln Leu Ser

145 150 155 160

Asn Asp Asn Arg Thr Leu Thr Leu Leu Ser Val Thr Arg Asn Asp Val

165 170 175

Gly Pro Tyr Glu Cys Gly Ile Gln Asn Lys Leu Ser Val Asp His Ser

180 185 190

Asp Pro Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Asp Pro Thr Ile

195 200 205

Ser Pro Ser Tyr Thr Tyr Tyr Arg Pro Gly Val Asn Leu Ser Leu Ser

210 215 220

Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Leu Ile Asp

225 230 235 240

Gly Asn Ile Gln Gln His Thr Gln Glu Leu Phe Ile Ser Asn Ile Thr

245 250 255

Glu Lys Asn Ser Gly Leu Tyr Thr Cys Gln Ala Asn Asn Ser Ala Ser

260 265 270

Gly His Ser Arg Thr Thr Val Lys Thr Ile Thr Val Ser Ala Leu Ser

275 280 285

Pro Val Val Ala Lys Pro Gln Ile Lys Ala Ser Lys Thr Thr Val Thr

290 295 300

Gly Asp Lys Asp Ser Val Asn Leu Thr Cys Ser Thr Asn Asp Thr Gly

305 310 315 320

Ile Ser Ile Arg Trp Phe Phe Lys Asn Gln Ser Leu Pro Ser Ser Glu

325 330 335

Arg Met Lys Leu Ser Gln Gly Asn Ile Thr Leu Ser Ile Asn Pro Val

340 345 350

Lys Arg Glu Asp Ala Gly Thr Tyr Trp Cys Glu Val Phe Asn Pro Ile

355 360 365

Ser Lys Asn Gln Ser Asp Pro Ile Met Leu Asn Val Asn Tyr Asn Ala

370 375 380

Leu Pro Gln Glu Asn Leu Ile Asn Val Asp Gly Ser Gly Leu Asn Asp

385 390 395 400

Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Ala Arg Ala His His

405 410 415

His His His His

420

<210> 149

<211> 420

<212> PRT

<213> Artificial sequence

<220>

<223> NA(B2)A-avi-His

<400> 149

Gln Leu Thr Thr Glu Ser Met Pro Phe Asn Val Ala Glu Gly Lys Glu

1 5 10 15

Val Leu Leu Leu Val His Asn Leu Pro Gln Gln Leu Phe Gly Tyr Ser

20 25 30

Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Val Gly Tyr

35 40 45

Ala Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Asn Ser Gly Arg

50 55 60

Glu Thr Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Val Thr Gln

65 70 75 80

Asn Asp Thr Gly Phe Tyr Thr Leu Gln Val Ile Lys Ser Asp Leu Val

85 90 95

Asn Glu Glu Ala Thr Gly Gln Phe His Val Tyr Pro Glu Leu Pro Lys

100 105 110

Pro Ser Ile Ser Ser Asn Asn Ser Asn Pro Val Glu Asp Lys Asp Ala

115 120 125

Met Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Thr Thr Tyr Leu Trp

130 135 140

Trp Ile Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln Leu Ser

145 150 155 160

Asn Gly Asn Arg Thr Leu Thr Leu Leu Ser Val Thr Arg Asn Asp Thr

165 170 175

Gly Pro Tyr Glu Cys Glu Ile Gln Asn Pro Val Ser Ala Asn Arg Ser

180 185 190

Asp Pro Val Thr Leu Asn Val Thr Tyr Gly Pro Asp Asp Pro Thr Ile

195 200 205

Ser Pro Ser Tyr Thr Tyr Tyr Arg Pro Gly Val Asn Leu Ser Leu Ser

210 215 220

Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Leu Ile Asp

225 230 235 240

Gly Asn Ile Gln Gln His Thr Gln Glu Leu Phe Ile Ser Asn Ile Thr

245 250 255

Glu Lys Asn Ser Gly Leu Tyr Thr Cys Gln Ala Asn Asn Ser Ala Ser

260 265 270

Gly His Ser Arg Thr Thr Val Lys Thr Ile Thr Val Ser Ala Leu Ser

275 280 285

Pro Val Val Ala Lys Pro Gln Ile Lys Ala Ser Lys Thr Thr Val Thr

290 295 300

Gly Asp Lys Asp Ser Val Asn Leu Thr Cys Ser Thr Asn Asp Thr Gly

305 310 315 320

Ile Ser Ile Arg Trp Phe Phe Lys Asn Gln Ser Leu Pro Ser Ser Glu

325 330 335

Arg Met Lys Leu Ser Gln Gly Asn Ile Thr Leu Ser Ile Asn Pro Val

340 345 350

Lys Arg Glu Asp Ala Gly Thr Tyr Trp Cys Glu Val Phe Asn Pro Ile

355 360 365

Ser Lys Asn Gln Ser Asp Pro Ile Met Leu Asn Val Asn Tyr Asn Ala

370 375 380

Leu Pro Gln Glu Asn Leu Ile Asn Val Asp Gly Ser Gly Leu Asn Asp

385 390 395 400

Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Ala Arg Ala His His

405 410 415

His His His His

420

<210> 150

<211> 90

<212> DNA

<213> Artificial sequence

<220>

<223> A5H1EL1D_H1_rev_TN

<220>

<221> misc_feature

<222> (40)..(42)

<223> N is a, c, g or t and the codons are 50% N, 20% S, 3%

D/E/Q/G/Y/V/T/H/A/L

<220>

<221> misc_feature

<222> (46)..(48)

<223> n is a, c, g or t and the codons are 50% Y, 20% A, 3.75%

G/V/T/H/L/I/R/F

<220>

<221> misc_feature

<222> (49)..(51)

<223> N is a, c, G or t and the codon is 60% Y, 4% G/V/H/S/E/Q/N/D/R/F

<220>

<221> misc_feature

<222> (52)..(54)

<223> n is a, c, g or t and the codons are 50% D, 20% S, 4.3%

G/Y/T/N/A/E/Q

<220>

<221> misc_feature

<222> (55)..(57)

<223> n is a, c, g or T and the codons are 50% T, 20% S, 4.3%

A/G/Y/N/D/E/Q

<220>

<221> misc_feature

<222> (61)..(62)

<223> N is a, c, G or T and the codon is 60% T, 5% A/S/G/Y/N/D/E/Q

<220>

<221> misc_feature

<222> (63)..(63)

<223> n is a, c, g or t

<400> 150

cagccactcg aggcctttac ccggtgcttg gcgtacccan nncatnnnnn nnnnnnngaa 60

nnngaagcca gaagccgcgc agctgagacg 90

<210> 151

<211> 84

<212> DNA

<213> Artificial sequence

<220>

<223> A5H1EL1D_H2_for_TN

<220>

<221> misc_feature

<222> (37)..(39)

<223> n is a, c, G or t and the codons are 60% F, 10% A, 6% Y/V/L/I/G

<220>

<221> misc_feature

<222> (43)..(45)

<223> n is a, c, G or t and the codons are 50% G, 20% S, 3%

A/K/T/V/N/D/E/Q/L/I

<220>

<221> misc_feature

<222> (46)..(48)

<223> N is a, c, G or t and the codons are 650% N, 20% G, 3.75%

D/E/Q/S/Y/T/H/A

<220>

<221> misc_feature

<222> (49)..(51)

<223> N is a, c, g or T and the codon is 60% K, 5% A/T/Y/N/D/E/Q/R

<220>

<221> misc_feature

<222> (52)..(54)

<223> N is a, c, G or t and the codon is 60% A, 4% V/G/D/P/H/N/E/Q/L/I

<220>

<221> misc_feature

<222> (55)..(57)

<223> N is a, c, g or t and the codons are 60% N, 5% D/E/Q, 4.17%

G/T/H/S/A/R

<400> 151

cgccaagcac cgggtaaagg cctcgagtgg ctgggtnnna tcnnnnnnnn nnnnnnngcg 60

tacaccacgg aatactccgc ctcc 84

<210> 152

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> LMB3 Length

<400> 152

caggaaacag ctatgaccat gattac 26

<210> 153

<211> 53

<212> DNA

<213> Artificial sequence

<220>

<223> HCDR 3-rev-invariable

<400> 153

aacggtcacc gtggtaccct ggccccagta gtcgaaatag aagcgcagac cac 53

<210> 154

<211> 84

<212> DNA

<213> Artificial sequence

<220>

<223> A5H1EL1D _L1_rev_TN

<220>

<221> misc_feature

<222> (37)..(39)

<223> n is a, c, g or t and the codons are 50% H, 20% A, 3.33%

R/K/G/S/T/Q/Y/N/V

<220>

<221> misc_feature

<222> (40)..(42)

<223> n is a, c, g or t and the codon is 70% I, 30% L

<220>

<221> misc_feature

<222> (43)..(45)

<223> N is a, c, G or T and the codon is 60% Y, 4% F/G/A/V/T/H/S/N/Q/R

<220>

<221> misc_feature

<222> (46)..(48)

<223> n is a, c, g or T and the codons are 50% T, 20% S, 2.72%

A/G/Y/V/P/H/N/D/E/Q/R

<220>

<221> misc_feature

<222> (49)..(51)

<223> n is a, c, g or t and the codons are 50% V, 20% S, 3.33%

T/A/G/N/Q/F/Y/P/H

<220>

<221> misc_feature

<222> (52)..(54)

<223> n is a, c, g or t and the codons are 50% S, 20% V, 3.33%

T/A/G/N/D/E/Q/Y/H

<400> 154

ggaacgcggg gcctggcctg gtttttgctg ataccannnn nnnnnnnnnn nnnngctgga 60

tgcgcggcaa gacagggtag cacg 84

<210> 155

<211> 84

<212> DNA

<213> Artificial sequence

<220>

<223> A5H1EL1D _L2_for_TN

<220>

<221> misc_feature

<222> (37)..(39)

<223> N is a, c, g or t and the codons are 60% Y, 10% F, 7.5% H/K/N/S

<220>

<221> misc_feature

<222> (40)..(42)

<223> n is a, c, g or t and the codons are 50% A, 20% D, 3.33%

V/G/S/T/Y/H/N/E/Q

<220>

<221> misc_feature

<222> (43)..(45)

<223> n is a, c, g or T and the codons are 50% T, 20% A, 3.33%

S/G/V/P/H/N/D/E/Q

<220>

<221> misc_feature

<222> (46)..(48)

<223> N is a, c, G or T and the codon is 60% S, 4% T/A/G/N/D/E/Q/Y/V/H

<220>

<221> misc_feature

<222> (49)..(51)

<223> N is a, c, g or T and the codon is 60% N, 4% D/E/Q/Y/K/T/H/S/A/R

<400> 155

cagcaaaaac caggccaggc cccgcgttcc tggatcnnnn nnnnnnnnnn nctcgcttct 60

ggtatcccgg cacgtttctc cggc 84

<210> 156

<211> 84

<212> DNA

<213> Artificial sequence

<220>

<223> A5H1EL1D _L3_for_TN

<220>

<221> misc_feature

<222> (31)..(33)

<223> n is a, c, g or t and the codons are 90% Q, 10% H

<220>

<221> misc_feature

<222> (34)..(36)

<223> N is a, c, g or t and the codon is 60% H, 5% R/K/Q/E/Y/F/N/D

<220>

<221> misc_feature

<222> (37)..(39)

<223> n is a, c, g or t and the codon is 65% W, 7% F/Y/V/L/I

<220>

<221> misc_feature

<222> (40)..(42)

<223> N is a, c, G or T and the codon is 58% S, 4% T/A/G/N/D/E/Q, 2%

Y/V/P/H/L/I/R

<220>

<221> misc_feature

<222> (43)..(45)

<223> N is a, c, G or T and the codon is 58% S, 4% T/A/G/N/D/E/Q, 2%

Y/V/P/H/L/I/R

<220>

<221> misc_feature

<222> (46)..(48)

<223> n is a, c, g or t and the codons are 60% K, 5% R/H, 2.72%

A/V/T/P/Y/N/D/E/Q/L/I

<220>

<221> misc_feature

<222> (49)..(51)

<223> n is a, c, g or T and the codon is 70% P, 5% A/S/T/R/S/L

<220>

<221> misc_feature

<222> (52)..(54)

<223> n is a, c, G or t and the codons are 60% P, 5% L/G/R/M, 2.86%

A/V/L/I/F/S/R

<400> 156

gagcctgaag attttgccgt atactattgt nnnnnnnnnn nnnnnnnnnn nnnnactttc 60

ggtcagggca ccaagctgga aatc 84

<210> 157

<211> 90

<212> DNA

<213> Artificial sequence

<220>

<223> A5H1EL1D _H3_rev_TN

<220>

<221> misc_feature

<222> (34)..(36)

<223> n is a, c, g or t and the codon is 80% F, 10% I/L

<220>

<221> misc_feature

<222> (37)..(39)

<223> n is a, c, g or t and the codons are 60% Y, 5% F/W, 2.14%

G/A/V/T/P/H/S/N/D/E/Q/L/I/R

<220>

<221> misc_feature

<222> (40)..(42)

<223> n is a, c, G or t and the codon is 65% F, 5% Y/W/A/V/L/I/G

<220>

<221> misc_feature

<222> (43)..(45)

<223> n is a, c, g or t and the codons are 60% R, 5% K/H, 2.72%

A/V/T/P/Y/N/D/E/Q/L/I

<220>

<221> misc_feature

<222> (46)..(48)

<223> n is a, c, g or t and the codon is 60% L, 4% I/V/A/F, 2.4%

G/Y/T/P/H/S/N/D/E/Q

<220>

<221> misc_feature

<222> (49)..(51)

<223> n is a, c, G or T and the codons are 60% G, 5% A/S/T, 2.5%

Y/V/P/H/N/D/E/Q/L/I

<220>

<221> misc_feature

<222> (52)..(54)

<223> n is a, c, g or t and the codons are 60% R, 5% K/H, 2.72%

A/V/T/P/Y/N/D/E/Q/L/I

<220>

<221> misc_feature

<222> (55)..(57)

<223> n is a, c, g or t

<220>

<221> misc_feature

<222> (58)..(60)

<223> n is a, c, g or t and the codon is 80% F, 10% I/L

<220>

<221> misc_feature

<222> (58)..(60)

<223> n is a, c, g or t and the codons are 60% R, 10% K, 2.72%

A/V/T/P/Y/N/D/E/Q/L/H

<400> 157

aacggtcacc gtggtaccct ggccccagta gtcnnnnnnn nnnnnnnnnn nnnnnnnnnn 60

agtacagtag taggtggcgg tgtcttctgc 90

<210> 158

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> LCDR 3-rev-invariable

<400> 158

acaatagtat acggcaaaat cttcaggctc 30

<210> 159

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> HCDR3 amplification

<400> 159

agaaacggtc accgtggtac cctggcccca gtagtc 36

<210> 160

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038)- CDR-H1

<400> 160

Gly Phe Thr Phe Thr Asp Tyr Tyr Met Asn

1 5 10

<210> 161

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038)- CDR-H2

<400> 161

Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 162

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038)- CDR-H3

<400> 162

Asp Arg Gly Ile Arg Phe Gly Phe Asp Tyr

1 5 10

<210> 163

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038)- CDR-L1

<400> 163

Arg Ala Ser Ser Ser Val Thr Tyr Ile His

1 5 10

<210> 164

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038)- CDR-L2

<400> 164

Ala Thr Ser Asn Leu Ala Ser

1 5

<210> 165

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038)- CDR-L3

<400> 165

Gln His Trp Ser Ser Val Pro Pro Thr

1 5

<210> 166

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.097)- CDR-H1

<400> 166

Gly Phe Thr Phe Thr Asp Tyr Tyr Met Asn

1 5 10

<210> 167

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.097)- CDR-H2

<400> 167

Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 168

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.097)- CDR-H3

<400> 168

Asp Arg Gly Leu Arg Phe Ser Phe Asp Tyr

1 5 10

<210> 169

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.097)- CDR-L1

<400> 169

Arg Ala Ser Ser Ser Val Thr Tyr Ile His

1 5 10

<210> 170

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.097)- CDR-L2

<400> 170

Ala Thr Ser Asn Leu Ala Ser

1 5

<210> 171

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.097)- CDR-L3

<400> 171

Gln His Trp Ser Ser Gln Pro Pro Thr

1 5

<210> 172

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103)- CDR-H1

<400> 172

Gly Phe Thr Phe Thr Asp Tyr Tyr Met Asn

1 5 10

<210> 173

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103)- CDR-H2

<400> 173

Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 174

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103)- CDR-H3

<400> 174

Asp Arg Gly Ile Arg Phe Tyr Phe Asp Tyr

1 5 10

<210> 175

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103)- CDR-L1

<400> 175

Arg Ala Ser Ser Ser Val Thr Tyr Ile His

1 5 10

<210> 176

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103)- CDR-L2

<400> 176

Ala Thr Ser Asn Leu Ala Ser

1 5

<210> 177

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103)- CDR-L3

<400> 177

Gln His Trp Ser Ser Ile Ser Pro Thr

1 5

<210> 178

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P002.139)- CDR-H1

<400> 178

Gly Phe Tyr Phe Thr Asp Tyr Ala Met Asn

1 5 10

<210> 179

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P002.139)- CDR-H2

<400> 179

Val Ile Ser Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 180

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P002.139)- CDR-H3

<400> 180

Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr

1 5 10

<210> 181

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P002.139)- CDR-L1

<400> 181

His Ala Ser Ser Ser Val Thr Tyr Ile His

1 5 10

<210> 182

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P002.139)- CDR-L2

<400> 182

Ala Thr Ser Asn Leu Ala Ser

1 5

<210> 183

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P002.139)- CDR-L3

<400> 183

Gln His Trp Ser Ser Lys Pro Pro Thr

1 5

<210> 184

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P001.177)- CDR-H1

<400> 184

Gly Phe Tyr Phe Thr Asp Tyr Tyr Met Asn

1 5 10

<210> 185

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P001.177)- CDR-H2

<400> 185

Phe Ile Ser Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 186

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P001.177)- CDR-H3

<400> 186

Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr

1 5 10

<210> 187

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P001.177)- CDR-L1

<400> 187

Arg Ala Ser Ser Ser Val Thr Tyr Ile His

1 5 10

<210> 188

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P001.177)- CDR-L2

<400> 188

Ala Thr Ser Asn Leu Ala Ser

1 5

<210> 189

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P001.177)- CDR-L3

<400> 189

Gln His Trp Ser Ser Lys Pro Pro Thr

1 5

<210> 190

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102)- CDR-H1

<400> 190

Gly Phe Thr Phe Thr Asp Tyr Tyr Met Asn

1 5 10

<210> 191

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102)- CDR-H2

<400> 191

Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 192

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102)- CDR-H3

<400> 192

Asp Arg Gly Ile Arg Phe Gln Phe Asp Tyr

1 5 10

<210> 193

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102)- CDR-L1

<400> 193

Arg Ala Ser Ser Ser Val Thr Tyr Ile His

1 5 10

<210> 194

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102)- CDR-L2

<400> 194

Ala Thr Ser Asn Leu Ala Ser

1 5

<210> 195

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102)- CDR-L3

<400> 195

Gln His Trp Ser Ser Lys Ser Pro Thr

1 5

<210> 196

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102-combo1)- CDR-H1

<400> 196

Gly Phe Tyr Phe Thr Asp Tyr Tyr Met Asn

1 5 10

<210> 197

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102-combo1)- CDR-H2

<400> 197

Val Ile Ser Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 198

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102-combo1)- CDR-H3

<400> 198

Asp Arg Gly Ile Arg Phe Gln Phe Asp Tyr

1 5 10

<210> 199

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102-combo1)- CDR-L1

<400> 199

Arg Ala Ser Ser Ser Val Thr Tyr Ile His

1 5 10

<210> 200

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102-combo1)- CDR-L2

<400> 200

Ala Thr Ser Asn Leu Ala Ser

1 5

<210> 201

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102-combo1)- CDR-L3

<400> 201

Gln His Trp Ser Ser Lys Ser Pro Thr

1 5

<210> 202

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102-combo2)- CDR-H1

<400> 202

Gly Phe Tyr Phe Ser Asp Tyr Tyr Met Asn

1 5 10

<210> 203

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102-combo2)- CDR-H2

<400> 203

Val Ile Ser Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 204

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102-combo2)- CDR-H3

<400> 204

Asp Arg Gly Ile Arg Phe Gln Phe Asp Tyr

1 5 10

<210> 205

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102-combo2)- CDR-L1

<400> 205

Arg Ala Ser Ser Ser Val Thr Tyr Ile His

1 5 10

<210> 206

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102-combo2)- CDR-L2

<400> 206

Ala Thr Ser Asn Leu Ala Ser

1 5

<210> 207

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.102-combo2)- CDR-L3

<400> 207

Gln His Trp Ser Ser Lys Ser Pro Thr

1 5

<210> 208

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103-combo1)- CDR-H1

<400> 208

Gly Phe Thr Phe Thr Asp Tyr Tyr Met Asn

1 5 10

<210> 209

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103-combo1)- CDR-H2

<400> 209

Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 210

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103-combo1)- CDR-H3

<400> 210

Asp Arg Gly Ile Arg Phe Ser Phe Asp Tyr

1 5 10

<210> 211

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103-combo1)- CDR-L1

<400> 211

Arg Ala Ser Ser Ser Val Thr Tyr Ile His

1 5 10

<210> 212

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103-combo1)- CDR-L2

<400> 212

Ala Thr Ser Asn Leu Ala Ser

1 5

<210> 213

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103-combo1)- CDR-L3

<400> 213

Gln His Trp Ser Ser Ile Ser Pro Thr

1 5

<210> 214

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103-combo2)- CDR-H1

<400> 214

Gly Phe Tyr Phe Thr Asp Tyr Tyr Met Asn

1 5 10

<210> 215

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103-combo2)- CDR-H2

<400> 215

Val Ile Ser Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 216

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103-combo2)- CDR-H3

<400> 216

Asp Arg Gly Ile Arg Phe Ser Phe Asp Tyr

1 5 10

<210> 217

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103-combo2)- CDR-L1

<400> 217

Arg Ala Ser Ser Ser Val Thr Tyr Ile His

1 5 10

<210> 218

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103-combo2)- CDR-L2

<400> 218

Ala Thr Ser Asn Leu Ala Ser

1 5

<210> 219

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P005.103-combo2)- CDR-L3

<400> 219

Gln His Trp Ser Ser Ile Ser Pro Thr

1 5

<210> 220

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038-combo1)- CDR-H1

<400> 220

Gly Phe Tyr Phe Thr Asp Tyr Ala Met Asn

1 5 10

<210> 221

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038-combo1)- CDR-H2

<400> 221

Val Ile Ser Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 222

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038-combo1)- CDR-H3

<400> 222

Asp Arg Gly Ile Arg Phe Gly Phe Asp Tyr

1 5 10

<210> 223

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038-combo1)- CDR-L1

<400> 223

Arg Ala Ser Ser Ser Val Thr Tyr Ile His

1 5 10

<210> 224

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038-combo1)- CDR-L2

<400> 224

Ala Thr Ser Asn Leu Ala Ser

1 5

<210> 225

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038-combo1)- CDR-L3

<400> 225

Gln His Trp Ser Ser Val Pro Pro Thr

1 5

<210> 226

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038-combo2)- CDR-H1

<400> 226

Gly Phe Thr Phe Ser Asp Tyr Glu Met Asn

1 5 10

<210> 227

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038-combo2)- CDR-H2

<400> 227

Phe Ile Ser Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 228

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038-combo2)- CDR-H3

<400> 228

Asp Arg Gly Ile Arg Phe Gly Phe Asp Tyr

1 5 10

<210> 229

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038-combo2)- CDR-L1

<400> 229

Arg Ala Ser Ser Ser Val Thr Tyr Ile His

1 5 10

<210> 230

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038-combo2)- CDR-L2

<400> 230

Ala Thr Ser Asn Leu Ala Ser

1 5

<210> 231

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (P006.038-combo2)- CDR-L3

<400> 231

Gln His Trp Ser Ser Val Pro Pro Thr

1 5

<210> 232

<211> 96

<212> PRT

<213> Artificial sequence

<220>

<223> IGHV1-2-02 human receptor sequence

<400> 232

Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val

1 5 10 15

Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr Tyr Met

20 25 30

His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Trp

35 40 45

Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln Gly

50 55 60

Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu

65 70 75 80

Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg

85 90 95

<210> 233

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<223> IGHV1-69-01 human receptor sequence

<400> 233

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210> 234

<211> 98

<212> PRT

<213> Artificial sequence

<220>

<223> IGHV1-69-05 human receptor sequences

<400> 234

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Thr Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210> 235

<211> 95

<212> PRT

<213> Artificial sequence

<220>

<223> IGKV1-39-01

<400> 235

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro

85 90 95

<210> 236

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (A5B7) Fc mortar chain

<400> 236

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Gln Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Ser Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 237

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (A5B7) light chain

<400> 237

Gln Thr Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 238

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (A5H1EL1D) -Fc mortar chain

<400> 238

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 239

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (A5H1EL1D) light chain

<400> 239

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 240

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P006.038) Fc mortar chain

<400> 240

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Ile Arg Phe Gly Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 241

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P006.038) light chain

<400> 241

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Val Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 242

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P005.097) Fc mortar chain

<400> 242

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Ser Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 243

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P005.097) light chain

<400> 243

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Gln Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 244

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P005.103) Fc mortar chain

<400> 244

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Ile Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 245

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P005.103) light chain

<400> 245

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Ile Ser Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 246

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P002.139) Fc mortar chain

<400> 246

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Tyr Phe Thr Asp Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Ser Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 247

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P002.139) light chain

<400> 247

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys His Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 248

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P001.177) Fc mortar chain

<400> 248

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Tyr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Ser Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 249

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P001.177) light chain

<400> 249

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 250

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P005.102) Fc mortar chain

<400> 250

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Ile Arg Phe Gln Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 251

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P005.102) light chain

<400> 251

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Ser Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 252

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P005.103-combo1) Fc mortar chain

<400> 252

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Ile Arg Phe Ser Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 253

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P005.103-combo1) light chain

<400> 253

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Ile Ser Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 254

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P005.103-combo2) Fc mortar chain

<400> 254

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Tyr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Ser Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Ile Arg Phe Ser Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 255

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P005.103-combo2) light chain

<400> 255

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Ile Ser Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 256

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P005.102-combo1) Fc mortar chain

<400> 256

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Tyr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Ser Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Ile Arg Phe Gln Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 257

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P005.102-combo1) light chain

<400> 257

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Ser Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 258

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P005.102-combo2) Fc mortar chain

<400> 258

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Tyr Phe Ser Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Ser Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Ile Arg Phe Gln Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 259

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P005.102-combo2) light chain

<400> 259

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Ser Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 260

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P006.038-combo1) Fc mortar chain

<400> 260

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Tyr Phe Thr Asp Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Ser Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Ile Arg Phe Gly Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 261

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P006.038-combo1) light chain

<400> 261

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Val Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 262

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P006.038-combo2) Fc mortar chain

<400> 262

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Glu Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Ser Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Ile Arg Phe Gly Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 263

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (P006.038-combo2) light chain

<400> 263

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Val Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 264

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (MFE23) Fc mortar chain

<400> 264

Gln Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Thr

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Leu Arg Gln Gly Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Lys Ala Thr Phe Thr Thr Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 265

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (MFE23) light chain

<400> 265

Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 266

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (humFE23-L28-H24) Fc kemel chain

<400> 266

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 267

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (humFE23-L28-H24) light chain

<400> 267

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Leu Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 268

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (humFE23-L28-H28) Fc kemel chain

<400> 268

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 269

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (humFE23-L28-H25) Fc kemel chain

<400> 269

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 270

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (humFE23-L27-H29) Fc kemel chain

<400> 270

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Thr Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 271

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (humFE23-L27-H29) light chain

<400> 271

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 272

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (humFE23-L27-H28) Fc kemel chain

<400> 272

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 273

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (humFE23-L27-H26) Fc kemel chain

<400> 273

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 274

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CEA (humFE23-L27-H24) Fc kemel chain

<400> 274

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 275

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> CD3-HCDR1

<400> 275

Thr Tyr Ala Met Asn

1 5

<210> 276

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> CD3-HCDR2

<400> 276

Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser

1 5 10 15

Val Lys Gly

<210> 277

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> CD3-HCDR3

<400> 277

His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr

1 5 10

<210> 278

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> CD3-LCDR1

<400> 278

Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn

1 5 10

<210> 279

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CD3-LCDR2

<400> 279

Gly Thr Asn Lys Arg Ala Pro

1 5

<210> 280

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CD3-LCDR3

<400> 280

Ala Leu Trp Tyr Ser Asn Leu Trp Val

1 5

<210> 281

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> CD3 VH

<400> 281

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 282

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> CD3 VL

<400> 282

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 283

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (CH1A1A 98/99)- CDR-H1

<400> 283

Glu Phe Gly Met Asn

1 5

<210> 284

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (CH1A1A 98/99)- CDR-H2

<400> 284

Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe Lys

1 5 10 15

Gly

<210> 285

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (CH1A1A 98/99)- CDR-H3

<400> 285

Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr

1 5 10

<210> 286

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (2F1)- CDR-L1

<400> 286

Lys Ala Ser Ala Ala Val Gly Thr Tyr Val Ala

1 5 10

<210> 287

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (2F1)- CDR-L2

<400> 287

Ser Ala Ser Tyr Arg Lys Arg

1 5

<210> 288

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (2F1)- CDR-L3

<400> 288

His Gln Tyr Tyr Thr Tyr Pro Leu Phe Thr

1 5 10

<210> 289

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> VH (CEA CH1A1A 98/99)

<400> 289

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 290

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> VL (CEA 2F1)

<400> 290

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Ala Ala Val Gly Thr Tyr

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Lys Arg Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu

85 90 95

Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 291

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (T84.66-LCHA)- CDR-H1

<400> 291

Asp Thr Tyr Met His

1 5

<210> 292

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (T84.66-LCHA)- CDR-H1

<400> 292

Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe Gln

1 5 10 15

Gly

<210> 293

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (T84.66-LCHA)- CDR-H1

<400> 293

Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr

1 5 10

<210> 294

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (T84.66-LCHA)- CDR-L1

<400> 294

Arg Ala Gly Glu Ser Val Asp Ile Phe Gly Val Gly Phe Leu His

1 5 10 15

<210> 295

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (T84.66-LCHA)- CDR-L2

<400> 295

Arg Ala Ser Asn Arg Ala Thr

1 5

<210> 296

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (T84.66-LCHA)- CDR-L3

<400> 296

Gln Gln Thr Asn Glu Asp Pro Tyr Thr

1 5

<210> 297

<211> 254

<212> PRT

<213> Intelligent people

<400> 297

Met Glu Tyr Ala Ser Asp Ala Ser Leu Asp Pro Glu Ala Pro Trp Pro

1 5 10 15

Pro Ala Pro Arg Ala Arg Ala Cys Arg Val Leu Pro Trp Ala Leu Val

20 25 30

Ala Gly Leu Leu Leu Leu Leu Leu Leu Ala Ala Ala Cys Ala Val Phe

35 40 45

Leu Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser

50 55 60

Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp

65 70 75 80

Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val

85 90 95

Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp

100 105 110

Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu

115 120 125

Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe

130 135 140

Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser

145 150 155 160

Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala

165 170 175

Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala

180 185 190

Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala

195 200 205

Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His

210 215 220

Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val

225 230 235 240

Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu

245 250

<210> 298

<211> 199

<212> PRT

<213> Intelligent people

<400> 298

Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala

1 5 10 15

Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro

20 25 30

Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala

35 40 45

Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro

50 55 60

Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp

65 70 75 80

Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe

85 90 95

Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val

100 105 110

Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala

115 120 125

Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg

130 135 140

Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly

145 150 155 160

Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala

165 170 175

Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr

180 185 190

Pro Glu Ile Pro Ala Gly Leu

195

<210> 299

<211> 107

<212> PRT

<213> Intelligent people

<400> 299

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Trp Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

35 40 45

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

50 55 60

Glu Ser Thr Tyr Arg Trp Ser Val Leu Thr Val Leu His Gln Asp Trp

65 70 75 80

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

85 90 95

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

100 105

<210> 300

<211> 106

<212> PRT

<213> Intelligent people

<400> 300

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

1 5 10 15

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

35 40 45

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

65 70 75 80

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 301

<211> 98

<212> PRT

<213> Intelligent people

<400> 301

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val

<210> 302

<211> 5

<212> PRT

<213> Intelligent people

<400> 302

Glu Pro Lys Ser Cys

1 5

<210> 303

<211> 10

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> X is S or P

<400> 303

Asp Lys Thr His Thr Cys Pro Xaa Cys Pro

1 5 10

<210> 304

<211> 7

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X is S or P

<400> 304

His Thr Cys Pro Xaa Cys Pro

1 5

<210> 305

<211> 5

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> X is S or P

<400> 305

Cys Pro Xaa Cys Pro

1 5

<210> 306

<211> 330

<212> PRT

<213> Intelligent people

<400> 306

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 307

<211> 330

<212> PRT

<213> Intelligent people

<400> 307

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 308

<211> 326

<212> PRT

<213> Intelligent people

<400> 308

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 309

<211> 377

<212> PRT

<213> Intelligent people

<400> 309

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro

100 105 110

Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg

115 120 125

Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys

130 135 140

Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

145 150 155 160

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

165 170 175

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

180 185 190

Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr

195 200 205

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

210 215 220

Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His

225 230 235 240

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

245 250 255

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln

260 265 270

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

275 280 285

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

290 295 300

Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn

305 310 315 320

Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

325 330 335

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile

340 345 350

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln

355 360 365

Lys Ser Leu Ser Leu Ser Pro Gly Lys

370 375

<210> 310

<211> 327

<212> PRT

<213> Intelligent people

<400> 310

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 311

<211> 88

<212> PRT

<213> Intelligent people

<400> 311

Pro Lys Pro Phe Ile Thr Ser Asn Asn Ser Asn Pro Val Glu Asp Glu

1 5 10 15

Asp Ala Val Ala Leu Thr Cys Glu Pro Glu Ile Gln Asn Thr Thr Tyr

20 25 30

Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln

35 40 45

Leu Ser Asn Asp Asn Arg Thr Leu Thr Leu Leu Ser Val Thr Arg Asn

50 55 60

Asp Val Gly Pro Tyr Glu Cys Gly Ile Gln Asn Lys Leu Ser Val Asp

65 70 75 80

His Ser Asp Pro Val Ile Leu Asn

85

<210> 312

<211> 88

<212> PRT

<213> Intelligent people

<400> 312

Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro Val Glu Asp Lys

1 5 10 15

Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Ala Thr Tyr

20 25 30

Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln

35 40 45

Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn Val Thr Arg Asn

50 55 60

Asp Thr Ala Ser Tyr Lys Cys Glu Thr Gln Asn Pro Val Ser Ala Arg

65 70 75 80

Arg Ser Asp Ser Val Ile Leu Asn

85

<210> 313

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE-L27, L28, L29)- CDR-L1

<400> 313

Arg Ala Ser Ser Ser Val Pro Tyr Met His

1 5 10

Claims

1. An antigen binding molecule comprising a trimer of 4-1BBL, comprising

An antigen binding domain capable of specifically binding to CEA,

2. The antigen binding molecule comprising a 4-1BBL trimer according to claim 1, wherein the 4-1BBL ectodomain or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO 87, 88, 89, 90, 91, 92, 93 and 94, in particular the amino acid sequence of SEQ ID NO 91.

3. The antigen binding molecule comprising a trimer of 4-1BBL according to claim 1 or 2, comprising

(a) An antigen binding domain capable of specifically binding to CEA,

(c) an Fc domain comprising a first subunit and a second subunit capable of stably associating, wherein the antigen binding domain capable of specifically binding to CEA comprises

4. The antigen binding molecule comprising a trimer of 4-1BBL according to any one of claims 1 to 3, wherein the Fc domain comprises a knob and hole modification that facilitates association of the first subunit with the second subunit of the Fc domain.

5. The antigen binding molecule comprising a trimer of 4-1BBL according to any one of claims 1 to 4, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, in particular to an fey receptor.

6. The antigen binding molecule comprising a trimer of 4-1BBL according to any one of claims 1 to 5 wherein the Fc domain is an IgG1 Fc domain comprising the amino acid substitutions L234A, L235A and P329G (numbering according to Kabat EU numbering).

7. The antigen binding molecule comprising a trimer of 4-1BBL according to any one of claims 1 to 6, wherein the antigen binding domain capable of specifically binding to CEA is a Fab molecule capable of specifically binding to CEA.

8. The antigen binding molecule comprising a trimer of 4-1BBL according to any one of claims 1 to 7, wherein the antigen binding domain capable of specifically binding to CEA comprises

9. The antigen binding molecule comprising a trimer of 4-1BBL according to any one of claims 1 to 9, wherein the antigen binding domain capable of specifically binding to CEA comprises

10. The antigen binding molecule comprising a trimer of 4-1BBL according to any one of claims 1 to 7, wherein the antigen binding domain capable of specifically binding to CEA comprises a VH domain comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:65, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:66 or SEQ ID NO:67, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 68; and a VL domain comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:69 or SEQ ID NO:70 or SEQ ID NO:313, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:71 or SEQ ID NO:72 or SEQ ID NO:73, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 74.

11. The antigen binding molecule comprising a trimer of 4-1BBL according to any one of claims 1 to 7 or claim 10, wherein the antigen binding domain capable of specifically binding to CEA comprises

A heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO 75, 76, 77, 78, 79 or 80; and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85 or SEQ ID NO: 86.

12. The antigen binding molecule comprising a trimer of 4-1BBL according to any one of claims 1 to 7 or claims 10 to 11, wherein the antigen binding domain capable of specifically binding to CEA comprises

13. The 4-1BBL trimer containing antigen binding molecule according to any one of claims 1 to 12, wherein a first peptide comprising two 4-1BBL ectodomains or fragments thereof linked to each other by a first peptide linker is fused at its C-terminus to a CL domain that is part of a heavy chain by a second peptide linker,

and a second peptide comprising one of the 4-1BBL extracellular domains or a fragment thereof is fused at its C-terminus to the CH1 domain that is part of the light chain via a third peptide linker.

14. The antigen binding molecule comprising a trimer of 4-1BBL according to any one of claims 1 to 13, wherein the antigen binding molecule comprises

15. The antigen binding molecule comprising a trimer of 4-1BBL according to any one of claims 1 to 9, wherein the antigen binding molecule comprises

16. The antigen binding molecule comprising a trimer of 4-1BBL according to any one of claims 1 to 7 or claims 10 to 12, wherein the antigen binding molecule comprises

17. A humanized antibody that binds to carcinoembryonic antigen (CEA), comprising

18. The humanized antibody of claim 17, wherein the antibody comprises

19. A humanized antibody that binds to carcinoembryonic antigen (CEA), comprising

A VH domain comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:65, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:66 or SEQ ID NO:67, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 68; and a VL domain comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:69 or SEQ ID NO:70 or SEQ ID NO:313, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:71 or SEQ ID NO:72 or SEQ ID NO:73, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 74.

20. The humanized antibody of claim 21, wherein the antibody comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO 75, 76, 77, 78, 79 or 80; and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85 or SEQ ID NO: 86.

21. The humanized antibody of claim 19 or 20, wherein the antigen binding domain capable of specifically binding to CEA comprises

22. The antibody according to any one of claims 17 to 18 or claims 19 to 21, wherein the antibody is an antibody fragment, in particular a Fab molecule, that specifically binds to CEA.

23. The antibody of any one of claims 17-18 or 19-21, wherein the antibody is a full length IgG1 antibody.

24. An isolated nucleic acid encoding the antigen binding molecule comprising a 4-1BBL trimer according to any one of claims 1 to 16 or the antibody according to any one of claims 17 to 23.

25. A host cell comprising the nucleic acid of claim 24.

26. A method of producing the 4-1BBL trimer containing antigen binding molecule of any one of claims 1 to 16 or the antibody of any one of claims 17 to 23, the method comprising culturing the host cell of claim 25 under conditions suitable for expression of the 4-1BBL trimer containing antigen binding molecule or the antibody.

27. The method of claim 26, further comprising recovering the antigen binding molecule comprising a trimer of 4-1BBL or the antibody from the host cell.

28. An antigen binding molecule or antibody comprising a trimer of 4-1BBL produced by the method of claim 27.

29. A pharmaceutical composition comprising the antigen binding molecule comprising a 4-1BBL trimer according to any one of claims 1 to 16 or the antibody according to any one of claims 17 to 23 and at least one pharmaceutically acceptable excipient.

30. The pharmaceutical composition of claim 31, further comprising an additional therapeutic agent.

31. The pharmaceutical composition of claim 29 or 30, further comprising a T cell activating anti-CD 3 bispecific antibody.

32. The antigen binding molecule containing a trimer of 4-1BBL according to any one of claims 1 to 16 or the pharmaceutical composition according to any one of claims 29 to 31 for use as a medicament.

33. The antigen binding molecule containing a trimer of 4-1BBL according to any one of claims 1 to 16 or the pharmaceutical composition according to any one of claims 29 to 31 for use in the treatment of cancer.

34. The 4-1BBL trimer containing antigen binding molecule according to any one of claims 1 to 16 or the pharmaceutical composition according to claim 29 for use according to claim 33, wherein the 4-1BBL trimer containing antigen binding molecule is used in combination with another therapeutic agent.

35. The 4-1BBL trimer containing antigen binding molecule according to any one of claims 1 to 16 for use in the treatment of cancer, wherein the 4-1BBL trimer containing antigen binding molecule is used in combination with a T cell activating anti-CD 3 bispecific antibody, and wherein the T cell activating anti-CD 3 bispecific antibody is administered simultaneously with or before or after the 4-1BBL trimer containing antigen binding molecule.

36. Use of the antigen binding molecule containing a 4-1BBL trimer according to any one of claims 1 to 16 or the antibody according to any one of claims 17 to 23 for the manufacture of a medicament for the treatment of cancer.

37. A method of treating an individual having cancer, the method comprising administering to the individual an effective amount of the antigen binding molecule comprising a 4-1BBL trimer according to any one of claims 1 to 16 or the pharmaceutical composition according to any one of claims 29 to 31.

38. A method for treating an individual having cancer, the method comprising administering to a subject an effective amount of the antigen-binding molecule comprising a 4-1BBL trimer according to any one of claims 1 to 16 or the pharmaceutical composition according to any one of claims 29 to 31, and an effective amount of a T cell activating anti-CD 3 bispecific antibody.

39. A method of up-regulating or prolonging cytotoxic T cell activity in an individual having cancer, the method comprising administering to the individual an effective amount of the antigen binding molecule comprising a 4-1BBL trimer according to any one of claims 1 to 16 or the pharmaceutical composition according to claim 29.