Bispecific antigen-binding molecules comprising lipocalin muteins

Abstract

The present invention relates to a bispecific antigen-binding molecule capable of bivalently binding to 4-1BB and monovalently binding to a target cell antigen and its use in treating cancer or infectious diseases, wherein the bispecific antigen-binding molecule comprises two (two) lipocalin mutant proteins capable of specifically binding to 4-1BB.

Description

Technical Field

[0001] This invention relates to bispecific antigen-binding molecules capable of bivalently binding to 4-1BB and monovalently binding to target cell antigens, and their use in the treatment of cancer or infectious diseases, said bispecific antigen-binding molecules comprising two (two types) of lipid carrier mutant proteins capable of specifically binding to 4-1BB. The invention further relates to methods for producing these molecules and methods for using them. Background Technology

[0002] 4-1BB (CD137) is a member of the TNF receptor superfamily and was first identified as an inducible molecule expressed by T cell activation (Kwon and Weissman, 1989, Proc Natl Acad Sci USA 86, 1963-1967). 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 non-hematopoietic cells such as endothelial cells and smooth muscle cells (Vinay and Kwon, 2011, Cell Mol Immunol 8, 281-284). 4-1BB expression is mostly inducible across different cell types and is driven by a variety of stimuli, such as T-cell receptor (TCR) or B-cell receptor triggering and signal transduction induced by receptors of co-stimulatory molecules or pro-inflammatory cytokines (Diehl et al., 2002, J Immunol 168, 3755-3762; Zhang et al., 2010, Clin Cancer Res 13, 2758-2767).

[0003] The 4-1BB ligand (4-1BBL or CD137L) was identified in 1993 (Goodwin et al., 1993, Eur J Immunol 23, 2631-2641). It has been shown that 4-1BBL expression is limited to professional antigen-presenting cells (APCs) such as B cells, dendritic cells (DCs), and macrophages. Inducible expression of 4-1BBL is characteristic of T cells (including both αβ and γδ T cell subsets) and endothelial cells (Shao and Schwarz, 2011, J Leukoc Biol 89, 21-29).

[0004] T cells (CD4) were activated by co-stimulation of the 4-1BB receptor (e.g., via 4-1BBL linkage). + and CD8 +Multiple signaling cascades within both subsets strongly enhance T cell activation (Bartkowiak and Curran, 2015, Front Oncol 5, 117). Combined with TCR triggering, agonistic 4-1BB-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 was further developed as the first evidence of a cancer immunotherapy concept. In a preclinical model of tumor-bearing mice, administration of agonistic anti-4-1BB antibodies produced potent antitumor activity (Melero et al., 1997, Nat Med 3, 682-685). Later, mounting evidence suggested that 4-1BB typically only exhibits its antitumor potency when administered in combination with other immunomodulatory compounds, chemotherapy agents, tumor-specific vaccines, or radiotherapy (Bartkowiak and Curran, 2015, Front Oncol 5, 117).

[0005] Signal transduction of the TNFR superfamily requires cross-linking of trimer ligands to bind to receptors, as do 4-1BB agonist antibodies that require wild-type Fc binding (Li and Ravetch, 2011, Science 333, 1030-1034). However, systemic administration of 4-1BB-specific agonist antibodies with functionally active Fc domains leads to CD8 toxicity associated with hepatotoxicity. + The influx of T cells (Dubrot et al., 2010, Cancer Immunol Immunother 59, 1223-1233) resulted in a reduction or significant improvement in hepatotoxicity in mice in the absence of functional Fc receptors. Clinically, the Fc-competent 4-1BB agonist ab (BMS-663513) (NCT00612664) induced grade 4 hepatitis, leading to trial termination (Simeone and Ascierto, 2012, J Immunotoxicol 9, 241-247). Therefore, there is a need for effective and safer 4-1BB agonists.

[0006] Human fibroblast activating protein (FAP; GenBank accession number AAC51668), also known as Seprase, is a 170 kDa integrated membrane serine peptidase (EC 3.4.21.B28). FAP belongs to the dipeptidyl peptidase IV family along with dipeptidyl peptidase IV (also known as CD26; GenBank accession number P27487) (a closely related cell surface enzyme) and other peptidases (Yu et al., FEBS J 277, 1126-1144 (2010)). It is a homodimer containing two N-glycosylated subunits with a large C-terminal extracellular domain in which the enzyme's catalytic domain is located (Scanlan et al., Proc Natl Acad Sci USA 91, 5657-5661 (1994)). FAP, in its glycosylated form, possesses both prolyl dipeptidyl peptidase and gelatinase activities (Sun et al., Protein Expr Purif 24, 274-281 (2002)). Due to its expression in many common cancers and its limited expression in normal tissues, FAP has been considered a promising antigenic target for imaging, diagnosis, and treatment of various cancers. Consequently, several monoclonal antibodies against FAP have been developed for research, diagnostic, and therapeutic purposes.

[0007] Human epidermal growth factor receptor-2 (HER2; ErbB2) is a receptor tyrosine kinase and a member of the epidermal growth factor receptor (EGFR) family of transmembrane receptors. HER2 is overexpressed in a range of tumor types and is associated with disease occurrence and progression. It is associated with poor prognosis. For example, HER2 overexpression is observed in approximately 30% of human breast cancers and is associated with aggressive growth and poor clinical outcomes associated with these tumors (Slamon et al., (1987) Science 235:177-182).

[0008] Humanized anti-HER2 monoclonal antibody trastuzumab (CAS 180288-69-1) huMAb4D5-8 and rhuMAb HER2 (Genentech) target the extracellular domain of HER2 (US 5677171; US ​​5821337; US6054297; US 6165464; US 6339142; US 6407213; US 6639055; US 6719971; US ​​6800738; US7074404; Coussens et al., (1985) Science 230:1132-9; Slamon et al., (1989) Science 244:707-12; Slamon et al., (2001) New Engl.J.Med.344:783-792). Trastuzumab has been shown to inhibit the proliferation of human tumor cells overexpressing HER2 and to act as a mediator of antibody-dependent cytotoxic ADCC (Hudziak et al., (1989) Mol Cell Biol 9:1 165-72; Lewis et al., (1993) Cancer Immunol Immunother; 37:255-63; Baselga et al., (1998) Cancer Res. 58:2825-2831; Hotaling et al., (1996) [Abstract]. Proc. Annual Meeting Am Assoc Cancer Res; 37:471; Pegram MD, et al., (1997) [Abstract]. Proc Am Assoc Cancer Res; 38:602; Sliwkowski et al., (1999) Seminars in Oncology 26(4), Suppl 12:60-70; Yarden Y. and Sliwkowski, M. (2001) Nature Reviews: Molecular Cell Biology, Macmillan Magazines, Ltd., Volume 2: 127-137).

[0009] Trastuzumab (Genentech) was approved in 1998 for the treatment of patients with metastatic breast cancer that overexpresses HER2 (Baselga et al., (1996) J. Clin. Oncol. 14:737-744). In 2006, the FDA approved... It is used as part of a treatment regimen containing doxorubicin, cyclophosphamide, and paclitaxel as adjuvant therapy for patients with HER2-positive, lymph node-positive breast cancer.

[0010] Pertuzumab (also known as recombinant humanized monoclonal antibody 2C4, rhuMAb 2C4) Genentech, Inc. (South San Francisco) is another antibody therapy targeting HER2. Pertuzumab is a Her dimerization inhibitor (HDI) and functions to inhibit the ability of HER2 to form active heterodimers or homodimers with other Her receptors such as EGFR / HER1, HER2, HER3, and HER4. See, for example, Harari and Yarden Oncogene 19:6102-14 (2000); Yarden and Sliwkowski. Nat Rev Mol Cell Biol 2:127-37 (2001); Sliwkowski, Nat Struct Biol 10:158-9 (2003); Cho et al., Nature 421:756-60 (2003); and Malik et al., Pro Am Soc Cancer Res 44:176-7 (2003); US 7560111. In 2012, it was first approved in combination with trastuzumab and docetaxel for the treatment of patients with advanced or late-stage (metastatic) HER2-positive breast cancer. Simultaneously, the combination therapy of trastuzumab and pertuzumab has also been approved for neoadjuvant (preoperative) treatment of HER2-positive, locally advanced, inflammatory, or early-stage breast cancer and for adjuvant (postoperative) treatment of HER2-positive early-stage breast cancer (EBC) at high risk of recurrence. The mechanisms of action of perjeta and herceptin are believed to be complementary, as both bind to the HER2 receptor, but to different sites. The combination of perjeta and herceptin is thought to provide a more comprehensive dual blockade of the HER signaling pathway, thereby preventing tumor cell growth and survival.

[0011] Bispecific bivalent HER2 antibodies targeting domains II, III, and IV of human ErbB2 are disclosed in WO 2012 / 143523. A bispecific HER-2 antibody containing optimized variants of the antibodies rhuMab 2C4 and hu4D5, termed Herceptarg, has been described in WO 2015 / 091738. Although trastuzumab has demonstrated good efficacy in breast cancer, many patients do not benefit from it due to resistance. Given the lack of effective anti-HER2 therapies in specific cancers expressing low levels of HER2, resistance to current therapies, and the prevalence of HER2-related cancers, new therapies are needed to treat these cancers.

[0012] The bispecific antigen-binding molecules of the present invention are characterized by their binding to target cell antigens, particularly tumor targets such as FAP or HER2, and their binding specificity to 4-1BB. The antigen-binding domain capable of specifically binding to 4-1BB is represented by an anticalin mutant protein. Anticalins are non-antibody scaffolds derived from natural human anticalins and offer several advantages, such as small size, stable folding, and significant target specificity (Rothe C, Skerra A., BioDrugs 2018, 32, 233-243). Anticalins with specificity for CD137 (4-1BB) are described in WO 2016 / 177762 and WO 2018 / 087108. A fusion protein including binding specificity for CD137 and binding specificity for HER2 / neu is disclosed in WO 2016 / 177802. Based on their Fc domains, these fusion proteins form symmetrical antibody-like dimers, which bind divalently to CD137 and HER2.

[0013] The antigen-binding molecules of the present invention are characterized by providing monovalent binding to target cell antigens and bivalent binding to 4-1BB. Surprisingly, a 1:2 ratio of tumor target binding to effector cell target binding has been found to lead to improved cross-linking of 4-1BB agonists on effector cells, stronger downstream signaling of the 4-1BB receptor, and thus improved therapeutic efficacy. Summary of the Invention

[0014] In one aspect, the present invention provides a bispecific antigen-binding molecule capable of bivalently binding to 4-1BB and monovalently binding to a target cell antigen, comprising...

[0015] (a) An antigen-binding domain that can specifically bind to target cell antigens.

[0016] (b) The Fc domain, comprising a first subunit and a second subunit capable of stable association, and

[0017] (c) Two lipid transporter mutant proteins capable of specifically binding to 4-1BB, wherein one of the lipid transporter mutant proteins is fused to the C-terminus of the first subunit of the Fc domain, and the other is fused to the C-terminus of the second subunit of the Fc domain.

[0018] In one particular aspect, the present invention provides a bispecific antigen-binding molecule wherein each of the 4-1BB lipid transporter mutant proteins capable of specifically binding is a lipid transporter mutant protein derived from the mature human neutrophil gelatinase-associated lipid transporter protein (huNGAL) of SEQ ID NO:1.

[0019] In a further aspect, the present invention provides a bispecific antigen-binding molecule as defined above, wherein each of the 4-1BB lipid transporter mutant proteins specifically binding to comprises the amino acid sequence of SEQ ID NO:2 or wherein one or more of the following amino acids are mutated to the amino acid sequence of SEQ ID NO:2 as follows:

[0020] (a) Q at position 20 is replaced by R, or

[0021] (b) N at position 25 is replaced by Y or D, or

[0022] (c) H at position 28 is replaced by Q, or

[0023] (d) Q at position 36 is replaced by M, or

[0024] (e) The I at position 40 is replaced by N, or

[0025] (f) R at position 41 is replaced by L or K, or

[0026] (g) E at position 44 is replaced by V or D, or

[0027] (h) K at position 46 is replaced by S, and amino acids at positions 47 to 49 are deleted, or

[0028] (i) The I at position 49 is replaced by H, N, V ​​or S, or

[0029] (j) M at position 52 is replaced by S or G, or

[0030] (k) K at position 59 is replaced by N, or

[0031] (l) D at position 65 is replaced by N, or

[0032] (m) M at position 68 is replaced by D, G, or A, or

[0033] (n) K at position 70 is replaced by M, T, A, or S, or

[0034] (o) F at position 71 is replaced by L, or

[0035] (p) D at position 72 is replaced by L, or

[0036] (q) M at position 77 is replaced by Q, H, T, R, or N, or

[0037] (s) D at position 79 is replaced by I or A, or

[0038] (t) I at position 80 is replaced by N, or

[0039] (u) The W at position 81 is replaced by Q, S, or M, or

[0040] (v) T at position 82 is replaced by P, or

[0041] (w) F at position 83 is replaced by L, or

[0042] (y) F at position 92 is replaced by L or S, or

[0043] (z) L at position 94 is replaced by F, or

[0044] (za) K at position 96 is replaced by F, or

[0045] (zb) F at position 100 is replaced by D, or

[0046] (zc) P at position 101 is replaced by L, or

[0047] (zd) At position 103, H is replaced by P, or

[0048] (ze) S at position 106 is replaced by Y, or

[0049] F at position 122 (zf) is replaced by Y, or

[0050] (zg) At position 125, F is replaced by S, or

[0051] (zh) The F at position 127 is replaced by I, or

[0052] (zi) The E at position 132 is replaced by W, or

[0053] Y at position 134 (zj) is replaced by G.

[0054] In one aspect, the lipotransferase mutant protein capable of specifically binding to 4-1BB comprises the amino acid sequence of SEQ ID NO:2, wherein 4 to 10 amino acids have been mutated as defined above. In another aspect, each of the lipotransferase mutant proteins capable of specifically binding to 4-1BB comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. In one aspect, each of the lipotransferase mutant proteins capable of specifically binding to 4-1BB comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. In a further aspect, each of the lipotransferase mutant proteins capable of specifically binding to 4-1BB comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. In one aspect, each of the lipotransferase mutant proteins capable of specifically binding to 4-1BB comprises the amino acid sequence of SEQ ID NO:2. In one aspect, all the lipotransferase mutant proteins comprise the same amino acid sequence.

[0055] In one aspect, the Fc domain is IgG, particularly the IgG1 Fc domain or the IgG4 Fc domain. More specifically, the Fc domain is the IgG1 Fc domain. In a particular aspect, the Fc domain includes modifications that promote association between the first and second subunits of the Fc domain. In a particular aspect, a bispecific antigen-binding molecule is provided, wherein the Fc domain includes a knock-in-hole modification that promotes association between the first and second subunits of the Fc domain. In a particular aspect, a bispecific antigen-binding molecule is provided, wherein the first subunit of the Fc domain includes amino acid substitutions S354C and T366W (according to Kabat's EU designation), and the second subunit of the Fc domain includes amino acid substitutions Y349C, T366S, L368A, and Y407V (according to Kabat's EU designation).

[0056] On the other hand, the present invention relates to a bispecific antigen-binding molecule as defined herein, comprising (b) an Fc domain including 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 Fc receptors, particularly Fcγ receptors. Specifically, the Fc domain comprises amino acid substitutions at positions 234 and 235 (according to Kabat's EU number) and / or 329 (according to Kabat's EU number) of the IgG heavy chain. Specifically, a bispecific antigen-binding molecule is provided wherein the Fc domain is the human IgG1 Fc domain, comprising amino acid substitutions L234A, L235A, and P329G (according to Kabat's EU number). In a further aspect, a bispecific antigen-binding molecule is provided, wherein the Fc domain is the human IgG4 Fc domain, comprising one or more amino acid substitutions selected from the group consisting of: S228P, N297A, F234A, and L235A (according to Kabat's EU number), particularly the amino acid substitutions S228P, F234A, and L235A (according to Kabat's EU number), and more particularly the amino acid substitution S228P (according to Kabat's EU number).

[0057] In one aspect, the present invention provides a bispecific antigen-binding molecule comprising two lipid carrier mutant proteins capable of specifically binding to 4-1BB, wherein one of the lipid carrier mutant proteins is fused to the C-terminus of a first subunit of the Fc domain via a peptide linker, and the other is fused to the C-terminus of a second subunit of the Fc domain via a peptide linker. In one aspect, the peptide linker has an amino acid sequence selected from the group consisting of: SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120 and SEQ ID NO:121. In one aspect, the peptide linker has an amino acid sequence selected from the group consisting of: SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89. Specifically, the peptide linker has the amino acid sequence of SEQ ID NO:78, namely (G4S)3.

[0058] In one particular aspect, the present invention provides a bispecific antigen-binding molecule capable of bivalently binding to 4-1BB and monovalently binding to a target cell antigen, wherein the antigen-binding domain capable of specifically binding to the target cell antigen is a Fab fragment capable of specifically binding to the target cell antigen. Therefore, the present invention provides a bispecific antigen-binding molecule comprising:

[0059] (a) Fab fragments that can specifically bind to target cell antigens;

[0060] (b) The Fc domain, comprising a first subunit and a second subunit capable of stable association, and

[0061] (c) Two lipid transporter mutant proteins capable of specifically binding to 4-1BB, wherein one of the lipid transporter mutant proteins is fused to the C-terminus of the first subunit of the Fc domain, and the other is fused to the C-terminus of the second subunit of the Fc domain.

[0062] In one aspect, a bispecific antigen-binding molecule is provided that is capable of bivalently binding to 4-1BB and monovalently binding to a target cell antigen, wherein the target cell antigen is fibroblast activation protein (FAP). Thus, a bispecific antigen-binding molecule as defined above is provided, wherein the Fab fragment capable of specifically binding to the target cell antigen is a Fab fragment capable of specifically binding to fibroblast activation protein (FAP).

[0063] In one respect, the Fab fragment that can specifically bind to fibroblast activation protein (FAP) includes: (a) a heavy chain variable region (V H FAP), comprising: (i) CDR-H1, comprising the amino acid sequence of SEQ ID NO:21, (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO:22, and (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO:23; and a light chain variable region (V L FAP), comprising: (iv) CDR-L1, comprising the amino acid sequence of SEQ ID NO:24, (v) CDR-L2, comprising the amino acid sequence of SEQ ID NO:25, and (vi) CDR-L3, comprising the amino acid sequence of SEQ ID NO:26; or (b) heavy chain variable region (V H FAP), comprising: (i) CDR-H1, comprising the amino acid sequence of SEQ ID NO:29, (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO:30, and (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO:31; and a light chain variable region (V L The FAP comprises: (iv) CDR-L1, which contains the amino acid sequence of SEQ ID NO:32; (v) CDR-L2, which contains the amino acid sequence of SEQ ID NO:33; and (vi) CDR-L3, which contains the amino acid sequence of SEQ ID NO:34. Specifically, the Fab fragment capable of specifically binding to the FAP comprises: a heavy chain variable region (V... HFAP), comprising: (i) CDR-H1, comprising the amino acid sequence of SEQ ID NO:21, (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO:22, and (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO:23; and a light chain variable region (V L FAP), comprising: (iv) CDR-L1, which comprises the amino acid sequence of SEQ ID NO:24, (v) CDR-L2, which comprises the amino acid sequence of SEQ ID NO:25, and (vi) CDR-L3, which comprises the amino acid sequence of SEQ ID NO:26.

[0064] In one respect, the Fab fragment that can specifically bind to fibroblast activation protein (FAP) includes: (a) a heavy chain variable region (V H FAP), comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:27; and a light chain variable region (V L FAP), which contains at least about 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO:28; or (b) heavy chain variable region (V H FAP), comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:35; and a light chain variable region (V L FAP), comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:36. Specifically, the Fab fragment capable of specifically binding to FAP comprises: a heavy chain variable region (V H FAP, which contains the amino acid sequence of SEQ ID NO:27, and the light chain variable region (V L FAP), which contains the amino acid sequence of SEQ ID NO:28; or (b) the heavy chain variable region (V H FAP, which contains the amino acid sequence of SEQ ID NO:35, and the light chain variable region (V L FAP, which contains the amino acid sequence of SEQ ID NO:36. More specifically, the Fab fragment that specifically binds to FAP includes: a heavy chain variable region (V H FAP), which contains the amino acid sequence of SEQ ID NO:27; and the light chain variable region (V L FAP), which contains the amino acid sequence of SEQ ID NO:28.

[0065] In one aspect, the present invention provides a bispecific antigen-binding molecule capable of bivalently binding to 4-1BB and monovalently binding to FAP, comprising a first heavy chain of SEQ ID NO:37, a second heavy chain of SEQ ID NO:38, and a light chain of SEQ ID NO:39.

[0066] On the other hand, a bispecific antigen-binding molecule capable of bivalently binding to 4-1BB and monovalently binding to a target cell antigen, wherein the target cell antigen is HER2, is provided. Therefore, a bispecific antigen-binding molecule as defined above is provided, wherein the Fab fragment capable of specifically binding to the target cell antigen is a Fab fragment capable of specifically binding to HER2.

[0067] In one aspect, the Fab fragment capable of specifically binding to HER2 comprises: (a) a VH domain comprising: (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:40, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:41, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:42; and a VL domain comprising: (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:43, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:44, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:45; or (b) a VH domain comprising: (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:48, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:49, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:42. The amino acid sequence of SEQ ID NO:50; and the VL domain comprising: (iv) CDR-L1, comprising the amino acid sequence of SEQ ID NO:51, (v) CDR-L2, comprising the amino acid sequence of SEQ ID NO:52, and (vi) CDR-L3, comprising the amino acid sequence of SEQ ID NO:53; or (c) the VH domain comprising: (i) CDR-H1, comprising the amino acid sequence of SEQ ID NO:56, (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO:57, and (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO:58; and the VL domain comprising: (iv) CDR-L1, comprising the amino acid sequence of SEQ ID NO:59, (v) CDR-L2, comprising the amino acid sequence of SEQ ID NO:60, and (vi) CDR-L3, comprising the amino acid sequence of SEQ ID NO:61. In one aspect, the Fab fragment capable of specifically binding to HER2 comprises: a VH domain comprising: (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:40, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:41, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:42; and a VL domain comprising: (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:43, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:44, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:45.In one aspect, the Fab fragment capable of specifically binding to HER2 comprises: a VH domain comprising: (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:48, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:49, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:50; and a VL domain comprising: (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:51, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:52, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:53.

[0068] In one respect, the Fab fragments that can specifically bind to HER2 include: (a) the heavy chain variable region (V H HER2), comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:46; and a light chain variable region (V L HER2), which contains at least about 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO:47; or (b) a heavy chain variable region (V H HER2), comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:54; and a light chain variable region (V L HER2), which contains at least about 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO:55; or (c) heavy chain variable region (V H HER2), comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:62; and a light chain variable region (V L HER2), comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:63. In one aspect, the Fab fragment capable of specifically binding to HER2 comprises: (a) a heavy chain variable region (V H HER2), which contains the amino acid sequence of SEQ ID NO:46, and the light chain variable region (V L HER2), which contains the amino acid sequence of SEQ ID NO:47, or (b) the heavy chain variable region (V HHER2), which contains the amino acid sequence of SEQ ID NO:54, and the light chain variable region (V L HER2), which contains the amino acid sequence of SEQ ID NO:55, or (c) the heavy chain variable region (V H HER2), which contains the amino acid sequence of SEQ ID NO:62, and the light chain variable region (V L HER2), which contains the amino acid sequence of SEQ ID NO:63. In one particular aspect, the Fab fragment capable of specifically binding to HER2 includes: a heavy chain variable region (V H HER2), which contains the amino acid sequence of SEQ ID NO:46, and the light chain variable region (V L HER2), which contains the amino acid sequence of SEQ ID NO:47. In one aspect, the Fab fragment capable of specifically binding to HER2 includes: a heavy chain variable region (V H HER2), which contains the amino acid sequence of SEQ ID NO:54, and the light chain variable region (V L HER2), which contains the amino acid sequence of SEQ ID NO:55.

[0069] In one aspect, the present invention provides a bispecific antigen-binding molecule capable of bivalently binding to 4-1BB and monovalently binding to HER2, comprising a first heavy chain of SEQ ID NO:64, a second heavy chain of SEQ ID NO:65, and a light chain of SEQ ID NO:66.

[0070] According to another aspect of the invention, an isolated nucleic acid encoding a bispecific antigen-binding molecule as defined herein is provided. The invention further provides a vector, particularly an expression vector, comprising the isolated nucleic acid of the invention; and 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.

[0071] In another aspect, a method for producing the bispecific antigen-binding molecule of the present invention is provided, comprising culturing host cells of the present invention under conditions suitable for expressing the bispecific antigen-binding molecule, and further comprising recovering the bispecific antigen-binding molecule from the host cells. The present invention also covers bispecific antigen-binding molecules produced by the method of the present invention.

[0072] Further, a pharmaceutical composition is provided comprising the bispecific antigen-binding molecule of the present invention and at least one pharmaceutical excipient. In another aspect, a pharmaceutical composition is provided comprising the bispecific antigen-binding molecule of the present invention and at least one pharmaceutical excipient, further comprising additional therapeutic agents, such as chemotherapeutic agents for cancer immunotherapy and / or other agents.

[0073] This invention also encompasses the bispecific antigen-binding molecule of the present invention or the pharmaceutical composition of the present invention, used as a medicine. In one aspect, the bispecific antigen-binding molecule of the present invention or the pharmaceutical composition of the present invention is provided for treating a disease in an individual of need. In a specific aspect, the bispecific antigen-binding molecule of the present invention or the pharmaceutical composition of the present invention is provided for treating cancer or an infectious disease. In another aspect, the bispecific antigen-binding molecule of the present invention or the pharmaceutical composition of the present invention is provided for upregulating or prolonging cytotoxic T cell activity.

[0074] Additionally, the use of the bispecific antigen-binding molecule of the present invention in the preparation of medicaments for treating diseases in individuals in need is provided, particularly in the manufacture of medicaments for treating cancer or infectious diseases, and a method of treating a disease in an individual comprising administering to the individual a therapeutically effective amount of a composition comprising the bispecific antigen-binding molecule as disclosed herein, said composition being in pharmaceutical form. In one aspect, the disease is cancer or an infectious disease. In a specific aspect, the disease is cancer. A method of upregulating or prolonging cytotoxic T-cell activity in an individual suffering from cancer is also provided, comprising administering to the individual an effective amount of the bispecific antigen-binding molecule of the present invention or a pharmaceutical composition of the present invention. In any of the above embodiments, the individual is preferably a mammal, particularly a human. Attached Figure Description

[0075] Figure 1A and 1B A bispecific antigen-binding molecule comprising two fusion proteins is shown, which specifically bind to 4-1BB and are targeted to tumor antigens (TA). Figure 1A In this context, bispecific antigen-binding molecules are bivalent for both tumor target antigen (TA1) and 4-1BB, also known as the 2+2 format. Figure 1B The diagram shows the bispecific antigen-binding molecule of the present invention, which is monovalent for TA1 and bivalent for 4-1BB, also known as the 1+2 format. Both antigen-binding molecules are in the huIgG1 P329GLALA format.

[0076] Figure 2AThe setup for an SPR experiment is shown, designed for simultaneous binding to a FAP-targeting bispecific antigen-binding molecule comprising two fusion proteins (TA1 being FAP) capable of specifically binding to 4-1BB. Figure 2B and 2C The study demonstrated the simultaneous binding of a bispecific anti-FAP, anti-4-1BB lipotransferase huIgG1 PGLALA antigen-binding molecule (analyte 1) to immobilized human 4-1BB and human FAP (analyte 2). The simultaneous binding of the bispecific bivalent 2+2 anti-FAP, anti-4-1BB lipotransferase huIgG1 PGLALA (referred to as 2+2) was also shown. Figure 2B middle. Figure 2C The study demonstrated that the bispecific monovalent 1+2 anti-FAP and anti-4-1BB lipid transporter huIgG1 PGLALA (referred to as 1+2) simultaneously binds to human 4-1BB and human FAP.

[0077] Figure 3A The setup for the SPR experiment is shown, designed for simultaneous binding to the HER2-targeting bispecific 4-1BB lipid transporter (TA1 is HER2). Figure 3B The study showed that bispecific anti-HER2 and anti-4-1BB huIgG1PGLALA antigen-binding molecules (analyte 1) in 2+2 and 1+2 formats simultaneously bind to immobilized human 4-1BB and human HER2 (analyte 2).

[0078] Figure 4This diagram illustrates the binding of FAP to 4-1BB lipocalin to FAP expressed in the NIH / 3T3-huFAP clone 19 cell line, which expresses human FAP. The geometric mean (gMFI) of fluorescence intensity against PE-conjugated secondary detection antibodies was plotted at concentrations of bispecific divalent 2+2 anti-FAP, anti-4-1BB lipocalin huIgG1PGLALA (referred to as FAP(4B9) x 4-1BB lipocalin huIgG1 PG LALA 2+2, hollow downturned triangle and dashed line) or bispecific monovalent 1+2 anti-FAP, anti-4-1BB lipocalin huIgG1 PGLALA (referred to as FAP(4B9) x 4-1BB lipocalin huIgG1 PG LALA 1+2, solid black triangle and line) or their controls. All values ​​were baseline corrected by subtracting the baseline value from the blank control (e.g., containing only the secondary detection antibody, with no primary detection antibody). Constructs containing only the FAP-binding domain, such as FAP(4B9)x 4-1BB lipotransferin huIgG1 PG LALA 2+2 (hollow downward triangle and dashed line), FAP(4B9)x 4-1BB lipotransferin huIgG1 PG LALA 1+2 (solid black triangle and line), FAP(4B9)x 4-1BB lipotransferin huIgG4 SP 2+2 (semi-solid black circle and line-dashed line), or FAP(4B9)huIgG1 PG LALA antibody (gray star and line), effectively bind to FAP-expressing cells.

[0079] Figure 5The binding of FAP to 4-1BB lipotransferrin to the reporter cell line Jurkat-hu4-1BB-NFkB-luc2, which expresses human 4-1BB (CD137), is illustrated. The geometric mean (gMFI) of fluorescence intensity against PE-conjugated secondary detection antibodies is plotted at concentrations of bispecific divalent 2+2 anti-FAP, anti-4-1BB lipotransferrin huIgG1 PGLALA (referred to as FAP(4B9) x 4-1BB lipotransferrin huIgG1 PG LALA 2+2, hollow downturned triangle and dashed line) or bispecific monovalent 1+2 anti-FAP, anti-4-1BB lipotransferrin huIgG1 PGLALA (referred to as FAP(4B9) x 4-1BB lipotransferrin huIgG1 PG LALA 1+2, solid black triangle and line) or their controls. All values ​​were baseline corrected by subtracting the baseline value from the blank control (e.g., containing only the secondary detection antibody and no primary detection antibody). The binding of anti-4-1BB(20H4.9)x anti-FAP(4B9)2+1 H2H (black solid circle and line) to 4-1BB is similar to that of its control anti-4-1BB(20H4.9)huIgG1 P329G LALA (gray star and line).

[0080] Activation of the NFκB signaling pathway was demonstrated by measuring NFκB-mediated luciferase activity in the Jurkat-hu4-1BB-NFκB-luc2 reporter cell line. Figures 6A to 6CIn order to test bispecific bivalent 2+2 anti-FAP, anti-4-1BB lipotransferase huIgG1 PGLALA (referred to as FAP(4B9) x 4-1BB lipotransferase huIgG1 PG LALA 2+2, hollow down-facing black triangle and dashed line) or bispecific monovalent 1+2 anti-FAP, anti-4-1BB lipotransferase huIgG1 PGLALA (referred to as FAP(4B9) x 4-1BB lipotransferase huIgG1 PG LALA 1+2, solid black triangle and line) or control molecule bispecific bivalent 2+2 anti-FAP, anti-4-1BB lipotransferase huIgG4 PGLALA (referred to as FAP(4B9) x 4-1BB lipotransferase huIgG4 SP) The functionality of 2+2, semi-solid black hexamers (hexagonal) and line-dashed lines (or monospecific control molecules) was evaluated by incubation at different titration concentrations with the reporter cell line Jurkat-hu4-1BB-NFκB-luc2 in the absence or presence of FAP-expressing cell lines WM-266-4 or NIH / 3T3-huFAP clone 19. In the absence of FAP-expressing cells, all molecules failed to activate 4-1BB signaling because no cross-linking occurred. In the presence of FAP-expressing cells, only the bispecific molecules binding to FAP and 4-1BB led to NFκB activation in the reporter cell line. Results in the absence of FAP+ cells showed... Figure 6A In the presence of the cell line WM-266-4, which expresses human FAP, it was shown that... Figure 6B In the presence of the cell line NIH / 3T3-huFAP clone 19 expressing human FAP, or in the presence of this cell line, it was shown to... Figure 6C middle.

[0081] Figure 7A and 7B This demonstrates that HER2 targets 4-1BB lipid transporter protein and is derived from the human gastric cancer cell line NCI-N87 ( Figure 7B ) or breast cancer cell line KPL4 ( Figure 7AThe binding of HER2 expressed on the cell surface was plotted. The geometric mean (gMFI) of fluorescence intensity against PE-conjugated secondary detection antibodies was plotted at concentrations of bispecific divalent 2+2 anti-HER2, anti-4-1BB lipotransferrin huIgG1 PGLALA (referred to as HER2(TRAS)x 4-1BB lipotransferrin huIgG1 PGLALA 2+2, black hollow downward-pointing triangle, dashed line) or bispecific monovalent 1+2 anti-HER2, anti-4-1BB lipotransferrin huIgG1 PGLALA (referred to as HER2(TRAS)x 4-1BB lipotransferrin huIgG1 PG LALA 1+2, black solid triangle and line) or their controls. All values ​​were baseline corrected by subtracting the baseline value of the blank control (e.g., containing only the secondary detection antibody and no primary detection antibody). Constructs containing only the HER2-binding domain, such as bivalent 2+2 anti-HER2, anti-4-1BB huIgG1PGLALA (referred to as HER2(TRAS)x4-1BB lipotransferin huIgG1 PG LALA 2+2, black hollow downward triangle, dashed line), or bispecific monovalent 1+2 anti-HER2, anti-4-1BB lipotransferin huIgG1 PGLALA (referred to as HER2(TRAS)x4-1BB lipotransferin huIgG1 PG LALA 1+2, black solid triangle and line), or HER2(TRAS)huIgG1 PGLALA antibody (gray star and line), or HER2(TRAS)x4-1BB lipotransferin huIgG4 SP (semi-solid black hexamer, black dashed line), effectively bind to HER2-expressing cells.

[0082] Figure 8 The binding of HER2-targeting 4-1BB lipotransferase to the reporter cell line Jurkat-hu4-1BB-NFκB-luc2, which expresses human 4-1BB (CD137), is illustrated. The geometric mean (gMFI) of fluorescence intensity against PE-conjugated secondary detection antibodies is plotted at concentrations of bispecific divalent 2+2 anti-HER2, anti-4-1BB lipotransferase huIgG1 PGLALA (referred to as HER2(TRAS)x 4-1BB lipotransferase huIgG1 PG LALA 2+2, hollow downturned triangle and dashed line) or bispecific monovalent 1+2 anti-HER2, anti-4-1BB lipotransferase huIgG1 PGLALA (referred to as HER2(TRAS)x 4-1BB lipotransferase huIgG1 PG LALA 1+2, solid black triangle and line) or their controls. All values ​​were baseline corrected by subtracting the baseline values ​​of the blank control (e.g., containing only the secondary detection antibody and no primary detection antibody).

[0083] Measuring NFκB-mediated luciferase activity in the Jurkat-hu4-1BB-NFkB-luc2 reporter cell line revealed activation of the NFκB signaling pathway. Figures 9A to 9D In order to test the bispecific bivalent 2+2 anti-HER2, anti-4-1BB lipotransferase huIgG1 PG LALA (referred to as HER2(TRAS)x 4-1BB lipotransferase huIgG1 PG LALA 2+2, hollow black triangle with downturn and dashed line) or the bispecific monovalent 1+2 anti-HER2, anti-4-1BB lipotransferase huIgG1 PG LALA (referred to as HER2(TRAS)x 4-1BB lipotransferase huIgG1 PG LALA 1+2, solid black triangle and line) or the control molecule bispecific bivalent 2+2 anti-HER2, anti-4-1BB lipotransferase huIgG4 SP (referred to as HER2(TRAS)x 4-1BB lipotransferase huIgG4 SP) The functionality of the 2+2 (solid black hexamer and dashed line) or control molecule was assessed by incubation at different titration concentrations with the reporter cell line Jurkat-hu4-1BB-NFκB-luc2 in the absence or presence of HER2-expressing cell lines NCI-N87, KPL4, or SK-Br3. In the absence of HER2-expressing cells, none of the molecules activated 4-1BB signaling because no cross-linking occurred. In the presence of HER2-expressing cells, only the bispecific molecule binding to HER2 and 4-1BB led to NFκB activation in the reporter cell line. Results in the absence of HER2+ cells showed... Figure 9A In the presence of the HER2-expressing cell line SK-Br3, the results showed... Figure 9B In the presence of the HER2-expressing cell line KPL4, the results showed... Figure 9C Results, or those in the presence of the HER2-expressing cell line NCI-N87, showed... Figure 9D middle. Detailed Implementation

[0084] definition

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the field to which this invention pertains. For purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural, and vice versa.

[0086] As used herein, the term "antigen-binding molecule" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules are antibodies, antibody fragments, and scaffold antigen-binding proteins.

[0087] The term "antigen-binding domain" refers to a portion of an antigen-binding molecule that contains a region that specifically binds to and is complementary to a portion or all of the antigen. In cases where the antigen is large, the antigen-binding molecule may bind only to a specific portion of the antigen, called an epitope. Antigen-binding domains can be provided by, for example, one or more variable domains (also called variable regions). Preferably, the antigen-binding domain comprises a variable region (VL) of the antibody light chain and a variable region (VH) of the antibody heavy chain, but it can also be provided by scaffold antigen-binding proteins, particularly lipid transporter mutant proteins.

[0088] As used herein, the terms "antigen-binding domain capable of specifically binding to a target cell antigen" or "partial capable of specifically binding to a target cell antigen" refer to a polypeptide molecule that specifically binds to a target cell antigen. In one aspect, an antigen-binding domain can guide an attached entity (e.g., a lipotransferase mutant protein capable of specifically binding to 4-1BB) to a target site, such as to a specific type of tumor cell carrying the target cell antigen. Antigen-binding domains capable of specifically binding to target cell antigens include antibodies and fragments thereof as further defined herein. Furthermore, portions capable of specifically binding to target cell antigens include scaffold antigen-binding proteins as further defined herein. Regarding antibodies or fragments thereof, the term "antigen-binding domain capable of specifically binding to a target cell antigen" includes the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH).

[0089] As used herein, the term "Fab fragment capable of specifically binding to a target cell antigen" refers to a Fab molecule that specifically binds to a target cell antigen. In one aspect, the antigen-binding moiety is capable of activating signal transduction via its target cell antigen. In a particular aspect, the antigen-binding moiety is capable of directing the attached entity (e.g., a lipid transporter mutant protein) to a target site, such as to a specific type of tumor cell or tumor stroma carrying the target cell antigen.

[0090] The term “antibody” is used in the broadest sense and covers a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0091] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, meaning that, apart from possible variant antibodies (e.g., containing naturally occurring mutations or generated during the production of the monoclonal antibody formulation, such variants are typically present in small amounts), the individual antibodies comprising said group are identical and / or bind to the same epitopes. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen.

[0092] As used herein, the term "monospecific" antibody refers to an antibody having one or more binding sites, each binding to the same epitope of the same antigen. The term "bispecific" means that an antigen-binding molecule is capable of specifically binding to at least two different antigenic determinants (targets). Typically, a bispecific antigen-binding molecule contains two antigen-binding sites, each of which is specific for a different antigenic determinant. In one particular aspect, a bispecific antigen-binding molecule contains three antigen-binding sites, where two antigen-binding sites are specific for a first antigenic determinant and one antigen-binding site is specific for a second antigenic determinant. In some embodiments, a bispecific antigen-binding molecule is capable of binding to two antigenic determinants simultaneously, particularly two antigenic determinants expressed on two distinct cell types.

[0093] As used in this application, the term "valence" indicates the presence of a specified number of binding sites in an antigen-binding molecule. Therefore, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" respectively indicate the presence of one, two, four, and six binding sites in an antigen-binding molecule.

[0094] As used in this application, the term "antigen monovalent" means that only the antigen is present in the antigen-binding molecule. one Binding site. As used herein, the term "monovalent to target cell antigen" means that only the target cell antigen is present in the antigen-binding molecule. one Binding site.

[0095] The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably in this document and refer to antibodies with a structure substantially similar to that of natural antibodies. “Natural antibody” refers to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. Each heavy chain has a variable region (VH) (also called a variable heavy chain domain or heavy chain variable domain) from the N-terminus to the C-terminus, followed by three constant domains (CH1, CH2, and CH3) (also called heavy chain constant regions). Similarly, each light chain has a variable region (VL) (also called a variable light chain domain or light chain variable domain) from the N-terminus to the C-terminus, followed by a light chain constant domain (CL) (also called light chain constant regions). Antibody heavy chains can be classified into one of five types, referred to as α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further subdivided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Antibody light chains, based on the amino acid sequence of their constant domains, can be classified into one of two types, referred to as kappa (κ) and lamuda (λ).

[0096] "Antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen bound by the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bisomatic antibodies, trisomatic antibodies, tetrasomatic antibodies, cross-Fab fragments; linear antibodies; 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, 129-134 (2003). For a review of scFv fragments, see, for example, Plückthun in The harmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patents 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and having an increased in vivo half-life, see U.S. Patent 5,869,046. A bispecific antibody is an antibody fragment having two antigen-binding sites; the bispecific antibody may be bivalent or bispecific, see, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9,129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90,6444-6448 (1993). Trisomic and tetrasomic antibodies are also described in Hudson et al., Nat Med 9,129-134 (2003). A single-domain antibody is an antibody fragment containing all or part of the variable domain of the heavy chain or all or part of the variable domain of the light chain. In some embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent 6,248,516 B1). Antibody fragments can be prepared using various techniques, including but not limited to proteolytic digestion of intact antibodies and production from recombinant host cells (e.g., E. coli or bacteriophages), as described herein.

[0097] Papain digestion of an intact antibody produces two identical antigen-binding fragments called “Fab” fragments. Each “Fab” fragment contains a heavy chain variable domain and a light chain variable domain, as well as a light chain constant domain and a first constant domain (CH1) of the heavy chain. Therefore, as used herein, the term “Fab fragment” refers to an antibody fragment containing a light chain fragment along with the VH domain and the first constant domain (CH1) of the heavy chain, the light chain fragment containing the VL domain and the light chain constant domain (CL). The Fab’ fragment differs from the Fab fragment in that the Fab’ fragment has residues added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. Fab’-SH is a Fab’ fragment in which the cysteine ​​residues of the constant domain have a free thiol group. Pepsin treatment produces the F(ab’)2 fragment, which has two antigen-binding sites (two Fab fragments) and a portion of the Fc region.

[0098] The terms "cross-Fab fragment," "xFab fragment," or "exchangeable Fab fragment" refer to Fab fragments in which the variable or constant regions of the heavy and light chains are exchanged. Two different chain compositions are possible for exchangeable Fab molecules and are included in the bispecific antibodies of this invention: in one aspect, the variable regions of the Fab heavy and light chains are exchanged, i.e., the exchangeable Fab molecule comprises a peptide chain consisting of a light chain variable region (VL) and a heavy chain constant region (CH1), and a peptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL). This exchangeable 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 exchangeable Fab molecule comprises a peptide chain including a heavy chain variable region (VH) and a light chain constant region (CL), and a peptide chain including a light chain variable region (VL) and a heavy chain constant region (CH1). This exchangeable Fab molecule is also known as CrossFab. (CLCH1) In one respect, the term "Fab fragment" also includes cross-Fab fragments.

[0099] “Scaffold antigen-binding proteins” are known in the art. For example, fibronectin and engineered ankylosing repeat protein (DARPin) have been used as alternative scaffolds for antigen-binding domains. See, for example, Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin ChemBiol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapyeutics. Drug Discovery Today 13:695-701 (2008). In one aspect of the invention, the scaffold antigen-binding protein is selected from the group consisting of: CTLA-4 (Evibody), anticalin, protein A-derived molecules (such as the Z domain (affinity) of protein A), A domain (Avimer / giant antibody), serum transferrin (trans form); designed ankyrin repeat protein (DARPin), variable domains of antibody light or heavy chains (single-domain antibody, sdAb), variable domains of antibody heavy chains (nanobodies, aVH), V NAR Fragments, fibronectin (AdNectin), C-type lectin domain (tetraligin); variable domain of neoantigen receptor β-lactamase (V NAR Fragments), human γ-crystal proteins or ubiquitin proteins (Affilin molecules); kunitz-type domains of human protease inhibitors, microbodies (such as proteins from the knottin family), aptamers, and adnectin. CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) is mainly found in CD4+. +CD28 family receptors are expressed on T cells. Their extracellular domains have variable domain-like Ig folds. The loops corresponding to the antibody CDR can be replaced with heterologous sequences to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also called Evibody (e.g., US7166697B1). The variable regions of Evibody and antibody (e.g., domain-specific antibodies) are roughly the same size. For further details, see Journal of Immunological Methods 248(1-2), 31-45 (2001). Lipid carriers are a family of extracellular proteins that transport small hydrophobic molecules such as steroids, choline, retinoids, and lipids. They have a rigid β-sheet secondary structure with numerous loops at the open ends of the cone-shaped structure, and can be engineered to bind to different target antigens. Anticalins are between 160 and 180 amino acids in size and are derived from lipid carriers. For further details, see Biochim Biophys Acta 1482:337-350 (2000), US7250297B1, and US20070224633. The affinity protein is a scaffold derived from protein A of Staphylococcus aureus, which can be engineered to bind to antigens. This domain consists of a triple-helix bundle of approximately 58 amino acids. Libraries have been formed through randomization of surface residues. For further details, see Protein Eng. Des. Sel. 2004, 17, 455-462 and EP1641818A1. Avimer is a multi-domain protein derived from the A-domain scaffold family. The native domain of approximately 35 amino acids employs a defined disulfide bond structure. Diversity arises from natural variations exhibited by recombinant A-domain families. For further details, see Nature Biotechnology 23(12), 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007). Transferrin is a monomeric serum transport glycoprotein. Transferrin can be engineered to bind to different target antigens by inserting peptide sequences into permitted surface loops. Examples of engineered transferrin scaffolds include the trans form. For further details, see J. Biol. Chem 274, 24066-24073 (1999). The designed ankyrin repeat sequence protein (DARPin) is derived from ankyrin, a family of proteins that mediate the attachment of integrated membrane proteins to the cellular scaffold. A single ankyrin repeat sequence is a 33-residue motif consisting of two α-helices and a β-turn.They can be engineered to bind different target antigens by randomizing residues in the first α-helix and β-turn of 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 US20040132028A1. Single-domain antibodies are antibody fragments composed of a single monomeric variable antibody domain. The first single domain is derived from the variable domain of the antibody heavy chain of cameloids (nanobodies or V). H H fragment). Furthermore, the term "single-domain antibody" encompasses either an autologous human heavy chain variable domain (aVH) or a V domain derived from sharks. NAR Fragments. Fibronectin can be engineered to form a scaffold for binding antigens. Adnectin consists of a backbone of the native amino acid sequence of the 10th domain of a 15-repetitive unit of human type III fibronectin (FN3). Three loops at one end of the β-dissection can be engineered to enable Adnectin to specifically recognize the target therapeutic target. For further details, see Protein Eng. Des. Sel. 18, 435-444 (2005), US20080139791, WO2005056764, and US6818418B1. Peptide aptamers are composite recognition molecules composed of a constant scaffold protein, typically thioredoxin (TrxA), containing constrained variable peptide loops inserted at the active site. For further details, see Expert Opin. Biol. Ther. 5, 783-797 (2005). Microsomes are derived from naturally occurring microproteins containing 3-4 cysteine ​​bridges and 25-50 amino acids in length. Examples of microproteins include KalataBI, cone snail toxins, and knottin. Microproteins have rings that can be engineered to include up to 25 amino acids without affecting the overall folding of the microprotein. For further details on engineered knottin domains, see WO2008098796.

[0100] Lipocarriers are a family of extracellular proteins that transport small hydrophobic molecules, such as steroids, choline, retinoids, and lipids. Lipocarriers are monomeric proteins weighing approximately 18-20 kDa that exhibit highly structurally flexible binding sites, including four peptide loops mounted on a stable β-cell scaffold (Skerra, FEBS Journal 2008, 275, 2677-2683). Therefore, they possess a rigid β-sheet secondary structure with numerous loops at the open ends of the cone-shaped structure, which can be engineered to bind to different target antigens. This results in lipocrier mutant proteins specific to a particular target antigen. A “lipocrier mutant protein” is a mutated protein in which one or more amino acids are exchanged, deleted, or inserted compared to the naturally occurring (wild-type) lipocrier protein. The term lipocrier mutant protein also includes fragments or variants of wild-type lipocrier proteins. As described herein, lipocrier mutant proteins are between 160 and 180 amino acids in size. In one particular aspect, a lipid transporter mutant protein is a polypeptide defined by its supersecondary structure, namely a cylindrical β-sheet-like supersecondary structure region containing eight β-chains linked in pairs at one end by four rings to define a binding pocket, wherein at least one amino acid in each of at least three of the four rings has been mutated and wherein the lipid transporter protein binds 4-1BB efficiently with detectable affinity.

[0101] In one aspect, the lipidotransferase mutant protein disclosed herein is a mutant protein derived from human tear lipidotransferase (TLPC or Tlc), also known as tear prealbumin or von Ebner gland protein. As used herein, the term "human tear lipidotransferase" or "Tlc" refers to mature human tear lipidotransferase having SWISS-PROT / UniProt database accession number P31025 (isotype 1). This type of lipidotransferase mutant protein is therefore derived from the amino acid sequence of SEQ ID NO: 90. Specifically, the lipidotransferase mutant protein disclosed herein is a mutant protein derived from mature human neutrophil gelatinase-associated lipidotransferase (huNGAL) having SWISS-PROT / UniProt database accession number P80188. This type of lipidotransferase mutant protein can be designated as "huNGAL mutant protein" and is a polypeptide derived from the amino acid sequence of SEQ ID NO: 1. In some aspects, a lipidotransferase mutant protein capable of binding specifically to 4-1BB with detectable affinity may include at least one amino acid substitution, wherein a native cysteine ​​residue is replaced by another amino acid (e.g., a serine residue). In some other aspects, a lipidotransferase mutant protein capable of binding specifically to 4-1BB with detectable affinity may include one or more non-native cysteine ​​residues that replace one or more amino acids of the wild-type lipidotransferase. In another particular aspect, a lipidotransferase mutant protein capable of specifically binding to 4-1BB includes at least two amino acid substitutions, wherein a native amino acid is replaced by a cysteine ​​residue, thereby forming one or more cysteine ​​bridges. In some embodiments, the cysteine ​​bridge may connect at least two loop regions. In related aspects, this disclosure teaches one or more lipidotransferase mutant proteins capable of activating downstream signaling pathways of 4-1BB by binding to 4-1BB.

[0102] "Antigen-binding molecules that bind to the same epitope" as a reference molecule refers to such antigen-binding molecules that, in a competitive assay, block the binding of the reference molecule to its antigen by 50% or more, and conversely, in a competitive assay, block the binding of the antigen-binding molecule to its antigen by 50% or more.

[0103] As used herein, the term “antigenic determinant” is synonymous with “antigen” and “epitope”, and refers to a site on a polypeptide macromolecule (e.g., a continuous amino acid sequence or a conformational configuration consisting of different regions of non-continuous amino acids) to which an antigen-binding moiety binds, thereby forming an antigen-binding moiety-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, and / or in the extracellular matrix (ECM). Unless otherwise stated, proteins used herein as antigens can be any naturally occurring form of protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In one specific embodiment, the antigen is a human protein. When referring to a particular protein herein, the term covers “full-length,” unprocessed protein, and any form of protein produced by intracellular processing. The term also covers naturally occurring protein variants, such as splice variants or allelic variants.

[0104] "Specific binding" refers to binding that is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art (e.g., surface plasmon resonance (SPR) technique (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, for example as measured by SPR, the degree of binding of the antigen-binding molecule to unrelated proteins is less than about 10% of the degree of binding of the antigen-binding molecule to the antigen. In some embodiments, the dissociation constant (Kd) of the antigen-binding molecule is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 μM). -8 M or lower, such as 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 M).

[0105] “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). Unless otherwise stated, as used herein, “binding affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can typically be expressed using the dissociation constant (K0). D The dissociation constant is expressed as the ratio of the dissociation rate constant to the association rate constants (koff and kon, respectively). Affinity can be measured by conventional methods known in the art, including those described herein. A specific method for measuring affinity is surface plasmon resonance (SPR).

[0106] "Affinity-mature" antibodies are those that have one or more alterations in one or more complementarity-determining regions (CDRs) that result in improved affinity of the antibody for the antigen compared to parental antibodies that do not have such alterations.

[0107] As used herein, “target cell antigen” refers to an antigenic determinant present on the surface of a target cell, such as a cell in a tumor (e.g., a cancer cell or a cell in the tumor stroma). In some embodiments, the target cell antigen is an antigen on the surface of tumor cells. In one embodiment, the target cell antigen is selected from the group consisting of: fibroblast activation protein (FAP), HER2, carcinoembryonic antigen (CEA), melanoma-associated chondroitin sulfate proteoglycan (MCSP), epidermal growth factor receptor (EGFR), CD19, CD20, and CD33. Specifically, the target cell antigen is either fibroblast activation protein (FAP) or HER2.

[0108] The term "fibroblast activating protein (FAP)," also known as prolyl endopeptidase FAP or Seprase (EC 3.4.21), refers to any naturally occurring FAP from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The term includes "full-length" unprocessed FAP, as well as any form of FAP produced through cellular processing. The term also covers naturally occurring variants of FAP, such as splice variants or allelic variants. In one embodiment, the antigen-binding molecule of the present invention is capable of specifically binding to human, mouse, and / or cynomolgus monkey FAP. The amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession number Q12884 (version 149, SEQ ID NO: 91) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_004451.2. The extracellular domain (ECD) of human FAP extends from amino acid position 26 to amino acid position 760. The amino acid sequence of His-labeled human FAP ECD is shown in SEQ ID NO:92. The amino acid sequence of mouse FAP is shown in UniProt accession number P97321 (version 126, SEQ ID NO:93) or NCBI RefSeq NP_032012.1. The extracellular domain (ECD) of mouse FAP extends from amino acid position 26 to amino acid position 761. SEQ ID NO:94 shows the amino acid sequence of His-labeled mouse FAP ECD. SEQ ID NO:95 shows the amino acid sequence of His-labeled cynomolgus monkey FAP ECD. Preferably, the anti-FAP binding molecule of the present invention binds to the extracellular domain of FAP. Exemplary anti-FAP binding molecules are described in International Patent Application No. WO 2012 / 020006 A2.

[0109] The term "capable of specifically binding to FAP" refers to an antigen-binding molecule that can bind to FAP with sufficient affinity, such that the antigen-binding molecule can be used as a diagnostic and / or therapeutic agent targeting FAP. Antigen-binding molecules include, but are not limited to, antibodies, Fab molecules, exchangeable Fab molecules, single-chain Fab molecules, Fv molecules, scFv molecules, single-domain antibodies, and VH and scaffold antigen-binding proteins. In one aspect, as measured, for example, by surface plasmon resonance (SPR), the binding degree of anti-FAP antigen-binding molecules to unrelated, non-FAP proteins is less than about 10% of the binding of the antigen-binding molecule to FAP. Specifically, antigen-binding molecules capable of specifically binding to FAP have the following dissociation constant (K... d): ≤1μM, ≤100nM, ≤10nM, ≤1nM, ≤0.1nM, ≤0.01nM or ≤0.001nM (e.g., 10 -8 M or lower, such as 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 In some respects, anti-FAP antigen-binding molecules bind to FAP from different species. Specifically, anti-FAP antigen-binding molecules bind to human and cynomolgus monkey FAP or human, cynomolgus monkey, and mouse FAP.

[0110] The term “carcinoembryonic antigen (CEA)”, also known as carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5), refers to any naturally occurring CEA from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The amino acid sequence of human CEA is shown in UniProt accession number P06731 (version 151, SEQ ID NO: 96). CEA has long been identified as a tumor-associated antigen (Gold and Freedman, J Exp Med., 121:439-462, 1965; Berinstein NL, J Clin Oncol., 20:2197-2207, 2002). Initially classified as a protein expressed only in fetal tissues, CEA has now been identified in a variety of normal adult tissues. These tissues are primarily epithelial, including cells from the gastrointestinal tract, respiratory tract, and urogenital tract, as well as cells from 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). Epithelial-derived tumors and their metastases all contain CEA as a tumor-associated antigen. The presence of CEA itself does not indicate transformation into cancer cells, but the distribution of CEA is indicative. In normal tissues, CEA is typically expressed on the apical surface of cells (…). S.,Semin Cancer Biol.9(2):67-81(1999)), making it impossible for antibodies in the bloodstream to absorb it. Compared with normal tissue, CEA tends to be expressed on the entire surface of cancer cells ( S.,SeminCancerBiol.9(2):67-81(1999)). This change in expression pattern makes CEA more susceptible to binding to antibodies in cancer cells. In addition, CEA expression is increased in cancer cells. Furthermore, the increase in CEA expression promotes increased intercellular adhesion, which may lead to metastasis (Marshall J.,SeminOncol.,30(a Suppl.8):30-6,2003). CEA expression is generally very high in various tumor entities. Based on published data, our own analysis in tissue samples confirmed the high prevalence of CEA, with an incidence of approximately 95% in colorectal cancer (CRC), 90% in pancreatic cancer, 80% in gastric cancer, 60% in non-small cell lung cancer (NSCLC, co-expressed with HER3), and 40% in breast cancer; and low expression levels were found in small cell lung cancer and glioblastoma.

[0111] CEA readily cleaves from the cell surface and flows directly from the tumor into the bloodstream, either through lymphatic vessels or elsewhere. Due to this property, serum CEA levels have been used as a clinical indicator for diagnosing cancer and screening for cancer recurrence, 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; Flamini et al., Clin Cancer Res; 12(23):6985-6988, 2006).

[0112] The term "melanoma-associated chondroitin sulfate proteoglycan (MCSP)," also known as chondroitin sulfate proteoglycan 4 (CSPG4), refers to any naturally occurring MCSP from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The amino acid sequence of human MCSP is shown in UniProt accession number Q6UVK1 (version 103, SEQ ID NO: 97). The term "epidermal growth factor receptor (EGFR)," also known as proto-oncogene c-ErbB-1 or receptor tyrosine protein kinase erbB-1, refers to any naturally occurring EGFR 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 amino acid sequence of human EGFR is shown in UniProt accession number P00533 (version 211, SEQ ID NO: 98).

[0113] The term “CD19” refers to B lymphocyte antigen CD19, also known as B lymphocyte surface antigen B4 or T cell surface antigen Leu-12, and unless otherwise stated, the term includes any naturally occurring CD19 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The amino acid sequence of human CD19 is shown in UniProt accession number P15391 (version 160, SEQ ID NO: 99). The term encompasses “full-length” unprocessed human CD19 as well as any form of human CD19 produced by cell processing, provided that an antibody as reported herein binds to it. CD19 is a structurally distinct cell surface receptor expressed on the surface of human B cells, including but not limited to pre-B cells, early-developing B cells (i.e., immature B cells), mature B cells that have terminally differentiated into plasma cells, and malignant B cells. CD19 is expressed in most pre-B-cell acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, B-cell chronic lymphocytic leukemia (CLL), pre-lymphocytic leukemia, hairy cell leukemia, common acute lymphoblastic leukemia, and some null-acute acute lymphoblastic leukemias. The expression of CD19 on plasma cells further suggests its potential expression in differentiated B-cell tumors such as multiple myeloma. Therefore, the CD19 antigen is a target for immunotherapy in the treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and / or acute lymphoblastic leukemia.

[0114] “CD20” refers to B lymphocyte antigen CD20, also known as transmembrane 4-domain subfamily A member 1 (MS4A1), B lymphocyte surface antigen B1, or leukocyte surface antigen Leu-16, and unless otherwise stated, the term includes any naturally occurring CD20 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 amino acid sequence of human CD20 is shown in UniProt accession number P11836 (version 149, SEQ ID NO:100). “CD33” refers to myeloid cell surface antigen CD33, also known as SIGLEC3 or gp67, and unless otherwise stated, the term includes any naturally occurring CD33 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 amino acid sequence of human CD33 is shown in UniProt accession number P20138 (version 157, SEQ ID NO:101).

[0115] The term “HER2,” also known as “ErbB2,” “ErbB2 receptor,” or “c-Erb-B2,” refers to any naturally occurring, mature HER2 produced from the processing of the HER2 precursor protein in cells. Unless otherwise specified, the term includes HER2 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats). The term also includes naturally occurring variants of HER2, such as splice variants or allelic variants. An exemplary amino acid sequence of the human HER2 protein is shown in SEQ ID NO:102.

[0116] The term "capable of specifically binding to HER2" refers to an antigen-binding molecule that can bind to HER2 with sufficient affinity, such that the antigen-binding molecule can be used as a diagnostic and / or therapeutic agent targeting HER2. Antigen-binding molecules include, but are not limited to, antibodies, Fab molecules, exchangeable Fab molecules, single-chain Fab molecules, Fv molecules, scFv molecules, single-domain antibodies, and VH and scaffold antigen-binding proteins. In one aspect, as measured, for example, by surface plasmon resonance (SPR), the binding degree of anti-HER2 antigen-binding molecules to unrelated, non-HER2 proteins is less than about 10% of the binding degree of antigen-binding molecules to HER2. Specifically, antigen-binding molecules capable of specifically binding to HER2 have the following dissociation constant (K... d ): ≤1μM, ≤100nM, ≤10nM, ≤1nM, ≤0.1nM, ≤0.01nM or ≤0.001nM (e.g., 10 -8 M or lower, such as 10-8 M to 10 -13 M, for example, 10 -9 M to 10 -13 M). In some respects, anti-HER2 antigen-binding molecules bind to HER2 from different species. Specifically, anti-HER2 antigen-binding molecules bind to HER2 in humans and cynomolgus monkeys.

[0117] The term "epitope" refers to a site on a protein or non-protein antigen that binds to an anti-[[PRO]] antibody. Epitopes can be formed from consecutive amino acid segments (linear epitopes) or contain discontinuous amino acids (conformal epitopes), for example, due to antigen folding, i.e., spatial proximity through the tertiary folding of protein antigens. Linear epitopes typically remain bound to the antibody after exposure to a denaturing agent, while conformational epitopes are usually destroyed upon treatment with a denaturing agent. Epitopes contain at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique stereoconformation.

[0118] "Epitope 4D5" or "4D5 epitope" or "4D5" refers to the region of the extracellular domain of HER2 that binds to antibodies 4D5 (ATCC CRL10463) and trastuzumab. This epitope is located near the transmembrane domain of HER2 and within domain IV of HER2. To screen for antibodies that bind to the 4D5 epitope, routine cross-blocking assays are required, as described in *Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988)*. Alternatively, epitope mapping can be performed to assess whether the antibody binds to the 4D5 epitope of HER2 (e.g., any one or more residues in the region from about residue 550 to about residue 610 (including the terminal value) of human HER2 (SEQ ID NO: 102)).

[0119] "Epitope 2C4" or "2C4 epitope" is the region in the extracellular domain of HER2 that binds to antibody 2C4. To screen for antibodies that bind to the 2C4 epitope, routine cross-blocking assays can be performed, as described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Alternatively, epitope mapping can be performed to assess whether an antibody binds to the 2C4 epitope of HER2. Epitope 2C4 contains residues of domain II from the extracellular domain of HER2. 2C4 antibodies and pertuzumab bind to the extracellular domain of HER2 at the junction of domains I, II, and III (Franklin et al., Cancer Cell 5:317-328 (2004)).

[0120] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in the binding of the antigen-binding molecule to the antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WHFreeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0121] As used herein, the term “hypervariant region” or “HVR” refers to the individual regions of an antibody variable domain that are hypervariable in sequence and determine antigen-binding specificity, such as the “complementarity-determining region” (“CDR”).

[0122] Typically, an antibody contains six CDRs; three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs in this document include:

[0123] (a) Highly variable rings occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987));

[0124] (b) CDRs present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and

[0125] (c) Antigen contact sites present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2) and 93-101 (H3) (MacCallum et al., J.Mol.Biol.262:732-745 (1996)).

[0126] Unless otherwise stated, the CDR is determined according to the method described by Kabat et al. (ibid.). Those skilled in the art will understand that the CDR name can also be determined according to the methods described by Chothia (ibid.), McCallum (ibid.), or any other scientifically accepted naming system.

[0127] "Frame" or "FR" refers to the variable domain residues other than the complementarity-determining region (CDR). A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the CDR and FR sequences usually appear in the VH (or VL) as follows: FR1-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)-FR4.

[0128] The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably in this document and refer to antibodies that have a structure substantially similar to that of natural antibodies or that have a heavy chain containing an Fc region as defined herein.

[0129] The "human common framework" is a framework that represents the most frequently occurring amino acid residues in the selection of the human immunoglobulin VL or VH framework sequence. Generally, the selection of the human immunoglobulin VL or VH sequence is derived from a subgroup of variable domain sequences. Generally, the subgroups of sequences are those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one aspect, for VL, this subgroup is subgroup κI as described in Kabat et al., ibid. In another aspect, for VH, this subgroup is subgroup III as described in Kabat et al., ibid.

[0130] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0131] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of them can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In some respects, an antibody is an IgG1 isotype. In some respects, an antibody is an IgG1 isotype with P329G, L234A, and L235A mutations to reduce the function of the Fc region effector. In other respects, an antibody is an IgG2 isotype. In some respects, an antibody is an IgG4 isotype with an S228P mutation in the hinge region to improve the stability of IgG4 antibodies. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The light chain of an antibody, based on the amino acid sequence of its constant domain, can be classified into one of two types, called kappa (κ) and lamuda (λ).

[0132] As used in this application, the term "human-derived constant region" or "human constant region" refers to the constant heavy chain region and / or constant light chain κ or λ region of a human antibody of subclass IgG1, IgG2, IgG3, or IgG4. Such constant regions are well known in the art and, for example, described by: Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also, e.g., Johnson, G., and Wu, TT., Nucleic Acids Res. 28 (2000) 214-218; Kabat, EA, et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788). Unless otherwise specified herein, the amino acid residues in the constant region are numbered according to the EU numbering system, also known as Kabat’s EU index, as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0133] "Humanized" antibodies refer to chimeric antibodies comprising amino acid residues from nonhuman HVRs and amino acid residues from human FRs. In some embodiments, a humanized antibody will substantially comprise at least one of all, typically two, variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to the HVRs of the nonhuman antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Humanized antibodies may optionally comprise at least a portion of the antibody constant region derived from a human antibody. Antibodies in a "humanized form," such as nonhuman antibodies, refer to antibodies that have undergone humanization. Other forms of "humanized antibodies" covered by this invention are antibodies in which the constant regions, relative to the original antibody, have been further modified or altered to produce the properties according to the invention, particularly with respect to C1q binding and / or Fc receptor (FcR) binding.

[0134] A "human" antibody is an antibody whose amino acid sequence corresponds to that of an antibody produced by a human or human cell, or derived from a non-human source using a human antibody library or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues.

[0135] The term "Fc domain" or "Fc region" as used herein is used to define the C-terminal region of an antibody heavy chain containing at least a portion of a constant region. This term includes both native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, antibodies produced by host cells by expressing a specific nucleic acid molecule encoding the full-length heavy chain may comprise the full-length heavy chain, or said antibodies may comprise cleaved variants of the full-length heavy chain. This could be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, according to the Kabat EU index). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified, the amino acid sequence of the heavy chain containing the Fc region is represented herein as a C-terminal glycine-lysine dipeptide. In one embodiment, a heavy chain including the Fc region as specified herein is included in an antibody according to the invention, the heavy chain containing additional C-terminal glycine-lysine dipeptides (G446 and K447, according to Kabat's EU index number). In another embodiment, a heavy chain including the Fc region as specified herein is included in an antibody according to the invention, the heavy chain containing additional C-terminal glycine residues (G446, according to Kabat's EU index number). 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, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. The IgG Fc region contains an IgG CH2 domain and an IgG CH3 domain. The “CH2 domain” of the human IgG Fc region typically extends from approximately amino acid residue 231 to approximately amino acid residue 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein can be a native sequence CH2 domain or a variant CH2 domain. The “CH3 domain” comprises a segment of residues from the C-terminus of the Fc region to the CH2 domain (i.e., from amino acid residue approximately 341 to amino acid residue approximately 447 of the IgG).The CH3 region described herein can be a natural sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having an introduced “bump” (“protrusion”) in one chain and a corresponding introduced “cavity” (“pore”) in another chain; see U.S. Patent No. 5,821,333, which is explicitly incorporated herein by reference). Such variant CH3 domains can be used to facilitate heterodimerization of two distinct antibody heavy chains as described herein.

[0136] The term "wild-type Fc domain" refers to an amino acid sequence identical to that of an Fc domain found in nature. Wild-type human Fc domains include the natural human IgG1 Fc region (non-A and A allotypes), the natural human IgG2 Fc region, the natural human IgG3 Fc region, and the natural human IgG4 Fc region, as well as their naturally occurring variants. Wild-type Fc regions are represented in SEQ ID NO:122 (IgG1, Caucasian allotype), SEQ ID NO:123 (IgG1, African American allotype), SEQ ID NO:124 (IgG2), SEQ ID NO:125 (IgG3), and SEQ ID NO:126 (IgG4).

[0137] The term "variant (human) Fc domain" indicates an amino acid sequence that differs from the "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 compared to the natural Fc region, such as a mutation of about 1 to about 10 amino acids in the natural Fc region, and in another aspect, a mutation of about 1 to about 5 amino acids. In another aspect, the (variant) Fc region shares at least about 95% homology with the wild-type Fc region.

[0138] The “knob-into-hole” technique is described, for example, in 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). Typically, this method involves introducing a protrusion (“protrusion”) at the interface of a first polypeptide and a corresponding cavity (“pore”) at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote the formation of a heterodimer and inhibit the formation of a homodimer. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensating cavity having the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). Protrusions and cavities can be prepared by altering the nucleic acids encoding the polypeptide, for example, through site-specific mutagenesis or peptide synthesis. In one specific embodiment, the protrusion modification comprises an amino acid substitution of T366W in one of the two subunits of the Fc domain, while the cavity modification comprises an amino acid substitution of T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In another specific embodiment, the subunit of the Fc domain containing the protrusion modification further comprises an amino acid substitution of S354C, while the subunit of the Fc domain containing the cavity modification further comprises an amino acid substitution of Y349C. The introduction of these two cysteine ​​residues results in the formation of a disulfide bridge between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). Numbering is based on the EU index of Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0139] "Region equivalent to the Fc region of immunoglobulins" is intended to include naturally occurring allelic variants of the Fc region of immunoglobulins, as well as modified variants with the ability to produce substitutions, additions, or deletions that substantially do not diminish the function of immunoglobulin-mediated effectors such as antibody-dependent cytotoxicity. For example, one or more amino acids may be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantially losing biological function. Such variants may be selected according to general rules known in the art to minimize the impact on activity (see, for example, Bowie, JU et al., Science 247:1306-10 (1990)).

[0140] The term "effective function" refers to those biological activities attributable to the Fc region of an antibody that vary with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, antigen uptake by immune complex-mediated antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0141] "Activated Fc receptors" are Fc receptors that, upon binding to the Fc region of an antibody, trigger signal transduction events that stimulate receptor-carrying cells to perform effector functions. Activated Fc receptors include FcγRⅢa (CD16a), FcγRⅠ (CD64), FcγRⅡa (CD32), and FcαRⅠ (CD89). The specific activated Fc receptor is human FcγRⅢa (see UniProt accession number P08637, version 141).

[0142] The "tumor necrosis factor receptor superfamily," or "TNF receptor superfamily," currently comprises 27 receptors. It is a group of cytokine receptors characterized by their ability to bind tumor necrosis factor (TNF) via extracellular cysteine-rich domains (CRDs). These pseudorepetitions are defined by intrachain disulfides generated from highly conserved cysteine ​​residues within the receptor chain. Except for nerve growth factor (NGF), all TNF receptors are homologous to the proto-TNF-α. Most TNF receptors form trimeric complexes in their active form within the plasma membrane. Therefore, most TNF receptors contain a transmembrane domain (TMD). Many of these receptors also contain intracellular death domains (DDs), which, upon ligand binding, recruit proteins that interact with caspases, initiating an extrinsic pathway of caspase activation. Other TNF superfamily receptors lacking death domains bind to TNF receptor-associated factors and activate intracellular signaling pathways, leading to proliferation or differentiation. These receptors can also initiate apoptosis, but they do so via indirect mechanisms. In addition to regulating apoptosis, multiple TNF superfamily receptors are involved in regulating immune cell function, such as B cell homeostasis and activation, natural killer cell activation, and T cell co-stimulation. They also regulate responses in specific cell types, such as hair follicle development and osteoclast development.Members of the TNF receptor superfamily include: tumor necrosis factor receptor 1 (1A) (TNFRSF1A, CD120a), tumor necrosis factor receptor 2 (1B) (TNFRSF1B, CD120b), lymphotoxin β receptor (LTBR, ​​CD18), OX40 (TNFRSF4, CD134), CD40 (Bp50), Fas receptor (Apo-1, CD95, FAS), decoy receptor 3 (TR6, M68, TNFRSF6B), CD40, CD120, CD13 ...30, CD120, CD130, CD130, CD140, CD130, CD130, CD140, CD130, CD140, CD130, CD140, CD130, CD140, CD130, CD140, CD130, CD140, CD130, CD140, CD130, CD140, CD130, CD140, CD130, CD140, CD130, CD140, CD130, CD140, CD130, CD140, CD130, CD14 27 (S152, Tp55), CD30 (Ki-1, TNFRSF8), 4-1BB (CD137, TNFRSF9), DR4 (TRAILR1, Apo-2, CD261, TNFRSF10A), DR5 (TRAILR2, CD262, TNFRSF10B), decoy receptor 1 (TRAILR3, CD263, TNFRSF10C), decoy receptor 2 (TRAILR4, CD264, TNFRSF9), DR5 (TRAILR2, CD262, TNFRSF10B), decoy receptor 1 (TRAILR3, CD263, TNFRSF10C), decoy receptor 2 (TRAILR4, CD264, TNFRSF9), DR5 (TRAILR2, CD262, TNFRSF9), DR5 (TRAILR2, CD262, TNFRSF9), DR5 (TRAILR2, CD263, TNFRSF9), DR5 (TRAILR2, CD264, TNFRSF9), DR5 (TRAILR2, CD262 ... F10D, RANK (CD265, TNFRSF11A), osteoprotegerin (OCIF, TR1, TNFRSF11B), TWEAK receptor (Fn14, CD266, TNFRSF12A), TACI (CD267, TNFRSF13B), BAFF receptor (CD268, TNFRSF13C), herpesvirus invasion mediator (HVEM, TR2, CD270, TNFRSF14), nerve growth factor receptor (p75NTR, CD271, NGFR), B cell maturation antigen (CD269, TNFRSF17), glucocorticoid-induced TNFR-associated receptors (GITR, AITR, CD357, TNFRSF18), TROY (TNFRSF19), DR6 (CD358, TNFRSF21), DR3 (Apo-3, TRAMP, WS-1, TNFRSF25), and ectodermal dysplasia A2 receptor (XEDAR, EDA2R).

[0143] Following initial T cell activation, multiple members of the tumor necrosis factor receptor (TNFR) family maintain the T cell response. The term "co-stimulatory TNF receptor family member" or "co-stimulatory TNF family receptor" refers to a subgroup of TNF receptor family members that synergistically stimulate T cell proliferation and cytokine production. Unless otherwise stated, this term refers to any naturally occurring TNF family receptor from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). In specific embodiments of the invention, the co-stimulatory TNF receptor family member is selected from the group consisting of: OX40 (CD134), 4-1BB (CD137), CD27, HVEM (CD270), CD30, and GITR, all of which have co-stimulatory effects on T cells. More specifically, the co-stimulatory TNF receptor family member is 4-1BB.

[0144] Unless otherwise specified, as used herein, the term "4-1BB" means any naturally occurring 4-1BB from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term includes "full-length" unprocessed 4-1BB, as well as any form of 4-1BB produced through cellular processing. The term also covers naturally occurring variants of 4-1BB, such as splice variants or allelic variants. The amino acid sequence of an exemplary human 4-1BB is shown in SEQ ID NO:103 (Uniprot accession number Q07011), the amino acid sequence of an exemplary mouse 4-1BB is shown in SEQ ID NO:104 (Uniprot accession number P20334), and the amino acid sequence of an exemplary cynomolgus monkey 4-1BB (from a rhesus macaque) is shown in SEQ ID NO:105 (Uniprot accession number F6W5G6).

[0145] The term "peptide linker" refers to a peptide containing one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides are, for example, (G4S). n (SG4) n Or G4 (SG4) nPeptide linker, where "n" is typically a number between 1 and 10, typically between 1 and 4, and particularly 2, i.e., peptides selected from the group consisting of: GGGGS (SEQ ID NO:75), GGGGSGGGGS (SEQ ID NO:76), SGGGGSGGGG (SEQ ID NO:77), (G4S)3 or GGGGSGGGGSGGGGS (SEQ ID NO:78), GGGGSGGGGSGGG or G4(SG4)2 (SEQ ID NO:79), and (G4S)4 or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:80), but also includes sequences GSPGSSSSGS (SEQ ID NO:81), GSGSGSGS (SEQ ID NO:82), GSGSGNGS (SEQ ID NO:83), GGSGSGSG (SEQ ID NO:84), GGSGSG (SEQ ID NO:85), GGSG (SEQ ID NO:86), GGSG (SEQ ID NO:87), GGGGSGGG (SEQ ID NO:88), GGSG (SEQ ID NO:89), GGSG (SEQ ID NO:80), GGSG (SEQ ID NO:80), GGSG (SEQ ID NO:81), GGSGSGSG (SEQ ID NO:82), GGSGSGNGS (SEQ ID NO:83), GGSGSGSG (SEQ ID NO:84), GGSGSG (SEQ ID NO:85), GGSG (SEQ ID NO:86), GGSG (SEQ ID NO:87), GGSG (SEQ ID NO:88), GGSG (SEQ ID NO:89), GGSG (SEQ ID NO:80 ... (SEQ ID NO:86), GGSGNGSG (SEQ ID NO:87), GGNGSGSG (SEQ ID NO:88), and GGNGSG (SEQ ID NO:89). Of particular interest are (G4S)2 or GGGGSGGGGGS (SEQ ID NO:76), (G4S)3 (SEQ ID NO:78), and (G4S)4 (SEQ ID NO:80), more specifically (G4S)3 (SEQ ID NO:78). Other peptide linkers are selected from the group consisting of: SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, and SEQ ID NO:121.

[0146] As used in this application, the term "amino acid" refers to the group of naturally occurring carboxylated α-amino acids including: alanine (three-letter code: ala, single-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).

[0147] As used herein, a "fusion polypeptide" or "fusion protein" refers to a single-chain polypeptide comprising an antibody fragment and a peptide not derived from the antibody. In one aspect, the fusion polypeptide comprises a lipidotransferase mutant protein linked to the Fc region of an antibody via a peptide bond, optionally via a peptide linker. Fusion can occur by directly linking an N- or C-terminal amino acid of the lipidotransferase mutant protein to a C- or N-terminal amino acid of the heavy chain via a peptide linker.

[0148] "Fusion" or "linking" means that components (such as peptides and the extracellular domains of the TNF ligand family members) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0149] The "percentage of amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after aligning the candidate sequence with the reference polypeptide sequence and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity, and without considering any conservation substitutions as part of sequence identity for alignment purposes. Alignment used to determine the percentage of amino acid sequence identity can be performed in various ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared. Alternatively, the identity percentage value can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been submitted with the user documentation to the US Copyright Office, Washington DC, 20559, where it is registered under US Copyright Registry No. TXU510087 and as described in WO 2001 / 007611.

[0150] Unless otherwise specified, for the purposes of this paper, the percentage values ​​of amino acid sequence identity were generated using the ggsearch program of FASTA package version 36.3.8c or later with the BLOSUM50 comparison matrix. The FASTA package was created by WRPearson and DJ Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448; WRPearson (1996) “Effective protein sequence comparison” Meth. Enzymol. 266:227-258; and Pearson et al., (1997) Genomics 46:24-36, and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, sequences can be compared using a public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi, using the ggsearch (global protein: protein) program with default options (BLOSUM50; open: -10; ext: -2; Ktup=2) to ensure a global rather than local alignment. The percentage of amino acid identity is given in the output alignment header.

[0151] The term "amino acid sequence variant" includes substantial variants in which amino acid substitutions are present in one or more hypervariable residues of a parent antigen-binding molecule (e.g., humanized or human antibody). Typically, one or more resulting variants selected for further research will alter (e.g., improve) 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 it. Exemplary substituted variants are affinity-matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. In short, one or more CDR residues are mutated and the variant antigen-binding molecule is displayed on a phage and screened for a specific biological activity (e.g., binding affinity). In some embodiments, substitution, insertion, or deletion may occur within one or more CDRs, as long as such changes do not substantially reduce the antigen-binding molecule's ability to bind antigens. For example, conserved changes (e.g., conserved substitutions as provided herein) that do not substantially reduce binding affinity can be made in the CDRs. A method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scan mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or a group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine if the antibody-antigen interaction is affected. Additional substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the contact points between the antibody and antigen can be identified using the crystal structure of the antigen-antigen binding molecule complex. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine if they possess the desired properties. Amino acid sequence insertions include amino and / or carboxyl terminus fusions of polypeptides ranging in length from one residue to one hundred or more residues, and intra-sequence insertions of one or more amino acid residues. Examples of terminal insertions include bispecific antigen-binding molecules with an N-terminal methionine residue.

[0152] In some respects, the bispecific antigen-binding molecules provided herein can be modified to increase or decrease the degree of antibody glycosylation. Glycosylated variants of the molecule can be conveniently obtained by altering the amino acid sequence to create or remove one or more glycosylation sites. When the bispecific antigen-binding molecule contains an Fc region, the carbohydrates attached thereto can be modified. Naturally occurring antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, which are typically N-linked to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose of GlcNAc linked to the “stem” of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the bispecific antigen-binding molecule can be modified to produce variants with certain improved properties. In one aspect, variants of the bispecific antigen-binding molecule are provided, having a carbohydrate structure lacking fucosylation (directly or indirectly) attached to the Fc region. Such fucosylated variants may have improved ADCC function, see, for example, U.S. Patent Publication No. US 2003 / 0157108 (Presta, L.) or US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Other variants of the bispecific antigen-binding molecule of the present invention include variants having bispecific oligosaccharides, such as wherein the biantennary oligosaccharide attached to the Fc region is bispecificated via GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function, see, for example, WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. 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.).

[0153] In some aspects, it is desirable to generate cysteine-engineered variants of the bispecific antigen-binding molecule of the present invention, such as "thioMAb," wherein one or more residues of the molecule are substituted with cysteine ​​residues. In certain embodiments, the substituted residues are located at accessible sites of the molecule. By substituting those residues with cysteine, a reactive thiol group is thereby positioned at an accessible site of the antibody and can be used to conjugate the antibody with other parts, such as a pharmaceutical part or a linker-pharmaceutical part, to produce an immunoconjugate. In some embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat number) of the light chain; A118 (EU number) of the heavy chain; and S400 (EU number) of the Fc region of the heavy chain. Cysteine-engineered antigen-binding molecules may be formed as described, for example, in U.S. Patent No. 7,521,541.

[0154] In some respects, the bispecific antigen-binding molecules provided herein can be further modified to include additional non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization 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, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-triane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers) and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethyleneized polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. PEG-propionaldehyde may be advantageous in manufacturing due to its stability in water. Polymers can have any molecular weight and can be branched or unbranched. 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. Typically, the number and / or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the bispecific antibody derivative will be used for a defined therapeutic purpose, etc. On the other hand, conjugates of antibodies and non-protein fractions that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-protein fraction is carbon nanotubes (Kam, NW et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation can have any wavelength and includes, but is not limited to, wavelengths that do not harm ordinary cells but heat the non-protein fraction to a temperature at which cells near the antibody-non-protein fraction are killed.

[0155] On the other hand, immunoconjugates of the bispecific antigen-binding molecules provided herein can be obtained. An “immunoconjugate” is an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

[0156] The terms “nucleic acid” or “polynucleotide” include any compound and / or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, particularly 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. Typically, nucleic acid molecules are described by a base sequence, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is typically represented from 5' to 3'. In this document, 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 containing two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes sense and antisense strands, as well as single-stranded and double-stranded forms. Additionally, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone bonds or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct in vitro and / or in vivo (e.g., in a host or patient) expression of antibodies used in this invention. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoding molecule, enabling the mRNA to be injected into a subject to generate in vivo antibodies (see, for example, Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or EP 2 101 823 B1).

[0157] "Isolated" nucleic acids refer to nucleic acid molecules that have been isolated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules that are contained in cells that normally contain said nucleic acid molecules, but which are located outside chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0158] "Isolated nucleic acid encoding a bispecific antigen-binding molecule" refers to one or more nucleic acid molecules that encode the heavy and light chains (or fragments thereof) of a bispecific antigen-binding molecule, including such nucleic acid molecules in a single vector or in separate vectors, and such nucleic acid molecules present at one or more locations in a host cell.

[0159] The term "expression cassette" refers to a polynucleotide generated through recombination or synthesis, which has a specific set of nucleic acid elements that allow a particular nucleic acid to be transcribed in target cells. Recombinant expression cassettes can be incorporated into plasmids, chromosomes, mitochondrial DNA, plasmid DNA, viruses, or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, the nucleic acid sequence to be transcribed and a promoter. In some embodiments, the expression cassette of the present invention comprises a polynucleotide sequence encoding the bispecific antigen-binding molecule of the present invention or a fragment thereof.

[0160] The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule used to introduce a specific gene into a target cell with which it is operatively associated and to direct the expression of said gene. This term includes vectors as self-replicating nucleic acid structures, as well as vectors integrated into the genome of a host cell into which they have been introduced. The expression vector of the present invention comprises an expression cassette. The expression vector allows for the transcription of a large amount of stable mRNA. Once the expression vector is inside the target cell, a ribonucleic acid molecule or protein encoded by the gene is produced through cellular transcription and / or translation mechanisms. In one embodiment, the expression vector of the present invention comprises an expression cassette containing a multinucleotide sequence encoding a bispecific antigen-binding molecule or a fragment thereof of the present invention.

[0161] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and progeny derived from said primary transformed cells, regardless of passage number. Progeny may not be identical to the nucleic acid contents of the parent cells and may contain mutations. This includes mutant progeny with the same function or biological activity as screened or selected in the original transformed cells. Host cells are any type of cell system that can be used to generate the bispecific antigen-binding molecule of the present invention. Host cells include cultured cells, for example, cultured mammalian cells such as CHO cells, BHK cells, NSO 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.

[0162] The "effective amount" of a drug refers to the amount required to produce physiological changes in the cells or tissues to which it is applied.

[0163] A "therapeuticly effective amount" of a pharmaceutical agent (such as a pharmaceutical composition) refers to the amount that effectively achieves the desired therapeutic or preventative outcome at the necessary dosage and time. A therapeutically effective amount of a pharmaceutical agent may eliminate, reduce, delay, minimize, or prevent adverse effects of disease, for example.

[0164] "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). Specifically, the individual or subject is a human.

[0165] The term "pharmaceutical composition" refers to a formulation in which the bioactive ingredient contained therein is in a form in which it is bioactively effective and does not contain any additional components that would have unacceptable toxicity to a subject to whom the formulation will be administered.

[0166] "Pharmaceutically acceptable excipients" refer to components in a pharmaceutical composition other than the active ingredient that are non-toxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, or preservatives.

[0167] The term "packaging insert" is used to refer to the instruction leaflet typically included in the commercial packaging of a therapeutic product, which contains information concerning the indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings related to the use of such therapeutic products.

[0168] As used herein, “treatment” (and its grammatical variations, such as “treat” or “treating”) refers to a clinical intervention that attempts to alter the natural course of a treated individual, and may be for prevention or in the course of clinicopathology. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis. In some embodiments, the molecules of the present invention are used to delay the development of disease or to slow its progression.

[0169] As used in this article, the term "cancer" refers to proliferative diseases such as various lymphomas, carcinomas, lymphomas, blastomas, sarcomas, leukemia, lymphocytic leukemia, lung cancer, non-small cell lung cancer (NSCL), bronchoalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, gastric 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, endocrine system cancers, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvis cancer, mesothelioma, hepatocellular carcinoma, bile duct cancer, and central nervous system (CNS) tumors. Spinal axis tumors, brainstem gliomas, glioblastoma multiforme, astrocytomas, schwannomas, ependymomas, myeloma, meningiomas, squamous cell carcinomas, pituitary adenomas and Ewing's sarcomas, melanomas, multiple myelomas, B-cell cancers (lymphomas), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloid leukemia, including any refractory form of the above cancers, or a combination of one or more cancers.

[0170] HER2-positive cancers include cancer cells with HER2 levels higher than normal. Examples of HER2-positive cancers include HER2-positive breast cancer and HER2-positive gastric cancer. Optionally, HER2-positive cancers have an immunohistochemical (IHC) score of 2+ or 3+ and / or an in situ hybridization (ISH) amplification rate >2.0.

[0171] The term “early-stage breast cancer (EBC)” or “early-stage breast cancer” is used in this article to refer to breast cancer that has not yet spread beyond the breast or axillary lymph nodes. This includes ductal carcinoma in situ and stage I, IIA, IIB, and IIIA breast cancer.

[0172] Referring to tumors or cancers as "Stage 0," "Stage I," "Stage II," "Stage III," or "Stage IV," and the various sub-stages within this classification, indicates a classification of tumors or cancers using overall stage grouping or Roman numeral staging methods known in the art. Although the actual stage of cancer depends on the type of cancer, generally speaking, Stage 0 cancer is an in situ lesion, Stage I cancer is a small localized tumor, Stage II and III cancers are locally advanced tumors exhibiting local lymph node involvement, and Stage IV cancer represents metastatic cancer. The specific stage for each type of tumor is known to a skilled clinician.

[0173] The term "metastatic breast cancer" refers to a condition in which cancer cells spread from the original site to one or more other sites in the body via blood vessels or lymphatic vessels, thereby forming one or more secondary tumors in one or more organs other than the breast.

[0174] "Late-stage" cancer refers to cancer that has spread beyond its original site or organ due to local invasion or metastasis. Therefore, the term "late-stage" cancer includes both locally advanced and metastatic disease.

[0175] “Recurrent” cancer refers to cancer that recurs at the initial site or distantly after responding to an initial treatment (such as surgery). “Locally recurrent” cancer refers to cancer that recurs at the same location as previously treated cancer after treatment. “Operable” or “resectable” cancer is cancer that is confined to a major organ and is suitable for surgical (removal). “Non-resectable” or “unresectable” cancer cannot be removed (resected) surgically.

[0176] The bispecific antigen-binding molecule of the present invention

[0177] This invention provides a novel bispecific antigen-binding molecule capable of bivalently binding to 4-1BB and monovalently binding to target cell antigens. The bispecific antigen-binding molecule comprises two lipid carrier mutant proteins capable of specifically binding to 4-1BB and has particularly advantageous properties such as manufacturability, stability, binding affinity, biological activity, targeting efficiency, reduced toxicity, and reduced immunity.

[0178] The bispecific antigen-binding molecule of the present invention comprises two lipid carrier mutant proteins capable of specifically binding to 4-1BB, each fused to the C-terminus of one of the subunits of the Fc domain. The geometry of the bispecific antigen-binding molecule, and specifically the distance between the two distinct binding sites of 4-1BB and the target cell antigen, is crucial for optimal tumor-local activation of the co-stimulated TNF receptor (i.e., 4-1BB) (M. Rothe and A. Skerrra, BioDrugs 2018, 32, 233-243). It has also been found that impressively better activation can be achieved when only one antigen-binding domain targeting the target cell antigen is present in the molecule. A lower 1:2 ratio of tumor target binding to effector cell target binding, such as the 1:2 ratio of antigen-binding domains that can specifically bind to target cell antigens to lipotransferase mutants that can specifically bind to 4-1BB, results in a higher occupancy density on tumor cells. This leads to dense cross-linking of 4-1BB agonists on effector cells and ultimately stronger downstream signaling of the 4-1BB receptor.

[0179] In a first aspect, a bispecific antigen-binding molecule capable of bivalently binding to 4-1BB and monovalently binding to target cell antigens is provided, comprising:

[0180] (a) Antigen-binding domains that can specifically bind to target cell antigens, particularly Fab fragments that can specifically bind to target cell antigens.

[0181] (b) The Fc domain, comprising a first subunit and a second subunit capable of stable association, and

[0182] (c) Two lipid transporter mutant proteins capable of specifically binding to 4-1BB, wherein one of the lipid transporter mutant proteins is fused to the C-terminus of the first subunit of the Fc domain, and the other is fused to the C-terminus of the second subunit of the Fc domain.

[0183] In a further aspect, a bispecific antigen-binding molecule capable of bivalently binding to 4-1BB and monovalently binding to target cell antigens is provided, comprising:

[0184] (a) Antigen-binding domains, particularly the Fab fragment capable of specifically binding to target cell antigens.

[0185] (b) The Fc domain, comprising a first subunit and a second subunit capable of stable association, and

[0186] (c) Two lipid transporter mutant proteins that can specifically bind to 4-1BB, wherein one of the lipid transporter mutant proteins is fused to the C-terminus of the first subunit of the Fc domain and the other is fused to the C-terminus of the second subunit of the Fc domain, and wherein each of the lipid transporter mutant proteins that can specifically bind to 4-1BB is derived from mature human neutrophil gelatinase-associated lipid transporter protein (huNGAL) of SEQ ID NO: 1.

[0187] In one aspect, a bispecific antigen-binding molecule as defined above is provided, wherein each of the 4-1BB lipid transporter mutant proteins comprises the amino acid sequence of SEQ ID NO:2 or one or more of the following amino acids mutated as follows:

[0188] (a) Q at position 20 is replaced by R, or

[0189] (b) N at position 25 is replaced by Y or D, or

[0190] (c) H at position 28 is replaced by Q, or

[0191] (d) Q at position 36 is replaced by M, or

[0192] (e) The I at position 40 is replaced by N, or

[0193] (f) R at position 41 is replaced by L or K, or

[0194] (g) E at position 44 is replaced by V or D, or

[0195] (h) K at position 46 is replaced by S, and amino acids at positions 47 to 49 are deleted, or

[0196] (i) The I at position 49 is replaced by H, N, V ​​or S, or

[0197] (j) M at position 52 is replaced by S or G, or

[0198] (k) K at position 59 is replaced by N, or

[0199] (l) D at position 65 is replaced by N, or

[0200] (m) M at position 68 is replaced by D, G, or A, or

[0201] (n) K at position 70 is replaced by M, T, A, or S, or

[0202] (o) F at position 71 is replaced by L, or

[0203] (p) D at position 72 is replaced by L, or

[0204] (q) M at position 77 is replaced by Q, H, T, R, or N, or

[0205] (s) D at position 79 is replaced by I or A, or

[0206] (t) I at position 80 is replaced by N, or

[0207] (u) The W at position 81 is replaced by Q, S, or M, or

[0208] (v) T at position 82 is replaced by P, or

[0209] (w) F at position 83 is replaced by L, or

[0210] (y) F at position 92 is replaced by L or S, or

[0211] (z) L at position 94 is replaced by F, or

[0212] (za) K at position 96 is replaced by F, or

[0213] (zb) F at position 100 is replaced by D, or

[0214] (zc) P at position 101 is replaced by L, or

[0215] (zd) At position 103, H is replaced by P, or

[0216] (ze) S at position 106 is replaced by Y, or

[0217] F at position 122 (zf) is replaced by Y, or

[0218] (zg) At position 125, F is replaced by S, or

[0219] (zh) The F at position 127 is replaced by I, or

[0220] (zi) The E at position 132 is replaced by W, or

[0221] Y at position 134 (zj) is replaced by G.

[0222] In one aspect, the lipid transporter mutant protein capable of specifically binding to 4-1BB comprises the amino acid sequence of SEQ ID NO:2, wherein 4 to 10 amino acids have been mutated as defined above. In other aspects, the lipid transporter mutant protein capable of specifically binding to 4-1BB comprises one or more of the following amino acid mutations:

[0223] (d) Q at position 36 is replaced by M, or

[0224] (e) The I at position 40 is replaced by N, or

[0225] (f) R at position 41 is replaced by L or K, or

[0226] (i) The I at position 49 is replaced by H, N, V ​​or S, or

[0227] (j) M at position 52 is replaced by S or G, or

[0228] (m) M at position 68 is replaced by D, G, or A, or

[0229] (n) K at position 70 is replaced by M, T, A, or S, or

[0230] (p) D at position 72 is replaced by L, or

[0231] (q) M at position 77 is replaced by Q, H, T, R, or N, or

[0232] (s) D at position 79 is replaced by I or A, or

[0233] (u) The W at position 81 is replaced by Q, S, or M, or

[0234] (za) K at position 96 is replaced by F, or

[0235] (zb) F at position 100 is replaced by D, or

[0236] (zd) At position 103, H is replaced by P, or

[0237] (zg) At position 125, F is replaced by S, or

[0238] (zh) The F at position 127 is replaced by I, or

[0239] (zi) The E at position 132 is replaced by W, or

[0240] Y at position 134 (zj) is replaced by G.

[0241] On the other hand, lipid transporter mutants capable of specifically binding to 4-1BB contain one or more of the following amino acid mutations:

[0242] (a) Q at position 20 is replaced by R, or

[0243] (b) N at position 25 is replaced by Y or D, or

[0244] (g) E at position 44 is replaced by V or D, or

[0245] (k) K at position 59 is replaced by N, or

[0246] (o) F at position 71 is replaced by L, or

[0247] (t) I at position 80 is replaced by N, or

[0248] (v) T at position 82 is replaced by P, or

[0249] (y) F at position 92 is replaced by L or S, or

[0250] (zc) P at position 101 is replaced by L, or

[0251] F at position 122 (zf) is replaced by Y.

[0252] In one aspect, each of the lipotransfer protein mutants capable of specifically binding to 4-1BB comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. In another aspect, each of the lipotransfer protein mutants capable of specifically binding to 4-1BB comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. In a further aspect, each of the lipid transporter mutant proteins capable of specifically binding to 4-1BB comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. In one aspect, each of the lipid transporter mutant proteins capable of specifically binding to 4-1BB comprises the amino acid sequence of SEQ ID NO:2. In another aspect, all the lipid transporter mutant proteins comprise the same amino acid sequence.

[0253] In a further aspect, a bispecific antigen-binding molecule capable of bivalently binding to 4-1BB and monovalently binding to target cell antigens is provided, comprising:

[0254] (a) Fab fragments that can specifically bind to target cell antigens;

[0255] (b) The Fc domain, comprising a first subunit and a second subunit capable of stable association, and

[0256] (c) Two lipid transporter mutant proteins capable of specifically binding to 4-1BB, wherein one of the lipid transporter mutant proteins is fused to the C-terminus of the first subunit of the Fc domain and the other is fused to the C-terminus of the second subunit of the Fc domain, and wherein each of the lipid transporter mutant proteins capable of specifically binding to 4-1BB is derived from human tear lipid transporter protein (Tlc) of SEQ ID NO: 90.

[0257] In one respect, each of the lipotransporter mutant proteins capable of specifically binding to 4-1BB comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111 and SEQ ID NO:112.

[0258] In one aspect, the present invention provides a bispecific antigen-binding molecule comprising two lipid carrier mutant proteins capable of specifically binding to 4-1BB, wherein one of the lipid carrier mutant proteins is fused to the C-terminus of a first subunit of the Fc domain via a peptide linker, and the other is fused to the C-terminus of a second subunit of the Fc domain via a peptide linker. In one aspect, the peptide linker has an amino acid sequence selected from the group consisting of: SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120 and SEQ ID NO:121. In one aspect, the peptide linker has an amino acid sequence selected from the group consisting of: SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89. In another aspect, the peptide linker has an amino acid sequence selected from the group consisting of: SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, and SEQ ID NO:121. Specifically, the peptide linker has the amino acid sequence of SEQ ID NO:78, namely (G4S)3.

[0259] In a further aspect, the Fc domain is IgG, particularly the IgG1 Fc domain or the IgG4 Fc domain. More specifically, the Fc domain is the IgG1 Fc domain. In one particular aspect, the Fc domain includes modifications that promote association between the first subunit and the second subunit of the Fc domain.

[0260] Fc domain modification that promotes heterodimerization

[0261] In one aspect, the bispecific antigen-binding molecule of the present invention comprises: an Fc domain including a first and a second subunit capable of stable association; a Fab fragment capable of specifically binding to a target cell antigen, said Fab fragment being fused to the N-terminus of the first subunit of the Fc domain; and two lipid carrier mutant proteins capable of specifically binding to 4-1BB, wherein one of the lipid carrier mutant proteins is fused to the C-terminus of the first subunit of the Fc domain, and the other is fused to the C-terminus of the second subunit of the Fc domain. Therefore, the bispecific antigen-binding molecule of the present invention comprises: two distinct polypeptide chains (“heavy chains”) comprising the first and second subunits of the Fc domain, respectively, and a light chain. Recombinant co-expression and subsequent dimerization of these polypeptides result in several possible combinations of the two distinct heavy chains. To improve the yield and purity of the bispecific antigen-binding molecule in recombinant production, it is therefore advantageous to introduce modifications into the Fc domain of the bispecific antigen-binding molecule that promote the association of the desired polypeptide.

[0262] Therefore, the Fc domain of the bispecific antigen-binding molecule of the present invention includes modifications that promote association between the first and second subunits of the Fc domain. The most extensive protein-protein interaction site between the two subunits of the human IgG Fc domain is located in the CH3 domain of the Fc domain. Therefore, the modification is particularly located in the CH3 domain of the Fc domain.

[0263] In one specific aspect, the modification is a so-called "mortar and pestle structure" modification, comprising 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. Therefore, in one particular aspect, the present invention relates to a bispecific antigen-binding molecule as described above, comprising an IgG molecule, wherein the Fc portion of the first heavy chain comprises a first dimerizing module and the Fc portion of the second heavy chain comprises a second dimerizing module that allows heterodimerization of the two heavy chains of the IgG molecule, and the first dimerizing module comprises a protrusion and the second dimerizing module comprises a hole according to the mortar and pestle structure technique.

[0264] The mortar and pestle structure technique is described, for example, in 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). Typically, this method involves introducing a protrusion (“protrusion”) at the interface of a first polypeptide and a corresponding cavity (“pore”) at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote the formation of a heterodimer and inhibit the formation of a homodimer. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensating cavity having the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0265] Therefore, in one particular aspect, in the CH3 domain of the first subunit of the Fc domain of the bispecific antigen-binding molecule disclosed herein, amino acid residues are replaced by amino acid residues having a larger side chain volume, thereby creating a protrusion within the CH3 domain of the first subunit, the protrusion being localized in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, amino acid residues are replaced by amino acid residues having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit, the protrusion within the CH3 domain of the first subunit being localized in the cavity.

[0266] Protrusions and cavities can be created by altering the nucleic acids encoding polypeptides, for example, through site-specific mutagenesis or peptide synthesis.

[0267] In one specific aspect, in the CH3 domain of the first subunit of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V). More specifically, in the second subunit of the Fc domain, the threonine residue at position 366 is further replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A). More specifically, in the first subunit of the Fc domain, the serine residue at position 354 is further replaced by a cysteine ​​residue (S354C), and in the second subunit of the Fc domain, the tyrosine residue at position 349 is further replaced by 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)).

[0268] On the other hand, modifications that promote association between the first and second subunits of the Fc domain include modifications that mediate electrostatic reorientation effects, such as those described in PCT Publication WO 2009 / 089004. Typically, this method involves replacing one or more amino acid residues at the interface between the two Fc domain subunits with charged amino acid residues, making homodimer formation electrostatically unfavorable but heterodimerization electrostatically favorable.

[0269] Fc domain modifications that reduce Fc receptor binding and / or effector function

[0270] The Fc domain of the bispecific antigen-binding molecule of the present 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 contains CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain can stably associate with each other.

[0271] The Fc domain endows the antigen-binding molecules of the present invention with favorable pharmacokinetic properties, including a long serum half-life that facilitates good accumulation in target tissues and a favorable tissue-blood partition ratio. However, at the same time, it may cause the bispecific antibodies of the present invention to undesirably target cells expressing Fc receptors, rather than preferred antigen-carrying cells. Therefore, in another specific aspect, compared with the natural IgG1 Fc domain, the Fc domain of the bispecific antigen-binding molecules of the present invention exhibits reduced binding affinity to Fc receptors and / or reduced effector function. In one aspect, the Fc domain substantially does not bind to the Fc receptor and / or does not induce effector function. In one specific aspect, the Fc receptor is an Fcγ receptor. In one aspect, the Fc receptor is a human Fc receptor. In a particular aspect, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. In one aspect, the Fc domain does not induce effector function. Reduced effector functions may include, but are not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent phagocytosis (ADCP), reduced cytokine secretion, reduced antigen uptake by immune complex-mediated antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signal transduction-induced apoptosis, reduced dendritic cell maturation, or reduced T cell initiation.

[0272] In some respects, one or more amino acid modifications may be introduced into the Fc region of the bispecific antigen-binding molecule provided herein, thereby generating an Fc region variant. The Fc region variant may contain a human Fc region sequence (e.g., the human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0273] In one particular aspect, the present invention provides a substance capable of bivalently binding to 4-1BB and monovalently binding to a target cell antigen, comprising...

[0274] (a) Fab fragments that can specifically bind to target cell antigens;

[0275] (b) The Fc domain, comprising a first subunit and a second subunit capable of stable association, and

[0276] (c) Two lipid transporter mutant proteins capable of specifically binding to 4-1BB, wherein one of the lipid transporter mutant proteins is fused to the C-terminus of the first subunit of the Fc domain and the other is fused to the C-terminus of the second subunit of the Fc domain, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to the Fc receptor, particularly the Fcγ receptor.

[0277] In one aspect, the Fc domain of the bispecific antigen-binding molecule of the present invention comprises one or more amino acid mutations that reduce the binding affinity of the Fc domain to the Fc receptor and / or the effector function. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. Specifically, the Fc domain contains amino acid substitutions at positions E233, L234, L235, N297, P331, and P329 (EU numbers). Specifically, the Fc domain contains amino acid substitutions at positions 234 and 235 (EU numbers) and / or 329 (EU number) of the IgG heavy chain. More specifically, an antigen-binding molecule comprising a trimer TNF family ligand according to the present invention is provided, comprising an Fc domain having amino acid substitutions L234A, L235A, and P329G (“P329G LALA”, EU number) in the IgG heavy chain. The amino acid substitutions L234A and L235A refer to the so-called LALA mutation. The amino acid-substituted “P329G LALA” combination almost completely eliminates Fcγ receptor binding of the human IgG1 Fc domain and is described in International Patent Application Publication No. WO 2012 / 130831A1, which also describes methods for preparing such mutant Fc domains and methods for determining their properties (such as Fc receptor binding or effector function). “EU number” refers to the numbering based on the EU index of Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0278] In one particular aspect, the Fc domain comprising a first subunit and a second subunit capable of stable association includes: a first subunit comprising the amino acid sequence of SEQ ID NO:128, and a second subunit comprising the amino acid sequence of SEQ ID NO:129.

[0279] Fc domains with reduced Fc receptor binding and / or effector function also include those with substitutions of one or more of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant, in which residues 265 and 297 are substituted with alanine (US Patent No. 7,332,581).

[0280] On the other hand, the Fc domain is the IgG4 Fc domain. Compared to IgG1 antibodies, IgG4 antibodies exhibit reduced binding affinity to the Fc receptor and reduced effector function. More specifically, the Fc domain is the IgG4 Fc domain containing an amino acid substitution at position S228 (Kabat designation), specifically the amino acid substitution S228P. More specifically, the Fc domain is the IgG4 Fc domain containing amino acid substitutions L235E and S228P, as well as P329G (EU designation). Such IgG4 Fc domain mutants and their binding properties to the Fcγ receptor are also described in WO 2012 / 130831.

[0281] Mutant Fc domains can be prepared using genetic or chemical methods well-known in the art, through amino acid deletion, substitution, insertion, or modification. Genetic methods may include site-specific mutagenesis of coding DNA sequences, PCR, gene synthesis, etc. Correct nucleotide changes can be verified, for example, by sequencing.

[0282] 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, through recombinant expression. Suitable such binding assays are described herein. Alternatively, the binding affinity of Fc domains or cell-activating bispecific antigen-binding molecules containing Fc domains to Fc receptors can be assessed using cell lines known to express specific Fc receptors, such as human NK cells expressing the FcγIIIa receptor.

[0283] The effector function of the Fc domain, or the bispecific antibody of the present invention containing the Fc domain, 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 the ADCC activity of a target molecule are described in: U.S. Patent No. 5,500,362; Hellstrom et al., ProcNatl Acad Sci USA 83,7059-7063 (1986) and Hellstrom et al., ProcNatl Acad Sci USA 82,1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166,1351-1361 (1987). Alternatively, non-radioactive assays can be used (see, for example, ACTI for flow cytometry). TM Non-radioactive cytotoxicity assay (Cell Technology, Inc., Mountain View, CA); and CytoTox Non-radioactive cytotoxicity assays (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the target molecule can be assessed in vivo in animal models such as those disclosed in Clynes et al., Proc Natl Acad Sci USA 95,652-656 (1998).

[0284] In some embodiments, the binding of the Fc domain to complement components, particularly C1q, is reduced. Therefore, in some embodiments, the Fc domain is engineered to have reduced effector functions, including reduced CDC. C1q binding assays can be performed to determine whether the bispecific antibody of the present invention is capable of binding C1q and thus possessing CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

[0285] Specific bispecific antigen-binding molecules

[0286] In one aspect, the present invention provides a bispecific antigen-binding molecule capable of bivalently binding to 4-1BB and monovalently binding to a target cell antigen, comprising...

[0287] (a) The Fab fragment that can specifically bind to fibroblast activation protein (FAP)

[0288] (b) The Fc domain, comprising a first subunit and a second subunit capable of stable association, and

[0289] (c) Two lipid transporter mutant proteins capable of specifically binding to 4-1BB, wherein one of the lipid transporter mutant proteins is fused to the C-terminus of the first subunit of the Fc domain, and the other is fused to the C-terminus of the second subunit of the Fc domain.

[0290] In one respect, the Fab fragment capable of specifically binding to fibroblast activation protein (FAP) includes:

[0291] (a) Heavy chain variable region (V H FAP), comprising: (i) CDR-H1, comprising the amino acid sequence of SEQ ID NO:21, (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO:22, and (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO:23; and a light chain variable region (V L FAP), comprising: (iv) CDR-L1, comprising the amino acid sequence of SEQ ID NO:24, (v) CDR-L2, comprising the amino acid sequence of SEQ ID NO:25, and (vi) CDR-L3, comprising the amino acid sequence of SEQ ID NO:26; or

[0292] (b) Heavy chain variable region (V) H FAP), comprising: (i) CDR-H1, comprising the amino acid sequence of SEQ ID NO:29, (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO:30, and (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO:31; and a light chain variable region (V L FAP), comprising: (iv) CDR-L1, which comprises the amino acid sequence of SEQ ID NO:32, (v) CDR-L2, which comprises the amino acid sequence of SEQ ID NO:33, and (vi) CDR-L3, which comprises the amino acid sequence of SEQ ID NO:34.

[0293] In one respect, the Fab fragment that can specifically bind to fibroblast activation protein (FAP) includes: a heavy chain variable region (V...H FAP), comprising: (i) CDR-H1, comprising the amino acid sequence of SEQ ID NO:21, (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO:22, and (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO:23; and a light chain variable region (V L FAP), comprising: (iv) CDR-L1, which comprises the amino acid sequence of SEQ ID NO:24, (v) CDR-L2, which comprises the amino acid sequence of SEQ ID NO:25, and (vi) CDR-L3, which comprises the amino acid sequence of SEQ ID NO:26.

[0294] In one aspect, a Fab fragment capable of specifically binding to fibroblast activation protein (FAP) is provided, comprising:

[0295] (a) Heavy chain variable region (V H FAP), comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:27; and a light chain variable region (V L FAP), which contains at least about 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO:28; or

[0296] (b) Heavy chain variable region (V) H FAP), comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:35; and a light chain variable region (V L FAP), which contains at least about 95%, 96%, 97%, 98%, 99% or 100% the same amino acid sequence as the amino acid sequence of SEQ ID NO:36.

[0297] In one aspect, a Fab fragment capable of specifically binding to fibroblast activation protein (FAP) is provided, comprising: a heavy chain variable region (V... H FAP), which contains the amino acid sequence of SEQ ID NO:27, and the light chain variable region (V L FAP), which contains the amino acid sequence of SEQ ID NO:28; or the heavy chain variable region (V H FAP, which contains the amino acid sequence of SEQ ID NO:35, and the light chain variable region (V LFAP), which contains the amino acid sequence of SEQ ID NO:36. In one aspect, the Fab fragment capable of specifically binding to fibroblast activation protein (FAP) includes: a heavy chain variable region (V H FAP), which contains the amino acid sequence of SEQ ID NO:27; and the light chain variable region (V L FAP), which contains the amino acid sequence of SEQ ID NO:28.

[0298] In one respect, the bispecific antigen-binding molecule provided herein comprises a first heavy chain of SEQ ID NO: 37, a second heavy chain of SEQ ID NO: 38, and a light chain of SEQ ID NO: 39.

[0299] On the other hand, the present invention provides a bispecific antigen-binding molecule capable of bivalently binding to 4-1BB and monovalently binding to target cell antigens, comprising...

[0300] (a) A Fab fragment that can specifically bind to HER2;

[0301] (b) The Fc domain, comprising a first subunit and a second subunit capable of stable association, and

[0302] (c) Two lipid transporter mutant proteins capable of specifically binding to 4-1BB, wherein one of the lipid transporter mutant proteins is fused to the C-terminus of the first subunit of the Fc domain, and the other is fused to the C-terminus of the second subunit of the Fc domain.

[0303] In one respect, the Fab fragments that can specifically bind to HER2 include:

[0304] (a) A VH domain comprising: (i) CDR-H1, comprising the amino acid sequence of SEQ ID NO:40, (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO:41, and (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO:42; and a VL domain comprising: (iv) CDR-L1, comprising the amino acid sequence of SEQ ID NO:43, (v) CDR-L2, comprising the amino acid sequence of SEQ ID NO:44, and (vi) CDR-L3, comprising the amino acid sequence of SEQ ID NO:45; or

[0305] (b) A VH domain comprising: (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:48, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:49, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:50; and a VL domain comprising: (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:51, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:52, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:53; or

[0306] (c) A VH domain comprising: (i) CDR-H1, comprising the amino acid sequence of SEQ ID NO:56, (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO:57, and (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO:58; and a VL domain comprising: (iv) CDR-L1, comprising the amino acid sequence of SEQ ID NO:59, (v) CDR-L2, comprising the amino acid sequence of SEQ ID NO:60, and (vi) CDR-L3, comprising the amino acid sequence of SEQ ID NO:61.

[0307] In one aspect, the Fab fragment capable of specifically binding to HER2 comprises: (a) a VH domain comprising: (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:40, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:41, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:42; and a VL domain comprising: (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:43, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:44, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:45. In one aspect, the Fab fragment capable of specifically binding to HER2 comprises: a VH domain comprising: (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:48, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:49, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:50; and a VL domain comprising: (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:51, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:52, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:53.

[0308] In one aspect, a Fab fragment capable of specifically binding to HER2 is provided, comprising:

[0309] (a) Heavy chain variable region (V H HER2), comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:46; and a light chain variable region (V L HER2), which contains an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:47; or

[0310] (b) Heavy chain variable region (V) H HER2), comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:54; and a light chain variable region (V L HER2), which contains an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:55; or

[0311] (c) Heavy chain variable region (V) H HER2), comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:62; and a light chain variable region (V L HER2), which contains an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:63.

[0312] In one aspect, a Fab fragment capable of specifically binding to HER2 is provided, comprising: a heavy chain variable region (V H HER2), which contains the amino acid sequence of SEQ ID NO:46, and the light chain variable region (V L HER2), which contains the amino acid sequence of SEQ ID NO:47; or the heavy chain variable region (V H HER2), which contains the amino acid sequence of SEQ ID NO:54, and the light chain variable region (V L HER2), which contains the amino acid sequence of SEQ ID NO:55; or the heavy chain variable region (V H HER2), which contains the amino acid sequence of SEQ ID NO:62, and the light chain variable region (V LHER2), which contains the amino acid sequence of SEQ ID NO:63. In one aspect, the Fab fragment capable of specifically binding to HER2 includes: a heavy chain variable region (V H HER2), which contains the amino acid sequence of SEQ ID NO:46, and the light chain variable region (V L HER2), which contains the amino acid sequence of SEQ ID NO:47. In one aspect, the Fab fragment capable of specifically binding to HER2 includes: a heavy chain variable region (V H HER2), which contains the amino acid sequence of SEQ ID NO:54, and the light chain variable region (V L HER2), which contains the amino acid sequence of SEQ ID NO:55.

[0313] In one respect, the bispecific antigen-binding molecule provided herein comprises a first heavy chain of SEQ ID NO: 64, a second heavy chain of SEQ ID NO: 65, and a light chain of SEQ ID NO: 66.

[0314] Polynucleotides

[0315] The present invention further provides isolated nucleic acids encoding bispecific antigen-binding molecules or fragments thereof as described herein.

[0316] The isolated polynucleotide encoding the bispecific antigen-binding molecule of the present invention can be expressed as a single polynucleotide encoding the complete antigen-binding molecule, or as multiple (e.g., two or more) polynucleotides co-expressed. The polypeptides encoded by the co-expressed polynucleotides can 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 can 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.

[0317] In some respects, the isolated nucleic acids encode the complete bispecific antigen-binding molecule according to the invention as described herein. Specifically, the isolated polynucleotides encode polypeptides contained in the bispecific antigen-binding molecule according to the invention as described herein.

[0318] In one aspect, the present invention relates to isolated nucleic acids encoding bispecific antigen-binding molecules, wherein the nucleic acid molecule comprises: (a) a sequence encoding an antigen-binding domain capable of specifically binding to a target cell antigen, (b) a sequence encoding an Fc domain comprising a first and second subunit capable of stable association, and (c) a sequence encoding a lipotransferase mutant protein capable of specifically binding to 4-1BB.

[0319] On the other hand, a separate polynucleotide encoding a bispecific antigen-binding molecule is provided, wherein the polynucleotide comprises encoding the following sequence: (a) a Fab fragment capable of specifically binding to a target cell antigen; (b) an Fc domain comprising a first and second subunit capable of stable association; and (c) two lipid transporter mutant proteins capable of specifically binding to 4-1BB, wherein one of the lipid transporter mutant proteins is fused to the C-terminus of the first subunit of the Fc domain, and the other is fused to the C-terminus of the second subunit of the Fc domain.

[0320] In some respects, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA). The RNA of the present invention can be single-stranded or double-stranded.

[0321] Recombination method

[0322] The bispecific antigen-binding molecules of the present invention 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 encoding a bispecific antigen-binding molecule or a polypeptide fragment thereof, as described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such polynucleotides can be readily isolated and sequenced using conventional methods. In one aspect of the invention, a vector, preferably an expression vector, is provided containing one or more polynucleotides of the polynucleotides of the present invention. Expression vectors containing the coding sequence of a bispecific antigen-binding molecule (fragment) and appropriate transcription / translation control signals can be constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. See, for example, the techniques described in the following literature: Maniatis et al., MOLECULARCLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, NY (1989); and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, NY (1989), which label antibodies with a ligand reagent that binds, chelates, or otherwise conjugates a radioisotope metal, wherein the reagent reacts with an engineered cysteine ​​thiol of the antibody. The expression vector may be a plasmid, a portion of a virus, or a fragment of nucleic acid. The expression vector comprises an expression cassette into which a polynucleotide (i.e., a coding region) encoding a bispecific antigen-binding molecule or a polypeptide fragment thereof is operatively associated with a promoter and / or other transcriptional or translational control element is cloned. As used herein, a “coding region” is a portion of a nucleic acid consisting of codons that translate into amino acids. Although the "stop codon" (TAG, TGA, or TAA) is not translated into amino acids, it (if present) can be considered part of the coding region, while any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, 5' and 3' untranslated regions, are not part of the coding region. 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). Furthermore, any vector may contain a single coding region or may contain two or more coding regions; for example, the vectors of the present invention may encode one or more polypeptides that are cleaved into the final protein post-translation or during translation via proteolytic cleavage.Furthermore, the vectors, polynucleotides, or nucleic acids of the present invention may encode heterologous coding regions, which may or may not be fused with a polynucleotide or its variants or derivatives encoding a polypeptide fragment of the bispecific antigen-binding molecule of the present invention or thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretory signal peptides or heterologous functional domains. Operable association is when the coding region of a gene product (e.g., a polypeptide) is associated in a way that brings the expression of the gene product under the influence or control of the regulatory sequences. Two DNA fragments (such as a polypeptide coding region and its associated promoter) are “operably associated” if the induction of promoter function leads to transcription of mRNA encoding the desired gene product, and if the nature of the connection between the two DNA fragments does not interfere with the ability of the expression regulatory sequence to direct the expression of the gene product or the ability to interfere with the gene template to be transcribed. Therefore, if the promoter can influence the transcription of the nucleic acid, the promoter region will operateably associate with the nucleic acid encoding the polypeptide. The promoter may be a cell-specific promoter that directs the substantial transcription of DNA only in a predetermined cell. In addition to promoters, other transcriptional control elements, such as enhancers, operons, repressors, and transcription termination signals, can be operatively associated with polynucleotides to guide cell-specific transcription.

[0323] This document discloses suitable promoters and other transcriptional control regions. 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 cytomegaloviruses (e.g., immediate early promoter binding intron-A), simian virus 40 (e.g., early promoter), and retroviruses (e.g., Raoult's sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes (such as actin, heat shock protein, bovine growth hormone, and rabbit...). The transcriptional control regions of globins, and other sequences capable of controlling gene expression in eukaryotic cells. Other suitable transcriptional control regions include tissue-specific promoters and enhancers, as well as inducible promoters (e.g., tetracycline-inducible promoters). Similarly, various translational control elements are known to those skilled in the art. These translational 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). Expression cassettes may also include other features such as origins of replication, and / or chromosomal integration elements, such as retroviral long terminal repeats (LTRs) or adeno-associated virus (AAV) inverted terminal repeats (ITRs).

[0324] The polynucleotide and nucleic acid coding regions of the present invention can associate with additional coding regions encoding secretory peptides or signal peptides, which in turn direct the secretion of polypeptides encoded by the polynucleotides of the present invention. For example, if secretion of a bispecific antigen-binding molecule or a polypeptide fragment thereof is desired, DNA encoding a signal sequence can be placed upstream of a nucleic acid encoding the bispecific antigen-binding molecule 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 begun to export across the rough endoplasmic reticulum. Those skilled in the art know 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 produce the secretory or “mature” form of the polypeptide. In some embodiments, a natural signal peptide (e.g., an immunoglobulin heavy or light chain signal peptide) or a functional derivative of the sequence that retains the ability to direct the secretion of a polypeptide with which it operably associates is used. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof may be used. For example, the wild-type leader sequence can be replaced by the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0325] DNA encoding short protein sequences (e.g., histidine tags) that can be used to facilitate subsequent purification or to help label fusion proteins may be contained within or at the end of a polynucleotide encoding the bispecific antigen-binding molecule of the present invention or a polypeptide fragment thereof.

[0326] In another aspect of the invention, a host cell comprising one or more of the polynucleotides of the invention is provided. In some embodiments, a host cell comprising one or more vectors of the invention is provided. The polynucleotides and vectors may be incorporated individually or in combination into any features described herein with respect to the polynucleotides and vectors, respectively. In one aspect, the host cell comprises (e.g., transformed or transfected) a vector containing a polynucleotide encoding a portion of the bispecific antigen-binding molecule of the invention. As used herein, the term “host cell” refers to any kind of cellular system that can be engineered to produce the fusion protein or fragments thereof of the invention. 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 specific expression vectors, and large quantities of vector-containing cells can be grown for inoculation of large-scale fermenters to obtain sufficient quantities of antigen-binding molecules for clinical applications. Suitable host cells include prokaryotic microorganisms, such as *Escherichia coli*, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, etc. For example, peptides can be produced in bacteria, particularly when glycosylation is not required. After expression, peptides can be separated from bacterial cell paste in a soluble fraction and can be further purified. Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for vectors encoding peptides, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in peptides with partial or complete human glycosylation patterns. See Gerngross, Nat Biotech 22, 1409-1414 (2004) and Li et al., Nat Biotech 24, 210-215 (2006).

[0327] Suitable host cells for expressing (glycosylated) peptides also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLATNIBODIES for generating antibodies in transgenic plants). TM(Technology). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines include the monkey kidney CV1 line (COS-7) transformed from SV40; human embryonic kidney lines (293 or 293T cells, as described, for example, in Graham et al., J Gen Virol 36, 59 (1977)); young hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, in Mather, Biol Reprod 23, 243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor cells (MMT060562); TRI cells (as described, for example, in Mather et al., Annals NYAcad Sci 383, 44-68 (1982)); and MRC. 5 cells, as well as FS4 cells. 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, NSO, P3X63, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by BKCLo, 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 just a few, as well as 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 Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or lymphocytes (e.g., Y0, NSO, Sp20 cells). Standard techniques for expressing exogenous genes in these systems are known in the art. Cells expressing polypeptides containing heavy or light chains of immunoglobulins can be engineered to also express another immunoglobulin chain, such that the expressed product is an immunoglobulin having both heavy and light chains.

[0328] In one aspect, a method for producing the bispecific antigen-binding molecule of the present invention or a polypeptide fragment thereof is provided, wherein the method comprises culturing a host cell containing a polynucleotide encoding the bispecific antigen-binding molecule of the present invention or a polypeptide fragment thereof as provided herein, under conditions suitable for expressing the bispecific antigen-binding molecule of the present invention or a polypeptide fragment thereof, and recovering the bispecific antigen-binding molecule of the present invention or a polypeptide fragment thereof from the host cell (or host cell culture medium).

[0329] In the bispecific antigen-binding molecule of the present invention, the components (at least one portion capable of specifically binding to target cell antigens, a subunit containing an Fc domain, and a polypeptide containing a lipid transporter mutant protein) do not genetically fuse with each other. The polypeptide is designed such that its components fuse with each other directly or via a linker 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 different components of the antigen-binding molecule of the present invention are provided herein. If desired, additional sequences (e.g., endopeptidase recognition sequences) may be included to incorporate cleavage sites to separate the individual components of the fusion protein.

[0330] In some embodiments, the antigen-binding domain (such as the Fab fragment) that forms part of the antigen-binding molecule and is capable of specifically binding to the target cell antigen includes at least one immunoglobulin variable region capable of binding to the antigen. The variable region can form part of and be derived from naturally occurring or non-naturally occurring antibodies and fragments thereof. Methods for generating polyclonal and monoclonal antibodies are well known in the art (see, for example, Harlow and Lane, "Antibodies, a laboratory manual", Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid-phase peptide synthesis, can be recombinantly generated (e.g., as described in U.S. Patent No. 4,186,567), or can be obtained, for example, by screening a combined library containing variable heavy and variable light chains (see, for example, U.S. Patent No. 5,969,108 to McCafferty).

[0331] Immunoglobulins from any animal species can be used in this invention. Non-limiting immunoglobulins that can be used in this invention can be mouse, primate, or human. If the fusion protein is intended for human use, a chimeric form of immunoglobulin can be used, wherein the constant region of the immunoglobulin is derived from a human. Humanized or fully human forms of immunoglobulins can also be prepared according to methods well known in the art (see, for example, U.S. Patent No. 5,565,332 to Winter). Humanization can be achieved by a variety of methods, including but not limited to (a) transplanting a non-human (e.g., donor antibody) CDR onto a human (e.g., receptor antibody) architecture and constant region, with or without retaining key architectural residues (e.g., key architectural residues important for maintaining good antigen-binding affinity or antibody function), (b) transplanting only the non-human specificity-determining region (SDR or a-CDR; residues essential for antibody-antigen interaction) onto the human architecture and constant region, or (c) transplanting the entire non-human variable domain, but “hiding” them with human-like segments by replacing surface residues.Humanized antibodies and their preparation methods are reviewed, for example, in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), and further described, for example: Riechmann et al., Nature 332, 323-329 (1988); Queen et al., ProcNatl Acad Sci USA 86, 10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones et al., Nature 321, 522-525 (1986); Morrison et al., Proc Natl Acad Sci 81, 6851-6855 (1984); Morrison and Oi, Adv Immunol 44, 65-92 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan, Molec Immun 31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005) (describes SDR(a-CDR) transplantation); Padlan, Mol Immunol 28, 489-498 (1991) (describes “surface reshaping”); 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, 252-260 (2000) (describes a “guided selection” approach for FR shuffling). The specific immunoglobulin according to the invention is a human immunoglobulin. Human antibodies and human variable regions can be produced using various techniques known in the art. 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, 450-459 (2008). The human variable region can form part of and be derived from a human monoclonal antibody prepared by a hybridoma method (see, for example, 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 administering an immunogen to a transgenic animal that has been modified to produce a complete human antibody or a complete antibody with a human variable region in response to an antigen challenge (see, for example, Lonberg, Nat Biotech 23, 1117-1125 (2005)). Human antibodies and human variable regions can also be generated by isolating variable region sequences of Fv clones selected from human phage display libraries (see, for example, Hoogenboom et al. in Methods in Molecular Biology 178, 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001); McCafferty et al., Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments.

[0332] In some aspects, antigen-binding domains (such as Fab fragments) in the antigen-binding molecules of the present invention that are capable of specifically binding to target cell antigens are engineered to have enhanced binding affinity, for example, according to the method disclosed in PCT Publication WO 2012 / 020006 (see examples related to affinity maturation) or U.S. Patent Application Publication No. 2004 / 0132066. The ability of the antigen-binding molecules of the present invention to bind to specific antigenic determinants can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). Competition assays can be used to identify antigen-binding molecules that compete with a reference antibody for binding to a specific antigen. In some embodiments, such competing antigen-binding molecules bind to the same epitopes (e.g., linear or conformational epitopes) bound by the reference antigen-binding molecule. Detailed exemplary methods for mapping epitopes bound by antigen-binding molecules are provided in: Morris (1996), “Epitope Mapping Protocols,” Methods in Molecular Biology, Vol. 66 (Humanities). Press, Totowa, NJ). In an exemplary competitive assay, immobilized antigen is incubated in a solution containing a first labeled antigen-binding molecule that binds to the antigen and a second unlabeled antigen-binding molecule whose ability to compete with the first antigen-binding molecule for binding to the antigen is being tested. The second antigen-binding molecule may be present in the hybridoma supernatant. As a control, immobilized antigen is incubated in a solution containing 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 labeled molecule associated with the immobilized antigen is measured. If the amount of labeled molecule associated with the immobilized antigen is substantially reduced relative to the control sample in the test sample, it indicates that the second antigen-binding molecule competes with the first antigen-binding molecule for binding to the antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manual, Chapter 14 (ColdSpring Harbor Laboratory, Cold Spring Harbor, NY).

[0333] The bispecific antigen-binding molecules of the present invention prepared as described herein can be purified using techniques known in the art, such as high-performance liquid chromatography (HPLC), ion-exchange chromatography (IEC), gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The specific conditions used for purifying a particular protein will depend in part on factors such as net charge, hydrophobicity, and hydrophilicity, and will be apparent to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors, or antigens bound to the bispecific antigen-binding molecules can be used. For example, for affinity chromatography purification of the fusion protein of the present invention, a matrix containing protein A or protein G can be used. Essentially as described in the examples, antigen-binding molecules can be separated using sequential protein A or G affinity chromatography and size exclusion chromatography. The purity of the bispecific antigen-binding molecules or fragments thereof can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high-performance liquid chromatography, etc. For example, the expressed bispecific antigen-binding molecules described in the examples are shown to be fully and properly assembled, as shown by reduced and non-reduced SDS-PAGE.

[0334] Measurement

[0335] The physical / chemical properties and / or biological activities of the antigen-binding molecules described herein can be identified, screened, or characterized by various assays known in the art. Biological activities may include, for example, the ability to enhance the activation and / or proliferation of different immune cells, particularly T cells. They may also enhance the secretion of immunomodulatory cytokines. Other immunomodulatory cytokines that they enhance or can enhance include IL-2, granzyme B, etc. Biological activities may also include cynomolgus monkey binding cross-reactivity and binding to different cell types. Antigen-binding molecules exhibiting such biological activities in vivo and / or in vitro are also provided.

[0336] 1. Affinity test

[0337] The affinity of the bispecific antigen-binding molecule provided herein for 4-1BB (CD137) can be determined by surface plasmon resonance (SPR) using standard instruments such as BIAcore instruments (GE Healthcare) and receptors or target proteins that can be obtained through recombinant expression, according to the methods described in the examples. Specific conditions for determining the affinity for 4-1BB are also described in WO 2018 / 087108. The affinity of the bispecific antigen-binding molecule for target cell antigens (such as FAP or HER2) can also be determined by surface plasmon resonance (SPR) using standard instruments such as BIAcore instruments (GE Healthcare) and receptors or target proteins that can be obtained through recombinant expression. Specific illustrative and exemplary embodiments for measuring binding affinity are described in Examples 1.2 and 2.2. According to one aspect, at 25°C... The T100 instrument (GE Healthcare) measures K using surface plasmon resonance. D .

[0338] 2. Combined measurement with other measurements

[0339] The binding of the bispecific antigen-binding molecule provided herein to cells expressing the corresponding receptor can be evaluated using cell lines expressing specific receptors or target antigens, for example, by flow cytometry (FACS). In one aspect, fresh peripheral blood mononuclear cells (PBMCs) expressing 4-1BB can be used for binding assays. These cells can be used directly after isolation (primary PMBCs) or after stimulation (activated PMBCs). In another aspect, activated mouse spleen cells (expressing 4-1BB) can be used to demonstrate the binding of the bispecific antigen-binding molecule of the present invention to cells expressing 4-1BB.

[0340] In a further step, cell lines expressing FAP or HER2 were used to demonstrate the binding of antigen-binding molecules to this target cell antigen.

[0341] On the other hand, competitive assays can be used to identify antigen-binding molecules that compete with specific antibodies or antigen-binding molecules for binding to FAP, HER2, or 4-1BB. In some respects, such competitive antigen-binding molecules bind to the same epitope (e.g., linear or conformational epitope) bound by a specific anti-FAP antibody, anti-HER2 antibody, or specific anti-4-1BB antibody. Detailed exemplary methods for locating the epitope to which the antibody binds are provided in: Morris (1996), “Epitope Mapping Protocols,” Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ).

[0342] 3. Activity Assay

[0343] In one aspect, an assay method is provided for identifying bispecific antigen-binding molecules that bind to FAP or HER2 and to biologically active 4-1BB. Biological activity may include, for example, agonistic signaling via 4-1BB in cancer cells expressing FAP or HER2. Bispecific antigen-binding molecules exhibiting such in vitro biological activity are also provided by assay.

[0344] In some aspects, such biological activity of the bispecific antigen-binding molecules of the present invention is tested. Assays for detecting the biological activity of the molecules of the present invention are those described in Examples 3.3 and 4.3. Furthermore, 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 a TUNEL assay) are well known in the art. Additionally, the biological activity of such complexes can be assessed by evaluating their effects on the survival, proliferation, and lymphokine secretion of various lymphocyte subsets such as NK cells, NKT cells, or γδT cells, or by assessing their ability to regulate the phenotype and function of antigen-presenting cells such as dendritic cells, monocytes / macrophages, or B cells.

[0345] Pharmaceutical compositions, formulations and routes of administration

[0346] In a further aspect, the present invention provides pharmaceutical compositions comprising any of the bispecific antigen-binding molecules provided herein, for example, in any of the following treatment methods. In one embodiment, the pharmaceutical composition comprises any of the bispecific antigen-binding molecules provided herein and at least one pharmaceutical excipient. In another embodiment, the pharmaceutical composition comprises any of the bispecific antigen-binding molecules provided herein and at least one additional therapeutic agent as described below.

[0347] The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of one or more bispecific antigen-binding molecules dissolved or dispersed in a pharmaceutical excipient. The term “pharmaceuticalally or pharmacologically acceptable” means that the molecular entity and composition are generally non-toxic to the recipient at the doses and concentrations employed, i.e., do not produce adverse, allergic, or other adverse reactions when administered to animals (e.g., humans) as appropriate. Preparation of pharmaceutical compositions containing at least one bispecific antigen-binding molecule and optionally an additional active ingredient will be as known to those skilled in the art in light of this disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th 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, “pharmaceuticalally 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 those skilled in the art.

[0348] Parenteral compositions include those designed for injection (e.g., subcutaneous, intradermal, intralesional, intravenous, intra-arterial, intramuscular, intrathecal, or intraperitoneal injection). For injection, the bispecific antigen-binding molecule of the invention can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hanks' solution, Ringer's solution, or physiological saline. The solution may contain a formulatory agent, such as a suspending agent, stabilizer, and / or dispersant. Alternatively, the fusion protein may be in powder form for use with a suitable medium (e.g., sterile pyrogen-free water) prior to use. As needed, a sterile injectable solution is prepared by incorporating the fusion protein of the invention in the desired amount with various other components listed below into a suitable solvent. For example, sterility can be readily achieved by filtration through a sterile filter membrane. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile medium containing a base dispersion medium and / or other components. In the case of preparing sterile powders for sterile injectable solutions, suspensions, or emulsions, the preferred preparation method is vacuum drying or lyophilization, which produces a powder of the active ingredient from a previously sterile filtered liquid medium, plus any additional desired ingredients. If necessary, the liquid medium should be appropriately buffered, and the liquid diluent should be isotonicized with sufficient saline or glucose before injection. The composition must be stable under the manufacturing and storage conditions and preserved against contamination by microorganisms such as bacteria and fungi. It should be understood that endotoxin contamination should be kept to a safe level, for example, below 0.5 ng / mg protein. Suitable pharmaceutically acceptable 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; hexamethyl diammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight (less than about 10 residues) peptides; proteins. The suspension may contain compounds 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 dextrin; 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 nonionic surfactants 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, etc. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of high-concentration solutions.In addition, suspensions of the active compounds can be prepared into suitable oily injectable suspensions. Suitable lipophilic solvents or carriers include fatty oils, such as sesame oil; or synthetic fatty acid esters, such as ethyl oleate or triglycerides; or liposomes.

[0349] The active ingredient can be encapsulated in microcapsules (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared by, for example, cohesive drug delivery systems (e.g., liposomes, albumin, microspheres, microemulsions, nanoparticles, and nanocapsules); or in crude emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th edition, Mack Printing Company, 1990). Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include a semi-permeable matrix containing a solid hydrophobic polymer of a polypeptide, said matrix being in the form of a molded article, such as a membrane or microcapsule. In certain embodiments, prolonged absorption of the injectable composition can be achieved by using a delayed-absorption agent (e.g., aluminum monostearate, gelatin, or combinations thereof) in said composition.

[0350] Exemplary pharmaceutically acceptable excipients also include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (…). Baxter International, Inc. Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycans (such as chondroitinase). Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent Nos. 6,171,586 and WO2006 / 044908, the latter containing a histidine-acetate buffer. In addition to the compositions previously described, bispecific antigen-binding molecules can also be formulated into long-acting preparations. Such long-acting preparations can be administered by implantation (e.g., subcutaneous or intramuscular implantation) or by intramuscular injection. Thus, for example, fusion proteins can be formulated with suitable polymeric or hydrophobic materials (e.g., as emulsions in acceptable oils) or ion exchange resins, or formulated as microsoluble derivatives (e.g., formulated as microsoluble salts).

[0351] Pharmaceutical compositions comprising the bispecific antigen-binding molecule of the present invention can be produced by conventional mixing, dissolving, emulsifying, encapsulating, embedding, or lyophilizing processes. The pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants that facilitate the processing of proteins into pharmaceutically usable preparations. Appropriate formulation depends on the chosen route of administration.

[0352] Bispecific antigen-binding molecules can be formulated into compositions in the form of free acids or bases, neutral or salts. Pharmaceutically acceptable salts are those that essentially retain the biological activity of the free acid or free base. These pharmaceutically acceptable salts include acid addition salts, such as those formed from the free amino groups of protein-based compositions, or those formed from inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, or mandelic acid). Salts formed from free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents compared to their corresponding free base forms.

[0353] The compositions described herein may also contain more than one active ingredient essential for the specific indication being treated, preferably active ingredients having complementary activities that do not adversely affect each other. Such active ingredients are suitably combined in amounts effective for the intended purpose.

[0354] Formulations intended for in vivo administration are typically sterile. For example, sterility can be easily achieved through filtration using a sterile filter membrane.

[0355] Treatment methods and compositions

[0356] Any of the bispecific antigen-binding molecules provided in this article that can bind bivalently to 4-1BB and monovalently to target cell antigens can be used for treatment.

[0357] For use in treatment methods, the bispecific antigen-binding molecule of the present invention can be formulated, dispensed, and administered in accordance with good medical practice. Factors to be considered in this context include the specific disease being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disease, the site of delivery, the method of administration, the timing of administration, and other factors known to the practicing physician.

[0358] In one aspect, a bispecific antigen-binding molecule capable of bivalently binding to 4-1BB and monovalently binding to a target cell antigen is provided for use as a medicament. In a further aspect, the bispecific antigen-binding molecule of the present invention is provided for treating diseases, particularly for treating cancer or infectious diseases. In some aspects, the bispecific antigen-binding molecule of the present invention is provided for use in treatment methods. In one aspect, the present invention provides a bispecific antigen-binding molecule as described herein for treating a disease in an individual with this need. In some aspects, the present invention provides a bispecific antigen-binding molecule for use in a method of treating an individual suffering from a disease, the method comprising administering a therapeutically effective amount of the bispecific antigen-binding molecule to the individual.

[0359] In some respects, the disease to be treated is cancer. The term "cancer" according to the invention also includes cancer metastasis. "Metastasis" means the spread of cancer cells from their original site to another part of the body. Tumor metastasis often occurs even after removal of the primary tumor because tumor cells or components may remain and develop metastatic potential. In one aspect, bispecific antigen-binding molecules capable of bivalently binding to 4-1BB and monovalently binding to target cell antigens are used to treat solid tumors. Representative examples of solid tumors include colon cancer, prostate cancer, breast cancer, lung cancer, skin cancer, liver cancer, bone cancer, ovarian cancer, pancreatic cancer, brain cancer, head and neck cancer, and lymphoma. Therefore, bispecific antigen-binding molecules capable of bivalently binding to 4-1BB and monovalently binding to FAP, as described herein, are provided for the treatment of solid tumors.

[0360] In some respects, the disease to be treated is HER2-positive cancer. Examples of HER2-positive cancers include breast cancer, ovarian cancer, stomach cancer, bladder cancer, salivary gland cancer, endometrial cancer, pancreatic cancer, and non-small cell lung cancer (NSCLC). Therefore, bispecific antigen-binding molecules, as described herein, capable of bivalently binding to 4-1BB and monovalently binding to HER2, are provided for the treatment of these cancers. The subjects, patients, or “individuals” requiring treatment are typically mammals, and more specifically, humans.

[0361] On the other hand, bispecific antigen-binding molecules, as described herein, are provided for the treatment of infectious diseases, particularly viral infections. The term "infectious disease" refers to any disease that can be transmitted between individuals or between organisms and is caused by a microbial pathogen. In a further aspect, bispecific antigen-binding molecules, as described herein, are provided for the treatment of autoimmune diseases, such as lupus. In some aspects, the infectious disease to be treated is a chronic viral infection, such as HIV (human immunodeficiency virus), HBV (hepatitis B virus), HCV (hepatitis C virus), HSV1 (herpes simplex virus type 1), CMV (cytomegalovirus), LCMV (lymphocytic meningitis virus), or EBV (Epstein-Barr virus).

[0362] In a further aspect, the present invention relates to the use of a bispecific antigen-binding molecule capable of bivalently binding to 4-1BB and monovalently binding to a target cell antigen for the production or preparation of a medicament for treating a disease in an individual in need. In one aspect, a method of using a medicament to treat a disease includes administering a therapeutically effective amount of the medicament to an individual having said disease. In some aspects, the disease to be treated is a proliferative disorder, particularly cancer. Therefore, in one aspect, the present invention relates to the use of the bispecific binding molecule of the present invention in the production or preparation of a medicament for treating cancer. In one aspect, the use of the bispecific binding molecule of the present invention in the production or preparation of a medicament for treating solid tumors is provided. In one aspect, the use of the bispecific binding molecule of the present invention in the production or preparation of a medicament for treating HER2-positive cancers is provided. Examples of HER2-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 some aspects, the cancer to be treated is HER2-positive breast cancer, particularly HER2-positive metastatic breast cancer. Those skilled in the art will recognize that in some cases, the bispecific antigen-binding molecule may not provide a cure, but only partial benefit. In some respects, physiological changes that have certain benefits are also considered to have therapeutic benefits. Therefore, in some respects, the amount of bispecific antigen-binding molecules that provide physiological changes is considered an “effective amount” or a “therapeutic effective amount.”

[0363] In some aspects, the use of the bispecific binding molecule of the present invention in the production or preparation of medicaments for treating infectious diseases is provided. In one aspect, the infectious disease is a chronic viral infection such as HIV (human immunodeficiency virus), HBV (hepatitis B virus), HCV (hepatitis C virus), HSV1 (herpes simplex virus type 1), CMV (cytomegalovirus), LCMV (lymphocytic meningitis virus), or EBV (Epstein-Barr virus).

[0364] In a further aspect, the present invention provides a method for treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of a bispecific antigen-binding molecule of the present invention capable of bivalently binding to 4-1BB and monovalently binding to a target cell antigen. In one aspect, administering to the individual a composition comprising the bispecific antigen-binding molecule of the present invention in a pharmaceutical form. In some aspects, the disease to be treated is a proliferative disorder. In a particular aspect, the disease is cancer. In one aspect, the disease to be treated is an infectious disease. In some 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, such as an anticancer agent. In some aspects, the method comprises further administering to the individual a therapeutically effective amount of a cytotoxic agent or another immunotherapy. The “individual” according to any of the above embodiments may be a mammal, preferably a human.

[0365] For the prevention or treatment of disease, the appropriate dose of the bispecific 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 weight, the type of antigen-binding molecule, the severity and progression of the disease, whether the bispecific antigen-binding molecule is administered for preventive or therapeutic purposes, prior or concurrent therapeutic interventions, the patient's clinical history and response to the fusion protein, and the attending physician's discretion. In any case, the practitioner responsible for administration will determine the concentration and appropriate dose of the active ingredient in the composition for the individual subject. Various dosing schedules are considered herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulse infusion. The bispecific antigen-binding molecule is appropriately administered to the patient once or in a series of treatments. Depending on the type and severity of the disease, a bispecific antigen-binding molecule of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg-10 mg / kg) may be an initial candidate dose administered to the patient, for example, by single or multiple administrations alone or by continuous infusion. Depending on the factors described above, a typical daily dose can range from about 1 μg / kg to 100 mg / kg or more. For repeated administration over several days or longer, depending on the condition, treatment typically continues until the desired suppression of disease symptoms occurs. An exemplary dose range for a bispecific antigen-binding molecule is from about 0.005 mg / kg to about 10 mg / kg. In other examples, doses may also include administrations of about 1 μg / kg body weight, about 5 μg / kg body weight, about 10 μg / kg body weight, about 50 μg / kg body weight, about 100 μg / kg body weight, about 200 μg / kg body weight, about 350 μg / kg body weight, about 500 μg / kg body weight, about 1 mg / kg body weight, about 5 mg / kg body weight, about 10 mg / kg body weight, about 50 mg / kg body weight, about 100 mg / kg body weight, about 200 mg / kg body weight, about 350 mg / kg body weight, about 500 mg / kg body weight to about 1000 mg / kg body weight or more, and any range derived therefrom. In examples of ranges that can be derived from the figures listed herein, a range of approximately 5 mg / kg body weight to approximately 100 mg / kg body weight, approximately 5 μg / kg body weight to approximately 500 mg / kg body weight, etc., can be administered based on the figures above. Therefore, one or more doses of approximately 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg, or 10 mg / kg (or any combination thereof) can be administered to the patient. Such doses can be administered intermittently, for example weekly or every three weeks (e.g., so that the patient receives approximately 2 to approximately 20, or for example, approximately 6, doses of the bispecific antigen-binding molecule). An initial higher loading dose can be administered, followed by one or more lower doses. However, other dosing regimens may be useful. Progression of the therapy can be easily monitored using routine techniques and assays.

[0366] The bispecific antigen-binding molecule of the present invention will generally be used in an amount that effectively achieves the intended purpose. For the treatment or prevention of a condition, the bispecific antigen-binding molecule of the present invention or a pharmaceutical composition thereof will be administered or applied in a therapeutically effective amount. The determination of the therapeutically effective amount is entirely within the competence of those skilled in the art, particularly based on the detailed disclosure provided herein. For systemic administration, the therapeutically effective dose can initially be estimated based on in vitro assays (such as cell culture assays). The dose can then be formulated in animal models to achieve an IC50 including, as determined in cell culture. 50 Circulating concentration range. This information can be used to more accurately determine the effective dose for humans. Initial doses can also be estimated based on in vivo data (e.g., animal models) using techniques well known in the art. Those skilled in the art can readily optimize human administration based on animal data. Dosage and intervals can be individually adjusted to provide sufficient plasma levels of the bispecific antigen-binding molecule to maintain therapeutic efficacy. Common patient doses administered by injection range from about 0.1 to 50 mg / kg / day, typically about 0.5 to 1 mg / kg / day. Therapeuticly effective plasma levels can be achieved by administering multiple doses daily. Plasma levels can be measured, for example, by HPLC. In cases of topical application or selective uptake, the effective local concentration of the bispecific antigen-binding molecule may be independent of plasma concentration. Those skilled in the art will be able to optimize therapeutically effective local doses without undue experimentation.

[0367] The therapeutically effective doses of bispecific antigen-binding molecules described in this article generally provide therapeutic benefit without significant toxicity. The toxicity and therapeutic efficacy of bispecific antigen-binding molecules can be determined using standard pharmaceutical procedures in cell culture or laboratory animals. Cell culture assays and animal studies can be used to determine the LD50. 50 (50% of the lethal dose) and ED 50 (The dose that is effective in 50% of the population). The dose ratio between toxicity and efficacy is the therapeutic index, which can be expressed as the ratio LD50. 50 / ED 50 Bispecific antigen-binding molecules exhibiting a high therapeutic index are preferred. In one aspect, the bispecific antigen-binding molecule according to the invention exhibits a high therapeutic index. Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages suitable for humans. Dosages are preferably within the range of ED (excessive toxicity) including those with little or no toxicity. 50The dosage can vary within this range depending on various factors, such as the dosage form used, the route of administration, and the patient's condition. The exact formulation, route of administration, and dosage can be selected by an individual physician based on the patient's condition (see, for example, Fingl et al., 1975, in: The Pharmacological 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 the fusion protein of this invention will know how and when to terminate, interrupt, or adjust 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 a higher level. The dosage administered in the management of the target disease will vary depending on the severity of the disease being treated, the route of administration, etc. For example, the severity of the disease can be assessed in part by standard prognostic assessment methods. Furthermore, the dosage and possible dosing frequency will also vary based on the individual patient's age, weight, and response.

[0368] Other medications and treatments

[0369] The bispecific antigen-binding molecule of the present invention, capable of bivalently binding to 4-1BB and monovalently binding to target cell antigens, can be administered in combination with one or more other agents in therapy. For example, the bispecific antigen-binding molecule of the present invention can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" includes any agent that can be administered to treat symptoms or diseases in an individual requiring such treatment. Such additional therapeutic agents may contain any active ingredient suitable for the specific indication being treated, preferably active ingredients having complementary activities that do not adversely affect each other. In some aspects, the additional therapeutic agent is another anticancer agent, such as a cytotoxic agent, a chemotherapeutic agent, or an antiangiogenic agent.

[0370] In one aspect, the bispecific antigen-binding molecule of the present invention, capable of bivalently binding to 4-1BB and monovalently binding to target cell antigens, can be administered in combination with an agent that blocks PD-L1 / PD-1 interaction. Specifically, the agent blocking PD-L1 / PD-1 interaction is an anti-PD-L1 antibody or an anti-PD1 antibody. More specifically, the agent blocking PD-L1 / PD-1 interaction is selected from the group consisting of atezolizumab, durvalumab, pembrolizumab, and nivolumab. In a particular aspect, the agent blocking PD-L1 / PD-1 interaction is atezolizumab.

[0371] Other such agents are appropriately combined in amounts effective for the intended purpose. The effective amount of these other agents depends on the amount of fusion protein used, the type of disease or treatment, and other factors discussed above. Bispecific antigen-binding molecules are generally used at the same dosage and route of administration as described herein, or at about 1% to 99% of the dosage described herein, or at any dosage and route determined empirically / clinically to be appropriate.

[0372] The aforementioned combination therapies cover combined administration (where two or more therapeutic agents are contained in the same composition or separate compositions) and separate administration, in which case the administration of the bispecific antigen-binding molecule of the present invention may occur before, simultaneously with and / or after the administration of additional therapeutic agents and / or adjuvants.

[0373] Products

[0374] In another aspect of the invention, an article is provided containing a substance that can be used to treat, prevent, and / or diagnose the aforementioned conditions. The article includes a container and a label or package insert on or associated with said container. Suitable containers include, for example, bottles, vials, syringes, intravenous (IV) solution bags, etc. The container can be formed from a variety of materials such as glass or plastic. The container contains a composition, alone or in combination with another composition effective for treating, preventing, and / or diagnosing said conditions, and the container may have a sterile inlet (e.g., the container may be an IV solution bag or a vial with a stopper that can be punctured by a hypodermic needle). At least one active agent in the composition is a bispecific antigen-binding molecule of the present invention.

[0375] A label or packaging insert indicates that the composition is intended for the treatment of a selected condition. Furthermore, the article may include: (a) a first container having a composition contained therein, wherein the composition comprises the antigen-binding molecule of the present invention containing a 4-1BBL trimer; and (b) a second container having a composition contained therein, wherein the composition comprises additional cytotoxic agents or other therapeutic agents. The article in this embodiment of the invention may further include a packaging insert indicating that the composition is intended for the treatment of a specific condition.

[0376] Alternatively or additionally, the article may also include a second (or third) container comprising a pharmaceutically acceptable buffer solution, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. The article may also include other substances required from a commercial and user perspective, including additional buffers, diluents, filters, needles, and syringes.

[0377] Table B (Sequence):

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393] General information on the nucleotide sequences of the light and heavy chains of human immunoglobulins is given in: Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991). The amino acids of the antibody chains are numbered and referenced according to the EU numbering system defined above by Kabat (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

[0394] ***

[0395] Example

[0396] The following are examples of the methods and compositions of the present invention. It should be understood that various other embodiments may be practiced given the general description provided above.

[0397] Recombinant DNA technology

[0398] Use standard methods to manipulate DNA, as described in Sambrook et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Use molecular biology reagents according to the manufacturer's instructions. General information about the nucleotide sequences of the light and heavy chains of human immunoglobulins is given in the following reference: Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242.

[0399] DNA sequencing

[0400] DNA sequences were determined using double-strand sequencing.

[0401] Gene synthesis

[0402] The desired gene segment is generated by PCR using an appropriate template, or synthesized from synthetic oligonucleotides and PCR products by Geneart AG (Regensburg, Germany) through automated gene synthesis. When the exact gene sequence is unavailable, oligonucleotide primers are designed based on the sequence of the closest homolog, and the gene is isolated from RNA derived from a suitable tissue by RT-PCR. The gene segment flanked by a single restriction endonuclease cleavage site is cloned into a standard cloning / sequencing vector. Plasmid DNA is purified from transformed bacteria, and its concentration is determined by UV spectroscopy. The DNA sequence of the subcloned gene fragment is confirmed by DNA sequencing. Gene segments with appropriate restriction sites are designed to allow subcloning into the corresponding expression vector. All constructs are designed with a 5' DNA sequence encoding a leader peptide that targets a protein secreted by eukaryotic cells.

[0403] Cell culture technology

[0404] Use standard cell culture techniques as described in Current Protocols in Cell Biology (2000), Bonifacino, JS, Dasso, M., Harford, JB, Lippincott-Schwartz, J., and Yamada, KM (eds.), John Wiley & Sons, Inc.

[0405] Protein purification

[0406] Proteins were purified from filtered cell culture supernatant according to standard protocols. Briefly, antibodies were applied to a Protein A agarose column (GE Healthcare) and washed with PBS. Antibody elution was achieved at pH 2.8, followed by immediate sample neutralization. Aggregated proteins were separated from monomeric antibodies by size exclusion chromatography (Superdex 200, GE Healthcare) in PBS or in 20 mM histidine, 150 mM NaCl, pH 6.0. Monomeric antibody fractions were pooled and concentrated (if necessary) using a centrifuge such as a MillIPOREAmicon Ultra (30 MWCO), then frozen and stored at -20°C or -80°C. Fractions of the sample were provided for subsequent protein analysis and analytical characterization, for example, by SDS-PAGE, size exclusion chromatography (SEC), or mass spectrometry.

[0407] SDS-PAGE

[0408] Use according to the manufacturer's instructions. Pre-formed gel system (Invitrogen). Specifically, use 10% or 4-12%. Bis-TRIS pre-prepared gel (pH 6.4) and MES (reducing gel, with...) Antioxidant electrophoresis buffer additives or MOPS (non-reducing gel) electrophoresis buffer.

[0409] Analytical size exclusion chromatography

[0410] Size exclusion chromatography (SEC) was performed by HPLC to determine the aggregate and oligomeric states of the antibody. Briefly, the protein A purified antibody was applied to a Tosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH2PO4 / K2HPO4, pH 7.5 on an Agilent HPLC 1100 system, or to a Superdex 200 column (GE Healthcare) in 2×PBS on a Dionex HPLC system. The eluted protein was quantified by UV absorbance and peak area integration. BioRad gel filtration standard 151-1901 was used as a standard.

[0411] Example 1

[0412] Preparation, purification, and characterization of bispecific antibodies with bivalent binding to 4-1BB and monovalent / bivalent binding to FAP.

[0413] 1.1 Generation of bispecific antibodies with bivalent binding to 4-1BB and monovalent or bivalent binding to FAP

[0414] Methods for preparing bispecific agonistic 4-1BB antibodies that bind bivalently to 4-1BB and monovalently or bivalently to FAP include: Figure 1A and 1B As shown. The FAP conjugate (clone 4B9, generated and prepared as described in WO 2012 / 020006 A2, which is incorporated herein by reference) and the 4-1BB conjugate (anticalin, generated and prepared as described in WO 2016 / 177802) were used to prepare... Figure 1A and 1B The molecule described in [the document], where TA1 is FAP. According to the method described in International Patent Application Publication No. WO2012 / 130831A1, mutations in Pro329Gly, Leu234Ala, and Leu235Ala are introduced into the Fc constant region of the heavy chain to eliminate binding to the Fcγ receptor.

[0415] The variable regions of the heavy and light chain DNA sequences encoding the FAP(4B9) conjugate were subcloned together with the constant heavy chain of the well or the constant light chain of human IgG1 within the frame.

[0416] The following clones have bivalent FAP-binding constructs: each containing two heavy chains of the VH(FAP)-Fc(huIgG1)-(G4S)3 linker-4-1BB-binding lipid transporter and two light chains of VL(FAP)-Cκ. The amino acid sequences of the bispecific bivalent 2+2 anti-FAP, anti-4-1BB huIgG1 PGLALA are shown in Table 1.

[0417] The following clones possess a construct with monovalent binding to FAP: a heavy chain containing a VH(FAP)Fc protrusion (hu IgG1)-(G4S)3 linker 4-1BB-binding lipid transporter, a heavy chain Fc pore (hu IgG1)-(G4S)3 linker 4-1BB-binding lipid transporter, and a light chain containing VL(FAP)-Cκ. The combination of an Fc protrusion heavy chain containing the S354C / T366W mutation and an Fc pore heavy chain containing the Y349C / T366S / L368A / Y407V mutation, along with an anti-FAP light chain, allows for the generation of a heterodimer comprising two 4-1BB-binding lipid transporters. The amino acid sequences of the bispecific monovalent 2+1 anti-FAP, anti-4-1BB huIgG1 PGLALA are shown in Table 2.

[0418] Table 1: Amino acid sequences of mature bispecific bivalent 2+2 anti-FAP and anti-4-1BB lipotransporter huIgG1 PGLALA antigen-binding molecules

[0419]

[0420] Table 2: Amino acid sequences of mature bispecific monovalent 1+2 anti-FAP and anti-4-1BB lipid transport protein huIgG1PGLALA antigen-binding molecules

[0421]

[0422] Bispecific antibodies were generated via transient transfection of HEK293 EBNA cells. Cells were centrifuged and the medium was replaced with preheated CD CHO medium. The expression vector was mixed in CD CHO medium, PEI was added, the solution was vortexed, and incubated at room temperature for 10 minutes. Then, cells were mixed with the DNA / PEI solution, transferred to shake flasks, and incubated at 37°C for 3 hours under a 5% CO2 atmosphere. After incubation, Excell medium with supplementation was added. One day after transfection, 12% feed was added. The cell supernatant was harvested after 7 days and purified using standard methods. For constructs a) and b), cells were transfected with the corresponding expression vector at a 1:1 or 1:1:1 ratio, respectively.

[0423] Proteins were purified from filtered cell culture supernatant according to a standard protocol. In short, Fc-containing proteins were purified from the cell culture supernatant using affinity chromatography of protein A. Elution was achieved at pH 3.0, followed by immediate sample neutralization. The proteins were concentrated, and aggregated proteins were separated from monomeric proteins by size exclusion chromatography in 20 mM histidine and 140 mM sodium chloride (pH 6.0).

[0424] The protein concentration of the purified construct was determined by measuring the optical density (OD) at 280 nm using the molar extinction coefficient calculated based on the amino acid sequence, as per Pace et al., Protein Science, 1995, 4, 2411-1423. Protein purity and molecular weight were analyzed by CE-SDS using LabChip GXII in the presence and absence of reducing agents. Aggregate content was determined by HPLC using an analytical size exclusion column (TSKgel G3000SW XL) equilibrated in running buffer (25 mM K₂HPO₄, 125 mM NaCl, 200 mM L-arginine monohydrochloride (pH 6.7)) at 25 °C.

[0425] Table 3 summarizes the yield and final monomer content of bispecific FAP(4B9) targeting 4-1BB-binding antigen-binding molecules.

[0426] Table 3: Biochemical analysis of bispecific 4-1BB binding antigen-binding molecules

[0427]

[0428] For comparison, a 2+2FAP(4B9)x 4-1BB lipid transporter huIgG4 SP molecule containing the amino acid sequences of SEQ ID NO:69 and SEQ ID NO:70 was also produced, along with a non-targeted 2+2DP47 x 4-1BB lipid transporter huIgG4 SP control molecule containing SEQ ID NO:71 and SEQ ID NO:72.

[0429] 1.2 Surface plasmon resonance-mediated bonding with divalent 4-1BB and monovalent or divalent FAP Functional characterization of combined bispecific and trispecific antibodies

[0430] The ability to simultaneously bind human 4-1BB Fc(kih) and human FAP was evaluated using surface plasmon resonance (SPR). All SPR experiments were performed using a Biacore T200 at 25 °C with HBS-EP as the run buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, Biacore, Freiburg / Germany). Biotinylated human 4-1BB Fc(kih) was directly coupled to the flow cell of the streptavidin (SA) sensor chip. The maximum immobilization level used was 500 resonance units (RU).

[0431] Bispecific FAP-targeting anti-4-1BB lipotransporter protein was injected through a flow cell at a concentration of 200 nM at a flow rate of 30 μL / min for 90 seconds, with dissociation set to 0 seconds. Human FAP, as a second analyte, was injected through the flow cell at a concentration of 500 nM at a flow rate of 30 μL / min for 90 seconds. Figure 2A Dissociation was monitored for 120 seconds. Bulk refractive index differences were corrected by subtracting the responses obtained in a reference flow cell where the protein was not immobilized.

[0432] As in Figure 2B and 2C As can be seen from the figure, the bispecific FAP-targeting anti-4-1BB lipid transport protein can bind to both human 4-1BB and human FAP simultaneously.

[0433] Example 2

[0434] Preparation, purification, and characterization of bispecific antibodies with bivalent binding to 4-1BB and monovalent / bivalent binding to HER2.

[0435] 2.1 Generation of bispecific antibodies with bivalent binding to 4-1BB and monovalent or bivalent binding to HER2

[0436] Methods for preparing bispecific agonistic 4-1BB antibodies that bind bivalently to 4-1BB and monovalently or bivalently to HER2 include: Figure 1A and 1B As shown. HER2 conjugate (corresponding to trastuzumab) and 4-1BB conjugate (lipotransferase, generated and prepared as described in WO 2016 / 177802) are used to prepare... Figure 1A and 1B The molecule described in [the document], where TA1 is HER2. According to the method described in International Patent Application Publication No. WO2012 / 130831A1, mutations in Pro329Gly, Leu234Ala, and Leu235Ala are introduced into the Fc constant region of the heavy chain to eliminate binding to the Fcγ receptor.

[0437] The variable regions of the heavy and light chain DNA sequences encoding the FAP(4B9) conjugate were subcloned together with the constant heavy chain of the well or the constant light chain of human IgG1 within the frame.

[0438] The following clones have bivalent FAP-binding constructs: each containing two heavy chains of the VH(HER2)-Fc(huIgG1)-(G4S)3 linker-4-1BB-binding lipid transporter and two light chains of VL(HER2)-Cκ. The amino acid sequences of the bispecific bivalent 2+2 anti-HER2, anti-4-1BB huIgG1 PGLALA are shown in Table 4.

[0439] The following clones possess a construct with monovalent binding to FAP: a heavy chain containing a VH(HER2)Fc protrusion (hu IgG1)-(G4S)3 linker 4-1BB-binding lipid transporter, a heavy chain Fc pore (hu IgG1)-(G4S)3 linker 4-1BB-binding lipid transporter, and a light chain containing VL(HER2)-Cκ. The combination of an Fc protrusion heavy chain containing the S354C / T366W mutation and an Fc pore heavy chain containing the Y349C / T366S / L368A / Y407V mutation, along with an anti-HER2 light chain, allows for the generation of a heterodimer comprising two 4-1BB-binding lipid transporters. The amino acid sequences of the bispecific monovalent 2+1 anti-HER2, anti-4-1BB huIgG1PGLALA are shown in Table 5.

[0440] Table 4: Amino acid sequences of mature bispecific bivalent 2+2 anti-HER2 and anti-4-1BB lipotransporter huIgG1PGLALA antigen-binding molecules

[0441]

[0442] Table 5: Amino acid sequences of mature bispecific monovalent 1+2 anti-HER2 and anti-4-1BB lipid transport protein huIgG1PGLALA antigen-binding molecules

[0443]

[0444]

[0445] Bispecific antibodies were produced and purified as described in Example 1.

[0446] Table 6 summarizes the yield and final monomer content of bispecific HER2 targeting 4-1BB antigen-binding molecules.

[0447] Table 6: Biochemical analysis of bispecific 4-1BB binding antigen-binding molecules

[0448]

[0449] For comparison, the previously described fusion peptide 2+2HER2(TRAS)-anticalin-4-1BB human IgG4 SP (WO2016 / 177802), containing the amino acid sequences of SEQ ID NO:73 and SEQ ID NO:74, was also prepared.

[0450] 2.2 Surface plasmon resonance-mediated synthesis of a compound with divalent 4-1BB and monovalent or divalent HER2 Functional characterization of valence-binding bispecific and trispecific antibodies

[0451] The ability to simultaneously bind human 4-1BB Fc(kih) and human HER2 was evaluated using surface plasmon resonance (SPR). All SPR experiments were performed using a Biacore T200 at 25 °C with HBS-EP as the run buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, Biacore, Freiburg / Germany). Biotinylated human 4-1BB Fc(kih) was directly coupled to the flow cell of the streptavidin (SA) sensor chip. The highest immobilization level used was 500 resonance units (RU).

[0452] Bispecific HER2-targeting anti-4-1BB lipotransferase was injected through the flow cell at a concentration of 200 nM at a flow rate of 30 μL / min for 90 seconds, with dissociation set to 0 seconds. Human FAP, as a second analyte, was injected through the flow cell at a concentration of 500 nM at a flow rate of 30 μL / min for 90 seconds. Figure 3A Dissociation was monitored for 120 seconds. Bulk refractive index differences were corrected by subtracting the responses obtained in a reference flow cell where the protein was not immobilized.

[0453] As in Figure 3B As can be seen from the figure, both the bispecific HER2-targeting anti-4-1BB lipid transport protein and human 4-1BB can bind to human HER2 simultaneously.

[0454] Example 3

[0455] Functional characterization of FAP targeting 4-1BB lipid transporter antigen-binding molecules

[0456] 3.1 Binding with cell lines expressing human FAP

[0457] To facilitate binding to human fibroblast activation protein (FAP) expressed on the cell surface, NIH / 3T3-huFAP clone 19 cells were used. NIH / 3T3-huFAP clone 19 was formed by transfecting mouse embryonic fibroblast NIH / 3T3 cells (ATCC CRL-1658) with the expression plasmid pETR4921 encoding human FAP in the presence of the CMV promoter. Cells were maintained in DMEM (GIBCO, Life Technologies, catalog 16000-044, batch 941273, γ-ray irradiated, mycoplasma-free, heat-inactivated) supplemented with fetal bovine serum (FBS, GIBCO, catalog 16000-044, batch 941273, provided by Life Technologies, γ-ray irradiated, mycoplasma-free, heat-inactivated), 2 mM L-alanyl-L-glutamine dipeptide (Glutqa-MAX-I, GIBCO, catalog 35050-038, provided by Life Technologies), and 1.5 μg / mL puromycin (InvivoGen, catalog ant-pr-5). In the binding assay, 2 × 10⁶ cells were... 5NIH / 3T3-huFAP clone 19 cells were added to each well of a 96-well round-bottom suspension cell plate (Greiner bio-one, Cellstar, catalog number 650185). Cells were washed once with 200 μL of DPBS, and the pellet was resuspended in 100 μL / well of 4°C cold DPBS buffer (containing 1:5000 dilution of the immobilizable active dye eFluor 450 (eBioscience, catalog number 65 0863 18)). The plate was incubated at 4°C for 30 minutes, followed by a wash with 200 μL of 4°C cold DPBS buffer. Cells were then resuspended in 50 μL / well of 4°C cold FACS buffer containing different titration concentrations (starting at 300 nM, diluted 1:6 in eight dilution steps) of bispecific divalent 2+2 anti-FAP, anti-4-1BB lipotransferrin huIgG1 PGLALA antigen-binding molecule (referred to as 2+2), or bispecific monovalent 1+2 anti-FAP, anti-4-1BB lipotransferrin huIgG1 PGLALA antigen-binding molecule (referred to as 1+2), or a control molecule, and incubated in the dark at 4°C for 1 hour. After washing four times with 200 μL / well of DPBS, cells were stained at 4°C for 30 minutes with 50 μL / well of 4°C cold FACS buffer containing 2.5 μg / mL of PE-conjugated AffiniPure anti-human IgG Fc fragment-specific goat F(ab')2 fragment (Jackson ImmunoResearch, catalog number 109-116-098). Cells were washed twice with 200 μL of DPBS buffer at 4°C and then resuspended in 50 μL / well of DPBS containing 1% formaldehyde for fixation. Cells from the same or second day were resuspended in 100 μL of FACS buffer and collected using a MACSQuant Analyzer 10 (Miltenyi Biotec) or Canto II (BD). Data were analyzed using FlowJo 10.4.2 (FlowJo LLC), Microsoft Office Excel Professional 2010 (Microsoft Software Inc.), and GraphPad Prism (GraphPad Software Inc.).

[0458] like Figure 4As shown, the bispecific bivalent 2+2 anti-FAP, anti-4-1BB huIgG1 PGLALA (referred to as FAP(4B9)x 4-1BB lipotransferase huIgG1 PG LALA 2+2) binds with similar affinity to FAP(4B9)huIgG1 PG LALA, since both molecules are bivalent to FAP. Therefore, the C-terminal fusion of the 4-1BB-binding lipotransferase does not affect binding to FAP. The bispecific bivalent 2+2 anti-FAP, anti-4-1BB huIgG4 SP molecule (FAP(4B9)x 4-1BB lipotransferase huIgG4 SP 2+2) exhibits a lower gMFI than other bivalent FAP-binding molecules. This can be explained by the different isotypes of the Fc fragment. Because we used a polyclonal anti-human Fc fragment-specific goat IgG F(ab`)2 fragment, the epitopes in the Fc portion may differ, resulting in less binding of the second detection fragment and a lower gMFI. The bispecific monovalent 1+2 anti-FAP, anti-4-1BB huIgG1 PG LALA molecule (FAP(4B9) x 4-1BB lipotransporter huIgG1 PG LALA 1+2, solid black triangles and lines) showed a higher gMFI than the bispecific divalent 2+2 anti-FAP, anti-4-1BB huIgG1 PG LALA (referred to as FAP(4B9) x 4-1BB lipotransporter huIgG1 PG LALA 2+2). This can be explained by its monovalent binding to FAP, resulting in a higher occupancy rate on the cell surface, since one molecule occupies only one FAP monomer instead of two. Because FAP(4B9) exhibits very high affinity, loss of affinity (e.g., EC50) could not be detected in this binding assay. 50 (Increase in value). EC of a single binding curve 50 The values ​​and the area under the curve (AUC) are listed in Tables 7 and 8, respectively.

[0459] Table 7: ECG binding curves of FAP-expressing cell line NIH / 3T3-huFAP clone 19 50 Values ​​such as Figure 4 As shown

[0460]

[0461] Table 8: Area under the curve (AUC) of the binding curve with FAP-expressing cell line NIH / 3T3-huFAP clone 19. Figure 4 As shown

[0462]

[0463] 3.2 Binding with the reporter cell line Jurkat-hu4-1BB-NFκB-luc2 expressing human 4-1BB

[0464] To enable binding to human 4-1BB (CD137) expressed on the cell surface, the Jurkat-hu4-1BB-NFkB-luc2 reporter cell line (Promega, Germany) was used. Cells were maintained as suspension cells in RPMI 1640 medium (GIBCO, catalog 42401-042, provided by Life Technologies), supplemented with 10% (v / v) fetal bovine serum (FBS, GIBCO, catalog 16000-044, batch 941273, gamma-ray irradiated, mycoplasma-free, heat-inactivated), 2 mM L-alanyl-L-glutamine dipeptide (Glutqa-MAX-I, GIBCO, catalog 35050-038, provided by Life Technologies), 1 mM sodium pyruvate (SIGMA-Aldrich, catalog S8636), and 1% (v / v) MEM-nonessential amino acid solution 100x (SIGMA-Aldrich, catalog M7145), 600 μg / ml. G-418 (Roche, catalog number 04727894001), 400 μg / ml hygromycin B (Roche, catalog number 10843555001), and 25 mM HEPES (Sigma LifeSience, catalog number H0887-100 mL). In the binding assay, 2 × 10⁻⁶ 5Jurkat-hu4-1BB-NFkB-luc2 was added to each well of a 96-well round-bottom suspension cell plate (Greiner bio-one, Cellstar, catalog number 650185). Cells were washed once with 200 μL of DPBS, and the pellet was resuspended in 100 μL / well of 4°C cold DPBS buffer (containing 1:5000 dilution of the immobilizable active dye eFluor 450 (eBioscience, catalog number 65 0863 18)). The plate was incubated at 4°C for 30 minutes, followed by a wash with 200 μL of 4°C cold DPBS buffer. Cells were then resuspended in 50 μL / well of 4°C cold FACS buffer containing different titration concentrations (starting at 300 nM, diluted 1:6 in eight dilution steps) of bispecific divalent 2+2 anti-FAP, anti-4-1BB huIgG1 PGLALA (referred to as 2+2), or bispecific monovalent 1+2 anti-FAP, anti-4-1BB huIgG1 PGLALA (referred to as 1+2), or a control molecule, and incubated in the dark at 4°C for 1 hour. After washing four times with 200 μL / well of DPBS, cells were stained at 4°C for 30 minutes with 50 μL / well of 4°C cold FACS buffer containing 2.5 μg / mL PE-conjugated AffiniPure anti-human IgG Fc fragment-specific goat F(ab')2 fragment (Jackson ImmunoResearch, catalog number 109-116-098). Cells were washed twice with 200 μL of FACS buffer at 4°C and then resuspended in 50 μL / well of DPBS containing 1% formaldehyde for fixation. Cells from the same or second day were resuspended in 100 μL of FACS buffer and collected using a MACSQuant Analyzer 10 (Miltenyi Biotec) or CantoII (BD). Data were analyzed using FlowJo 10.4.2 (FlowJo LLC), Microsoft Office Excel Professional 2010 (Microsoft Software Inc.), and GraphPad Prism (GraphPad Software Inc.).

[0465] like Figure 5 As shown, all anti-4-1BB lipid transporter bispecific molecules bind with similar affinity to the human 4-1BB-expressing transgenic human T-cell lymphoma cell line Jurkat-hu4-1BB-NFkB-luc2. This is in contrast to their binding affinity to FAP-expressing cells during binding to human 4-1BB. Figure 4Unlike FAP, we did not observe a difference in binding affinity (gMFI) between molecules containing Fc-huIgG1 PG LALA or Fc-huIgG4SP. This could involve lower expression levels of 4-1BB compared to FAP, and therefore the gMFI values ​​would be much lower, making such assays insufficiently sensitive to detect the difference. EC50 of the binding curves 50 The values ​​and AUC are listed in Tables 9 and 10, respectively.

[0466] Table 9: Figure 5 The EC50 curves showing the binding of human 4-1BB expressed in cells are shown. 50 Summary of Values

[0467]

[0468] Table 10: Summary of the area under the curve (AUC) values ​​of the binding curves of human 4-1BB expressed in cells. Figure 5 As shown

[0469]

[0470] 3.3 In the reporter cell line Jurkat-hu4-1BB-NFκB expressing human 4-1BB and NFκB-luciferase reporter genes NF-κB activation in B-luc2

[0471] The agonistic binding of the 4-1BB (CD137) receptor to its ligand (4-1BBL) induces downstream 4-1BB signaling by activating nuclear factor kappa B (NFkB), and promotes the survival and activity of CD8 T cells (Lee HW, Park SJ, Choi BK, Kim HH, Nam KO, Kwon BS. 4-1BB promotes the survival of CD8(+) T lymphocytes by increasing expression of Bcl-x(L) and Bfl-1. J Immunol 2002; 169:4882-4888). To monitor this NFκB activation mediated by bispecific bivalent 2+2 anti-FAP, anti-4-1BB huIgG1 PGLALA molecules (referred to as 2+2), or bispecific monovalent 1+2 anti-FAP, anti-4-1BB huIgG1 PGLALA molecules (referred to as 1+2), the Jurkat-hu4-1BB-NFκB-luc2 reporter cell line was purchased from Promega (Germany). Cell culture methods were as described above (in conjunction with the human 4-1BB-expressing reporter cell line Jurkat-hu4-1BB-NFκB-luc2). For the assay, cells were harvested and resuspended in RPMI 1640 assay medium supplemented with 10% (v / v) FBS and 1% (v / v) GlutaMAX-I. 10 μl of the medium containing 2 × 10⁻⁶ cells was used. 3 Jurkat-hu4-1BB-NFκB-luc2 reporter cells were transferred to each well of a sterile white 384-well flat-bottomed tissue culture plate with a cap (Corning, catalog number 3826). 10 μL of assay medium containing titrated concentrations of bispecific bivalent 2+2 anti-FAP, anti-4-1BB huIgG1 PGLALA (referred to as 2+2), or bispecific monovalent 1+2 anti-FAP, anti-4-1BB huIgG1 PGLALA (referred to as 1+2), or a control molecule was added. Finally, 10 μL of assay medium alone or containing 1×10⁻⁶ cells was provided. 4Assay medium was prepared for FAP-expressing cells (human melanoma cell line WM-266-4 (ATCC CRL-1676) or NIH / 3T3-huFAP clone 19 (as described above), and the plates were incubated in a cell culture incubator at 37°C and 5% CO2 for 6 hours. 6 μl of freshly thawed One-Glo luciferase assay solution (Promega, catalog number E6110) was added to each well, and the luminescence intensity was immediately measured using a Tecan microplate reader (integration time 500 ms, no filter, acquiring signals at all wavelengths). Data were analyzed using Microsoft Office Excel Professional 2010 (Microsoft Software Inc.) and GraphPad Prism (GraphPad Software Inc.).

[0472] like Figure 6A As shown, in the absence of FAP-expressing cells, no molecule was able to induce strong human 4-1BB receptor activation in the urkat-hu4-1BB-NFκB-luc2 reporter cell line, leading to NFκB activation and consequently luciferase expression. In the presence of FAP-expressing cells (such as WM-266-4)... Figure 6B Human melanoma cell line, intermediate FAP expression) or NIH / 3T3-huFAP clone 19 ( Figure 6CHuman FAP transgenic mouse fibroblast cell line), bispecific bivalent 2+2 anti-FAP, anti-4-1BB huIgG1 PGLALA antigen-binding molecule (referred to as FAP(4B9)x 4-1BB lipotransporter huIgG1PG LALA 2+2, hollow downward black triangle and dashed line), or bispecific monovalent 1+2 anti-FAP, anti-4-1BB huIgG1 PGLALA antigen-binding molecule (referred to as FAP(4B9)x 4-1BB lipotransporter huIgG1 PG LALA 1+2, solid black triangle and line) or bispecific control molecule, bispecific bivalent 2+2 anti-FAP, anti-4-1BB huIgG4 PGLALA antigen-binding molecule (referred to as FAP(4B9)x 4-1BB lipotransporter huIgG4 SP) Crosslinking (2+2, semi-solid black hexamer and dashed line) resulted in a strong increase in NFκB-activated luciferase activity in the Jurkat-hu4-1BB-NFκB-luc2 reporter cell line. The bispecific monovalent 1+2 anti-FAP, anti-4-1BB huIgG1 PGLALA antigen-binding molecule (referred to as FAP(4B9)x 4-1BB lipid carrier huIgG1 PG LALA 1+2, solid black triangle and line) showed the best performance by the highest area under the curve (AUC) of the activation curve. A lower 1:2 ratio of tumor target binding to effector cell target binding, such as a 1:2 ratio of FAP binding to 4-1BB binding, appears to lead to higher occupancy density, thus resulting in dense crosslinking of the 4-1BB agonist on effector cells and ultimately stronger downstream signaling of the 4-1BB receptor. EC50 of the activation curve... 50 The values ​​and the area under the curve (AUC) are listed in Table 11 and Table 12, respectively.

[0473] Table 11: Figure 6B and 6C EC of the activation curve shown 50 value

[0474]

[0475] Table 12: As Figure 6B and 6C Summary of the area under the curve (AUC) values ​​of the activation curves shown

[0476]

[0477] Example 4

[0478] Functional characterization of HER2 targeting 4-1BB lipid transporter antigen-binding molecules

[0479] 4.1 Binding with cell lines expressing human HER2

[0480] To bind to HER2 expressed on the cell surface, the human gastric cancer cell line NCI-N87 (ATCC CRL-5822) and the human breast cancer cell line KPL4 (Kawasaki Medical School) were used. NCI-N87 cells were cultured as adherent cells in RPMI 1640 medium (Life Technologies GIBCO, catalog 42401-042), which provided 10% (v / v) FBS (Life Technologies GIBCO, catalog 16000-044, batch 941273, gamma-irradiated mycoplasma-free, heat-inactivated at 56°C for 35 min) and 2 mM L-alanyl-L-glutamine (GlutaMAX-I, GIBCO, Invitrogen, catalog 35050-038). KPL4 cells were cultured as adherent cells in DMEM medium (GIBCO, Life Technologies, catalog number 42430082) provided with 10% (v / v) FBS and 2 ml L-alanyl-L-glutamine. In the binding assay, 2 × 10⁻⁶ cells were... 5One NCI-N87 and one KPL4 were added to each well of a 96-well round-bottom suspension cell plate (Greiner bio-one, Cellstar, catalog number 650185). Cells were washed once with 200 μL of DPBS, and the pellet was resuspended in 100 μL / well of 4°C cold DPBS buffer (containing 1:5000 dilution of the immobilizable active dye eFluor 450 (eBioscience, catalog number 65 0863 18)). The plate was incubated at 4°C for 30 minutes, and then washed once with 200 μL of 4°C cold DPBS buffer. Cells were then resuspended in 50 μL / well of 4°C cold FACS buffer containing different titration concentrations (starting at 300 nM, diluted 1:6 in eight dilution steps) of bispecific divalent 2+2 anti-HER2, anti-4-1BB huIgG1 PGLALA antigen-binding molecules (referred to as 2+2), or bispecific monovalent 1+2 anti-HER2, anti-4-1BB huIgG1 PGLALA antigen-binding molecules (referred to as 1+2), or a control molecule, and incubated in the dark at 4°C for 1 hour. After washing four times with 200 μL / well of DPBS, cells were stained at 4°C for 30 minutes with 50 μL / well of 4°C cold FACS buffer containing 2.5 μg / mL PE-conjugated AffiniPure anti-human IgG Fc fragment-specific goat F(ab')2 fragment (Jackson ImmunoResearch, catalog number 109-116-098). Cells were washed twice with 200 μL of DPBS buffer at 4°C and then resuspended in 50 μL / well of DPBS containing 1% formaldehyde for fixation. Cells from the same or second day were resuspended in 100 μL of FACS buffer and collected using a MACSQuant Analyzer 10 (Miltenyi Biotec) or CantoII (BD). Data were analyzed using FlowJo 10.4.2 (FlowJo LLC), Microsoft Office Excel Professional 2010 (Microsoft Software Inc.), and GraphPad Prism (GraphPad Software Inc.).

[0481] like Figure 7A and 7BAs shown, the bispecific bivalent 2+2 anti-HER2, anti-4-1BB huIgG1 PGLALA antigen-binding molecule (referred to as HER2(TRAS)x 4-1BB lipotransferase huIgG1 PG LALA 2+2) binds with a similar affinity to HER2(TRAS)huIgG1PG LALA because both molecules are bivalently bound to HER2. Therefore, the C-terminal fusion of the 4-1BB-binding lipotransferase does not affect binding to HER2. The bispecific bivalent 2+2 anti-HER2, anti-4-1BB huIgG4 SP molecule (HER2(TRAS)x 4-1BB lipotransferase huIgG4 SP 2+2) exhibits a lower MFI than other bivalent HER2-binding molecules. This can be explained by the different isotypes of the Fc fragment. Because we used a polyclonal anti-human Fc fragment-specific goat IgG F(ab`)2 fragment, the epitopes in the Fc portion may differ, resulting in less binding of the second detection fragment and a lower gMFI. The bispecific monovalent 1+2 anti-HER2, anti-4-1BB huIgG1 PG LALA molecule (HER2(TRAS) x 4-1BB lipotransporter huIgG1 PG LALA 1+2, solid black triangles and lines) showed a higher gMFI than the bispecific divalent 2+2 anti-HER2, anti-4-1BB huIgG1 PG LALA antigen-binding molecule (referred to as HER2(TRAS) x 4-1BB lipotransporter huIgG1 PG LALA 2+2). This can be explained by its monovalent binding to HER2, resulting in a higher occupancy rate on the cell surface, since one molecule binds only one HER2 receptor instead of two. EC50 of the single binding curve 50 The values ​​and the area under the curve (AUC) are listed in Tables 13 and 14, respectively.

[0482] Table 13: EC binding curves of HER2-expressing cell lines NCI-N87 and KPL4 50 Values ​​such as Figure 7A and 7B As shown

[0483]

[0484] Table 14: Area under the curve (AUC) values ​​of binding curves with HER2-expressing cell lines NCI-N87 and KPL4. Figure 7A and 7B As shown

[0485]

[0486] 4.2 Binding with the reporter cell line Jurkat-hu4-1BB-NFκB-luc2 expressing human 4-1BB

[0487] To enable binding to human 4-1BB (CD137) expressed on the cell surface, the Jurkat-hu4-1BB-NFkB-luc2 reporter cell line (Promega, Germany) was used. Cells were maintained as suspension cells in RPMI 1640 medium (GIBCO, catalog 42401-042, provided by Life Technologies), supplemented with 10% (v / v) fetal bovine serum (FBS, GIBCO, catalog 16000-044, batch 941273, gamma-ray irradiated, mycoplasma-free, heat-inactivated), 2 mM L-alanyl-L-glutamine dipeptide (GlutaMAX-I, GIBCO, catalog 35050-038, provided by Life Technologies), 1 mM sodium pyruvate (SIGMA-Aldrich, catalog S8636), and 1% (v / v) MEM-nonessential amino acid solution 100x (SIGMA-Aldrich, catalog M7145), 600 μg / ml. G-418 (Roche, catalog number 04727894001), 400 μg / ml hygromycin B (Roche, catalog number 10843555001), and 25 mM HEPES (SigmaLife Sience, catalog number H0887-100 mL). In the binding assay, 2 × 10⁻⁶ μg / ml of HEPES was used. 5Jurkat-hu4-1BB-NFkB-luc2 was added to each well of a 96-well round-bottom suspension cell plate (Greiner bio-one, Cellstar, catalog number 650185). Cells were washed once with 200 μL of DPBS, and the pellet was resuspended in 100 μL / well of 4°C cold DPBS buffer (containing 1:5000 dilution of the immobilizable active dye eFluor 450 (eBioscience, catalog number 65 0863 18)). The plate was incubated at 4°C for 30 minutes, followed by a wash with 200 μL of 4°C cold DPBS buffer. Cells were then resuspended in 50 μL / well of 4°C cold FACS buffer containing different titration concentrations (starting at 300 nM, diluted 1:6 in eight dilution steps) of bispecific divalent 2+2 anti-HER2, anti-4-1BB huIgG1 PGLALA antigen-binding molecules (referred to as 2+2), or bispecific monovalent 1+2 anti-HER2, anti-4-1BB huIgG1 PGLALA antigen-binding molecules (referred to as 1+2), or a control molecule, and incubated in the dark at 4°C for 1 hour. After washing four times with 200 μL / well of DPBS, cells were stained at 4°C for 30 minutes with 50 μL / well of 4°C cold FACS buffer containing 2.5 μg / mL PE-conjugated AffiniPure anti-human IgG Fc fragment-specific goat F(ab')2 fragment (Jackson ImmunoResearch, catalog number 109-116-098). Cells were washed twice with 200 μL of FACS buffer at 4°C and then resuspended in 50 μL / well of DPBS containing 1% formaldehyde for fixation. Cells from the same or second day were resuspended in 100 μL of FACS buffer and collected using a MACSQuantAnalyzer 10 (Miltenyi Biotec) or CantoII (BD). Data were analyzed using FlowJo 10.4.2 (FlowJo LLC), Microsoft Office Excel Professional 2010 (Microsoft Software Inc.), and GraphPad Prism (GraphPad Software Inc.).

[0488] like Figure 8 As shown, all anti-4-1BB lipid transporter bispecific molecules bind with similar affinity to the human 4-1BB-expressing transgenic human T-cell lymphoma cell line Jurkat-hu4-1BB-NFkB-luc2. This is in contrast to their binding affinity to HER2-expressing cells during binding to human 4-1BB. Figure 7A and7B Unlike HER2, we did not observe a difference in binding affinity (gMFI) between molecules containing Fc-huIgG1 PG LALA or Fc-huIgG4 SP. This may involve lower expression levels of 4-1BB compared to HER2, and therefore significantly lower gMFI values, making such assays insufficiently sensitive to detect the difference. EC50 of the binding curves... 50 The values ​​and AUC are listed in Tables 15 and 16, respectively.

[0489] Table 15: Figure 8 The EC50 curves showing the binding of human 4-1BB expressed in cells are shown. 50 Summary of Values

[0490]

[0491] Table 16: Area under the curve (AUC) values ​​of binding curves with HER2-expressing cell lines NCI-N87 and KPL4. Figure 8 As shown

[0492]

[0493] 4.3 In the reporter cell line Jurkat-hu4-1BB-NFκB expressing human 4-1BB and NFκB-luciferase reporter genes NF-κB activation in B-luc2

[0494] The agonistic binding of the 4-1BB (CD137) receptor to its ligand (4-1BBL) induces downstream 4-1BB signaling by activating nuclear factor kappa B (NFkB), and promotes the survival and activity of CD8 T cells (Lee HW, Park SJ, Choi BK, Kim HH, Nam KO, Kwon BS. 4-1BB promotes the survival of CD8(+) T lymphocytes by increasing expression of Bcl-x(L) and Bfl-1. J Immunol 2002; 169:4882-4888). To monitor this NFκB activation mediated by bispecific bivalent 2+2 anti-HER2, anti-4-1BB huIgG1 PGLALA antigen molecules (referred to as 2+2) or bispecific monovalent 1+2 anti-HER2, anti-4-1BB huIgG1 PGLALA antigen-binding molecules (referred to as 1+2), the Jurkat-hu4-1BB-NFκB-luc2 reporter cell line was purchased from Promega (Germany). Cell culture methods were as described above (with the conjugation of the human 4-1BB-expressing reporter cell line Jurkat-hu4-1BB-NFκB-luc2). For the assay, cells were harvested and resuspended in RPMI 1640 assay medium supplemented with 10% (v / v) FBS and 1% (v / v) GlutaMAX-I. 10 μl of the medium containing 2 × 10⁻⁶ cells was used. 3 Jurkat-hu4-1BB-NFκB-luc2 reporter cells were transferred to each well of a sterile white 384-well flat-bottomed tissue culture plate (Corning, catalog number 3826) with a top cap. 10 μL of assay medium containing titrated concentrations of bispecific bivalent 2+2 anti-HER2, anti-4-1BB huIgG1 PGLALA antigen-binding molecule (referred to as 2+2), or bispecific monovalent 1+2 anti-HER2, anti-4-1BB huIgG1 PGLALA antigen-binding molecule (referred to as 1+2), or a control molecule were added. Finally, 10 μL of assay medium alone, or containing 1 × 10⁻⁶ cells, was provided. 4 The assay medium for HER2-expressing cells KPL4, NCI-N87 (as described above), or SK-Br3 (human breast cancer, ATCC HTB-30) was prepared, and the plate was incubated in a cell culture incubator at 37°C and 5% CO2 for 6 hours. 6 μl of freshly thawed One-Glo luciferase assay solution (Promega, catalog number E6110) was added to each well, and the luminescence intensity was immediately measured using a Tecan microplate reader (integration time 500 ms, no filter, acquiring signals at all wavelengths).

[0495] like Figures 9A to 9D As shown, in the absence of cells expressing HER2 ( Figure 9A No molecule was able to induce strong human 4-1BB receptor activation in the Jurkat-hu4-1BB-NFkB-luc2 reporter cell line, leading to NFkB activation and consequently luciferase expression. In cells expressing HER2 (such as SK-Br3...), no molecule was able to induce strong human 4-1BB receptor activation, leading to NFkB activation and consequently luciferase expression. Figure 9B KPL4 Figure 9C ) and NCI-N87 ( Figure 9D In the case of bispecific monovalent 2+1 anti-HER2, anti-4-1BB huIgG1 PGLALA antigen-binding molecules (referred to as HER2(TRAS)x 4-1BB lipotransporter huIgG1 PG LALA 2+1, solid black triangle and solid line), crosslinking showed a good activation curve, with its height and / or EC 50 The value is correlated with the intensity of HER2 expression in cross-linked cells. Bispecific bivalent 2+2 anti-HER2 and anti-4-1BB huIgG1 PGLALA antigen-binding molecules (referred to as HER2(TRAS)x 4-1BB lipotransferrin huIgG1 PG LALA 2+2, solid black triangles and lines), and their control molecule HER2(TRAS)x 4-1BB lipotransferrin huIgG4 SP (semi-solid black hexamer and dashed line) all bind to HER2 bivalently and induce similar activation curves. Therefore, the activation of these two molecules is much lower than that of HER2(TRAS)x 4-1BB lipotransferrin huIgG1 PG LALA 2+1 (solid black triangles and lines). Based on the highest area under the curve (AUC) of the activation curve, the bispecific monovalent 1+2 anti-HER2, anti-4-1BB huIgG1 PGLALA (referred to as HER2(TRAS)x 4-1BB lipid carrier huIgG1 PG LALA 1+2, solid black triangle and line) showed the best performance. We believe that a lower 1:2 ratio of tumor target binding to effector cell target binding, such as the 1:2 ratio of the HER2 binding moiety to the 4-1BB binding moiety, leads to a higher occupancy density on tumor cells, thus resulting in dense cross-linking of 4-1BB agonists on effector cells, and ultimately leading to stronger downstream signaling of the 4-1BB receptor. EC50 of the activation curve 50 The values ​​and the area under the curve (AUC) are listed in Tables 17 and 18, respectively.

[0496] Table 17: As Figure 9B , 9C EC and the activation curve shown in 9D 50 Summary of Values

[0497]

[0498] Table 18: As Figure 9B , 9C The area under the curve (AUC) of the activation curve shown in Figure 9D

[0499] sequence list <110> Hofmeister Roche Ltd. <120> Bispecific antigen-binding molecules containing lipid transporter mutant proteins <130> P35474-WO <150> EP19169022.1 <151> 2019-04-12 <160> 129 <170> PatentIn version 3.5 <210> 1 <211> 178 <212> PRT <213> Homo sapiens <400> 1 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe Gln Gly Lys Trp Tyr 20 25 30 Val Val Gly Leu Ala Gly Asn Ala Ile Leu Arg Glu Asp Lys Asp Pro 35 40 45 Gln Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asn Val Thr Ser Val Leu Phe Arg Lys Lys Lys Cys Asp Tyr Trp Ile 65 70 75 80 Arg Thr Phe Val Pro Gly Cys Gln Pro Gly Glu Phe Thr Leu Gly Asn 85 90 95 Ile Lys Ser Tyr Pro Gly Leu Thr Ser Tyr Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Lys Val Ser Gln 115 120 125 Asn Arg Glu Tyr Phe Lys Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> 2 <211> 178 <212> PRT <213> Artificial sequence <220> <223> Lipid transporter mutant protein variant 13 <400> 2 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30 Val Val Gly Gln Ala Gly Asn Ile Arg Leu Arg Glu Asp Lys Asp Pro 35 40 45 Ile Lys Met Met Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asp Val Thr Met Val Lys Phe Asp Asp Lys Lys Cys Met Tyr Asp Ile 65 70 75 80 Trp Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Lys 85 90 95 Ile Lys Ser Phe Pro Gly His Thr Ser Ser Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Phe Val Phe Gln 115 120 125 Asn Arg Glu Glu Phe Tyr Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> 3 <211> 178 <212> PRT <213> Artificial Sequence <220> <223> Lipocalin mutant protein variant 12 <400> 3 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30 Val Val Gly Gln Ala Gly Asn Ile Lys Leu Arg Glu Asp Lys Asp Pro 35 40 45 Asn Lys Met Met Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asn Val Thr Gly Val Thr Phe Asp Asp Lys Lys Cys Thr Tyr Ala Ile 65 70 75 80 Ser Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Lys 85 90 95 Ile Lys Ser Phe Pro Gly His Thr Ser Ser Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Phe Val Phe Gln 115 120 125 Asn Arg Glu Glu Phe Tyr Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> 4 <211> 178 <212> PRT <213> Artificial sequence <220> <223> Lipid transport protein mutant variant 14 <400> 4 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30 Val Val Gly Gln Ala Gly Asn Ile Arg Leu Arg Glu Asp Lys Asp Pro 35 40 45 Asn Lys Met Met Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asp Val Thr Ala Val Ala Phe Asp Asp Lys Lys Cys Thr Tyr Asp Ile 65 70 75 80 Trp Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Lys 85 90 95 Ile Lys Ser Phe Pro Gly His Thr Ser Ser Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Phe Val Phe Gln 115 120 125 Asn Arg Glu Glu Phe Tyr Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> 5 <211> 178 <212> PRT <213> Artificial sequence <220> <223> Lipid transporter mutant protein variant 15 <400> 5 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30 Val Val Gly Gln Ala Gly Asn Ile Lys Leu Arg Glu Asp Lys Asp Pro 35 40 45 Asn Lys Met Met Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asp Val Thr Ala Val Ala Phe Asp Asp Lys Lys Cys Thr Tyr Asp Ile 65 70 75 80 Trp Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Lys 85 90 95 Ile Lys Ser Phe Pro Gly His Thr Ser Ser Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Phe Val Phe Gln 115 120 125 Asn Arg Glu Glu Phe Tyr Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> 6 <211> 175 <212> PRT <213> Artificial sequence <220> <223> Lipid transporter mutant protein variant 16 <400> 6 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30 Val Val Gly Gln Ala Gly Asn Ile Lys Leu Arg Glu Asp Ser Lys Met 35 40 45 Met Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr Asp Val Thr 50 55 60 Gly Val Ser Phe Asp Asp Lys Lys Cys Thr Tyr Ala Ile Met Thr Phe 65 70 75 80 Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Lys Ile Lys Ser 85 90 95 Phe Pro Gly His Thr Ser Ser Leu Val Arg Val Val Ser Thr Asn Tyr 100 105 110 Asn Gln His Ala Met Val Phe Phe Lys Phe Val Phe Gln Asn Arg Glu 115 120 125 Glu Phe Tyr Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu Thr Ser Glu 130 135 140 Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly Leu Pro Glu 145 150 155 160 Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile Asp Gly 165 170 175 <210> 7 <211> 178 <212> PRT <213> Artificial sequence <220> <223> Lipid transport protein mutant variant 17 <400> 7 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30 Val Val Gly Gln Ala Gly Asn Ile Lys Leu Arg Glu Asp Lys Asp Pro 35 40 45 Val Lys Met Met Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asp Val Thr Gly Val Thr Phe Asp Asp Lys Lys Cys Arg Tyr Asp Ile 65 70 75 80 Ser Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Phe Gly Lys 85 90 95 Ile Lys Ser Phe Pro Gly His Thr Ser Ser Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Phe Val Phe Gln 115 120 125 Asn Arg Glu Glu Phe Tyr Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> 8 <211> 178 <212> PRT <213> Artificial sequence <220> <223> Lipid transporter mutant protein variant 18 <400> 8 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30 Val Val Gly Gln Ala Gly Asn Ile Arg Leu Arg Glu Asp Lys Asp Pro 35 40 45 His Lys Met Met Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asp Val Thr Gly Val Thr Phe Asp Asp Lys Lys Cys Thr Tyr Ala Ile 65 70 75 80 Ser Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Lys 85 90 95 Ile Lys Ser Phe Pro Gly His Thr Ser Ser Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Phe Val Phe Gln 115 120 125 Asn Arg Glu Glu Phe Tyr Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> 9 <211> 178 <212> PRT <213> Artificial sequence <220> <223> Lipid transport protein mutant variant 19 <400> 9 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30 Val Val Gly Gln Ala Gly Asn Ile Lys Leu Arg Glu Asp Lys Asp Pro 35 40 45 Asn Lys Met Met Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asp Val Thr Gly Val Thr Phe Asp Asp Lys Lys Cys Thr Tyr Ala Ile 65 70 75 80 Ser Thr Leu Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Phe Gly Lys 85 90 95 Ile Lys Ser Phe Pro Gly His Thr Ser Ser Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Phe Val Phe Gln 115 120 125 Asn Arg Glu Glu Phe Tyr Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> 10 <211> 178 <212> PRT <213> Artificial sequence <220> <223> Lipid transport protein mutant variant 20 <400> 10 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30 Val Val Gly Gln Ala Gly Asn Ile Arg Leu Arg Glu Asp Lys Asp Pro 35 40 45 Ser Lys Met Met Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asp Val Thr Ala Val Thr Phe Asp Asp Lys Lys Cys Asn Tyr Ala Ile 65 70 75 80 Ser Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Lys 85 90 95 Ile Lys Ser Phe Pro Gly His Thr Ser Ser Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Phe Val Phe Gln 115 120 125 Asn Arg Glu Glu Phe Tyr Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> 11 <211> 178 <212> PRT <213> Artificial sequence <220> <223> Lipid transport protein mutant variant 47 <400> 11 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30 Val Val Gly Met Ala Gly Asn Asn Leu Leu Arg Glu Asp Lys Asp Pro 35 40 45 His Lys Met Ser Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asn Val Thr Asp Val Met Phe Leu Asp Lys Lys Cys Gln Tyr Ile Ile 65 70 75 80 Trp Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Phe 85 90 95 Ile Lys Ser Asp Pro Gly His Thr Ser Tyr Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Ser Val Ile Gln 115 120 125 Asn Arg Glu Trp Phe Gly Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> 12 <211> 178 <212> PRT <213> Artificial sequence <220> <223> Lipid transport protein mutant variant 48 <400> 12 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe Gln Gly Lys Trp Tyr 20 25 30 Val Val Gly Met Ala Gly Asn Asn Leu Leu Arg Glu Asp Lys Asp Pro 35 40 45 His Lys Met Ser Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asn Val Thr Asp Val Met Phe Leu Asp Lys Lys Cys Gln Tyr Ile Ile 65 70 75 80 Trp Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Leu Thr Leu Gly Phe 85 90 95 Ile Arg Ser Asp Leu Gly His Thr Ser Tyr Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Ser Val Ile Gln 115 120 125 Asn Arg Glu Trp Phe Gly Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> 13 <211> 178 <212> PRT <213> Synthetic Sequence <220> <223> Lipocalin mutant protein variant 49 <400> 13 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Tyr Gln Phe Gln Gly Lys Trp Tyr 20 25 30 Val Val Gly Met Ala Gly Asn Asn Leu Leu Arg Glu Asp Lys Asp Pro 35 40 45 His Lys Met Gly Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asn Val Thr Asp Val Met Leu Leu Asp Lys Lys Cys Gln Tyr Ile Ile 65 70 75 80 Gln Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Ser Thr Leu Gly Phe 85 90 95 Ile Lys Ser Asp Pro Gly His Thr Ser Tyr Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Ser Val Ile Gln 115 120 125 Asn Arg Glu Trp Phe Gly Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> 14 <211> 178 <212> PRT <213> Artificial sequence <220> <223> Lipid transport protein mutant variant 50 <400> 14 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe Gln Gly Lys Trp Tyr 20 25 30 Val Val Gly Met Ala Gly Asn Asn Leu Leu Arg Glu Asp Lys Asp Pro 35 40 45 His Lys Met Gly Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asn Val Thr Asp Val Met Phe Leu Asp Lys Lys Cys Gln His Ile Ile 65 70 75 80 Trp Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Leu Thr Leu Gly Phe 85 90 95 Ile Lys Ser Asp Pro Gly His Thr Ser Tyr Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Ser Val Ile Gln 115 120 125 Asn Arg Glu Trp Phe Gly Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> 15 <211> 178 <212> PRT <213> Artificial sequence <220> <223> Lipid transport protein mutant variant 51 <400> 15 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asp Gln Phe Gln Gly Lys Trp Tyr 20 25 30 Val Val Gly Met Ala Gly Asn Asn Leu Leu Arg Glu Asp Lys Asp Pro 35 40 45 His Lys Met Gly Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asn Val Thr Asp Val Met Phe Leu Asp Lys Lys Cys Gln Tyr Ile Ile 65 70 75 80 Trp Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Leu Thr Leu Gly Phe 85 90 95 Ile Lys Ser Asp Pro Gly His Thr Ser Tyr Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Ser Val Ile Gln 115 120 125 Asn Arg Glu Trp Phe Gly Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> 16 <211> 178 <212> PRT <213> Artificial sequence <220> <223> Lipid transport protein mutant variant 52 <400> 16 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe Gln Gly Lys Trp Tyr 20 25 30 Ile Val Gly Met Ala Gly Asn Asn Leu Leu Arg Glu Asp Lys Asp Pro 35 40 45 His Lys Met Gly Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asn Val Thr Asp Val Met Phe Leu Asp Lys Lys Cys Gln Tyr Ile Ile 65 70 75 80 Trp Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Leu Thr Leu Gly Phe 85 90 95 Ile Lys Ser Asp Pro Gly His Thr Ser Tyr Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Ser Val Ile Gln 115 120 125 Asn Arg Glu Trp Phe Gly Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> 17 <211> 178 <212> PRT <213> Artificial sequence <220> <223> Lipid transport protein mutant variant 53 <400> 17 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Arg Asn Phe Gln Asp Asn Gln Phe Gln Gly Lys Trp Tyr 20 25 30 Val Val Gly Met Ala Gly Asn Asn Leu Leu Arg Val Asp Lys Asp Pro 35 40 45 His Lys Met Gly Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asn Val Thr Asp Val Met Phe Leu Asp Lys Lys Cys Gln Tyr Ile Ile 65 70 75 80 Trp Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Leu Thr Leu Gly Phe 85 90 95 Ile Lys Ser Asp Pro Gly His Thr Ser Tyr Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Tyr Phe Lys Ser Val Ile Gln 115 120 125 Asn Arg Glu Trp Phe Gly Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> 18 <211> 178 <212> PRT <213> Artificial sequence <220> <223> Lipid transport protein mutant variant 54 <400> 18 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe Gln Gly Lys Trp Tyr 20 25 30 Val Val Gly Met Ala Gly Asn Asn Leu Leu Arg Glu Asp Lys Asp Pro 35 40 45 His Lys Met Ser Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asn Val Thr Asp Val Met Phe Leu Asp Lys Lys Cys Gln Tyr Ile Asn 65 70 75 80 Trp Pro Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Phe 85 90 95 Ile Lys Ser Asp Leu Gly Pro Thr Ser Tyr Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Ser Val Ile Gln 115 120 125 Asn Arg Glu Trp Phe Gly Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> 19 <211> 178 <212> PRT <213> Artificial sequence <220> <223> Lipid transporter mutant protein variant 55 <400> 19 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe Gln Gly Lys Trp Tyr 20 25 30 Val Val Gly Met Ala Gly Asn Asn Leu Leu Arg Glu Asp Lys Asp Pro 35 40 45 His Lys Met Gly Ala Thr Ile Tyr Glu Leu Asn Glu Asp Lys Ser Tyr 50 55 60 Asn Val Thr Asp Val Met Phe Leu Asp Lys Lys Cys Gln Tyr Ile Ile 65 70 75 80 Trp Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Leu Thr Leu Gly Phe 85 90 95 Ile Lys Ser Asp Pro Gly His Thr Ser Tyr Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Ser Val Ile Gln 115 120 125 Asn Arg Glu Trp Phe Gly Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> 20 <211> 178 <212> PRT <213> Artificial sequence <220> <223> Lipid transport protein mutant variant 56 <400> 20 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe Gln Gly Lys Trp Tyr 20 25 30 Val Val Gly Met Ala Gly Asn Asn Leu Leu Arg Asp Asp Lys Asp Pro 35 40 45 His Lys Met Ser Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asn Val Thr Asp Val Met Leu Leu Asp Lys Lys Cys His Tyr Ile Ile 65 70 75 80 Trp Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Leu Thr Leu Gly Phe 85 90 95 Ile Lys Ser Asp Pro Gly His Thr Ser Tyr Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Ser Val Ile Gln 115 120 125 Asn Arg Glu Trp Phe Gly Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> 21 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> FAP(4B9) CDR-H1 <400> 21 Ser Tyr Ala Met Ser 1 5 <210> 22 <211> 17 <212> PRT[[ID=Y37]] <213> Artificial Sequence <220> <223> FAP(4B9) CDR-H2 <400> 22 Ala Ile Ile Gly Ser Gly Ala Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 23 <211> 8 <212> PRT [[ID=Y58]]<213> Artificial Sequence <220> <223> FAP(4B9) CDR-H3 <400> 23 Gly Trp Phe Gly Gly Phe Asn Tyr 1 5 <210> twenty four <211> 12 <212> PRT <213> Artificial sequence <220> <223> FAP(4B9) CDR-L1 <400> twenty four Arg Ala Ser Gln Ser Val Thr Ser Ser Tyr Leu Ala 1 5 10 <210> 25 <211> 7 <212> PRT <213> Artificial sequence <220> <223> FAP(4B9) CDR-L2 <400> 25 Val Gly Ser Arg Arg Ala Thr 1 5 <210> 26 <211> 9 <212> PRT <213> Artificial sequence <220> <223> FAP(4B9) CDR-L3 <400> 26 Gln Gln Gly Ile Met Leu Pro Pro Thr 1 5 <210> 27 <211> 117 <212> PRT <213> Artificial sequence <220> <223> FAP(4B9) VH <400> 27 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 Ile Gly Ser Gly Ala Ser Thr Tyr Tyr Ala 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 Gly Trp Phe Gly Gly Phe Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 28 <211> 108 <212> PRT <213> artificial sequence <220> <223> FAP(4B9) VL <400> 28 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Asn Val Gly Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Ile Met Leu Pro 85 90 95 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 29 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> FAP (28H1) CDR-H1 <400> 29 Ser His Ala Met Ser 1 5 <210> 30 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> FAP (28H1) CDR-H2 <400> 30 Ala Ile Trp Ala Ser Gly Glu Gln Tyr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 31 <211> 8 <212> PRT <213> Artificial sequence <220> <223> FAP (28H1) CDR-H3 <400> 31 Gly Trp Leu Gly Asn Phe Asp Tyr 1 5 <210> 32 <211> 12 <212> PRT <213> Artificial sequence <220> <223> FAP (28H1) CDR-L1 <400> 32 Arg Ala Ser Gln Ser Val Ser Arg Ser Tyr Leu Ala 1 5 10 <210> 33 <211> 7 <212> PRT <213> Artificial sequence <220> <223> FAP (28H1) CDR-L2 <400> 33 Gly Ala Ser Thr Arg Ala Thr 1 5 <210> 34 <211> 9 <212> PRT <213> Artificial sequence <220> <223> FAP (28H1) CDR-L3 <400> 34 Gln Gln Gly Gln Val Ile Pro Pro Thr 1 5 <210> 35 <211> 116 <212> PRT <213> Artificial sequence <220> <223> FAP(28H1) 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 Ser Ser His 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 Trp Ala Ser Gly Glu Gln Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Gly Trp Leu Gly Asn Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 36 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> FAP(28H1) VL <400> 36 Glu Ile Val Leu Thr Gln Ser Pro Gly 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 Arg Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Ile Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Gln Val Ile Pro 85 90 95 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 37 <211> 419 <212> PRT <213> Artificial sequence <220> <223> Fc pore huIgG1 PGLALA - 4-1BB lipid transporter heavy chain <400> 37 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 225 230 235 240 Ser Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys 245 250 255 Val Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp 260 265 270 Tyr Val Val Gly Gln Ala Gly Asn Ile Arg Leu Arg Glu Asp Lys Asp 275 280 285 Pro Ile Lys Met Met Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser 290 295 300 Tyr Asp Val Thr Met Val Lys Phe Asp Asp Lys Lys Cys Met Tyr Asp 305 310 315 320 Ile Trp Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly 325 330 335 Lys Ile Lys Ser Phe Pro Gly His Thr Ser Ser Leu Val Arg Val Val 340 345 350 Ser Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Phe Val Phe 355 360 365 Gln Asn Arg Glu Glu Phe Tyr Ile Thr Leu Tyr Gly Arg Thr Lys Glu 370 375 380 Leu Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu 385 390 395 400 Gly Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys 405 410 415 Ile Asp Gly <210> 38 <211> 639 <212> PRT <213> Artificial sequence <220> <223> VH (FAP 4B9) Fc protrusion huIgG1 PGLALA 4-1BB lipid transporter heavy chain <400> 38 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 Ile Gly Ser Gly Ala Ser Thr Tyr Tyr Tyr Ala 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 Gly Trp Phe Gly Gly Phe Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly 435 440 445 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp Ser 450 455 460 Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val Pro Leu Gln 465 470 475 480 Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr Val Val Gly 485 490 495 Gln Ala Gly Asn Ile Arg Leu Arg Glu Asp Lys Asp Pro Ile Lys Met 500 505 510 Met Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr Asp Val Thr 515 520 525 Met Val Lys Phe Asp Asp Lys Lys Cys Met Tyr Asp Ile Trp Thr Phe 530 535 540 Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Lys Ile Lys Ser 545 550 555 560 Phe Pro Gly His Thr Ser Ser Leu Val Arg Val Val Ser Thr Asn Tyr 565 570 575 Asn Gln His Ala Met Val Phe Phe Lys Phe Val Phe Gln Asn Arg Glu 580 585 590 Glu Phe Tyr Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu Thr Ser Glu 595 600 605 Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly Leu Pro Glu 610 615 620 Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile Asp Gly 625 630 635 <210> 39 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> VL (FAP 4B9) Cκ Light Chain <400> 39 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Asn Val Gly Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Ile Met Leu Pro 85 90 95 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 40 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR-H1, pertuzumab <400> 40 Gly Phe Thr Phe Thr Asp Tyr Thr Met Asp 1 5 10 <210> 41 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR-H2, pertuzumab <400> 41 Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe Lys 1 5 10 15 Gly <210> 42 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR-H3, pertuzumab <400> 42 Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr 1 5 10 <210> 43 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Light chain CDR-L1, pertuzumab <400> 43 Lys Ala Ser Gln Asp Val Ser Ile Gly Va...

Claims

1. A bispecific antigen-binding molecule capable of bivalently binding to 4-1BB and monovalently binding to a target cell antigen, comprising (a) only one Fab fragment that can specifically bind to the target cell antigen, (b) an IgG1 Fc domain comprising a first subunit and a second subunit capable of stably associating and comprising amino acid substitutions L234A, L235A and P329G according to EU numbering according to Kabat, and (c) two lipocalin muteins capable of specifically binding to 4-1BB, wherein one of the lipocalin muteins is fused to the C-terminus of the first subunit of the Fc domain, and the other is fused to the C-terminus of the second subunit of the Fc domain, and Each of the lipocalin mutant proteins capable of specifically binding to 4-1BB is as shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO:

20.

2. The bispecific antigen binding molecule of claim 1, wherein each of the lipocalin mutant proteins capable of specifically binding to 4-1BB is as shown in SEQ ID NO:

2.

3. The bispecific antigen binding molecule of claim 1, wherein the Fc domain comprises a knob-to-hole modification that promotes the association of the first subunit and the second subunit of the Fc domain. 4 . The bispecific antigen-binding molecule according to claim 1 , wherein the Fab fragment capable of specifically binding to a target cell antigen is a Fab fragment capable of specifically binding to fibroblast activation protein (FAP).

5. The bispecific antigen binding molecule according to claim 4, wherein the Fab fragment capable of specifically binding to fibroblast activation protein (FAP) comprises: (a) Heavy chain variable region (V H FAP), which comprises: (i) CDR-H1, which is shown in SEQ ID NO: 21, (ii) CDR-H2, which is shown in SEQ ID NO: 22, and (iii) CDR-H3, which is shown in SEQ ID NO: 23; and a light chain variable region (V L FAP), which comprises: (iv) CDR-L1, which is shown in SEQ ID NO:24, (v) CDR-L2, which is shown in SEQ ID NO:25, and (vi) CDR-L3, which is shown in SEQ ID NO:26; or (b) Heavy chain variable region (V H FAP), which comprises: (i) CDR-H1, which is shown in SEQ ID NO: 29, (ii) CDR-H2, which is shown in SEQ ID NO: 30, and (iii) CDR-H3, which is shown in SEQ ID NO: 31; and a light chain variable region (V L FAP), which comprises: (iv) CDR-L1, which is shown in SEQ ID NO:32, (v) CDR-L2, which is shown in SEQ ID NO:33, and (vi) CDR-L3, which is shown in SEQ ID NO:

34.

6. The bispecific antigen binding molecule according to claim 5, wherein the Fab fragment capable of specifically binding to fibroblast activation protein (FAP) comprises: (a) Heavy chain variable region (V H FAP), which comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 27; and a light chain variable region (V L FAP), which comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 28; or (b) Heavy chain variable region (V H FAP), which comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 35; and a light chain variable region (V L FAP), which comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

36.

7. The bispecific antigen binding molecule according to claim 5, wherein the Fab fragment capable of specifically binding to fibroblast activation protein (FAP) comprises: (a) Heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 27; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 28; or (b) Heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 35; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO:

36.

8. The bispecific antigen binding molecule of claim 5, comprising: The first heavy chain is SEQ ID NO:37, the second heavy chain is SEQ ID NO:38 and the light chain is SEQ ID NO:

39.

9. The bispecific antigen binding molecule according to claim 1, wherein the Fab fragment capable of specifically binding to a target cell antigen is a Fab fragment capable of specifically binding to HER2.

10. The bispecific antigen binding molecule according to claim 9, wherein the Fab fragment capable of specifically binding to HER2 comprises: (a) a VH domain comprising: (i) CDR-H1, which is shown in SEQ ID NO:40, (ii) CDR-H2, which is shown in SEQ ID NO:41, and (iii) CDR-H3, which is shown in SEQ ID NO:42; and a VL domain comprising: (iv) CDR-L1, which is shown in SEQ ID NO:43, (v) CDR-L2, which is shown in SEQ ID NO:44, and (vi) CDR-L3, which is shown in SEQ ID NO:45; or (b) a VH domain comprising: (i) CDR-H1, which is shown in SEQ ID NO:48, (ii) CDR-H2, which is shown in SEQ ID NO:49, and (iii) CDR-H3, which is shown in SEQ ID NO:50; and a VL domain comprising: (iv) CDR-L1, which is shown in SEQ ID NO:51, (v) CDR-L2, which is shown in SEQ ID NO:52, and (vi) CDR-L3, which is shown in SEQ ID NO:53; or (c) a VH domain comprising: (i) CDR-H1, which is shown in SEQ ID NO:56, (ii) CDR-H2, which is shown in SEQ ID NO:57, and (iii) CDR-H3, which is shown in SEQ ID NO:58; and a VL domain comprising: (iv) CDR-L1, which is shown in SEQ ID NO:59, (v) CDR-L2, which is shown in SEQ ID NO:60, and (vi) CDR-L3, which is shown in SEQ ID NO:

61.

11. The bispecific antigen binding molecule according to claim 10, wherein the Fab fragment capable of specifically binding to HER2 comprises: (a) Heavy chain variable region (V H HER2), which comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 46; and a light chain variable region (V L HER2), which comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:47; or (b) Heavy chain variable region (V H HER2), which comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 54; and a light chain variable region (V L HER2), which comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:55; or (c) Heavy chain variable region (V H HER2), which comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 62; and a light chain variable region (V L HER2), which comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

63.

12. The bispecific antigen binding molecule according to claim 10, wherein the Fab fragment capable of specifically binding to HER2 comprises: (a) Heavy chain variable region (V H HER2), comprising the amino acid sequence of SEQ ID NO:46; and the light chain variable region (V L HER2), which comprises the amino acid sequence of SEQ ID NO: 47; or (b) Heavy chain variable region (V H HER2), which comprises the amino acid sequence of SEQ ID NO: 54; and the light chain variable region (V L HER2), which comprises the amino acid sequence of SEQ ID NO: 55; or (c) Heavy chain variable region (V H HER2), comprising the amino acid sequence of SEQ ID NO: 62; and the light chain variable region (V L HER2), which comprises the amino acid sequence of SEQ ID NO:

63.

13. The bispecific antigen binding molecule of claim 10, comprising a first heavy chain of SEQ ID NO: 64, a second heavy chain of SEQ ID NO: 65, and a light chain of SEQ ID NO:

66.

14. An isolated nucleic acid encoding the bispecific antigen binding molecule according to any one of claims 1 to 13.

15. An expression vector comprising the isolated nucleic acid according to claim 14.

16. A host cell comprising the nucleic acid according to claim 14 or the vector according to claim 15.

17. A method for producing a bispecific antigen binding molecule according to any one of claims 1 to 13, comprising culturing the host cell according to claim 16 under conditions suitable for expressing the bispecific antigen binding molecule.

18. The method of claim 17, further comprising recovering the bispecific antigen binding molecule from the host cell.

19. A bispecific antigen binding molecule produced by the method according to claim 17 or 18.

20. A pharmaceutical composition comprising the bispecific antigen binding molecule according to any one of claims 1 to 13 and 19 and at least one pharmaceutically acceptable excipient.

21. The pharmaceutical composition of claim 20, further comprising an additional therapeutic agent.

22. Use of the bispecific antigen binding molecule according to any one of claims 1 to 13 and 19 or the pharmaceutical composition according to claim 20 or 21 for the manufacture of a medicament for treating cancer or an infectious disease.

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