Antibodies that bind fibroblast-activating protein alpha and death receptor 4.

Abstract

The present invention relates to a multispecific antibody comprising a FAPα-binding region comprising at least a first heavy chain variable region and a first light chain variable region, and a DR4-binding region comprising a second heavy chain variable region and a second light chain variable region. The invention further provides pharmaceutical compositions comprising the antibody, and uses of the antibody in therapeutic and diagnostic procedures, particularly in cancer treatment.

Description

[Technical Field]

[0001] This invention relates to antibodies that bind to fibroblast-activating protein alpha (FAPα) and death receptor 4 (DR4). The invention further provides pharmaceutical compositions comprising antibodies, as well as therapeutic and diagnostic procedures, such as the use of antibodies for the treatment of cancer. [Background technology]

[0002] Therapeutic monoclonal antibodies are a promising type of immunotherapy due to their specific characteristics, including target specificity, immunomodulation, and generally low toxicity. Monoclonal antibodies are constructed from two distinct functional units: an antigen-binding fragment (Fab) that binds to a target antigen, and a constant fragment (Fc) that mediates antibody-dependent immune effector function. The primary Fc domain-mediated mechanism of action is the binding of Fc-gamma receptors (specific to IgG) on various immune cells, resulting in complement-dependent cell-mediated cytotoxicity (CDC) and, for example, antibody-dependent cell-mediated cytotoxicity (ADCC). Furthermore, Fab binding to targets can lead to signaling perturbations.

[0003] Knowledge of antibody-based therapeutic strategies has advanced dramatically in recent years, leading to breakthroughs in target biology, mechanisms of action, as well as antibody formats and development. This has resulted in antibody-based therapies aimed at improving clinical efficacy and, consequently, target specificity (and therefore safety).

[0004] Apoptosis is a form of programmed cell death. Tumor necrosis factor (TNF)-associated apoptosis-inducing ligand (TRAIL) can induce apoptosis through binding to its agonist receptor, which contains an intracellular death domain (DD). Tumor necrosis factor receptor superfamily member 10A (TNFRSF10A), TRAIL receptor 1 (TRAIL-R1), and DR4, also known as CD261, is a cell surface receptor of the TNF receptor superfamily that binds TRAIL and mediates apoptosis. DR4 shares 60% homology with death receptor 5 (DR5), another known TRAIL receptor that can induce apoptosis. DR4 is a single-pass type I membrane protein with at least three extracellular cysteine-rich domains (CRDs), a transmembrane domain (TM), and cytoplasmic DDs. TRAIL binding leads to DR4 activation via receptor trimerization, resulting in DD clustering, followed by recruitment of Fas-associated death domain (FADD) adapter proteins. FADD then recruits caspases-8 and -10 to form the death-inducing signaling complex (DISC). Active caspases-8 and -10 are then released into the cytosol, where they activate downstream effector caspases such as caspase-3. Effector caspase activation culminates in apoptotic cell death.

[0005] FAPα is a type II transmembrane (homodimerated) serine protease that is overexpressed in pathological conditions including fibrosis, arthritis, and cancer. FAPα is primarily expressed by activated stromal fibroblasts, e.g., CAFs. FAPα can also be effluxed from the cell membrane, forming soluble FAPα. FAPα is a member of the prolyl peptidase family and shares 70% amino acid sequence homology with the well-described dipeptidyl peptidase 4 (DPP4). While both peptidases possess dipeptidyl peptidase enzymatic activity, the endopeptidase activity is FAPα-specific, targeting substrates including denatured collagen and α-2 anti-plasmin. For most cancers, elevated FAPα expression is associated with worsening outcomes, but the underlying biological mechanisms are not well understood.

[0006] The limited antitumor effects shown by some first-generation DR agonist antibodies being tested clinically are likely due to their inability to induce efficient receptor cluster formation, which is essential for inducing apoptosis (Dubuisson and Micheau, Antibodies (Basel) 6(4), 2017). One such example is mapatumumab (HGS-ETR1), a DR4-specific agonist monoclonal antibody, which showed limited clinical activity when investigated in multiple phase 1 / 2 trials (Snajdauf et al., Front Mol Biosci 8:628332, 2021). Next-generation drugs such as the TRAIL-R agonist Fc fusion protein eftozanermin alfa (ABBV-621) showed promising clinical activity, but also induced side effects such as hepatotoxicity (Papadopoulos et al, Cancer Chemother Pharmacol 75(5):887-895, 2015; LoRusso et al., Invest New Drugs 40(4):762-772, 2022; Di Cristofano et al., Biochem Soc Trans 51(1):57-70, 2023). Dual targeting of DR and FAPα is being explored using RG7386, an optimized tetravalent bispecific antibody that targets FAPα and DR5 (US Patent No. 9926379B2). However, clinical development for solid malignancies was discontinued in 2018. Therefore, there is a clear unmet need for the development of novel therapeutics with improved safety and efficacy. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] U.S. Patent No. 9926379B2 [Non-patent literature]

[0008] [Non-Patent Document 1] Dubuisson and Micheau,Antibodies(Basel)6(4),2017 [Non-Patent Document 2] Snajdauf et al.,Front Mol Biosci 8:628332,2021 [Non-Patent Document 3] Papadopoulos et al, Cancer Chemother Pharmacol 75(5):887-895,2015 [Non-Patent Document 4] LoRusso et al.,Invest New Drugs 40(4):762-772,2022 [Non-Patent Document 5] Di Cristofano et al.,Biochem Soc Trans 51(1):57-70,2023 [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] The objective of the present invention is to provide a multispecific antibody with improved safety and efficacy. [Means for solving the problem]

[0010] The multispecific antibodies according to the present invention include a DR4-binding domain and an FAPα-binding domain. Therefore, the proposed mechanism of action for the multispecific antibodies according to the present invention is the conditional transactivation of DR4 as a result of simultaneous binding to DR4-expressing tumor cells and FAPα on CAFs in the tumor microenvironment (TME). Furthermore, it has been found that some DR4-binding antibodies bind well to DR4-expressing cells, and some FAPα-binding antibodies bind well to FAPα-expressing cells in a bivalent (monoclonal) format, but show reduced binding in a monovalent format. Therefore, another object of the present invention relates to monoclonal antibodies capable of strong binding in either a monovalent or bivalent format. This could form the basis for, for example, the production of bispecific antibodies exhibiting strong monovalent binding to DR4 and FAPα.

[0011] In the present invention, as demonstrated, for example by Examples 10-13 and 15, the trans-binding mechanism of multispecific antibodies is advantageous when the death of DR4-expressing tumor cells may depend on the simultaneous binding of multispecific antibodies to FAPα-expressing cells such as CAFs. Thus, FAPα-dependent DR4 trans-activation leads to improved tumor-specific targeting due to the high expression of FAPα on CAFs in TMEs. Furthermore, multispecific antibodies exhibit an expanded therapeutic window through trans-binding-dependent DR4 activation, as opposed to cis-binding, when DR4 is not expressed on fibroblasts (e.g., Example 9). Since fibroblasts do not express DR4, they are not susceptible to induction of cell death by FAP-dependent DR4 agonism (e.g., Examples 9 and 11). In one embodiment, the present invention relates to a multispecific antibody comprising at least (i) an FAPα-binding domain capable of binding to FAPα comprising a first heavy chain variable region and a first light chain variable region, and (ii) a DR4-binding domain capable of binding to DR4 comprising a second heavy chain variable region and a second light chain variable region.

[0012] In a further embodiment, the present invention relates to nucleic acid constructs or combinations of nucleic acid constructs that encode antibodies as defined herein.

[0013] In another aspect, the present invention relates to a composition comprising a nucleic acid construct or a combination of nucleic acid constructs as defined herein.

[0014] In yet another aspect, the present invention relates to a delivery vehicle comprising one or more nucleic acid constructs described herein.

[0015] In yet another aspect, the present invention relates to a recombinant host cell capable of producing the antibodies described herein, wherein the host cell comprises one or more nucleic acid constructs encoding the antibodies described herein.

[0016] In further embodiments, the present invention relates to a pharmaceutical composition comprising a multispecific antibody as defined herein and a pharmaceutically acceptable carrier.

[0017] In further embodiments, the present invention relates to a multispecific antibody as described herein, one or more nucleic acid constructs as described herein, a delivery vehicle as described herein, or a pharmaceutical composition as described herein, for use in the treatment of cancer.

[0018] In a further embodiment, the present invention relates to a method for producing a multispecific antibody according to the present invention.

[0019] Finally, the present invention also provides monospecific antibodies targeting DR4 and FAPα, respectively.

[0020] These aspects and other aspects and embodiments are described in more detail in the following sections. [Brief explanation of the drawing]

[0021] [Figure 1]Binding to human lung fibroblasts and CAFs. (A-B) The binding of BisG1-FAPα-FERL / DR4-FERR, BisG1-FAPα-FERL / b12-FERR, BisG1-b12-FERL / b12-FERR, and IgG1-FAPα-FERL to human lung fibroblasts (A) and CAFs (B) was evaluated by flow cytometry. The data shown are geometric mean fluorescence intensity (gMFI) values ​​for one of three representative experiments. The concentration (μg / mL) at which 50% of the maximum effect (EC50) was observed was derived from the fitted curve. For CAFs (B), only the top four concentrations of the IgG1-FAPα-FERL antibody were included. (C) The binding of two FAPα-targeted antibody clones, FAPα-FEAL and FAP5 (antibodies of BisG1-FAPα-FEAL / b12-FEAR, BisG1-FAP5-FEAL / b12-FEAR, IgG1-FAPα-FEAL, and IgG1-FAP5-FEAL) and IgG1-b12-FEAR to human lung fibroblasts was evaluated by flow cytometry. The data shown are the gMFI values ​​determined by flow cytometry for one of the two representative experiments. [Figure 2-1]The binding of DR4-specific antibodies to cell surface-expressed DR4 was evaluated in six different cell lines: (A-F) BisG1-FAPα-FERL / DR4-FERR, BisG1-b12-FERL / DR4-FERR, IgG1-DR4-FERR, and IgG1-b12 in six different cell lines: colorectal adenocarcinoma cell lines DLD-1 and HCT-15, lung adenocarcinoma A549, colorectal carcinoma HCT-116, breast carcinoma MDA-MB-231, and pancreatic ductal adenocarcinoma PANC-1. The data shown are gMFI values ​​determined by flow cytometry for one of three representative experiments. The binding of (G)BisG1-b12-FEAL / DR4-T1014A04-FEAR, BisG1-b12-FEAL / DR4-chCTB007-FEAR, IgG1-DR4-chCTB007-FEAR, IgG1-DR4-T1014A04-FEAR, and IgG1-b12-FEAL to DR4 was evaluated in the multiple myeloma cell line OPM-2. The data shown are the gMFI values ​​determined by flow cytometry for one experiment. The binding of (H)IgG1-DR4-FERR, BisG1-b12-FERL / DR4-FERR, BisG1-FAPα-FERL / DR4-FERR, IgG1-DR4-chCTB007-FEAR, BisG1-b12-FEAL / DR4-chCTB007-FEAR, IgG1-DR4-T1014A04-FEAR, BisG1-b12-FEAL / DR4-T1014A04-FEAR, and BisG1-b12-FERL / b12-FERR to DR4 was evaluated on the mammary cancer cell line MDA-MB-231. Data are expressed as mean (±SD) gMFI values ​​determined by flow cytometry in two independent experiments. [Figure 2-2]The binding of DR4-specific antibodies to cell surface-expressed DR4 was evaluated in six different cell lines: (A-F) BisG1-FAPα-FERL / DR4-FERR, BisG1-b12-FERL / DR4-FERR, IgG1-DR4-FERR, and IgG1-b12 in six different cell lines: colorectal adenocarcinoma cell lines DLD-1 and HCT-15, lung adenocarcinoma A549, colorectal carcinoma HCT-116, breast carcinoma MDA-MB-231, and pancreatic ductal adenocarcinoma PANC-1. The data shown are gMFI values ​​determined by flow cytometry for one of three representative experiments. The binding of (G)BisG1-b12-FEAL / DR4-T1014A04-FEAR, BisG1-b12-FEAL / DR4-chCTB007-FEAR, IgG1-DR4-chCTB007-FEAR, IgG1-DR4-T1014A04-FEAR, and IgG1-b12-FEAL to DR4 was evaluated in the multiple myeloma cell line OPM-2. The data shown are the gMFI values ​​determined by flow cytometry for one experiment. The binding of (H)IgG1-DR4-FERR, BisG1-b12-FERL / DR4-FERR, BisG1-FAPα-FERL / DR4-FERR, IgG1-DR4-chCTB007-FEAR, BisG1-b12-FEAL / DR4-chCTB007-FEAR, IgG1-DR4-T1014A04-FEAR, BisG1-b12-FEAL / DR4-T1014A04-FEAR, and BisG1-b12-FERL / b12-FERR to DR4 was evaluated on the mammary cancer cell line MDA-MB-231. Data are expressed as mean (±SD) gMFI values ​​determined by flow cytometry in two independent experiments. [Figure 3] Species cross-reactivity to FAPα orthologs. Antibodies BisG1-FAPα-FERL / DR4-FERR, BisG1-FAPα-FEAL / b12-FEAR, IgG1-FAPα-FERL, and IgG1-b12 were tested. (A-F) Binding to Expi293F cells expressing human, cynomolgus monkey, mouse, rat, pig, and dog FAPα, respectively. The data shown are gMFI values ​​determined by flow cytometry for one of the three representative experiments. [Figure 4] Species cross-reactivity to DR4 or mouse / rat DR orthologues. Antibodies BisG1-FAPα-FERL / DR4-FERR, BisG1-DR4-FERL / b12-FEAR, IgG1-DR4-FERR, and IgG1-b12 were tested. (A) Binding to ExpiCHO-S cells expressing human DR4. (B) Binding to ExpiCHO-S cells expressing cynomolgus monkey DR4. Binding of BisG1-FAPα-FERL / DR4-FERR versus IgG1-DR4-FERR and IgG1-b12 to ExpiCHO-S cells expressing porcine DR4 (C), rat DR (D), mouse DR (E), canine DR4 (F), or rabbit DR4 (G). The data shown are gMFI values ​​determined by flow cytometry for one of three representative experiments. [Figure 5] Quantification of DR4, DR5, and FAPα surface expression and evaluation of fibroblast cell death. (A-B) Surface expression of DR4, DR5, and FAPα in human lung fibroblasts and CAFs. Summary data (mean ± SEM) from two experiments are shown. The horizontal dotted line indicates the limit of quantification (LLOQ). (C) Human lung fibroblasts cultured with BisG1-FAPα-FEAL / DR4-FEAR, BisG1-FAPα-FEAL / b12-FEAR, BisG1-b12-FEAL / DR4-FEAR, IgG1-FAPα-FEAL (negative control), or RG7386. Percentage of viable cells normalized to the no-antibody condition is plotted against antibody concentration, with dual mean ± SEM shown. (D) Human lung fibroblasts cultured with 10 μg / mL BisG1-FAPα-FEAL / DR4-FEAR, BisG1-FAPα-FEAL / b12-FEAR, BisG1-b12-FEAL / DR4-FEAR, or negative control IgG1-b12-FEAR. Staurosporine was used as a positive control. The graph shows the fluorescence signal representing the number of dead cells (double ± SEM) plotted against time. (E) CAF cultured with BisG1-FAPα-FEAL / DR4-FEAR. The graph shows the double surviving tumor cell % ± SEM, normalized to the no-antibody condition and plotted against antibody concentration. [Figure 6] Transactivation ability of BisG1-FAPα-FEAL / DR4-FEAR. (A) Targeted cell death via DR4 transactivation was evaluated in the cancer cell line MDA-MB-231 co-cultured with human lung fibroblasts using DR4-targeted bispecific antibodies containing FAPα or FAP5-binding arms. Negative control antibodies included BisG1-FAPα-FEAL / b12-FEAR, BisG1-FAP5-FEAL / b12-FEAR, and BisG1-b12-FEAL / DR4-FEAR. 1 μM staurosporine was included as a positive control. The data shown are dual mean viable tumor cell surface (μm2 / image) ± SEM, plotted against antibody concentration. (B-C) To evaluate DR4 or DR5-mediated cell death in the absence of fibroblasts, monocultures of MDA-MB-231(B) or DLD-1(C) cells were tested using BisG1-FAPα-FEAL / DR4-FEAR, BisG1-b12-FERL / b12-FERR (top four concentrations), or RG7386. (D-E) DR4 transactivation-mediated cell death using BisG1-FAPα-FERL / DR4-FERR, BisG1-b12-FERL / DR4-FERR, BisG1-b12-FERL / b12-FERR (top four concentrations), and BisG1-FAPα-FERL / b12-FERR was evaluated in MDA-MB-231(D) and DLD-1(E) cancer cell lines co-cultured with the FAPα-expressing cell line NIH / 3T3-FAPα. Results B-D show dual mean viable cell percentages ± SEM and are plotted against antibody concentration for one representative experiment out of three. [Figure 7-1]Caspase-8 activation in co-cultures of tumor cells and NIH / 3T3-FAPα cells. Caspase-Glo 8 luminescence signaling for measurement of caspase-8 activation in co-cultures of DLD-1(A), MDA-MB-231(C), A549(E), and SNU-1076(G) tumor cells and NIH / 3T3-FAPα cells, or in tumor cell monocultures (B, D, F, H), in the presence of BisG1-FAPα-FERL / DR4-FERR, BisG1-b12-FERL / DR4-FERR, or RG7386. Recombinant human TRAIL (natural ligand for DR4) was included as a positive control. The data presented are mean ± SEM luminescence (RLU) plotted against antibody concentration for two or three independent experiments. [Figure 7-2] Caspase-8 activation in co-cultures of tumor cells and NIH / 3T3-FAPα cells. Caspase-Glo 8 luminescence signaling for measurement of caspase-8 activation in co-cultures of DLD-1(A), MDA-MB-231(C), A549(E), and SNU-1076(G) tumor cells and NIH / 3T3-FAPα cells, or in tumor cell monocultures (B, D, F, H), in the presence of BisG1-FAPα-FERL / DR4-FERR, BisG1-b12-FERL / DR4-FERR, or RG7386. Recombinant human TRAIL (natural ligand for DR4) was included as a positive control. The data presented are mean ± SEM luminescence (RLU) plotted against antibody concentration for two or three independent experiments. [Figure 8]Targeted cell death of patient-derived organoids (PDOs) via DR4 transactivation in the presence of CAF. (A-C) The ability of BisG1-FAPα-FEAL / DR4-FEAR and IgG1-FAPα-FEAL to induce DR4 transactivation-mediated cell death in three colorectal cancer (CRC) PDOs was investigated in the presence and absence of CAF. Data show the survival of PDOs, Hub096 (A), p18T (B), and p19B (C), cultured with or without CAF in the presence of the indicated antibodies. Cell survival (%) is plotted against antibody concentration (double mean ± SEM). (D) Apoptosis induction in co-cultures of fluorescently labeled Hub096 (CellBrite Blue) and CAF (CellBrite Orange) in the presence of BisG1-FAPα-FEAL / DR4-FEAR or IgG1-FAPα-FEAL was separately evaluated in both cell populations using flow cytometry with annexin V staining. Data are presented as mean (±SD) percentages of annexin V-positive cells from two independent experiments. [Figure 9-1]Evaluation of BisG1-FAPα-FEAL / DR4-FEAR antitumor activity in vivo. (A) FAPα staining in formalin-fixed paraffin-embedded (FFPE) tissue obtained from PDX models. (A) CTG-1234 (gastric cancer), CTG-1150 (pancreatic cancer), and positive control (invasive ductal carcinoma) are shown as percentages of tissue surface area and categorized from 1+ (low) to 3+ (high). (B) Mean (±SEM) tumor volume in gastric PDX tumor model CTG-1234 after treatment with BisG1-FAPα-FEAL / DR4-FEAR (0.5-8 mg / kg, administered once weekly for 3 weeks) or BisG1-DR4-FEAL / b12-FEAR (8 mg / kg). (C) Kaplan-Meier curve showing progression-free survival (PFS) of CTG-1234 tumor-bearing mice, where PFS is defined as the percentage of mice with a tumor volume of less than 1000 mm3. (D) Individual mouse tumor volume and mean ± SEM for each treatment group in the gastric cancer PDX CTG-1234 model on day 42, the final day when all groups completed the procedure. *** = p < 0.001 vs control BisG1-DR4-FEAL / b12-FEAR treatment group (Mann-Whitney). (E) Mean (± SEM) mouse body weight for all treatment groups. (F) Individual mouse tumor volume and mean ± SEM for each treatment group in the pancreatic PDX tumor model CTG-1150 on day 25, the final day when all groups completed the procedure. * = p < 0.05 vs control BisG1-DR4-FEAL / b12-FEAR treatment group (Mann-Whitney). (G) Kaplan-Meier curve showing PFS in CTG-1150 tumor-bearing mice, where PFS is defined as the percentage of mice with a tumor volume of less than 500 mm3. (H) Mean (±SEM) tumor volume per treatment group over time in the pancreatic PDX tumor model CTG-1150. (I) Mean (±SEM) body weight for all treatment groups. [Figure 9-2]Evaluation of BisG1-FAPα-FEAL / DR4-FEAR antitumor activity in vivo. (A) FAPα staining in formalin-fixed paraffin-embedded (FFPE) tissue obtained from PDX models. (A) CTG-1234 (gastric cancer), CTG-1150 (pancreatic cancer), and positive control (invasive ductal carcinoma) are shown as percentages of tissue surface area and categorized from 1+ (low) to 3+ (high). (B) Mean (±SEM) tumor volume in gastric PDX tumor model CTG-1234 after treatment with BisG1-FAPα-FEAL / DR4-FEAR (0.5-8 mg / kg, administered once weekly for 3 weeks) or BisG1-DR4-FEAL / b12-FEAR (8 mg / kg). (C) Kaplan-Meier curve showing progression-free survival (PFS) of CTG-1234 tumor-bearing mice, where PFS is defined as the percentage of mice with a tumor volume of less than 1000 mm3. (D) Individual mouse tumor volume and mean ± SEM for each treatment group in the gastric cancer PDX CTG-1234 model on day 42, the final day when all groups completed the procedure. *** = p < 0.001 vs control BisG1-DR4-FEAL / b12-FEAR treatment group (Mann-Whitney). (E) Mean (± SEM) mouse body weight for all treatment groups. (F) Individual mouse tumor volume and mean ± SEM for each treatment group in the pancreatic PDX tumor model CTG-1150 on day 25, the final day when all groups completed the procedure. * = p < 0.05 vs control BisG1-DR4-FEAL / b12-FEAR treatment group (Mann-Whitney). (G) Kaplan-Meier curve showing PFS in CTG-1150 tumor-bearing mice, where PFS is defined as the percentage of mice with a tumor volume of less than 500 mm3. (H) Mean (±SEM) tumor volume per treatment group over time in the pancreatic PDX tumor model CTG-1150. (I) Mean (±SEM) body weight for all treatment groups. [Figure 10]Evaluation of in vivo follow-up of BisG1-FAPα-FERL / DR4-FERR antitumor activity. Mice with established tumors were administered the indicated antibody concentrations by IV injection in 3 quarter weeks (n=8 mice per group). (A) Mean (±SEM) tumor volume in the gastric PDX tumor model CTG-1234 after treatment with BisG1-b12-FERL / DR4-FERR (2 mg / kg), BisG1-FAPα-FERL / DR4-FERR (0.1, 0.5, or 2 mg / kg) or equimolar doses of RG7386 (0.16, 0.8, or 3.2 mg / kg). (B) Tumor volume of individual mice and mean ±SEM for each group in the gastric cancer PDX CTG-1234 model on day 12, the final day when all groups completed the procedure. ***=p<0.001 vs control BisG1-b12-FERL / DR4-FERR treatment group (Mann-Whitney)**p<0.01 vs BisG1-FAPα-FERL / DR4-FERR 0.5 or 2 mg / kg treatment group (Mann-Whitney). (C) Kaplan-Meier curves showing progression-free survival (PFS) of CTG-1234 tumor-bearing mice (PFS is defined as the percentage of mice with a tumor volume of less than 750 mm3). [Figure 11] Antitumor activity (A-E) in a multi-organ metastatic mouse model was plotted for each organ for the indicated treatment group, based on ex vivo bioluminescence imaging (BioLI) measurements (counts per minute (cpm) / cm2, Log10 scale) of tumor volume from each mouse. For statistical analysis, paired t-tests were performed, with p<0.05 considered statistically significant. The data shown are box plots containing individual data with median values ​​for all animals for each treatment group (n=9), including whiskers from minimum to maximum. *p≦0.05, **p≦0.01(F) DR4 activation was measured by cleavage caspase-3 IHC staining of FFPE sections from resected primary tumors of the cecum and metastatic tissues of the peritoneal wall and liver. Mean percentage (±SEM) of annotated tumor area scored as positive for cleavage caspase-3 in all analyzed sections is shown for each treatment group. ns = No significance, ***P≦0.001 ****P≦0.0001 (Mann-Whitney). [Figure 12] Evaluation of hepatotoxicity using human liver spheroids. Liver spheroid survival was determined by measuring LDH release (an indicator of plasma membrane damage) after 4 days of culture with BisG1-FAPα-FEAL / DR4-FEAR, ABBV-621-Fc fusion, RG7386, IgG1-b12-FEAR, or IgG1-b12 antibody (A and C), and intracellular ATP (an indicator of metabolically active cells) levels after 6 days (B) or 7 days (D) of culture. The dashed line represents the limit of detection (LLOD) of LDH. The data shown are the mean ± SEM of 4 technical replicates for each condition of bioluminescence signaling. [Figure 13] Cytotoxicity in co-cultures of tumor cells with different ratios of NIH / 3T3-FAPα cells. CellTiter-Glo survival data of (A) DLD-1 or (B) MDA-MB-231 tumor cells co-cultured with NIH / 3T3-FAPα cells in different ratios of tumor cells to NIH / 3T3-FAPα cells in the presence of BisG1-FAPα-FERL / DR4-FERR. Data are shown as mean (±SD)% viable cells plotted against antibody concentration, with overlaps from one representative of three independent experiments. [Figure 14] Cytotoxicity in co-cultures of PDOs with FAPα knockdown CAFs. Co-cultures of CRC-derived PDOs (A) Tor9, (B) p19B, (C) Hub096, and (D) Hub098 with CAFs transduced with FAPα shRNA (CAF#34) or non-target shRNA (CAF#Scr) were treated with BisG1-FAPα-FERL / DR4-FERR or the negative control antibody BisG1-b12-FERL / b12-FERR. CellTiter-Glo survival data are shown as mean ± SD cell survival (%) plotted against antibody concentration for each experiment. [Figure 15]Cytotoxicity and DR4 transactivation in the presence of soluble FAPα. CellTiter-Glo cytotoxicity data of (A) DLD-1 and (B) MDA-MB-231 tumor cell lines in monocultures in the presence of soluble FAPα (29.4 nM) and BisG1-FAPα-FERL / DR4-FERR. Data are shown as mean ± SEM% viable cells plotted against antibody concentration for 2-3 independent experiments. [Figure 16] Effects on FAPα Enzyme Activity: Fluorescent dipeptidyl peptidase substrates were incubated in the presence of recombinant human FAPα with BisG1-FAPα-FERL / DR4-FERR, BisG1-b12-FERL / b12-FERR, or the positive control, the chemical dipeptidyl peptidase inhibitor Talabostat. Fluorescence 7-amino-4-methylcoumarin (AMC) was measured as a readout of FAPα dipeptidyl peptidase activity. The data shown are AMC concentrations plotted against antibody concentrations for one representative of the two experiments. [Figure 17] C1q binding to cell membrane-bound BisG1-FAPα-FERL / DR4-FERR: (A) The binding of BisG1-FAPα-FERL / DR4-FERR and IgG1-b12-FER unbound control antibodies to MDA-MB-231 cells, and (B) the binding of C1q to BisG1-FAPα-FERL / DR4-FERR bound to the cell surface, were determined by flow cytometry using MDA-MB-231 cells in the presence of 20% NHS. The data shown are gMFI values ​​determined by flow cytometry for one representative experiment out of three independent experiments. [Figure 18] Binding of BisG1-FAPα-FERL / DR4-FERR to immobilized FcγRs: The binding of BisG1-FAPα-FERL / DR4-FERR to immobilized recombinant human FcγRs (FcγRIa, FcγRIIa-H131, FcγRIIa-R131, FcγRIIb, FcγRIIIa-F158, FcγRIIIa-V158) was analyzed by SPR. For each experiment, the relative binding response was plotted against antibody concentration. [Figure 19] Binding of BisG1-FAPα-FERL / DR4-FERR to immobilized FcRn The binding of BisG1-FAPα-FERL / DR4-FERR to immobilized recombinant human FcRn at pH 6.0 (A) and pH 7.4 (B) was analyzed by SPR. The sensogram shows raw data from one representative run out of two runs (pH 6.0) or four runs (pH 7.4) performed as a dashed line, and the curve fitting as a solid black line. [Figure 20] Pharmacokinetic Profile of BisG1-FAPα-FERL / DR4-FERR in Non-Tumor-Bearing Mice A single dose of 2 mg / kg of BisG1-FAPα-FERL / DR4-FERR was intravenously injected into mice, and the total human IgG concentration in plasma samples was determined by ECLIA. The data shown are the mean plasma IgG concentrations over time after treatment in C57BL / 6 SCID mice (n=1) and hFcRn SCID mice (n=3). The 95% confidence interval for the pharmacokinetic profile of wild-type IgG1 in hFcRn SCID mice is shown in the gray shaded area. [Modes for carrying out the invention]

[0022] definition As used herein, the term “antibody” is intended to refer to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or any derivative thereof, which, under typical physiological and / or tumor-specific conditions, has the ability to specifically bind to an antigen for a significant period, e.g., a half-life of at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours or more, at least about 48 hours or more, or any other relevant functionally defined period, e.g., a period sufficient to induce, promote, enhance and / or modulate a physiological response associated with the binding of the antibody to the antigen. The binding region (or binding domain as may be used herein, both having the same meaning) that interacts with the antigen includes variable regions of both the heavy and light chains of the immunoglobulin molecule. The constant region (Ab) of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system, such as C1q, the first component in the classical pathway of complement activation. Alternatively, the constant region of an antibody may be silenced, for example, by mutation, thereby preventing them from activating the complement system, or at least activating it less efficiently. The term “antibody” includes antibody-like polypeptides such as monoclonal antibodies (mAbs), chimeric antibodies, and humanized antibodies that retain the ability to specifically bind to an antigen (antigen-binding fragment) and to be conjugated to a toxin, provided by any known technology, e.g., enzymatic cleavage, peptide synthesis, and recombinant technology, as well as “antibody fragment” or “its fragment.” An antibody as defined herein may have any isotype unless otherwise disclosed herein. As stated above, the term “antibody” as used herein includes a fragment of an antibody that specifically interacts with, for example, an antigen, or retains the ability to bind to an antigen, unless otherwise specified or unless otherwise clearly inconsistent with the context. It has been shown that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies.Examples of binding fragments encompassed by the term "antibody" include: (i) monovalent fragments consisting of Fab' or Fab fragments, light chain variable domain (VL), heavy chain variable domain (VH), light chain constant region (CL), and heavy chain constant region domain 1 (CH1) domain, or monovalent antibodies described in International Publication No. 2007 / 059782; (ii) bivalent fragments containing F(ab')2 fragments, two Fab fragments linked by disulfide crosslinking at the hinge region; (iii) Fd fragments essentially consisting of the VH domain and CH1 domain; (iv) Fv fragments essentially consisting of the VL domain and VH domain of a single arm of the antibody; (v) fragments essentially consisting of the VH domain, also called domain antibodies (Holt et al; Trends Biotechnol. 2003 Nov; 21(11): 484-90); and dAb fragments (Ward et al., Nature (vi) camelids or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5(1): 111-24), and (vii) isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they may be linked by a synthetic linker that allows the VL and VH regions to be paired and produced as a single protein chain forming a monovalent molecule (known as a single-chain antibody or single-chain Fv (scFv)) using recombination. See, for example, Revets et al; Expert Opin Biol Ther. 2005 Jan; 5(1): 111-24, and Bird et al., Science 242, 423-426 (1988). Such single-chain antibodies are encompassed by the term antibody unless otherwise specified or clearly indicated by the context. While such fragments generally fall within the scope of the meaning of antibody, they are unique features of the present invention, exhibiting different biological properties and utility responsibilities both collectively and independently. These, and other useful antibody fragments in the context of the present invention, will be discussed further herein.Antibodies can be produced and recovered from different in vitro or ex vivo expression or production systems, such as recombinant modified host cells, from hybridomas or systems that use cell extracts that assist in in vitro transcription and / or translation of nucleic acid sequences encoding antibodies. It should be understood that a number of different antibodies, as defined in the context of this invention, can be provided by separately producing each antibody in the aforementioned production systems and then mixing the antibodies, or by producing several antibodies in the same production system.

[0023] As used herein, the terms “immunoglobulin heavy chain” or “heavy chain of immunoglobulin” are intended to refer to one of the heavy chains of immunoglobulins. A heavy chain typically consists of a variable region (abbreviated herein as VH) and a constant region (abbreviated herein as CH) that define the isotype of the immunoglobulin. The constant region typically consists of three domains: CH1, CH2, and CH3. As used herein, the term “immunoglobulin” is intended to refer to a structurally related class of glycoproteins consisting of two pairs of polypeptide chains, all four of which are potentially interconnected by disulfide bonds: one pair of light (L) low molecular weight chains and one pair of heavy (H) chains. The structure of immunoglobulins is well characterized (e.g., Fundamental Immunology Ch.7 (Paul, W., 2nd ed. Raven)). See Press, NY (1989). Within the structure of immunoglobulins, two heavy chains are linked to each other via disulfide bonds in a so-called “hinge region.” Similar to the heavy chains, each light chain typically consists of several regions: a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically consists of one domain, CL. Furthermore, the VH and VL regions can be further subdivided into hypervariable regions (or hypervariable regions that can be hypervariable in the form of sequencely and / or structurally defined loops), also called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The CDR sequence is IMGT (Lefranc MP. et al., Nucleic Acids). It is defined according to Research, 27, 209-212, 1999 and Brochet X. Nucl. Acids Res. 36, W503-508 (2008).

[0024] As used herein, the terms “halving,” “Fab arm,” and “arm” refer to a single heavy-light chain pair. Where a bispecific antibody is described as comprising a hapmon antibody “derived from” a first antibody and a hapmon antibody “derived from” a second antibody, the term “derived from” indicates that the bispecific antibody was produced by recombining the hapmones from each of the first and second antibodies into the resulting bispecific antibody by any known method. In this context, “recombination” is not intended to be limited to any particular recombination method and therefore includes all methods for producing bispecific antibodies described below herein, including, for example, recombination by hapmon exchange, as well as recombination at the nucleic acid level, and / or recombination by co-expression of two hapmones in the same cell.

[0025] As used herein, the terms “antigen-binding region” or “binding region” refer to a region of an antibody that can bind to an antigen and contains an epitope. The antigen may be any molecule, such as a polypeptide present on a cell, bacterium, or virion. The terms “antigen” and “target” may be used interchangeably in the context of this invention, provided that the context does not conflict with the terms. The terms “antigen-binding region” and “antigen-binding site” may be used interchangeably in the context of this invention, provided that the context does not conflict with the terms.

[0026] The term "epitope" refers to an antigenic determinant that is specifically bound by an antibody. Epitopes typically consist of surface groupings of molecules, such as amino acids, sugar side chains, or combinations thereof, and usually possess specific three-dimensional structural and charge properties. Conformational and non-conformational epitopes are distinguished in that binding to the former is lost in the presence of a denaturing solvent, while binding to the latter is not. Epitopes may include amino acid residues directly involved in binding, and other amino acid residues not directly involved in binding, such as those that, when bound to the antigen, are effectively blocked or coated by the antibody (in other words, the amino acid residues are within or close to the footprint of the specific antibody).

[0027] The antibody-binding region can be determined by epitope binning using biolayer interferometry, by alanine scanning, or by a shuffle assay (using an antigen construct in which a region of the antigen is exchanged with a region of another species to determine whether the antibody still binds to the antigen). The amino acids within the antibody-binding region involved in the interaction with the antibody can be determined by hydrogen / deuterium exchange mass spectrometry and the crystallography of the antibody bound to the antigen.

[0028] The terms "blocking binding," "blocking antibody binding," "cross-blocking binding," or "cross-blocking binding" refer to a situation where one antibody bound to a particular antigen prevents another antibody from binding to the same antigen. In the absence of the other antibody, each antibody has the ability to bind to the antigen as determined by a significant binding response; however, in the presence of the other antibody, one of the antibodies lacks a binding response. This type of behavior indicates that both antibodies are binding to substantially overlapping epitopes on the antigen. The ability of one antibody to block the binding of another can be determined, for example, by biolayer interference in a classic sandwich epitope binning assay format, as described in Abdiche et al. (Abdiche YN, Malashock DS, Pinkerton A, Pons J. Exploring blocking assays using Octet, ProteOn, and Biacore biosensors. Anal Biochem. 2009;386(2):172-180). In short, a sandwich epitope binning assay tests the binding of an antibody in solution to its specific antigen, which is initially captured via an immobilized antibody. In the context of this invention, one antibody does not block the binding of another antibody if it can "substitute" for another antibody; that is, one antibody dissociates from the antigen when another antibody binds to it. The terms "block binding," "blocking antibody binding," "cross-blocking binding," and "cross-blocking binding" may be used interchangeably in the context of this invention, provided that the context does not contradict each other. Preferably, the ability of one antibody to block the binding of another antibody is determined using a full-length antibody.

[0029] As used herein, the term “bonding” is typically 1E when determined by biolayer interferometry. -6 M or less, for example, 5E -7 M or less, 1E -7 M or less, for example, 5E -8 M or less, for example, 1E -8Less than M, for example 5E -9 Less than M, for example 1E -9 Less than M, for example 5E -10 Less than M, for example 1E -10 Less than M, for example 5E -11 Less than M, for example 1E -11 Less than M, for example 5E -12 Less than M, or for example 1E -12 K less than M D Refers to the binding of an antibody to a predetermined antigen or target with a binding affinity corresponding thereto.

[0030] As used herein, the term "K" D [[ID=2,4]]"(M)" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, and is obtained by dividing k d by k[[ID=2,7]] a

[0031] As used herein, the term "k" d "(sec" -1 )" refers to the dissociation rate constant of a specific antibody-antigen interaction. The said value is also called the k off value or off-rate.

[0032] As used herein, the term "k" a "(M" -1 x sec" -1 )" refers to the association rate constant of a specific antibody-antigen interaction. The said value is also called the k on value or on-rate.

[0033] As used herein, the term "cis binding" refers to the simultaneous binding of a multispecific antibody to different targets on the same cell. For example, a bispecific antibody can bind to its two targets on the same cell.

[0034] As used herein, the term "trans binding" refers to the simultaneous binding of a multispecific antibody to different targets on different cells. For example, a bispecific antibody can bind two cells by binding to one target on a first cell and another target on a second cell.

[0035] ​As used herein, the term "FAPα" refers to a protein titled fibroblast-activating protein α, which is an enzyme encoded by the FAP gene and is also known as a surface-expressed protease (seplase), serine endometrial protease (SIMP), dipeptidyl peptidase FAP, prolyl endopeptidase FAP, and endometrial serine protease. It is a 170 kDa transmembrane protein. In humans (Homo sapiens), the FAPα protein has the amino acid sequence shown in SEQ ID NO: 33 ([Prolyl Endopeptidase FAP]: Uniprot accession number [Q12884]). In the amino acid sequence shown in SEQ ID NO: 33, amino acid residues [1-4] are cytoplasmic peptides, amino acid residues [5-25] are transmembrane peptides, and amino acid residues [26-760] are extracellular polypeptides. In cynomolgus monkeys (Macaca fascicularis), the FAPα protein has the amino acid sequence shown in SEQ ID NO: 39 (Uniprot accession number A0A2K5VGF4), with amino acid residues [1-760] being a mature polypeptide. In mice (Mus musculus), the FAPα protein has the amino acid sequence shown in SEQ ID NO: 35 (Uniprot accession number P97321), with amino acid residues [1-761] being a mature polypeptide. In rats (Rattus norvegicus), the FAPα protein has the amino acid sequence shown in SEQ ID NO: 36 (Uniprot accession number Q8R492), with amino acid residues [1-761] being a mature polypeptide. In dogs (Canis lupus familiaris), the FAPα protein has the amino acid sequence shown in SEQ ID NO: 37 (Uniprot accession number A0A8C0NKP1), with amino acid residues [1-760] being a mature polypeptide. In pigs (Sus scrofa), the FAPα protein has the amino acid sequence shown in SEQ ID NO: 38 (Uniprot accession number K7GQN2), and amino acid residues [1-760] are mature polypeptides.

[0036] As used herein, the term "DR4" refers to the protein titled Death Receptor 4, which is the receptor for the cytotoxic ligand TNFSF10 / TRAIL and is also known as Tumor Necrosis Factor Receptor Superfamily Member 10A, APO2, CD261, and TNF-Related Apoptosis-Inducing Ligand Receptor 1 (TRAIL Receptor 1; TRAIL-R1). It is a 56 kDa transmembrane protein. In humans (Homo sapiens), the DR4 protein has the amino acid sequence shown in SEQ ID NO: 68 (Tumor Necrosis Factor Receptor Superfamily Member 10A). In the amino acid sequence shown in SEQ ID NO: 68, amino acid residues [1-23] are the signal peptide and amino acid residues [24-468] are the mature polypeptide. In cynomolgus monkeys (Macaca fascicularis), the DR4 protein has the amino acid sequence shown in SEQ ID NO: 69. In the amino acid sequence shown in SEQ ID NO: 69, amino acid residues [1-23] are the signal peptide, and amino acid residues [24-471] are the mature polypeptide.

[0037] As used herein, the term "CAF" refers to cancer-associated fibroblasts, which are a heterogeneous population of stromal cells with mesenchymal cell lineages present in the tumor microenvironment and coexisting with the growing tumor mass. CAFs are spindle-shaped cells that construct and reconstruct the extracellular matrix structure. The definition of CAF is based on a combination of morphological features, biomarkers, and genetic mutations.

[0038] As used herein, the term “tumor microenvironment” or “TME” refers to the ecosystem surrounding a tumor in the body. The TME is a complex and dynamic environment that influences tumor growth, invasion, and metastasis. The tumor and TME constantly interact and influence each other both positively and negatively. The TME includes immune cells, extracellular matrix, blood vessels, and stromal cells.

[0039] As used herein, terms such as “monoclonal antibody,” “monoclonal Ab,” “monoclonal antibody composition,” and “mAb” refer to preparations of antibody molecules in single-molecule compositions. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a specific epitope. Therefore, the term “human monoclonal antibody” refers to an antibody exhibiting single binding specificity, having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced by hybridomas, including B cells obtained from transgenic mice, which are transgenic or transchromosomal non-human animals having a genome containing human heavy and light chain transgenes fused to immortalized cells. Monoclonal antibodies can also be produced from recombinantly modified host cells or from systems using cell extracts that assist in in vitro transcription and / or translation of nucleic acid sequences encoding antibodies.

[0040] As used herein, the term “isotype” refers to an immunoglobulin class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by a heavy chain constant region gene, or any allotype thereof, e.g., IgG1m(za) and IgG1m(f)). Furthermore, each heavy chain isotype can be combined with either a kappa (κ) or lambda (λ) light chain.

[0041] As used herein, the term “allotype” refers to an amino acid variation within a single isotype class of the same species. The main allotypes of antibody isotypes differ among ethnic individuals. Known allotype variations within the heavy chain IgG1 isotype result from four amino acid substitutions within the antibody frame. In one embodiment, the antibody of the present invention is of the IgG1m(f) allotype as defined by SEQ ID NO: 21. In one embodiment of the present invention, the first and second antibodies of the present invention are of the IgG1m(f) allotype as defined by SEQ ID NO: 21, with at least one amino acid substitution introduced. In one embodiment of the present invention, the first and second antibodies of the present invention are of the IgG1m(f) allotype as defined by SEQ ID NO: 21, with up to five amino acid substitutions introduced, e.g., four amino acid substitutions, e.g., three amino acid substitutions, e.g., two amino acid substitutions.

[0042] When used in relation to an antibody, the term "full-length" indicates that the antibody is not a fragment but contains all of the specific isotype domains typically found in its native isotype, e.g., the VH, CH1, CH2, CH3, hinge, VL, and CL domains of an IgG1 antibody. In some embodiments, when used herein in relation to an antibody, the term "full-length" refers to an antibody (e.g., a parent antibody or a variant antibody) that comprises one or two pairs of heavy and light chains, each containing all of the heavy and light chain constant and variable domains typically found in the heavy-light chain pair of a wild-type antibody of that isotype. In a full-length variant antibody, the heavy and light chain constant and variable domains may include amino acid substitutions that improve the functional properties of the antibody compared to a full-length parent antibody or wild-type antibody. These include substitutions to reduce antibody effector function and substitutions to facilitate the construction of multispecific antibodies, such as bispecific antibodies. The full-length antibody according to the present invention can be prepared by a method comprising (i) cloning a CDR sequence into a suitable vector containing a complete heavy chain sequence and a complete light chain sequence, and (ii) expressing the complete heavy chain and light chain sequences in a suitable expression system. Preparing a full-length antibody starting from either a CDR sequence or a complete variable region sequence is within the knowledge of those skilled in the art.

[0043] As used herein, the term “human antibody” is intended to include antibodies having variable regions and framework regions derived from human germline immunoglobulin sequences and human immunoglobulin constant domains. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions, or deletions introduced by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo). However, as used herein, the term “human antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another non-human species, such as mouse, are grafted onto human framework sequences.

[0044] As used herein, the term “Fc-mediated effector function” is intended to refer to the function that is the consequence of a polypeptide or antibody binding to its target or antigen on the cell membrane, and Fc effector function is attributable to the Fc region of the polypeptide or antibody. Examples of Fc effector functions include (i) C1q binding, (ii) complement activation, (iii) complement-dependent cytotoxicity (CDC), (iv) antibody-dependent cell-mediated cytotoxicity (ADCC), (v) Fc-gamma receptor binding, (vi) antibody-dependent cytophagocytosis (ADCP), (vii) complement-dependent cytotoxicity (CDCC), (viii) complement-enhancing cytotoxicity, (ix) antibody-mediated binding of opsonized antibodies to complement receptors, (x) opsonization, and (xi) any combination of (i) to (x).

[0045] As used herein, the term “hinge region” refers to the hinge region of an immunoglobulin heavy chain. For example, the hinge region of a human IgG1 antibody corresponds to amino acids 216–230 according to the Eu numbering shown in Kabat (Kabat, EA et al., Sequences of proteins of immunological interest. 5th Edition - U.S. Department of Health and Human Services, NIH Publication No. 91-3242, pp. 662, 680, 689 (1991)). However, the hinge region may be any of the other subtypes described herein.

[0046] As used herein, the terms “CH1 region” or “CH1 domain” refer to the CH1 region of an immunoglobulin heavy chain. For example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118–215 by Eu numbering as shown in Kabat (ibid.). However, the CH1 region may be any of the other subtypes described herein.

[0047] As used herein, the terms “CH2 region” or “CH2 domain” refer to the CH2 region of an immunoglobulin heavy chain. For example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231–340 by Eu numbering as shown in Kabat (ibid.). However, the CH2 region may be any of the other subtypes described herein.

[0048] As used herein, the terms “CH3 region” or “CH3 domain” refer to the CH3 region of an immunoglobulin heavy chain. For example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341–447 by Eu numbering as shown in Kabat (ibid.). However, the CH3 region may be any of the other subtypes described herein.

[0049] As used herein, the terms “inactive,” “inactive,” or “deactivated” refer to an Fc region that is unable to bind to any Fc gamma receptor (FcgR), unable to induce Fc-mediated crosslinking of FcgR, or unable to induce FcgR-mediated crosslinking of a target antigen via two Fc regions of an individual antibody, or unable to bind to C1q. The inactivity of an antibody’s Fc region can be tested using antibodies in monospecific or bispecific format.

[0050] In the context of this invention, the term "monovalent antibody" refers to an antibody molecule that can interact with a specific epitope on an antigen by only one antigen-binding domain (e.g., one Fab arm). In the context of bispecific antibodies, "monovalent antibody binding" refers to the binding of a bispecific antibody to a single specific epitope on an antigen by only one antigen-binding domain (e.g., one Fab arm).

[0051] In the context of this invention, the term "monospecific antibody" refers to an antibody that has binding specificity to only one epitope. An antibody may be a monovalent antibody of monospecificity (i.e., having only one antigen-binding region) or a bivalent antibody of monospecificity (i.e., having two identical antigen-binding regions). Therefore, an antibody may be a monospecific antibody with monovalent binding (i.e., having only one antigen-binding region) or a monospecific antibody with bivalent binding (i.e., having two identical antigen-binding regions).

[0052] The term "bispecific antibody" refers to an antibody having two non-identical antigen-binding domains, for example, two non-identical Fab arms or two Fab arms having non-identical CDR regions. In the context of this invention, a bispecific antibody has specificity for at least two different epitopes. Such epitopes may lie on the same or different antigens or targets. If the epitopes lie on different antigens, such antigens may lie on the same or different cells, cell types, or structures, such as the extracellular matrix or vesicles and soluble proteins. Therefore, a bispecific antibody can crosslink multiple antigens, for example, two different cells.

[0053] The term "bivalent antibody" refers to an antibody having two antigen-binding regions that bind to one or two epitopes on a target or antigen, or to one or two epitopes on the same antigen. Therefore, a bivalent antibody can be a monospecific bivalent antibody or a bispecific bivalent antibody; that is, a bivalent antibody can be a monospecific antibody with a bivalent bond or a bispecific antibody with a bivalent bond. In one embodiment, a bispecific antibody with a bivalent bond is a bispecific antibody having a monovalent bond to a first target and a monovalent bond to a second target.

[0054] The term "multispecific antibody" refers to an antibody having two or more non-identical antigen-binding domains, for example, two or more non-identical Fab arms or two or more Fab arms having non-identical CDR regions. In the context of the present invention, a multispecific antibody has specificity for at least two different epitopes. Such epitopes may lie on the same or different antigens or targets. If the epitopes lie on different antigens, such antigens may lie on the same or different cells, cell types, or structures, such as the extracellular matrix or vesicles and soluble proteins. Therefore, a multispecific antibody can crosslink multiple antigens, for example, two different cells.

[0055] The terms “amino acid” and “amino acid residue” may be used interchangeably herein and should not be understood as limiting. Amino acids are organic compounds containing amine (-NH2) and carboxyl (-COOH) functional groups, along with side chains (R groups) specific to each amino acid. In the context of this invention, amino acids can be classified based on their structure and chemical properties. Therefore, the classes of amino acids may be reflected in one or both of the following tables.

[0056] [Table 1]

[0057] [Table 2]

[0058] The substitution of one amino acid with another can be classified as either a conserved or non-conserved substitution. In the context of this invention, a “conserved substitution” is the substitution of one amino acid with another amino acid having similar structural and / or chemical characteristics, and such substitution of one amino acid residue is a substitution with another amino acid residue of the same class as defined in either of the two tables above. For example, leucine can be substituted with isoleucine, since both are aliphatic branched hydrophobic. Similarly, aspartic acid can be substituted with glutamic acid, since both are small, negatively charged residues.

[0059] In the context of the present invention, substitution in an antibody is expressed as follows:

[0060] Original amino acid - position - substituted amino acid Referring to the well-known nomenclature for amino acids, a three-letter or one-letter code is used that includes the code "Xaa" or "X" to indicate any amino acid residue. Thus, Xaa or X can typically represent any of the 20 naturally occurring amino acids. As used herein, the term "naturally occurring" refers to any one of the following amino acid residues: glycine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, asparagine, glutamic acid, glutamine, proline, tryptophan, phenylalanine, tyrosine, methionine, and cysteine. Therefore, the notation "K409R" or "Lys409Arg" means that the antibody contains a substitution of lysine with arginine at amino acid position 409.

[0061] Substitution of an amino acid at a given position with any other amino acid is, The original amino acid-position, or, for example, "K409".

[0062] In modifications where the original amino acid and / or substituted amino acid may contain multiple but not all amino acids, the multiple amino acids are separated by a comma or a slash. For example, the substitution of lysine with arginine, alanine, or phenylalanine at position 409 is: The sequence is either "Lys409Arg, Ala, Phe" or "Lys409Arg / Ala / Phe" or "K409R, A, F" or "K409R / A / F" or "K409 to R, A or F".

[0063] Such designations may be used interchangeably in the context of the present invention, but they have the same meaning and purpose.

[0064] Furthermore, the term “substitution” encompasses substitutions to any one or any other 19 native amino acids, or to other amino acids such as non-natural amino acids. For example, substitutions of amino acid K at position 409 include each of substitutions 409A, 409C, 409D, 409E, 409F, 409G, 409H, 409I, 409L, 409M, 409N, 409Q, 409R, 409S, 409T, 409V, 409W, 409P, and 409Y. This is, by the way, equivalent to the symbol 409X, where X represents any amino acid other than the original amino acid. These substitutions may also be called K409A, K409C, et al., or K409A, C, et al., or K409A / C / et al. The same applies equally to all positions each referred to herein, and any one of such substitutions is specifically included herein.

[0065] As used herein, the term “amino acid corresponding to position…” refers to the amino acid position number of the human IgG1 heavy chain. The corresponding amino acid positions in other immunoglobulins can be found by alignment with human IgG1. Therefore, an amino acid or segment in one sequence that “corresponds” to an amino acid or segment in another sequence is aligned with the other amino acid or segment using a standard sequence alignment program. Methods for aligning sequences or segments within sequences to determine the positions in the sequence corresponding to the amino acid positions according to the present invention are considered well known in the art.

[0066] As used herein, the term “host cell” is intended to refer to a cell into which an expression vector has been introduced. It should be understood that such a term is intended to refer not only to a specific target cell but also to the offspring of such a cell. Such offspring may not be identical to the parent cell in fact, as certain modifications may occur in subsequent generations due to either mutation or environmental influences, but they are still included within the scope of the term “host cell” as used herein. Examples of recombinant host cells include transfectomas, e.g., CHO cells, HEK-293 cells, Expi293F cells, PER.C6 cells, NS0 cells, and lymphocytic cells, as well as prokaryotic cells, e.g., Escherichia coli, and other eukaryotic hosts, e.g., plant cells and fungi.

[0067] As used herein, the term “transfectoma” includes recombinant eukaryotic host cells expressing antibodies or target antigens, such as CHO cells, PER.C6 cells, NS0 cells, HEK-293 cells, Expi293F cells, plant cells, or fungi including yeast cells.

[0068] For the purposes of this invention, the "sequence identity" between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453), which is implemented in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 5.0.0 or later of the Needle program. The parameters used are a gap-open penalty of 10, a gap-extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output labeled "longest identity" (obtained using the -nobrief option) is used as the identity percentage and is calculated as follows:

[0069] (Number of identical residues × 100) / (Length of alignment - Total number of gaps in the alignment) The retention of similar residues may further, or alternatively, be measured by a similarity score determined by using the BLAST program (e.g., BLAST 2.2.8, available from NCBI, using standard settings BLOSUM62, Open Gap=11, Extended Gap=1). A suitable variant typically exhibits similarity to the parent sequence of at least about 45%, e.g., at least about 55%, at least about 65%, at least about 75%, at least about 85%, at least about 90%, at least about 95%, or higher (e.g., about 99%).

[0070] As used herein, the terms “internalized” or “internalization” refer to the biological process by which molecules such as antibodies according to the present invention are engulfed by the cell membrane and drawn into the interior of the cell. Internalization is sometimes also called “endocytosis.”

[0071] multispecific antibodies In a first embodiment, the present invention relates to a multispecific antibody comprising at least (i) an FAPα-binding region including a first heavy chain variable region and a first light chain variable region, and (ii) a DR4-binding region including a second heavy chain variable region and a second light chain variable region. In one embodiment, the FAPα-binding region can bind to FAPα. In a further embodiment, the DR4-binding region can bind to DR4.

[0072] In a further embodiment, the present invention relates to a multispecific antibody comprising at least (i) an FAPα-binding region capable of binding to FAPα including a first heavy chain variable region and a first light chain variable region, and (ii) a DR4-binding region capable of binding to DR4 including a second heavy chain variable region and a second light chain variable region.

[0073] The present invention further provides a multispecific antibody as described herein, which is a bispecific antibody having monovalent binding to FAPα and monovalent binding to DR4. In a further embodiment, the present invention relates to a bispecific antibody having monovalent binding to FAPα (e.g., one Fab arm binding to FAPα) and monovalent binding to DR4 (e.g., one Fab arm binding to DR4).

[0074] As is known to those skilled in the art, each antigen-binding region of an antibody generally comprises a heavy chain variable region (VH) and a light chain variable region (VL), each of which may contain three CDR sequences, CDR1, CDR2, and CDR3, and four framework sequences, FR1, FR2, FR3, and FR4, respectively. This structure is preferably also found in antibodies according to the present invention. In one embodiment, one, two, three, or all of the four framework sequences are human framework sequences. The CDR1, CDR2, and CDR3 regions can be identified from the variable heavy chain region and the variable light chain region using methods known in the art.

[0075] The FAPα-binding region of a multispecific antibody may include a heavy chain variable region (VH) containing three complementarity-determining regions, CDR1, CDR2, and CDR3, located within the amino acid sequence shown in SEQ ID NO: 13. The FAPα-binding region of a multispecific antibody may also include a light chain variable region (VL) containing three complementarity-determining regions, CDR1, CDR2, and CDR3, located within the amino acid sequence shown in SEQ ID NO: 14. In one embodiment, the FAPα-binding region of a multispecific antibody described herein includes a heavy chain variable region (VH) containing three complementarity-determining regions, CDR1, CDR2, and CDR3, located within the amino acid sequence shown in SEQ ID NO: 13, and a light chain variable region (VL) containing three complementarity-determining regions, CDR1, CDR2, and CDR3, located within the amino acid sequence shown in SEQ ID NO: 14.

[0076] This specification further discloses multispecific antibodies in which the FAPα-binding region comprises a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. Also disclosed here are multispecific antibodies in which the FAPα-binding region comprises a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. In one embodiment, the FAPα-binding region of a multispecific antibody comprises a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 1, 2, and 3, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 4, 5, and 6, respectively. The CDR regions from the aforementioned variable heavy chain region and variable light chain region are annotated according to IMGT (see Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, Developmental and Comparative Immunology, 27(1), 55-77 (2003)).

[0077] The Disclosure further provides a multispecific antibody in which the VH sequence of the FAPα-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 13. The Disclosure also provides a multispecific antibody in which the VL sequence of the FAPα-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 14. In a further embodiment, the VH sequence of the FAPα-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 13, and the VL sequence of the FAPα-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 14.

[0078] Furthermore, the multispecific antibody may further include a framework region of the VH sequence of the FAPα-binding region having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 13. Furthermore, the multispecific antibody may further include a framework region of the VL sequence of the FAPα-binding region having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 14. In a further embodiment, the framework region of the VH sequence of the FAPα-binding domain has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 13, and the framework region of the VL sequence of the FAPα-binding domain has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 14.

[0079] In further embodiments, the framework region of the VH sequence of the FAPα-binding domain of the multispecific antibody described herein has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the framework region of the amino acid sequence shown in SEQ ID NO: 13. In further embodiments, the framework region of the VL sequence of the FAPα-binding domain of the multispecific antibody described herein has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the framework region of the amino acid sequence shown in SEQ ID NO: 14. In further embodiments, the framework region of the VH sequence of the FAPα-binding domain has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the framework region of the amino acid sequence shown in SEQ ID NO: 13, and the framework region of the VL sequence of the FAPα-binding domain has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the framework region of the amino acid sequence shown in SEQ ID NO: 14.

[0080] In one further embodiment, the multispecific antibody includes a VH sequence of the FAPα-binding region outside the CDR region, having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 13. In a further embodiment, the multispecific antibody includes a VL sequence of the FAPα-binding region outside the CDR region, having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 14. In a further embodiment, the multispecific antibody includes a VH sequence of the FAPα binding region outside the CDR region having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 13, and a VL sequence of the FAPα binding region outside the CFR region having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 14.

[0081] In a further embodiment, the VH sequence of the FAPα binding region shown in SEQ ID NO: 13 includes up to 10 substitutions, for example, up to 9 substitutions, for example, up to 8 substitutions, for example, up to 7 substitutions, for example, up to 6 substitutions, for example, up to 5 substitutions, for example, up to 4 substitutions, for example, up to 3 substitutions, for example, up to 2 substitutions, for example, up to 1 substitution. In a further embodiment, the VL sequence of the FAPα binding region shown in SEQ ID NO: 14 includes up to 10 substitutions, for example, up to 9 substitutions, for example, up to 8 substitutions, for example, up to 7 substitutions, for example, up to 6 substitutions, for example, up to 5 substitutions, for example, up to 4 substitutions, for example, up to 3 substitutions, for example, up to 2 substitutions, for example, up to 1 substitution.

[0082] Alternatively, the VH sequence of the FAPα binding region differs from sequence number 13 by up to 10 substitutions, for example, up to 9 substitutions, for example, up to 8 substitutions, for example, up to 7 substitutions, for example, up to 6 substitutions, for example, up to 5 substitutions, for example, up to 4 substitutions, for example, up to 3 substitutions, for example, up to 2 substitutions, for example, up to 1 substitution. In a further embodiment, the VL sequence of the FAPα binding region differs from sequence number 14 by up to 10 substitutions, for example, up to 9 substitutions, for example, up to 8 substitutions, for example, up to 7 substitutions, for example, up to 6 substitutions, for example, up to 5 substitutions, for example, up to 4 substitutions, for example, up to 3 substitutions, for example, up to 2 substitutions, for example, up to 1 substitution.

[0083] In further embodiments, the VH sequence of the FAPα binding region shown in SEQ ID NO: 13 includes substitutions 1 to 10, for example substitutions 1 to 9, for example substitutions 1 to 8, for example substitutions 1 to 7, for example substitutions 1 to 6, for example substitutions 1 to 5, for example substitutions 1 to 4, for example substitutions 1 to 3, for example substitutions 1 to 2. In further embodiments, the VL sequence of the FAPα binding region shown in SEQ ID NO: 14 includes substitutions 1 to 10, for example substitutions 1 to 9, for example substitutions 1 to 8, for example substitutions 1 to 7, for example substitutions 1 to 6, for example substitutions 1 to 5, for example substitutions 1 to 4, for example substitutions 1 to 3, for example substitutions 1 to 2.

[0084] In further embodiments, the VH and VL sequences of the FAPα binding region differ only in the framework region. In one embodiment, in the VH sequence of SEQ ID NO: 13, FR1 is defined by amino acid residues 1-25, FR2 by amino acid residues 34-50, FR3 by amino acid residues 58-96, and FR4 by amino acid residues 111-121. In another embodiment, in the VL sequence of SEQ ID NO: 14, FR1 is defined by amino acid residues 1-26, FR2 by amino acid residues 33-49, FR3 by amino acid residues 53-88, and FR4 by amino acid residues 98-107.

[0085] The Disclosure further provides a multispecific antibody in which the VH sequence of the FAPα-binding region includes or comprises the VH sequence shown in SEQ ID NO: 13. The Disclosure further provides a multispecific antibody in which the VL sequence of the FAPα-binding region includes or comprises the VL sequence shown in SEQ ID NO: 14. In a further embodiment, the VH and VL sequences of the FAPα-binding region include or comprise the VH sequence shown in SEQ ID NO: 13 and the VL sequence shown in SEQ ID NO: 14. In a further embodiment, the VH sequence of the FAPα-binding region includes, essentially comprises, or comprises the VH sequence shown in SEQ ID NO: 13. In a further embodiment, the VL sequence of the FAPα-binding region includes, essentially comprises, or comprises the VL sequence shown in SEQ ID NO: 14. In a further embodiment, the VH and VL sequences of the FAPα-binding region include, essentially comprises, or comprises the VH sequence shown in SEQ ID NO: 13 and the VL sequence shown in SEQ ID NO: 14.

[0086] In the context of the present invention, the antibody may include an FAPα-binding region capable of binding to FAPα, where FAPα is human FAPα, e.g., the mature polypeptide of SEQ ID NO: 33 or the soluble FAPα of SEQ ID NO: 34; FAPα is mouse FAPα, e.g., the mature polypeptide of SEQ ID NO: 35; FAPα is rat FAPα, e.g., the mature polypeptide of SEQ ID NO: 36; FAPα is canine FAPα, e.g., the mature polypeptide of SEQ ID NO: 37; FAPα is porcine FAPα, e.g., the mature polypeptide of SEQ ID NO: 38; or cynomolgus monkey FAPα, e.g., the mature polypeptide of SEQ ID NO: 39. In further embodiments, FAPα is human FAPα, e.g., the mature polypeptide of SEQ ID NO: 33 or the soluble FAPα of SEQ ID NO: 34; or cynomolgus monkey FAPα, e.g., the mature polypeptide of SEQ ID NO: 39. In preferred embodiments, FAPα is human FAPα, e.g., the mature polypeptide of SEQ ID NO: 33.

[0087] The antibody according to the present invention has an equilibrium dissociation constant K between the antigen-binding region that binds to human FAPα and human FAPα.D It can be joined with K D This includes levels below 1000 pM, for example below 900 pM, for example below 800 pM, for example below 700 pM, for example below 600 pM, for example below 500 pM, for example below 400 pM, for example below 300 pM, for example below 200 pM, for example below 100 pM, for example below 90 pM, for example below 80 pM, for example below 70 pM, for example below 60 pM, for example below 50 pM, for example below 40 pM, for example below 3 The binding affinity is either 0 pM or less, or within the range of 0.1 pM to 1000 pM, e.g., 0.5-900 pM, e.g., 1 pM to 800 pM, e.g., 2 pM to 700 pM, e.g., 3 pM to 600 pM, e.g., 4 pM to 500 pM, e.g., 5 pM to 400 pM, e.g., 6 pM to 300 pM, e.g., 7 pM to 200 pM, e.g., 8 pM to 100 pM, e.g., 9 pM to 75 pM, e.g., 10 pM to 50 pM. Binding affinity can be determined by biolayer interferometry.

[0088] In further embodiments, the equilibrium dissociation constant K of the antigen-binding region for human FAPα D When bonded in a monovalent state, the concentration is 1000 pM or less, for example 900 pM or less, for example 800 pM or less, for example 700 pM or less, for example 600 pM or less, for example 500 pM or less, for example 400 pM or less, for example 300 pM or less, for example 200 pM or less, for example 100 pM or less, for example 90 pM or less, for example 80 pM or less, for example 70 pM or less, for example 60 pM or less, for example 50 pM or less, for example 40 pM or less. For example, it is 30 pM or less, or within the range of 0.1 pM to 1000 pM, for example 0.5 to 900 pM, for example 1 pM to 800 pM, for example 2 pM to 700 pM, for example 3 pM to 600 pM, for example 4 pM to 500 pM, for example 5 pM to 400 pM, for example 6 pM to 300 pM, for example 7 pM to 200 pM, for example 8 pM to 100 pM, for example 9 pM to 75 pM, for example 10 pM to 50 pM. Binding affinity can be determined by biolayer interferometry.

[0089] When a multispecific antibody binds to human lung fibroblasts or CAF, the assay, for example, as described in Example 2 of this specification, yields EC for FAPα binding. 50 Multispecific antibodies having a concentration in the range of 0.01 to 0.5 μg / mL, for example, in the range of 0.02 to 0.4 μg / mL, for example, in the range of 0.03 to 0.3 μg / mL are further disclosed herein.

[0090] When a multispecific antibody binds monovalently to human lung fibroblasts, for example, as described in Example 2 of this specification, the EC for FAPα binding is obtained. 50 Multispecific antibodies having a concentration in the range of 0.01 to 0.5 μg / mL, for example, in the range of 0.02 to 0.1 μg / mL, for example, in the range of 0.03 to 0.5 μg / mL, are further disclosed herein.

[0091] When a multispecific antibody binds to human lung fibroblasts in a divalent state, for example, when assayed as described in Example 2 of this specification, the EC for FAPα binding is obtained. 50 Multispecific antibodies having a concentration in the range of 0.01 to 0.05 μg / mL, for example, in the range of 0.01 to 0.03 μg / mL, for example, in the range of 0.01 to 0.02 μg / mL, are further disclosed herein.

[0092] In the case of a multispecific antibody that conjugates monovalently to CAF, the assay, for example, as described in Example 2 of this specification, yields EC for FAPα binding. 50 Multispecific antibodies having a concentration in the range of 0.1 to 0.5 μg / mL, for example, in the range of 0.15 to 0.4 μg / mL, for example, in the range of 0.2 to 0.3 μg / mL, are further disclosed herein. The DR4 binding region of a multispecific antibody may include a heavy chain variable region (VH) containing three complementarity-determining regions, CDR1, CDR2, and CDR3, located within the amino acid sequence shown in SEQ ID NO: 15.

[0093] The DR4 binding region of a multispecific antibody may include a light chain variable region (VL) containing three complementarity-determining regions, CDR1, CDR2, and CDR3, located within the amino acid sequence shown in SEQ ID NO: 16. In one embodiment, the DR4 binding region of a multispecific antibody includes a heavy chain variable region (VH) containing three complementarity-determining regions, CDR1, CDR2, and CDR3, located within the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region (VL) containing three complementarity-determining regions, CDR1, CDR2, and CDR3, located within the amino acid sequence shown in SEQ ID NO: 16.

[0094] This specification further discloses multispecific antibodies in which the DR4 binding region comprises a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. Also disclosed here are multispecific antibodies in which the DR4 binding region comprises a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. In further embodiments, the DR4 binding region of a multispecific antibody comprises a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 7, 8, and 9, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 10, 11, and 12, respectively. The CDR regions from the aforementioned variable heavy chain region and variable light chain region are annotated according to IMGT (see Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, Developmental and Comparative Immunology, 27(1), 55-77 (2003)).

[0095] The Disclosure further provides a multispecific antibody in which the VH sequence of the DR4 binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 15. The Disclosure also provides a multispecific antibody in which the VL sequence of the DR4 binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 16. In a further embodiment, the VH sequence of the DR4 binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 15, and the VL sequence of the DR4 binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 16.

[0096] Furthermore, the multispecific antibody may further include a framework region of the VH sequence of the DR4 binding region having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 15. Furthermore, the multispecific antibody may further include a framework region of the VL sequence of the DR4 binding region having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 16. In a further embodiment, the framework region of the VH sequence of the DR4 binding domain has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 15, and the framework region of the VL sequence of the DR4 binding domain has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 16.

[0097] In further embodiments, the framework region of the VH sequence of the DR4 binding domain of the multispecific antibody described herein has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the framework region of the amino acid sequence shown in SEQ ID NO: 15. In further embodiments, the framework region of the VL sequence of the DR4 binding domain described herein has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the framework region of the amino acid sequence shown in SEQ ID NO: 16. In a further embodiment, the framework region of the VH sequence of the DR4 binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the framework region of the amino acid sequence shown in SEQ ID NO: 15, and the framework region of the VL sequence of the DR4 binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the framework region of the amino acid sequence shown in SEQ ID NO: 16.

[0098] In a further embodiment, the VH sequence of the DR4 binding region shown in SEQ ID NO: 15 includes up to 10 substitutions, for example, up to 9 substitutions, for example, up to 8 substitutions, for example, up to 7 substitutions, for example, up to 6 substitutions, for example, up to 5 substitutions, for example, up to 4 substitutions, for example, up to 3 substitutions, for example, up to 2 substitutions, for example, up to 1 substitution. In a further embodiment, the VL sequence of the DR4 binding region shown in SEQ ID NO: 16 includes up to 10 substitutions, for example, up to 9 substitutions, for example, up to 8 substitutions, for example, up to 7 substitutions, for example, up to 6 substitutions, for example, up to 5 substitutions, for example, up to 4 substitutions, for example, up to 3 substitutions, for example, up to 2 substitutions, for example, up to 1 substitution. In a further embodiment, the VH sequence of the DR4 binding region differs from SEQ ID NO: 15 by only up to 10 substitutions, for example, up to 9 substitutions, for example, up to 8 substitutions, for example, up to 7 substitutions, for example, up to 6 substitutions, for example, up to 5 substitutions, for example, up to 4 substitutions, for example, up to 3 substitutions, for example, up to 2 substitutions, for example, up to 1 substitution. In further embodiments, the VL sequence of the DR4 binding region differs from sequence number 16 by a maximum of 10 substitutions, for example, a maximum of 9 substitutions, for example, a maximum of 8 substitutions, for example, a maximum of 7 substitutions, for example, a maximum of 6 substitutions, for example, a maximum of 5 substitutions, for example, a maximum of 4 substitutions, for example, a maximum of 3 substitutions, for example, a maximum of 2 substitutions, or for example, a maximum of 1 substitution.

[0099] In further embodiments, the VH and VL sequences of the DR4 binding region differ only in the framework region. In one embodiment, in the VH sequence of SEQ ID NO: 15, FR1 is defined by amino acid residues 1-25, FR2 by amino acid residues 35-51, FR3 by amino acid residues 59-96, and FR4 by amino acid residues 106-116. In another embodiment, in the VL sequence of SEQ ID NO: 16, FR1 is defined by amino acid residues 1-25, FR2 by amino acid residues 35-51, FR3 by amino acid residues 55-90, and FR4 by amino acid residues 101-110.

[0100] The Disclosure further provides a multispecific antibody in which the VH sequence of the DR4 binding region includes or consists of the VH sequence shown in SEQ ID NO: 15. The Disclosure further provides a multispecific antibody in which the VL sequence of the DR4 binding region includes or consists of the VL sequence shown in SEQ ID NO: 16. In a further embodiment, the VH and VL sequences of the DR4 binding region include or consist of the VH sequence shown in SEQ ID NO: 15 and the VL sequence shown in SEQ ID NO: 16. In a further embodiment, the VH sequence of the DR4 binding region includes, essentially consists of, or consists of the VH sequence shown in SEQ ID NO: 15. In a further embodiment, the VL sequence of the DR4 binding region includes, essentially consists of, or consists of the VL sequence shown in SEQ ID NO: 16. In a further embodiment, the VH and VL sequences of the DR4 binding region include, essentially consists of, or consists of the VH sequence shown in SEQ ID NO: 15 and the VL sequence shown in SEQ ID NO: 16.

[0101] In the context of the present invention, the antibody may include a DR4-binding region capable of binding to DR4, where DR4 is a human DR4, e.g., the mature polypeptide of SEQ ID NO: 68, or a cynomolgus monkey DR4, e.g., the mature polypeptide of SEQ ID NO: 69. In further embodiments, DR4 is a human DR4, e.g., the mature polypeptide of SEQ ID NO: 68.

[0102] The antibody according to the present invention has an equilibrium dissociation constant K between the antigen-binding region that binds to human DR4 and human DR4. D It can be joined with K DFor example, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, etc. For example, it is 0.5 nM or less, or within the range of 0.01 nM to 10 nM, e.g., 0.02 nM to 9 nM, e.g., 0.03 nM to 8 nM, e.g., 0.04 nM to 7 nM, e.g., 0.05 nM to 6 nM, e.g., 0.075 nM to 5 nM, e.g., 0.1 nM to 4 nM, e.g., 0.15 nM to 3 nM, e.g., 0.2 nM to 2 nM, e.g., 0.25 nM to 1 nM, e.g., 0.3 nM to 0.75 nM (monovalent bond). Binding affinity can be determined by biolayer interferometry.

[0103] In further embodiments, the equilibrium dissociation constant K of the antigen-binding region that binds to human DR4 is D When bonding in a monovalent state, the minimum nucleotides are 100 nM or less, for example 90 nM or less, for example 80 nM or less, for example 70 nM or less, for example 60 nM or less, for example 50 nM or less, for example 40 nM or less, for example 30 nM or less, for example 20 nM or less, for example 10 nM or less, for example 9 nM or less, for example 8 nM or less, for example 7 nM or less, for example 6 nM or less, for example 5 nM or less, for example 4 nM or less, for example 3 nM or less, for example 2 nM or less, for example The binding affinity is 1 nM or less, for example, 0.5 nM or less, or within the range of 0.01 nM to 10 nM, for example, 0.02 nM to 9 nM, for example, 0.03 nM to 8 nM, for example, 0.04 nM to 7 nM, for example, 0.05 nM to 6 nM, for example, 0.075 nM to 5 nM, for example, 0.1 nM to 4 nM, for example, 0.15 nM to 3 nM, for example, 0.2 nM to 2 nM, for example, 0.25 nM to 1 nM, for example, 0.3 nM to 0.75 nM. Binding affinity can be determined by biolayer interferometry.

[0104] If a multispecific antibody binds to DLD-1, A549, HCT-116, HCT-15, MDA-MB-231, or PANC-1, then, as described in Example 3 of this specification, an EC for binding to DR4 is obtained. 50 Multispecific antibodies having a concentration in the range of 0.1 to 3.0 μg / mL, for example, in the range of 0.2 to 2.5 μg / mL, for example, in the range of 0.3 to 2.0 μg / mL, are further disclosed herein.

[0105] If a multispecific antibody conjugates monovalently to DLD-1, A549, HCT-116, HCT-15, MDA-MB-231, or PANC-1, then, as described in Example 3 of this specification, an EC for binding to DR4 is obtained. 50 However, multispecific antibodies in the range of 0.1 to 3.0 μg / mL, for example, in the range of 0.25 to 2.0 μg / mL, for example, in the range of 0.3 to 1.75 μg / mL, are further disclosed herein.

[0106] If a multispecific antibody conjugates to DLD-1, A549, HCT-116, HCT-15, MDA-MB-231, or PANC-1 in a divalent state, then, as described in Example 3 of this specification, an EC for binding to DR4 is obtained. 50 However, multispecific antibodies in the range of 0.1 to 1.0 μg / mL, for example, in the range of 0.1 to 0.75 μg / mL, for example, in the range of 0.1 to 0.5 μg / mL, are further disclosed herein.

[0107] This disclosure further provides a multispecific antibody which is a bispecific antibody. In one embodiment, the antibody includes (i) a FAPα binding region including a first heavy chain variable region and a first light chain variable region, wherein the heavy chain variable region (VH) includes the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 1, 2, and 3, respectively, and the light chain variable region (VL) includes the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 4, 5, and 6, respectively; and (ii) a DR4 binding region including a second heavy chain variable region and a second light chain variable region, wherein the heavy chain variable region (VH) includes the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 7, 8, and 9, respectively, and the light chain variable region (VL) includes the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 10, 11, and 12, respectively. In a further embodiment, the antibody comprises (i) an FAPα binding region comprising or consisting of the VH sequence shown in SEQ ID NO: 13 and the VL sequence shown in SEQ ID NO: 14, and (ii) a DR4 binding region comprising or consisting of the VH sequence shown in SEQ ID NO: 15 and the VL sequence shown in SEQ ID NO: 16. In another embodiment, the bispecific antibody comprises (i) an FAPα binding region comprising, essentially consisting of, or consisting of the VH sequence shown in SEQ ID NO: 13 and the VL sequence shown in SEQ ID NO: 14, and (ii) a DR4 binding region comprising, essentially consisting of, or consisting of the VH sequence shown in SEQ ID NO: 15 and the VL sequence shown in SEQ ID NO: 16.

[0108] The antibody according to the present invention has an equilibrium dissociation constant K between the antigen-binding region that binds to human FAPα and human FAPα, which is 100 pM or less. D It can bind, and the equilibrium dissociation constant K is less than 1 nM between the antigen-binding region that binds to human DR4 and human DR4. D It can bind to it. The binding affinity can be determined by biolayer interferometry.

[0109] The antibody according to the present invention has an equilibrium dissociation constant K between the antigen-binding region that binds to human FAPα and human FAPα, which is 100 pM or less. DIt can bind (monovalent bond), and the equilibrium dissociation constant K is less than 1 nM between the antigen-binding region that binds to human DR4 and human DR4. D They can bind (monovalent bond). Binding affinity can be determined by biolayer interferometry.

[0110] The antibody according to the present invention has an equilibrium dissociation constant K of 50 pM or less between the antigen-binding region that binds to human FAPα and human FAPα. D It can bind (monovalent binding), and the equilibrium dissociation constant K is 0.5 nM or less between the antigen-binding region that binds to human DR4 and human DR4. D They can bind (monovalent bond). Binding affinity can be determined by biolayer interferometry.

[0111] In a further embodiment, the present invention relates to a multispecific antibody comprising at least one antigen-binding region capable of binding to FAPα, wherein the antibody can compete for binding to FAPα with antibody FAP-ESC11 comprising, for example, a heavy chain (HC) comprising the sequence shown in SEQ ID NO: 50, disclosed in International Publication No. 2011040972, and a light chain (LC) comprising the sequence shown in SEQ ID NO: 51, and / or can simultaneously bind to FAPα as antibody FAP5 comprising a heavy chain (HC) comprising the sequence shown in SEQ ID NO: 48, disclosed in, for example, U.S. Patent Application Publication No. 20090304718, and a light chain (LC) comprising the sequence shown in SEQ ID NO: 49.

[0112] Antibody format The multispecific antibodies of the present invention may have two or more specificities, for example, two or three or more specificities. Furthermore, the multispecific antibodies may have multiple copies of the antigen-binding region for FAPα and / or DR4. For example, in one embodiment, the antibody has two antigen-binding regions that can bind to FAPα, for example, two identical binding regions that bind to FAPα. For example, in another embodiment, the antibody has two antigen-binding regions that bind to DR4, for example, two identical binding regions that bind to DR4. Further antigen-binding regions may exist, for example, in the form of scFv covalently bound to the constant region.

[0113] In preferred embodiments, the multispecific antibody of the present invention is a bispecific antibody. A variety of formats and uses of bispecific antibodies are known in the art and have been outlined by Kontermann; Drug Discov Today, 2015 Jul;20(7):838-47, and MAbs, 2012 Mar-Apr;4(2):182-97, and by Labrijn et al. 2019 Nat Rev Drug Discov 18(8)585-608. The bispecific antibodies of the present invention are not limited to any particular bispecific format or method for producing the same.

[0114] Examples of bispecific antibody molecules that can be used in the present invention include: (i) a single antibody having two arms containing different antigen-binding regions; (ii) a single-chain antibody specific to two different targets via two scFvs linked in tandem, for example, by an additional peptide linker; and (iii) a bivariable domain antibody (DVD-Ig) in which each of the light and heavy chains contains two variable regions in tandem via short peptide bonds (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig®) Molecule, In: Antibody Engineering, Springer Berlin). Heidelberg (2010), (iv) a chemically bonded bispecific (Fab')2 fragment, (v) a Tandab which is a fusion of two single-stranded diabodies resulting in a tetravalent bispecific antibody having two binding sites for each of the target antigens, (vi) a flexibody which is a combination of scFv and diabodies resulting in a multivalent molecule, (vii) a so-called "dock and lock" molecule based on the "dimerization and docking domain" of protein kinase A which, when applied to Fab, can result in a trivalent bispecific binding protein consisting of two identical Fab fragments linked to different Fab fragments, (viii) a so-called Scorpion molecule which includes, for example, two scFv fused to both ends of a human Fab arm, and (ix) a diabodies.

[0115] Further examples of different classes of bispecific antibodies include, but are not limited to, (i) IgG-like molecules having a complementary CH3 domain and forcing heterodimerization, (ii) recombinant IgG-like bitargeted molecules in which both sides of the molecule each contain Fab fragments or portions of Fab fragments of at least two different antibodies, (iii) IgG fusion molecules in which a full-length IgG antibody is fused to an additional Fab fragment or portion of a Fab fragment, (iv) Fc fusion molecules in which a single-chain Fv molecule or a stabilizing diabody is fused to a heavy chain constant domain, Fc region or portion thereof, (v) Fab fusion molecules in which different Fab fragments are fused together and fused to a heavy chain constant domain, Fc region or portion thereof, and (vi) scFv-based and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) in which different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule fused to a heavy chain constant domain, Fc region or portion thereof.

[0116] Examples of IgG-like molecules having complementary CH3 domain molecules include, but are not limited to, Triomab / Quadroma molecules (Trion Pharma / Fresenius Biotech; Roche, International Publication No. 2011069104), so-called Knobs-into-Holes molecules (Genentech, International Publication No. 9850431), CrossMAb (Roche, International Publication No. 2011117329), and electrostatically-matched molecules (Amgen, European Patent No. 1870459 and International Publication No. 2009089004; Chugai, US Patent Application Publication No. 201000155133; Oncomed, International Publication No. 2010129304), and LUZ-Y molecules (Genentech, Wranik et al.). al.J.Biol.Chem.2012,287(52):43331-9,doi:10.1074 / jbc.M112.397869.Epub 2012 Nov 1), DIG body and PIG body molecules (Pharmabcine, International Publication No. 2010134666, International Publication No. 2014081202), Strand Exchange Engineered Domain body (SEEDbody) molecule (EMD Examples include Serono (International Publication No. 2007110205), Biclonics molecules (Merus, International Publication No. 2013157953), FcΔAdp molecules (Regeneron, International Publication No. 201015792), bispecific IgG1 and IgG2 molecules (Pfizer / Rinat, International Publication No. 11143545), Azymetric scaffold molecules (Zymeworks / Merck, International Publication No. 2012058768), mAb-Fv molecules (Xencor, International Publication No. 2011028952), bivalent bispecific antibodies (International Publication No. 2009080254), and DuoBody® molecules (Genmab A / S, International Publication No. 2011131746).

[0117] Examples of recombinant IgG-like dual-targeting molecules include, but are not limited to, Dual Targeting (DT)-Ig molecule (International Publication No. 2009058383), Two-in-one antibody (Genentech; Bostrom, et al, 2009. Science 323, 1610-1614), cross-linked Mab (Karmanos Cancer Center), mAb2 (F-Star, International Publication No. 2008003116), Zybody molecule (Zyngenia; LaFleur et al. MAbs. 2013 Mar-Apr; 5(2):208-18), common light chain approach (Crucell / Merus, U.S. Patent No. 7,262,028), kappa / lambda body™ molecule (NovImmune, International Publication No. 2012023053), and CovX body (CovX / Pfizer; Doppalapudi, VR, et al. (2007. Bioorg. Med. Chem. Lett. 17, 501-506) is one example.

[0118] Examples of IgG fusion molecules include, but are not limited to, the bivariable domain (DVD)-Ig molecule (Abbott, U.S. Patent No. 7,612,181), bidomain double-head antibody (Unilever; Sanofi Aventis, International Publication No. 20100226923), IgG-like bispecificity molecule (ImClone / Eli Lilly, Lewis et al. Nat Biotechnol. 2014 Feb;32(2):191-8), Ts2Ab (MedImmune / AZ; Dimasi et al. J Mol Biol. 2009 Oct 30;393(3):672-92) and BsAb molecule (Zymogenetics, International Publication No. 2010111625), HERCULES molecule (Biogen Idec, U.S. Patent No. 007951918), scFv fusion molecule (Novartis), scFv fusion molecule (Changzhou Adam Biotech Examples include Roche Inc. (Chinese Patent No. 102250246) and the TvAb molecule (Roche, International Publication No. 2012025525, International Publication No. 2012025530).

[0119] Examples of Fc fusion molecules include, but are not limited to, scFv / Fc fusions (Pearce et al., Biochem Mol Biol Int. 1997 Sep;42(6):1179-88), the SCORPION molecule (Emergent BioSolutions / Trubion, Blankenship JW, et al. AACR 100th Annual Meeting 2009 (Abstract #5465); Zymogenetics / BMS, International Publication No. 2010111625), Dual Affinity Retargeting Technology (Fc-based DART) molecule (MacroGenics, International Publication No. 2008157379, Publication No. 2010080538), and Dual(scFv)2-Fab molecule (National Research Center for Antibody Medicine-China).

[0120] Examples of Fab-fusion bispecific antibodies include, but are not limited to, F(ab)2 molecules (Medarex / AMGEN; Deo et al J Immunol. 1998 Feb 15; 160(4): 1677-86), dual-acting or Bis-Fab molecules (Genentech, Bostrom, et al 2009. Science 323, 1610-1614), Dock-and-Lock (DNL) molecules (ImmunoMedics, International Publication No. 2003074569, International Publication No. 2005004809), divalent bispecific molecules (Biotecnol, Schoonjans, J Immunol. 2000 Dec 15; 165(12): 7050-7), and Fab-Fv molecules (UCB-Celltech, International Publication No. 2009040562 A1).

[0121] Examples of scFv-based, diabody-based, and domain antibodies include, but are not limited to, bispecific T cell engager (BiTE) molecules (Micromet, International Publication No. 2005061547), tandem diabody molecules (TandAb) (Affimed) (Le Gall et al., Protein Eng Des Sel. 2004 Apr;17(4):357-66), DART molecules (MacroGenics, International Publication No. 2008157379, International Publication No. 2010080538), single-chain diabody molecules (Lawrence, FEBS Lett. 1998 Apr 3;425(3):479-84), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin scFv fusions (Merrimack, International Publication No. 2010059315), and COMBODY molecules (Epigen Biotech, Zhu et al.). Examples include al.Immunol Cell Biol.2010 Aug;88(6):667-75.), bi-targeted nanobodies (Ablynx, Hmila et al., FASEB J.2010), and bi-targeted heavy chain monodomain antibodies.

[0122] In one embodiment, the bispecific antibody of the present invention is a bispecific antibody obtained via diabody, crossbody, or controlled Fab arm exchange (for example, as described in International Publication No. 2011131746 (Genmab)).

[0123] In one embodiment, the antibody of the present invention is a bispecific DuoBody® molecule (Genmab A / S, International Publication No. 2011131746).

[0124] The multispecific, or bispecific, antibody of the present invention may be of any isotype. Exemplary isotypes include, but are not limited to, any of the human IgG1, IgG2, IgG3, and IgG4 isotypes. Preferably, the antibody may be selected to be of the human IgG1 isotype, as shown in the examples. Thus, in one embodiment, the multispecific antibody is an IgG1 antibody. A human light chain constant region, either kappa or lambda, or both, such as the sequences described in SEQ ID NOs. 27 and 28, may be used. In one embodiment, the multispecific antibody comprises kappa (κ) and lambda (λ) light chains. For example, in one embodiment, the light chain involved in FAPα binding comprises a kappa constant region, and the light chain involved in DR4 binding comprises a lambda constant region. In a further embodiment, the multispecific antibody comprises a heavy chain containing an FAPα binding region and a kappa (κ) light chain, and a heavy chain containing a DR4 binding region and a lambda (λ) light chain. In one embodiment, both heavy chains of the antibody of the present invention are of the IgG1 isotype. In a further embodiment, the two heavy chains of the bispecific antibody are of the IgG1 and IgG4 isotypes, respectively. In a further embodiment, the DR4 binding region is included in the heavy chain and light chain, the heavy chain includes the VH region and the IgG1 heavy chain constant region, the light chain includes the VL region and the lambda light chain constant region, the FAPα binding region is included in the heavy chain and light chain, the heavy chain includes the VH region and the IgG1 heavy chain constant region, and the light chain includes the VL region and the kappa light chain constant region. In a further embodiment, one of the IgG1 heavy chain constant regions is as defined in SEQ ID NO: 26, the other is as defined in SEQ ID NO: 70, the kappa light chain constant region is as defined in SEQ ID NO: 27, and the lambda light chain constant region is as defined in SEQ ID NO: 28.

[0125] Preferably, the bispecific antibody may be selected to be of a human IgG1 isotype, as shown in the examples. Optionally, and preferably, the heavy chain of the selected isotype and its Fc region sequence may be modified, preferably in the hinge, CH2 and / or CH3 regions, to enable and / or introduce inactivation of the bispecific antibody.

[0126] In one embodiment, the multispecific antibody of the present invention includes an Fc region comprising a first and a second Fc polypeptide.

[0127] In one embodiment, the first Fc polypeptide and the first heavy chain variable region are contained within the same polypeptide chain, and the second Fc polypeptide and the second heavy chain variable region are contained within the same polypeptide chain.

[0128] The first and second Fc polypeptides may each be any isotype, including any human isotype, such as IgG1, IgG2, IgG3, IgG4, IgE, IgD, IgM, or IgA isotypes or mixed isotypes. Preferably, the Fc region is a human IgG1, IgG2, IgG3, IgG4 isotype or mixed isotype. In one embodiment, the Fc region is a human IgG1 Fc region.

[0129] In further embodiments, the multispecific antibody is a full-length antibody as defined herein. In further embodiments, the multispecific antibody is a full-length IgG1 antibody. In further embodiments, the first and second Fc regions include the sequence of SEQ ID NO: 21 (IgG1m(f)), except for certain mutations as defined herein.

[0130] The antibodies according to the present invention may include modifications in the Fc region to make the antibody inactive or deactivated. Therefore, in the antibodies disclosed herein, one or both heavy chains may be modified to reduce the degree of Fc-mediated effector function induced by the antibody compared to an antibody that is identical except for the lack of such modifications. Thus, in one embodiment, the antibody comprises a first heavy chain and a second heavy chain, and one or both heavy chains are modified to reduce the degree of Fc-mediated effector function induced by the antibody compared to an antibody that is identical except for the presence of unmodified first and second heavy chains. Fc-mediated effector function can be measured by binding to the Fcγ receptor, by binding to C1q, or by induction of Fc-mediated crosslinking of FcγR. In particular, modifications of the heavy chain constant sequence and the light chain constant sequence can also result in a reduction of C1q binding to the antibody. Compared to the unmodified antibody, the reduction may be at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, and C1q binding can be determined by ELISA. Furthermore, the Fc region may be modified to reduce the Fc-mediated T cell proliferation mediated by the antibody by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to the unmodified antibody, for example in the linear portion of the curve, and the T cell proliferation is measured by a PBMC-based functional assay. In one embodiment, the multispecific antibody induces Fc-mediated effector function to less than 95%, e.g., less than 90%, less than 85%, e.g., less than 80%, e.g., less than 75%, e.g., less than 70%, e.g., less than 65%, e.g., less than 60%, e.g., less than 55%, e.g., less than 5050%, e.g., less than 95%, e.g., less than 90%, less than 85%, e.g., less than 75%, e.g., less than 70%, e.g., less than 65%, e.g., less than 60%, e.g., less than 55%, e.g., less than 5

[0131] A wide range of inactivating antibody formats have been developed by introducing amino acid substitutions and combinations thereof into the constant heavy chain region of IgG1 isotype antibodies to eliminate Fc-mediated effector function (e.g., Chiu et al., Antibodies 2019 Dec;8(4):55; Liu et al., Antibodies, 2020 Nov 17;9(4):64;29(10):457-66; Shields et al., J Biol Chem. 2001 Mar 2;276(9):6591-604). In one embodiment, a multispecific antibody comprises a first heavy chain and a second heavy chain, wherein in at least one of the first and second heavy chains, one or more amino acids at positions corresponding to L234, L235, G236, D265, N297, and P331 of the human IgG1 heavy chain according to Eu numbering are not L, L, G, D, N, and P, respectively.

[0132] For example, examples of amino acid positions that can be modified in an IgG1 isotype antibody include positions L234 and L235. Therefore, in one embodiment, the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain by Eu numbering are F and E in the first and / or second heavy chain, respectively.

[0133] It is understood that, in addition to modifications at amino acid positions L234 and L235, further positions may be modified. Therefore, in further embodiments, the first and second Fc polypeptides include substitutions of amino acids to F and E, respectively, corresponding to the amino acids at positions L234 and L235, and the first and / or second Fc polypeptides further include substitutions of amino acids, preferably to R, corresponding to the amino acid at position G236 of the human IgG1 heavy chain.

[0134] In another embodiment, the first and second Fc polypeptides include substitutions of amino acids to F and E, respectively, corresponding to the amino acids at positions L234 and L235, and the first and second Fc polypeptides further include substitutions of amino acids, preferably to R, corresponding to the amino acid at position G236 of the human IgG1 heavy chain.

[0135] In another embodiment, the first and second Fc polypeptides include substitutions of amino acids to F and E, respectively, corresponding to the amino acids at positions L234 and L235, and the first and / or second Fc polypeptides further include substitutions of amino acids, preferably to A, corresponding to the amino acid at position D265 of the human IgG1 heavy chain.

[0136] In another embodiment, the first and second Fc polypeptides include substitutions of amino acids to F and E, respectively, corresponding to the amino acids at positions L234 and L235, and the first and second Fc polypeptides further include substitutions of amino acids, preferably to A, corresponding to the amino acid at position D265 of the human IgG1 heavy chain.

[0137] In another embodiment, one of the first Fc polypeptide and the second Fc polypeptide contains substitutions of amino acids to F, E, and R corresponding to the amino acids at positions L234, L235, and G236, respectively, while the other Fc polypeptide contains substitutions of amino acids to F, E, and A corresponding to the amino acids at positions L234, L235E, and D265, respectively.

[0138] For example, constant regions having such Fc region substitutions are provided in ia SEQ ID NOs. 22-23, which can be compared with SEQ ID NO. 21, which does not have such substitutions. In one embodiment, the antibody of the present invention comprises a sequence selected from the group consisting of SEQ ID NOs. 22-23.

[0139] In one embodiment, the multispecific or bispecific antibody of the present invention comprises an Fc region containing different first and second CH3 regions, wherein the heterodimer interaction between the first and second CH3 regions is stronger than each of the homodimer interactions between the first and second CH3 regions. Further details on these interactions and how they can be achieved are provided in International Publication Nos. 2011131746 and 2013060867 (Genmab), which are incorporated herein by reference. Stable heterodimer antibodies can be obtained in high yield based on two homodimer starting antibodies containing only slight asymmetric mutations in the CH3 region by so-called Fab arm exchange, for example, provided in International Publication Nos. 2008 / 119353 and 2011 / 131746.

[0140] Accordingly, in one embodiment, in the first Fc polypeptide, at least one amino acid is substituted at a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 of the human IgG1 heavy chain, and in the second Fc polypeptide, at least one amino acid is substituted at a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 of the human IgG1 heavy chain, and the substitutions in the first and second Fc polypeptides are not at the same position, and the amino acid positions are as defined by Eu numbering. For example, constant regions having such Fc region substitutions are provided in ia SEQ ID NOs. 24-25, which can be compared with SEQ ID NO. 21, which does not have such substitutions. In one embodiment, the antibody of the present invention contains a sequence selected from the group consisting of SEQ ID NOs. 24-25.

[0141] Furthermore, a multispecific antibody is disclosed herein, (i) wherein the antibody comprises a first heavy chain and a second heavy chain, the first heavy chain comprising the FAPα binding region and the second heavy chain comprising the DR4 binding region, (ii) wherein each of the first and second heavy chains comprises at least a hinge region, CH2 and CH3 regions, and (iii) wherein in the first heavy chain, at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 (by Eu numbering) is substituted, and in the second heavy chain, at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 (by Eu numbering) is substituted.

[0142] Furthermore, a multispecific antibody is disclosed herein, (i) wherein the antibody comprises a first heavy chain and a second heavy chain, the first heavy chain comprising the VH region of the FAPα binding region and the second heavy chain comprising the VH region of the DR4 binding region, (ii) wherein each of the first and second heavy chains comprises at least a hinge region, CH2 and CH3 regions, and (iii) wherein the first heavy chain comprises (human IgG1 heavy chain by Eu numbering) In the second heavy chain, at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (in the human IgG1 heavy chain according to Eu numbering) is substituted.

[0143] Furthermore, multispecific antibodies are disclosed herein, wherein the first and second heavy chains are not substituted at the same position. Furthermore, multispecific antibodies are disclosed wherein (i) the amino acid at the position corresponding to F405 (according to Eu numbering) is L in the first heavy chain and the amino acid at the position corresponding to K409 (according to Eu numbering) is R in the second heavy chain, or (ii) the amino acid at the position corresponding to K409 (according to Eu numbering) is R in the first heavy chain and the amino acid at the position corresponding to F405 (according to Eu numbering) is L in the second heavy chain. Preferably, the amino acid at the position corresponding to F405 is L in the first Fc polypeptide and the amino acid at the position corresponding to K409 is R in the second Fc polypeptide, or vice versa. Accordingly, the present invention provides an antibody in which the amino acid at the position corresponding to F405 of the human IgG1 heavy chain is L in the first Fc polypeptide and the amino acid at the position corresponding to K409 of the human IgG1 heavy chain is R in the second Fc polypeptide, or vice versa. In a further embodiment, a multispecific antibody is disclosed in which (i) the amino acid at the position corresponding to F405 (in the human IgG1 heavy chain by Eu numbering) is L in the first heavy chain and the amino acid at the position corresponding to K409 (in the human IgG1 heavy chain by Eu numbering) is R in the second heavy chain, or (ii) the amino acid at the position corresponding to K409 (in the human IgG1 heavy chain by Eu numbering) is R in the first heavy chain and the amino acid at the position corresponding to F405 (in the human IgG1 heavy chain by Eu numbering) is L in the second heavy chain.

[0144] Therefore, in one embodiment, one of the first Fc polypeptide and the second Fc polypeptide contains substitutions of amino acids to F, E, R, and L corresponding to the amino acids at positions L234, L235, G236, and F405, respectively, and the other Fc polypeptide contains substitutions of amino acids to F, E, A, and R corresponding to the amino acids at positions L234, L235E, D265, and K409, respectively.

[0145] In another embodiment, one of the first Fc polypeptide and the second Fc polypeptide contains substitutions of amino acids to F, E, R, and R, respectively, corresponding to the amino acids at positions L234, L235, G236, and K409, and the other Fc polypeptide contains substitutions of amino acids to F, E, A, and L, respectively, corresponding to the amino acids at positions L234, L235E, D265, and F405.

[0146] In another embodiment, one of the first Fc polypeptide and the second Fc polypeptide contains substitutions of amino acids to F, E, R, and L corresponding to the amino acids at positions L234, L235, G236, and F405, respectively, and the other Fc polypeptide contains substitutions of amino acids to F, E, R, and R corresponding to the amino acids at positions L234, L235E, G236, and K409, respectively.

[0147] In further embodiments, the first Fc polypeptide comprises substitutions of amino acids to F, E, R, and L corresponding to the amino acids at positions L234, L235, G236, and F405, respectively, and the second Fc polypeptide comprises substitutions of amino acids to F, E, R, and R corresponding to the amino acids at positions L234, L235E, G236, and K409, respectively.

[0148] In another embodiment, one of the first Fc polypeptide and the second Fc polypeptide comprises substitutions of amino acids to F, E, A, and L corresponding to the amino acids at positions L234, L235, D265, and F405, respectively, and the other Fc polypeptide comprises substitutions of amino acids to F, E, A, and R corresponding to the amino acids at positions L234, L235E, D265, and K409, respectively.

[0149] In further embodiments, the first Fc polypeptide comprises substitutions of amino acids to F, E, A, and L corresponding to the amino acids at positions L234, L235, D265, and F405, respectively, and the second Fc polypeptide comprises substitutions of amino acids to F, E, A, and R corresponding to the amino acids at positions L234, L235E, D265, and K409, respectively.

[0150] In a further embodiment, a multispecific antibody is disclosed, wherein the antibody is a bispecific antibody comprising a first heavy chain and a second heavy chain, wherein the positions of both the first and second heavy chains corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively, wherein the position of the first heavy chain corresponding to F405 in the human IgG1 heavy chain according to Eu numbering is L, and the position of the second heavy chain corresponding to K409 in the human IgG1 heavy chain according to Eu numbering is R.

[0151] In further embodiments, one of the first and second heavy chains includes substitutions of amino acids F, E, and R corresponding to the amino acids at positions L234, L235, and G236, respectively, and the other heavy chain includes substitutions of amino acids F, E, and A corresponding to the amino acids at positions L234, L235, and D265, respectively, where the amino acid positions are as defined by Eu numbering.

[0152] In further embodiments, the antibody is a bispecific antibody comprising a first heavy chain and a second heavy chain, wherein one heavy chain comprises substitutions of amino acids F, E, and R corresponding to the amino acids at positions L234, L235, and G236, respectively, and the other heavy chain comprises substitutions of amino acids F, E, and A corresponding to the amino acids at positions L234, L235, and D265, respectively, wherein (i) the position of the first heavy chain corresponding to F405 of the human IgG1 heavy chain according to Eu numbering is L, and the position of the second heavy chain corresponding to K409 of the human IgG1 heavy chain according to Eu numbering is R, or (ii) the position of the first heavy chain corresponding to K409 of the human IgG1 heavy chain according to Eu numbering is R, and the position of the second heavy chain corresponding to F405 of the human IgG1 heavy chain according to Eu numbering is L.

[0153] In one embodiment, the antibody of the present invention comprises or consists of the heavy chain sequences shown in SEQ ID NOs: 17 and 19 and the light chain sequences shown in SEQ ID NOs: 18 and 20.

[0154] The present invention further discloses a multispecific antibody as described herein, comprising (ii) a first heavy chain and a first light chain linked via a disulfide crosslink forming a first binding region for binding to FAPα, and (ii) a second heavy chain and a second light chain linked via a disulfide crosslink forming a second binding region for binding to DR4, wherein i) the first heavy chain comprises the sequence shown in SEQ ID NO: 17, and the first light chain comprises the sequence shown in SEQ ID NO: 18, and ii) the second heavy chain comprises the sequence shown in SEQ ID NO: 19, and the second light chain comprises the sequence shown in SEQ ID NO: 20.

[0155] The constant region sequences listed in SEQ ID NOs: 21-26 and 70-72 do not contain C-terminal lysine (K). However, in naturally occurring human sequences from which these Fc regions originate, such C-terminal lysine may be present as part of the open reading frame. During cell culture preparation of recombinant antibodies, this terminal lysine may be cleaved by proteolysis by endogenous carboxypeptidases, resulting in a constant region with the same sequence but lacking C-terminal lysine. For the purpose of antibody production, the DNA encoding this terminal lysine may be removed from the sequence so that antibodies can be produced without lysine. Removing C-terminal lysine from the antibody-coding sequence can improve the uniformity of antibodies with respect to the presence of C-terminal lysine. Antibodies produced from nucleic acid sequences encoding or not encoding terminal lysine are substantially identical in sequence and function. This is because, for example, when using antibodies produced in CHO-based production systems, the degree of C-terminal lysine processing is typically high (Dick, L. Wet al. Biotechnol. Bioeng. 2008;100:1132-1143). Therefore, it is understood that antibodies according to the present invention may be produced without encoding or having C-terminal lysine as enumerated herein. For manufacturing purposes, antibodies can be produced in this manner without C-terminal lysine.

[0156] In one embodiment, the multispecific antibody is a bispecific antibody comprising a first heavy chain and a second heavy chain, where the positions of both the first and second heavy chains corresponding to positions L234 and L235 of the human IgG1 heavy chain according to Eu numbering are F and E, respectively, and where (i) the position of the first heavy chain corresponding to F405 of the human IgG1 heavy chain according to Eu numbering is L, and the position of the second heavy chain corresponding to K409 of the human IgG1 heavy chain according to Eu numbering is R, or (ii) the position of the first heavy chain corresponding to K409 of the human IgG1 heavy chain according to Eu numbering is R, and the position of the second heavy chain corresponding to F405 of the human IgG1 heavy chain according to Eu numbering is L.

[0157] In a further embodiment, the multispecific antibody is a bispecific antibody comprising a first heavy chain and a second heavy chain, where the positions of both the first and second heavy chains corresponding to positions L234, L235, and D265 of the human IgG1 heavy chain according to Eu numbering are F, E, and A, respectively, and where (i) the position of the first heavy chain corresponding to F405 of the human IgG1 heavy chain according to Eu numbering is L, and the position of the second heavy chain corresponding to K409 of the human IgG1 heavy chain according to Eu numbering is R, or (ii) the position of the first heavy chain corresponding to K409 of the human IgG1 heavy chain according to Eu numbering is R, and the position of the second heavy chain corresponding to F405 of the human IgG1 heavy chain according to Eu numbering is L.

[0158] In a further embodiment, the multispecific antibody is a bispecific antibody comprising a first heavy chain and a second heavy chain, where the positions of both the first and second heavy chains corresponding to positions L234, L235, and G236 of the human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively, and where (i) the position of the first heavy chain corresponding to F405 of the human IgG1 heavy chain according to Eu numbering is L, and the position of the second heavy chain corresponding to K409 of the human IgG1 heavy chain according to Eu numbering is R, or (ii) the position of the first heavy chain corresponding to K409 of the human IgG1 heavy chain according to Eu numbering is R, and the position of the second heavy chain corresponding to F405 of the human IgG1 heavy chain according to Eu numbering is L.

[0159] In further embodiments, the multispecific antibody is a bispecific antibody comprising a first heavy chain and a second heavy chain, where the positions corresponding to the amino acids at positions L234, L235, and G236 are F, E, and R, respectively, and the other Fc polypeptide comprises substitutions of the amino acids corresponding to the amino acids at positions L234, L235, and D265 to F, E, and A, respectively, where (i) the position of the first heavy chain corresponding to F405 of the human IgG1 heavy chain according to Eu numbering is L, and the position of the second heavy chain corresponding to K409 of the human IgG1 heavy chain according to Eu numbering is R, or (ii) the position of the first heavy chain corresponding to K409 of the human IgG1 heavy chain according to Eu numbering is R, and the position of the second heavy chain corresponding to F405 of the human IgG1 heavy chain according to Eu numbering is L.

[0160] In the context of the present invention, a multispecific antibody is a bispecific antibody comprising: (i) a first heavy chain and a first light chain including an FAPα binding region, wherein the FAPα binding region comprises a first heavy chain variable region and a first light chain variable region, and the first heavy chain variable region (VH) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 1, 2, and 3, respectively, and the first light chain variable region (VL) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 4, 5, and 6, respectively; and (ii) a second heavy chain and a second light chain including a DR4 binding region, wherein the DR4 binding region comprises a second heavy chain variable region and a second light chain variable region, and the second heavy chain variable region (VH) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 7, 8, respectively. The antibody may include a second heavy chain and a second light chain, each containing the CDR1, CDR2, and CDR3 sequences of sequence 9, respectively, and a second light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences of sequence numbers 10, 11, and 12, respectively, (iii) where the positions of both the first and second heavy chains corresponding to positions L234, L235, and G236 of the human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively, and (iv) the position of the first heavy chain corresponding to position F405 of the human IgG1 heavy chain according to Eu numbering is L, and the position of the second heavy chain corresponding to K409 of the human IgG1 heavy chain according to Eu numbering is R.

[0161] In the context of the present invention, a multispecific antibody may include, or consist of, a bispecific antibody comprising (i) the FAPα heavy chain sequence shown in SEQ ID NO: 17 and the FAPα light chain sequence shown in SEQ ID NO: 18, and (ii) the DR4 heavy chain sequence shown in SEQ ID NO: 19 and the DR4 light chain sequence shown in SEQ ID NO: 20.

[0162] In one embodiment, the multispecific antibody described herein is a bispecific bivalent antibody. In a further embodiment, the multispecific antibody is a bispecific bivalent antibody having monovalent binding to FAPα and monovalent binding to DR4.

[0163] In alternative embodiments, the multispecific antibody according to the present invention is not a classical full-length antibody containing an Fc region. For example, in one embodiment, the multispecific antibody is an antibody fragment. In further embodiments, (i) the FAPα binding region and / or the DR4 binding region is Fab, (ii) the FAPα binding region and / or the DR4 binding region is scFv, (iii) the FAPα binding region is Fab and the DR4 binding region is scFv, or (iv) the FAPα binding region is scFv and the DR4 binding region is Fab.

[0164] Binding and transactivation Multispecific antibodies, such as bispecific antibodies, described herein, that can bind to FAPα and DR4, for example, human FAPα and human DR4, can advantageously target DR4 and FAPα-positive cells on tumor cells, such as CAFs, thereby specifically inducing apoptosis in tumor cells. In one embodiment, the multispecific antibody is a transactivating antibody. In a further embodiment, the multispecific antibody induces DR4-mediated activation regulated by binding to FAPα, such as trans-binding to FAPα, which leads to apoptosis, for example. Thus, the multispecific antibody is capable of FAPα-dependent DR4 transactivation. This means that the apoptotic effect induced by DR4 activation upon binding of the multispecific antibody to DR4 is observed only by binding of the antibody to both DR4 and FAPα.

[0165] As described above, preferably, the multispecific antibody according to the present invention lacks or has reduced Fc-mediated effector function, and furthermore, the antibody i) FAPα-expressing fibroblasts, for example, cancer-associated fibroblasts (CAFs) as described in Example 2 herein, ii) Can bind to DR4-expressing human tumor cell lines such as those described in Example 3 herein, iii) For example, when assayed as described in Example 3 of this specification, it can be bound in vitro in the presence of one or more DR4-expressing human tumor cell lines selected from the group consisting of DLD-1, A549, HCT-116, HCT-15, MDA-MB-231 and PANC-1. iv) When assayed as described in Example 10 of this specification, cell death can be mediated in DR4-expressing human tumor cell lines in the presence of FAPα-expressing cells such as FAPα-expressing fibroblasts. v) When assayed as described in Example 10 of this specification, concentration-dependent cell death can be mediated in DR4-expressing human tumor cell lines in the presence of FAPα-expressing cells such as FAPα-expressing fibroblasts. vi) When assayed in the presence of FAPα-expressing fibroblasts as described in Example 10 herein, concentration-dependent cell death can be mediated in one or more human DR4-expressing tumor cell lines selected from the group consisting of DLD-1 and MDA-MB-231. vii) When assayed in the presence of FAPα-expressing fibroblasts as described in Example 10 herein, caspase-8 activation can be induced in one or more human DR4-expressing tumor cell lines selected from the group consisting of DLD-1 and MDA-MB-231. viii) When the assay is performed in the presence of FAPα-expressing CAF, for example as described in Example 11 of this specification, CRC-derived organoids can be killed. ix) For example, when assayed as described in Example 12 of this specification, antitumor activity against pancreatic and gastric tumors is possible. x) For example, when assayed as described in Example 13 of this specification, antitumor and antimetastatic activity is possible, xi) For example, when assayed as described in Example 13 of this specification, antitumor and anti-metastatic activity against CRC is possible, xii) For example, when the assay was performed as described in Example 14 of this specification, it did not show hepatotoxicity. xiii) For example, when the assay is performed as described in Example 18 of this specification, C1q cannot be bound. xiv) For example, if the assay is performed as described in Example 18 of this specification, it may not be possible to bind to FcγR, e.g., FcγRIa, FcγRIIa, FcγRIIb and / or FcγRIIIa. xv) For example, if the assay is performed as described in Example 18 of this specification, it may bind to FcRn, and / or xvi) For example, when assayed as described in Example 19 of this specification, it exhibits pharmacokinetic properties similar to wild-type IgG1.

[0166] Furthermore, the antibody according to the present invention can induce transactivation-mediated cell death, and the cytotoxicity is i) Provide transfected NIH / 3T3 cells that express FAPα mature polypeptide, ii) To provide DR4-expressing tumor cells such as MDA-MB-231 or DLD-1, iii) The NIH / 3T3 cells are combined with the DR4-expressing tumor cells in a ratio of 1:2 between the number of NIH / 3T3 cells and the number of selected tumor cells. iv) For example, providing the antibody in a dilution series in the range of 0.28 ng / mL to 14,400 ng / mL to the sample. v) The sample obtained in step iv) is incubated at, for example, 37°C for 72 hours, and then, vi) Evaluate the survival of DR4-expressing tumor cells. vii) For example, using luminescence readout to determine the percentage of living cells in each diluted sample, and viii) Determining the percentage of living cells, It is evaluated in in vitro survival assays, including those that include this assay.

[0167] In one embodiment, the antibody can induce cell death, such as transactivation-mediated cell death, by at least 20% at an antibody concentration of 28 ng / ml, and / or at least 45%, for example, at least 50%, at an antibody concentration of 1800 ng / ml.

[0168] In further embodiments, the multispecific antibodies according to the present invention do not induce detectable hepatotoxicity. This can be monitored, for example, by measuring lactate dehydrogenase on day 4, and / or intracellular adenosine triphosphate on day 6 and / or day 7, after exposing liver spheroids consisting of primary human hepatocytes and non-parenchymal hepatocyte types to the multispecific antibodies.

[0169] nucleic acid constructs and expression vectors Further aspects of the present invention provide nucleic acid constructs or combinations of nucleic acid constructs encoding antibodies as defined herein. For example, in one embodiment, the combination of nucleic acid constructs includes a first construct encoding a first heavy chain, a second construct encoding a second heavy chain, a third construct encoding a first light chain, and a fourth construct encoding a second light chain. Alternatively, the combination of nucleic acid constructs includes a first construct encoding a first heavy chain and a first light chain, and a second construct encoding a second heavy chain and a second light chain.

[0170] Another aspect of the present invention provides an expression vector comprising one or more nucleic acid constructs described herein, or a combination of expression vectors. An expression vector in the context of the present invention can be any suitable vector comprising chromosomes, non-chromosomes, and synthetic nucleic acid vectors (nucleic acid sequences comprising a suitable set of expression regulatory elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid encoding an anti-FAPα antibody and / or an anti-DR4 antibody is contained in a naked DNA or RNA vector, e.g., a linear expression element (e.g., as described in Sykes and Johnston, Nat Biotech 17, 355-59 (1997)), a compressed nucleic acid vector (e.g., as described in U.S. Patent No. 6,077,835 and / or International Publication No. 00 / 70087), a plasmid vector, e.g., pBR322, pUC 19 / 18 or pUC 118 / 119, the smallest size nucleic acid vector "midge" (e.g., as described in Schakowski et al., Mol Ther 3, 793-800 (2001)), or a precipitated nucleic acid vector construct, e.g., a CaPO4 precipitated construct (e.g., International Publication No. 00 / 46147, Benvenisty and Reshef, PNAS USA) These include those described in 83,9551-55 (1986), Wigler et al., Cell 14,725 (1978), and Coraro and Pearson, Somatic Cell Genetics 7,603 (1981). Such nucleic acid vectors and their uses are well known in the art (see, for example, U.S. Patent Nos. 5,589,466 and 5,973,972).

[0171] In one embodiment, the vector is suitable for the expression of anti-FAPα antibodies and / or anti-DR4 antibodies in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors such as Van Heeke & Schuster, J Biol Chem 264, 5503 5509 (1989), and pET vectors such as Novagen, Madison WI.

[0172] The expression vector may, or may be alternatively, a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system can be used. Suitable vectors include, for example, vectors containing constitutive or inductive promoters such as alpha factor, alcohol oxidase, and PGH (referred to below: F. Ausubel et al., eds., Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987), and Grant et al., Methods in Enzymol 153, 516 544 (1987)).

[0173] Nucleic acid constructs and / or vectors may also contain nucleic acid sequences encoding secretory / localization sequences, thereby enabling the targeting of polypeptides, such as nascent polypeptide chains, into the periplasmic space or cell culture medium. Such sequences are known in the art and include secretory leader or signal peptides, organelle targeting sequences (e.g., nuclear localization sequences, ER retention signals, mitochondrial localization sequences, chloroplast localization sequences), membrane localization / anchor sequences (e.g., transport termination sequences, GPI anchor sequences), and the like.

[0174] Nucleic acid and / or expression vectors may also contain nucleic acid sequences encoding secretory / localization sequences, thereby enabling the targeting of polypeptides, such as nascent polypeptide chains, within the periplasmic space or cell culture medium. Such sequences are known in the art and include secretory leaders or signal peptides. Nucleic acid and / or expression vectors may contain any suitable elements that promote expression, i.e., transcription and / or translation of nucleic acids, so that components of a (bispecific) antibody are expressed. Nucleic acid and / or vectors associate with any suitable promoters, enhancers, and other expression-promoting elements. Examples of such elements include potent expression promoters (e.g., human CMV IE promoter / enhancer as well as RSV, SV40, SL3 3, MMTV, and HIV LTR promoters), effective poly(A) termination sequences, origins of replication for plasmid products in E. coli, antibiotic resistance genes as selection markers, and / or convenient cloning sites (e.g., polylinkers). Nucleic acid may also contain inductive promoters, as opposed to constitutive promoters such as CMV IE.

[0175] In a further embodiment, the present invention provides compositions comprising nucleic acid constructs or combinations of nucleic acid constructs as defined herein.

[0176] Delivery vehicle In a further embodiment, the present invention relates to the administration of nucleic acid constructs encoding antibodies of the present invention for in vivo expression. For in vivo expression of nucleic acids encoding antibodies, the nucleic acid is typically administered in a form suitable for entry into target cells. Various methods of nucleic acid delivery for in vivo expression exist, including both mechanical and chemical methods. For example, such methods may include electroporation or tattooing of nucleic acids onto the skin (Patel et al., 2018, Cell Reports 25, 1982-1993). Other methods suitable for administering nucleic acids to a subject include administration of nucleic acids in a suitable formulation.

[0177] Accordingly, the present invention also relates to a delivery vehicle containing the nucleic acid construct described herein. In one embodiment, the delivery vehicle may be particles. In some embodiments, the delivery vehicle may be a lipid formulation. The lipids of the formulation may be particles such as lipid nanoparticles (LNPs). The nucleic acids or combinations of nucleic acids of the present invention may be encapsulated within the particles, for example, within the LNPs. Various lipid formulations suitable for administering nucleic acids to subjects for in vivo expression are well known to those skilled in the art. For example, the lipid formulation may typically include lipids, ionizable aminolipids, PEG-lipids, cholesterol, or any combination thereof.

[0178] Various forms and methods for preparing lipid formulations suitable for administering nucleic acids to targets for the expression of therapeutic antibodies are well known in the art. Examples of such lipid formulations include, but are not limited to, those described in U.S. Patent No. 20180170866 (Arcturus), European Patent No. 2391343 (Arbutus), International Publication No. 2018 / 006052 (Protiva), International Publication No. 2014152774 (Shire Human Genetics), European Patent No. 2972360 (Translate Bio), U.S. Patent No. 10195156 (Moderna), and U.S. Patent No. 20190022247 (Acuitas).

[0179] Therefore, in a further embodiment, the present invention relates to (a) one or more nucleic acid constructs or delivery vehicles according to the present invention for use as pharmaceuticals, preferably for use in the treatment of cancer, for example, solid tumors.

[0180] Cells and host cells In further embodiments, the present invention provides recombinant host cells capable of producing multispecific antibodies as defined herein, comprising one or more nucleic acid constructs encoding antibodies as defined herein, or expression vectors as defined herein. It should be understood that the cells may be obtained by transfecting host cells, such as recombinant host cells, with the nucleic acid constructs or expression vectors. In one embodiment, the host cells are isolated host cells.

[0181] The host cells may be of human origin, such as human fetal kidney (HEK) cells, including HEK / Expi cells. Alternatively, they may be of rodent origin, such as Chinese hamster ovary cells, including CHO / N50 cells or CHO cells. Furthermore, the host cells may be of bacterial origin.

[0182] The host cell may contain a nucleic acid sequence encoding the antibody of the present invention or a portion thereof, which is stably integrated into the cell genome. Alternatively, the cell may contain a linear expression element comprising a non-integrated nucleic acid, such as a plasmid, cosmid, phagemide, or sequence encoding the expression of the anti-FAPα antibody and / or anti-DR4 antibody or a portion thereof. In particular, the host cell may contain a linear expression element comprising a non-integrated nucleic acid, such as a plasmid, cosmid, phagemide, or sequence encoding the expression of the anti-FAPα antibody and / or anti-DR4 antibody or a portion thereof.

[0183] Composition, (medical) use, and therapeutic application Furthermore, the present invention provides compositions comprising antibodies as defined herein. Preferably, such compositions are pharmaceutical compositions, i.e., the antibodies are contained in a pharmaceutically acceptable carrier.

[0184] Pharmaceutical compositions may be formulated in accordance with the prior art, for example, as described in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed. Mack Publishing Co., Easton, PA, 1995. The pharmaceutical compositions of the present invention may include, for example, diluents, fillers, salts, buffers, surfactants (e.g., nonionic surfactants such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and / or other materials suitable for inclusion in the pharmaceutical composition.

[0185] The pharmaceutical composition or multispecific antibody may be administered in a therapeutically effective dose by any suitable route and mode. In one embodiment, the pharmaceutical composition and / or multispecific antibody are administered by intravenous injection or infusion.

[0186] In further embodiments, the multispecific antibodies, one or more nucleic acid constructs, delivery vehicles, compositions, or pharmaceutical compositions described herein are intended for use as pharmaceuticals.

[0187] In further embodiments, the multispecific antibodies, one or more nucleic acid constructs, delivery vehicles, compositions, or pharmaceutical compositions described herein are intended for use in the treatment of diseases such as cancer.

[0188] In particular, the bispecific antibodies of the present invention can be used to treat various forms of cancer.

[0189] Furthermore, multispecific antibodies, nucleic acid constructs, delivery vehicles, compositions, or pharmaceutical compositions are disclosed herein for use in the treatment of primary tumors, and / or for the prevention and / or treatment of metastases.

[0190] In one embodiment, the present invention relates to multispecific antibodies, one or more nucleic acid constructs, delivery vehicles, or pharmaceutical compositions for use in the treatment of cancer which is a solid tumor. In one embodiment, solid tumor is a malignant solid tumor, such as a malignant solid tumor. In a further embodiment, malignant solid tumor is an advanced solid tumor and / or a metastatic solid tumor. In a further embodiment, malignant solid tumor is a metastatic solid tumor. In one embodiment, solid tumor is a metastatic cancer. In a further embodiment, the present invention relates to the use of multispecific antibodies, one or more nucleic acid constructs, delivery vehicles, or pharmaceutical compositions. In one embodiment, cancer is a carcinoma. Examples of cancers that can be treated are cancers selected from the group of colorectal cancers (CRCs), e.g., colorectal adenocarcinoma; breast cancer, e.g., triple-negative breast cancer; pancreatic cancer, e.g., pancreatic ductal adenocarcinoma; gastric cancer and lung cancer, e.g., non-small cell lung cancer. In particular, cancers selected from the group of pancreatic cancer, gastric cancer, and CRCs. In further embodiments, the cancer is selected from the group consisting of colorectal cancer [CRC], breast cancer, e.g., triple-negative breast cancer [TNBC], pancreatic cancer, e.g., pancreatic ductal adenocarcinoma [PDAC], esophageal and gastric cancer, e.g., gastric and esophageal cancer, head and neck squamous cell carcinoma [HNSCC], cervical cancer, and lung cancer, e.g., non-small cell lung cancer [NSCLC].

[0191] In one embodiment, the present invention provides a method for treating a target cancer, the method comprising administering a therapeutically effective amount of the multispecific antibody of the present invention. In a further embodiment, the present invention provides a method for treating a disorder in which DR4-expressing cells adjacent to FAPα-expressing cells are involved, the method comprising administering a therapeutically effective amount of the multispecific antibody of the present invention. DR4-expressing cells adjacent to FAPα-expressing cells enable trans-binding of the multispecific antibody according to the present invention.

[0192] As described above, a preferred disease that may be intended in the methods and uses of the present invention is cancer. The cancer is most preferably characterized by the expression of DR4. The expression of DR4 in cancer can be readily determined using methods known in the art, such as PCR, immunohistochemistry, or FACS analysis, i.e., the detection of DR4 transcript and / or protein expression. Antibodies described herein that can bind to human DR4 can be used, for example, immunohistochemistry and / or FACS analysis. Furthermore, FAPα-expressing cells, such as CAFs, should preferably be detected in the TME. Thus, in one embodiment, the TME comprises CAFs, and preferably, the CAFs express FAPα. The expression of FAPα in the TME can be readily determined using methods known in the art, such as PCR, immunohistochemistry, or FACS analysis, i.e., the detection of DR4 transcript and / or protein expression. Preferably, a multispecific antibody, one or more nucleic acid constructs, a delivery vehicle, or a pharmaceutical composition is used for treatment when the cancer expresses DR4, the tumor microenvironment comprises CAFs, and the CAFs express FAPα.

[0193] In further embodiments, patients diagnosed with cancer can be subjected to evaluation of DR4 expression in cancer cells and FAPα-expressing cells in TMEs, and if DR4 and FAPα, which may be in a low to high range, are detected, such patients can be selected for antibody therapy according to the present invention. However, including such evaluations when selecting patients for treatment may not always be necessary.

[0194] In further embodiments, the present invention relates to a method for treating cancer, comprising administering a therapeutically effective amount of a multispecific antibody as defined herein, one or more nucleic acid constructs as defined herein, a delivery vehicle as defined herein, a composition as defined herein, or a pharmaceutical composition as defined herein, to a subject requiring such treatment. In particular, the method may be for treating solid tumors, primary tumors and / or metastases. Examples of cancers to be treated may be selected from the group consisting of CRC, breast cancer, pancreatic cancer, gastric cancer and lung cancer. In particular, selection may be made from the group consisting of pancreatic cancer, gastric cancer and colorectal cancer.

[0195] In a further embodiment, the present invention relates to the use of the multispecific antibodies described herein in the manufacture of pharmaceuticals for the treatment of cancer.

[0196] kit The present invention further provides a parts kit comprising the antibody disclosed above, for example, a kit for use as a companion diagnostic / for identifying patients in a patient population who are inclined to respond to treatment with the antibody as defined above, or for predicting the efficacy or antitumor activity of the antibody when used in the treatment of a patient, the kit comprising the antibody as defined above, and instructions for use of the kit.

[0197] In one embodiment, the present invention provides a kit for diagnosing cancer, comprising a container containing a multispecific FAPαxDR4 antibody and one or more reagents for detecting crosslinking between FAPα-expressing cells and DR4-expressing cells. The reagents may include, for example, fluorescent tags, enzyme tags, or other detectable tags. The reagents may also include secondary or tertiary antibodies, or reagents for enzymatic reactions, the enzymatic reactions producing products that can be visualized.

[0198] In a further embodiment, the present invention provides a diagnostic composition comprising an antibody as defined herein. The diagnostic composition may further comprise a dilution buffer.

[0199] In a further embodiment, the present invention relates to a method for detecting whether crosslinking occurs between FAPα-expressing cells and DR4-expressing cells in a patient-derived sample by administration of a multispecific antibody according to any one embodiment disclosed herein, the method comprising: (i) contacting the sample with a multispecific antibody according to any one embodiment disclosed herein under conditions that enable the formation of a complex between the multispecific antibody and FAPα-expressing cells and DR4-expressing cells; and (ii) analyzing whether a complex has been formed.

[0200] FAPα antibody In a further embodiment, the present invention relates to a monospecific antibody and provides an anti-FAPα antibody comprising at least one FAPα-binding region, wherein the FAPα-binding region comprises a heavy chain variable region (VH) comprising three complementarity-determining regions CDR1, CDR2, and CDR3 located in the amino acid sequence shown in SEQ ID NO: 13, and a light chain variable region (VL) comprising three complementarity-determining regions CDR1, CDR2, and CDR3 located in the amino acid sequence shown in SEQ ID NO: 14. In one embodiment, the FAPα-binding region can bind to FAPα.

[0201] In a further embodiment, the present invention relates to a monospecific antibody and provides an anti-FAPα antibody comprising at least one FAPα-binding region capable of binding to FAPα, wherein the FAPα-binding region comprises a heavy chain variable region (VH) comprising three complementarity-determining regions CDR1, CDR2, and CDR3 located in the amino acid sequence shown in SEQ ID NO: 13, and a light chain variable region (VL) comprising three complementarity-determining regions CDR1, CDR2, and CDR3 located in the amino acid sequence shown in SEQ ID NO: 14.

[0202] In the context of the present invention, the anti-FAPα antibody relates, in a further embodiment, to an anti-FAPα antibody in which the apparent affinity of monovalent binding of the anti-FAPα antibody to human FAPα expressed by, for example, human lung fibroblasts is higher compared to the monovalent reference antibody anti-FAP5, for example, at least 5 times higher and for example at least 10 times higher when measured as illustrated in Example 2. As is known to those skilled in the art, EC 50 A lower value indicates a higher apparent affinity.

[0203] In further embodiments, the maximum binding (max gMFI) of the anti-FAPα antibody at a concentration of 10 μg / mL is higher than that of the reference antibody anti-FAP5, for example, by at least 25% and for example by at least 40% when the anti-FAPα antibody and the anti-FAP5 antibody bind monovalently to human FAPα expressed by, for example, human lung fibroblasts, as measured, for example, as illustrated in Example 2.

[0204] In further embodiments, the apparent affinity of the divalent binding of an anti-FAPα antibody to human FAPα expressed by, for example, human lung fibroblasts, is higher than that of the divalent binding reference antibody anti-FAP5, for example, at least 5 times higher and at least 10 times higher when measured as illustrated in Example 2. As is known to those skilled in the art, EC 50 A lower value indicates a higher apparent affinity.

[0205] Further disclosures herein include anti-FAPα antibodies comprising heavy chain variable regions (VH) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 1, 2, and 3, respectively. Further disclosures herein also include anti-FAPα antibodies comprising light chain variable regions (VL) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 4, 5, and 6, respectively. In one embodiment, the anti-FAPα antibody comprises a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 1, 2, and 3, respectively, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 4, 5, and 6, respectively. The CDR regions from the aforementioned variable heavy chain region and variable light chain region are annotated according to IMGT (see Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, Developmental and Comparative Immunology, 27(1), 55-77 (2003)).

[0206] The Disclosure further provides an anti-FAPα antibody in which the VH sequence of the FAPα-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 13. The Disclosure also provides an anti-FAPα antibody in which the VL sequence of the FAPα-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 14. In one embodiment, the VH sequence of the FAPα-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 13, and the VL sequence of the FAPα-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 14.

[0207] In a further embodiment, the VH sequence of the FAPα binding region shown in SEQ ID NO: 13 includes up to 10 substitutions, for example, up to 9 substitutions, for example, up to 8 substitutions, for example, up to 7 substitutions, for example, up to 6 substitutions, for example, up to 5 substitutions, for example, up to 4 substitutions, for example, up to 3 substitutions, for example, up to 2 substitutions, for example, up to 1 substitution. In a further embodiment, the VL sequence of the FAPα binding region shown in SEQ ID NO: 14 includes up to 10 substitutions, for example, up to 9 substitutions, for example, up to 8 substitutions, for example, up to 7 substitutions, for example, up to 6 substitutions, for example, up to 5 substitutions, for example, up to 4 substitutions, for example, up to 3 substitutions, for example, up to 2 substitutions, for example, up to 1 substitution. In a further embodiment, the VH sequence and VL sequence of the anti-FAPα antibody differ only in the framework region. In further embodiments, the VH sequence of the FAPα binding region differs from sequence number 13 by a maximum of 10 substitutions, for example, a maximum of 9 substitutions, for example, a maximum of 8 substitutions, for example, a maximum of 7 substitutions, for example, a maximum of 6 substitutions, for example, a maximum of 5 substitutions, for example, a maximum of 4 substitutions, for example, a maximum of 3 substitutions, for example, a maximum of 2 substitutions, for example, a maximum of 1 substitution. In further embodiments, the VL sequence of the FAPα binding region differs from sequence number 14 by a maximum of 10 substitutions, for example, a maximum of 9 substitutions, for example, a maximum of 8 substitutions, for example, a maximum of 7 substitutions, for example, a maximum of 6 substitutions, for example, a maximum of 5 substitutions, for example, a maximum of 4 substitutions, for example, a maximum of 3 substitutions, for example, a maximum of 2 substitutions, for example, a maximum of 1 substitution.

[0208] The anti-FAPα antibody according to the present invention may be monovalent or bivalent. In one embodiment, the anti-FAPα antibody is monovalent. In another embodiment, the anti-FAPα antibody is a bivalent antibody having two antigen-binding regions capable of binding to human FAPα, preferably the two antigen-binding regions having the same variable region sequence.

[0209] In the context of the present invention, an anti-FAPα antibody may include one or more substitutions in the first and / or second heavy chain, thereby including an Fc region comprising different first and second CH3 regions and resulting in a heterodimer interaction between the first and second CH3 regions, or comprising similar first and second CH3 regions and resulting in an Fc region that results in a homodimer interaction between the first and second CH3 regions. The first and / or second heavy chain of the anti-FAPα antibody may also include modifications in the Fc region to make the antibody inactive or deactivating, similar to the substitutions and modifications described for the multispecific antibodies described above. Accordingly, the present disclosure further provides an anti-FAPα antibody comprising (i) a first heavy chain and a second heavy chain, (ii) each of the first and second heavy chains comprising at least a hinge region, CH2 and CH3 regions, and (iii) in the first heavy chain, at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 (by Eu numbering) is substituted, and in the second heavy chain, at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 (by Eu numbering) is substituted. This disclosure also provides an anti-FAPα antibody as described herein, wherein (i) the anti-FAPα antibody comprises a first heavy chain and a second heavy chain, (ii) each of the first and second heavy chains comprises at least a hinge region, CH2 and CH3 regions, (iii) in the first heavy chain, at least one amino acid is substituted at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 (of the human IgG1 heavy chain by Eu numbering), and in the second heavy chain, at least one amino acid is substituted at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 (of the human IgG1 heavy chain by Eu numbering).In further embodiments, the first and second heavy chains are substituted at the same position. In further embodiments, an anti-FAPα antibody is provided in which (i) the amino acid at the position corresponding to F405 (according to Eu numbering) is L, or (ii) the amino acid at the position corresponding to K409 (according to Eu numbering) is R. In further embodiments, an anti-FAPα antibody is provided in which (i) the amino acid at the position corresponding to F405 (according to Eu numbering) is L. In further embodiments, (i) the amino acid at the position corresponding to F405 (of the human IgG1 heavy chain according to Eu numbering) is L, or (ii) the amino acid at the position corresponding to K409 (of the human IgG1 heavy chain according to Eu numbering) is R.

[0210] This disclosure further provides an anti-FAPα antibody comprising a first heavy chain and a second heavy chain, wherein one or both heavy chains are modified to reduce the degree of Fc-mediated effector function induced by the antibody compared to an identical antibody, except that it comprises unmodified first and second heavy chains.

[0211] The anti-FAPα antibody may comprise a first heavy chain and a second heavy chain, wherein in at least one of the first and second heavy chains, one or more amino acids at positions corresponding to L234, L235, G236, D265, N297, and P331 of the human IgG1 heavy chain according to Eu numbering are not L, L, G, D, N, and P, respectively. In one embodiment, the anti-FAPα antibody has positions corresponding to L234 and L235 of the human IgG1 heavy chain according to Eu numbering as F and E in the first and second heavy chains, respectively. In a further embodiment, the positions corresponding to L234, L235, and D265 of the human IgG1 heavy chain according to Eu numbering as F, E, and A in the first and / or second heavy chains, respectively. In a further embodiment, the positions corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to Eu numbering are F, E, and R in the first and second heavy chains, respectively.

[0212] This disclosure further provides an anti-FAPα antibody comprising a first heavy chain and a second heavy chain, wherein the positions of both the first and second heavy chains corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively, wherein (i) the position corresponding to F405 in the human IgG1 heavy chain according to Eu numbering is L, or (ii) the position of the first heavy chain corresponding to K409 in the human IgG1 heavy chain according to Eu numbering is R.

[0213] In the context of the present invention, an anti-FAPα antibody may include an FAPα-binding region in which (i) the heavy chain variable region (VH) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 1, 2, and 3, respectively, and the light chain variable region (VL) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 4, 5, and 6, respectively; (ii) the anti-FAPα antibody comprises a first heavy chain and a second heavy chain, wherein the positions of both the first and second heavy chains corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; and (iii) the position of the first heavy chain corresponding to position F405 in the human IgG1 heavy chain according to Eu numbering is L, or the position of the first heavy chain corresponding to position K409 in the human IgG1 heavy chain according to Eu numbering is L. In one embodiment, the antibody includes an FAPα-binding region in which (i) the heavy chain variable region (VH) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 1, 2, and 3, respectively, and the light chain variable region (VL) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 4, 5, and 6, respectively; (ii) the anti-FAPα antibody comprises a first heavy chain and a second heavy chain, and the positions of both the first and second heavy chains corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; and (iii) the position of the first heavy chain corresponding to position F405 in the human IgG1 heavy chain according to Eu numbering is L.

[0214] In the context of the present invention, the anti-FAPα antibody may include the heavy chain sequence shown in SEQ ID NO: 17 and the light chain sequence shown in SEQ ID NO: 18.

[0215] This disclosure further provides an anti-FAPα antibody, wherein the EC of the binding of the anti-FAPα antibody to FAPα is described. 50 When it binds to human lung fibroblasts or CAFs, the assay, for example, as described in Example 2 herein, is in the range of 0.005 to 0.1 μg / mL, for example, in the range of 0.01 to 0.05 μg / mL, for example, in the range of 0.012 to 0.2 μg / mL.

[0216] This disclosure further provides an anti-FAPα antibody, wherein the EC of the binding of the anti-FAPα antibody to FAPα is described. 50 When bound to human lung fibroblasts or CAFs in a divalent state, the assay, for example, as described in Example 2 herein, is in the range of 0.005 to 0.1 μg / mL, for example, in the range of 0.01 to 0.05 μg / mL, for example, in the range of 0.012 to 0.2 μg / mL.

[0217] DR4 antibody In a further embodiment, the present invention relates to a monospecific antibody and provides an anti-DR4 antibody comprising at least one DR4-binding region, wherein the DR4-binding region comprises a heavy chain variable region (VH) comprising three complementarity-determining regions CDR1, CDR2, and CDR3 located in the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region (VL) comprising three complementarity-determining regions CDR1, CDR2, and CDR3 located in the amino acid sequence shown in SEQ ID NO: 16. In one embodiment, the DR4-binding region can bind to DR4.

[0218] In a further embodiment, the present invention relates to a monospecific antibody and provides an anti-DR4 antibody comprising at least one DR4-binding region capable of binding to DR4, wherein the DR4-binding region comprises a heavy chain variable region (VH) comprising three complementarity-determining regions CDR1, CDR2, and CDR3 located in the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region (VL) comprising three complementarity-determining regions CDR1, CDR2, and CDR3 located in the amino acid sequence shown in SEQ ID NO: 16.

[0219] In the context of the present invention, the anti-DR4 antibody relates to an anti-DR4 antibody in a further embodiment, for example, the apparent affinity of an anti-DR4 antibody monovalently bound to human DR4 expressed by the human tumor cell line MDA-MB-231 is (i) When measured as illustrated in Example 3, for example, it is, for example, at least twice as high, for example at least three times as high, and / or compared to the monovalent reference antibody IgG1-DR4-chCTB007. (ii) For example, when measured as exemplified in Example 3, it is, for example, at least 5 times higher, for example, at least 8 times higher compared to the monovalently binding reference antibody IgG1-DR4-T1014A04.

[0220] As is known to those skilled in the art, the lower the EC 50 the higher the apparent affinity.

[0221] In a further embodiment, the maximum binding (max gMFI) of the anti-DR4 antibody at a concentration of 90 μg / mL is (i) When the anti-DR4 antibody and the IgG1-DR4-chCTB007 antibody bind monovalently to human DR4 expressed by, for example, the human tumor cell line MDA-MB-231, for example, when measured as exemplified in Example 3, for example, at least 2 times higher, for example, at least 3 times higher, and / or (ii) When the anti-DR4 antibody and the IgG1-DR4-T1014A04 antibody bind monovalently to human DR4 expressed by, for example, the human tumor cell line MDA-MB-231, for example, when measured as exemplified in Example 3, for example, at least 5 times higher, for example, at least 9 times higher.

[0222] In a further embodiment, the apparent affinity of the anti-DR4 antibody for bivalent binding to human DR4 expressed by, for example, the human tumor cell line MDA-MB-231 is higher compared to the bivalent binding reference antibody anti-IgG1-DR4-T1014A04, for example, when measured as exemplified in Example 3, for example, at least 2 times higher, for example, at least 5 times higher. As is known to those skilled in the art, the lower the EC 50 the higher the apparent affinity.

[0223] In a further embodiment, the maximum gMFI of the anti-DR4 antibody at a concentration of 90 μg / mL is, for example, at least twice and for example at least three times higher than the reference antibody IgG1-DR4-T1014A04, when the anti-DR4 antibody and the IgG1-DR4-T1014A04 antibody are monovalently bound to human DR4 expressed by, for example, the human tumor cell line MDA-MB-231, as measured, for example, as illustrated in Example 3.

[0224] Further disclosure herein are anti-DR4 antibodies in which the DR4 binding region comprises a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. Further disclosure herein are anti-DR4 antibodies in which the DR4 binding region comprises a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. In one embodiment, the DR4 binding region comprises a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 7, 8, and 9, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 10, 11, and 12, respectively. The CDR regions from the aforementioned variable heavy chain region and variable light chain region are annotated according to IMGT (see Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, Developmental and Comparative Immunology, 27(1), 55-77 (2003)).

[0225] The present disclosure further provides an anti-DR4 antibody in which the VH sequence of the DR4-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 15. The present disclosure also further provides an anti-DR4 antibody in which the VL sequence of the DR4-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 16. In one embodiment, the VH sequence of the DR4-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 15, wherein the VL sequence of the DR4-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 16.

[0226] In a further embodiment, the VH sequence of the DR4 binding region shown in SEQ ID NO: 15 includes up to 10 substitutions, for example, up to 9 substitutions, for example, up to 8 substitutions, for example, up to 7 substitutions, for example, up to 6 substitutions, for example, up to 5 substitutions, for example, up to 4 substitutions, for example, up to 3 substitutions, for example, up to 2 substitutions, for example, up to 1 substitution. In a further embodiment, the VL sequence of the DR4 binding region shown in SEQ ID NO: 16 includes up to 10 substitutions, for example, up to 9 substitutions, for example, up to 8 substitutions, for example, up to 7 substitutions, for example, up to 6 substitutions, for example, up to 5 substitutions, for example, up to 4 substitutions, for example, up to 3 substitutions, for example, up to 2 substitutions, for example, up to 1 substitution. In a further embodiment, the VH sequence and the VL sequence differ only in the framework region. In further embodiments, the VH sequence of the DR4 binding region differs from sequence number 15 by a maximum of 10 substitutions, for example, a maximum of 9 substitutions, for example, a maximum of 8 substitutions, for example, a maximum of 7 substitutions, for example, a maximum of 6 substitutions, for example, a maximum of 5 substitutions, for example, a maximum of 4 substitutions, for example, a maximum of 3 substitutions, for example, a maximum of 2 substitutions, for example, a maximum of 1 substitution. In further embodiments, the VL sequence of the DR4 binding region differs from sequence number 16 by a maximum of 10 substitutions, for example, a maximum of 9 substitutions, for example, a maximum of 8 substitutions, for example, a maximum of 7 substitutions, for example, a maximum of 6 substitutions, for example, a maximum of 5 substitutions, for example, a maximum of 4 substitutions, for example, a maximum of 3 substitutions, for example, a maximum of 2 substitutions, for example, a maximum of 1 substitution.

[0227] The anti-DR4 antibody according to the present invention may be monovalent or bivalent. In one embodiment, the anti-DR4 antibody is monovalent. In another embodiment, the anti-DR4 antibody is a bivalent antibody having two antigen-binding regions capable of binding to human DR4, preferably the two antigen-binding regions having the same variable region sequence.

[0228] In the context of the present invention, an anti-DR4 antibody may include one or more substitutions in the first and / or second heavy chain, thereby including an Fc region comprising different first and second CH3 regions and resulting in a heterodimer interaction between the first and second CH3 regions, or comprising similar first and second CH3 regions and resulting in an Fc region that results in a homodimer interaction between the first and second CH3 regions. The first and / or second heavy chain of the anti-DR4 antibody may also include modifications in the Fc region to make the antibody inactive or deactivating, similar to the substitutions and modifications described for the multispecific antibodies described above. Accordingly, the present disclosure further provides an anti-DR4 antibody, wherein (i) the anti-DR4 antibody comprises a first heavy chain and a second heavy chain, (ii) each of the first and second heavy chains comprises at least a hinge region, CH2 and CH3 regions, (iii) in the first heavy chain, at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 (by Eu numbering) is substituted, and in the second heavy chain, at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 (by Eu numbering) is substituted. The disclosure also further provides an anti-DR4 antibody, (i) wherein the anti-DR4 antibody comprises a first heavy chain and a second heavy chain, (ii) wherein each of the first and second heavy chains comprises at least a hinge region, CH2 and CH3 regions, (iii) wherein in the first heavy chain, at least one amino acid is substituted at a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 (of the human IgG1 heavy chain by Eu numbering), and in the second heavy chain, at least one amino acid is substituted at a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 (of the human IgG1 heavy chain by Eu numbering). In further embodiments, the first and second heavy chains are substituted at the same position.In a further embodiment, an anti-DR4 antibody is provided in which (i) the amino acid at the position corresponding to F405 (according to Eu numbering) is L, or (ii) the amino acid at the position corresponding to K409 (according to Eu numbering) is R.

[0229] This disclosure further provides an anti-DR4 antibody comprising a first heavy chain and a second heavy chain, wherein one or both heavy chains are modified to reduce the degree of Fc-mediated effector function induced by the antibody compared to the same antibody, except that the first and second heavy chains are unmodified.

[0230] The anti-DR4 antibody may comprise a first heavy chain and a second heavy chain, wherein in at least one of the first and second heavy chains, one or more amino acids at positions corresponding to L234, L235, G236, D265, N297, and P331 of the human IgG1 heavy chain according to Eu numbering are not L, L, G, D, N, and P, respectively. In one embodiment, the anti-DR4 antibody comprises a first heavy chain and / or second heavy chain where positions corresponding to L234 and L235 of the human IgG1 heavy chain according to Eu numbering are F and E, respectively. In a further embodiment, positions corresponding to L234, L235, and D265 of the human IgG1 heavy chain according to Eu numbering are F, E, and A, respectively, in the first heavy chain and / or second heavy chain. In a further embodiment, the positions corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain by Eu numbering are F, E, and R in the first and / or second heavy chains, respectively.

[0231] This disclosure further provides an anti-DR4 antibody comprising a first heavy chain and a second heavy chain, wherein the positions of both the first and second heavy chains corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively, and where (i) the position corresponding to F405 in the human IgG1 heavy chain according to Eu numbering is L, or (ii) the position of the first heavy chain corresponding to K409 in the human IgG1 heavy chain according to Eu numbering is R.

[0232] In the context of the present invention, an anti-DR4 antibody may include a DR4 binding region comprising (i) a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 7, 8, and 9, respectively, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 10, 11, and 12, respectively; (ii) wherein the anti-DR4 antibody comprises a first heavy chain and a second heavy chain, and the positions of both the first and second heavy chains corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; and (iii) wherein in the first and second heavy chains, the position corresponding to K409 in the human IgG1 heavy chain according to Eu numbering is R, or in the first and second heavy chains, the position corresponding to F405 in the human IgG1 heavy chain according to Eu numbering is L. In one embodiment, the antibody may include a DR4 binding region comprising (i) a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 7, 8, and 9, respectively, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 10, 11, and 12, respectively; (ii) where the anti-DR4 antibody comprises a first heavy chain and a second heavy chain, the positions of both the first and second heavy chains corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; and (iii) where, in the first and second heavy chains, the position corresponding to K409 in the human IgG1 heavy chain according to Eu numbering is R.

[0233] In the context of the present invention, the anti-DR4 antibody may include the heavy chain sequence shown in SEQ ID NO: 19 and the light chain sequence shown in SEQ ID NO: 20.

[0234] This disclosure further provides an anti-DR4 antibody, wherein the EC of the binding of the anti-DR4 antibody to DR4 is described. 50 When bound to DLD-1, A549, HCT-116, HCT-15, MDA-MB-231, or PANC-1, assays performed as described in Example 3 of this specification result in concentrations in the range of 0.01 to 1.0 μg / mL, for example, 0.05 to 0.75 μg / mL, for example, 0.1 to 0.5 μg / mL.

[0235] This disclosure further provides an anti-DR4 antibody, wherein the EC of the binding of the anti-DR4 antibody to DR4 is described. 50 When conjugated monovalently to DLD-1, A549, HCT-116, HCT-15, MDA-MB-231, or PANC-1, assays performed as described in Example 3 of this specification yield values ​​in the range of 0.1 to 2.0 μg / mL, for example, in the range of 0.25 to 1.75 μg / mL, for example, in the range of 0.4 to 1.75 μg / mL, for example, in the range of 0.5 to 1.5 μg / mL.

[0236] This disclosure further provides an anti-DR4 antibody, wherein the EC of the binding of the anti-DR4 antibody to DR4 is described. 50 When conjugated divalently to DLD-1, A549, HCT-116, HCT-15, MDA-MB-231, or PANC-1, assays performed as described in Example 3 of this specification yield values ​​in the range of 0.1 to 1.0 μg / mL, for example, in the range of 0.15 to 0.75 μg / mL, for example, in the range of 0.2 to 0.5 μg / mL, for example, in the range of 0.25 to 0.4 μg / mL.

[0237] Antibody production Conventional methods, such as hybrid hybridoma and chemical conjugation methods (Marvin and Zhu (2005) Acta Pharmacol Sin 26:649), can be used to prepare the antibodies of the present invention, including multispecific and bispecific antibodies. Antibodies can be produced by a method comprising (a) culturing recombinant host cells described herein under conditions that produce antibodies, and (b) isolating the antibodies produced from the culture. Co-expression of two antibodies consisting of different heavy and light chains in host cells yields a mixture of possible antibody products, in addition to the desired bispecific antibody, which can then be isolated, for example, by affinity chromatography or similar methods.

[0238] As described, strategies supporting the formation of functionally bispecific products through co-expression of different antibody constructs can also be used, e.g., the method described by Lindhofer et al. (1995 J Immunol 155:219). In the fusion of rat and mouse hybridomas producing different antibodies, the number of heterodimeric proteins is limited by preferential species-limited heavy / light chain pairing. Another strategy to promote heterodimer formation over homodimer formation is the "knob-into-hole" strategy, where a protrusion is introduced onto the first heavy chain polypeptide, and a corresponding cavity into the second heavy chain polypeptide, so that the protrusion can enter the cavity at the interface of these two heavy chains, thereby promoting heterodimer formation and hindering homodimer formation. The "protrusion" is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain. Compensatory "caves" of the same or similar size as the protrusions are created at the interface of the second polypeptide by replacing larger amino acid side chains with smaller ones (U.S. Patent No. 5,731,168). European Patent No. 1870459 (Chugai) and International Publication No. 2009089004 (Amgen) describe other strategies for supporting heterodimerization by co-expression of different antibody domains in host cells. These methods involve substituting one or more residues constituting the CH3-CH3 interface of both CH3 domains with charged amino acids so that they are electrostatically unfavorable for homodimerization and electrostatically favorable for heterodimerization. International Publication No. 2007110205 (Merck) describes yet another strategy that leverages differences between IgA and IgG CH3 domains to promote heterodimerization.

[0239] Another in vitro method for producing bispecific antibodies is described in International Publication No. 2008119353 (Genmab), in which bispecific antibodies are formed by "Fab arm" or "half-arm" exchange (exchange of heavy and binding light chains) between two monospecific IgG4 or IgG4-like antibodies under reducing conditions. The resulting product is a bispecific antibody having two Fab arms that may contain different sequences.

[0240] A preferred method for preparing the bispecific FAPαxDR4 antibody of the present invention includes the method described in International Publication No. 2011131746 and International Publication No. 13060867 (Genmab), which comprises the following steps: a) Provide a first antibody comprising an Fc region, wherein the Fc region comprises a first CH3 region; b) Provide a second antibody comprising a second Fc region, wherein the Fc region comprises a second CH3 region, Here, the first antibody is an FAPα antibody and the second antibody is a DR4 antibody, or vice versa; Here, The sequences of the first and second CH3 regions are different, thereby resulting in a heterodimer interaction between the first and second CH3 regions that is stronger than the homodimer interaction between each of the first and second CH3 regions; c) Incubate the first antibody together with the second antibody under reducing conditions; and d) Obtain the bispecific FAPαxDR4 antibody. Process.

[0241] Similarly, the present invention relates to a method for producing a multispecific, for example, bispecific antibody according to the present invention, the method being a) Provided are a first homodimer antibody comprising an FAPα binding region as described herein, and a second homodimer antibody comprising a DR4 binding region as described herein, wherein the antibody comprises an Fc region and may contain further features as described herein. Here, the sequences of the first CH3 region and the second CH3 region of the first antibody and the second antibody are different, whereby the heterodimer interaction between the first and second CH3 regions is stronger than each of the homodimer interactions of the first CH3 region and the second CH3 region; b) incubating the first antibody together with the second antibody under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization; and c) obtaining the heterodimeric multispecific antibody of the invention described herein, comprising the first immunoglobulin heavy chain and the first immunoglobulin light chain of the first antibody, and the second immunoglobulin heavy chain and the second immunoglobulin light chain of the second antibody; comprising the steps of.

[0242] In one embodiment, the first antibody is incubated with the second antibody under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization, wherein the heterodimer interaction between the first and second antibodies in the resulting heterodimeric antibody is such that Fab arm exchange does not occur with 0.5 mM GSH after 24 hours at 37°C.

[0243] Although not limited to theory, in step c), the heavy chain disulfide bond in the hinge region of the parent antibody is reduced, and the resulting cysteine ​​can then form an inter-heavy chain disulfide bond with a cysteine ​​residue of another parent antibody molecule (which originally has different specificities). In one embodiment of this method, the reducing conditions in step c) include the addition of a reducing agent, for example, selected from the group consisting of 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and beta-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. In a preferred embodiment, the reducing agent is 2-mercaptoethylamine. In a further embodiment, step c) includes restoring the conditions to non-reducing or low-reducing, for example by removing the reducing agent, for example by desalting.

[0244] In a further embodiment, the present invention relates to a method for producing a multispecific antibody, the method comprising: (a)(i) A first antibody which is a monospecific anti-FAPα antibody as described herein, and A second antibody comprising a DR4 binding region comprising a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 7, 8, and 9, respectively, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 10, 11, and 12, respectively, Here, the antibodies comprise a first heavy chain and a second heavy chain, wherein the positions of both the first and second heavy chains, corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to Eu numbering, are F, E, and R, respectively; (ii) A second antibody which is a monospecific anti-DR4 antibody as described herein, and A first antibody comprising a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 1, 2, and 3, respectively, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 4, 5, and 6, respectively, and a FAPα-binding region, Here, the antibody comprises a first heavy chain and a second heavy chain, wherein the positions of both the first and second heavy chains, corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to Eu numbering, are F, E, and R, respectively; or (iii) A first antibody which is a monospecific anti-FAPα antibody as described herein and a second antibody which is a monospecific anti-DR4 antibody as described herein; Provided; Here, the sequences of the first and second CH3 regions of the first and second antibodies are different, thereby the heterodimer interaction between the first and second CH3 regions is stronger than the homodimer interaction of the first CH3 region and the homodimer interaction of the second CH3 region, preferably the amino acid at the position corresponding to F405 in the first CH3 region is L and the amino acid at the position corresponding to K409 in the second CH3 region is R, or vice versa; (b) Incubate the first antibody together with the second antibody under reducing conditions sufficient to allow the cysteine ​​in the hinge region to undergo disulfide bond isomerization; and (c) Obtain a multispecific antibody comprising the first immunoglobulin heavy chain and the first immunoglobulin light chain of the first antibody, and the second immunoglobulin heavy chain and the second immunoglobulin light chain of the second antibody.

[0245] In a further embodiment, the present invention relates to a method for producing a multispecific antibody, the method comprising: (a)(i) A first antibody which is a monospecific anti-FAPα antibody as described herein, and A second antibody comprising a DR4 binding region, wherein the heavy chain variable region (VH) contains the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 7, 8, and 9, respectively, and the light chain variable region (VL) contains the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 10, 11, and 12, respectively. Here, the antibodies comprise a first heavy chain and a second heavy chain, wherein the positions of both the first and second heavy chains, corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to Eu numbering, are F, E, and R, respectively; (ii) A second antibody which is a monospecific anti-DR4 antibody as described herein, and A first antibody comprising a FAPα-binding region, wherein the heavy chain variable region (VH) contains the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 1, 2, and 3, respectively, and the light chain variable region (VL) contains the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs. 4, 5, and 6, respectively. Here, the antibody comprises a first heavy chain and a second heavy chain, wherein the positions of both the first and second heavy chains, corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to Eu numbering, are F, E, and R, respectively; or (iii) Provides a first antibody which is a monospecific anti-FAPα antibody as described herein and a second antibody which is a monospecific anti-DR4 antibody as described herein; Here, the sequences of the first and second CH3 regions of the first and second antibodies are different, thereby the heterodimer interaction between the first and second CH3 regions is stronger than each of the homodimer interactions between the first and second CH3 regions, preferably the amino acid at the position corresponding to F405 in the first CH3 region is L, and the amino acid at the position corresponding to K409 in the second CH3 region is R; (b) Incubate the first antibody together with the second antibody under reducing conditions sufficient to allow the cysteine ​​in the hinge region to undergo disulfide bond isomerization; and (c) Obtain a multispecific antibody comprising the first immunoglobulin heavy chain and the first immunoglobulin light chain of the first antibody, and the second immunoglobulin heavy chain and the second immunoglobulin light chain of the second antibody.

[0246] In a further embodiment, the present invention relates to a method comprising the following steps: (a) A step of culturing host cells containing an expression vector comprising (i) a nucleic acid sequence encoding the heavy chain sequence of the FAPα binding region as defined herein, and (ii) a nucleic acid sequence encoding the light chain sequence of the FAPα binding region as defined herein, and purifying a first antibody from the culture medium; (b) A step of culturing host cells containing an expression vector comprising (iii) a nucleic acid sequence encoding the heavy chain sequence of the DR4 binding region as defined herein, and (iv) a nucleic acid sequence encoding the light chain sequence of the DR4 binding region as defined herein, and purifying a second antibody from the culture medium; (c) A step of incubating the first antibody with the second antibody under reducing conditions sufficient to allow cysteine ​​in the hinge region to undergo disulfide bond isomerization, thereby obtaining a bispecific antibody.

[0247] In one embodiment, step c) includes the addition of a reducing agent. In a further embodiment, step c) includes the addition of a reducing agent selected from the group consisting of 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and beta-mercaptoethanol. In a further embodiment, step c) includes the addition of a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. In a further embodiment, step c) includes restoring the conditions to non-reducing or low-reducing by, for example, removal of the reducing agent. In a further embodiment, the reducing agent is removed by desalting.

[0248] In a further embodiment, the present invention relates to multispecific antibodies obtained by the method described herein.

[0249] Anti-idiotype antibody In further embodiments, the present invention relates to an antibody comprising at least one antigen-binding region capable of binding to DR4 and / or FAPα, i.e., an anti-idiotype antibody that binds to the antibody according to the present invention as described herein. In certain embodiments, the anti-idiotype antibody binds to an antigen-binding region capable of binding to DR4 and / or FAPα.

[0250] Anti-idiotype (Id) antibodies are antibodies that recognize specific determinants that typically associate with the antigen-binding site of an antibody. Anti-Id antibodies can be prepared by immunizing animals of the same species and genotype as the source of the monoclonal antibody with the monoclonal antibody from which the anti-Id is prepared. The immunized animals can typically recognize and respond to the idiotype determinants of the immunized antibody by producing antibodies against these idiotype determinants (anti-Id antibodies). Such antibodies are described, for example, in U.S. Patent No. 4,699,880. Such antibodies are a further feature of the present invention.

[0251] The anti-Id antibody can also be used as an "immunogen" to induce an immune response in another animal, thereby producing a so-called anti-anti-Id antibody. The anti-anti-Id antibody may be epitopeically identical to the original monoclonal antibody that induced the anti-Id antibody. Therefore, by using an antibody against the idiotype determinant of the monoclonal antibody, it is possible to identify other clones that express antibodies of the same specificity. The anti-Id antibody can be modified (thereby producing an anti-Id antibody variant) and / or derivatized by any suitable technique, for example, those described elsewhere herein with respect to the DR4-specific antibody and / or FAPα-specific antibody of the present invention. For example, a monoclonal anti-Id antibody can be conjugated to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize BALB / c mice. Serum derived from these mice typically contains an anti-anti-Id antibody with binding properties similar, if not identical, to the original / parental antibody. [Table 3] TIFF0007852147000004.tif234154TIFF0007852147000005.tif231153TIFF0007852147000006.tif220154 TIFF0007852147000007.tif219153TIFF0007852147000008.tif232153TIFF0007852147000009.tif223153 TIFF0007852147000010.tif232153TIFF0007852147000011.tif232154TIFF0007852147000012.tif219153 TIFF0007852147000013.tif232154TIFF0007852147000014.tif232154TIFF0007852147000015.tif219153 TIFF0007852147000016.tif231155TIFF0007852147000017.tif233155TIFF0007852147000018.tif232153 TIFF0007852147000019.tif233153TIFF0007852147000020.tif233154TIFF0007852147000021.tif232154 TIFF0007852147000022.tif234155TIFF0007852147000023.tif235154TIFF0007852147000024.tif232153 TIFF0007852147000025.tif232154TIFF0007852147000026.tif197154TIFF0007852147000027.tif173154

[0252] Further items in this disclosure The present invention is further illustrated by the following embodiments, which should not be construed as further limitations.

[0253] [Examples] [Example 1] Antibody production a. FAPα antibody Immunization and hybridoma generation were performed at Aldevron GmbH (Freiburg, Germany). The construct used for immunization was a cDNA encoding full-length human FAPα (SEQ ID NO: 33) cloned into an Aldevron-owned immunization vector. Anti-FAPα antibodies were generated by gene (DNA) immunization of OmniRat animals (transgenic rats expressing a diverse repertoire of antibodies with a complete human idiotype; Ligand Pharmaceuticals Inc.) using a gene gun. Serum samples were collected after a series of immunizations and tested by flow cytometry using mammalian cells transiently transfected with an expression plasmid stably expressing human FAPα (SEQ ID NO: 33). Antibody-producing cells were isolated from rat spleens and fused with mouse myeloma cells (Ag8) following a standard procedure. RNA from hybridomas producing FAPα-specific antibodies was extracted for sequencing. The variable regions (VH domain and VL domain) of the target heavy and light chains were synthesized and cloned into expression vectors containing the skeletal sequence of either the constant region of the human IgG1 heavy chain (SEQ ID NO: 21) or the constant region (LC) of the human kappa light chain (SEQ ID NO: 27) of the human IgG1m(f) allotype, depending on the selected binding domain. The FAPα-specific IgG1 kappa antibody IgG1-FAPα was selected along with the variable domain sequences of SEQ ID NO: 14(VL) and SEQ ID NO: 13(VH).

[0254] Other FAPα-targeted antibodies used in the examples: FAP5 disclosed in U.S. Patent Publication No. 20090304718A1, ESC11 disclosed in International Publication No. 2011040972A1, and RG7386 disclosed in U.S. Patent Publication No. 9926379B2. Antibodies were produced according to standard procedures (described in sections d-f).

[0255] b.DR4 antibody Immunization and hybridoma generation were performed at Aldevron GmbH (Freiburg, Germany). The constructs used for immunization were cDNA encoding full-length human DR4 (SEQ ID NO: 68) cloned into an Aldevron-owned immunization vector, and cDNA encoding the extracellular domain (ECD) of human DR4 (aa 24-239 of SEQ ID NO: 68) cloned into an Aldevron-owned immunization vector with an N-terminal tag sequence derived from the vector. Anti-DR4 antibodies were generated by gene (DNA) immunization of OmniRat animals using a gene gun. Serum samples were collected after a series of immunizations and tested by flow cytometry using mammalian cells transiently transfected with an expression plasmid for human DR4 expression. Antibody-producing cells were isolated from rat spleens and fused with mouse myeloma cells (Sp2.0) following a standard procedure. RNA from hybridomas producing DR4-specific antibodies was extracted for sequencing. The variable regions (VH domain and VL domain) of the target heavy and light chains were synthesized and cloned into expression vectors containing the skeletal sequence of either the constant region of the human IgG1 heavy chain (SEQ ID NO: 21) or the constant region (LC) of the human lambda light chain (SEQ ID NO: 28) of the human IgG1m(f) allotype, depending on the selected binding domain. The DR4-specific IgG1 lambda antibody IgG1-DR4 was selected along with the variable domain sequences of SEQ ID NO: 16 (VL) and SEQ ID NO: 15 (VH).

[0256] Other DR4-targeted antibodies used in the experiment: T1014A04 disclosed in U.S. Patent No. 7,361,341,B2; chCTB007 disclosed in U.S. Patent Application Publication No. 2009,0136503,A1; and ABBV-621, a TRAIL-R fusion agonist disclosed in International Publication No. 2019,178,438,A1. Antibodies were generated according to standard procedures (described in sections d-f).

[0257] c. Control antibody A human IgG1 antibody having the same antigen-binding domain as the HIV1 gp120-specific antibody b12 was used as a negative, unbound control in several experiments (Barbas et al., J Mol Biol. 1993 Apr 5;230(3):812-2). The VH and VL domains of b12 were generated by de novo gene synthesis (GeneArt gene synthesis; ThermoFisher Scientific, Germany) and cloned into an expression vector containing the skeletal sequence of the human IgG1 heavy chain constant region of the human IgG1 m(f) allotype. The heavy chain and light chain sequences of the b12 control antibody are included in section g below.

[0258] d. Antibody expression Antibodies were obtained by transfection of heavy and light chain expression vectors in producing cell lines, and purified from the culture supernatant by protein A affinity chromatography for functional characterization. IgG concentration was measured by absorbance at 280 nm. The purified antibodies were stored in phosphate-buffered saline (PBS) at 4°C.

[0259] e. Generation of bispecific antibodies Bispecific antibodies were obtained by controlled Fab arm exchange (DuoBody® platform technology), specifically by 2-MEA-induced controlled Fab arm exchange (cFAE) as described in International Publication Nos. 2011147986, 2011131746, and 2013060867 (Genmab), and by Labrijn et al. (Labrijn et al., PNAS 2013, 110:5145~50; Gramer et al., Mabs 2013, 5:962~973). In short, two parental antibodies containing a single matched point mutation in the CH3 domain (one F405L and the other K409R [Eu numbering (Kabat, NIH publication no 91-3242, 5th edition ed. National Institutes of Public Health, Bethesda, MD, USA. 662, 680, 689)]) were prepared separately, mixed, and subjected to controlled reducing conditions. The reducing conditions degrade the interchain disulfide bonds of the molecules, while the matched CH3 domains (including F405L and K409R) drive heterodimerization of the Fab arm and the formation of a bispecific molecule. Subsequent re-oxidation of the disulfide bonds yields a highly pure bispecific antibody preparation with a regular IgG1 structure.

[0260] f.Fc mutation To minimize interaction with the Fcγ receptor and complement system component C1q, mutations L234F, L235E, and D265A (FEA; Engelberts et al., EBiomedicine, 2020; SEQ ID NO: 23) or L234F, L235E, and G236R (FER, International Publication No. 2022 / 189667, SEQ ID NO: 22) were introduced into the heavy chain constant domain using Eu numbering.

[0261] Antibodies introducing both the FEA and F405L or K409R mutations are referred to as FEAL or FEAR, respectively, in the experiments shown. Antibodies introducing both the FER and F405L or K409R mutations are referred to as FERL or FERR, respectively, in the experiments shown.

[0262] To generate bispecific antibodies, two parent antibodies are mixed in PBS buffer (phosphate-buffered saline; 8.7 mM HPO4). 2- , 1.8 mM H2PO4 - , 163.9 mM Na + , 140.3 mM Cl - Equal volumes were mixed in pH 7.4. 2-mercaptoethylamine-HCl (2-MEA) was added to a final concentration of 75 mM, and the reaction mixture was incubated at 31°C for 5 hours. To reoxidize the interchain disulfide bonds and form intact bispecific antibodies, 2-MEA was removed by dialyzing with PBS buffer using a 10 kDa cutoff Slide-A-Lyzer carrier (Thermo Fisher Scientific) according to the manufacturer's protocol.

[0263] Overview of antibodies used in the examples. The amino acid sequences of the parent antibodies used in the following experiments, such as bispecific antibodies and / or monospecific antibodies, are shown in the following sequence numbers.

[0264] IgG1-FAPα-FERL: SEQ ID NO: 17(HC) and SEQ ID NO: 18(LC) IgG1-FAPα-FEAL: SEQ ID NO: 29(HC) and SEQ ID NO: 18(LC) IgG1-DR4-FERR: SEQ ID NO: 19(HC) and SEQ ID NO: 20(LC) IgG1-DR4-FEAR: SEQ ID NO: 30(HC) and SEQ ID NO: 20(LC) IgG1-DR4-FEAL: SEQ ID NO: 32(HC) and SEQ ID NO: 20(LC) IgG1-b12-FERL: SEQ ID NO: 60(HC) and SEQ ID NO: 57(LC) IgG1-b12-FEAL: SEQ ID NO: 58(HC) and SEQ ID NO: 57(LC) IgG1-b12-FERR: SEQ ID NO: 61(HC) and SEQ ID NO: 57(LC) IgG1-b12-FEAR: SEQ ID NO: 59(HC) and SEQ ID NO: 57(LC) IgG1-b12: SEQ ID NO: 56(HC) and SEQ ID NO: 57(LC) IgG1-FAP5-FEAL: SEQ ID NO: 47(HC) and SEQ ID NO: 48(LC) IgG1-FAP5: SEQ ID NO: 49(HC) and SEQ ID NO: 48(LC) IgG1-FAPα-F405L:SEQ ID NO: 31(HC) and SEQ ID NO: 18(LC) IgG1-FAP-ESC11-F405L:SEQ ID NO: 50(HC) and SEQ ID NO: 51(LC) IgG1-DR4-T1014A04-FEAR:Sequence ID 52(HC) and Sequence ID 53(LC) IgG1-DR4-chCTB007-FEAR:SEQ ID NO: 54(HC) and SEQ ID NO: 55(LC) ABBV-621-Fc Fusion: Sequence ID No. 65 RG7386: Sequence ID 62 (HC) and Sequence IDs 63-64 (LC) IgG1-b12-FER: SEQ ID NO: 73(HC) and SEQ ID NO: 57(LC)

[0265] [Example 2] Binding to human lung fibroblasts and CAFs a. Binding of BisG1-FAPα-FERL / DR4-FERR to FAPα expressed on the cell surface. The binding of bispecific or monoclonal antibodies (BisG1-FAPα-FERL / DR4-FERR, BisG1-FAPα-FERL / b12-FERR, IgG1-FAPα-FERL) with anti-FAPα arms derived from IgG1-FAPα-FERL to human FAPα expressed on the cell surface was analyzed by flow cytometry using human lung fibroblasts and CAFs that endogenously express FAPα. BisG1-b12-FERL / b12-FERR was used as a negative control.

[0266] Cell viability of human lung fibroblasts (Coriell Institute, catalog number GM05389) and CAF (proliferated from primary human CRC biopsy; Strating et al., Front.Immunol.2023, 16:14:1053920) was measured using acridine orange / propidium iodide (AO / PI; Nexcelom, catalog number CS2-0106). Human lung fibroblasts and CAF (50,000 cells / well) were seeded into 96-well round-bottom plates (Greiner Bio-one, catalog number 650101). Antibody dilutions were prepared using a fluorescence-activated cell sorting (FACS) buffer consisting of phosphate-buffered saline (PBS, Lonza, catalog no. BE17-517Q) + 1% bovine serum albumin (BSA, Roche, catalog no. 10735086001) + 0.02% sodium azide (Bio-World, catalog no. 41920044-3). The plates were centrifuged, the supernatant was removed, and the cells were resuspended in 50 μL of human Fc Block (BD, catalog no. 564220, diluted 1:100 with FACS buffer) and 50 μL of viability stain TO-PRO-3 Iodide (Thermo Fisher, catalog no. T3605, diluted 1:25,000 with FACS buffer), and incubated at 4°C for 15 minutes. The plates were washed three times with FACS buffer. The plates were centrifuged, the supernatant was removed, and the cells were resuspended in 50 μL of antibody dilutions of BisG1-FAPα-FERL / DR4-FERR, BisG1-FAPα-FERL / b12-FERR, BisG1-b12-FERL / b12-FERR (top 6 concentrations for CAF cultures only), or IgG1-FAPα-FERL (top 4 concentrations for CAF cultures only) (concentration range 90-0.0005 μg / mL, 3-fold serial dilution with FACS buffer), and incubated at 4°C for 30 minutes. The cells were washed three times with FACS buffer and resuspended in 50 μL of fluorescein isothiocyanate (FITC)-labeled polyclonal antibody goat anti-mouse IgG1 (Jackson Immuno Research, catalog no. 109-096-097, 1:100 dilution with FACS buffer).After incubation at 4°C for 30 minutes, cells were washed twice with FACS buffer and resuspended in FACS buffer. Antibody binding to viable cells (TO-PRO-3 negative) was analyzed by flow cytometry using FACSCelesta® (BD biosciences), and the data were processed using FlowJo_v10.8.1 (FlowJo LLC). Geometric mean fluorescence intensity (gMFI) was determined and visualized using GraphPad Prism. Binding curves were analyzed using nonlinear regression analysis. Maximum effect was 50% (EC). 50 The concentration (μg / mL) at which the observed effect was found was derived from the fitted curve.

[0267] result Similar dose-dependent human FAPα binding profiles were observed for both bispecific antibodies containing FAPα-specific arms (i.e., BisG1-FAPα-FERL / DR4-FERR and BisG1-FAPα-FERL / b12-FERR) against human lung fibroblasts and CAFs (EC from 3 experiments). 50 (See Figures 1A-1B and Table 1). A lower maximum binding was observed for the bivalent monoclonal antibody IgG1-FAPα-FERL. No binding was observed for the negative control BisG1-b12-FERL / b12-FERR antibody.

[0268] [Table 4]

[0269] b. Superior monovalent bonding of FAPα clones compared to FAP5 clones. The binding of FAPα-targeted monoclonal antibody clones, IgG1-FAPα-FEAL and IgG1-FAP5-FEAL, and bispecific antibodies with binding arms derived from the two FAPα-binding clones (BisG1-FAPα-FEAL / b12-FEAR and BisG1-FAP5-FEAL / b12-FEAR) to human lung fibroblasts endogenously expressing FAPα was compared and analyzed by flow cytometry. IgG1-b12-FEAR was used as a negative control.

[0270] The binding assays and analyses were performed as detailed in Section A, including the following differences: 20,000 fibroblasts / well seeded, no viability staining performed, and primary antibody dilutions tested (final concentrations of 10–0.0001 μg / mL, 5-fold serial dilutions using FACS buffer).

[0271] result All antibodies containing FAPα-binding Fab arms showed dose-dependent binding to human FAPα in human lung fibroblasts (Figure 1C and Table 2). The apparent affinity (EC) of monovalent and bivalent antibodies containing FAPα-binding domains derived from the FAPα-FEAL clone was also observed. 50 ) was superior to those with a FAPα-binding domain derived from the FAP5 clone (lower EC 50 (As shown by...). Furthermore, in contrast to FAP5, the bivalent antibody IgG1-FAPα-FEAL showed lower maximum binding compared to the monovalent antibody BisG1-FAPα-FEAL / b12-FEAR, indicating that binding was more similar in the monovalent format compared to the bivalent format. No binding was observed for the negative control IgG1-b12-FEAR.

[0272] [Table 5]

[0273] [Example 3] Binding of DR4-specific antibodies to DR4 expressed on the cell surface. a. Efficient monovalent and bivalent conjugation of anti-DR4 antibodies against multiple DR4-expressing tumor cell lines. The binding of BisG1-FAPα-FERL / DR4-FERR, BisG1-b12-FERL / DR4-FERR, and IgG1-DR4-FERR to human DR4 expressed on the cell surface was analyzed by flow cytometry using six human cancer cell lines derived from endogenously expressing DR4: colorectal adenocarcinoma (DLD-1, HCT-15), non-small cell lung cancer (A549), colon cancer (HCT-116), triple-negative breast cancer (MDA-MB-231), and pancreatic ductal adenocarcinoma (PANC-1). IgG-b12 was included as a negative control.

[0274] Cancer cell lines (DLD-1:ATCC, catalog number CCL-221; HCT-15:ATCC, catalog number CCL-225; HCT-116:ATCC, catalog number CCL-247; A549:ATCC, catalog number CCL-185; MDA-MB-231:ATCC, catalog number HTB-26; PANC-1:ATCC, catalog number CRL-1469) were detached from cell culture flasks using trypsin solution (Gibco, catalog number 25300-054), washed with PBS (GE Healthcare, catalog number SH3A3830.03), counted, and resuspended at the desired concentration.

[0275] The binding assay was performed as detailed in Section a of Example 2, except for the use of the IgG1-b12 antibody, and only the top three concentrations were tested. Data were processed using FlowJo_v10.8.1. gMFI was determined and visualized using GraphPad Prism.

[0276] result DR4-specific antibodies exhibited dose-dependent binding to all human DR4-expressing cancer cell lines (Figures 2A-2F). Mean EC values ​​from three independent experiments. 50The values ​​are shown in Table 3. As observed with binding to FAPα, the maximum binding capacity for all cell lines was higher for the monovalent antibody variant compared to the bivalent antibody variant. The bivalent IgG1-DR4-FERR antibody showed lower EC values ​​for all cell lines compared to the monovalent antibody BisG1-FAPα-FERL / DR4-FERR. 50 This demonstrated that strong binding to DR4 is observed in both monovalent and divalent bond formats.

[0277] BisG1-b12-FERL / DR4-FERR exhibits similar binding and average EC compared to BisG1-FAPα-FERL / DR4-FERR. 50 However, the negative control antibody IgG1-b12 did not show binding, confirming that the observed binding was dependent on the DR4-specific arm.

[0278] [Table 6]

[0279] b. Monovalent binding of chCTB007 and T1014A04 antibodies to cell surface-expressed DR4 is reduced compared to bivalent binding. The binding of DR4-specific antibodies BisG1-b12-FEAL / DR4-T1014A04-FEAR, BisG1-b12-FEAL / DR4-chCTB007-FEAR, IgG1-DR4-chCTB007-FEAR, and IgG1-DR4-T1014A04-FEAR to human DR4 expressed on the cell surface was analyzed by flow cytometry using the human cancer cell line OPM-2 (DSMZ, catalog number ACC 50), which endogenously expresses DR4. IgG1-b12 was used as a negative control.

[0280] The binding assay was performed as detailed in section a of Example 2, except for the following modifications: no viability staining and Fc Block were added; an antibody concentration series (final concentrations of 10 to 0.0001 μg / mL, 4-fold serial dilutions in FACS buffer) was used; a secondary antibody: R-phycoerythrin (PE)-labeled polyclonal antibody goat anti-human IgG1 (Jackson Immuno Research, catalog no. 109-116-098, 1:500 dilution in FACS buffer) was used; and the entire concentration curve of the negative control antibody was included, using an iQue Plus flow cytometer.

[0281] Furthermore, the binding of DR4-specific antibodies IgG1-DR4-FERR, BisG1-b12-FERL / DR4-FERR, BisG1-FAPα-FERL / DR4-FERR, IgG1-DR4-chCTB007-FEAR, BisG1-b12-FEAL / DR4-chCTB007-FEAR, IgG1-DR4-T1014A04-FEAR, and BisG1-b12-FEAL / DR4-T1014A04-FEAR to human DR4 expressed on the cell surface was analyzed by flow cytometry using the human cancer cell line MDA-MB-231, which endogenously expresses DR4. BisG1-b12-FERL / b12-FERR was used as a negative control.

[0282] The binding assay was performed as detailed in Section a of Example 2, with the following modification: the inclusion of the total concentration curve of the negative control antibody.

[0283] result IgG1-DR4-T1014A04-FEAR and IgG1-DR4-chCTB007-FEAR showed dose-dependent binding to human DR4 expressed by the human tumor cell line OPM-2 (Figure 2G). Decreased binding of IgG1-DR4-T1014A04-FEAR was observed compared to IgG1-DR4-chCTB007. Furthermore, low binding was observed for the monovalent variant BisG1-b12-FEAL / DR4-chCTB007-FEAR, while no binding or low binding was detected for the monovalent variant BisG1-b12-FEAL / DR4-T1014A04-FEAR.

[0284] IgG1-DR4-FERR demonstrated dose-dependent binding to human DR4 expressed by the human tumor cell line MDA-MB-231 with similar apparent affinity and maximal MFI as IgG1-DR4-chCTB007-FEAR, but with higher apparent affinity and maximal MFI compared to IgG1-DR4-T1014A04-FEAR. Monovalent binding of BisG1-b12-FERL / DR4-FERR and BisG1-FAPα-FERL / DR4-FERR to DR4 expressed by MDA-MB-231 cells was stronger (higher apparent affinity and maximal MFI) compared to BisG1-b12-FEAL / DR4-chCTB007-FEAR and BisG1-b12-FEAL / DR4-T1014A04-FEAR (Figure 2H, Table 4).

[0285] [Table 7]

[0286] [Example 4] Seed cross-reactivity to FAPα orthologs The binding of BisG1-FAPα-FERL / DR4-FERR, BisG1-FAPα-FEAL / b12-FEAR, and IgG1-FAPα-FERL to FAPα from species commonly used in nonclinical toxicology studies was evaluated by flow cytometry using Expi293F cells transiently expressing FAPα from various animal species. IgG1-b12 was included as a negative control.

[0287] ExpiFectamine® 293 transfection reagent (Thermo Fisher Scientific, catalog number A14525), Opti-MEM® low serum medium, GlutaMAX® supplement (Thermo Fisher Scientific, catalog number 51985026), and transfection enhancers 1 and 2 (Thermo Fisher Scientific, catalog number A14525) were used in accordance with the manufacturer's instructions to transfect Expi293F suspension cells (Thermo Fisher Scientific, catalog number A14527) with full-length human cells (UniProt ID Q12884, SEQ ID NO. 33), cynomolgus monkey cells (Macaca fascicularis, UniProt ID A0A2K5VGF4, SEQ ID NO. 39), dogs (Canis familiaris, UniProt ID A0A8C0NKP1, SEQ ID NO. 37), pigs (Sus scrofa, UniProt ID K7GQN2, SEQ ID NO. 38), rats (Rattus norvegicus, UniProt The mammalian expression vector pSB encoding an FAPα orthologue from either ID Q8R492 (SEQ ID NO: 36) or mouse (Mus musculus, UniProt ID P97321, SEQ ID NO: 35) was transiently transfected.

[0288] Expi293F cells expressing recombinant FAPα from various species (human, cynomolgus monkey, dog, pig, rat, or mouse) were seeded in 96-well round-bottom plates (Greiner Bio-one, catalog no. 650101) (20,000 cells / well). Antibody dilutions were prepared using FACS buffer consisting of PBS (Capricorn Scientific, catalog no. PBS-10XA, diluted to 1×PBS in distilled water) + 1% BSA (Roche, catalog no. 10735086001) + 0.02% sodium azide (Bio-World, catalog no. 41920044-3). The plates were centrifuged, the supernatant was removed, and the cells were resuspended in 100 μL of antibody dilutions of BisG1-FAPα-FERL / DR4-FERR, BisG1-FAPα-FEAL / b12-FEAR, IgG1-FAPα-FERL, and IgG1-b12 (concentration range of 30-0.00017 μg / mL by 3-fold serial dilution with FACS buffer) and incubated at 4°C for 30 minutes. The cells were washed with FACS buffer and resuspended in 50 μL of PE-conjugated goat anti-human IgG (Jackson ImmunoResearch, catalog no. 106-116-098; diluted 1:400 with FACS buffer). After incubation at 4°C for 30 minutes, the cells were washed with FACS buffer and resuspended in FACS buffer supplemented with TO-PRO-3 iodide survival marker (Invitrogen, catalog no. T3605, diluted 1:4,000). Antibody binding to viable cells (gMFI of PE on TO-PRO-3-negative cells) was analyzed by flow cytometry using FACS Celesta and FACS_Diva software (first two experiments) or iQue® 3 and FlowJo software (last experiment). gMFI was determined and visualized using GraphPad Prism. Binding curves were analyzed using nonlinear regression analysis. 50% of the maximum effect (EC) 50 The concentration (μg / mL) at which the observed effect was found was derived from the fitted curve.

[0289] result BisG1-FAPα-FERL / DR4-FERR showed dose-dependent binding to all species tested (Figures 3A-3F). Apparent affinity for human and cynomolgus monkey FAPα was similar (Figures 3A-3B), and the average EC2 was similar. 50 The values ​​were 0.24±0.04 μg / mL and 0.25±0.055 μg / mL, respectively (Table 5). All antibodies tested showed reduced binding to FAPα derived from mouse, rat, dog, and pig compared to human (Figures 3C-3F), therefore, EC of these species 50 The calculation could not be performed.

[0290] BisG1-FAPα-FEAL / b12-FEAR and BisG1-FAPα-FERL / DR4-FERR showed similar dose-dependent binding profiles, indicating that binding was FAPα-specific. Bivalent binding with IgG1-FAPα-FERL antibody was higher than monovalent binding in most species, with the exception of humans (similar binding) and cynomolgus monkeys (lower maximum binding). Binding of BisG1-FAPα-FERL / DR4-FERR to untransfected control cells was not observed (data not shown), and binding of IgG1-b12 to FAPα in any of the tested species was also not observed (Figures 3A-3F).

[0291] In summary, BisG1-FAPα-FERL / DR4-FERR and IgG1-FAPα-FERL showed cross-reactivity with all species tested, with the highest and most comparable binding to human and cynomolgus monkey FAPα.

[0292] [Table 8]

[0293] [Example 5] Binding affinity of FAPα clones to recombinant human and cynomolgus monkey FAPα. The binding affinity of BisG1-FAPα-FERL / DR4-FERR, BisG1-FAPα-FERL / b12-FERR, and the control BisG1-b12-FERL / DR4-FERR to recombinant human and cynomolgus monkey FAPα proteins was determined using label-free biolayer interferometry with an Octet HTX instrument (Sartorius).

[0294] The experiment was conducted at 30°C with shaking at 1,000 RPM. The amine-reactive second-generation (AR2G) biosensor (Sartorius, catalog no. 18-5092) was activated by reacting it with 20 mM EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) (Sigma-Aldrich, catalog no. 03449) and 10 mM s-NHS (N-hydroxysulfosuccinimide sodium salt) (Sigma-Aldrich, catalog no. 56485) for 300 seconds. An activated AR2G sensor was loaded with 2.5 μg / mL of His-tagged recombinant human FAPα (Acro Biosystems, catalog no. FAP-H5244-100ug) in 10 mM sodium acetate pH 5.0 (Sartorius, catalog no. 18-1069) or 2.5 μg / mL of His-tagged recombinant cynomolgus monkey FAPα (Acro Biosystems, catalog no. FAP-C52H3-100ug) in 10 mM sodium acetate pH 5.0 for 600 seconds, and then quenched for 300 seconds with 1 M ethanolamine pH 8.5 (Sartorius catalog no. 18-1071). After baseline measurement with Sample Diluent (1000 sec; Sartorius, catalog no. 18-1104), the association (100 sec) and dissociation (4,000 sec) of functional monovalent antibodies were determined using a concentration range of 0.78 nM to 800 nM in 2-fold serial dilutions in Sample Diluent. The molecular weight of the antibody used in the calculation was calculated from its sequence.

[0295] Data was acquired using Data Acquisition Software v12 (Sartorius) and analyzed using Data Analysis Software v12 (Sartorius). Antibody data traces were corrected by subtracting the sample diluent incubated with the reference sensor instead of the antibody.

[0296] The Y-axis was aligned to the last 10 seconds of the baseline. Interstep Correction alignment and Savitzky-Golay filtering were applied for dissociation. Data traces with a response <0.05 nm were excluded from the analysis. K below 50 nM D For antibodies with values, data traces with concentrations exceeding 100 nM were also excluded. The data were fitted to a 1:1 global full-fit model using a 100-second association and dissociation time set and a 4,000-second window of interest.

[0297] result BisG1-FAPα-FERL / DR4-FERR has equivalent picomolar K for human and cynomolgus monkey FAPα D The values ​​are shown (Table 6). The bispecific antibody with the unbound control arm BisG1-FAPα-FERL / b12-FERR showed FAPα-specific binding affinity to human and cynomolgus monkey FAPα at a similar level to BisG1-FAPα-FERL / DR4-FERR (Table 6). The control antibody BisG1-b12-FERL / DR4-FERR did not show binding (data not shown).

[0298] [Table 9]

[0299] [Example 6] Binding competition between anti-FAPα antibodies to recombinant human FAPα in BLI-based classical sandwich cross-block assays. Antibody cross-block analysis (epitope binning) was performed using biolayer interferometry (BLI) on an Octet HTX instrument (ForteBio) to determine binding competition for recombinant human FAPα between IgG1-FAPα-F405L, the benchmark FAPα-specific antibody IgG1-FAP-ESC11-F405L, and IgG1-FAP5. IgG1-b12 was included as a negative control.

[0300] Sandwich cross-block experiments were performed at 30°C with shaking at 1,000 RPM. Amine-reactive biosensors (AR2G) (ForteBio, catalog no. 18-5092) were activated for 300 seconds with a solution of 20 mM EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) (ForteBio, catalog no. 18-1033) and 10 mM s-NHS (N-hydroxysulfosuccinimide sodium salt) (ForteBio, catalog no. 18-1067). The activated AR2G sensors were loaded with 10 μg / mL of antibody 1 in 10 mM sodium acetate pH 6.0 (ForteBio, catalog no. 18-1070) for 600 seconds and quenched for 300 seconds with 1 M ethanolamine pH 8.5 (ForteBio, catalog no. 18-1071). After baseline measurement in Sample Diluent (30 seconds; ForteBio, catalog number #18-1048), His-tagged recombinant human FAPα (R&D Systems, catalog number 3715-SE, 100 nM in Sample Diluent) was loaded onto an AR2G biosensor containing immobilized antibody for 200 seconds. The theoretical molecular weight of the His-tagged recombinant human FAPα protein based on its amino acid sequence (86 kDa) was used for calculation. The association of a second antibody (5 μg / mL in Sample Diluent) (200 seconds) was determined. The sensor was regenerated by exposure to 10 mM glycine pH 2.5 (Riedl-deHaen, catalog number #15527) for 5 seconds, followed by neutralization in Sample Diluent for 5 seconds. Both steps were repeated twice. Subsequently, the sensor containing the immobilized first antibody was used again, starting from the baseline step.

[0301] Data was acquired using Data Acquisition Software v9 (ForteBio) and analyzed using Data Analysis HT Software v9 (ForteBio). The Y-axis was aligned to the start of the association phase, and Savitzky-Golay filtering was applied. The association response of the second antibody was plotted in matrix format. For each immobilized antibody, the response was corrected for the dissociation of His-tagged recombinant human FAPα protein from the immobilized first antibody by subtracting the average response of two reference sensors incubated with Sample Diluent instead of the second antibody. In general, a 0.21 nm cutoff based on the typical response to self-blocking was used to distinguish between blocking antibody pairs (<0.21 nm) and non-blocking antibody pairs (>0.21 nm).

[0302] result Sandwich cross-blocking experiments using BLI showed that IgG1-FAPα-F405L did not bind to FAPα simultaneously with the benchmark antibody IgG1-FAP-ESC11-F405L (response below cutoff, Table 7), indicating that these antibodies block each other's binding in this particular assay setting. IgG1-FAP5 antibody was able to bind to FAPα simultaneously with either IgG1-FAPα-F405L antibody or IgG1-FAP-ESC11-F405L antibody (response above the 0.21 nm cutoff), suggesting that the benchmark IgG1-FAP5 antibody targets a different epitope on FAPα than IgG1-FAPα-F405L. The negative control antibody IgG1-b12 did not show binding.

[0303] [Table 10]

[0304] [Example 7] Species cross-reactivity to DR4 or mouse / rat DR. The binding of BisG1-FAPα-FERL / DR4-FERR, BisG1-DR4-FEAL / b12-FEAR, and IgG1-DR4-FERR to various species of DR4 commonly used in nonclinical toxicology studies was evaluated by flow cytometry using ExpiCHO-S cells transiently expressing DR4 from various animal species. In contrast to other species, rodents express only one DR with the highest homology to human DR5. Therefore, ExpiCHO-S cells transiently expressing rat and mouse DR proteins were used instead. All DR constructs lack intracellular DD to avoid apoptosis of transfected cells upon DR(4) activation. IgG1-b12 was used as a negative control.

[0305] ExpiCHO-S cells (Life Technologies, catalog number EXX8120-3605-036) were transfected using ExpiFectamine® CHO transfection reagent (Thermo Fisher Scientific, catalog number A29131), OptiPro® serum-free medium (Thermo Fisher Scientific, catalog number 12309019), and ExpiFectamine CHO® enhancer (Thermo Fisher Scientific, catalog number A29131) according to the manufacturer's instructions, in humans (UniProt ID: O00220, lacking aa 365-448 [DD deletion]; SEQ ID NO: 40), cynomolgus monkeys (Macaca fascicularis; Uniprot ID: 15309893.2, lacking aa 370-457 [DD deletion]; SEQ ID NO: 41), and rabbits (Oryctolagus cuniculus; UniProt DR4 protein with a C-terminal HA tag bound to the snorkel domain in dogs (ID:17195576.1, lacking aa 331-418 [DD deletion]; SEQ ID:46), dogs (Canis familiari; UniProt ID:38280584.1, lacking aa 350-433 [DD deletion]; SEQ ID:44), pigs (Sus scrofa; UniProt ID:5670488.1, lacking aa 336-423 [DD deletion]; SEQ ID:45), or rats (Rattus norvegicus; UniProt ID:B8YBG7, lacking aa 254-323 [DD deletion]; SEQ ID:43) and mice (Mus musculus; UniProt ID:Q9QZM4, aa The protein was transiently transfected with a mammalian expression vector pSB encoding a DR protein with a C-terminal HA tag bound to the snorkel domain, lacking 273-356 [DD deletion] (SEQ ID NO: 42).

[0306] The binding assay was performed as described in Example 4, with the following modification: 50,000 cells / well were seeded. The binding curves were analyzed using nonlinear regression analysis (4-parameter dose-response curve fitting) in GraphPad Prism.

[0307] result The BisG1-FAPα-FERL / DR4-FERR and BisG1-DR4-FEAL / b12-FEAR antibodies, which can only bind in a monovalent state, showed binding to ExpiCHO-S cells transfected with human DR4 and cynomolgus monkey DR4 (Figures 4A-4B). The apparent affinity of BisG1-FAPα-FERL / DR4-FERR for human DR4 was quite high, and the mean EC for binding to human DR4 was high. 50 The ±SEM was 0.50±0.26 μg / mL for BisG1-FAPα-FERL / DR4-FERR (Table 8), but for cynomolgus monkey DR4, it did not reach a plateau, therefore the average EC2 was not reached. 50 It was not possible to calculate. Monoclonal antibody (bivalent conjugation, IgG1-DR4-FERR) also showed results for cynomolgus monkey DR4 (mean EC2). 50 Compared to ±SEM 0.14±0.09 μg / mL, human DR4 (mean EC4) 50 It showed a higher apparent affinity to (±SEM 1.84±0.45 μg / mL). BisG1-FAPα-FERL / DR4-FERR and BisG1-DR4-FEAL / b12-FEAR showed similar dose-dependent binding, and EC 50 This confirmed that the observed binding was not via the FAPα-specific arm, but rather via the DR4-specific arm.

[0308] Binding to rabbit, dog, or pig-derived DR4, or to mouse / rat DR, was not observed for BisG1-FAPα-FERL / DR4-FERR or IgG1-DR4-FERR (Figures 4C-4G).

[0309] IgG1-b12, used as a negative control, did not show binding to DR4 in any of the tested species (Figures 4A-4G).

[0310] In conclusion, the DR4-specific antibody clones used to construct the bispecific antibodies BisG1-FAPα-FERL / DR4-FERR (and BisG1-DR4-FEAL / b12-FEAR) showed cross-reactivity with cynomolgus monkey DR4. However, the apparent affinity for cynomolgus monkey DR4 was lower than the apparent affinity for human DR4. Binding to DR4 of other species (or mouse / rat DR) was not observed (Figures 4C-4G).

[0311] [Table 11]

[0312] [Example 8] Binding affinity of DR4 clones to recombinant human and cynomolgus monkey DR4. The binding affinity of BisG1-FAPα-FERL / DR4-FERR, BisG1-b12-FERL / DR4-FERR, and the control BisG1-FAPα-FERL / b12-FERR to recombinant human and cynomolgus monkey DR4 proteins was determined using label-free biolayer interferometry with an Octet HTX instrument (Sartorius).

[0313] The protocol detailed in Example 5 was used with the following differences: An anti-Penta-HIS biosensor (Sartorius, catalog no. 18-5120) was used. The sensor was pre-treated by exposing it to 10 mM glycine (Sigma-Aldrich, catalog no. 15527) buffer pH 1.5 for 5 seconds, followed by neutralization in Sample Diluent (Sartorius, catalog no. 18-1104) for 5 seconds. Both steps were repeated twice. The sensor was loaded with 50 nM human DR4HsECD-FcHisCtag (SEQ ID NO: 66) or cynomolgus monkey DR4MfECD-FcHisCtag (SEQ ID NO: 67) for 600 seconds. The dissociation time was 1,000 seconds. The window of interest for the dissociation time was set to 100 seconds for the cynomolgus monkey antigen and 1,000 seconds for the human antigen.

[0314] result BisG1-FAPα-FERL / DR4-FERR showed significantly higher binding affinity to humans compared to cynomolgus monkey DR4 (Table 9). A bispecific antibody with an unbound control arm, BisG1-b12-FERL / DR4-FERR, showed a similar level of nanomolar K₂. D DR4-specific binding with a value was confirmed (Table 9). The negative control antibody BisG1-FAPα-FERL / b12-FERR did not show binding (data not shown).

[0315] [Table 12]

[0316] [Example 9] Quantification of DR4, DR5, and FAPα surface expression, and evaluation of fibroblast cell death. a. The surface expression patterns of FAPα and DR4 on fibroblasts support the trans binding of BisG1-FAPα-FERL / DR4-FERR. The proposed mechanism of action of BisG1-FAPα-FERL / DR4-FERR is the trans binding of FAPα expressed on CAFs in TME and DR4 expressed on tumor cells, resulting in DR4 transactivation-mediated tumor cell death. If cis binding of BisG1-FAPα-FERL / DR4-FERR to fibroblasts occurs, this may potentially reduce antitumor activity by directly targeting fibroblasts instead of tumor cells. Example 2 demonstrated efficient FAPα binding to lung fibroblasts and CAFs by BisG1-FAPα-FERL / DR4-FERR and IgG1-FAPα-FERL.

[0317] The expression of human FAPα, DR4, and DR5 was evaluated in human lung fibroblasts and CAFs by semi-quantitative analysis using indirect immunofluorescence staining (Qifi assay). The following primary antibodies were used: mouse anti-human DR4 (Biolegend, catalog number 307202), mouse anti-human DR5 (Diaclone antibody for human lung fibroblasts, catalog number 854.860.000; Invitrogen antibody for CAFs, catalog number 14-9908-82), and mouse anti-human FAPα (USBiological, catalog number F4208-57E). Human lung fibroblasts were cultured in MEM medium (Lonza, catalog no. M5650) supplemented with 10% fetal bovine serum (FBS, ATCC, catalog no. 30-2020), 1% L-glutamine (Lonza, catalog no. BE17-605E), and 1% penicillin / streptomycin (Pen / Strep, Lonza, catalog no. DE17-603E). CAF cells were cultured in DMEM high-glucose medium (Sigma-Aldrich, catalog no. D6429) supplemented with 50 U / mL penicillin / streptomycin, 2 mM GlutaMAX, and 10% FBS (Bodinco BV, catalog no. 5067V20002).

[0318] Cell viability was confirmed using AO / PI (Nexcelom, catalog number CS2-0106). Human lung fibroblasts and CAF (50,000 cells / well) were seeded in 96-well round-bottom plates (Greiner Bio-one, catalog number 650101). Antibody dilutions were prepared using FACS buffer consisting of PBS (Lonza, catalog number BE17-517Q) supplemented with 1% BSA (Roche, catalog number 10735086001) and 0.02% sodium azide (Bio-World, catalog number 41920044-3). The following steps were performed for experiments using CAF. The plates were centrifuged, the supernatant was removed, and the cells were resuspended in 50 μL of human Fc Block (BD, catalog no. 564220, diluted 1:100 with FACS buffer) and 50 μL of viability stain TO-PRO-3 Iodide (Thermo Fisher, catalog no. T3605, diluted 1:25,000 with FACS buffer), and incubated at 4°C for 15 minutes. The plates were washed once with FACS buffer. Plates containing either human lung fibroblasts or CAF were centrifuged, the supernatant was removed, and the cells were resuspended in 50 μL of primary antibody (final concentration of 10 μg / mL in FACS buffer), and incubated at 4°C for 30 minutes. Cells were washed three times with FACS buffer and resuspended in 50 μL of FITC-labeled polyclonal antibody goat anti-mouse IgG1 (Dako, catalog number F047902-2, diluted 1:50 in experiments using CAF cells and 1:100 in experiments using human lung fibroblasts, in FACS buffer). In parallel, 15 μL of human-constituted Qifi beads (Biocytex, catalog number CP010) and 15 μL of calibration Qifi beads (Dako, catalog number K0078) were added to empty wells. After incubation at 4°C for 30 minutes, cells were washed twice with FACS buffer and resuspended in FACS buffer. All samples were analyzed using an iQue flow cytometer (Sartorius) or a FACSCelesta flow cytometer (BD biosciences), and data were processed using FlowJo_v10.8.1 (FlowJo LLC). Data were analyzed using GraphPad Prism.

[0319] In the Qifi assay, the primary antibody was used at saturation concentration, and the number of bound primary antibody molecules corresponded to the number of antigen sites present on the cell surface. The FITC-conjugated secondary antibody was also used at saturation concentration, and its fluorescence intensity was correlated with the number of bound primary antibody molecules on the cells and beads. The fluorescence values ​​recorded from the calibration beads, along with a clearly defined number of IgG monoclonal antibodies per bead, were used to generate a standard curve using GraphPad Prism software. The software then used the standard curve equation to calculate the specific antibody-binding capacity (sABC, corresponding to the average number of accessible antigen or molecular sites per cell) of antibody-stained cells.

[0320] result Human lung fibroblasts expressed high levels of human FAPα and low levels of DR5 (25,387 ± 2,564 and 5,841 ± 479 molecules per cell, respectively; mean ± SEM from two independent experiments) (Figure 5A). DR4 expression was below the lower limit of quantification (LLOQ, dashed line, Figure 5A). Similar results were observed for CAFs: high FAPα expression (109,759 ± 25,397 molecules / cell, mean ± SEM from three independent experiments), low DR5 expression (11,610 ± 502 molecules / cell, mean ± SEM from two independent experiments), and DR4 expression below LLOQ were observed (Figure 5B). In conclusion, the binding data from Example 2 and these results suggest that BisG1-FAPα-FERL / DR4-FERR targets only CAFs with the FAPα arm, but DR4 activation on fibroblasts as a result of simultaneous binding to DR4 and FAPα is unlikely.

[0321] b. BisG1-FAPα-FEAL / DR4-FEAR does not induce fibroblast cell death. Next, we evaluated the ability of BisG1-FAPα-FEAL / DR4-FEAR, BisG1-FAPα-FEAL / b12-FEAR, and BisG1-b12-FEAL / DR4-FEAR to induce death of human lung fibroblasts and CAFs. IgG1-b12-FEAR (negative control), IgG1-FAPα-FEAL, and RG7386 were also tested.

[0322] Human lung fibroblasts were detached using trypsin / EDTA (Lonza, catalog number 17-161E), and their viability was confirmed using AO / PI (Nexcelom, catalog number CS2-0106-25ml). 100 μL containing 5,000 cells / well was added to a 96-well flat-bottom plate (Greiner bio-one, catalog number 655180), incubated at 37°C and 5% CO2 for 24 hours, and then 50 μL / well of antibody dilution (10-0.0000256 μg / mL, 5-fold dilution using FACS buffer) was added. After 72 hours at 37°C and 5% CO2, 15 μL / well of Cell-Titer Glo (Promega, catalog number G7571) was added. The plates were incubated at 37°C in 5% CO2 for 1.5 hours, and then 100 μL of supernatant was transferred to a 96-well white OptiPlate (Perkin Elmer, catalog number 6005299). Luminescence (viability readout) was measured using an Envision instrument. The data were analyzed and visualized using GraphPad Prism. The percentage of viable cells, normalized to the no-antibody condition, was plotted against antibody concentration.

[0323] Follow-up experiments using human lung fibroblasts were performed by applying the following differences. Cell viability was confirmed using trypan blue (Sigma, catalog number T8154-100ml). The 96-well flat-bottom plate was manufactured by Falcon® (catalog number 353072). After incubating the cells for 3 hours, serial dilutions of the antibody (1 to 0.000002 μg / mL, 5-fold serial dilution using M0130 medium) were added. Reagents were diluted using M0130 medium consisting of RPMI (Gibco, catalog number A10491-01) supplemented with 10% heat-inactivated donor bovine serum (Gibco, catalog number 20371-030). 3 μM staurosporine / well was added as a positive control for cell death, and antibody IgG1-b12 was used as a negative control for cell death. Next, 50 μL / well of 100 nM Cytotox Green (Essenbio, catalog number 4633, diluted with M0130) was added, and the plates were incubated in Incucyte for 72 hours (37°C, 5% CO2), with images taken every 3 hours. Data were generated and processed using Incucyte software 2021B. The data were analyzed and visualized using GraphPad Prism. The data were fitted to a nonlinear four-parameter logistic curve. For each condition at each time point, the Incucyte software calculated the number of Cytotox Green-positive dead cells and plotted this against time.

[0324] For experiments with CAF, CAF was detached using trypsin (Lonza, catalog number BE02-007E) and counted using trypan blue (Fluka, catalog number 93590). 100 μL containing 10,000 CAF / well was seeded onto a collagen (Ibidi, catalog number 50204) monolayer (7.5 μg / mL collagen in 17.5 mM acetic acid, VWR, catalog number 30010.292) in a 96-well plate, incubated at 37°C and 5% CO2 for 24 hours, and then 12 μL / well of BisG1-FAPα-FEAL / DR4-FEAR antibody dilution (20-0.05 μg / mL, 4-fold dilution using FACS buffer) was added. Cell viability at 5% CO2 was assessed by adding 80 μL / well of preheated cell TiterGlo3D (Promega, catalog no. G9681) to each well after 72 hours at 37°C. Luminescence was read using a Spectramax plate reader (Molecular Devices) after 30 minutes (with gentle agitation for the first 15 minutes). Data were processed and visualized using GraphPad Prism. Data were fitted to a nonlinear four-parameter logistic curve. The graph shows the %±SEM of surviving tumor cells for technical replicates, normalized to the no-antibody condition and plotted against antibody concentration.

[0325] result In two separate experiments using human lung fibroblasts, BisG1-FAPα-FEAL / DR4-FEAR, IgG1-FAPα-FEAL, BisG1-FAPα-FEAL / b12-FEAR, or BisG1-b12-FEAL / DR4-FEAR did not alter lung fibroblast survival compared to the negative control IgG1-b12-FEAR (Figures 5C-5D). However, treatment with high concentrations of RG7386 did show a decrease in fibroblast survival (Figure 5C). CAF survival was also unaffected by treatment with BisG1-FAPα-FEAL / DR4-FEAR (Figure 5E). These results indicate that the BisG1-FAPα-FEAL / DR4-FEAR antibody does not induce fibroblast cell death.

[0326] [Example 10] Transactivation capability of BisG1-FAPα-FEAL / DR4-FEAR a. Comparison of FAPα-binding clones in a bispecific format for inducing DR4 transactivation-mediated cell death. The ability of BisG1-FAPα-FEAL / DR4-FEAR and BisG1-FAP5-FEAL / DR4-FEAR to induce DR4 transactivation-mediated cell death in the DR4-expressing human cancer cell line MDA-MB-231 in the presence of human lung fibroblasts was evaluated. BisG1-FAPα-FEAL / b12-FEAR, BisG1-FAP5-FEAL / b12-FEAR, and BisG1-b12-FEAL / DR4-FEAR were used as negative controls.

[0327] An in vitro survival assay was performed using the human cancer cell line MDA-MB-231 (breast cancer, ATCC, catalog number HTB26) cultured in the presence of human lung fibroblasts. MDA-MB-231 cells (cultured in M0089 medium consisting of high-glucose DMEM and HEPES[N'-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid] (Lonza, catalog number BE12-709F), supplemented with 10% iron-containing donor bovine serum (Life Technologies, catalog number 20371), 1% L-glutamine (Lonza, catalog number BE17-605E), 1% 100 mM sodium pyruvate (Lonza, catalog number BE13-115E), and 1% 100× non-essential amino acid solution (Life Technologies, catalog number 11140)) were subjected to 0.3 μM CytoLight Rapid Red (Essen The cells were incubated with Bio (catalog no. 4706, stock 1 μM) at 37°C for 20 minutes and washed three times with culture medium M0130 (replenished with RPMI (Gibco, catalog no. A10491-01) and 10% thermo-inactivated donor bovine serum (Gibco, catalog no. 20371-030)). The viability of MDA-MB-231 cells was confirmed with trypan blue (Sigma-Aldrich, catalog no. T8154-100 ml). Cells (5,000 CytoLight Rapid Red labeled cells / well and 2,500 human lung fibroblasts / well) were seeded in M0130 medium in 96-well flat-bottom plates (Falcon, catalog number 353072), incubated at 37°C and 5% CO2 for 24 hours, and then serial dilutions of antibodies (final concentrations of 1000 to 0.00051 ng / mL, 5-fold serial dilution using M0130 medium) were added. Negative control antibodies (BisG1-FAPα-FEAL / b12-FEAR, BisG1-FAP5-FEAL / b12-FEAR, and BisG1-b12-FEAL / DR4-FEAR) were fixed at a concentration of 1000 ng / mL, and staurosporine (Sigma, catalog number S6942, diluted in M0130 medium) at a final concentration of 1 μM was used as a positive control for cell death.Next, 50 μL / well of 100 nM Cytotox Green (Essen Bio, catalog number 4633, diluted in M0130 medium) was added, and the co-culture plates were incubated in an Incucyte® (Essenbio) system at 37°C and 5% CO2 for 72 hours, with imaging performed every 4 hours. Data were generated and processed using Incucyte software 2021B. The data were analyzed and visualized using GraphPad Prism. The data were fitted to a nonlinear 4-parameter logistic curve. For each condition, the Incucyte software generated a signal corresponding to the number of Cytotox Green-positive dead cells at each time point (overlap area, μm). 2 (image) and signals corresponding to the total number of cells (red area, μm²) 2 The area under the curve (AUC) was calculated using GraphPad Prism and plotted against antibody concentration.

[0328] result In co-cultures of MDA-MB-231 cells and human lung fibroblasts, BisG1-FAPα-FEAL / DR4-FEAR and BisG1-FAP5-FEAL / DR4-FEAR induced dose-dependent cell death (Figure 6A). BisG1-FAPα-FEAL / DR4-FEAR induced maximal DR4-dependent MDA-MB-231 cell death at lower concentrations than BisG1-FAP5-FEAL / DR4-FEAR (Table 10). As expected, the control bispecific antibodies BisG1-FAPα-FEAL / b12-FEAR, BisG1-FAP5-FEAL / b12-FEAR, and BisG1-b12-FEAL / DR4-FEAR did not induce cell death.

[0329] [Table 13]

[0330] b. BisG1-FAPα-FERL / DR4-FERR induces DR4 transactivation-mediated cell death only in the presence of FAPα-expressing cells. The ability of BisG1-FAPα-FERL / DR4-FERR to induce DR4 transactivation-mediated cell death in DR4-expressing human cancer cells in the presence and absence of FAPα-expressing cells was evaluated. BisG1-b12-FERR / b12-FERL antibody was used as a negative control. RG7386 was included in experiments using cancer cells alone, while the control antibodies BisG1-b12-FERL / DR4-FERR and BisG1-FAPα-FERL / b12-FERR were included in co-culture experiments.

[0331] For co-culture experiments, cancer cells were cultured in the presence of the NIH / 3T3 cell line (mouse fibroblasts, ATCC, catalog number CRL-1658) transfected to express the FAPα mature polypeptide (SEQ ID NO: 33) as follows: On the day of transfection, NIH / 3T3 cells were harvested using 0.5% trypsin / EDTA (Gibco, catalog number 25300-062), and their viability was measured using a Vicell-BLU instrument (Beckman Coulter). A total of 1 million cells were transferred to 15 mL tubes (Greiner Bio-one, catalog number 188271), washed with Hanks equilibrium salt solution (HBSS, Gibco, catalog number 14175-053), resuspended in 100 μL of 4D-nucleofector solution (500 μL of supplement 1 mixed with 2.25 mL of SG Cell Line Solution; both reagents are from SG Cell Line Solution boxes, Lonza, catalog number PBC3-02250), and transferred to cuvettes (Lonza, catalog number PCK-2005) containing 4.7 μL of pGENPGK-FAP-puro plasmid DNA. The pGENPGK-FAP-puro expression vector (size approximately 7,000 bp) contained the following key elements: full-length human FAP expressed by a phosphoglycerate kinase (PGK) promoter, a puromycin-selective marker expressed by a Simian virus 40 (SV40) promoter, and a Kozak sequence upstream of the PGK promoter and an ampicillin-selective marker expressed by a beta-lactamase (BLA) promoter. Electroporation was performed using a 4D-Nucleofector (X unit, Lonza). Next, 400 μL of M0057-05 medium consisting of DMEM high glucose and HEPES (Lonza, catalog number BE12-709F), supplemented with 10% iron-containing thermoinactivated donor bovine serum (DBSI, Life Technologies, catalog number 20371) and 1% 200 mM L-glutamine (Life Technologies, catalog number 25030-081), was added to the cuvette.After incubation at 37°C and 5% CO2 for 10 minutes, the contents of the cuvette were transferred using a Pasteur pipette (from the Lonza kit, catalog number V4XC-3024) to a 24-well plate (Cellstar, catalog number 662 160) already containing 500 μL / well of M0057-05 medium. The plate was stored in an incubator (37°C, 5% CO2) to allow the cells to recover and proliferate. After 72 hours, the medium was replaced with 1 mL / well of medium consisting of 20 mL of a 1:1 mixture of fresh M0057-05 medium and M0057-05 medium recovered from a flask containing untransfected 100% confluent NIH / 3T3 cells, to which 3.5 μL of puromycin (stock solution 10 mg / mL, Sigma, catalog number P9620) was added. The culture medium was changed every four days until a sufficient number of cells were available for evaluation of transfection efficiency (FAPα expression) by flow cytometry.

[0332] To measure FAPα expression, 10,000 NIH / 3T3-FAPα cells were seeded in a round-bottom 96-well plate, centrifuged, and resuspended in 20 μL / well of IgG1-FAPα-FERL primary antibody (stock concentration of 5 mg / mL diluted 1:4 with FACS buffer). After incubation at 4°C in the dark for 20 minutes, the cells were washed twice with FACS buffer and resuspended in 20 μL of secondary FITC-labeled polyclonal antibody goat anti-mouse IgG1 (diluted 1:4 with FACS buffer), followed by a further 20-minute incubation (at 4°C in the dark). After washing the cells twice with FACS buffer and resuspending them in FACS buffer, data were read out using CellStream (Luminex). FAPα expression measured by gMFI was comparable to that measured in a positive control (HEK293F cells transiently expressing FAPα) (data not shown).

[0333] In vitro survival assays were performed using two human cancer cell lines: DLD-1 (colorectal adenocarcinoma, ATCC, catalog number CCL221) cultured in M0130 medium and MDA-MB-231 (cultured in M0089 medium). Cells were harvested using trypsin (Gibco, catalog number 25300-054), and cell viability was measured using AO / PI (Nexcelom, catalog number CS2-0106). 6,600 cancer cells per well (50 μL each), either containing 3,300 NIH / 3T3-FAPα cells per well or not, were seeded into 96-well plates (Perkin Elmer, catalog number 6005680). Cells were incubated for 4 hours (37°C, 5% CO2) to adhere to plates, and then an antibody concentration series (final concentrations from 14.4 to 0.000007 μg / mL, serially diluted 8-fold in M0130 medium) was added at 50 μL / well. 50 μL / well of phenylarsine oxide (PAO, Sigma-Aldrich, catalog number P3075, stock concentration 50 mg / mL diluted 1:1,000 in FACS buffer) was used as a positive control for cell death. After incubating the plates for 72 hours (37°C, 5% CO2), viability was read out. For viability readout, 20 μL / well of cell TiterGlo (Promega, catalog number G755A) was added, and after incubating the plates for 1.5 hours (37°C, 5% CO2), luminescence was read out using EnVision. The percentage of viable cells is calculated using the following formula: ([Signal sample - Signal PAO control] - [Signal fibroblasts only - Signal PAO control]) / ([Signal cancer cells only - Signal PAO control] - [Signal fibroblasts - Signal PAO control]). The data was analyzed and visualized using GraphPad Prism. The data was fitted to a nonlinear 4-parameter logistic curve. The percentage of viable cells (double) is plotted against antibody concentration.

[0334] result In monocultures of MDA-MB-231 and DLD-1 cancer cells, survival after treatment with BisG1-FAPα-FERL / DR4-FERR was comparable to that of the control BisG1-b12-FERL / b12-FERR. On the other hand, treatment with the highest concentration of RG7386 resulted in decreased cancer cell survival (Figures 6B-6C; Table 11). Both MDA-MB-231 and DLD-1 cancer cells express DR5 (data not shown).

[0335] [Table 14]

[0336] In co-cultures of cancer cells and NIH / 3T3-FAPα effector cells, BisG1-FAPα-FERL / DR4-FERR induced dose-dependent cell death (Figures 6D-6E; Table 12 for the top four concentrations). As expected, BisG1-b12-FERL / DR4-FERR, BisG1-FAPα-FERL / b12-FERR, and BisG1-b12-FERL / b12-FERR (all containing at least one unbound control Fab arm) did not induce substantial tumor cell death. Therefore, DR4 transactivation-mediated cancer cell death depends on the targeting of DR4 and FAPα by bispecific antibodies.

[0337] [Table 15]

[0338] c. Caspase-8 activation To evaluate downstream signaling of DR4 transactivation, caspase-8 activation was assessed in co-cultures of tumor cell lines and NIH / 3T3-FAPα cells in the presence or absence of BisG1-FAPα-FERL / DR4-FERR, RG7386, positive control recombinant human TRAIL (Biolegend, catalog number 752906), and negative control antibody BisG1-b12-FERL / DR4-FERR.

[0339] DR4-expressing tumor cells (MDA-MB-231, A549, DLD-1, and SNU-1076 [Head and Neck Cancer; Creative Bioarray catalog number CSC-C9620L]) were harvested as described in Example 3. NIH-3T3-FAPα cells were harvested as described in section b of Example 10. Tumor cells and fibroblasts were seeded in a 2:1 ratio (13,300 tumor cells and 6,700 fibroblasts / well) in a White Opaque 96-well microplate (PerkinElmer, catalog number 6005680) and incubated overnight at 37°C and 5% CO2 to adhere the cells to the plate. Next, an antibody concentration series (0.0003 to 100 nM in 5-fold increments) was added. The samples were mixed in a plate shaker (300 RPM) for 2 minutes, and the plates were incubated at 37°C and 5% CO2 for 5 hours.

[0340] Activation of the exogenous apoptotic pathway was determined by measuring caspase-8 activation using a homogeneous Caspase-Glo® 8 luminescence assay (Promega, catalog no. G8202). Plates were first left at room temperature for 30 minutes. Then, 100 μL / well of Caspase-Glo 8 reagent from the kit was added, mixed for 2 minutes on a plate shaker (300 RPM), and incubated at room temperature in the dark for 1 hour. Luminescence was measured using an EnVision Multiplate Reader. Luminescence data were processed with GraphPad Prism software to generate dose-response curves using nonlinear regression analysis (sigmoid dose-response with variable gradient).

[0341] result Dose-dependent caspase-8 activation for BisG1-FAPα-FERL / DR4-FERR was observed in co-cultures of DLD-1, MDA-MB-231, A549, and SNU-1076 tumor cells with NIH / 3T3-FAPα cells (Figures 7A, 7C, 7E, and 7G), but not in monocultures (Figures 7B, 7D, 7F, and 7H). These data suggest that BisG1-FAPα-FERL / DR4-FERR-induced cytotoxicity is mediated by caspase-8 activation. The maximum caspase-8 activation levels observed were comparable between BisG1-FAPα-FERL / DR4-FERR and TRAIL, which was included as a positive control. Furthermore, dose-dependent caspase-8 activation was observed for RG7386 in all tumor cell lines (Figure 7).

[0342] The absence of BisG1-FAPα-FERL / DR4-FERR-induced caspase-8 activation in tumor cell monocultures or co-cultures in the presence of BisG1-b12-FERL / DR4-FERR indicates that caspase-8 activation by BisG1-FAPα-FERL / DR4-FERR is conditional and dependent on the binding of both DR4 and FAPα. In contrast, the unconditional agonist TRAIL induced caspase-8 activation in tumor cell monocultures, regardless of the presence of FAPα. Further limited caspase-8 activation was observed in tumor cell monocultures containing RG7386 (possibly due to its ability to divalently bind to DR5), but not with BisG1-FAPα-FERL / DR4-FERR (Figures 7A-7H).

[0343] In summary, BisG1-FAPα-FERL / DR4-FERR exhibits effective caspase activation in all tumor cell lines tested, depending on the presence of FAPα-expressing fibroblasts. This is consistent with the conclusions of transactivation-mediated cell death presented in sections a and b of this example.

[0344] [Example 11] PDO-targeted cell death via DR4 transactivation in the presence of CAF The ability of BisG1-FAPα-FEAL / DR4-FEAR to induce DR4 trans-activation-mediated cell death via trans-binding of CRC PDO was investigated in the presence and absence of CAF. IgG1-FAPα-FEAL was used as a negative control.

[0345] In vitro survival assays were performed using PDOs derived from three patients with CRC: Hub096 (primary tumor, ascending colon), p19B (primary tumor, ascending colon), and p18T (primary tumor, sigmoid). The PDOs were incubated in a basement membrane extract (BME) matrix (Amsbio, catalog no. 3533-010-02) with the following supplements: 10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) buffer (Lonza, catalog no. 17737E), 50 U / mL penicillin / streptomycin (Gibco, catalog no. 15070-063), 2 mM GlutaMAX (Gibco, catalog no. 35050-038), 20% R-spongin-conditioned medium (293T-HA-Rspol-F cell line), 100 ng / mL. Noggin-conditioned medium (293T-mNoggin-Fc cell line), 1×B27 (Invitrogen, catalog number 17504-044), 10mM nicotinamide (Sigma-Aldrich, catalog number N0636), 10nM prostaglandin E2 (Tocris, catalog number 2296-10), 10nM gastrin (Sigma-Aldrich, catalog number G9145), 0.5mM N-acetylcysteine ​​(NAC, Sigma-Aldrich, catalog number A9165), 500nM A83-01 (SignalChem, catalog number A09-900-05), 50ng / mL human recombinant epidermal growth factor (EGF, Sigma-Aldrich, catalog number A9165), 10μM SB202190 (p38 inhibitor, Gentaur, catalog number A1632), and 10mM CAFs were cultured in a 2:1 ratio using PDO medium consisting of Advanced DMEM / F12 medium (Gibco, catalog number 12634-010) containing Y27632 (Rock kinase inhibitor, Abmole Bioscience, catalog number HY-10583). CAFs were cultured in CAF medium consisting of DMEM high-glucose medium (Sigma-Aldrich, catalog number D6429) supplemented with 50 U / mL penicillin / streptomycin, 2 mM GlutaMAX, and 10% FBS (Bodinco BV, catalog number 5067V20002).

[0346] CellTiter-Glo survival assay using PDO and CAF The day before the co-culture experiment, CAF was detached using trypsin (Lonza, catalog number BE02-007E) and counted using trypan blue (Fluka, catalog number 93590). 10,000 CAF per well was seeded onto a collagen (Ibidi, catalog number 50204) monolayer (7.5 μg / mL collagen in 17.5 mM acetic acid, VWR, catalog number 30010.292) in a 96-well plate (Thermo Fisher Scientific, catalog number 165306) and incubated at 37°C in 5% CO2. The following day, PDO was collected and dissociated using TripLE® Express (Gibco, catalog number 12604021) at 37°C for 5 minutes. The cells were washed with PBS (Corning, catalog number 21-0310CVR), counted using trypan blue, and resuspended on 5% Matrigel (Corning, catalog number 354234) diluted in a co-culture medium consisting of Advanced DMEM / F12 supplemented with HEPES buffer, penicillin / streptomycin, GlutaMAX, B27, EGF, Y27632, A83-01 and NAC (as described above), 10 ng / mL human insulin (Sigma-Aldrich, catalog number 19278), 10 ng / mL human fibroblast growth factor (FGF)-basic (Prepotech, catalog number 100-18B), and 25 ng / mL platelet-derived growth factor receptor (PDGFR) α / β (Sigma-Aldrich, catalog number p3326). 10,000 PDO was added to empty wells or CAF monolayers, and the plates were incubated at 37°C and 5% CO2 for 4 hours. Then, 12 μL / well of antibody concentration series (10–0.001 μg / mL, 5-fold serial dilution in co-culture medium) were added. All conditions were tested by technical replication. After 72 hours at 37°C, 5% CO2 cell viability was assessed by adding 80 μL / well of preheated TiterGlo3D (Promega, catalog no. G9681) to each well. After 30 minutes (with gentle agitation for the first 15 minutes), luminescence was read using a Spectramax plate reader (Molecular Devices). Data were processed and visualized using GraphPad Prism.The data were fitted to a nonlinear four-parameter logistic curve. The graph shows dual viable tumor cell percentages ± SEM, normalized for the PDO-only condition (without CAF or antibody) and plotted against antibody concentration.

[0347] Annexin V Survival Assay The day before the start of the co-culture experiment, CAF was collected as described for the CellTiter-Glo assay and incubated with the cytoplasmic membrane dye CellBrite Orange (Biotium, catalog no. 30022) at 37°C and 5% CO2 for 1.5–2 hours. Next, the cells were washed three times with CAF medium and placed in 4 × 10⁶ well plates coated with collagen (Costar®, catalog no. 3506). 5 Cells were seeded in wells (see CellTiter-Glo assay description for coating procedure). The following day, PDO Hub096 cells were harvested as described in the CellTiter-Glo assay, and the single-cell suspension was incubated with the cytoplasmic membrane dye CellBrite Blue (Biotium, catalog no. 30024) at 37°C and 5% CO2 for 1.5–2 hours. The cells were then washed and resuspended in PDO medium supplemented with 5% Matrigel, and 5 × 10⁶ cells were placed on adhesive CellBrite Orange-labeled CAF. 5 Cells were seeded in wells and incubated overnight at 37°C and 5% CO2 with antibody samples (0.1 μg / mL or 0.02 μg / mL).

[0348] After incubation overnight, cells were harvested using trypsin, washed with cold PBS, and gently resuspended in 200 μL of Annexin-V-FITC master mix consisting of 125 μL of Annexin-V-FITC (BD Pharmingen®, catalog no. 556419) and 2.5 mL of 1× binding buffer (BD Pharmingen, catalog no. 556454). Cells were incubated with the Annexin-V mix in the dark at room temperature for 30 minutes. Next, 400 μL of 1× binding buffer was added to each tube and transferred to 5 mL FACS tubes (Falcon®, catalog no. 352008). Annexin-V positivity was measured by flow cytometry using a FACSCelesta Cell Analyzer (BD Biosciences), and FITC was detected in CellBrite Orange-positive CAF and CellBrite Blue-positive PDO cell populations using FACSCelesta Cell Analyzer Software. + The cells were analyzed by gating.

[0349] result BisG1-FAPα-FEAL / DR4-FEAR induced dose-dependent cell death in all three PDOs, although sensitivity to BisG1-FAPα-FEAL / DR4-FEAR varied among the tested PDOs. Cell death was observed only in the presence of CAF. Treatment of PDO monocultures did not induce DR4 transactivation-mediated cell death, confirming that the bispecific antibody requires double binding to DR4 and FAPα to induce cell death. Treatment with the negative control IgG1-FAPα-FEAL antibody did not result in PDO cell death (Figures 8A-8C; Tables 13-15).

[0350] Co-cultures of fluorescently labeled PDO strain Hub096 and fluorescently labeled CAF confirmed that BisG1-FAPα-FEAL / DR4-FEAR induced apoptotic phenotypes only in PDO Hub096 (as assessed by annexin V positivity) without affecting CAF survival (Figure 8D, Table 16).

[0351] [Table 16]

[0352] [Table 17]

[0353] [Table 18]

[0354] [Table 19]

[0355] [Example 12] Evaluation of BisG1-FAPα-FEAL / DR4-FEAR antitumor activity in vivo a. Tumor tissue derived from patients expresses various levels of FAPα. To demonstrate its mechanism of action, BisG1-FAPα-FEAL / DR4-FEAR requires the expression of both FAPα and DR4 in the tumor mesenter to enable trans-binding-dependent DR4 agonism that leads to tumor cell death. For an in vivo proof-of-concept study, two patient-derived xenograft (PDX) models CTG-1234 (stomach) and CTG-1150 (pancreas) (available from vendors) with known DR4 mRNA expression were evaluated for FAPα expression using immunohistochemistry (IHC) on formalin-fixed paraffin-embedded (FFPE) tissue slides.

[0356] FFPE tissue from patient-derived invasive ductal carcinoma (Avaden Biosciences) was used as a positive control. Tissue sections were transferred to Superfrost Plus glass slides (Fisher Scientific; catalog no. 10149870) and subjected to IHC staining using the Ventana Discovery-Ultra (Roche) platform, along with FFPE tissue slides from CTG-1234 and CTG-1150 (both from Champions Oncology). Tissue staining was initiated by warming the slides with incubation at 37°C, followed by a firing process (60°C for 12 minutes). Three deparaffinization cycles were then performed, each cycle consisting of incubation at 70°C for 8 minutes, rinsing with EZ Prep (Roche, catalog no. 05279755001, 1 / 10 dilution), 4 minutes of coverslip coating, and EZ Prep coating. After the third cycle, the slides were washed again with EZ Prep and EZ Prep was added. The slides were incubated at 37°C, washed, and then treated with Discovery Cell Conditioner 1 (CC1; Roche, catalog number 06414575001) reagent for antigen retrieval. The slides were incubated at 95°C for 40 minutes. Next, the slides were incubated in CC Medium Coverslip (LCS; Roche, catalog number 05264839001) for 16 minutes. The slides were left at 37°C, washed three times with reaction buffer (Roche, catalog number 05353955001), then treated with one drop of inhibitor CM (Roche, catalog number 07017944001) and incubated for 8 minutes. After washing the slides twice with reaction buffer, the primary antibody rabbit anti-FAPα clone EPR20021 (reactive with both human and mouse FAPα: final concentration 5 μg / mL, Abcam, catalog number ab207178) or rabbit IgG isotype control (final concentration 5 μg / mL, Cell Signaling Technology, catalog number 3900S) was added.The slides were incubated for 32 minutes, washed twice, and then incubated with OmniMap anti-rabbit HRP conjugate secondary antibody (Roche, catalog no. 05269679001) for 16 minutes. After three washes, the slides were incubated with ChromoMap 3,3'-diaminobenzidine (DAB) and ChromoMap H2O2 for 8 minutes, followed by incubation with ChromoMap Copper for 4 minutes (all included in the Roche ChromoMap DAB kit, catalog no. 05266645001). The slides were washed twice, followed by incubation with hematoxylin II (Roche, catalog no. 05277965001) for 12 minutes and Bluing reagent (Roche, catalog no. 05266769001) for 8 minutes. The slides were washed three times and covered with coverslips using Epredia® ClearVue® mounting medium (Fisher Scientific, catalog number 23-425-401).

[0357] Immunostained FFPE tissue slides were scanned at 20x magnification using an AxioScan slide scanner (Zeiss). For scoring of FAPα-positive PDX tumor tissue, the image scans were analyzed with HALO software (Indica Labs), and the percentage of FAPα-positive tissue surface area and IHC intensity were quantified using the pre-designed Area Quantification v2.4.2 image analysis algorithm.

[0358] result FAPα positivity was demonstrated in both PDX tissues (IHC intensity staining was low (1+) to moderate (2+) across most of the surface area, Figure 9A), indicating that both PDX models were deemed suitable for in vivo proof-of-concept studies.

[0359] b. Antitumor activity in the gastric PDX CTG-1234 model The ability of BisG1-FAPα-FEAL / DR4-FEAR to induce antitumor activity in a gastric cancer-derived PDX model was evaluated in mice. BisG1-DR4-FEAL / b12-FEAR was used as a negative control.

[0360] Stock mice (Envigo's Athymic Nude-Foxn1nu) were bilaterally transplanted with fragments of Champions TumorGraft® CTG-1234, originally derived from human gastric cancer biopsies. The tumors were 1000-1500 mm. 3 After reaching this stage, the tumor fragments were collected and subcutaneously transplanted into the left flank of female test mice (Envigo athymoid Nude-Foxn1nu, 6-8 weeks old at the start of the experiment).

[0361] Use a digital caliper to monitor tumor growth twice a week, using the formula 0.52 × (length × width). 2 Tumor volume (TV) was calculated using ). The study was conducted with a mean tumor volume (MTV) of 1500 mm² in the control group of surviving mice (uncensored). 3 It ended when it reached that point.

[0362] The TV is approximately 200mm 3 Once the target was reached, the animals were matched by tumor size and assigned to either a control or treatment group (n=8 / group), and administration was started on day 0. On the treatment day, mice were intravenously (IV) injected with either BisG1-FAPα-FEAL / DR4-FEAR (0.5 mg / kg, 2 mg / kg, or 8 mg / kg, administered once weekly for 3 weeks [QW×3]) or the control BisG1-DR4-FEAL / b12-FEAR (8 mg / kg; QW×3) (Table 17).

[0363] Mice were monitored daily for clinical signs of disease, and their body weight was measured twice a week using a digital scale. Data including individual gram body weight, mean gram body weight, and mean percentage change in body weight relative to day 0 (%vD0) were recorded for each group. Animal deaths, if any, were recorded. Groups reporting mean %vD0 loss greater than 20 and / or mortality greater than 10% were considered to have exceeded the maximum tolerated dose (MTD) for their treatment with the evaluated regimen. Additional test toxicity endpoints were when mice were mortally injured, exhibited a net body weight loss of >20% over a continuous 7-day period, or exhibited a net body weight loss of >30%.

[0364] Tumor growth inhibition (TGI) was determined by calculating the percentage TGI (100% × [1 - (final MTV - initial MTV of the treatment group) / (final MTV - initial MTV of the control group)]). Tumor volumes of the treatment group during the study and at the end of the study were compared to those of the control group. Statistical differences between the various treatment groups were analyzed using the mean tumor volume on day 42 (the final day when all groups had completed treatment) (Mann-Whitney).

[0365] One additional endpoint used to assess efficacy was progression-free survival (PFS). Kaplan-Meier curves were analyzed using the log-rank (Mantel-Cox) test to assess statistical significance of PFS time (500mm). 3 (Using the tumor size cutoff).

[0366] [Table 20]

[0367] result In tumor-bearing mice treated with the negative control BisG1-DR4-FEAL / b12-FEAR, rapid tumor outgrowth was observed (Figure 9B). BisG1-FAPα-FEAL / DR4-FEAR significantly inhibited tumor growth of gastric cancer PDX tumors in athymic nude mice at all tested doses (Table 18 and Figure 9D, p=0.0002 for all tested doses). PFS in this model was significantly extended by BisG1-FAPα-FEAL / DR4-FEAR treatment, even at the lowest dose of 0.5 mg / kg, demonstrating strong antitumor activity (p<0.0001; Mantel-Cox; Figure 9C). Finally, longitudinal weight measurements showed no change compared to the negative control group, indicating that the treatment was well tolerated (Figure 9E).

[0368] [Table 21]

[0369] c. Antitumor activity in the pancreatic PDX CTG-1150 model Parallel experiments were conducted in which Champions TumorGraft® CTG-1150, originally derived from human pancreatic cancer biopsies, was transplanted into athymic nude mice. The same treatment regimens, protocols, and statistical tests described in Section b (and Table 17) were applied, with the following differences: statistical comparison of tumor volume was performed on day 25 (the final day when all groups completed the study), and PFS was 1000 mm. 3 The tumor size cutoff was used.

[0370] result Rapid tumor outgrowth was observed in mice with pancreatic PDX treated with BisG1-DR4-FEAL / b12-FEAR antibody, as observed in the gastric PDX model (Figure 9H). BisG1-FAPα-FEAL / DR4-FEAR administered at 2 mg / kg significantly inhibited tumor growth in the pancreatic PDX model of athymic nude mice compared to BisG1-DR4-FEAL / b12-FEAR controls (Table 19 and Figure 9F, p=0.038). BisG1-FAPα-FEAL / DR4-FEAR treatment did not prolong PFS compared to BisG1-DR4-FEAL / b12-FEAR controls (Figure 9G). Finally, longitudinal weight measurements showed no change, indicating that the treatment was well tolerated (Figure 9I).

[0371] [Table 22]

[0372] d. Follow-up of antitumor activity in the gastric PDX CTG-1234 model. The ability of BisG1-FAPα-FERL / DR4-FERR to induce antitumor activity was evaluated in a low-dose range in a mouse gastric cancer-derived PDX model CTG-1234, compared with RG7386. BisG1-b12-FERL / DR4-FERR was used as a negative control, and RG7386 was tested to compare the antitumor effects between BisG1-FAPα-FERL / DR4-FERR and RG7386. The experimental procedure was as described in section b of Example 12, but modified as follows: Randomization of mice into the control and treatment groups was performed at approximately 270 mm intervals. 2 This was performed when establishing the mean tumor volume (MTV) (Table 20).

[0373] [Table 23]

[0374] result BisG1-FAPα-FERL / DR4-FERR induced tumor regression and long-term tumor suppression in CTG-1234 tumors. Pairwise analysis of tumor volume (TV) on the final day (day 12), when all groups completed the treatment, showed that treatment with 2.0 and 0.5 mg / kg of BisG1-FAPα-FERL / DR4-FERR significantly reduced TV compared to 2.0 mg / kg of BisG1-b12-FERL / DR4-FERR control antibody and equimolar levels of RG7386 (Figures 10A-10B, Table 21).

[0375] PFS was significantly prolonged in the group treated with 2.0 mg / kg of BisG1-FAPα-FERL / DR4-FERR compared to the BisG1-b12-FERL / DR4-FERR control group and RG7386-treated mice (Figure 10C).

[0376] [Table 24]

[0377] [Example 13] Antitumor activity in a mouse model of multi-organ metastasis The antitumor activity of BisG1-FAPα-FEAL / DR4-FEAR was investigated using a mouse model of CRC PDO with multi-organ metastasis, which uses mouse fibroblasts as a source of FAPα. IgG1-b12-FEAL was used as a negative control.

[0378] Hub096 PDO (described in Example 11) was transduced using a lentivirus encoding luciferase linked to the green fluorescent protein (GFP) gene (PLV-luciferase-IRES-GFP lentiviral vector). The lentivirus encoding GFP-linked luciferase was produced by human embryonic kidney (HEK) 293T cells according to a calcium phosphate transfection protocol.

[0379] On the day of transfection, HEK293T cells were detached using trypsin (Lonza, catalog number BE02-007E), viability was confirmed using trypan blue (Fluka, catalog number 93590), and seeding was performed on a 10cm culture dish (Greiner, catalog number 664160) in 9mL of DMEM high-glucose medium (Sigma-Aldrich, catalog number D6429) supplemented with 50U / mL penicillin / streptomycin (Gibco, catalog number 15070-063), 2mM GlutaMAX (Gibco, catalog number 35050-038), and 10% thermally inactivated FBS (Bodinco BV, ID 5067V20002) at a density that would result in approximately 60% cell density the following day. The cells were incubated at 37°C and 5% (vol / vol) CO2 for 24 hours to adhere to the plate. The following day, the culture medium was gently washed twice with PBS (Corning, catalog number 21-0310CVR). Fresh DMEM high-glucose medium (supplemented with 50 U / mL penicillin / streptomycin, 2 mM GlutaMAX, and 10% thermally inactivated FBS) was added. 500 μL of 2×HEPES-buffered saline (HBS), pH 7.05, consisting of 280 mM sodium chloride (Riedel-de-Haen, catalog number 31434), 1.5 mM dibasic sodium phosphate (Sigma, catalog number S0876), 12 mM (d) glucose (Sigma, catalog number G8270), 10 mM potassium chloride (Riedel-de-Haen, catalog number 31248), and 50 mM HEPES (Sigma, catalog number H3375), is mixed with 50 μL of 3 M calcium chloride dihydrate (Riedel-de-Haen, catalog number 31307), and 20 μg of plasmid DNA (10 μg of PLV-luciferase-IRES-GFP, 5 μg of psPAX (Addgene) [plasmid #12260] and 5 μg of pCMV-VSV-G [Addgene, plasmid #12260]. Mix with 450 μL of

[8454] ) and desalinated water (Aqua B. Braun, catalog number 0082479E). Quickly add 1 mL of the transfection mix to a 10 cm culture dish, and incubate overnight at 37°C and 5% CO2.The following day, the HEK293T cells were washed twice again with PBS, and 6 mL of fresh DMEM high-glucose medium supplemented with penicillin / streptomycin, GlutaMAX, and heat-inactivated 10% FBS was added to a 10 cm culture dish, followed by incubation overnight. The following day, Hub096 PDO was dissociated using TrypLE Express Stable Trypsin-Like Enzyme (Gibco, catalog number 12604021), and 1 to 2,000,000 PDO cells were seeded into 6 mL of lentivirus-containing medium (recovered from a virus-producing HEK293 T cell culture plate and filtered through a 0.22 μm polyethersulfone filter (Sarstedt BV, catalog number 83.1826.001)) supplemented with 6 μg / mL of polyblen (Sigma-Aldrich, catalog number TR-1003), 0.5 mM N-acetylcysteine ​​(Sigma-Aldrich, catalog number A9165), and 10 μM ROCK inhibitor Y-27632 (Abmole bioscience, catalog number HY-10583) in a non-adherent 6-well plate (Corning, catalog number 3471). PDO was incubated overnight at 37°C and 5% CO2. After 24 hours of incubation, the PDO was collected in a 15 mL tube (Corning, catalog no. 430791), washed twice with PBS (Corning, catalog no. 21-031-CV), and resuspended in PDO medium (see Example 11 for medium composition) in a 2:1 ratio in basement membrane extract (BME) matrix (Amsbio, catalog no. 3533-010-02). PDO was seeded in 150 μL droplets (containing approximately 2,500-5,000 cells / droplet), solidified at 37°C and 5% CO2, then 2 mL / well of PDO medium was added and incubated at 37°C and 5% CO2. After at least two passages to grow the cells, GFP-positive PDO was sorted using a fluorescence-activated cell sorting (FACS) Aria II (BD Biosciences) instrument.

[0380] The day before transplantation, Hub096 PDO was collected, washed with PBS (Corning, catalog number 21-031-CVR), and dissociated into single cells using TrypLE (Thermofisher, catalog number 12604013). Next, the cells were washed, resuspended in PDO medium, and viability was measured with trypan blue. The cells were washed and resuspended in a pre-cooled solution of rat tail high-concentration type I collagen (Corning, catalog number 354249) mixed in a 4:1 ratio with 5× neutralizing buffer (1 g AlphaMEM powder 5X (Life Technologies, catalog number 12000~014); 5 mL 1 M HEPES pH 7.5 (Lonza, catalog number 17737E); 1 g sodium bicarbonate (Sigma, catalog number 31437)). A 10 μL droplet containing 300,000 single cells was added to a preheated 6-well plate (Corning, catalog no. 3506). The plate was incubated for 40-60 minutes (37°C, 5% CO2) to solidify the droplet, after which 2 mL / well of PDO medium was added. The PDO was recovered overnight at 37°C, 5% CO2.

[0381] For cecal transplantation, male test mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl Animals from SzJ NSG (registered trademark) (Charles River Laboratories), strain #005557 (8-9 weeks old at the start of the experiment), were treated with a subcutaneous injection of carprofen (5 mg / kg, Rimady®) 30 minutes before surgery. To perform the surgery, the animals were sedated (isoflurane inhalation anesthesia: approximately 2% [vol / vol] isoflurane / O2 mixture), the cecum was exposed by a midline abdominal incision, and a drop of collagen containing luciferase-tagged PDO was surgically transplanted into the submucosal tissue of the cecum.

[0382] Mice were randomized into groups (n=9 / group). Treatment began two weeks after cecal transplantation and was administered once at weeks 2, 5, and 6, and twice a week at weeks 3 and 4. On the treatment day, mice were intraperitoneally injected with antibodies (2 mg / kg in 200 μL of PBS).

[0383] Mice were monitored daily for clinical signs of disease and weighed once a week using a digital scale. Data including individual gram weights and mean gram weights, and the average weight change rate (%vD0) relative to day 0 were recorded for each group. If any, the death of animals was recorded. The test toxicity endpoints were reported as an average loss of %vD0 > 20 within 2 days and / or a net weight loss > 15%, the finding of moribund or mice showing abnormal behavior and posture, and the appearance of a combination of clinical signs (abdomen enlargement, ascites) that may indicate excessive tumor growth and metastasis.

[0384] Before the end of the test, mice were intraperitoneally injected with 100 μL (1.25 mg) of luciferin (VivoGlo™ luciferin, in vivo grade, Promega, catalog number P1041) and euthanized after 十分钟. Individual organs (cecum, peritoneal wall, liver, lung, and brain) were collected and measured for tumor volume using ex vivo bioluminescence imaging (BioLI). Data were analyzed using GraphPad Prism 9, and the BioLI measurements (cpm / cm 2 , Log 10 scale) of organs from each mouse were plotted for both treatment groups. The differences in BioLI were evaluated by comparing the treatment groups with the control group using paired t-tests and unpaired non-parametric t-tests on log-transformed data.

[0385] Furthermore, DR4 activation was evaluated by cleavage-type caspase-3 IHC staining of FFPE sections from the cecum, peritoneal wall, and liver tissue. After dissection and BioLI measurement, the cecum, peritoneal wall, and liver tissue were fixed with 4% (w / v) formaldehyde (Added Pharma, catalog number ROL.1642810) and embedded in Surgipath Paraplast paraffin (Leica Biosystems, catalog number #39602012) in a laser biopsy green cassette (FA-Tech Diagnostics Europe BV, catalog number #215-05-10LM) using HistoCore Arcadia H (Leica Biosystems, catalog number #14039357258). Tissue blocks were cut into 4 μm serial sections parallel to the longitudinal axis of each tissue using a microtome (Leica Biosystems, catalog number #RM2255), transferred to the surface of a water bath (KLINIPATH, catalog number #WB28040), and mounted on X-tra Slides (Leica Biosystems, catalog number #3800203AE). The slides were then dried on a slide warmer (Adamas Instruments BV catalog number #SW85). Subsequently, FFPE sections of cecum, peritoneal wall, and liver tissue were deparaffinized with xylene (Klinipath, catalog number #4055-9005) and rehydrated with serial dilutions of ethanol (Klinipath, catalog number #4096-9005) and water. Endogenous peroxidase activity was blocked for 20 minutes at room temperature with 5% H2O2 (Merck, catalog no. 1072091000) diluted with PBS (1.87 M NaCl (Merck, catalog no. 1064041000), 0.28 M NaH2PO4 (Sigma-Aldrich, catalog no. 7558-79-4), and 0.0366 M NaH2PO4 (Merck, catalog no. 1063451000)). Slides were incubated for 20 minutes in boiling 10 mM citrate antigen retrieval buffer pH 6.0 (ThermoFisher Scientific, catalog no. 36439).After cooling for 10 minutes, the slides were washed with 0.05% Tween 20 buffer (VWR, catalog number M147-1L) diluted in PBS and incubated with cleaved caspase-3 (CCASP3) antibody (Cell Signaling Technology, catalog number 9661, 1:300 in PBS supplemented with 1% BSA and 0.2% sodium azide) at room temperature for 1 hour. The slides were washed three times with Tween 20 buffer and incubated with BrightVision+ polyHRP conjugate anti-rabbit IgG (Immunologic, catalog number VWRKDPVR110HRP) at room temperature for 30 minutes. After three washes with PBS, the slides were incubated with 3,3'-diaminobenzidine (DAB)H2O2 solution (0.03% DAB [Sigma-Aldrich, catalog no. 91-95-2] and 0.03% H2O2 [Merck, catalog no. 7722-84-1] in 0.05 M Tris HCl buffer pH 7.6 [Biosolve, catalog no. 20092391]) at 37°C for 10 minutes. The slides were washed with water and stained with hematoxylin (Merck, catalog no. HHS32; 1:4 in water) at room temperature for 30 seconds. After washing with water, followed by 96% ethanol, the slides were air-dried and coverslips were attached using ClearVue® coverslippers (Thermo Scientific). Stained cecal, liver, and peritoneal wall slides were scanned at 40x magnification and a resolution of 0.25 μm / pixel using a NanoZoomer-XR digital slide scanner (Hamamatsu). QuPath software was used for automated recognition of background, tissue (hematoxylin), and CCASP3-stained (DAB-positive) areas. Subsequently, each tumor area (excluding necrotic areas) was manually outlined and annotated. Percentage CCASP3-positive tumor area was determined using a QuPath-trained pixel classifier.

[0386] result Ex vivo BioLI measurements of tumor volume in individual organs showed that BisG1-FAPα-FEAL / DR4-FEAR antibody significantly reduced tumor volume at the primary tumor site (cecum) and in metastases to the brain, liver, peritoneal wall, and lungs, compared to IgG1-b12-FEAL antibody, using a paired t-test (Figures 11A-11E; Table 22). Using an unpaired t-test (Mann-Whitney), a reduction in tumor volume was demonstrated at the primary tumor site (cecum) and metastatic sites, reaching statistical significance at metastatic sites including the brain, liver, peritoneal wall, and lungs (Table 22).

[0387] In mice treated with BisG1-FAPα-FEAL / DR4-FEAR, DR4 activation was observed in primary tumors and metastatic sites, as demonstrated by a significant increase in the percentage of tumor area positively stained for cleavage caspase-3 on FFPE sections of cecal and peritoneal wall tissue compared to mice treated with IgG1-b12-FEAL (Figure 11F, Table 23). Compared to cecal and peritoneal wall tissue, caspase-3 activity in the liver was lower and did not significantly increase with treatment with BisG1-FAPα-FEAL / DR4-FEAR.

[0388] [Table 25]

[0389] [Table 26]

[0390] In this study, NSG mice transplanted with CRC-derived PDO were treated with BisG1-FAPα-FEAL / DR4-FEAR, which showed stronger antitumor activity against primary tumors and metastases compared to the negative control IgG1-b12-FEAL.

[0391] [Example 14] Evaluation of hepatotoxicity using human liver spheroids TRAIL-R agonists have been shown to induce hepatotoxicity in some patients in clinical studies. Therefore, we developed a tumor-specific agonist (BisG1-FAPα-FEAL / DR4-FEAR) that should reduce the risk of hepatotoxicity. Hepatotoxicity assays were performed to evaluate the potential of BisG1-FAPα-FEAL / DR4-FEAR, ABBV-621-Fc fusion, and RG7386 to induce hepatotoxicity in vitro. IgG1-b12-FEAR and IgG1-b12 were included as negative controls.

[0392] The safety evaluation of Drug-Induced Liver Injury (DILI-Bio) was performed using Human 3D InSight® Human Liver Microtissues (InSphero, catalog number MT-02-302-04), which are liver spheroids composed of primary human hepatocytes, Kupffer cells, and non-parenchymal hepatocyte types such as hepatic endothelial cells. Two experiments were conducted, and the survival of the liver spheroids was determined by measuring the release of lactate dehydrogenase (LDH), an indicator of plasma membrane damage, on day 4, and intracellular adenosine triphosphate (ATP) levels, an indicator of metabolically active cells, on days 6 and 7.

[0393] The first experiment was performed according to InSphero's internal technical operating procedures (TOP). Briefly, antibody dilutions (final concentrations of 2 μg / mL, 10 μg / mL, and 50 μg / mL using TOX microtissue culture medium (Insphero, catalog number CS-07-001-01)) were added to 96-well Insphero Plates (catalog number MT-02-302-04). The plates were incubated at 37°C in 5% CO2. Extracellular LDH release was measured on day 4 of treatment using a bioluminescence LDH release toxicity assay kit (Promega, catalog number J2380), and intracellular ATP content was measured on day 6 of treatment using a CellTiter-Glo 2.0 cell viability assay (Promega, catalog number G9243).

[0394] The second experiment was performed as follows: Insphero Plates were centrifuged to ensure that the microtissues were at the bottom of the wells, and then the antibody dilution (the same as in the first experiment, diluted in TOX medium) was added. The plates were incubated at 37°C and 5% CO2 for up to 7 days. For LDH measurement, the supernatant was collected after 4 days and used in conjunction with a bioluminescence LDH release toxicity assay (Promega, catalog number J2381) according to the manufacturer's protocol. For ATP measurement, the supernatant was removed after 7 days and 50 μL / well of CellTiter-Glo 3D (Promega, catalog number G9681) reagent (diluted 1:1 with PBS (Hyclone GE Healthcare, catalog number SH3A3830.03)) was added. After mixing, the total volume was transferred to a 96-well white OptiPlate (Perkin Elmer, catalog number 6005299). After incubation at room temperature in the dark for 30 minutes, bioluminescence was measured using EnVision (Perkin Elmer). Data from both experiments were analyzed using Microsoft Excel and GraphPad Prism. The data shown are the mean ± SEM of four technical replicates.

[0395] result Treatment of liver spheroids with the ABBV-621-Fc fusion induced toxicity in hepatocytes, as indicated by increased LDH release into the culture supernatant (Figure 12A). Treatment with higher doses of RG7386 also induced significant LDH release (Figure 12C). In contrast, BisG1-FAPα-FEAL / DR4-FEAR did not induce toxicity in this liver spheroid model, as LDH levels in the culture supernatant were below LLOD (dashed line), similar to the negative controls IgG1-b12-FEAR or IgG1-b12 (Figure 12A / Figure 12C).

[0396] Cellular ATP levels in liver spheroids treated with low doses of ABBV-621-Fc fusion and RG7386 were similar to those treated with BisG1-FAPα-FEAL / DR4-FEAR or control IgG1-b12-FEAR antibody (Figure 12B). However, treatment with the highest doses of ABBV-621-Fc fusion and RG7386 resulted in decreased cellular ATP levels (Figure 12B / Figure 12D), indicating impaired hepatocyte survival.

[0397] In summary, the BisG1-FAPα-FEAL / DR4-FEAR antibody did not induce hepatic spheroid toxicity (survival indicators were comparable to the negative control IgG1-b12-FEAR / IgG1-b12). In contrast, the ABBV-621-Fc fusion and RG7386 showed dose-dependent toxicity.

[0398] [Example 15] Cell damage in co-cultures with reduced FAPα availability To investigate the relationship between FAPα expression levels and BisG1-FAPα-FERL / DR4-FERR-induced cytotoxicity, we examined the role of the tumor cell to FAPα-expressing fibroblast ratio in BisG1-FAPα-FERL / DR4-FERR efficacy.

[0399] Cytotoxicity in co-cultures of tumor cells DLD-1 and MDA-MB-231 with varying amounts of NIH / 3T3-FAPα cells was evaluated using the CellTiter Glo assay. DR4-expressing tumor cells (MDA-MB-231 and DLD-1) were harvested as described in Example 3. NIH-3T3-FAPα cells were harvested as described in Example 10. Tumor cells and fibroblasts were seeded in CELLSTAR® flat-bottom 96-well plates (Greiner Bio, catalog number #655180) in the indicated ratios (6,600 tumor cells and various numbers [100 to 3,300 cells / well in 2-fold increments] of fibroblasts) and incubated at 37°C and 5% CO2 for 4 hours to adhere the cells to the plates. In parallel, control wells were prepared using monocultures of tumor cells or NIH / 3T3-FAPα cells. Next, the supernatant is removed, and the cells are subjected to an antibody concentration series (6.91 × 10⁶) in RPMI 1640 (Gibco, catalog number A1049101) containing 10% DBSI (Gibco, catalog number #10371029). -6 The cells were incubated with ~14.5 μg / mL (8-fold increment) at 37°C and 5% CO2 for 72 hours. The viability of the cultured cells was then evaluated using a homogeneous CellTiter-Glo® luminescence cell viability assay (Promega, catalog number #G7571), as described in section a of Example 10. The percentage of remaining surviving tumor cells was calculated, and the data was processed using GraphPad Prism software to generate fitted cell viability curves (nonlinear regression analysis with 4-parameter logistic curve fitting). The graph shows the percentage of surviving tumor cells plotted against antibody concentration.

[0400] Cytotoxicity was further evaluated in co-cultures of PDO with CAF transduced with FAPα shRNA or non-target shRNA. Different FAPα shRNA clones were generated by a calcium phosphatase transfection-based protocol for lentiviral vectors in HEK293T cells and used for transduction of CAF. Lentiviral generation in HEK293T cells was performed as described in Example 13, with the following modifications. The transfection mix contained the following vectors: 15 μg of MISSION pLKO.1-puro-based plasmid (Sigma-Aldrich, catalog number, #Scr:SHC016, #34:TRCN 0000006802), 7.5 μg of psPAX2 (Addgene, plasmid #12260), and 7.5 μg of pCMV-VSV-G (Addgene, plasmid #8454). After collecting the lentivirus-containing medium, the transduction medium was filtered using a 0.22 μm polyethersulfone filter (Sarstedt BV catalog 83.1826.001) and supplemented with 3 μg / mL polyblen (Sigma-Aldrich catalog #H9268). CAF was collected, counted, and seeded in lentivirus-containing filtration medium (2.5 mL / well) in a 6-well plate coated with collagen as described in Example 11, and incubated at 37°C and 5% CO2 for growth (2.5 mL / well). Transduced CAF was collected and resuspended in medium supplemented with 0.5 μg / mL puromycin dihydrochloride (Santa Cruz Biotechnology, catalog number sc-108071A). To continue puromycin selection, the medium was replaced with fresh medium and puromycin every 2-3 days. Subsequently, a CellTiter-Glo survival assay was performed using PDO and CAF as described in Example 11.

[0401] result To investigate the relationship between FAPα expression levels and BisG1-FAPα-FERL / DR4-FERR-induced cytotoxicity, we examined the role of tumor cell-to-FAPα-expressing fibroblast ratios in cytotoxicity. DLD-1 or MDA-MB-231 tumor cell lines were cultured with different amounts of NIH-3T3-FAPα cells (tumor cell-to-NIH-3T3-FAPα ratios of 2:1, 4:1, 8:1, and 64:1). As the ratio of tumor cells to fibroblasts increased (e.g., decreased NIH / 3T3-FAPα cells), BisG1-FAPα-FERL / DR4-FERR-mediated cytotoxicity decreased (Figures 13A-13B).

[0402] Furthermore, the effects of BisG1-FAPα-FERL / DR4-FERR were studied in co-cultures with fibroblasts expressing different levels of FAPα. CAFs were transduced with either FAPα shRNA (CAF#34) or non-target shRNA (CAF#Scr). In co-cultures with CAFs (CAF#34) engineered to express lower levels of surface FAPα from four PDO lines, the maximum BisG1-FAPα-FERL / DR4-FERR-mediated cytotoxic effect was unaffected in one PDO line, slightly reduced in two PDO lines, and completely lost in one PDO line (Figures 14A-14D). In all PDO lines, a bell-shaped concentration-response relationship was observed in co-cultures using engineered CAFs expressing reduced levels of FAPα. In conclusion, BisG1-FAPα-FERL / DR4-FERR-mediated cytotoxicity depends on the density and expression level of FAPα.

[0403] [Example 16] Cytotoxicity and DR4 transactivation in the presence of soluble FAPα Considering that FAPα protein can be released from the cell membrane and is found in human plasma (Xin et al., Front Oncol 2021 11:648187), the effect of soluble FAPα on BisG1-FAPα-FERL / DR4-FERR-mediated cytotoxicity was evaluated. Cytotoxicity of BisG1-FAPα-FERL / DR4-FERR and the control antibody BisG1-b12-FERL / DR4-FERR was evaluated in monocultures of DR4-expressing DLD-1 and MDA-MB-231 tumor cells as described in Example 15, but with the following modification: Tumor cells were cultured in monocultures, and recombinant human FAPα (BioLegend®, catalog number #76908, fixed concentration of 29.4 nM) was added simultaneously with the antibody sample, where indicated.

[0404] result In tumor ce...

Claims

1. at least (i) A FAPα binding region including a first heavy chain variable region and a first light chain variable region, and (ii) DR4 binding region including the second heavy chain variable region and the second light chain variable region A multispecific antibody containing, Here, the antibody is, (i) the heavy chain sequence of the anti-FAPα antibody shown in SEQ ID NO: 17 and the light chain sequence of the anti-FAPα antibody shown in SEQ ID NO: 18, and (ii) The heavy chain sequence of the anti-DR4 antibody shown in SEQ ID NO: 19 and the light chain sequence of the anti-DR4 antibody shown in SEQ ID NO: 20, A multispecific antibody containing [specific antibody].

2. The multispecific antibody according to claim 1, wherein the antibody is a bivalent antibody.

3. A nucleic acid construct encoding the antibody described in any one of claims 1 to 2, or a combination of nucleic acid constructs.

4. An expression vector comprising one or more nucleic acid constructs according to claim 3, or a combination of expression vectors.

5. A composition comprising a nucleic acid construct or a combination of nucleic acid constructs as described in claim 3.

6. A delivery vehicle comprising one or more nucleic acid constructs according to claim 3.

7. The delivery vehicle according to claim 6, wherein the delivery vehicle is a particle.

8. The delivery vehicle according to claim 7, wherein the particles are lipid nanoparticles.

9. The delivery vehicle according to claim 8, wherein the lipid nanoparticles comprise lipids, ionizable aminolipids, PEG-lipids, cholesterol, or any combination thereof.

10. Recombinant host cells capable of producing the antibody described in any one of claims 1 to 2, wherein the host cells comprise one or more nucleic acid constructs encoding the antibody as defined in any one of claims 1 to 2.

11. The recombinant host cell according to claim 10, which is a CHO cell.

12. A pharmaceutical composition comprising a multispecific antibody as defined in any one of claims 1 to 2 and a pharmaceutically acceptable carrier.

13. A pharmaceutical composition according to claim 12, for use as a pharmaceutical.

14. A multispecific antibody according to any one of claims 1 to 2, for use in the treatment of cancer.

15. One or more nucleic acid constructs according to claim 3 for use in the treatment of cancer.

16. A delivery vehicle according to claim 6 for use in the treatment of cancer.

17. A pharmaceutical composition according to claim 12 for use in the treatment of cancer.

18. A multispecific antibody according to claim 14, for use in the treatment of a primary tumor, and / or for use in the prevention and / or treatment of metastasis.

19. The pharmaceutical composition according to claim 17, for use in the treatment of a primary tumor, and / or for use in the prevention and / or treatment of metastasis.

20. The multispecific antibody according to claim 14, wherein the cancer is a solid tumor including a malignant solid tumor, or a progressive and / or metastatic solid tumor.

21. The pharmaceutical composition according to claim 17, wherein the cancer is a solid tumor including a malignant solid tumor, or a progressive and / or metastatic solid tumor.

22. The multispecific antibody according to claim 14, wherein the cancer expresses DR4 and / or the tumor microenvironment contains FAPα-expressing cells.

23. The pharmaceutical composition according to claim 17, wherein the cancer expresses DR4 and / or the tumor microenvironment contains FAPα-expressing cells.

24. The multispecific antibody according to claim 14, wherein the cancer expresses DR4, the tumor microenvironment contains cancer-associated fibroblasts (CAFs), and the CAFs express FAPα.

25. The pharmaceutical composition according to claim 17, wherein the cancer expresses DR4, and the tumor microenvironment contains cancer-associated fibroblasts (CAFs), wherein the CAFs express FAPα.

26. The multispecific antibody according to claim 14, wherein the cancer is selected from the group consisting of colorectal cancer [CRC], breast cancer including triple-negative breast cancer [TNBC], pancreatic cancer including pancreatic ductal adenocarcinoma [PDAC], gastric cancer and esophageal cancer including gastric cancer and esophageal cancer, head and neck squamous cell carcinoma [HNSCC], cervical cancer, and lung cancer including non-small cell lung cancer [NSCLC].

27. The pharmaceutical composition according to claim 17, wherein the cancer is selected from the group consisting of colorectal cancer [CRC], breast cancer including triple-negative breast cancer [TNBC], pancreatic cancer including pancreatic ductal adenocarcinoma [PDAC], gastric cancer and esophageal cancer including gastric cancer and esophageal cancer, head and neck squamous cell carcinoma [HNSCC], cervical cancer, and lung cancer including non-small cell lung cancer [NSCLC].

28. The multispecific antibody according to claim 14, wherein the cancer is selected from the group consisting of pancreatic cancer, gastric cancer, and CRC.

29. The pharmaceutical composition according to claim 17, wherein the cancer is selected from the group consisting of pancreatic cancer, gastric cancer, and CRC.

30. A method for producing an antibody as defined in any one of claims 1 to 2, wherein the method is: (a) Recombinant host cells capable of producing the multispecific antibody described in any one of claims 1 to 2 are cultured under conditions in which the antibody is produced. Here, the host cell comprises one or more nucleic acid constructs encoding the multispecific antibody, and (b) The multispecific antibodies produced are isolated from the culture. A method that includes doing so.

31. A method for producing an antibody as defined in any one of claims 1 to 2, wherein the method is: a) Providing a first antibody comprising a FAPα binding region as described in claim 1, and providing a second antibody comprising a DR4 binding region as described in claim 1; wherein the antibody comprises a further feature as described in claim 2, wherein the first and second antibodies comprise an Fc region, and wherein the sequences of the first and second CH3 regions of the first and second antibodies are different, thereby the heterodimer interaction between the first and second CH3 regions is stronger than the homodimer interaction between each of the first and second CH3 regions; b) Incubate the first antibody together with the second antibody under reducing conditions sufficient to allow the cysteine ​​in the hinge region to undergo disulfide bond isomerization; and c) Obtain an antibody comprising a first immunoglobulin heavy chain and a first immunoglobulin light chain of the first antibody, and a second immunoglobulin heavy chain and a second immunoglobulin light chain of the second antibody. A method that includes the act of doing so.

32. (a)(i) A nucleic acid sequence encoding the heavy chain sequence of the FAPα binding region as defined in claim 1, (ii) A nucleic acid sequence encoding the light chain sequence of the FAPα binding region as defined in claim 1. A host cell containing an expression vector is cultured, and The process of purifying the first antibody from the culture medium; (b) (iii) ) A nucleic acid sequence encoding the heavy chain sequence of the DR4 binding region as defined in claim 1, (iv) A nucleic acid sequence encoding the light chain sequence of the DR4 binding region as defined in claim 1. A host cell containing an expression vector is cultured, and The process of purifying the second antibody from the culture medium; (c) Incubating the first antibody with the second antibody under conditions of sufficient reducing to allow cysteine ​​in the hinge region to undergo disulfide bond isomerization, thereby obtaining a bispecific antibody; The method according to claim 31, including the method described in claim 31.

Images