Therapeutic binding molecules

Abstract

This invention relates to binding molecules (e.g., antibodies) and related antibody-drug conjugates for the treatment of cancer.

Description

Technical Field

[0001] This invention relates to binding molecules (e.g., antibodies) and related antibody-drug conjugates for the treatment of cancer. Prior Technology

[0002] Despite years of research and development of potential anti-cancer drugs, cancer remains one of the leading diseases worldwide, affecting one-third of all people in their lifetime. The two most common types of cancer are breast cancer and lung cancer.

[0003] The main treatments for cancer remain chemotherapy and radiation therapy. However, these treatments are associated with a variety of adverse side effects, ranging from fatigue to illness and hair loss. These problems are exacerbated by the typically long courses of chemotherapy.

[0004] Over the past few decades, numerous antibody therapies targeting cancer have been developed and marketed, leading to a decrease in demanding forms of treatment for a wide range of cancer types, such as surgery and chemotherapy. Although the availability of methods for producing antibodies (e.g., monoclonal antibodies) has greatly improved during this period, relatively few anticancer antibodies are clinically available, and even fewer are available for targeting a broad spectrum of cancer types. Furthermore, there is a need to increase the potency of therapeutic antibodies, which is often limited by the subsequent effects on cancer cells after the target antigen and antibody bind.

[0005] The present invention solves one or more of the above-mentioned problems. Summary of the Invention

[0006] The inventors have surprisingly discovered that the transmembrane polypeptide B7-H4 is highly expressed in various cell types (e.g., breast cancer, lung cancer, and pancreatic cancer), exhibiting effects consistent with cancer antigens. The inventors have successfully generated antibodies that show high binding to cells expressing B7-H4 both in vitro and in vivo (e.g., better binding than commercially available antibodies). Advantageously, these antibodies can target a variety of different cancer cell types expressing B7-H4, demonstrating their broad applicability as an anticancer therapy.

[0007] Furthermore, these antibodies can be advantageously linked / conjugated to suitable drugs / cytotoxins (e.g., to provide antibody-drug conjugates (ADCs)), thereby increasing the efficacy of these antibodies as a therapy by allowing targeted toxins to be delivered to cancer cells. Simple Explanation of the Diagram

[0008] The embodiments of the present invention will now be described by way of example only, with reference to the following figures and examples.

[0009] [[Figures 1A-1D]] show the overexpression of B7-H4 in breast cancer. (A) TNBC (re-treatment); (B) HR+ (before treatment); (C) HER2+ (before treatment, compliant with Herceptin treatment); (D) HER2+ve (herceptin-treated).

[0010] [[Figure 2A-2K]] shows the results of species cross-reactivity ELISA analysis.

[0011] [[Figures 3A-3G]] show the results of ELISA analysis of selective binding with B7-H4 family members and homologs.

[0012] [[Figures 4A-4B]] show the results of Western blot analysis of selected colonies (and commercial antibodies) bound to multiple cell types. E=E Biosciences 14-5949 Anti-human B7H4 mouse IgG; U=US biological B0000-35B Anti-human B7H4 mouse IgG; R=R and D systems AF2514 Anti-mouse B7H4 goat IgG1; S=Sigma SAB2500141 Anti-B7H4 goat IgG1

[0013] [[Figures 5A-5F]] show the results of in vitro cytotoxicity assays of selected colonies.

[0014] [[Figure 6]] shows the results of ELISA analysis of selected ZY0EQD_E02 (and its variants), demonstrating binding to human, cynomolgus monkey, mouse and rat B7-H4.

[0015] [[Figure 7]] shows the results of ELISA analysis of selected E02_GL, which, compared with 1D11, indicates that the binding of E02_GL to human B7-H4 is improved.

[0016] [[Figures 8A-8B]] show the results of flow cytometry analysis of selected E02_GL cells, indicating improved binding of E02_GL to human B7-H4 present on the cells compared to 1D11. Results for HT29 cells are shown in (A), and results for SKBR-3 cells are shown in (B). The symbol ◇ indicates the 'E02-GL fraction', and the symbol □ indicates the '1D11 fraction'. The symbol "●" indicates the (negative) control 'R347 fraction'.

[0017] [[Figures 9A-9B]] show the cytotoxicity of human B7-H4 transfected (and untransfected control) Ad293 cells after treatment with the E02-GL-SG3932 conjugate. Hollow circles = allotype control ADC (e.g., NIP228-SG3932); solid circles = E02-GL-SG3932.

[0018] [[Figures 10A-10B]] show bystander killing of tumor cells in vitro (E02-GL-SG3932).

[0019] [[Figure 11]] shows bystander killing of tumor cells in vivo (E02-GL-SG3932).

[0020] [[Figure 12]] shows that E02-GL-SG3932 has effective in vivo activity in a patient-derived xenograft (PDX) model. Hollow circles = mediator control only; solid circles = E02-GL-SG3932 (7 mg / kg).

[0021] [[Figure 13]] shows the antitumor activity of E02-GL-SG3932 in the PDX model after a single intravenous injection of 7 mg / kg.

[0022] [[Figures 14A-14B]] show that the E02-GL-topo I inhibitor ADC has similar potency in MX-1 cells in vitro and in vivo (A); and in HT29-derived models in vitro and in vivo (B).

[0023] [[Figures 15A-15B]] show the cytotoxicity of HEK293 cells transfected with B7-H4 of cynomolgus monkeys (and untransfected control) after treatment with the E02-GL-SG3249 conjugate.

[0024] [[Figures 16A-16C]] show the results of the internalization experiment, demonstrating the internalization of the bound E02-GL-antigen complex in living cells.

[0025] [[Figures 17A-17B]] show Western blot analysis, demonstrating that E02-GL-SG3932 treatment caused double-strand DNA breaks in vitro; [Figure 17C] shows Western blot analysis of key double-strand break markers after HCC1569 cells were treated with an anti-B7-H4 antibody conjugated to SG3249 (more specifically E02-GL-SG3249).

[0026] [[Figure 18]] shows the caspase 3 / 7 activity in SKBR3 cells after treatment with E02-GL-SG3249.

[0027] [[Figure 19]] shows the in vitro activity of E02-GL-SG3249 and warhead SG3199 against tumor cells.

[0028] [[Figures 20A-20D]] show bystander killing of tumor cells in vitro (E02-GL-SG3249).

[0029] [[Figures 21A-21C]] show that the E02_GL ADC has improved cytotoxicity / potency compared to the 1D11 ADC. Comparative cytotoxicity against the HT29 cell line (A), SKBR3 cell line (B), and HCC1569 cell line (C) expressing human B7-H4 is shown.

[0030] [[Figures 22A-22C]] show the in vivo activity of E02-GL-SG3249 on tumor grafts of (A) OVCAR4 cells (cisplatin-refractory ovarian cancer, high B7-H4), (B) HCC1569 cells (HER2+ breast cancer, heterologous expression of B7-H4), and (C) MDA-MB-468 cells (triple-negative breast cancer, low B7-H4 expression).

[0031] [[Figure 23]] shows bystander killing of tumor cells in vivo.

[0032] [[Figures 24A-24O]] show that E02-GL-SG3249 has effective in vivo activity in a patient-derived xenograft (PDX) model. Circle = mediator control only; Square = E02-GL-SG3249 (0.3 mg / kg); Triangle = E02-GL-SG3249 (1.0 mg / kg).

[0033] [[Figures 25A-25B]] show the results of (A) immunohistochemical staining (IHC) of γ-H2AX in HCC1954 tumor xenografts (with and without E02-GL-SG3249), quantified in (B) as the number of γ-H2AX-positive cells / mm2 (within the target tissue area) (+ / - SEM). Image analysis was performed using HALO software (using CRO-OracleBio). An increase in the number of γ-H2AX-positive tumor cells was observed up to 10 days after E02-GL-SG3249 treatment.

[0034] [[Figures 26A-26D]] show the retention of B7-H4 Ab in tumors (HT29 cells) expressing B7H4. Intensity scales show the epifluorescence intensity. Radiative efficiency = (p / sec / cm² / sr) / (μW / cm²); intensity scale, minimum = 1.20e8, maximum = 1.50e9.

[0035] [[Figures 27A-27D]] show the retention of B7-H4 Ab in tumors (CT26 cells) expressing B7H4. Intensity scales show the epifluorescence intensity. Radiative efficiency = (p / sec / cm² / sr) / (μW / cm²); intensity scale, minimum = 1.20e8, maximum = 1.50e9.

[0036] [[Figures 28A-28B]] show sequence alignments for five exemplary antibody selections.

[0037] [[Figure 29A]] is a schematic diagram of the TOP1i-ADC targeting B7-H4. [Figure 29B] shows the key characteristics of the TOP1i-ADC E02-GL-SG3249 targeting B7-H4.

[0038] [[Figure 30]] shows example images of representative immunohistochemical staining of B7-H4 expression in mammary gland, pancreas, cervix, endometrium, fallopian tube / oviduct, and kidney tissues of normal humans and normal cynomolgus monkeys.

[0039] [[Figure 31]] shows an example image of representative immunohistochemical staining of B7-H4 expression in human tumor tissues including breast cancer (TNBC and ER+, see individual Figures 1A-1B), cholangiocarcinoma, NSCLC-SCC, endometrial tumors and ovarian tumors.

[0040] [[Figure 32]] is a homologous comparison of B7-H4.

[0041] [[Figure 33]] shows the binding of antibody intermediates E02-INT and E02-GL-SG3932 to human B7 H4 via DELFIA-ELISA and anti-human IgG (H+L). E02-INT: antibody intermediate of E02-GL-SG3932; NIP228: isotype-matched control; huB7-H4: recombinant human B7-H4.

[0042] [[Figure 34]] shows the binding of antibody intermediate E02-INT to HEK 293 cells stably expressing human, mouse, or cynomolgus monkey B7-H4. E02-INT: antibody intermediate of E02-GL-SG3932; HEK293 JI TREX: untransduced HEK293 JI TREX cells; HEK293 JI TREX cynoB7-H4: HEK 293 cells stably expressing cynomolgus monkey B7-H4; HEK293 JI TREX huB7-H4: HEK 293 cells stably expressing human B7-H4; HEK293 JI TREX muB7-H4: HEK 293 cells stably expressing mouse B7-H4; MFI: mean fluorescence intensity. Geometric mean fluorescence intensity along the Y-axis. Data are presented as mean ± SD of three replicate measurements.

[0043] [[Figure 35]] shows the binding of antibody intermediates E02-INT and E02-GL-SG3932 to human breast cancer cell lines and to HT29 cells stably expressing human B7-H4. E02-INT: antibody intermediate of E02-GL-SG3932; HT29-huB7-H4 selector 4 and HT29-huB7-H4 selector 26: HT29 cells stably expressing human B7-H4; MFI: mean fluorescence intensity. Geometric mean fluorescence intensity along the y-axis.

[0044] [[Figures 36A-36C]] show the in vitro cytotoxic activity of E02-GL-SG3932 in cell lines HT29, HT29-huB7-H4 Selected 26, and MX-1. Data are presented as mean ± SD of three replicate assays.

[0045] [[Figure 37]] shows the in vitro antibody-dependent cell-mediated cytotoxicity of E02-GL-SG3932. The figure shows the mean fold change ± SEM for six experiments.

[0046] [[Figure 38]] shows the timeline of E02-INT internalization as an image sequence. Human colorectal cancer cells HT29-huB7-H4 selected 26 (first row) and human breast cancer cells MX-1 (second row) were labeled with 5 μg / mL of E02-INT conjugated with Alexa Fluor™ 568 (red). Scale bar is 20 μm.

[0047] [[Figures 39A-39C]] show live-cell imaging of E02-INT internalized cells in human cancer cell lines HT29-huB7-H4 selected 26 and MX-1. (A) Each point represents the mean percentage of internalization ± the standard deviation of 3 independent wells, spaced 10 minutes apart for 480 minutes. (B) Percentage of internalization after 8 hours, and (C) shows the predicted half-life of 3 independent wells. These equations were derived using a dissociation-single-phase exponential decay equation. Horizontal bars represent the within-group arithmetic mean; statistical significance was assessed by one-way ANOVA and Tukey's multiple comparison test. ns: not significant; p>0.05; **p<0.05.

[0048] [[Figures 40A-40B]] show the colocalization of antibody E02-INT with lysosomes in HT29-huB7-H4 selected 26 cells. (A) HT29-huB7-H4 selected 26 cells were incubated with E02-INT conjugated with 5 μg / mL Alexa Fluor™ 568 antibody for 24 h [(A) Red - top left and bottom left]. Lysosomes were stained with mouse anti-human LAMP1-Alexa Fluor™ 488 antibody [(A) Green - top center]. Integrities were stained with rabbit anti-human EEA1 antibody and detected with goat anti-rabbit IgG1-DyLight™ 650 [(A) Green - bottom center]. Colocalization of E02-INT with LAMP1 or EEA1 is shown in the merged image. (B) Colocalization of E02-INT with EEA1 and LAMP1 was analyzed using Zeiss Zen software by Pearson correlation coefficient. Each point represents a single-cell measurement. Statistical significance was assessed using one-way ANOVA and Tukey's multiple comparison test. ns: not significant; p>0.05; ****p<0.0001.

[0049] [[Figure 41]] shows gel images illustrating DNA damage response signals in MX-1 cells treated with E02-GL-SG3932 or TOP1i warhead SG3924. The data presented represent n:2 experiments.

[0050] [[Figure 42]] shows gel images illustrating DNA damage response signals in HT29-huB7-H4 cells treated with E02-GL-SG3932 or TOP1i warhead SG3924. The data presented represent n:2 experiments.

[0051] [[Figures 43A-43F]] show immunohistochemical (IHC) staining images of human IgG, γH2AX, and cleaved caspase-3 in HT29-huB7-H4 selected 26 xenograft tumors after treatment with E02-GL-SG3932. These images represent IHC staining of human IgG, γH2AX, and cleaved caspase-3 in the HT29-huB7-H4 selected 26 tumor xenograft model 168 hours after a single IV administration of 7 mg / kg E02-GL-SG3932 (43D-43F) or the allotype-matched control ADC NIP228-SG3932 (43A-43C).

[0052] [[Figures 44A-44D]] show image analysis data of human IgG, γH2AX, and lysed caspase-3 IHC staining across all time points and treatments in the HT29 huB7 H4 xenograft study. Top inset (44A): Epithelial cell analysis, showing the change in the proportion of human IgG-positive epithelial cells across all epithelial cells over time. Second inset (44B): γH2AX analysis, showing the proportion of epithelial cells found to be positive for lesions in γH2AX assays. Third inset (44C): Lysed caspase-3 (CC-3), showing the percentage of lysed caspase-3-positive tumor cells in the sample over time. Bottom inset (44D): Cell density of all epithelial cells in the sample over time, indicating cell death induced by E02-GL-SG3932 treatment compared to the allotype-matched control ADC NIP228-SG3932.

[0053] [[Figures 45A-45B]] show the efficacy of E02-GL-SG3932 in a B7-H4 negative HT29 xenograft model (see also Figure 11). Values ​​are mean ± SEM tumor volume for n: 8 animals per group. Dashed lines indicate the date of administration.

[0054] [[Figures 46A-46C]] show the efficacy of E02-GL-SG3932 in the HT29-huB7-H4 selected xenograft model. Values ​​are mean ± SEM tumor volume for n: 10 or 8 animals per group. Dashed lines indicate the administration date.

[0055] [[Figures 47A and 47B]] show the efficacy of E02-GL-SG3932, NIP228-SG3932, and E02-INT in the MX-1 xenograft model. Values ​​are mean ± SEM tumor volume for n: 8 animals per group. Dashed lines indicate the administration day.

[0056] [[Figure 48]] shows the efficacy of E02-GL-SG3932 and NIP228-SG3932 in the MX-1 xenograft model. Values ​​are mean ± SEM tumor volume for n: 3 or 6 animals per group.

[0057] [[Figure 49]] shows the efficacy of E02-GL-SG3932 and NIP228-SG3932 in the MDA-MB-468 xenograft model. Values ​​are mean ± SEM tumor volume for n: 3 or 6 animals per group.

[0058] [[Figure 50]] shows a quantitative image analysis of B7-H4 manifestation data in a patient-derived xenograft (PDX) model, sorted by case mean of mean cell membrane OD. Manifestation is color encoded by IHC cell intensity gradation (negative: black, 1+: white, 2+: light gray, 3+: dark gray).

[0059] [[Figure 51]] shows the antitumor activity produced by a single administration of 1.25 mg / kg E02-GL-SG3932 or NIP228-SG3932 in a patient-derived xenograft model.

[0060] [[Figures 52A and 52B]] show the B7-H4 performance in PDX models grouped according to tumor response to E02-GL-SG3932 or NIP228-SG3932 at a dose level of 1.25 mg / kg. A model was considered responsive (R) to the test agent if the percentage change in tumor volume from baseline was -30% to -100% (inclusive). A model was considered non-responsive (NR) if the percentage change in tumor volume from baseline was greater than -30%. The y-axis represents the level of B7-H4 in each model, as determined by the H score.

[0061] [[Figure 53A]] shows the antitumor activity produced by a single administration of 3.5 mg / kg E02-GL-SG3932 or NIP228-SG3932 in a patient-derived xenograft model. [Figures 53B-53E] show the results of a study protocol used to determine the relationship between E02-GL-SG3932 administration, B7-H4 performance level, and HR deficit at (A) 1.25 mg / kg E02-GL-SG3932, (B) 3.5 mg / kg E02-GL-SG3932, (C) 1.25 mg / kg isotype control ADC, and (D) 3.5 mg / kg isotype control ADC. In [Figures 53B-53E], the triangle "△" symbol represents a model with homologous recombination defects (such as those determined by BRCA mutations or RAD51 lesion assays). The circle "●" symbol represents a model without homologous recombination defects.

[0062] [[Figures 54A and 54B]] show the B7-H4 performance in PDX models grouped according to tumor response to E02-GL-SG3932 or NIP228-SG3932 at a dose level of 3.5 mg / kg. A model was considered responsive (R) to the test agent if the percentage change in tumor volume from baseline was -30% to -100% (inclusive). A model was considered NR if the percentage change in tumor volume from baseline was greater than -30%. The y-axis represents the level of B7 H4 in each model, as determined by the H score.

[0063] [[Figures 55A-55G]] show the mean tumor volume over time in the first group of cholangiocarcinoma PDX mouse models treated with a single dose of E02-GL-SG3932 at 1.25 mg / kg or 3.5 mg / kg, compared to untreated mice.

[0064] [[Figures 56A-56K]] show the mean tumor volume over time in a second group of cholangiocarcinoma PDX mouse models treated with a single dose of E02-GL-SG3932 at 1.25 mg / kg or 3.5 mg / kg, compared to untreated mice.

[0065] [[Figures 57A-57B]] show that, compared with ADCs 2-4, the ADC prepared with the cleavable mal-PEG8-val-ala linker-warhead has the highest in vivo activity. [Figure 57A is the key to Figure 57B].

[0066] [[Figure 58]] shows that, compared with ADC 2-4, the cleavable mal-PEG8-val-ala linker-warhead ADC exhibited the cleanest safety profile in rat toxicity studies.

[0067] [[Figures 59A-59B]] show that, compared with ADCs 2-4, the cleavable mal-PEG8-val-ala linker-warhead ADC exhibits superior PK characteristics and the widest relative TI. Relative TI = the ratio of AUC at the highest dose tested in rats (NOAEL rather than HNSTD) to the AUC that provides tumor arrest (MX-1 model). [Figure 59A is the key to Figure 59B].

[0068] [[Figures 60A-60B]] show that E02-GL-SG3932 has potent activity in the mammary and ovarian PDX mouse models.

[0069] [[Figure 61]] shows that E02-GL-SG3932 has potent activity in HR-deficient tumors and HR-proficient tumors with elevated B7-H4.

[0070] [[Figures 62A-62B]] show tumor responses in HR-deficient (A) and HR-proficient (B) PDX models grouped according to tumor response to E02-GL-SG3932 at a dose level of 3.5 mg / kg. (R) indicates that the model is considered to have responded to the test agent. (NR) indicates that the model is considered to be a non-responder. The y-axis represents the level of B7-H4 in each model, as determined by the H score.

[0071] [[Figures 62C-62D]] show tumor responses in HR-deficient (A) and HR-proficient (B) PDX models grouped according to tumor response to E02-GL-SG3932 at a dose level of 1.25 mg / kg. (R) indicates that the model is considered to have responded to the test agent. (NR) indicates that the model is considered to be a non-responder. The y-axis represents the level of B7-H4 in each model, as determined by the H score.

[0072] [[Figure 63]] shows the results of a 6-day cytotoxicity assay for different warheads in DLD1 wt or BRCA2- / - cells. MMAE = negative control, microtubule inhibitor warhead. Implementation Cross-references to related applications

[0073] This application claims priority to U.S. Provisional Patent Application No. 63 / 077,207, filed September 11, 2020, which is incorporated herein by reference in its entirety for all purposes. Materials submitted electronically are incorporated through citation.

[0074] By referencing the computer-readable nucleotide / amino acid sequence listing incorporated herein in its entirety, which is hereby submitted and identified as follows: an ASCII (text) file named "B7H4-100-WO-PCT.txt", created on September 8, 2021, consisting of 50,724 bytes.

[0075] Therefore, in one aspect, the present invention provides an antibody or antigen-binding fragment thereof that binds to B7-H4 (e.g., the B7-H4 epitope), the antibody or antigen-binding fragment thereof comprising: i. heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, or functional variations thereof; ii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, or functional variations thereof; iii. SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, or functional variations thereof; iv. amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, or functional variants thereof; or v. amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, or functional variants thereof; or v. amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, or functional variants thereof.

[0076] Antibodies or their antigen-binding fragments may be suitably included in pharmaceutical compositions, for example, in formulations suitable for administration to patients.

[0077] On the other hand, a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof is provided, the antibody or antigen-binding fragment comprising: i. heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, or functional variants thereof; ii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, or functional variants thereof; iii. SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:6, respectively. iv. amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO:17 and SEQ ID NO:18, or functional variants thereof; or v. amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, or functional variants thereof; or v. amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, or functional variants thereof.

[0078] The term "drug composition" refers to a formulation that is in a form that allows for the effective bioactivity of the active ingredient and does not contain any other components that would have unacceptable toxicity to the subject to whom the composition is to be administered. Such a composition may be sterile and may contain a pharmaceutically acceptable medium, such as physiological saline. Suitable drug compositions may contain one or more buffer solutions (e.g., acetate, phosphate, or citrate buffers), surfactants (e.g., polysorbate), stabilizers (e.g., human albumin), preservatives (e.g., benzyl alcohol), and absorption enhancers to improve bioavailability, and / or other conventional solubilizers or dispersants.

[0079] Furthermore, it has been demonstrated that the antibodies or antigen-binding fragments thereof of the present invention target and inhibit the growth of B7-H4-positive tumors in vivo. Therefore, the present invention includes the antibodies or antigen-binding fragments thereof as defined above and the pharmaceutical compositions as defined above for use in methods for treating cancer. Preferably, the cancer comprises cancer cells expressing B7-H4.

[0080] In one aspect, an antibody or antigen-binding fragment thereof is provided for treating cancer (e.g., said cancer comprising cancer cells expressing B7-H4), wherein the antibody or antigen-binding fragment thereof comprises: i. heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, or functional variants thereof; ii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, or functional variants thereof; iii. SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:6, respectively, or SEQ ID NO:6. iv. amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, or functional variants thereof; or v. amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, or functional variants thereof; or v. amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, or functional variants thereof.

[0081] In other words, one aspect of the present invention provides a method for treating cancer (e.g., said cancer comprising cancer cells expressing B7-H4), the method comprising administering to a subject an effective amount of an antibody or antigen-binding fragment comprising: i. heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, or functional variations thereof; ii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, or functional variations thereof; iii. SEQ ID NO:6, respectively, or SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, or functional variations thereof; iii. SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, or functional variations thereof; iv. amino acid sequences of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18 containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, or functional variants thereof; or v. amino acid sequences of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24 containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, or functional variants thereof; or v. amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30 containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, or functional variants thereof.

[0082] In other words, on another aspect, the present invention includes the use of an antibody or antigen-binding fragment thereof in the manufacture of a medicament for treating cancer (e.g., said cancer comprising cancer cells expressing B7-H4), said antibody or antigen-binding fragment comprising: i. heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, or functional variations thereof; ii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, or functional variations thereof; iii. SEQ ID NO:6, respectively, or SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, or functional variations thereof; iii. SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, or functional variations thereof; iv. amino acid sequences of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18 containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, or functional variants thereof; or v. amino acid sequences of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24 containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, or functional variants thereof; or v. amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30 containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, or functional variants thereof.

[0083] Certain definitions and preferred implementations will now be outlined. It should be understood that the following definitions and implementations may relate to any aspect described herein, such as any method, composition, and / or formulation used in the therapies described herein.

[0084] The term "epitope" refers to a target protein region (e.g., a polypeptide) that is capable of binding to (e.g., being bound to) the antibody or antigen-binding fragment of the present invention.

[0085] B7-H4 (also known as T-cell activation inhibitor 1 containing the group V domain, encoded by the VTCN1 gene) is a transmembrane polypeptide of the B7 family of costimulatory proteins. B7-H4 is understood to be expressed on the surface of antigen-presenting cells for ligand interaction with immune cells (e.g., T lymphocytes, with CD28 as a potential ligand). Not wishing to be bound by theory, the inventors have observed high expression of B7-H4 on cells of various cancer types, suggesting that this molecule is a tumor-associated antigen. Therefore, the claimed antibody's ability to target (and, as needed, deliver cytotoxins to) expression of B7-H4 makes the antibody particularly suitable for use in cancer therapy. Furthermore, the expression of B7-H4 is not limited to a specific cancer type, thus representing a target antigen for treating a broad spectrum of cancer types.

[0086] The RNA, DNA, and amino acid sequences of B7-H4 are known to those skilled in the art and can be found in many databases (e.g., the databases of the National Center for Biotechnology Information (NCBI) and UniProt). Examples of such sequences found in UniProt are Q7Z7D3 (VTCN1_HUMAN) of human B7-H4 and Q7TSP5 (VTCN1_MOUSE) of mouse B7-H4. The nucleotide sequence encoding human B7-H4 may be SEQ ID NO:53, more preferably SEQ ID NO:54. The polypeptide sequence of human B7-H4 is preferably SEQ ID NO:55.

[0087] In one embodiment, the antibody or its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, containing the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. The antibody or its antigen-binding fragment containing said sequences may be referred to herein as "ZY0EPQ-E02" or "EPQ-E02".

[0088] In one embodiment, the antibody or its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, containing the amino acid sequences of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively. The antibody or its antigen-binding fragment containing said sequences may be referred to herein as "ZY0EOB-F05" or "EOB-F05".

[0089] In one embodiment, the antibody or its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, containing the amino acid sequences of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively. The antibody or its antigen-binding fragment containing said sequences may be referred to herein as "ZY0EO5-E07" or "EO5-E07".

[0090] In one embodiment, the antibody or its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, containing the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively. The antibody or its antigen-binding fragment containing said sequences may be referred to herein as "ZY0EP0-C07" or "EP0-C07".

[0091] In a particularly preferred embodiment, the antibody or its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, containing the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively. The antibody or its antigen-binding fragment containing said sequences may be referred to as "ZY0EQD-E02" or "EQD-E02".

[0092] In other words, the antibody or its antigen-binding fragment may preferably comprise: - HCDR1 containing the amino acid sequence of SEQ ID NO:7, or a functional variant thereof; - HCDR2 containing the amino acid sequence of SEQ ID NO:8, or a functional variant thereof; - HCDR3 containing the amino acid sequence of SEQ ID NO:9, or a functional variant thereof; - LCDR1 containing the amino acid sequence of SEQ ID NO:10, or a functional variant thereof; - LCDR2 containing the amino acid sequence of SEQ ID NO:11, or a functional variant thereof; and - LCDR3 containing the amino acid sequence of SEQ ID NO:12, or a functional variant thereof.

[0093] In one embodiment, the antibody or its antigen-binding fragment comprises: i. HCDR1 containing the amino acid sequence of SEQ ID NO:1, or a functional variant thereof; ii. HCDR2 containing the amino acid sequence of SEQ ID NO:2, or a functional variant thereof; iii. HCDR3 containing the amino acid sequence of SEQ ID NO:3, or a functional variant thereof; iv. LCDR1 containing the amino acid sequence of SEQ ID NO:4, or a functional variant thereof; v. LCDR2 containing the amino acid sequence of SEQ ID NO:5, or a functional variant thereof; and vi. LCDR3 containing the amino acid sequence of SEQ ID NO:6, or a functional variant thereof.

[0094] In one embodiment, the antibody or its antigen-binding fragment comprises: i. HCDR1 containing the amino acid sequence of SEQ ID NO: 13, or a functional variant thereof; ii. HCDR2 containing the amino acid sequence of SEQ ID NO: 14, or a functional variant thereof; iii. HCDR3 containing the amino acid sequence of SEQ ID NO: 15, or a functional variant thereof; iv. LCDR1 containing the amino acid sequence of SEQ ID NO: 16, or a functional variant thereof; v. LCDR2 containing the amino acid sequence of SEQ ID NO: 17, or a functional variant thereof; and vi. LCDR3 containing the amino acid sequence of SEQ ID NO: 18, or a functional variant thereof.

[0095] In one embodiment, the antibody or its antigen-binding fragment comprises: i. HCDR1 containing the amino acid sequence of SEQ ID NO: 19, or a functional variant thereof; ii. HCDR2 containing the amino acid sequence of SEQ ID NO: 20, or a functional variant thereof; iii. HCDR3 containing the amino acid sequence of SEQ ID NO: 21, or a functional variant thereof; iv. LCDR1 containing the amino acid sequence of SEQ ID NO: 22, or a functional variant thereof; v. LCDR2 containing the amino acid sequence of SEQ ID NO: 23, or a functional variant thereof; and vi. LCDR3 containing the amino acid sequence of SEQ ID NO: 24, or a functional variant thereof.

[0096] In one embodiment, the antibody or its antigen-binding fragment comprises: i. HCDR1 containing the amino acid sequence of SEQ ID NO:25, or a functional variant thereof; ii. HCDR2 containing the amino acid sequence of SEQ ID NO:26, or a functional variant thereof; iii. HCDR3 containing the amino acid sequence of SEQ ID NO:27, or a functional variant thereof; iv. LCDR1 containing the amino acid sequence of SEQ ID NO:28, or a functional variant thereof; v. LCDR2 containing the amino acid sequence of SEQ ID NO:29, or a functional variant thereof; and vi. LCDR3 containing the amino acid sequence of SEQ ID NO:30, or a functional variant thereof.

[0097] Alternatively, the antibodies or antigen-binding fragments described herein may be described by their variable heavy (VH) chains and variable light (VL) chains.

[0098] Suitable variable heavy (VH) chain sequences (which may be included in antibodies or their antigen-binding fragments) are outlined in an individualized manner as follows:

[0099] - SEQ ID NO:31, or a functional variant thereof;

[0100] - SEQ ID NO:33, or a functional variant thereof

[0101] - SEQ ID NO:43, or a functional variant thereof

[0102] - SEQ ID NO:45, or a functional variant thereof

[0103] - SEQ ID NO:46, or a functional variant thereof

[0104] - SEQ ID NO:47, or a functional variant thereof

[0105] - SEQ ID NO:35, or a functional variant thereof

[0106] - SEQ ID NO:37, or a functional variant thereof

[0107] - SEQ ID NO:39, or a functional variant thereof

[0108] Particularly suitable variable heavy (VH) chain sequences (which may be included in antibodies or their antigen-binding fragments) are outlined in an individualized manner below:

[0109] - SEQ ID NO:45, or a functional variant thereof

[0110] - SEQ ID NO:33, or a functional variant thereof

[0111] - SEQ ID NO:43, or a functional variant thereof

[0112] - SEQ ID NO:46, or a functional variant thereof

[0113] - SEQ ID NO:47, or a functional variant thereof

[0114] Suitable variable light (VL) chain sequences (which may be included in antibodies or their antigen-binding fragments) are outlined in an individualized manner as follows:

[0115] - SEQ ID NO:32, or a functional variant thereof

[0116] - SEQ ID NO:34, or a functional variant thereof

[0117] - SEQ ID NO:36, or a functional variant thereof

[0118] - SEQ ID NO:38, or a functional variant thereof

[0119] - SEQ ID NO:40, or a functional variant thereof

[0120] A preferred variable light (VL) chain sequence (which may be contained in the antibody or its antigen-binding fragment) may contain the amino acid sequence (or a functional variant thereof) of SEQ ID NO:34.

[0121] For example, in one embodiment, the antibody or its antigen-binding fragment comprises: i. a variable heavy chain, or a functional variant thereof, containing an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 31, 33, 35, 37, or 39; and ii. a variable light chain, or a functional variant thereof, containing an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 32, 34, 36, 38, or 40.

[0122] For example, in one embodiment, the antibody or its antigen-binding fragment comprises: i. a variable heavy chain, or a functional variant thereof, containing an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 31, 33, 35, 37, 39, 43, 45, 46, or 47; and ii. a variable light chain, or a functional variant thereof, containing an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 32, 34, 36, 38, or 40.

[0123] Suitablely, the antibody or its antigen-binding fragment may comprise: i. a variable heavy chain, or a functional variant thereof, containing an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:33; and ii. a variable light chain, or a functional variant thereof, containing an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:34.

[0124] More suitably, the antibody or its antigen-binding fragment may comprise: i. a variable heavy chain, or a functional variant thereof, containing an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:45; and ii. a variable light chain, or a functional variant thereof, containing an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:34.

[0125] In one embodiment, the antibody or its antigen-binding fragment comprises: - a variable heavy (VH) chain and a variable light (VL) chain, or functional variants thereof, respectively containing the amino acid sequences of SEQ ID NO:31 and SEQ ID NO:32; - a VH chain and a VL chain, or functional variants thereof, respectively containing the amino acid sequences of SEQ ID NO:33 and SEQ ID NO:34; - a VH chain and a VL chain, or functional variants thereof, respectively containing the amino acid sequences of SEQ ID NO:43 and SEQ ID NO:34; - a VH chain and a VL chain, or functional variants thereof, respectively containing the amino acid sequences of SEQ ID NO:45 and SEQ ID NO:34; - a VH chain and a VL chain, or functional variants thereof, respectively containing the amino acid sequences of SEQ ID NO:46 and SEQ ID NO:34; - a VH chain and a VL chain, or functional variants thereof, respectively containing the amino acid sequences of SEQ ID NO:47 and SEQ ID NO:34; - a VH chain and a VL chain, or functional variants thereof, respectively containing the amino acid sequences of SEQ ID NO:35 and SEQ ID NO:34; - The VH and VL chains of the amino acid sequence NO:36, or functional variants thereof; - The VH and VL chains of the amino acid sequences SEQ ID NO:37 and SEQ ID NO:38, or functional variants thereof, respectively; or - The VH and VL chains of the amino acid sequences SEQ ID NO:39 and SEQ ID NO:40, or functional variants thereof, respectively.

[0126] In a preferred embodiment, the antibody or its antigen-binding fragment comprises: a variable heavy (VH) chain (or a functional variant thereof) containing the amino acid sequence of SEQ ID NO: 45, 33, 43, 46, or 47; and a variable light (VL) chain (or a functional variant thereof) containing the amino acid sequence of SEQ ID NO: 34. For example, the VH of SEQ ID NO: 33, 45, 46, and / or 47 may correspond to "germized" versions of the VH of SEQ ID NO: 33 (e.g., all having the same CDR sequence but with frame variations). Advantageously, each variant retains equivalent binding properties.

[0127] In one embodiment, the antibody or its antigen-binding fragment comprises: a variable heavy chain containing the amino acid sequence of SEQ ID NO:31, or a functional variant thereof; and a variable light chain containing the amino acid sequence of SEQ ID NO:32, or a functional variant thereof. An antibody or its antigen-binding fragment containing said sequences may be referred to as "ZY0EPD-E02" or "EPD-E02".

[0128] In one embodiment, the antibody or its antigen-binding fragment comprises: a variable heavy chain containing the amino acid sequence of SEQ ID NO:35, or a functional variant thereof; and a variable light chain containing the amino acid sequence of SEQ ID NO:36, or a functional variant thereof. An antibody or its antigen-binding fragment containing said sequences may be referred to as "ZY0EOB-F05" or "EOB-F05".

[0129] In one embodiment, the antibody or its antigen-binding fragment comprises: a variable heavy chain containing the amino acid sequence of SEQ ID NO:37, or a functional variant thereof; and a variable light chain containing the amino acid sequence of SEQ ID NO:38, or a functional variant thereof. An antibody or its antigen-binding fragment containing said sequences may be referred to as "ZY0EO5-E07" or "EO5-E07".

[0130] In one embodiment, the antibody or its antigen-binding fragment comprises: a variable heavy chain containing the amino acid sequence of SEQ ID NO:39, or a functional variant thereof; and a variable light chain containing the amino acid sequence of SEQ ID NO:40, or a functional variant thereof. An antibody or its antigen-binding fragment containing said sequences may be referred to as "ZY0EP0-C07" or "EP0-C07".

[0131] In one embodiment, the antibody or its antigen-binding fragment comprises: a variable heavy chain containing the amino acid sequence of SEQ ID NO:33, or a functional variant thereof; and a variable light chain containing the amino acid sequence of SEQ ID NO:34, or a functional variant thereof. An antibody or its antigen-binding fragment containing said sequences may be referred to as "ZY0EQD-E02" or "EQD-E02".

[0132] In one embodiment, the antibody or its antigen-binding fragment comprises: a variable heavy chain containing the amino acid sequence of SEQ ID NO:43, or a functional variant thereof; and a variable light chain containing the amino acid sequence of SEQ ID NO:34, or a functional variant thereof.

[0133] In one embodiment, the antibody or its antigen-binding fragment comprises: a variable heavy chain containing the amino acid sequence of SEQ ID NO:46, or a functional variant thereof; and a variable light chain containing the amino acid sequence of SEQ ID NO:34, or a functional variant thereof.

[0134] In one embodiment, the antibody or its antigen-binding fragment comprises: a variable heavy chain containing the amino acid sequence of SEQ ID NO:47, or a functional variant thereof; and a variable light chain containing the amino acid sequence of SEQ ID NO:34, or a functional variant thereof.

[0135] In a preferred embodiment, the antibody or its antigen-binding fragment comprises: a variable heavy chain containing the amino acid sequence of SEQ ID NO:45, or a functional variant thereof; and a variable light chain containing the amino acid sequence of SEQ ID NO:34, or a functional variant thereof. An antibody or its antigen-binding fragment containing said sequences may be referred to as "EQD-E02_GL".

[0136] In one embodiment, the antibody or its antigen-binding fragment comprises a variable heavy chain having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with a reference amino acid sequence SEQ ID NO:43. In another embodiment, the antibody or its antigen-binding fragment comprises a variable heavy chain containing the amino acid sequence of SEQ ID NO:43. For example, the antibody or its antigen-binding fragment may comprise a variable heavy chain containing the amino acid sequence of SEQ ID NO:43 and a variable light chain containing the amino acid sequence of SEQ ID NO:34.

[0137] Alternatively, the antibodies or antigen-binding fragments described herein may be described by their heavy and / or light chains.

[0138] In one embodiment, the antibody or its antigen-binding fragment comprises a light chain (e.g., comprising a VL and a constant light chain) having an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:44. In a preferred embodiment, the antibody or its antigen-binding fragment comprises a light chain (e.g., comprising a VL and a constant light chain) containing the amino acid sequence of SEQ ID NO:44.

[0139] In one embodiment, the antibody or its antigen-binding fragment comprises a heavy chain (e.g., comprising VH and a constant heavy chain) that contains an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with a reference amino acid sequence SEQ ID NO:48. For example, the antibody or its antigen-binding fragment may comprise a heavy chain containing the amino acid sequence of SEQ ID NO:48 (e.g., comprising VH and a constant heavy chain). Such a heavy chain may be referred to as "E02-GL-Maia-heavy chain".

[0140] In one embodiment, the antibody or its antigen-binding fragment comprises a heavy chain (e.g., comprising VH and a constant heavy chain) that contains an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with a reference amino acid sequence SEQ ID NO:49. For example, the antibody or its antigen-binding fragment may comprise a heavy chain (e.g., comprising VH and a constant heavy chain) containing the amino acid sequence of SEQ ID NO:49. Such a heavy chain may be referred to as "E02-GLY-Maia-heavy chain".

[0141] In one embodiment, the antibody or its antigen-binding fragment comprises a heavy chain (e.g., comprising VH and a constant heavy chain) that contains an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with a reference amino acid sequence SEQ ID NO:50. For example, the antibody or its antigen-binding fragment may comprise a heavy chain (e.g., comprising VH and a constant heavy chain) containing the amino acid sequence of SEQ ID NO:50. Such a heavy chain may be referred to as "E02-GLQ-Maia-heavy chain".

[0142] In a preferred embodiment, the antibody or its antigen-binding fragment comprises a heavy chain (e.g., comprising VH and a constant heavy chain) having an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the reference amino acid sequence SEQ ID NO:51. In a more preferred embodiment, the antibody or its antigen-binding fragment comprises a heavy chain (e.g., comprising VH and a constant heavy chain) containing the amino acid sequence of SEQ ID NO:51. Such a heavy chain may be referred to as "E02-GL-WT-heavy chain".

[0143] In one embodiment, the antibody or its antigen-binding fragment comprises a light chain constant region having an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the reference amino acid sequence SEQ ID NO:42. In a preferred embodiment, the antibody or its antigen-binding fragment comprises a light chain constant region containing the amino acid sequence of SEQ ID NO:42.

[0144] In one embodiment, the antibody or its antigen-binding fragment comprises a heavy chain constant region containing the amino acid sequence of SEQ ID NO:41. More preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region containing the amino acid sequence of SEQ ID NO:52.

[0145] In a preferred embodiment, the antibody or its antigen-binding fragment comprises a light chain containing the amino acid sequence of SEQ ID NO:44 (e.g., containing VL and constant light chain) and a heavy chain containing the amino acid sequence of SEQ ID NO:51 (e.g., containing VH and constant heavy chain).

[0146] Advantageously, the inventors have demonstrated that the claimed antibody or antigen-binding fragment can target a broader spectrum of cells expressing B7-H4 compared to reported existing (commercially available) antibodies targeting B7-H4. Therefore, the inventors have not only provided antibodies (or antigen-binding fragments thereof) with affinity and specificity for clinically relevant targets, but have also demonstrated unique advantages associated with them (e.g., unexpected technical effects).

[0147] For example, Figure 4 demonstrates that the claimed exemplary antibodies bind to a broad spectrum of cancer cell types with an affinity that the following antibodies cannot achieve: Biosciences 14-5949 anti-human B7H4 mouse IgG; US biological B0000-35B anti-human B7H4 mouse IgG; R and D systems AF2514 anti-mouse B7H4 goat IgG1; and Sigma SAB2500141 anti-B7H4 goat IgG1.

[0148] Preferably, the antibody or its antigen-binding fragment described herein can bind to B7-H4, which is a component of cancer cells (e.g., B7-H4 is a component of the cell membrane of cancer cells).

[0149] The antibodies or antigen-binding fragments described herein may bind to OVCAR4 cell lines and / or CHO cell lines (e.g., which may lack exogenous nucleic acids encoding B7-H4). For example, the antibody or antigen-binding fragment binds to the B7-H4 (e.g., the B7-H4 epitope) of OVCAR4 cell lines and / or CHO cell lines (e.g., which may lack exogenous nucleic acids encoding B7-H4). Suitably, the antibodies or antigen-binding fragments described herein may bind to OVCAR4 cell lines and CHO cell lines (e.g., which may lack exogenous nucleic acids encoding B7-H4).

[0150] In one embodiment, the antibody or its antigen-binding fragment binds to the OVCAR4 cell line and / or CHO cell line (e.g., which may lack exogenous nucleic acid encoding B7-H4) with higher affinity than one or more antibodies selected from E Biosciences 14-5949 anti-human B7H4 mouse IgG, US biological B0000-35B anti-human B7H4 mouse IgG, R and D systems AF2514 anti-mouse B7H4 goat IgG1, Sigma SAB2500141 anti-B7H4 goat IgG1, isotype 1 CAT004 SP06-003, isotype 2 R and D normal goat IgG control (AB-108C), AdD serotec MCA2632, Epitomics 2516-1, eBiosciences 145972-82, eBioscience 145970-85, or combinations thereof. For example, compared to one or more antibodies selected from E Biosciences 14-5949 anti-human B7H4 mouse IgG, US biological B0000-35B anti-human B7H4 mouse IgG, R and D systems AF2514 anti-mouse B7H4 goat IgG1 and Sigma SAB2500141 anti-B7H4 goat IgG1, or combinations thereof, this antibody or its antigen-binding fragment can bind to OVCAR4 cell lines and / or CHO cell lines (e.g., which may lack exogenous nucleic acids encoding B7-H4) with higher affinity.

[0151] In a preferred embodiment, the antibody or its antigen-binding fragment binds to the OVCAR4 cell line with higher affinity than E Biosciences 14-5949 anti-human B7H4 mouse IgG.

[0152] The reference to "E Biosciences 14-5949 anti-human B7H4 mouse IgG" is used interchangeably with the term "B7-H4 monoclonal antibody (H74), eBioscience" used herein. This antibody is available from Thermo Fisher Scientific (catalog number 14-5949-82).

[0153] In another preferred embodiment, the antibody or its antigen-binding fragment binds to the OVCAR4 cell line with higher affinity than US biological B0000-35B anti-human B7H4 mouse IgG.

[0154] The affinity (e.g., binding affinity) can be measured by any suitable method for measuring binding affinity described herein.

[0155] The OVCAR4 cell line is a human ovarian cancer cell line. It is available from the National Cancer Institute, specifically from the Division of Cancer Treatment and Diagnosis Tumor Repository. The Chinese hamster ovary (CHO) cell line is an epithelial cell line derived from the ovaries of Chinese hamsters and is widely available.

[0156] As described above, the antibody or its antigen-binding fragment of the present invention may be included in a pharmaceutical composition. This pharmaceutical composition may contain one or more pharmaceutically acceptable excipients. In one embodiment, the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable, non-toxic, sterile carrier, such as physiological saline, a non-toxic buffer, a preservative, etc. Suitable formulations used in the treatment methods disclosed herein are described in Remington's Pharmaceutical Sciences, 22nd edition, edited by Lloyd V. Allen, Jr. (2012).

[0157] In one embodiment, the pharmaceutical composition of the present invention may be contained in one or more formulations selected from capsules, tablets, aqueous suspensions, solutions, nasal aerosols, or combinations thereof.

[0158] In one embodiment, the pharmaceutical composition comprises more than one type of antibody or antigen-binding fragment of the present invention. For example, the pharmaceutical composition may comprise two or more selected from antibodies, antigen-binding fragments, antibodies conjugated to cytotoxicants or their antigen-binding fragments, or combinations thereof.

[0159] The term "pharmaceutically effective amount" of an antibody or antigen-binding fragment means an amount sufficient to achieve effective binding to a target and to provide benefits (e.g., improving symptoms of a disease or condition, or detecting a substance or cell).

[0160] In one embodiment, the pharmaceutical composition may include a buffer solution (e.g., acetate, phosphate, or citrate buffer), a surfactant (e.g., polysorbate), and a stabilizer reagent as needed (e.g., human albumin), etc.

[0161] Suitablely, the antibody or antigen-binding fragment of the present invention binds to the B7-H4 molecule with sufficient affinity, such that the antibody can be used as a therapeutic agent or diagnostic reagent targeting B7-H4.

[0162] In one embodiment, the antibody or its antigen-binding fragment binds to B7-H4 (preferably human B7-H4) with the following dissociation constant (KD): 1μM, 100nM 10nM 1nM, 0.1nM 10pM 1pM, or 0.1 pM. In one embodiment, the antibody or its antigen-binding fragment binds to B7-H4 (preferably human B7-H4) at the following KD concentrations: between about 0.1 nM and about 40 nM, between about 0.5 nM and about 30 nM, between about 1 nM and about 20 nM, or between about 1.5 nM and about 20 nM.

[0163] In a preferred embodiment, the antibody or its antigen-binding fragment binds to B7-H4 (preferably human B7-H4) with a KD of about 23 nM to about 27 nM. In a more preferred embodiment, the antibody or its antigen-binding fragment binds to B7-H4 (preferably human B7-H4) with a KD of about 1 nM to about 1.5 nM.

[0164] KD measurements (binding affinity) can be performed by any suitable assay known in the art. Suitable assays include affinity assays that can be performed via a KinExA system (e.g., KinExA 3100, KinExA 3200, or KinExA 4000) (Sapidyne Instruments, Idaho) or a ForteBio Octet system.

[0165] In one embodiment, the antibody or its antigen-binding fragment of the present invention binds to unrelated non-B7-H4 proteins to a degree that is about 10%, 5%, 2%, or 1% (preferably about 10%) less than the binding of the antibody (or its antigen-binding fragment) to B7-H4 (preferably human B7-H4). This binding can be measured, for example, by radioimmunoassay (RIA), BIACORE® (using recombinant B7-H4 as the analyte and the antibody as the ligand, or vice versa), KINEXA®, the ForteBio Octet system, or other binding assays known in the art.

[0166] In one embodiment, the antibody or its antigen-binding fragment does not bind to one or more molecules selected from human B7-H1, human B7-H2, human B7-H3, human BTN1A1, human HHLA2, human BTN3A2, or combinations thereof. In a preferred embodiment, the antibody or its antigen-binding fragment does not bind to one or more molecules selected from human B7-H1, human B7-H2, human B7-H3, or combinations thereof.

[0167] The term "non-binding" means that the antibody or its antigen-binding fragment described herein substantially does not bind to one or more of the described molecules (e.g., human B7-H1 molecule, human B7-H2 molecule, human B7-H3 molecule, human BTN1A1 molecule, human HHLA2 molecule, human BTN3A2 molecule, or combinations thereof). When used in the context of binding herein, the term "substantially none" may mean that less than 5%, 2%, 1%, 0.5%, or 0.1% of cells in a cell culture expressing one or more of the described molecules are bound (at contact) to the antibody or its antigen-binding fragment described herein. Suitably, when used in the context of binding herein, the term "substantially none" may mean that no such cells are bound.

[0168] In one embodiment, the antibody or its antigen-binding fragment does not bind to human B7-H1, human B7-H2, human B7-H3, human BTN1A1, human HHLA2, or human BTN3A2 molecules. In a preferred embodiment, the antibody or its antigen-binding fragment does not bind to human B7-H1, human B7-H2, or human B7-H3 molecules.

[0169] In one embodiment, the B7-H4 polypeptide is contained within a B7-H4 polypeptide sequence or a fragment thereof.

[0170] The "B7-H4 polypeptide" may comprise the full-length polypeptide sequence of B7-H4 (e.g., SEQ ID NO.: 55), or a B7-H4 fragment of any length (e.g., comprising 5%, 15%, 25%, 35%, 45%, 55%, 65%, 75%, 85%, or 95% of the full-length polypeptide sequence of B7-H4), the fragment comprising an epitope that can bind (e.g., be bound to) an antibody or antigen-binding fragment of the present invention. The B7-H4 polypeptide may comprise a sequence having 75%, 80%, 85%, 90%, or 90% sequence identity with the sequence of SEQ ID NO.: 55. Preferably, the B7-H4 polypeptide comprises the sequence of SEQ ID NO.: 55.

[0171] This antibody or antigen-binding fragment has a high affinity for B7-H4 both in vitro and in vivo, and therefore can be advantageously used in methods for detecting B7-H4 epitopes and related diagnostic methods.

[0172] "Treatment" refers to therapeutic measures that cure, slow down, alleviate the symptoms of a diagnosed pathological condition or disorder, and / or halt the progression of a diagnosed pathological condition or disorder. Therefore, those who require treatment include those who already have the disorder. In one implementation, if a patient shows, for example, complete, partial, or transient relief or elimination of symptoms associated with the disease or disorder (preferably cancer), then the subject has successfully "treated" the disease or disorder (preferably cancer) according to the methods provided herein.

[0173] In one embodiment, the method of the present invention can be used to prevent the onset of cancer comprising cancer cells expressing B7-H4. "Prevention" refers to defensive or preventative measures to prevent and / or slow the development of a target pathological condition or disorder. Therefore, those who need prevention include those who are predisposed to or susceptible to the disorder. In one embodiment, the method provided herein successfully prevents the disease or disorder (preferably cancer) if, compared to a patient who has not been subjected to the method of the present invention, the patient transiently or permanently exhibits, for example, fewer or less severe symptoms associated with the disease or disorder, or a later onset of symptoms associated with the disease or disorder.

[0174] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammalian subject. In one embodiment, the "subject" is a human, livestock, farm animal, racing animal, or zoo animal, such as a human, non-human primate, dog, cat, guinea pig, rabbit, rat, mouse, horse, cow, etc. In one embodiment, the subject is a cynomolgus monkey (Macaca fascicularis). In a preferred embodiment, the subject is a human. In the method of the present invention, the subject may not have previously been diagnosed with cancer. Alternatively, the subject may have previously been diagnosed with cancer. The subject may also be a person exhibiting disease risk factors or a person without cancer symptoms. The subject may also be a person with cancer or at risk of developing cancer. Thus, in one embodiment, the method of the present invention can be used to confirm the presence of cancer in a subject. For example, the subject may have previously been diagnosed with cancer by alternative means. In one embodiment, the subject has previously received cancer therapy.

[0175] In one embodiment, the treatment method of the present invention includes one or more administration steps selected from oral, intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal, inhalation, local, or combinations thereof. In a preferred embodiment, the administration is intravenous or intra-arterial (e.g., by injection or infusion) or a combination thereof.

[0176] In one embodiment, the antibody or its antigen-binding fragment is delivered directly to the site of a harmful cell population (e.g., thereby increasing the exposure of diseased tissue to the therapeutic agent). In another embodiment, it is administered directly into the airway, for example by inhalation or intranasal administration.

[0177] In a preferred embodiment, the cancer mentioned herein is characterized by the expression (preferably overexpression) of the B7-H4 molecule. In other words, the cancer mentioned herein may comprise cancer cells expressing B7-H4. These cancer cells may be contained within a tumor.

[0178] In one embodiment, the cancer is selected from one or more of breast cancer, ovarian cancer, endometrial cancer, bile duct cancer, NSCLC (squamous cell carcinoma and adenocarcinoma), pancreatic cancer, and gastric cancer.

[0179] In one embodiment, the cancer is selected from one or more of colorectal cancer, HNSCC, prostate cancer, lung cancer (e.g., NSCLC or SCLC), breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, bile duct cancer, melanoma, endometrial cancer, hematologic malignancies (AML, MM, DLBCL), and cancers containing CSC.

[0180] In a preferred embodiment, the cancer is lung cancer, breast cancer, or a combination thereof. For example, the cancer may be lung cancer. The cancer may be breast cancer. The cancer may be ovarian cancer.

[0181] In one embodiment, the cancer is one or more types of breast cancer, selected from hormone receptor (HR)-positive (HR+) breast cancer, human epidermal growth factor receptor 2-positive (HER2+) breast cancer, and triple-negative breast cancer (TNBC). The subject may be eligible for Herceptin treatment. The subject may have already received Herceptin treatment.

[0182] In one embodiment, the cancer is one or more non-small cell lung cancers (NSCLC), preferably selected from squamous NSCLC, adenocarcinoma NSCLC, or a combination thereof.

[0183] Antibodies or their antigen-binding fragments can also be used to detect cancer cells, for example, as part of a diagnostic method.

[0184] On the other hand, a method is provided for detecting the presence of a B7-H4 peptide (e.g., a B7-H4 peptide epitope) in a sample, the method comprising:

[0185] a. Contacting a sample with an antibody or an antigen-binding fragment thereof, or a pharmaceutical composition containing an antibody or an antigen-binding fragment thereof, to provide an antibody-antigen complex; wherein said antibody or antigen-binding fragment thereof comprises: i. heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, or functional variants thereof; ii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, or functional variants thereof; iii. SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, or functional variants thereof; iv. Containing the amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, or functional variants thereof; or v. Containing the amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, or functional variants thereof; or v. Containing the amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, or functional variants thereof.

[0186] b. Detecting the presence of the antibody-antigen complex; and c. wherein the presence of the antibody-antigen complex confirms the presence of the B7-H4 peptide (e.g., the B7-H4 peptide epitope); or d. wherein the absence of the antibody-antigen complex confirms the absence of the B7-H4 peptide (e.g., the B7-H4 peptide epitope).

[0187] In one related aspect, a method is provided for detecting the presence of cancer cells expressing a B7-H4 peptide (e.g., a B7-H4 peptide epitope) in a sample, the method comprising: a. contacting the sample with an antibody or an antigen-binding fragment thereof, or a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof, to provide an antibody-antigen complex; wherein said antibody or antigen-binding fragment thereof comprises: i. heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), or functional variants thereof, respectively containing the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; ii. respectively containing SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:6. iii. Containing the amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO:12, or functional variants thereof; iv. Containing the amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, or functional variants thereof; iv. Containing the amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, or functional variants thereof; or v. Containing the amino acid sequences SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:24, or functional variants thereof. NO:30 amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof; b. detecting the presence of the antibody-antigen complex; and c. wherein the presence of the antibody-antigen complex confirms the presence of a B7-H4 peptide (e.g., a B7-H4 peptide epitope); or d. wherein the absence of the antibody-antigen complex confirms the absence of a B7-H4 peptide (e.g., a B7-H4 peptide epitope).

[0188] This invention includes the use of the antibodies of this invention or antigen-binding fragments thereof for the detection of B7-H4 peptides (e.g., B7-H4 peptide epitopes).

[0189] In one implementation, the presence of an antibody-antigen complex indicates the presence of cancer cells, and the absence of an antibody-antigen complex indicates the absence of cancer cells. For example, the method may include confirming the presence of cancer upon detection of an antibody-antigen complex, or not confirming the presence of cancer upon the absence of an antibody-antigen complex.

[0190] In a preferred embodiment, the cancer cell line is a cancer cell line that expresses a B7-H4 peptide (e.g., a B7-H4 peptide epitope).

[0191] Therefore, the present invention includes the corresponding use of the method steps described herein in a method for diagnosing a subject with cancer, preferably wherein the cancer comprises cancer cells expressing B7-H4.

[0192] In one embodiment, the detection or diagnostic method may include measuring the performance level of B7-H4 in cells (or tissues) available from a subject, and comparing the measured performance level with standard B7-H4 performance in control cells (or tissues), wherein an increase in performance level compared to the control indicates the presence of cancer. Preferably, the control sample comprises non-cancerous (e.g., normal) cells.

[0193] "Antibody-antigen complex" refers to a complex (e.g., a macromolecular complex) containing B7-H4 antigen bound to an antibody. The term "antibody-antigen complex" can be used synonymously with the terms "bound B7-H4-antibody complex" and "antibody bound to B7-H4".

[0194] Antibody-antigen complexes can be detected by any method known to the art. In one embodiment, the antibody (or its antigen-binding fragment) is labeled with a detectable label. The label may be an epi-fluorescent label. In a preferred embodiment, the antibody is labeled with 800 CW.

[0195] In one implementation, the antibody-antigen complex is detected by a second (e.g., detection) antibody that binds to the antibody and / or the antibody-antigen complex.

[0196] Suitably, the second antibody includes a detection tool, such as a tag / label, to aid in detection. The detection tool is preferably conjugated to the second antibody. Examples of suitable labels include detectable labels, such as radioactive labels or fluorescent or colored molecules, enzyme labels, or chromogenic labels (e.g., dyes that provide a visible color change when detecting antibody-antigen binding). For example, the label may be fluorescein isothiocyanate (FITC), R-phycoerythrin, Alexa 532, CY3, or digoxigenin. The label may be a reporter molecule that is detected directly, such as by detecting its fluorescent signal, or by exposing the label to photographic or X-ray film. Alternatively, the label may not be detected directly, but may be detected, for example, in a two-phase system. An example of indirect label detection is the binding of the antibody to the label.

[0197] In a preferred embodiment, the second antibody comprises a fluorescent tag, and the antibody-antigen complex is detected by fluorescence emitted by the antibody-antigen-second antibody complex. "Antibody-antigen-second antibody complex" means a complex comprising an antigen (e.g., B7-H4) already bound to an antibody, wherein the complex is further bound by a second antibody that binds the antibody and / or the antibody-antigen complex.

[0198] Suitablely, an antibody-antigen complex is detected when the signal emitted by the detection marker (preferably fluorescence) is greater than the signal detected in a control that does not contain an antibody (e.g., does not contain an antibody binding to B7-H4). The control may alternatively contain B7-H4, but the sample should not be used for the control.

[0199] Suitablely, "sample" refers to a sample obtained from a subject (e.g., a biopsy), a cell line, a tissue culture, or other cellular sources that may potentially express B7-H4. In a preferred embodiment, the sample is derived from a biopsy of a subject. The biopsy may be taken from a tumor or a site at risk of developing a tumor.

[0200] In a preferred embodiment, the sample system can be obtained (e.g., obtained) from a separated sample from the subject.

[0201] In a preferred embodiment, the B7-H4 polypeptide (e.g., a B7-H4 polypeptide epitope) is a component of cancer cells, and more preferably a component of the cell membrane of cancer cells.

[0202] This invention covers antibodies (reference antibodies) (e.g., antibody or antigen-binding fragments) having the CDR sequence or variable heavy and light chain sequences as defined herein, and their functional variants. The functional variants bind to the same target antigen as the reference antibody and preferably exhibit the same antigen cross-reactivity as the reference antibody. These functional variants may have different affinities for the target antigen compared to the reference antibody, but substantially the same affinity is preferred.

[0203] In contrast, the term "reference antibody" is used conveniently to refer to the antibody or antigen of the present invention. Therefore, the term "reference antibody" refers to the antibody or antigen of the present invention. For example, a reference antibody may mean an antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively. More specifically, a reference antibody may mean an antibody or antigen-binding fragment thereof comprising a variable heavy chain containing the amino acid sequence of SEQ ID NO:33 and a variable light chain containing the amino acid sequence of SEQ ID NO:34. Preferably, a reference antibody may mean an antibody or antigen-binding fragment thereof comprising a variable heavy chain containing the amino acid sequence of SEQ ID NO:45 and a variable light chain containing the amino acid sequence of SEQ ID NO:34.

[0204] In one implementation, a functional variant of the reference antibody exhibits sequence variations at one or more CDRs when compared to a corresponding reference CDR sequence. Therefore, a functional antibody variant may comprise a functional variant of a CDR. When the term "functional variant" is used in the context of a CDR sequence, it means that the CDR has at most two, preferably at most one, amino acid differences when compared to a corresponding reference CDR sequence, and when combined with the remaining five CDRs (or variants thereof), enables the variant antibody to bind to the same target antigen as the reference antibody, and preferably exhibits the same antigenic cross-reactivity as the reference antibody. The functional variant may be referred to as a "variant antibody."

[0205] In one embodiment, the variant antibody (or its antigen-binding fragment) comprises: a light chain CDR1 having a difference of up to two amino acids compared to a corresponding reference CDR sequence; a light chain CDR2 having a difference of up to two amino acids compared to a corresponding reference CDR sequence; a light chain CDR3 having a difference of up to two amino acids compared to a corresponding reference CDR sequence; a heavy chain CDR1 having a difference of up to two amino acids compared to a corresponding reference CDR sequence; a heavy chain CDR2 having a difference of up to two amino acids compared to a corresponding reference CDR sequence; and a heavy chain CDR3 having a difference of up to two amino acids compared to a corresponding reference CDR sequence; wherein the variant antibody binds to the same target antigen as the reference antibody, and preferably exhibits the same antigen cross-reactivity as the reference antibody (or lacks antigen cross-reactivity).

[0206] Preferably, the variant antibody (or its antigen-binding fragment) comprises: a light chain CDR1 having at most one amino acid difference compared to the corresponding reference CDR sequence; a light chain CDR2 having at most one amino acid difference compared to the corresponding reference CDR sequence; a light chain CDR3 having at most one amino acid difference compared to the corresponding reference CDR sequence; a heavy chain CDR1 having at most one amino acid difference compared to the corresponding reference CDR sequence; a heavy chain CDR2 having at most one amino acid difference compared to the corresponding reference CDR sequence; and a heavy chain CDR3 having at most one amino acid difference compared to the corresponding reference CDR sequence; wherein the variant antibody binds to the same target antigen as the reference antibody, and preferably exhibits the same antigen cross-reactivity as the reference antibody (or lacks antigen cross-reactivity).

[0207] For example, the variant of the antibody or antigen-binding fragment may comprise: a heavy chain CDR1 having a difference of up to two amino acids compared to SEQ ID NO:7; a heavy chain CDR2 having a difference of up to two amino acids compared to SEQ ID NO:8; and a heavy chain CDR3 having a difference of up to two amino acids compared to SEQ ID NO:9; a light chain CDR1 having a difference of up to two amino acids compared to SEQ ID NO:10; a light chain CDR2 having a difference of up to two amino acids compared to SEQ ID NO:11; and a light chain CDR3 having a difference of up to two amino acids compared to SEQ ID NO:12; wherein the variant antibody binds to a B7-H4 peptide (e.g., a B7-H4 peptide epitope), and preferably exhibits the same antigen cross-reactivity as the reference antibody or antigen-binding fragment (or lacks antigen cross-reactivity).

[0208] For example, the variant of the antibody or antigen-binding fragment may (preferably) comprise: a heavy chain CDR1 having at most one amino acid difference compared to SEQ ID NO:7; a heavy chain CDR2 having at most one amino acid difference compared to SEQ ID NO:8; and a heavy chain CDR3 having at most one amino acid difference compared to SEQ ID NO:9; a light chain CDR1 having at most one amino acid difference compared to SEQ ID NO:10; a light chain CDR2 having at most one amino acid difference compared to SEQ ID NO:11; and a light chain CDR3 having at most one amino acid difference compared to SEQ ID NO:12; wherein the variant antibody binds to a B7-H4 peptide (e.g., a B7-H4 peptide epitope), and preferably exhibits the same antigen cross-reactivity as the reference antibody or antigen-binding fragment (or lacks antigen cross-reactivity).

[0209] The foregoing can be similarly applied to other antibody variants described herein, wherein the amino acid differences are defined relative to their CDR sequences, and wherein the variant antibody binds to the same target antigen as the antibody and preferably exhibits the same antigen cross-reactivity.

[0210] In one embodiment, when compared with the corresponding reference antibody, the variant antibody may have a total of up to 5, 4, or 3 amino acid differences in its CDR, provided that each CDR has at most 2 (preferably at most 1) amino acid differences. More preferably, when compared with the corresponding reference antibody, the variant antibody has a total of up to 2 (more preferably at most 1) amino acid differences in its CDR, provided that each CDR has at most 2 amino acid differences. Even more preferably, when compared with the corresponding reference antibody, the variant antibody has a total of up to 2 (more preferably at most 1) amino acid differences in its CDR, provided that each CDR has at most 1 amino acid difference.

[0211] The amino acid difference can be an amino acid substitution, insertion, or deletion. In one embodiment, the amino acid difference is a conserved amino acid substitution as described herein.

[0212] In one embodiment, the variant antibody has the same frame sequence as the exemplary antibody described herein. In another embodiment, the variant antibody may comprise a frame region having at most two, preferably at most one, amino acid differences (when compared to the corresponding reference frame sequence). Thus, each frame region may have at most two, preferably at most one, amino acid differences (when compared to the corresponding reference frame sequence).

[0213] In one embodiment, when compared with a corresponding reference antibody, the variant antibody may have a total of up to 5, 4, or 3 amino acid differences in its frame regions, provided that there are at most 2 (preferably at most 1) amino acid differences in each frame region. More preferably, when compared with a corresponding reference antibody, the variant antibody has a total of up to 2 (more preferably at most 1) amino acid differences in its frame regions, provided that there are at most 2 amino acid differences in each frame region. Even more preferably, when compared with a corresponding reference antibody, the variant antibody has a total of up to 2 (more preferably at most 1) amino acid differences in its frame regions, provided that there are at most 1 amino acid difference in each frame region.

[0214] Therefore, the variant antibody may comprise a variable heavy chain and a variable light chain as described herein, wherein: when compared with the heavy chain sequence herein, the heavy chain has a maximum of 14 amino acid differences (a maximum of 2 amino acid differences in each CDR and a maximum of 2 amino acid differences in each frame region); and when compared with the light chain sequence herein, the light chain has a maximum of 14 amino acid differences (a maximum of 2 amino acid differences in each CDR and a maximum of 2 amino acid differences in each frame region); wherein the variant antibody binds to the same target antigen as the reference antibody, and preferably exhibits the same antigenic cross-reactivity as the reference antibody (or lacks antigenic cross-reactivity).

[0215] The variable heavy chain or light chain can be referred to as a "functional equivalent" of the reference heavy chain or light chain.

[0216] In one embodiment, the variant antibody may comprise a variable heavy chain and a variable light chain as described herein, wherein: when compared with the heavy chain sequence herein, the heavy chain has at most 7 amino acid differences (at most 1 amino acid difference in each CDR and at most 1 amino acid difference in each frame region); and when compared with the light chain sequence herein, the light chain has at most 7 amino acid differences (at most 1 amino acid difference in each CDR and at most 1 amino acid difference in each frame region); wherein the variant antibody binds to the same target antigen as the reference antibody, and preferably exhibits the same antigen cross-reactivity as the reference antibody (or lacks antigen cross-reactivity).

[0217] Antibody-drug conjugates (ADCs)

[0218] Advantageously, the antibody or antigen-binding fragment of the present invention may comprise a heterologous drug agent. In one embodiment, the antibody or antigen-binding fragment of the present invention is linked to a heterologous drug agent. In a preferred embodiment, the antibody or antigen-binding fragment is conjugated to the heterologous drug agent. Suitably, "conjugation" means a connection via a covalent or ionic bond. Preferably, the heterologous drug agent is a cytotoxic agent.

[0219] Heterogeneous agents can be simply referred to as "agents" or "active agents." For example, in other words, the antibody or antigen-binding fragment of the present invention may contain an active agent. In one embodiment, the antibody or antigen-binding fragment of the present invention is linked to an active agent. In a preferred embodiment, the antibody or antigen-binding fragment is conjugated to an active agent.

[0220] The heterologous agent / active agent may be a drug. Preferably, the heterologous agent / active agent is a cytotoxin.

[0221] Particularly preferred is that the antibody or its antigen-binding fragment of the present invention is conjugated (e.g., conjugated) to a heterologous drug agent / active agent in a treatment method, as described below.

[0222] The pharmaceutical agents and / or cytotoxic agents of the present invention can be conjugated to the antibody or its antigen-binding fragment by means of a spacer (e.g., at least one spacer). In one embodiment, the spacer is a peptide spacer. In another embodiment, the spacer is a non-peptide (e.g., chemical) spacer.

[0223] Cytotoxic agents or cytotoxins can be any molecule known in the art that inhibits or prevents cell function and / or causes cell damage (cell death), and / or exerts antitumor / antiproliferative effects. Many classes of cytotoxic agents are known to have potential utility in ADC molecules. These include, but are not limited to: topoisomerase I inhibitors, muscarin, auristatin, daunomycin, doxorubicin, salicylate, enediyne, enediyne, lexitropsin, taxanes, puromycin, maytansinoids, vinblastine, tubulolysin, and pyrrolobenzodiazepines (PBD). Examples of such cytotoxic agents include AFP, MMAF, MMAE, AEB, AEB, oxalistatin E, paclitaxel, docetaxel, CC-1065, SN-38, and topotecan. Pyrrolidone, Rhizomycin, Cyanide Pyrrolizidine, dorastestatin-10, echinococin, compressorin, chalicheamicin, maytansin, DM-1, vincristine, methotrexate, and fuscin, and their derivatives and analogues. Further disclosures regarding cytotoxicants suitable for use in ADCs can be found, for example, in International Patent Application Publications Nos. WO 2015 / 155345 and WO 2015 / 157592, which are incorporated herein by reference in their entirety.

[0224] For example, the antibody or antigen-binding fragment can be conjugated to such heterologous drugs to provide an "antibody-drug conjugate" (ADC).

[0225] The agent is typically linked to or "loaded" onto the antibody or antigen-binding fragment. Agent loading (p) refers to the average amount of agent on each antibody or antigen-binding fragment (e.g., ligand unit).

[0226] The average amount of drug on each antibody (or antigen-binding fragment) in an ADC formulation derived from a conjugation reaction can be characterized by conventional methods such as UV, reverse-phase HPLC, HIC, mass spectrometry, ELISA, and electrophoresis. The quantitative distribution of ADCs based on p can also be determined. The average p value in a specific ADC formulation can be determined by ELISA (Hamblett et al. (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al. (2005) Clin. Cancer Res. 11:843-852). In some cases, the separation, purification, and characterization of homogeneous ADCs (where p is a determined value derived from an ADC with other drug loadings) can be achieved by, for example, reverse-phase HPLC or electrophoresis. Such techniques are also applicable to other types of conjugations.

[0227] Cysteine ​​amino acids can be engineered at reactive sites in antibodies (or their antigen-binding fragments), and preferably without forming intrachain or intermolecular disulfide bonds (Junutula et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al. (2009) Blood 114(13):2721-2729; US 7521541; US ​​7723485; WO 2009 / 052249). Engineered cysteine ​​thiols can react with linkers within a pharmaceutical agent (e.g., having the following formula I) to form an ADC of an antibody engineered with cysteine, the linker having a thiol-reactive electrophilic group (such as maleimide or α-haloacetylamine). Thus, the positions of pharmaceutical units can be designed, controlled, and known. Because engineered cysteine ​​thiol groups typically react with drug linker reagents in high yields, drug loading can be controlled. IgG antibodies are engineered to introduce cysteine ​​amino acids by substitution at a single site on the heavy or light chain, thus giving two new cysteines to the symmetrical antibody. Drug loading close to 2 can be achieved, with the conjugate product ADC exhibiting near-homogeneity.

[0228] When the antibody or its antigen-binding fragment reacts with more than one nucleophilic or electrophilic group with a pharmaceutical agent, the resulting product can be a mixture of ADC compounds in which the distribution of the pharmaceutical units attached to the antibody is, for example, 1, 2, 3, etc. Liquid chromatography (such as polymer reverse phase (PLRP) and hydrophobic interaction (HIC)) can separate the compounds in the mixture by means of the pharmaceutical loading value. ADC formulations having a single pharmaceutical loading value (p) can be separated.

[0229] Therefore, the antibody-drug conjugate composition of the present invention may include a mixture of antibody-drug conjugates, wherein the antibody or its antigen-binding fragment has one or more pharmaceutical portions, and wherein the pharmaceutical portions may be attached to the antibody or its antigen-binding fragment at various amino acid residues.

[0230] In one embodiment, the average amount of drug on each antibody (or its antigen-binding fragment) is in the range of 1 to 20. In some embodiments, this range is selected from 1 to 10, 2 to 10, 2 to 8, 2 to 6, and 4 to 10. In some embodiments, one drug is present on each antibody (or its antigen-binding fragment). In some embodiments, the amount of drug on each antibody (or its antigen-binding fragment) can be expressed as a ratio of drug (i.e., pharmacology) to antibody. This ratio is called the drug-to-antibody ratio (DAR). DAR is the average amount of pharmacology (i.e., pharmacology) attached to each antibody. In one embodiment of the invention, DAR is in the range of 1 to 20. In some embodiments, the range of DAR is selected from 1 to 10, 2 to 10, 2 to 8, 2 to 6, and 4 to 10. In a particular embodiment of the invention, DAR is approximately 8. In a particular embodiment of the invention, DAR is 8.

[0231] In one embodiment, the antibody or antigen-binding fragment is conjugated to one or more heterologous agents selected from the group consisting of: topoisomerase I inhibitors, tubulolysin derivatives, pyrrolobenzodiazepines, antimicrobial agents, therapeutic agents, precursors, peptides, proteins, enzymes, lipids, biological response modulators, pharmaceutical reagents, lymphatic mediators, heterologous antibodies, fragments of heterologous antibodies, detectable labels, polyethylene glycol (PEG), radioisotopes, or combinations thereof.

[0232] In one embodiment, the antibody-antigen-binding fragment is conjugated to one or more cytotoxins selected from topoisomerase I inhibitors, tubulolysin derivatives, pyrrolobenzodiazepines, or combinations thereof. For example, the antibody or its antigen-binding fragment is conjugated to one or more cytotoxins selected from the group consisting of: topoisomerase I inhibitors SG3932, SG4010, SG4057, or SG4052 (the structures of which are provided below); tubulolysin AZ1508; pyrrolobenzodiazepines SG3315; pyrrolobenzodiazepines SG3249; or combinations thereof.

[0233] Preferably, the antibody or its antigen-binding fragment can be conjugated to a topoisomerase I inhibitor. Topoisomerase inhibitors are chemical compounds that block the action of topoisomerases (topoisomerases I and II), a type of enzyme that controls DNA structural changes during the normal cell cycle by catalyzing the breaking and rejoining of the phosphodiester backbone of the DNA strand.

[0234] Typical examples of suitable topoisomerase I inhibitors are represented by the following compounds:

[0235] The compound is denoted as A* and may be referred to herein as a "pharmaceutical unit".

[0236] The compound (e.g., A*) preferably provides a linker for attachment (preferably conjugation) to an antibody or antigen-binding fragment (which may be referred to as a "ligand unit") described herein. Suitably, the linker is attached (e.g., conjugated) to an amino residue, such as an amino acid of the antibody or antigen-binding fragment described herein, in a cleavable manner.

[0237] More specifically, examples of suitable topoisomerase I inhibitors are represented by the following compounds having the formula "I":

[0238] and its salts and solvates, wherein RL is a linker for linking to the antibody or its antigen-binding fragment (e.g., ligand unit) described herein, wherein the linker is preferably selected from: (ia): Among them, the Q series: Where QX, Q is an amino acid residue, dipeptide residue, tripeptide residue, or tetrapeptide residue; X is: Where a = 0 to 5, b1 = 0 to 16, b2 = 0 to 16, c1 = 0 or 1, c2 = 0 or 1, d = 0 to 5, wherein at least b1 or b2 = 0 (i.e., only one of b1 and b2 may not be 0) and at least c1 or c2 = 0 (i.e., only one of c1 and c2 may not be 0); GL refers to a linker used to connect to the antibody or its antigen-binding fragment (e.g., ligand unit) described herein; or (ib): RL1 and RL2 are independently selected from H and methyl, or together with the carbon atoms they are bonded to form cyclopropene or cyclobutene groups; and the e-series is 0 or 1.

[0239] Those skilled in the art will understand that more than one of the agents (e.g., topoisomerase I inhibitors) can be conjugated to the antibody or its antigen-binding fragment.

[0240] For example, the conjugates of the present invention (e.g., antibody-drug conjugates) can have the general formula IV: L-(DL)p ( [IV])

[0241] Or a pharmaceutically acceptable salt or solvate thereof, wherein L is an antibody or antigen-binding fragment (e.g., a ligand unit) as described herein, DL is a topoisomerase I inhibitor having a linker (e.g., a drug linker unit) and has Formula III:

[0242] RLL is a linker that connects to the antibody or its antigen-binding fragment (e.g., ligand unit) described herein, wherein the linker is preferably selected from (ia'): Where Q and X are as defined above, and GLL is a linker connected to the antibody or its antigen-binding fragment (e.g., ligand unit) described herein; and (ib'): RL1 and RL2 are as defined above; and p is an integer from 1 to 20.

[0243] Drug loading is represented by p, which is the number of topoisomerase I inhibitors (e.g., drug units) on each antibody or its antigen-binding fragment (e.g., ligand unit). Drug loading can range from 1 to 20 drug units (D) / ligand unit. For compositions, p represents the average drug loading of the conjugate in the composition, and p ranges from 1 to 20.

[0244] Therefore, the present invention includes conjugates comprising an antibody or antigen-binding fragment thereof (e.g., a ligand unit) covalently linked to at least one topoisomerase I inhibitor (e.g., a pharmaceutical unit, such as A* described above). The inhibitor is preferably linked to the antibody or antigen-binding fragment thereof via a linker (e.g., a linker unit, such as the linkers described above as RL and / or RLL). In other words, the present invention includes an antibody or antigen-binding fragment thereof (e.g., a ligand unit) attached (preferably via a linker) to one or more topoisomerase I inhibitors (e.g., pharmaceutical-linker units). The antibody or antigen-binding fragment thereof (representing the ligand unit) described more fully above is a target agent that binds to a target portion. More specifically, the ligand unit may, for example, specifically bind to B7-H4 on a target cell, thereby delivering the pharmaceutical unit to the target cell. Therefore, the present invention also provides a method of treating, for example, various cancers and other disorders (e.g., cancers / disorders associated with the presence of cells expressing B7-H4, preferably cancer cells) with an ADC.

[0245] Further preferences

[0246] Certain features of the aforementioned topoisomerase I inhibitors are particularly preferred and can be defined in more detail as follows. For example, a preferred embodiment of feature QX (e.g., within the linker of 1a above) will be outlined.

[0247] The following preferences may apply to all aspects of the invention as described above, or may relate to a single aspect. These preferences can be combined in any combination.

[0248] Various definitions of some of the terms used in this section are provided under the heading "Definitions" below.

[0249] QX

[0250] In one embodiment, Q is an amino acid residue. The amino acid can be a natural amino acid or a non-natural amino acid. For example, Q can be selected from: Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp, wherein Cit is a citrulline.

[0251] In one embodiment, Q comprises a dipeptide residue. The amino acid in the dipeptide can be any combination of natural and non-natural amino acids. In some embodiments, the dipeptide comprises a natural amino acid. The site of action for dipeptide-mediated cleavage occurs when the linker cathepsin is unstable. Then, the recognition site of the dipeptide-mediated cathepsin.

[0252] In one embodiment, Q is selected from: NH-Phe-Lys-C=O, NH-Val-Ala-C=O, NH-Val-Lys-C=O, NH-Ala-Lys-C=O, NH-Val-Cit-C=O, NH-Phe-Cit-C=O, NH-Leu-Cit-C=O, NH-Ile-Cit-C=O, NH-Phe-Arg-C=O, NH-Trp-Cit-C=O, and NH-Gly-Val-C=O; wherein Cit is citrulline.

[0253] Preferably, Q is selected from: NH-Phe-Lys-C=O, NH-Val-Ala-C=O, NH-Val-Lys-C=O, NH-Ala-Lys-C=O, and NH-Val-Cit-C=O.

[0254] More preferably, Q is selected from NH-Phe-Lys-C=O, NH-Val-Cit-C=O, or NH-Val-Ala-C=O.

[0255] Other suitable dipeptide combinations include: NH-Gly-Gly-C=O, NH-Gly-Val-C=O, NH-Pro-Pro-C=O, and NH-Val-Glu-C=O.

[0256] Other dipeptide combinations may be used, including those described by Dubowchik et al., Bioconjugate Chemistry, 2002, 13, 855-869, which are incorporated herein by reference.

[0257] In some embodiments, Q-series tripeptide residues are used. The amino acids in the tripeptide can be any combination of native and non-native amino acids. In some embodiments, the tripeptide contains native amino acids. The action site for tripeptide-series cathepsin-mediated cleavage occurs when the linker is unstable. Then, the recognition site of the tripeptide-series cathepsin is located. Of particular interest is the tripeptide linker system: NH-Glu-Val-Ala-C=O.

[0258] NH-Glu-Val-Cit-C=O

[0259] NH-αGlu-Val-Ala-C=O

[0260] NH-αGlu-Val-Cit-C=O

[0261] In some embodiments, Q-series tetrapeptide residues. The amino acid in the tetrapeptide can be any combination of native and non-native amino acids. In some embodiments, the tetrapeptide contains a native amino acid. The tetrapeptide-series cathepsin-mediated cleavage site is the linker when the linker is unstable. Then, the tetrapeptide-series cathepsin recognition site. Particularly interesting tetrapeptide linker series are: NH-Gly-Gly-Phe-GlyC=O; and NH-Gly-Phe-Gly-GlyC=O.

[0262] In some embodiments, the tetrapeptide system is: NH-Gly-Gly-Phe-GlyC=O.

[0263] In the above representation of peptide residues, NH- represents the N-terminus of the residue, and -C=O represents the C-terminus of the residue. The C-terminus binds the NH of A*.

[0264] Glu represents glutamic acid residues, that is:

[0265] αGlu represents the residues of glutamic acid when bound via the α chain, i.e.:

[0266] In one embodiment, the amino acid side chain is chemically protected where appropriate. The side chain protecting group may be a group as discussed above. The protected amino acid sequence can be cleaved by an enzyme. For example, a dipeptide sequence containing a Lys residue protected by a Boc side chain can be cleaved by cathepsins.

[0267] The protecting groups of the amino acid side chains are well known in the art and are described in the Novabiochem catalogue, as stated above.

[0268] GL

[0269] GL can be selected from:

[0270]

[0271] Where Ar represents a C5-6 aryl group, such as arylphenyl, and X represents a C1-4 alkyl group.

[0272] In some embodiments, GL is selected from GL1-1 and GL1-2. In some of these embodiments, GL refers to GL1-1.

[0273] GLL

[0274] GLL can be selected from:

[0275]

[0276] Where Ar represents a C5-6 aryl group, such as arylphenyl, and X represents a C1-4 alkyl group.

[0277] In some embodiments, GLL is selected from GLL1-1 and GLL1-2. In some of these embodiments, GLL is GLL1-1.

[0278] X

[0279] X is better: Where a = 0 to 5, b1 = 0 to 16, b2 = 0 to 16, c = 0 or 1, d = 0 to 5, wherein at least b1 or b2 = 0 and at least c1 or c2 = 0.

[0280] a can be 0, 1, 2, 3, 4, or 5. In some embodiments, a refers to 0 to 3. In some of these embodiments, a refers to 0 or 1. In other embodiments, a refers to 0.

[0281] b1 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b1 is 0 to 12. In some of these embodiments, b1 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8.

[0282] b2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b2 is 0 to 12. In some of these embodiments, b2 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8. Preferably, only one of b1 and b2 can be non-0.

[0283] c1 can be 0 or 1. c2 can be 0 or 1. Preferably, only one of c1 and c2 can be non-zero.

[0284] d can be 0, 1, 2, 3, 4, or 5. In some embodiments, d is 0 to 3. In some of these embodiments, d is 1 or 2. In other embodiments, d is 2. In still other embodiments, d is 5.

[0285] In some embodiments of X, a is series 0, b1 is series 0, c1 is series 1, c2 is series 0, and d is series 2, and b2 can be from 0 to 8. In some of these embodiments, b2 is series 0, 2, 3, 4, 5, or 8. In some embodiments of X, a is series 1, b2 is series 0, c1 is series 0, c2 is series 0, and d is series 0, and b1 can be from 0 to 8. In some of these embodiments, b1 is series 0, 2, 3, 4, 5, or 8. In some embodiments of X, a is series 0, b1 is series 0, c1 is series 0, c2 is series 0, and d is series 1, and b2 can be from 0 to 8. In some of these embodiments, b2 is series 0, 2, 3, 4, 5, or 8. In some embodiments of X, b1 is series 0, b2 is series 0, c1 is series 0, c2 is series 0, and one of a and d is series 0. The other series of a and d is from 1 to 5. In some of these embodiments, the other series of a and d is series 1. In some of these embodiments, another of a and d is 5. In some embodiments of X, a is 1, b2 is 0, c1 is 0, c2 is 1, d is 2, and b1 can be from 0 to 8. In some of these embodiments, b2 is 0, 2, 3, 4, 5, or 8.

[0286] In some implementations, RL has formula Ib. In some implementations, RLL has formula Ib'.

[0287] RL1 and RL2 can be independently selected from H and methyl, or together with the carbon atoms to which they are bonded, to form cyclopropene or cyclobutene groups.

[0288] In some embodiments, both RL1 and RL2 are H. In some embodiments, RL1 is H and RL2 is methyl. In some embodiments, both RL1 and RL2 are methyl.

[0289] In some embodiments, RL1 and RL2, together with the carbon atoms they are bonded to, form a cyclopropylene group. In some embodiments, RL1 and RL2, together with the carbon atoms they are bonded to, form a cyclobutene group.

[0290] In group Ib, in some embodiments, e is 0. In other embodiments, e is 1 and the nitro group can be at any available position on the ring. In some of these embodiments, it is located at the ortho position. In others of these embodiments, it is located at the para position.

[0291] In some embodiments in which the compounds described herein are provided as a single mirror-image isomer or as an enrichment of mirror-image isomers, the enriched form has a mirror-image isomer ratio greater than 60:40, 70:30, 80:20, or 90:10. In other embodiments, the mirror-image isomer ratio is greater than 95:5, 97:3, or 99:1.

[0292] In some implementations, RL is selected from:

[0293]

[0294] In some embodiments, RLL is a group derived from the aforementioned RL group.

[0295] Having outlined the above preferences, we will now describe some preferred topoisomerase I-linkers (e.g., drug linker units).

[0296] In some implementations, having the formula The compound [I] has the formula [I] [P]: and its salts and solvates, wherein RLP is a linker for linking to the antibody or its antigen-binding fragment described herein, wherein the linker is selected from: (ia): Among them, QP series: QXP, where QP is an amino acid residue, dipeptide residue, or tripeptide residue; XP system: Where aP = 0 to 5, bP = 0 to 16, cP = 0 or 1, dP = 0 to 5; GL is a linker used to connect to the antibody or its antigen-binding fragment (e.g., ligand unit) described herein; (ib): RL1 and RL2 are independently selected from H and methyl, or together with the carbon atoms they are bonded to form cyclopropene or cyclobutene groups; and the e-series is 0 or 1.

[0297] aP can be 0, 1, 2, 3, 4, or 5. In some embodiments, aP is 0 to 3. In some of these embodiments, aP is 0 or 1. In other embodiments, aP is 0.

[0298] bP can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b refers to 0 to 12. In some of these embodiments, bP refers to 0 to 8, and can be 0, 2, 4, or 8.

[0299] cP can be 0 or 1.

[0300] dP can be 0, 1, 2, 3, 4, or 5. In some embodiments, dP is 0 to 3. In some of these embodiments, dP is 1 or 2. In other embodiments, dP is 2.

[0301] In some implementations of XP, aP is 0, cP is 1, and dP is 2, and bP can be from 0 to 8. In some of these implementations, bP is 0, 4, or 8.

[0302] The above applies to formulas The preference for QX of compounds [I] can be applied to QXP (e.g., where appropriate).

[0303] The above applies to formulas The preference for GL, RL1, RL2, and e of compounds [I] can be applied to compounds with formula [I]. [I] [P] compounds.

[0304] In some implementations, having the formula [IV] The conjugate has the formula [IV] [P]: L-(DLP)p ( [IV] [P])

[0305] Or a pharmaceutically acceptable salt or solvation thereof, wherein L is an antibody or its antigen-binding fragment (e.g., a ligand unit) described herein, DLP is a topoisomerase I inhibitor (e.g., a drug linker unit) and has the formula IIIP: RLLP is a linker connected to the antibody or its antigen-binding fragment (e.g., a ligand unit), wherein the linker is selected from (ia'): Wherein QP and XP are as defined above, and GLL is a linker connected to the antibody or its antigen-binding fragment (e.g., ligand unit) described herein; and (ib'): RL1 and RL2 are as defined above; and p is an integer from 1 to 20.

[0306] In some implementations, having the formula The compound [I] has the formula [I] [P2]: and its salts and solvates, wherein RLP2 is a linker for linking to the antibody or its antigen-binding fragment described herein, wherein the linker is selected from: (ia): Among them, the Q series: Where QX makes Q an amino acid residue, dipeptide residue, tripeptide residue, or tetrapeptide residue; XP2 system: Where aP2 = 0 to 5, b1P2 = 0 to 16, b2P2 = 0 to 16, cP2 = 0 or 1, dP2 = 0 to 5, wherein at least b1P2 or b2P2 = 0 (i.e., only one of b1 and b2 can be non-0); GL is a linker used to connect to the antibody or its antigen-binding fragment (e.g., ligand unit) described herein; (ib): RL1 and RL2 are independently selected from H and methyl, or together with the carbon atoms they are bonded to form cyclopropene or cyclobutene groups; and the e-series is 0 or 1.

[0307] aP2 can be 0, 1, 2, 3, 4, or 5. In some embodiments, aP2 is 0 to 3. In some of these embodiments, aP2 is 0 or 1. In other embodiments, aP2 is 0.

[0308] b1P2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b1P2 is 0 to 12. In some of these embodiments, b1P2 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8.

[0309] b2P2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b2P2 is 0 to 12. In some of these embodiments, b2P2 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8.

[0310] Ideally, only one of b1P2 and b2P2 can be non-zero.

[0311] cP2 can be 0 or 1.

[0312] dP2 can be 0, 1, 2, 3, 4, or 5. In some embodiments, dP2 is 0 to 3. In some of these embodiments, dP2 is 1 or 2. In other embodiments, dP2 is 2. In still other embodiments, dP2 is 5.

[0313] In some embodiments of XP2, aP2 is 0, b1P2 is 0, cP2 is 1, and dP2 is 2, and b2P2 can be from 0 to 8. In some of these embodiments, b2P2 is 0, 2, 3, 4, 5, or 8. In some embodiments of XP2, aP2 is 1, b2P2 is 0, cP2 is 0, and dP2 is 0, and b1P2 can be from 0 to 8. In some of these embodiments, b1P2 is 0, 2, 3, 4, 5, or 8. In some embodiments of XP2, aP2 is 0, b1P2 is 0, cP2 is 0, and dP2 is 1, and b2P2 can be from 0 to 8. In some of these embodiments, b2P2 is 0, 2, 3, 4, 5, or 8. In some embodiments of XP2, b1P2 is 0, b2P2 is 0, cP2 is 0, and one of aP2 and dP2 is 0. Another series in aP2 and d is from 1 to 5. In some of these embodiments, another series in aP2 and d is 1. In others of these embodiments, another series in aP2 and dP2 is 5.

[0314] The above applies to formulas The preference for QX of the compound [I] can be applied to QX in formula IaP2 (e.g., where appropriate).

[0315] The above applies to formulas The preference for GL, RL1, RL2, and e of compounds [I] can be applied to compounds with formula [I]. [I] [P2] compounds.

[0316] In some implementations, having the formula [IV] The conjugate has the formula [IV] [P2]:L-(DLP2)p ( [IV] [P2])

[0317] Or a pharmaceutically acceptable salt or solvation thereof, wherein L is the antibody described herein or its antigen-binding fragment (e.g., ligand unit), DLP2 is a topoisomerase I inhibitor (e.g., drug linker unit) and has the formula IIIP2: RLLP2 is a linker connected to the antibody or its antigen-binding fragment (e.g., a ligand unit), wherein the linker is selected from (ia'): Where Q and XP2 are as defined above, and GLL is a linker connected to the antibody or its antigen-binding fragment; and (ib'): RL1 and RL2 are as defined above; and p is an integer from 1 to 20.

[0318] Particularly suitable topoisomerase I inhibitors include those having the following formula: ; and / or

[0319] The SG3932 series is particularly preferred. Therefore, in a preferred embodiment, the one described herein...

[0320] Antibodies or their antigen-binding fragments are conjugated to topoisomerase I inhibitors having the following formula (e.g., SG3932):

[0321] To avoid ambiguity, the number "8" specifies that the structure within the square brackets is repeated eight times. Therefore, another representation of SG3932 is:

[0322] Another representation of SG4010:

[0323] Another representation of SG4057:

[0324] Another representation of SG4052:

[0325] Any antibody or its antigen-binding fragment described herein may be conjugated to one or more of the topoisomerase I inhibitors.

[0326] In a preferred aspect, an antibody or antigen-binding fragment thereof is provided that binds to a B7-H4 polypeptide (e.g., a B7-H4 polypeptide epitope), the antibody or antigen-binding fragment comprising: i. HCDR1 containing the amino acid sequence of SEQ ID NO:7, or a functional variant thereof; ii. HCDR2 containing the amino acid sequence of SEQ ID NO:8, or a functional variant thereof; iii. HCDR3 containing the amino acid sequence of SEQ ID NO:9, or a functional variant thereof; iv. LCDR1 containing the amino acid sequence of SEQ ID NO:10, or a functional variant thereof; v. LCDR2 containing the amino acid sequence of SEQ ID NO:11, or a functional variant thereof; and vi. LCDR3 containing the amino acid sequence of SEQ ID NO:12, or a functional variant thereof; wherein the antibody or antigen-binding fragment thereof is conjugated to SG3932.

[0327] Another preferred aspect provides an antibody or antigen-binding fragment thereof comprising: a variable heavy chain containing the amino acid sequence of SEQ ID NO: 33, or a functional variant thereof; and a variable light chain containing the amino acid sequence of SEQ ID NO: 34, or a functional variant thereof; wherein the antibody or antigen-binding fragment thereof is conjugated to SG3932.

[0328] A particularly preferred aspect provides an antibody or antigen-binding fragment thereof comprising: a variable heavy chain containing the amino acid sequence of SEQ ID NO:45, or a functional variant thereof; and a variable light chain containing the amino acid sequence of SEQ ID NO:34, or a functional variant thereof; wherein the antibody or antigen-binding fragment thereof is conjugated to SG3932.

[0329] Another preferred aspect provides an antibody or antigen-binding fragment thereof comprising: a heavy chain containing the amino acid sequence of SEQ ID NO: 51, or a functional variant thereof; and a light chain containing the amino acid sequence of SEQ ID NO: 44, or a functional variant thereof; wherein the antibody or antigen-binding fragment thereof is conjugated to SG3932.

[0330] Synthesis of topoisomerase I inhibitors

[0331] To complete this, some general synthetic routes for the preparation of one or more preferred topoisomerase I inhibitors will now be described. Further details can be found in the Examples section.

[0332] A compound having formula I (where RL has formula Ia) can be derived from a compound having formula 2: (where RL* series -QH) is connected by compounds having formula 3: Or its activated version for synthesis.

[0333] Such a reaction can be carried out under amide coupling conditions.

[0334] A compound having formula 2 can be derived from a compound having formula 4: (where RL*prot is a -Q-ProtN group, and ProtN is an amine protecting group) is deprotected to synthesize it.

[0335] Compounds having formula 4 can be derived from compounds having formula 5: The coupling with compound A3 was synthesized using the Friedlander reaction.

[0336] Compounds having formula 5 can be derived from compounds having formula 6: It is synthesized by removing the protecting group of trifluoroacetamide.

[0337] Compounds having Formula 6 can be synthesized by the following coupling: RL*prot-OH to compound I7.

[0338] Compounds having formula I (where RL has formula Ia or Ib) can be synthesized from compound I11 by coupling with compound RL-OH or its activated form.

[0339] Amine protecting group:

[0340] The amine protecting group is well known to those skilled in the art. In particular, refer to the disclosure of suitable protecting groups in the following literature: Greene's Protecting Groups in Organic Synthesis, 4th Edition, John Wiley & Sons, 2007 (ISBN 978-0-471-69754-1), pp. 696-871.

[0341] Other ADCs

[0342] While topoisomerase I inhibitors are preferred as described above, it should be noted that any suitable agent (e.g., drug / cytotoxin) can be linked to the antibody or its antigen-binding fragment of the present invention. Examples of other suitable agents are summarized below.

[0343] In one embodiment, the cytotoxin is tubulolysin or a tubulolysin derivative. In one embodiment, the cytotoxin is tubulolysin A, which has the following chemical structure:

[0344] Tubulolysins are members of a class of natural products isolated from myxobacterial species. As cytoskeleton interactors, tubulolysins are mitotic toxins that inhibit tubulin polymerization and lead to cell cycle arrest and apoptosis. As used herein, the term "tubulolysin" refers both commonly and individually to naturally occurring tubulolysins and their analogues and derivatives. Exemplary examples of tubolytics are disclosed, for example, in WO 2004005326 A2, WO 2012019123 A1, WO 2009134279 A1, WO 2009055562 A1, WO 2004005327 A1, US 7776841, US 7754885, US 20100240701, US 7816377, US 20110021568 and US 20110263650, which are incorporated herein by reference. It will be understood that such derivatives include, for example, tubolytic precursors or tubolytics containing one or more protecting groups and one or more linking portions.

[0345] In one embodiment, the cytotoxin is tubulolysin 1508, also referred to herein as "AZ1508" and described in more detail in WO 2015157594 (which is incorporated herein by reference), and has the following structure:

[0346] In another embodiment, the cytotoxin may be pyrrolobenzodiazepine (PBD) or a PBD derivative. PBD is transported to the nucleus where its cross-linked DNA is located, preventing replication during mitosis, damaging the DNA by inducing single-strand breaks, and subsequently leading to apoptosis. Some PBDs have the ability to recognize and bind to specific DNA sequences; the preferred sequence is PuGPu. PBDs have the following general structure:

[0347] PBDs differ in the number, type, and position of substituents, in both their aromatic A ring and pyrrolo C ring, and in the saturation of the C ring. In the B ring, an imine (N=C), ethanolamine (NH-CH(OH)), or ethanolamine methyl ether (NH-CH(OMe)) is present at the N10-C11 position, which is the electrophilic center responsible for alkylating DNA. All known natural products have a (S)-configuration at the chiral C11a position, which provides a right-handed twist when viewed from the C ring to the A ring. This provides them with a suitable three-dimensional shape to have isohelicity with the minor groove of B-type DNA, thus allowing for tight binding at the binding site. Their ability to form adducts in the minor groove enables them to interfere with DNA processing, thus allowing them to be used as antitumor agents.

[0348] The first PBD (proton pump inhibitor) antitumor antibiotic, anthramycin, was discovered in 1965. Since then, many naturally occurring PBDs have been reported, and more than 10 synthetic routes have been developed for various analogues. Family members include abbeymycin, chicamycin, DC-81, mazethramycin, neothramycin A and B, porothramycin, prothracarcin, sibanomicin (DC-102), siberiamycin, and tomathamycin. PBDs and ADCs containing these compounds are also described in WO 2015 / 155345 and WO 2015 / 157592, which are incorporated herein by reference in their entirety.

[0349] In one embodiment, the cytotoxin is PBD 3249, also referred to herein as "SG3249" and described in more detail in WO 2014 / 057074 (which is incorporated herein by reference), and has the following structure:

[0350] Therefore, the antibody or its antigen-binding fragment is conjugated to pyrrolobenzodiazepine SG3249 cytotoxin.

[0351] For example, in one aspect, an antibody or antigen-binding fragment thereof is provided that binds to a B7-H4 polypeptide (e.g., a B7-H4 polypeptide epitope), the antibody or antigen-binding fragment comprising: i. HCDR1 containing the amino acid sequence of SEQ ID NO:7, or a functional variant thereof; ii. HCDR2 containing the amino acid sequence of SEQ ID NO:8, or a functional variant thereof; iii. HCDR3 containing the amino acid sequence of SEQ ID NO:9, or a functional variant thereof; iv. LCDR1 containing the amino acid sequence of SEQ ID NO:10, or a functional variant thereof; v. LCDR2 containing the amino acid sequence of SEQ ID NO:11, or a functional variant thereof; and vi. LCDR3 containing the amino acid sequence of SEQ ID NO:12, or a functional variant thereof; wherein the antibody or antigen-binding fragment thereof is conjugated to pyrrolobenzodiazepine SG3249 cytotoxin.

[0352] On the other hand, an antibody or antigen-binding fragment thereof is provided that binds to a B7-H4 polypeptide (e.g., a B7-H4 polypeptide epitope), the antibody or antigen-binding fragment comprising: a variable heavy chain containing the amino acid sequence of SEQ ID NO: 33, or a functional variant thereof; and a variable light chain containing the amino acid sequence of SEQ ID NO: 34, or a functional variant thereof; wherein the antibody or antigen-binding fragment thereof is conjugated to pyrrolobenzodiazepine SG3249 cytotoxin.

[0353] On the other hand, an antibody or antigen-binding fragment thereof is provided that binds to a B7-H4 polypeptide (e.g., a B7-H4 polypeptide epitope), the antibody or antigen-binding fragment comprising: a variable heavy chain containing the amino acid sequence of SEQ ID NO: 45, or a functional variant thereof; and a variable light chain containing the amino acid sequence of SEQ ID NO: 34, or a functional variant thereof; wherein the antibody or antigen-binding fragment thereof is conjugated to pyrrolobenzodiazepine SG3249 cytotoxin.

[0354] In one embodiment, the cytotoxin is PBD 3315, also referred to herein as "SG3315" and described in more detail in WO 2015 / 052322 (which is incorporated herein by reference), and has the following structure:

[0355] The antibody or its antigen-binding fragment is preferably conjugated to pyrrolobenzodiazepine SG3249 cytotoxin via cysteine ​​residues.

[0356] The antibodies or antigen fragments thereof of the present invention can be conjugated to heterologous agents (preferably cytotoxins) using site-specific or non-site-specific conjugation methods. In one embodiment, the antibodies and antigen fragments thereof comprise one, two, three, four or more therapeutic portions. In one embodiment, all therapeutic portions are identical.

[0357] Conventional conjugation strategies for antibodies or their antigen-binding fragments rely on randomly conjugating the payload to the antibody or fragment via lysine or cysteine. In one embodiment, the antibody or its antigen-binding fragment is randomly conjugated to a heterologous agent (preferably a cytotoxic agent), for example, by partially reducing the antibody or fragment and then reacting it with the desired agent, wherein the linker portion is attached or not attached. The antibody or fragment can be reduced using DTT or a similar reducing agent. The agent, with or without the linker portion attached, can then be added in molar excess to the reduced antibody or fragment in the presence of DMSO. After conjugation, excess free cysteine ​​can be added to quench any unreacted agent. The reaction mixture can then be purified, and the buffer replaced with PBS.

[0358] In one embodiment, the agent (e.g., a cytotoxin) is conjugated to an antibody or its antigen-binding fragment by site-specific conjugation. In another embodiment, site-specific conjugation of the therapeutic portion with the antibody using reactive amino acid residues at a specific location produces a homogeneous ADC formulation with consistent stoichiometry.

[0359] This site-specific conjugation can be achieved via cysteine, a residue, or a non-natural amino acid. In a preferred embodiment, the heterologous agent (preferably a cytotoxic agent) is conjugated to the antibody or its antigen-binding fragment via at least one cysteine ​​residue.

[0360] In one embodiment, the heterologous agent (preferably a cytotoxin) is chemically conjugated to the side chain of an amino acid (preferably at a specific Kabat position in the Fc region). In one embodiment, the agent (e.g., a cytotoxic agent or imaging agent) is conjugated to the antibody or its antigen-binding fragment by a cysteine ​​substitution at at least one of positions 239, 248, 254, 273, 279, 282, 284, 286, 287, 289, 297, 298, 312, 324, 326, 330, 335, 337, 339, 350, 355, 356, 359, 360, 361, 375, 383, 384, 389, 398, 400, 413, 415, 418, 422, 440, 441, 442, 443, and 446, where the number corresponds to the EU index in the Kabat. In one embodiment, the specific Kabat position is 239, 442, or both. In one embodiment, the specific position is an amino acid insertion between Kabat positions 442, 239, and 240, or both. In one embodiment, the heterologous agent (preferably a cytotoxin) is conjugated to the antibody or its antigen-binding fragment via a thiol-maleimine bond. In some aspects, the amino acid side chain is a thiol side chain.

[0361] In one embodiment, the antibody or its antigen-binding fragment comprises a light chain containing the amino acid sequence of SEQ ID NO:44 (e.g., preferably containing VL and a constant light chain) and a heavy chain containing the amino acid sequence of SEQ ID NO:48 (e.g., containing VH and a constant heavy chain); wherein the antibody or its antigen-binding fragment is conjugated to pyrrolobenzodiazepine SG3249 cytotoxin; preferably, wherein the pyrrolobenzodiazepine SG3249 cytotoxin is conjugated to a cysteine ​​residue at amino acid position 240 of the heavy chain.

[0362] In one embodiment, the antibody or its antigen-binding fragment comprises a light chain containing the amino acid sequence of SEQ ID NO:44 (e.g., preferably containing VL and a constant light chain) and a heavy chain containing the amino acid sequence of SEQ ID NO:51 (e.g., containing VH and a constant heavy chain); wherein the antibody or its antigen-binding fragment is conjugated to pyrrolobenzodiazepine SG3249 cytotoxicity.

[0363] The antibody or antigen-binding fragment conjugated to cytotoxic agents mentioned in this article are synonymous with the terms "antibody-drug conjugate (ADC)" or "anti-B7-H4 ADC".

[0364] In one embodiment, the antibody or its antigen-binding fragment (e.g., anti-B7-H4 ADC) delivers a cytotoxic payload to cells (preferably cells expressing B7-H4) and inhibits or suppresses proliferation (e.g., tumor proliferation) by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or about 100% (preferably at least 40%), relative to the level of inhibition or suppression in the absence of the antibody or its antigen-binding fragment (e.g., anti-B7-H4 ADC). Cell proliferation can be determined using techniques recognized in the art that measure the rate of cell division, and / or the fraction of cells undergoing cell division in the cell population, and / or the rate of cell loss from the cell population due to terminal differentiation or cell death (e.g., thymidine incorporation).

[0365] In one embodiment, the antibody or antigen fragment of the present invention (e.g., anti-B7-H4 ADC) binds to B7-H4 on the cell surface and is internalized into the cell. In one embodiment, the antigen or antibody fragment is internalized into cells (preferably cells expressing B7-H4) at the following IC50 values ​​at 10 minutes: about 100 ng / ml to about 1 μg / ml, about 100 ng / ml to about 500 ng / ml, about 100 ng / ml to about 250 ng / ml, about 250 ng / ml to about 500 ng / ml, about 350 ng / ml to about 450 ng / ml, about 500 ng / ml to about 1 μg / ml, about 500 ng / ml to about 750 ng / ml, about 750 ng / ml to about 850 ng / ml, or about 900 ng / ml to about 1 μg / ml.

[0366] In one embodiment, the antibody or its antigen fragment (e.g., anti-B7-H4 ADC) is internalized into cells (preferably cells expressing B7-H4) at the following IC50 at 30 minutes: about 100 ng / ml to about 1 μg / ml, about 100 ng / ml to about 500 ng / ml, about 100 ng / ml to about 250 ng / ml, about 250 ng / ml to about 500 ng / ml, about 250 ng / ml to about 350 ng / ml, about 350 ng / ml to about 450 ng / ml, about 500 ng / ml to about 1 μg / ml, about 500 ng / ml to about 750 ng / ml, about 750 ng / ml to about 850 ng / ml, or about 900 ng / ml to about 1 μg / ml.

[0367] In one embodiment, the antibody or its antigen fragment (e.g., anti-B7-H4 ADC) is internalized into cells (preferably cells expressing B7-H4) at the following IC50 at 120 minutes: about 50 ng / ml to about 500 ng / ml, about 50 ng / ml to about 100 ng / ml, about 100 ng / ml to about 200 ng / ml, about 200 ng / ml to about 300 ng / ml, about 300 ng / ml to about 400 ng / ml, or about 400 ng / ml to about 500 ng / ml.

[0368] In some embodiments, the antibody or its antigen fragment (e.g., anti-B7-H4 ADC) is internalized into cells (preferably cells expressing B7-H4) at the following IC50 at 8 hours: about 5 ng / ml to about 250 ng / ml, about 10 ng / ml to about 25 ng / ml, about 25 ng / ml to about 50 ng / ml, about 50 ng / ml to about 100 ng / ml, about 100 ng / ml to about 150 ng / ml, about 150 ng / ml to about 200 ng / ml, or about 200 ng / ml to about 250 ng / ml.

[0369] To avoid ambiguity, the term "conjugate" as used herein refers to an antibody or antigen-binding fragment conjugated to a heterologous drug (including any such drug described above, preferably a cytotoxin).

[0370] In addition to the therapeutic applications of the antibodies or antigen-binding fragments of the present invention described above, the "conjugates" of the present invention can also be used in treatment methods. Therefore, treatment methods are also provided that involve administering a therapeutically effective amount of the conjugates described herein (e.g., conjugates having formula IV) to a subject requiring treatment. The term "therapeutically effective amount" refers to an amount sufficient to demonstrate benefit to the patient. This benefit may be the relief of at least one symptom. The actual amount administered, as well as the rate and duration of administration, will depend on the nature and severity of the disease being treated. Treatment prescription (e.g., dosage determination) falls under the responsibility of general practitioners and other physicians.

[0371] Depending on the condition being treated, the conjugate may be administered alone or in combination with other treatments, simultaneously or sequentially. Examples of treatments and therapies include, but are not limited to, chemotherapy (including, for example, the administration of active agents of drugs); surgery; and radiation therapy.

[0372] In addition to the active ingredient (i.e., the conjugate / ADC of the present invention, e.g., formula IV)), the pharmaceutical composition according to the present invention may also contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other materials will depend on the route of administration, which may be oral or by injection, such as through the skin, subcutaneous, or intravenous injection.

[0373] Pharmaceutical compositions intended for oral administration may be in tablet, capsule, powder, or liquid form. Tablets may contain a solid carrier or excipient. Liquid pharmaceutical compositions typically contain a liquid carrier such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. They may include physiological saline solutions, dextran or other sugar solutions, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol. Capsules may contain a solid carrier, such as gelatin.

[0374] For intravenous, subcutaneous, or subcutaneous injection, or injection at the site of pain, the active ingredient will be in a parenteral acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art can readily prepare suitable solutions using isotonic media such as sodium chloride injection, Ringer's solution, or lactate Ringer's solution. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included if necessary.

[0375] Preferably, the conjugate can be used to treat proliferative disorders. The term "proliferative disorder" refers to unwanted or uncontrolled cell proliferation of excessive or abnormal cells, whether in vitro or in vivo, which is undesirable, such as vesicular or hyperplastic growth. The term "proliferative disorder" can also be referred to as "cancer".

[0376] For appropriate proliferative diseases (such as cancer), the presence of cancer cells characterized as expressing B7-H4 would be preferable.

[0377] Examples of proliferative disorders include, but are not limited to, benign, precancerous, and malignant cell proliferations, including but not limited to vegetations and tumors (e.g., histiocytoma, glioma, astrocytoma, osteoma), cancers (e.g., lung cancer, small cell lung cancer, gastrointestinal cancer, colon cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemia, psoriasis, bone diseases, fibroproliferative disorders (e.g., connective tissue disorders), and atherosclerosis. Other cancers of concern include, but are not limited to, hematologic malignancies such as leukemia and lymphomas such as non-Hodgkin's lymphoma and its subtypes (e.g., DLBCL, marginal zone lymphoma, mantle lymphoma, and follicular lymphoma), Hodgkin's lymphoma, AML, and other cancers of B or T cell origin. It can treat any type of cell, including but not limited to lung, gastrointestinal (e.g., including intestine, colon), breast, mammary, ovary, prostate, liver, kidney, bladder, pancreas, brain, and skin cells.

[0378] The antibody-drug conjugate can be labeled, for example, to aid in the detection of cell binding (in vitro or in vivo). This labeling could be biotinylation. In another embodiment, the labeling could be a radioactive isotope.

[0379] On the other hand, a polynucleotide is provided that comprises a nucleic acid sequence encoding an antibody of the present invention or an antigen-binding fragment thereof.

[0380] In one embodiment, the polynucleotide may be a separate polynucleotide.

[0381] One or more sequences of the present invention (e.g., one or more polynucleotide sequences) include sequences that have been removed from their natural environment, recombinant or selected (e.g., DNA) isolates, and chemically synthesized analogs or analogs biosynthesized by heterologous systems.

[0382] One or more sequences of the present invention (e.g., one or more polynucleotide sequences) can be prepared by any method known in the art. For example, large quantities of the one or more sequences can be generated by replication and / or expression in suitable host cells. Natural or synthetic DNA fragments encoding the desired segment are typically incorporated into recombinant nucleic acid constructs, which are usually DNA constructs and capable of being introduced into and replicated in prokaryotic or eukaryotic cells. Typically, the DNA constructs will be adapted to replicate autonomously in single-celled hosts (such as yeast or bacteria), but can also be used to introduce into and integrate into the genome of cultured bacteria, insects, mammals, plants, or other eukaryotic cell lines.

[0383] One or more sequences of the present invention (e.g., one or more polynucleotide sequences) can also be produced by chemical synthesis (e.g., by phosphoramidite or triester synthesis of polynucleotides) and can be performed on a commercially available automated oligonucleotide synthesizer. Double-stranded (e.g., DNA) fragments can be obtained from chemically synthesized single-stranded products, which are performed by synthesizing complementary strands and annealing the strands together under appropriate conditions or by adding complementary strands using a DNA polymerase with appropriate primer sequences.

[0384] When applied to sequences (e.g., polynucleotide sequences) of the present invention, the term "isolated" preferably means that the sequence has been removed from its natural genetic environment and therefore does not contain other foreign or unwanted coding sequences (but may include naturally present 5' and 3' untranslated regions, such as promoters and terminators), and is in a form suitable for use in genetically engineered protein production systems. Such isolated molecules are those isolated from their natural environment.

[0385] Another aspect provided herein is a host cell containing a polynucleotide, said polynucleotide containing a nucleic acid sequence encoding an antibody of the present invention or an antigen-binding fragment thereof.

[0386] In one embodiment, the polynucleotide encodes the VH chain of an antibody or an antigen-binding fragment thereof. In one embodiment, the polynucleotide of the present invention may encode the VL chain of an antibody or an antigen-binding fragment thereof. In one embodiment, the polynucleotide may encode both the VH and VL chains of an antibody or an antigen-binding fragment thereof. In one embodiment, the polynucleotide may further encode a leader sequence (e.g., a secretory sequence that controls the transport of a polypeptide from a cell).

[0387] On the other hand, a carrier (e.g., a plastid) comprising the polynucleotide of the present invention is provided.

[0388] This invention includes variants of the aforementioned polynucleotides. Polynucleotide variants may contain alterations in coding regions, non-coding regions, or both. In one embodiment, a polynucleotide variant comprises alterations that produce silent substitutions, additions, or deletions without changing the properties or activity of the encoded polypeptide. In one embodiment, a polynucleotide variant is produced by silent substitutions attributable to the degeneracy of the genetic code. Polynucleotide variants can be produced for various reasons, such as to optimize codon performance for a particular host (changing codons in human mRNA to those preferred by bacterial hosts such as *E. coli*). Vectors and cells comprising said polynucleotide variants are also provided.

[0389] The present invention includes methods for generating antibodies or antigen-binding fragments thereof that bind to B7-H4 polypeptides (e.g., B7-H4 polypeptide epitopes), the methods comprising expressing polynucleotides in host cells, the polynucleotides comprising nucleic acid sequences encoding antibodies or antigen-binding fragments thereof of the present invention.

[0390] The present invention further includes antibodies or antigen-binding fragments thereof that can be obtained by the method described herein for generating antibodies or antigen-binding fragments thereof that bind to B7-H4 polypeptides (e.g., B7-H4 polypeptide epitopes).

[0391] In a preferred embodiment, a method for generating an antibody or an antigen-binding fragment thereof includes (a) culturing a host cell and (b) isolating the expressed antibody or antigen-binding fragment thereof from the cell.

[0392] Suitable host cells for expressing the antibodies or antigen-binding fragments of the present invention include prokaryotic cells, yeast cells, insect cells, or higher eukaryotic cells (preferably wherein the polynucleotide is under the control of a suitable promoter). Prokaryotic cells include Gram-negative or Gram-positive organisms, such as *Escherichia coli* or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin as described herein. Cell-free translation systems may also be used.

[0393] In one aspect, a kit comprising the antigen or antibody binding fragment described herein is provided. Further, the use of said kit in the method of the present invention is included.

[0394] In one embodiment, the kit contains an isolated (e.g., purified) antigen or antibody binding fragment of the present invention. In another embodiment, the kit contains an isolated (e.g., purified) antigen or antibody binding fragment of the present invention, wherein the antigen or antibody binding fragment contains a pharmaceutical agent (e.g., a conjugated cytotoxic agent) as described herein. In one embodiment, the kit contains one or more containers. The kit may provide the antigen or antibody binding fragment and the pharmaceutical agent separately (e.g., the agent is not conjugated to the antigen or antibody binding fragment but is in a form suitable for conjugation thereto); if desired, the kit may further provide instructions and / or reagents for conjugating the pharmaceutical agent to the antigen or antibody binding fragment. In one embodiment, the kit contains all the necessary and / or sufficient elements for performing the assay, including all controls, instructions for performing the assay, and any necessary software for analyzing and presenting the results.

[0395] The antibodies or antigen-binding fragments of the present invention can be used in assays of immune-specific binding by any method known in the art. Applicable immunoassays include, but are not limited to, competitive and non-competitive assay systems using techniques such as: Western blotting, RIA, ELISA, ELISPOT, sandwich immunoassay, immunoprecipitation assay, precipitin reaction, gel diffusion precipitin reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradioassay, fluorescence immunoassay, and protein A immunoassay.

[0396] The antibodies of this invention or their antigen-binding fragments can be used histologically, such as in immunofluorescence, immunoelectron microscopy, or non-immunologic assays, for example, for in situ detection of B7-H4 or its conserved variants or peptide fragments. In situ detection can be achieved by taking a histological sample from a patient and applying a labeled antibody of this invention or its antigen-binding fragment to it, for example, by coating the biological sample with the labeled antibody or its antigen-binding fragment. By using this procedure, not only the presence of B7-H4 or its conserved variants or peptide fragments can be determined, but also their distribution in the examined tissue. Using this invention, those skilled in the art will readily recognize that any of a variety of histological methods (such as staining procedures) can be modified to achieve this in situ detection.

[0397] Antibody

[0398] The term "antibody" encompasses monoclonal antibodies and fragments thereof (e.g., fragments exhibiting desired biological activity). In a preferred embodiment, the anti-antibody system of the present invention is a monoclonal antibody. In a more preferred embodiment, the anti-antibody system is a fully human monoclonal antibody. In one embodiment, the method of the present invention may use multiple antibodies.

[0399] Specifically, the anti-system comprises a protein containing at least one or two heavy (H) chain variable regions (VHC) and at least one or two light (L) chain variable regions (VLC). The VHC and VLC regions can be further subdivided into hypervariable regions called complementarity-determining regions ("CDRs"), interspersed with more conserved regions called framework regions (FRs). The extent of the framework regions and CDRs has been precisely defined (see, Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991; and Chothia, C. et al., J. Mol. Biol. 196:901-917, 1987). Preferably, each VHC and VLC consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, DR2, FR3, CDR3, FR4. The VHC or VLC chain of the antibody may further include all or part of the heavy chain or light chain constant regions. In one embodiment, the antibody system is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are interconnected by, for example, disulfide bonds. The heavy chain constant region includes three domains, namely CH1, CH2, and CH3. The light chain constant region consists of one domain CL. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The term "antibody" includes complete immunoglobulins of types IgA, IgG, IgE, IgD, IgM (and their subtypes), wherein the light chain of the immunoglobulin may be of type κ or λ. As used herein, the term antibody also refers to a portion of an antibody that binds to one of the aforementioned markers, for example, a molecule in which one or more immunoglobulin chains are not full-length but bind to the marker. Examples of the “binding portion” encompassed in the term antibody include (i) Fab fragments: monovalent fragments consisting of VLC, VHC, CL, and CH1 domains; (ii) F(ab')2 fragments: bivalent fragments comprising two Fab fragments linked by disulfide bridges in the hinge region; (iii) Fc fragments consisting of VHC and CH1 domains; (iv) Fv fragments consisting of VLC and VHC domains of an antibody single arm; (v) dAb fragments consisting of VHC domains (Ward et al., Nature 341:544-546, 1989); and (vi) separate complementarity-determining regions (CDRs) having a framework sufficient for binding to occur, such as antigen-binding portions of variable regions.The antigen-binding portions of the light chain variable region and the heavy chain variable region (e.g., the two domains VLC and VHC of the Fv fragment) can be linked using a recombinant approach via a synthetic linker that allows them to become a single protein chain, where the VLC and VHC regions pair to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 1A1-ATi-Alβ; and Huston et al. (1988) Proc. Proc. Natl. Acad. Sc. USA 85: 5879-5883). Such single-chain antibodies are also encompassed within the term antibody. These portions can be obtained using conventional techniques known to those skilled in the art, and are screened for practicality in the same manner as intact antibodies.

[0400] In one embodiment, the antibody or antigen-binding fragment is selected from one or more of the following: mouse antibody, humanized antibody, chimeric antibody, monoclonal antibody, polyclonal antibody, recombinant antibody, multispecific antibody, or a combination thereof.

[0401] In one embodiment, the antigen-binding fragment is selected from one or more of the following: Fv fragment, Fab fragment, F(ab')2 fragment, Fab' fragment, dsFv fragment, scFv fragment, sc(Fv)2 fragment, or a combination thereof.

[0402] In a preferred embodiment, the antibody or its antigen-binding fragment is a monoclonal antibody (mAb).

[0403] In one embodiment, the antibody or its antigen-binding fragment (e.g., mAb) of the present invention is scFV.

[0404] In one embodiment, the antibody or its antigen-binding fragment can bind to B7-H4 molecules across species; for example, the antibody or fragment can bind to mouse B7-H4, rat B7-H4, rabbit, human B7-H4, and / or cynomolgus monkey B7-H4. In one embodiment, the antibody or fragment can bind to both human B7-H4 and cynomolgus monkey B7-H4. In one embodiment, the antibody or antigen-binding fragment can also bind to mouse B7-H4.

[0405] In one embodiment, the antibody or its antigen-binding fragment may specifically bind to B7-H4, such as human B7-H4 and cynomolgus monkey B7-H4, but not specifically bind to human B7-H1, B7-H2 and / or B7-H3.

[0406] In one embodiment, the antibody or its antigen-binding fragment may include a heavy chain constant region or a fragment thereof in addition to VH and VL. In one embodiment, the heavy chain constant region is a human heavy chain constant region, such as the human IgG constant region, such as the human IgG1 constant region. In one embodiment (particularly in the case where the antibody or its antigen-binding fragment is conjugated to an agent (such as a cytotoxic agent), a cysteine ​​residue is inserted between amino acids S239 and V240 in the CH2 region of IgG1. This cysteine ​​is referred to as "239 insertion" or "239i".

[0407] In one embodiment, the antibody or its antigen-binding fragment may include a heavy chain constant region containing the amino acid sequence of SEQ ID NO:41. Preferably, the antibody or its antigen-binding fragment may include a heavy chain constant region containing the amino acid sequence of SEQ ID NO:52.

[0408] In one embodiment, the heavy chain constant region or a fragment thereof (e.g., the human IgG constant region or a fragment thereof) may include one or more amino acid substitutions relative to the wild-type IgG constant domain, wherein the modified IgG has an increased half-life compared to the half-life of IgG having the wild-type IgG constant domain. For example, the IgG constant domain may contain one or more amino acid substitutions at positions 251-257, 285-290, 308-314, 385-389, and 428-436, wherein the amino acid positions are numbered according to the EU index as illustrated in Kabat. In one embodiment, the IgG constant domain may contain one or more of the following substitutions: the amino acid at Kabat position 252 is substituted with tyrosine (Y), phenylalanine (F), tryptophan (W), or threonine (T); the amino acid at Kabat position 254 is substituted with threonine (T); the amino acid at Kabat position 256 is substituted with serine (S), arginine (R), glutamic acid (Q), glutamic acid (E), aspartic acid (D), or threonine (T); the amino acid at Kabat position 257 is substituted with leucine (L); and the amino acid at Kabat position 309... The amino acid is replaced by proline (P), the amino acid at position 311 of Kabat is replaced by serine (S), the amino acid at position 428 of Kabat is replaced by threonine (T), leucine (L), phenylalanine (F), or serine (S), the amino acid at position 433 of Kabat is replaced by arginine (R), serine (S), isoleucine (I), proline (P), or glutamic acid (Q), or the amino acid at position 434 of Kabat is replaced by tryptophan (W), methionine (M), serine (S), histidine (H), phenylalanine (F), or tyrosine. In a preferred embodiment, the IgG constant domain may contain amino acid substitutions relative to the wild-type human IgG constant domain, including substitution of the amino acid at Kabat position 252 with tyrosine (Y), substitution of the amino acid at Kabat position 254 with threonine (T), and substitution of the amino acid at Kabat position 256 with glutamic acid (E). In one embodiment, the antibody or its antigen-binding fragment comprises a heavy chain, wherein the heavy chain is a human IgG1 YTE mutant.

[0409] In one embodiment, the antibody or its antigen-binding fragment may, in addition to VH and VL, include, as needed, a heavy chain constant region or a fragment thereof, and a light chain constant region or a fragment thereof. In one embodiment, the light chain constant region is a κ-λ light chain constant region, for example, a human κ constant region or a human λ constant region.

[0410] In one embodiment, the antibody or its antigen-binding fragment comprises a light chain constant region containing the amino acid sequence of SEQ ID NO:42.

[0411] In one embodiment, the VH and / or VL amino acid sequences may have 85%, 90%, 95%, 96%, 97%, 98%, or 99% similarity to the sequences described herein. In one embodiment, the VH and / or VL amino acid sequences may contain 1, 2, 3, 4, 5, or more substitutions, such as conserved substitutions, relative to the sequences described herein. B7-H4 antibodies containing VH and VL regions with a certain percentage similarity to the VH or VL regions or with one or more substitutions (e.g., conserved substitutions) can be obtained by mutagenesis (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) of nucleic acid molecules encoding the VH and / or VL regions described herein, followed by testing the binding of the encoded modified antibody to B7-H4, and, if necessary, testing the retained function using the functional assays described herein.

[0412] The affinity or cohesion of an antibody or its antigen-binding fragment to an antigen can be determined experimentally using any suitable method well known in the art (e.g., flow cytometry, enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), or kinetic assay (e.g., KINEXA® or BIACORE™ assay)). Direct binding assays and competitive binding assays can be readily employed. (See, for example, Berzofsky et al., Antibody-Antigen Interactions, in Fundamental Immunology, edited by Paul, WE, Raven Press: New York, NY (1984); Kuby, Immunology, WH Freeman and Company Publishers: New York, NY (1992); and the methods described herein.) The measured affinity of a particular antibody-antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH, temperature). Therefore, affinity and other antigen binding parameters (e.g., KD or Kd, Kon, Koff) are measured using standardized solutions and standardized buffers of antibodies and antigens as known in the art.

[0413] In one embodiment, the antibody or its antigen-binding fragment can bind to cells expressing B7-H4 at the following IC50 values: below about 500 nM, below about 350 nM, below about 250 nM, below about 150 nM, below about 100 nM, below about 75 nM, below about 60 nM, below about 50 nM, below about 40 nM, below about 30 nM, below about 20 nM, below about 15 nM, below about 10 nM, below about 5 nM, below about 1 nM, below about 500 pM, below about 350 pM, below about 250 pM, below about 150 pM, below about 100 pM, below about 75 pM, below about 60 pM, below about 50 pM, below about 40 pM, below about 30 pM, below about 20 pM, below about 15 pM, below about 10 pM, or below about 5 pM. Preferably, the IC50 is measured by flow cytometry.

[0414] "Monoclonal antibody" (mAb) refers to a group of homologous antibodies that involve the highly specific recognition and binding of a single epitope or epitope. This contrasts with polyclonal antibodies, which typically include different antibodies targeting different epitopes. The term "monoclonal antibody" encompasses full-length and complete monoclonal antibodies, as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins containing antibody moieties, and any other modified immunoglobulin molecules containing antigen recognition sites. Furthermore, "monoclonal antibody" refers to such antibodies prepared in any number of ways, including but not limited to fusion tumors, phage selection, recombinant expression, and transgenic animals.

[0415] In a preferred embodiment, the antibody or antigen-binding fragment (e.g., mAb) of the present invention is a humanized antibody or antigen-binding fragment thereof. Suitably, the humanized antibody or antigen-binding fragment thereof is IgG.

[0416] The term "humanized antibody" refers to an antibody derived from a non-human (e.g., mouse) immunoglobulin, engineered to contain a minimal non-human (e.g., mouse) sequence. Typically, humanized antibodies are derived from human immunoglobulins, where residues from the complementarity-determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, or hamster) with the desired specificity, affinity, and capability (Jone et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536). In some cases, the Fv framework region (FW) residues of human immunoglobulins are replaced by corresponding residues from antibodies derived from non-human species with the desired specificity, affinity, and capability.

[0417] Humanized antibodies can be further modified by substitution of additional residues within the Fv frame region and / or replaced non-human residues to improve and optimize antibody specificity, affinity, and / or potency. Typically, humanized antibodies will contain substantially all at least one (and typically two or three) variable domains containing all or substantially all CDR regions corresponding to non-human immunoglobulins, and all or substantially all FR regions corresponding to the FR regions of the human immunoglobulin congruent sequence. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region or domain (Fc), typically at least a portion of the constant region or domain of human immunoglobulins. Examples of methods for generating humanized antibodies are described in U.S. Patent Nos. 5,225,539 or 5,639,641.

[0418] The "variable region" of an antibody refers to the variable region of a single antibody light chain, the variable region of an antibody heavy chain, or a combination thereof. Each of these variable regions in the heavy and light chains consists of four frame regions (FWs) connected by three complementarity-determining regions (CDRs) (also known as hypervariable regions). The CDRs in each chain are held together closely by the FW regions and, together with CDRs from the other chain, contribute to the formation of the antibody's antigen-binding site. At least two techniques exist for determining CDRs: (1) methods based on interspecies sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed., 1991, National Institutes of Health, Bethesda, MD)); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273: 927-948). Furthermore, a combination of these two methods is sometimes used in the art to determine CDRs.

[0419] When referring to residues in the variable domain (approximately residues 1-107 in the light chain and residues 1-113 in the heavy chain), the "Kabat numbering system" is usually used (e.g., Kabat et al., Sequences of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (1991)).

[0420] The amino acid position numbering in Kabat refers to the numbering system used to compile heavy chain or light chain variable domains of antibodies in Kabat et al., Sequences of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids, corresponding to truncation or insertion of the FW or CDR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (according to residue 52a in Kabat) and inserted residues after residue 82 of the heavy chain FW (e.g., residues 82a, 82b, and 82c, etc., according to Kabat).

[0421] The Kabat number of a given antibody residue can be determined by comparing the antibody sequence with homology regions of the "standard" Kabat numbering sequence. Chothia refers to the location of the structural loop (Chothia and Lesk, J. Mol. Biol. [Journal of Molecular Biology] 196:901-917 (1987)). The end of the Chothia CDR-H1 loop varies between H32 and H34 when numbered using the Kabat numbering convention, depending on the loop length (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if neither 35A nor 35B is present, the loop endpoint is at 32; if only 35A is present, the loop endpoint is at 33; if both 35A and 35B are present, the loop endpoint is at 34). The AbM hypervariable region represents a compromise between the Kabat CDR and the Chothia structural loop and is used by the Oxford Molecular AbM antibody modeling software. The table below lists the positions of the amino acids that make up the antibody variable region in each system.

[0422]

[0423] 1Kabat number

[0424] 2Chothia number

[0425] Immunogenetics (IMGT) also provides a numbering system for the variable regions of immunoglobulins, including CDRs. See, for example, Lefranc, MP et al., Dev. Comp. Immunol. [Developmental and Comparative Immunology] 27:55-77 (2003). The IMGT numbering system is based on alignments, structural data, and characterization of more than 5,000 sequences of hypervariable loops and allows for easy comparison of variable regions and CDRs across all species. According to the IMGT numbering scheme, VH-CDR1 is located at positions 26 to 35, VH-CDR2 at positions 51 to 57, VH-CDR3 at positions 93 to 102, VL-CDR1 at positions 27 to 32, VL-CDR2 at positions 50 to 52, and VL-CDR3 at positions 89 to 97.

[0426] As used throughout this specification, the VH CDR sequence corresponds to the classic Kabat numbering positions, i.e., Kabat VH-CDR1 is at positions 31-35, VH-CDR2 is at positions 50-65, and VH-CDR3 is at positions 95-102. VL-CDR1, VL-CDR2, and VL-CDR3 also correspond to the classic Kabat numbering positions, i.e., positions 24-34, 50-56, and 89-97, respectively.

[0427] In one embodiment, the present invention provides an anti-human antibody system.

[0428] The term "human antibody" means an antibody produced in the human body or an antibody having an amino acid sequence corresponding to an antibody produced in the human body using any technique known in the art. This definition of human antibody includes full-length or complete antibodies, fragments thereof, and / or antibodies containing at least one human heavy chain and / or light chain polypeptide, such as antibodies containing mouse light chain and human heavy chain polypeptides.

[0429] In one embodiment, the present invention provides a chimeric antibody against a system.

[0430] The term "chimeric antibody" refers to an antibody in which the amino acid sequence of an immunoglobulin molecule is derived from two or more species. Typically, the variable regions of the light and heavy chains correspond to the variable regions of antibodies derived from one mammal (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and ability, while the constant regions are homologous to sequences in antibodies derived from another mammal (usually human) to avoid inducing an immune response in that species.

[0431] The term "YTE" or "YTE mutant" refers to a mutation in the IgG1 Fc that results in increased binding to human FcRn and elevates the serum half-life of antibodies containing that mutation. YTE mutants comprise a combination of three mutations introduced into the heavy chain of IgG1: M252Y / S254T / T256E (EU No., Kabat et al. (1991), Sequences of Proteins of Immunological Interest, US Public Health Service, National Institutes of Health, Washington, D.C.). See U.S. Patent No. 7,658,921, which is incorporated herein by reference. Compared to the wild-type of the same antibody, the YTE mutant showed an approximately four-fold increase in the serum half-life of the antibody (Dall'Acqua et al., J. Biol. Chem. 281:23514-24 (2006); Robbie et al., (2013) Antimicrob. Agents Chemother. 57,6147-6153). See also U.S. Patent No. 7,083,784, which is incorporated herein by reference in its entirety.

[0432] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y is generally expressed as a dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein. Low-affinity antibodies typically bind antigens slowly and tend to dissociate easily, while high-affinity antibodies typically bind antigens more quickly and tend to remain bound for longer periods. Various methods for measuring binding affinity are known in the art, and any of these methods may be used for the purposes of this invention.

[0433] Unless otherwise stated, the potency of an antibody or its antigen-binding fragment is usually expressed as an IC50 value in ng / ml. IC50 is the half-maximal inhibitory concentration of an antibody molecule. In functional assays, IC50 is the concentration that reduces the biological response by 50% of its maximum value. In ligand binding studies, IC50 is the concentration that reduces receptor binding to 50% of the maximum specific binding level. IC50 can be calculated using any number of methods known in the art.

[0434] Compared with a reference antibody, the potency of the antibody or its antigen-binding fragment of the present invention can be improved by at least about 2 times, at least about 4 times, at least about 6 times, at least about 8 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, at least about 110 times, at least about 120 times, at least about 130 times, at least about 140 times, at least about 150 times, at least about 160 times, at least about 170 times, or at least about 180 times or more.

[0435] Unless otherwise stated, antibody binding potency is typically expressed as an EC50 value in nM or pM. EC50 is the median drug concentration that induces a response between baseline and maximum after a specific exposure time. EC50 can be calculated using any number of methods known in the art.

[0436] Antibody preparation

[0437] The antibodies of this invention can be obtained using conventional techniques known to those skilled in the art, and their efficacy is demonstrated by conventional binding studies—exemplary methods are described in Example 2. For instance, a simple binding assay involves incubating cells expressing an antigen with the antibody. If the antibody is labeled with a fluorophore, the binding of the antibody to the antigen can be detected by FACS analysis.

[0438] The antibodies of this invention can be produced in a variety of animals, including mice, rats, rabbits, goats, sheep, monkeys, or horses. Antibodies can be produced after immunization with a single capsular polysaccharide or with multiple capsular polysaccharides. Blood isolated from these animals contains polyclonal antibodies—multiple antibodies that bind to the same antigen. The antigen can also be injected into chickens to produce polyclonal antibodies in the egg yolk. To obtain monoclonal antibodies specific to a single epitope of the antigen, antibody-secreting lymphocytes are isolated from the animal and immortalized by fusing them with cancer cell lines. The fused cells are called fusion tumors and continuously grow and secrete antibodies in culture. Individual fusion tumor cells are isolated by dilution selection to generate cell colonies that all produce the same antibody; these antibodies are called monoclonal antibodies. The method used to produce monoclonal antibodies is a conventional technique known to those skilled in the art (see, for example, Making and Using Antibodies: A Practical Handbook, GC Howard, CRC Books, 2006, ISBN 0849335280). Multiselective and monoclonal antibodies are typically purified using protein A / G or antigen affinity chromatography.

[0439] The antibodies or antigen-binding fragments of the present invention can be prepared as monoclonal antibodies against B7-H4, which can be prepared using fusion tumor methods, such as those described in Kohler and Milstein, Nature 256:495 (1975). Using the fusion tumor method, mice, hamsters, or other suitable host animals are immunized as described above to induce lymphocytes to produce antibodies that specifically bind to the immunoantigen. Lymphocytes can also be immunized in vitro. After immunization, lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol, to form fusion tumor cells that can subsequently be selected from unfused lymphocytes and myeloma cells. The monoclonal antibodies can then be produced in vitro using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986) or in vivo as ascites tumors of animals that produce monoclonal antibodies specifically against selected antigens, as determined by immunoprecipitation, immunoblotting, or in vitro binding assays (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). The monoclonal antibodies can then be purified from the culture medium or ascites using known methods.

[0440] Alternatively, the antibody or its antigen-binding fragment (e.g., a monoclonal antibody) can also be prepared using a recombinant DNA method as described in U.S. Patent No. 4,816,567. Polynucleotides encoding monoclonal antibodies are isolated from mature B cells or fusion tumor cells, for example by RT-PCR using oligonucleotide primers (which specifically amplify genes encoding the heavy and light chains of the antibody), and their sequences are determined using standard procedures. The isolated polynucleotides encoding the heavy and light chains are then selected and colonized into suitable expression vectors, which, when transfected into non-immunoglobulin-producing host cells (such as *E. coli* cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells), enable the host cells to produce monoclonal antibodies. In addition, recombinant monoclonal antibodies of the desired species or their antigen-binding fragments can be isolated from phage display libraries expressing the CDR of the desired species, as described in the following literature: McCafferty et al., Nature 348:552-554 (1990); Clackson et al., Nature 352:624-628 (1991); and Marks et al., J. Mol. Biol 222:581-597 (1991).

[0441] One or more polynucleotides encoding the antibody or antigen-binding fragment thereof of the present invention may be further modified in a variety of different ways using recombinant DNA technology to produce alternative antibodies. In some embodiments, for example, the constant domains of the light and heavy chains of a mouse monoclonal antibody may be replaced by (1) regions such as those of a human antibody to produce a chimeric antibody, or by (2) non-immunoglobulin polypeptides to produce a fusion antibody. In some embodiments, such constant regions are truncated or removed to produce antibody fragments of the desired monoclonal antibody. Site-directed mutagenesis or high-density mutagenesis of variable regions may be used to optimize the specificity, affinity, etc., of the monoclonal antibody.

[0442] In one embodiment, the antibody or its antigen-binding fragment is a human antibody or its antigen-binding fragment. Human antibodies can be prepared directly using various techniques known in the art. Immortalized human B lymphocytes, either immunized in vitro or isolated from an immunized individual producing antibodies against the target antigen, can be generated. See, for example, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., J. Immunol. 147(1):86-95 (1991); U.S. Patent 5,750,373.

[0443] In one embodiment, the antibody or its antigen-binding fragment may be selected from a phage library containing human antibodies, as described in the following references: for example, Vaughan et al., Nat. Biotech. [Nature Biotechnology] 14:309-314 (1996); Sheets et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America], 95:6157-6162 (1998); Hoogenboom and Winter, J. Mol. Biol. [Journal of Molecular Biology] 227:381 (1991); and Marks et al., J. Mol. Biol. [Journal of Molecular Biology] 222:581 (1991). Techniques for generating and using antibody phage libraries are also described in U.S. Patent Nos. 5,969,108, 6,172,197, 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, 6,593,081, 6,300,064, 6,653,068, 6,706,484, and 7,264,963; and Rothe et al., J. Molec. Biol. [Journal of Molecular Biology] 376:1182-1200 (2008), each of which is incorporated herein by reference in its entirety.

[0444] Affinity maturation strategies and chain modification grouping principles are known in the art and can be used to generate high-affinity human antibodies or their antigen-binding fragments. See Marks et al., BioTechnology 10:779-783 (1992), which is incorporated herein by reference in its entirety.

[0445] In one embodiment, the antibody or its antigen-binding fragment (e.g., a monoclonal antibody) may be a humanized antibody. Methods for engineering, humanizing, or surface-repairing non-human or human antibodies may also be used and are well known in the art. Humanized, surface-repaired, or similarly engineered antibodies may have one or more amino acid residues from a non-human source, such as, but not limited to, mice, rats, rabbits, non-human primates, or other mammals. These non-human amino acid residues are replaced by residues commonly referred to as "input" residues, which are typically derived from the "input" variable domain, constant domain, or other domains of a known human sequence. Such input sequences can be used to reduce immunogenicity or decrease, enhance, or alter binding, affinity, binding rate, dissociation rate, specificity, half-life, or any other suitable characteristic as known in the art. Suitably, CDR residues may directly and most substantially participate in influencing B7-H4 binding. Therefore, it is preferable to retain some or all of the non-human or human CDR sequence while the non-human sequences in the variable and constant regions can be replaced by human amino acids or other amino acids.

[0446] Antibodies can also be humanized, surface-repaired, engineered, or engineered into human antibodies as needed, retaining high affinity for antigen B7-H4 and other favorable biological properties. To achieve this, humanized (or human) or engineered anti-B7-H4 antibodies and surface-repaired antibodies can be prepared as needed using methods that analyze parental sequences and various conceptual humanized and engineered products through three-dimensional modeling of parental, engineered, and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs illustrating and demonstrating the possible three-dimensional conformations of selected candidate immunoglobulin sequences are available. Examining these displays allows analysis of the possible roles of residues in the function of the candidate immunoglobulin sequence, i.e., analyzing residues that affect the ability of the candidate immunoglobulin to bind its antigen, such as B7-H4. In this way, free radical residues (FW residues) can be selected and combined from consistent sequences and input sequences, thus enabling the desired antibody characteristics (such as increased affinity for one or more target antigens) to be achieved.

[0447] The humanization, surface remodeling, or engineering of the anti-B7-H4 antibody or its antigen-binding fragment of the present invention can be performed using any known method, such as, but not limited to, those described in the following literature: Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988); Sims et al., J. Immunol. [Journal of Immunology] 151:2296 (1993); Chothia and Lesk, J. Mol. Biol. [Journal of Molecular Biology] 196:901 (1987); Carter et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 89:4285 (1992); Presta et al., J. Immunol. [Journal of Immunology] 151:2623 (1993); US Patent Nos. 5,639,641, 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539, 4,816,567, 7,557,189, 7,538,195 and 7,342,110; International application numbers PCT / US98 / 16280, PCT / US96 / 18978, PCT / US91 / 09630, PCT / US91 / 05939, PCT / US94 / 01234, PCT / GB89 / 01334, PCT / GB91 / 01134, PCT / GB92 / 01755; International patent application publication number WO 90 / 14443, WO 90 / 14424, WO 90 / 14430; and European Patent Publication No. EP 229246; the entirety of each of these, including any references cited therein, is incorporated herein by reference.

[0448] Humanized anti-B7-H4 antibodies and their antigen-binding fragments can also be prepared in transgenic mice containing human immunoglobulin loci that, upon immunization, generate a full suite of human antibodies in the absence of endogenous immunoglobulins. This method is described in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016.

[0449] In one embodiment, a fragment (e.g., an antibody fragment) of an antibody (e.g., an anti-B7-H4 antibody) is provided. Various techniques for producing antibody fragments are known. Conventionally, such fragments are derived by proteolytic hydrolysis of the intact antibody, as described, for example, by Morimoto et al., J. Biochem. Biophys. Meth. 24:107-117 (1993) and Brennan et al., Science 229:81 (1985). In one embodiment, recombinant synthesis produces the anti-B7-H4 antibody fragment. All Fab, Fv, and scFv antibody fragments can be expressed and secreted from *E. coli* or other host cells, thus allowing for the production of large quantities of such fragments. Such anti-B7-H4 antibody fragments can also be isolated from antibody phage libraries discussed above. These anti-B7-H4 antibody fragments can also be linear antibodies as described in U.S. Patent No. 5,641,870. Other techniques for producing antibody fragments will be apparent to those skilled in the art.

[0450] According to the present invention, the technique can be adapted to produce single-chain antibodies specific to B7-H4. See, for example, U.S. Patent No. 4,946,778. Furthermore, the method can be adapted to construct Fab expression libraries to allow for rapid and efficient identification of single-chain Fab fragments with desired specificity against B7-H4 or its derivatives, fragments, analogs, or homologs. See, for example, Huse et al., Science 246:1275-1281 (1989). Antibody fragments can be produced by techniques known in the art, including but not limited to: F(ab')2 fragments produced by digesting antibody molecules with pepsin; Fab fragments produced by reducing the disulfide bonds of F(ab')2 fragments; Fab fragments produced by treating antibody molecules with papain and a reducing agent; or Fv fragments.

[0451] In one embodiment, the antibody or its antigen-binding fragment of the present invention can be modified to increase its serum half-life. This can be achieved, for example, by incorporating a rescue receptor-binding epitope into the antibody or antibody fragment, by mutating an appropriate region of the antibody or antibody fragment, or by incorporating the epitope into a peptide tag subsequently fused to the end or middle of the antibody or antibody fragment (e.g., by DNA or peptide synthesis), or by YTE mutation. Other methods for increasing the serum half-life of the antibody or its antigen-binding fragment are known in the art, such as conjugation to a heterologous molecule like PEG.

[0452] The modified antibodies or antigen-binding fragments thereof provided herein can contain any type of variable region that enables association of the antibody or peptide with B7-H4. In this respect, the variable region can be formed from or derived from any type of mammal capable of inducing an increased humoral response and generating immunoglobulins against the desired antigen. Therefore, the variable region of an anti-B7-H4 antibody or antigen-binding fragment thereof can be derived from, for example, humans, mice, non-human primates (e.g., cynomolgus monkeys, macaques, etc.), or wolves (lupines). In one embodiment, both the variable and constant regions of the modified antibody or antigen-binding fragment thereof are human. In one embodiment, the variable region of a compatibility antibody (typically derived from a non-human source) can be engineered or specifically tailored to improve binding properties or reduce the immunogenicity of the molecule. In this respect, the variable region useful in this invention can be humanized or additionally modified by incorporating an input amino acid sequence.

[0453] In one embodiment, the variable domains in both the heavy and light chains of the antibody or its antigen-binding fragment are altered by at least partial replacement of one or more CDRs and / or by partial frame region substitution and sequence changes. While CDRs can be derived from the same class or even subclass of antibody as the frame region from which they are derived, it is envisioned that CDRs will be derived from different classes of antibodies and, in some embodiments, from antibodies of different species. It is not necessary to replace all CDRs with complete CDRs from the donor variable region to transfer the antigen-binding capacity of one variable domain to another. Rather, only those residues necessary to maintain the activity of the antigen-binding site need to be transferred. Considering the explanations set forth in U.S. Patent Nos. 5,585,089, 5,693,761, and 5,693,762, those skilled in the art are fully capable of obtaining functional antibodies with reduced immunogenicity through routine experiments.

[0454] Despite modifications to the variable regions, those skilled in the art will understand that the modified antibodies or antigen-binding fragments thereof of the present invention will comprise antibodies (e.g., full-length antibodies or antigen-binding fragments thereof) wherein portions of at least one or more constant region domains have been deleted or otherwise modified to provide desired biochemical characteristics, such as increased tumor localization or reduced serum half-life when compared to antibodies containing native or unmodified constant regions with approximately the same immunogenicity. In one embodiment, the constant region of the modified antibody will comprise a human constant region. Modifications to constant regions compatible with the present invention include the addition, deletion, or substitution of one or more amino acids in one or more domains. That is, the modified antibodies disclosed herein may comprise alterations or modifications to one or more of the three heavy chain constant domains (CH1, CH2, or CH3) and / or to the light chain constant domain (CL). In one embodiment, modified constant regions are contemplated in which one or more domains are partially or completely deleted. In one embodiment, the modified antibody will comprise a domain-deficient construct or variant in which the entire CH2 domain is removed (ΔCH2 construct). In one embodiment, the omitted constant region domain may be replaced by a short amino acid spacer (e.g., 10 residues) that provides a degree of molecular flexibility typically conferred by the absence of a constant region.

[0455] In addition to their conformation, constant regions are known in the art to mediate several effector functions. For example, antibodies bind to cells via Fc regions, where Fc receptor sites on the antibody's Fc region bind to Fc receptors (FcRs) on the cell. Many Fc receptors exist that are specific to different classes of antibodies, including IgG (γ receptor), IgE (η receptor), IgA (α receptor), and IgM (μ receptor). The binding of antibodies to Fc receptors on the cell surface triggers many important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, cell lysis of antibody-coated target cells (known as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production.

[0456] In one embodiment, the antibody or its antigen-binding fragment provides altered effector function, thereby affecting the biological properties of the administered antibody or its antigen-binding fragment. For example, deletion or inactivation of the constant region domain (via point mutation or other means) can reduce Fc receptor binding of circulating modified antibodies. In other cases, consistent with the present invention, constant region modification can mitigate complement binding and thus reduce the serum half-life and nonspecific association of conjugated cytotoxins. Further modifications to the constant region can be used to eliminate disulfide bonds or oligosaccharide moieties, thereby allowing enhanced localization due to increased antigen specificity or antibody flexibility. Similarly, modifications to the constant region according to the present invention can be readily performed using well-known biochemical or molecular engineering techniques within the scope of the art.

[0457] In one embodiment, the antibody or its antigen-binding fragment does not have one or more effector functions. For example, in one embodiment, the antibody or its antigen-binding fragment does not have antibody-dependent cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity. In one embodiment, the antibody or its antigen-binding fragment does not bind to Fc receptors and / or complement factors. In one embodiment, the antibody or its antigen-binding fragment does not have effector functions.

[0458] In one embodiment, the antibody or its antigen-binding fragment can be engineered to directly fuse the CH3 domain to the hinge region of the corresponding modified antibody or fragment. In other constructs, peptide spacers can be inserted between the hinge region and the modified CH2 and / or CH3 domains. For example, compatibility constructs can be exhibited where the CH2 domain is missing and the remaining CH3 domain (modified or unmodified) binds to the hinge region with spacers having 5-20 amino acids. This spacer can be added, for example, to ensure that the regulatory elements of the constant domain remain free and accessible, or that the hinge region remains flexible. In some cases, the amino acid spacers can be shown to be immunogenic and elicit an undesirable immune response against the construct. In one embodiment, any spacers added to the construct can be relatively non-immunogenic, or even omitted entirely, in order to maintain the desired biochemical properties of the modified antibody.

[0459] In addition to the deletion of the entire constant region domain, the antibodies or antigen-binding fragments provided herein can be modified by partial deletion or substitution of several or even a single amino acid in the constant region. For example, a mutation of a single amino acid in a selected region of the CH2 domain can be sufficient to substantially reduce Fc binding and thereby increase tumor localization. Similarly, one or more constant region domains controlling effector functions (e.g., complement C1Q binding) can be completely or partially deleted. Such partial deletion of the constant region can improve selected characteristics of the antibody or antigen-binding fragment (e.g., serum half-life) while keeping other desired functions associated with the subject's constant region domain intact. Furthermore, the constant region of the antibody and its antigen-binding fragment can be modified by mutation or substitution of one or more amino acids that enhance the properties of the resulting construct. In this respect, the activity provided by the conserved binding site (e.g., Fc binding) can be interfered with while substantially maintaining the conformation and immunogenic properties of the modified antibody or its antigen-binding fragment. In one embodiment, one or more amino acids can be added to the constant region to enhance desired characteristics, such as reducing or increasing effector function, or providing more cytotoxic or carbohydrate attachment. In one implementation, it may be desirable to insert or copy a specific sequence derived from a selected constant region structural domain.

[0460] The present invention further includes variants and equivalents substantially homologous to the antibody or antigen-binding fragments of the present invention (e.g., mouse, chimeric, humanized, or human antibodies or their antigen-binding fragments). These may contain, for example, conserved substitution mutations, i.e., substitution of one or more amino acids by similar amino acids. For example, a conserved substitution refers to the substitution of an amino acid with another amino acid within the same general category, such as, for example, the substitution of an acidic amino acid with another acidic amino acid, the substitution of a basic amino acid with another basic amino acid, or the substitution of a neutral amino acid with another neutral amino acid. The meaning of conserved amino acid substitution is well known in the art.

[0461] In one embodiment, the antibody or its antigen-binding fragment may be further modified to include additional chemical moieties that are not normally part of the protein. These derived moieties can improve the protein's solubility, biological half-life, or absorption. They can also reduce or eliminate any desired side effects of the protein. A review of these moieties can be found in Remington's Pharmaceutical Sciences, 22nd edition, edited by Lloyd V. Allen, Jr., (2012).

[0462] definition

[0463] The following definitions specifically refer to the description of the above topoisomerase I inhibitors, and may even more specifically refer to the section entitled "Further Preferences".

[0464] C5-6 aryl: As used herein, the term "C5-6 aryl" refers to the divalent portion obtained by removing two hydrogen atoms from an aromatic ring atom of an aromatic compound.

[0465] In this paper, prefixes (e.g., C5-6) indicate the number of ring atoms or the range of ring atoms, whether carbon atoms or heteroatoms.

[0466] The ring atoms can all be carbon atoms, as in the "carbon aryl group", in which case the group is phenyl (C6).

[0467] Alternatively, the ring atom may include one or more heteroatoms, as in "heteroaryl groups". Examples of heteroaryl groups include, but are not limited to, those derived from: N1: pyrrole (azole) (C5), pyridine (acetylene) (azine))(C6);O1:furan (oxole))(C5);S1:thiole (thiole))(C5);N1O1: azole (C5), isazole (C5), isosorbide azole (C5), isazole (C5), isosorbide (isoxazine)(C6);N2O1: Diazole (furazan) (C5); N3O1: Triazole (C5); N1S1: Thiazole (C5), Isothiazole (C5); N2: Imidazole (1,3-diazole) (C5), Pyrazole (1,2-diazole) (C5), Tadalafil (1,2-II) (C6), pyrimidine (1,3-di) (C6) (e.g., cytosine, thymine, uracil), pyridine (1,4-II) (C6); and N3: triazole (C5), triazole (C6)

[0468] C1-4 alkyl: As used herein, the term "C1-4 alkyl" refers to a monovalent portion obtained by removing hydrogen atoms from carbon atoms of a hydrocarbon having 1 to 4 carbon atoms, which may be aliphatic or alicyclic and may be saturated or unsaturated (e.g., partially unsaturated, fully unsaturated). As used herein, the term "C1-n alkyl" refers to a monovalent portion obtained by removing hydrogen atoms from carbon atoms of a hydrocarbon having 1 to n carbon atoms, which may be aliphatic or alicyclic and may be saturated or unsaturated (e.g., partially unsaturated, fully unsaturated). Thus, the term "alkyl" includes the subclasses discussed below: alkenyl, ynyl, cycloalkyl, etc.

[0469] Examples of saturated alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), propyl (C3), and butyl (C4).

[0470] Examples of saturated straight-chain alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), and n-butyl (C4).

[0471] Examples of saturated branched alkyl groups include isopropyl (C3), isobutyl (C4), secondary butyl (C4), and tertiary butyl (C4).

[0472] C2-4 alkenyl; as used herein, the term "C2-4 alkenyl" refers to an alkyl group having one or more carbon-carbon double bonds.

[0473] Examples of unsaturated alkenyl groups include, but are not limited to, ethenyl (-CH=CH2), 1-propenyl (-CH=CH-CH3), 2-propenyl (allyl, -CH-CH=CH2), isopropenyl (1-methyl vinyl, -C(CH3)=CH2), and butenyl (C4).

[0474] C2-4 ynyl group: As used herein, the term "C2-4 ynyl group" refers to an alkyl group having one or more carbon-carbon triple bonds.

[0475] Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (-C≡CH) and 2-propynyl (propynyl, -CH2-C≡CH).

[0476] C3-4 cycloalkyl: As used herein, the term "C3-4 cycloalkyl" refers to an alkyl group that is also a cycloalkyl group; that is, a monovalent portion obtained by removing hydrogen atoms from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, the portion having 3 to 7 carbon atoms, including 3 to 7 ring atoms.

[0477] Examples of cycloalkyl groups include, but are not limited to, those derived from: saturated monocyclic hydrocarbons: cyclopropane (C3) and cyclobutane (C4); and unsaturated monocyclic hydrocarbons: cyclopropylene (C3) and cyclobutene (C4).

[0478] Link tag: in style In this diagram, the superscripts C (=O) and NH indicate the groups to which the atoms are bonded. For example, the NH group shows a bond with a carbonyl group (which is not part of the part shown), and the carbonyl group shows a bond with an NH group (which is not part of the part shown).

[0479] Salt

[0480] Salts of active compounds / pharmaceuticals, such as pharmaceutically acceptable salts, can be conveniently or desirablely prepared, purified, and / or processed. Examples of pharmaceutically acceptable salts are found in Berge et al., J. Pharm. Sci. [Journal of Pharmaceutical Sciences].

[66] , 1-19 (1977) discusses this.

[0481] For example, if a compound is anionic or has a functional group that can be anionic (e.g., -COOH can be -COO-), it can form a salt with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na+ and K+, alkaline earth metal cations such as Ca2+ and Mg2+, and other cations such as Al3+. Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., NH4+) and substituted ammonium ions (e.g., NH3R+, NH2R2+, NHR3+, NR4+). Some examples of suitable substituted ammonium ions are derived from those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, etc. , benzylamine, phenylbenzylamine, choline, meglumine and aminebutanetriol, and amino acids (such as lysine and arginine). A common example of a quaternary ammonium ion is N(CH3)4+.

[0482] If the compound is cationic or has a functional group that can be cationic (e.g., -NH2 can be -NH3+), it can form a salt with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid.

[0483] Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, ethylenediaminetetraacetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthoic acid, hydroxyethanesulfonic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, mesylic acid, mucoic acid, oleic acid, oxalic acid, palmitic acid, dihydroxynaphthoic acid, pantothenic acid, phenylacetic acid, benzenesulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid, trifluoroacetic acid, and valeric acid. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid and carboxymethyl cellulose.

[0484] solvates

[0485] The corresponding solvates of active compounds can be conveniently or desirablely prepared, purified, and / or processed. The term "solvate" is used herein in its conventional sense, referring to a complex of a solute (e.g., an active compound, a salt of an active compound) and a solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, such as a monohydrate, dihydrate, trihydrate, etc.

[0486] Heteromers

[0487] Certain compounds / pharmaceuticals of this invention can exist in one or more specific geometric, optical, mirror-image, non-mirror-image, epimeric, trans-blocking, stereoisomer, tautomer, conformational, or anomeric forms, including but not limited to cis and trans forms; E- and Z- forms; c-, t-, and r- forms; meso- and exo- forms; R-, S-, and meso- forms; D- and L- forms; d- and l- forms; (+) and (-) forms; ketone-, enol-, and enolate- forms; cis- and trans- forms; synclinal- and anticline- forms; α- and β- forms; axial and equatorial forms; boat-, chair-, twisted-, envelope-, and semi-chair- forms; and combinations thereof, collectively referred to below as "isomers" (or "isoconfigurations").

[0488] The term "chirality" refers to a molecule that has a non-overlapping mirror pair, while the term "chirality" refers to a molecule that can be superimposed on its mirror pair.

[0489] The term "stereoisomer" refers to compounds that have the same chemical composition but differ in the spatial arrangement of atoms or groups.

[0490] "Non-mirror image isomers" refer to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Non-mirror image isomers have different physical properties, such as melting point, boiling point, spectral properties, and reactivity. Mixtures of non-mirror image isomers can be separated using high-resolution analytical procedures such as electrophoresis and chromatography.

[0491] "Mirror-image isomers" refers to two stereoisomers of a compound that are non-overlapping mirror images of each other.

[0492] The stereochemical definitions and conventions used herein generally follow those of SP Parker, ed., McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds, John Wiley & Sons, Inc., New York, 1994. The compounds of this invention may contain asymmetric or chiral centers and thus exist in different stereoisomers. It is intended that all stereoisomers of the compounds of this invention (including, but not limited to, non-mirror image isomers, mirror image isomers, and rotation-blocking isomers and mixtures thereof, such as racemic mixtures) constitute a part of this invention. Many organic compounds exist in an optically active form, i.e., they have the ability to rotate the plane of plane-polarized light. In describing optically active compounds, the prefixes D and L, or R and S, are used to indicate the absolute configuration of the molecule around one or more chiral centers. The prefixes d and l, or (+) and (-), are used to indicate that the compound rotates plane-polarized light, where (-) or l indicates that the compound is levorotatory. Compounds with the prefix (+) or d are dextrorotatory. For a given chemical structure, these stereoisomers are identical, only they are mirror images of each other. Specific stereoisomers can also be called mirror image isomers, and mixtures of such isomers are usually called mirror image isomer mixtures. A 50:50 mixture of mirror image isomers is called a racemic mixture or racemate, which can occur where there is no stereoselectivity or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equal molar mixture of two mirror image isomers that are not optically active.

[0493] "Mirror-image isomer enrichment" refers to a sample of chiral substances with a mirror-image isomer ratio greater than 50:50 but less than 100:0.

[0494] Note that, except as discussed below regarding tautomer configurations, the term "isomer" as used herein specifically excludes structural (or constitutive) isomers (i.e., isomers that differ in the connection between atoms, not merely in the spatial position of atoms). For example, a reference to a methoxy group (-OCH3) should not be interpreted as a reference to its structural isomer, the hydroxymethyl group (-CH2OH). Similarly, a reference to an o-chlorophenyl should not be interpreted as a reference to its structural isomer, the m-chlorophenyl. However, a reference to a class of structures may well include structural isomers belonging to that class (e.g., C1-7 alkyl includes n-propyl and isopropyl; butyl includes n-, iso, secondary, and tertiary butyl; methoxyphenyl includes o-, m-, and p-methoxyphenyl).

[0495] The above exclusions do not apply to tautomer forms, such as ketones, enols, and enol esters, such as the following tautomer pairs: ketone / enol (as shown below), imine / enamine, imino alcohol, amidine / enediamine, nitroso / oxime, thionone / enethiol, N-nitroso / hydroxyazo, and nitro / aci-nitro.

[0496]

[0497] The terms "tautomer" or "tautomer configuration" refer to structural isomers of different energies that can interconvert via a low energy barrier. For example, proton tautomers (also known as proton heterotautomers) include interconversions via proton migration, such as keto-enol and imine-enamine isomerization. Valence tautomers include interconversions via the recombination of some valence electrons.

[0498] Note that the term "isomer" specifically includes compounds with one or more isotopic substitutions. For example, H can be any isotopic form, including 1H, 2H(D), and 3H(T); C can be any isotopic form, including 12C, 13C, and 14C; O can be any isotopic form, including 16O and 18O; and so on.

[0499] Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as, but not limited to, 2H (deuterium, D), 3H (tritium), 11C, 13C, 14C, 15N, 18F, 31P, 32P, 35S, 36Cl, and 125I. Various isotopically labeled compounds of the present invention, such as those incorporated with radioactive isotopes like 3H, 13C, and 14C, are also possible. Such isotopically labeled compounds can be used in metabolic studies, reaction kinetic studies, detection or imaging techniques (e.g., positron emission tomography (PET) or single-photon emission computed tomography (SPECT), including drug or matrix tissue distribution assays), or for radiotherapy of patients. The deuterium-labeled or substituted therapeutic compounds of the present invention can have improved DMPK (drug metabolism and pharmacokinetics) properties related to distribution, metabolism, and excretion (ADME). Substitution with a heavier isotope (such as deuterium) may provide certain therapeutic advantages (due to greater metabolic stability), such as increased in vivo half-life or reduced dose requirement. 18F-labeled compounds can be used in PET or SPECT studies. The isotope-labeled compounds of the present invention and their precursors can generally be prepared by the methods disclosed in the embodiments or examples and preparations described below, wherein a non-isotope-labeled reagent is substituted with an readily available isotope-labeled reagent. Furthermore, substitution with a heavier isotope (especially deuterium (i.e., 2H or D)) may provide certain therapeutic advantages (due to greater metabolic stability), such as increased in vivo half-life or reduced dose requirement or improved therapeutic index. It should be understood that deuterium is considered a substituent herein. The concentration of such a heavier isotope (especially deuterium) can be defined by an isotope enrichment factor. In the compounds of the present invention, any atom not specifically designated as a particular isotope represents any stable isotope of that atom.

[0500] Unless otherwise stated, references to a particular compound include all such isomer forms, including (all or part) racemic mixtures and other mixtures thereof. Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallization and chromatography) of such isomer forms are known in the art or readily available by means of the methods taught herein or known methods in a known manner.

[0501] sequence homology

[0502] A variety of sequence alignment methods can be used to determine the percentage of identity, including but not limited to global, local, and hybrid methods such as segmental methods. The scheme for determining the percentage of identity is a standard procedure familiar to those skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the optimal alignment by summing the scores of individual residue pairs and applying a vacancy penalty. Non-restrictive methods include, for example, CLUSTAL W, see Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments [The accuracy of alignment of multiple protein sequences is significantly improved by iterative computational refinement using reference structure alignment], 264(4) J. MoI. Biol. [Journal of Molecular Biology] 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all input sequences.Non-restrictive methods include, for example, Match-box, see, for example, Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbs sampling, see, for example, C.E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, see, for example, Ivo Van Waille et al., Align-MA New Algorithm for Multiple Alignment of Highly Divergent Sequences [A Novel Algorithm for Multiple Alignment of Align-M Highly Divergent Sequences], 20(9) Bioinformatics: 1428-1435 (2004).

[0503] Therefore, the percentage of sequence identity is determined using conventional methods. See, for example, Altschul et al., Bull. Math. Bio. [Bulletin of Mathematical Biology] 48:603-16, 1986, and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 89:10915-19, 1992. In short, the alignment scores of two amino acid sequences are optimized by using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (as previously described) as shown below. (Amino acids are represented by standard single-letter codes.)

[0504] The percentage of sequence identity between two or more nucleic acid or amino acid sequences is a function of the number of common positions shared by the sequences. Therefore, % identity can be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids, then multiplied by 100. The calculation of % sequence identity can also take into account the number of vacancies, and the length of each vacancy that needs to be introduced to optimize the alignment of two or more sequences. Specific mathematical algorithms familiar to the technician (such as BLAST) can be used for sequence comparisons and determination of the percentage of identity between two or more sequences.

[0505] Alignment score used to determine sequence identity

[0506]

[0507]

[0508] Then calculate the percentage of identity:

[0509] Essentially homologous polypeptides are characterized by the presence of one or more amino acid substitutions, deletions, or additions. These variations are preferably small, i.e., conserved amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically from 1 to about 30 amino acids; and small amino or carboxyl terminal extensions, such as amino-terminal methionine residues, small linker peptides of up to about 20-25 residues, or affinity tags.

[0510] Conservative amino acid substitution

[0511] Alkaline: Arginine; Lysine; Histamine

[0512] Acids: glutamic acid; aspartic acid

[0513] Polarity: glutamic acid; aspartic acid

[0514] Hydrophobic: Leucine; Isoleucine; Valine

[0515] Aromatic acids: phenylalanine; tryptophan; tyrosine

[0516] Smaller types: glycine; alanine; serine; threonine; methionine

[0517] In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovalinic acid, and α-methylserine) can replace the amino acid residues of the polypeptides of the present invention. A limited number of non-conserved amino acids, amino acids not encoded by the genetic code, and non-natural amino acids can replace the amino acid residues of the polypeptides. The polypeptides of the present invention may also contain non-naturally occurring amino acid residues.

[0518] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-bridged methylene-proline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allethamine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamate, homoglutamate, hexahydronicotinic acid, tert-leucine, n-valine, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods for incorporating non-naturally occurring amino acid residues into proteins are known in the art. For example, an in vitro system can be used in which chemically amination-modified repressive tRNA is used to suppress nonsense mutations. Methods for synthesizing amino acids and amination-modified tRNA are known in the art. Transcription and translation of plastids containing nonsense mutations were carried out in a cell-free system containing *E. coli* S30 extract and commercially available enzymes and other reagents. The protein was purified by chromatography. See, for example, Robertson et al., *J. Am. Chem. Soc.* 113:2722, 1991; Ellman et al., *Methods Enzymol.* 202:301, 1991; Chung et al., *Science* 259:806-9, 1993; and Chung et al., *Proc. Natl. Acad. Sci. USA* 90:10145-9, 1993). In the second method, translation is performed in Xenopus oocytes by microinjection of mutant mRNA and chemically amination-modified repressive tRNA (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). In the third method, *E. coli* cells are cultured in the absence of the natural amino acid to be replaced (e.g., phenylalanine) and in the presence of one or more non-naturally occurring amino acids (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). Non-naturally occurring amino acids are incorporated into the polypeptide in place of their natural counterparts. See Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted into non-naturally occurring species through in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. [Protein Science] 2:395-403, 1993).

[0519] A limited number of non-conserved amino acids, amino acids not encoded by the genetic code, non-naturally occurring amino acids, and non-natural amino acids can replace the amino acid residues of the polypeptides of the present invention.

[0520] The essential amino acids in the polypeptides of this invention can be identified using methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244:1081-5, 1989). Biological interaction sites can also be determined by physical analysis of the structure, such as by techniques including nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutagenesis of the presumed contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identity of the essential amino acids can also be inferred from homology analysis of related components (e.g., translocation components or protease components) of the polypeptides of this invention.

[0521] Multiple amino acid substitutions can be synthesized and tested using known mutagenesis and screening methods, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). In short, these authors disclosed methods for simultaneously randomizing two or more positions in a peptide, selecting functional peptides, and then sequencing the mutagenized peptide to determine the range of permissible substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene. 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0522] Multiple amino acid substitutions can be synthesized and tested using known mutagenesis and screening methods, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). In short, these authors disclosed methods for simultaneously randomizing two or more positions in a peptide, selecting functional peptides, and then sequencing the mutagenized peptide to determine the range of permissible substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene. 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0523] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20th edition, John Wiley and Sons, New York (1994), and Hale and Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, New York (1991) provide general dictionaries for those skilled in the art of the art of the many terms used in this disclosure.

[0524] This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of embodiments of this disclosure. Numerical ranges include the digits defining the range. Unless otherwise specified, any nucleic acid sequence is written from left to right in a 5' to 3' direction; amino acid sequences are written from left to right in an amino-to-carboxyl direction.

[0525] The headings provided herein are not intended to limit any aspect of or implementation of this disclosure.

[0526] Amino acids are referred to herein by name, three-letter abbreviation, or single-letter abbreviation. As used herein, the term "protein" includes proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the terms "polypeptide" and / or "protein." In some cases, the term "amino acid sequence" is synonymous with the term "peptide." In some cases, the term "amino acid sequence" is synonymous with the term "enzyme." The terms "protein" and "polypeptide" are used interchangeably herein. Conventional one-letter and three-letter codes for amino acid residues may be used in this disclosure and the claims. The three-letter codes for amino acids follow the definition of the Joint Commission on Biochemical Nomenclature (JCBN) of IUPACIUB. It should also be understood that, due to the degeneracy of the genetic code, a polypeptide can be encoded by more than one nucleotide sequence.

[0527] Other definitions of terms may appear throughout this specification. Before describing exemplary embodiments in more detail, it should be understood that this disclosure is not limited to the described embodiments, and therefore such embodiments may vary. It should also be understood that the terminology used herein is for descriptive purposes only and is not intended to be restrictive, as the scope of this disclosure is limited only by the appended claims.

[0528] Where a numerical range is provided, it should be understood that each intermediate value between the upper and lower limits of the range (to one-tenth of the units digit of the lower limit, unless the context clearly specifies otherwise) is also specifically disclosed. Each smaller range between any specified value or intermediate value in the stated range and any other specified value or intermediate value in the stated range is covered within this disclosure. The upper and lower limits of such smaller ranges may be independently included or excluded from the range, and each range in which no one, and no one or both limit values ​​are included is also covered within this disclosure, depending on any limit values ​​specifically excluded from the stated range. Where a stated range contains one or both limit values, the range in which one or both of those included limit values ​​are excluded is also included in this disclosure.

[0529] It must be noted that, as used herein and in the appended claims, the singular forms "a / an" and "the" include plural indicators unless the context clearly specifies otherwise. Thus, for example, reference to "a medicine" includes multiple such medicines and reference to "the medicine" includes reference to one or more medicines and their equivalents known to those skilled in the art, and so on.

[0530] The publications discussed herein are provided only for their disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that such publications constitute prior art to the appended claims. Example

[0531] Materials and Methods

[0532] Protein reagents

[0533] The resulting protein reagents (e.g., constructs) are shown in Table 1. This table indicates the species from which the protein originated (human, mouse, or cynomolgus monkey), the vector used to select and colonize the construct, and whether the leader sequence used is natural or human CD33 leader sequence.

[0534]

[0535] The "triple mutant Fc" (TM) comprises a triple mutation L234F / L235E / P331S within the Fc region, as previously described in Acta Crystallogr D Biol Crystallogr. [Acta Crystallographica Part D: Biocrystals] 2008 June 1; 64(Pt 6):700-704 (incorporated herein by reference). To avoid any doubt, the inventors have ensured that the antibodies described below retain their advantageous binding properties / characteristics even in the absence of such a TM (e.g., in the presence of WT Fc).

[0536] All proteins in the table were purified using standard conditions. In short, they were expressed in HEK EBNA, concentrated using TFF concentration settings, purified using a His trap column or a Protein G column (depending on the presence of a 10His Flag tag or Fc tag), and then final polished using an SEC S200 column.

[0537] ELISA

[0538] The binding of anti-B7-H4 antibodies (e.g., intermediate ZY0EQD-E02-GL and anti-B7-H4 mAB D11) to B7-H4 (e.g., human and mouse B7-H4) was measured by ELISA. Recombinant B7-H4-Fc protein (e.g., human and mouse B7-H4-Fc) was diluted in DPBS to obtain a 5 μg / mL solution. 50 μL of the diluted stock solution was then added to a Nunc Maxisorp 96-well plate. 50 μL / well of DPBS was added to the control wells. The antigen was allowed to adsorb onto the plate overnight at 4°C, washed once with DPBS, and incubated for 1 hour at room temperature with blocking buffer (3% w / v Marvel, in DPBS). The plate was then washed once with PBS and incubated for 1 hour with 6.4 pM-100 nM anti-B7-H4 antibody (e.g., intermediate ZY0EQD-E02-GL or anti-B7-H4 mAb D11) diluted in DPBS containing 1% BSA and 0.3% Triton X-100. The plate was then washed three times with PBS containing 0.1% Tween and incubated for 1 hour at room temperature with goat anti-human light chain antibody conjugated to peroxidase (Sigma Aldrich, Poole, UK). The plate was then washed five times with PBS containing 0.1% Tween. The reaction was terminated by adding 0.5 M H2SO4 after 5 minutes of incubation with TMB peroxidase substrate. The absorbance at 450 nm (A450) was measured using an Envision multi-label microplate reader (PerkinElmer, Seer Green, UK).

[0539] Flow cytometry (for detecting antibodies that bind to B7-H4 positive cells)

[0540] Cells were isolated from tissue culture flasks using acutase (Gibco, Paisley, UK), precipitated by centrifugation, and resuspended in ice-cold DPBS (Gibco, Paisley, UK). Viable cells were counted using a hemocytometer with trypan blue exclusion. The cell density was adjusted to 5 x 10⁶ cells / mL in DPBS. 100 μL of cell suspension (5 x 10⁵ cells) was added to 96-well V-plates and placed on ice. The cells were then incubated on ice for 20 minutes with Live / Dead fixative purple stain (Thermo Fisher Scientific, Loughborough, UK). After washing with flow cytometry buffer (eBiosciences, Hatfield, UK), cells were incubated on ice for 30 min in 100 μL of flow cytometry buffer (unstained control cells) or supplemented with AF647-labeled anti-B7H4 antibody (e.g., E02-GL or 1D11) or isotype R347 at concentrations ranging from 10 μg / ml to 78 ng / ml. Cells were then washed three times with 200 μL of ice-cold flow cytometry buffer, fixed with 200 μL of 4% paraformaldehyde (Sigma-Aldrich, Poole, UK) for 20 min, and then resuspended on ice in DPBS for flow cytometry analysis on a FACSCanto II instrument (BD Biosciences, San Jose, CA, USA). The following uses FlowJo cell counting analysis software (Treestar, Ashland, Oregon, USA) to quantify antibody binding to cells. Live single cells were gated based on forward scattering, side scattering, and Live / Dead purple fluorescence intensity, and the geometric mean fluorescence intensity (MFI) of AF647 was determined.

[0541] Cytotoxicity assay

[0542] Cell lines were isolated from tissue culture flasks using acutase (Gibco, Paisley, UK), precipitated by centrifugation, and resuspended in growth media (McCoy's 5A, 10% FBS, 400 μg / mL G418 for HT29-hB7H4; McCoy's 5A, 10% FBS for SKBR3; and RPMI-1640, 10% FBS for HCC1569). Viable cells were counted using a hemocytometer with trypan blue exclusion. The cell density was adjusted to 2.7 x 10⁴ cells / mL in the growth medium. 75 μL of cell suspension (2 x 10³ cells) per well was added to 96-well clear-bottomed tissue culture plates and incubated overnight at 37°C in a humidified tissue culture incubator with 5% CO₂.

[0543] ADCs (e.g., 1D11-MMAE, E02-GL-MMAE, and E02-GL-SG3249) were diluted in DPBS to obtain stock solutions of 400 μg / mL or 16 μg / mL, respectively. Four-fold serial dilutions of stock solutions were prepared in DPBS. Then, 25 μL of the diluted stock solution was added to duplicated wells of cultured cells, while the antibody (e.g., 1D11-MMAE, E02-GL-MMAE, or E02-GL-SG3249) was serially diluted ten times to four-fold concentrations. 25 μL / well of DPBS was added to control cells that had undergone a simulated treatment.

[0544] Cells were cultured for six days in the presence of either ADC or DPBS (simulated control cells). Cell viability was assessed using the CellTiter-Glo® assay: 100 μL of CellTiter-Glo® (Promega, Southampton, UK) was added to each well. The plate was stirred on a benchtop shaker for 2 minutes, then incubated at room temperature for another 10 minutes. Luminescence was measured using an Envision multi-label microplate reader (PerkinElmer, Seer Green, UK). The potency of the test antibody (e.g., 1D11-MMAE, E02-GL-MMAE, or E02-GL-SG3249) was determined by generating the half-maximal inhibitory concentration (IC50) value using a nonlinear regression model [log agonist vs. response - variable slope (three parameters)] in GraphPad Prism version 7 (GraphPad Software, La Jolla, CA). The IC50 value was expressed as a percentage of cell viability relative to simulated control cells - ([(treated cells - background) / (simulated control cells - background)] x 100).

[0545] Protein blotting on cells

[0546] A method for Western blotting on cells was developed and used to run five exemplary antibody selections using the following cell types: SKBR3, A549, OVCAR4 (all minus transfection with B7-H4 vector), CHO and HEK cells (with and without transfection with full-length B7-H4 vector).

[0547] In addition to the exemplary antibody selection, the following antibodies were also used (for comparative purposes):

[0548] ˙E Biosciences 14-5949 Anti-human B7H4 mouse IgG

[0549] US biological B0000-35B anti-human B7H4 mouse IgG

[0550] ˙R and D systems AF2514 anti-mouse B7H4 goat IgG1

[0551] Sigma SAB2500141 Anti-B7H4 Goat IgG1

[0552] ˙Type 1 CAT004 SP06-003

[0553] ˙Isotype 2 R and D normal goat IgG control (AB-108C)

[0554] Affinity analysis KinExA 3200

[0555] EO2_GL Fab:E02_GL Fab SEC component (33.4-34.5 min, 07031802.D), B7H4:hB7H4-ECD-Flag-His10 (4.34 mg mL-1, J Watson, October 31, 2017).

[0556] KinExA buffer:

[0557] 1 liter of D-PBS (VWR / Merck 103692K, batch: K35580906) with 0.02% sodium azide added, sterile filtered at 0.20 μm.

[0558] D-PBS (VWR / Merck 103692K, batch: K35580906) with 0.02% sodium azide and 1 mg mL⁻¹ bovine serum albumin (Sigma A-2058, batch: 108H0573) was added. 1.0 L, sterile filtered through 0.20 μm.

[0559] Second test reagent:

[0560] Full IgG or Fab was detected using the DyLight 649-labeled mouse anti-human H+L chain secondary assay kit (Jackson Immunoresearch, 209-495-088, batch 91003). Vials were reconstituted with 800 μL Milli-Q water (approximately 1 mg).

[0561] Minimal amine biotinylation of r-B7H4 ECD:

[0562] Protein: r-human B7H4 ECD-FlagHis10 (4.34 mg mL-1, Batch 1, October 31, 2017)

[0563] Source: JWPur006

[0564] Volume / buffer: 0.100 mL / PBS

[0565] Protein quality (Da): 29,053.57 Da

[0566] The quality of biotinylated protein is 0.434 mg.

[0567] The amount of biotinylated protein required (picomoles) is 0.000434 g / 29,054 Da = 1.494 E-8 moles (14,938 picomoles).

[0568] Biotinylation:

[0569] Add 10 μL of saturated NaHCO3 to D-PBS.

[0570] Reagent: EZ link Sulfo-NHS-LC-Biotin (Perbio, product number 21335), in (1.0 mg mL⁻¹, in DMF).

[0571] First pulse protein:Biotin ratio: 1:0.5 14,938 × 0.5 = 14,938

[0572] ○7,468 / 1,797pmol μL-1=4.16μL

[0573] Start time: 16:07; Sampling time: 16:35

[0574] All of these were applied to a Dulbecco's PBS equilibrated PD-10 column.

[0575] In vivo imaging studies

[0576] Antibodies (e.g., E02_GL) were labeled with 800CW (LI-COR Biosciences). R347 labeled with 800CW was used in the control experiment.

[0577] CT26 / 4TI / HT29 cancer cells expressing B7-H4 were transplanted (e.g., subcutaneously) into the left ventral region of 3–5 day-old nude mice (Charles River Laboratories, Wilmington, Massachusetts (MA)), and CT26 / 4TI / HT29 cancer cells not expressing B7-H4 were transplanted into the right ventral region to provide an internal control. Mice were fed for one week to induce tumor formation and injected with 800CW-labeled E02_GL. 800CW-labeled R347 was injected into control mice. In vivo imaging of the tumors was performed on days 1, 3, 7, and 9 following injection of the labeled antibody by imaging radiation from the labeled antibody. Example 1 B7-H4 is overexpressed in multiple cell types.

[0578] Immunohistochemistry was performed on sections from multiple tumor tissue samples taken from human subjects, representing various tumor types (as described in Table 2). The presence of B7-H4 was found to be particularly pronounced in breast cancer (e.g., hormone receptor-positive (HR+) breast cancer) and non-small cell lung cancer (NSCLC) (see Figures 1A-1D). Interestingly, most tumors exhibited heterologous features.

[0579] The outcomes remained unchanged in a subset of patients after treatment (HER2+ breast cancer patients treated with Herceptin; and ovarian cancer patients treated with platinum-based chemotherapy).

[0580]

[0581] 1 Positive: Tumors stained with any intensity and frequency.

[0582] 2. High positivity: Tumors with membrane staining intensity >2+ in >50% of tumor cells.

[0583] 3. Low positivity: Membrane staining intensity in <50% of tumor cells. 2+ tumors

[0584] Data based on tissue microarray analysis Example 2 Production of anti-B7-H4 antibodies

[0585] A repeated multisite immunization (RIMS) strategy was employed, in which VelocImmune II mice (Regeneron, Tarrytown, New York) were immunized as follows:

[0586] - -4 days before bleeding

[0587] - Day 0: Initial Immunity

[0588] - 7 days: Second reinforcement

[0589] - Day 13: First bleeding

[0590] - 15 days: Third reinforcement

[0591] - Day 20: Second bleeding

[0592] - 22 days: Fourth buff

[0593] - 24 days: Fifth reinforcement

[0594] - Day 28: Last bleeding, and fusion of spl and ln

[0595] For immunization, sixteen V2 mice were divided into four groups of four animals each. The animals were immunized with recombinant human and mouse B7-H4 and SkBr3 cells (e.g., expressing B7-H4). Details of the immunogens can be found in Table 3 below (TT = tetanus toxin; DTA = diphtheria toxin; KLH = keyhole hemocyanin).

[0596] [Table 3].

[0597] The formation of fusion tumors

[0598] Lymphoid cells were harvested from 10 mice, and B cells were enriched from cells from mice 3 and 9. No selection was performed on cells from the other 8 mice. Lymphoid cells and Sp2 / 0 Ag14 myeloma cells were mixed at a 5:1 ratio, washed in serum-free medium, and fused manually or by a Tecan robot using PEG. After fusion, the cells were resuspended in 200 ml of complete HM20 medium, and 100 μl was added to columns 1-11 of 20 plates. After 3 days, another 100 μl of medium was added to each well. Fusion details for each mouse are shown in Table 4.

[0599]

[0600] Fusion tumor screening and selection

[0601] The supernatant harvested 13 days after fusion was screened using bead-based IgG / IgM screening by biochemical binding assays of B7H4 HTRF in the fusion tumor group, as well as in humans, cynomolgus monkeys, and mice, and further screened using HTS in SkBr3 FMAT assays.

[0602] Following screening, positive matches were selected, with 58 of them being transferred to semi-solid culture medium. IgG-positive isolates were selected from each well using ClonePix-FL and then screened in a primary assay. Up to four isolates were then selected from each well for growth and small-scale IgG purification (Phytip-protein A).

[0603] After biological screening of the Phytip material, five exemplary antibodies were selected for further characterization (details can be found in Table 5).

[0604]

[0605] ZY0EPQ-E02 (and its phylogenetic forms, such as those represented by the abbreviation "GL") contains CHDR1-3 (each) of SEQ ID NO:1-3 and CLDR1-3 (each) of SEQ ID NO:4-6. ZY0EPQ-E02 contains the VH chain of SEQ ID NO:31 and the VL chain of SEQ ID NO:32.

[0606] ZY0EQD-E02 contains CHDR1-3 (each) of SEQ ID NO:7-9 and CLDR1-3 (each) of SEQ ID NO:10-12. ZY0EQD-E02 contains the VH chain of SEQ ID NO:33 and the VL chain of SEQ ID NO:34. The phylogenetic form of ZY0EQD-E02 will later be referred to, for example, by including the abbreviation "GL", as EQD-E02_GL (with the VH chain of SEQ ID NO:45 and the VL chain of SEQ ID NO:34).

[0607] ZY0EOB-F05 contains CHDR1-3 (each) of SEQ ID NO:13-15 and CLDR1-3 (each) of SEQ ID NO:16-18. ZY0EOB-F05 contains the VH chain of SEQ ID NO:35 and the VL chain of SEQ ID NO:36.

[0608] ZY0EO5-E07 contains CHDR1-3 (each) of SEQ ID NO:19-21 and CLDR1-3 (each) of SEQ ID NO:22-24. ZY0EO5-E07 contains the VH chain of SEQ ID NO:37 and the VL chain of SEQ ID NO:38.

[0609] ZY0EP0-C07 contains CHDR1-3 (each) of SEQ ID NO:25-27 and CLDR1-3 (each) of SEQ ID NO:28-30. ZY0EP0-C07 contains the VH chain of SEQ ID NO:39 and the VL chain of SEQ ID NO:40.

[0610] These five exemplary antibodies were reformulated onto the backbones of human IgG1, human IgG1-TM (triple mutation), and mouse IgG1.

[0611] Sequence analysis of these five reformulated exemplary antibodies revealed the identity between ZY0EPQ-E02 and ZY0EQD-E02, as well as between the selected ZY0EOB-F05 and ZY0EO5-E07 (see Figure 28). Example 3 Antigen binding assay performed using selected strains

[0612] Concentration-effect binding of these five exemplary antibodies (such as mouse IgG1, human IgG1, and human IgG1-TM) was measured using human, cynomolgus monkey, and mouse B7-H4-Fc assays. For the mouse IgG1 antibody, detection was performed using anti-mouse IgG conjugated to Dylight-649. Similar assays were performed with human IgG1 and IgG1-TM antibodies; however, anti-human κ conjugated to Dylight-649 was used for detection.

[0613] Compared to binding to human or cynomolgus monkey B7-H4-Fc, antibodies ZY0EPQ-E02 and ZY0EQD-E02 exhibited higher EC50 and lower maximum binding to mouse B7-H4-Fc, indicating lower affinity for mouse B7-H4. All other antibodies showed similar EC50 and maximum binding to human, cynomolgus monkey, and mouse B7-H4-Fc (see Tables 6-8).

[0614]

[0615]

[0616]

[0617] Combined with affinity measurement

[0618] The affinity of antibodies for human, mouse, and splice variant B7-H4 ECD was measured using the ForteBio Octet system. Human IgG1-TM antibody was captured by protein G, and the binding of the monomeric substance B7-H4-FLAGECD to this antibody was measured. Affinity to human B7-H4 ranged from 10–25 nM, while affinity to mouse ranged from 10–600 nM. All antibodies except ZY0EP0-C07 bound the splice variant with affinity ranging from 200–1600 nM. Kinetic binding data are summarized in Table 9 below.

[0619]

[0620] Epitope grouping

[0621] Epitope grouping was performed using an exemplary antibody (IgG1-TM) conjugated with the monomer B7-H4-FLAG ECD and DyLight-649 via HTRF assay, and detected with europium-conjugated anti-FLAG antibody.

[0622] Group 1 was defined as antibodies ZY0EPQ-E02, ZY0EQD-E02, and ZY0E05-E07 completely inhibiting each other and only partially inhibiting ZY0EPO-C07. Group 2 was defined as antibody ZY0EOB-F05 completely competing with all antibodies and all antibodies completely competing with it. Group 3 was defined as ZY0EP0-C07 partially competing with antibodies ZY0EPQ-E02, ZY0EQD-E02, and ZY0E05-E07 (Group 1).

[0623]

[0624] Species Cross-Reactivity ELISA

[0625] Binding of each of the five exemplary anti-B7-H4 antibodies derived from VelocImmune to internally derived mouse B7-H4 (ECD) was tested. All exemplary antibodies were tested as human IgG1-TM and 4 / 5 mouse IgG1 (ZY0EP0-C07 was not available as mouse IgG1 at this time). Binding to monomeric and dimeric mouse and human B7-H4 variants (labeled with FlagHis10 or FcHis6, respectively) was compared.

[0626] Regardless of the isotype, all IgGs retained cross-reactivity with both mouse and human B7-H4 antigens. Binding curves generated by the dimer-like, FcHis6-labeled B7-H4 antigen shifted uniformly to the left of those curves provided by the corresponding monomer, FlagHis10, B7-H4 antigen. This effect was more pronounced with mouse B7-H4.

[0627] No significant binding to irrelevant antigen controls was observed using any of the exemplary VelocImmune antibodies. The allotype control IgGs did not bind nonspecifically to the B7-H4 antigens (NIP228-human IgG1-TM and MOPC-21-mouse IgG1) – see Figure 2A-2K.

[0628] Commercial anti-B7-H4 multi-select and single-select specific ELISA

[0629] The binding of a panel of commercially available single- and multi-selective antisera to human and mouse B7-H4 antigens was tested by ELISA. Binding of each antiserum to a truncated human B7-H4 splice variant (essentially lacking its extracellular IgV domain) was also tested. Equivalent data were obtained using both FlagHis10 and FcHis6 B7-H4 antigens. The different cross-reactivity profiles are summarized in Tables 11 and 12.

[0630]

[0631]

[0632] B7-H4 homolog-specific ELISA

[0633] Non-specific binding to B7-H4 family members and homologs was tested in each of five selected select (VelocImmune IgG) antigens (mouse IgG1 ZY0EP0-C07 was available for testing). The selection of antigens to be tested was guided by a BLASTP search using the human B7-H4 ECD as the test sequence. CLUSTALW analysis was then used to compare favorable hits from this list and to identify those antigens showing the highest percentage of identity at the primary amino acid level (see Table 13).

[0634] [Table 13].

[0635] The five exemplary antibodies did not bind to homologs huB7-H1, huB7-H2, huB7-H3, huBTN1A1, huHHLA2, or huBTN3A2 to a measurable extent, regardless of the antibody isotype. Note that no binding was observed when testing for huMOG1, huBTN2A1, or huBTNL3 antigens (see Figure 3).

[0636] Western blot binding analysis of proteins on cells

[0637] Cells were isolated from T175 flasks and counted using Accutase. The transfection method used was essentially the one described in the Invitrogen Lipofectamine LTX protocol (a high-throughput protocol using a reverse transfection method). 100 μl of cells (4.5 x 10⁴ cells / 100 μl) were added to 96-well plates containing the DNA-liposome transfection mixture. The plates were incubated at 37°C for 18 h and fixed overnight with 10% buffered formalin. The plates were blocked with Odyssey blocking buffer, incubated overnight with an appropriate primary antibody, washed, and detected with an appropriate Odyssey secondary antibody.

[0638] The plate was air-dried and then scanned using an Odyssey imager at the manufacturer's recommended scan level.

[0639] QD-E02 and PQ-E02 exhibited high levels of nonspecific binding, thus the antibodies unexpectedly bound to a wide range of cell types in a manner exceeding that of the control antibodies (lanes E, U, R, S) – see Figure 4. OB-F05 and O5-E07 produced good results with transfected HEK cells and also bound to transfected CHO cells. Exemplary antibodies QD-E02, OB-F05, and O5-E07, as well as PQ-E02, bind to OVCAR4 cells. Overview

[0640] Table 14 shows a summary of the properties of five exemplary antibodies.

[0641] Example 4 Screening of cell lines for B7-H4 surface expression using flow cytometry

[0642] The following human cell lines have been tested positive for B7-H4 using exemplary antibodies and / or commercial anti-B7-H4 antibodies (relative B7-H4 expression level is indicated by "+"):

[0643] - SK-BR-3(+++)

[0644] - T47D(+)

[0645] - MDA-MB-468(++)

[0646] - OVCAR4(+++)

[0647] - NIH:OVCAR3(+)

[0648] - Calu-3(+)

[0649] The following human cell lines were tested negative: NCI-H322, Raji (+ / - IFNg activation), Ramos (+ / - IFNg activation), and Du145. Example 5 In vitro cytotoxicity assay

[0650] Internalization of five exemplary human IgG1-TM antibodies (SK-BR-3, MDA-MB-468, OVCAR4, and JumpIn CHO Fl-B7H4) in B7-H4 cell lines has been experimentally confirmed. The ADC cytotoxicity of the five exemplary IgG1-TM antibodies was demonstrated using saponin-conjugated anti-human IgG secondary antibodies (see Figure 5). Cell viability was approximately 100% at the 20 μg / ml 'antibody alone' control. Example 6 ZY0EQD_E02 specific ELISA selection

[0651] Due to the superior performance of the selected strain ZY0EQD_E02, it was chosen for more detailed analysis. ZY0EQD_E02 contains CHDR1 of SEQ ID NO:7; CHDR2 of SEQ ID NO:8; CHDR3 of SEQ ID NO:9; CLDR1 of SEQ ID NO:10; CLDR1 of SEQ ID NO:11; and CLDR1 of SEQ ID NO:12. The selected strain contains the VH chain of SEQ ID NO:33 and the VL chain of SEQ ID NO:34. The selected strain contains the heavy chain of SEQ ID NO:48 and the light chain of SEQ ID NO:44. The germplasm form (E02-GL) contains the VH chain of SEQ ID NO:45 and the VL chain of SEQ ID NO:34; for example, the heavy chain of SEQ ID NO:51 and the light chain of SEQ ID NO:44.

[0652] ELISA analysis was performed to determine binding to B7-H4 from humans (including splice variants), cynomolgus monkeys, mice, and rats. The percentage of sequence identity of human B7-H4 from these species was as follows: domain FL – cynomolgus monkey (98.6%); rabbit (91.6%); mouse (87.9%); rat (86.9%); domain ECD – cynomolgus monkey (99.6%); rabbit (94.3%); mouse (90%); rat (89.6%).

[0653] Binding to selected Maia form mAbs (e.g., containing C insertions, such as SEQ ID NO.:41) was tested. Wild-type and several variants (germination (GL), GLQ, GLY) were measured, along with R347-maia human IgG1 as a control (see Figure 6).

[0654] The R347-maia isotype control showed no binding to any of the tested antigens. The binding profile of E02-maia was similar to previous experiments. E02-maia and E02-GL-maia had similar binding profiles. Advantageously, this demonstrates that both germline (GL) and non-germination (e.g., WT) forms of the antibody retain the favorable binding properties / profile of the antibody. Example 7 Comparison of E02-GL binding and mAb "1D11" binding

[0655] By means of ELISA, the binding affinity of germline selected ZY0EQD_E02 (hereinafter referred to as E02-GL) was directly compared with the binding affinity of the known anti-B7-H4 antibody "1D11" (Genentech; described in WO 2016040724, which is incorporated herein by reference) (see Materials and Methods above).

[0656] The "E02-GL" antibody has a CDR sequence of ZY0EQD-E02 (e.g., corresponding to it) ("GL" indicates that the antibody has been germinated). For example, E02-GL in such examples contains the VH chain sequence of SEQ ID NO:45, such as the germinated form of SEQ ID NO:43.

[0657] Direct comparisons showed that the selected E02-GL exhibited significantly better binding (affinity) than 1D11 (see Figure 7). Consistent with Example 7, the R347-maia isotype control showed no binding to any of the tested antigens.

[0658] Furthermore, compared to 1D11, E02-GL showed (through flow cytometry analysis, see Materials and Methods above) better binding to B7-H4 expressed on cancer cells. Therefore, E02-GL not only exhibited excellent binding but also excellent cancer cell targeting. Binding with HT29 cells and SK-BR-3 cells (expressing B7-H4) was tested. Binding was tested at antibody concentrations of 31 ng / ml, 156 ng / ml, and 78 ng / ml. Results for HT29 cells are shown in Figure 8A, and results for SK-BR-3 cells are shown in Figure 8B. The symbol ◇ indicates the 'E02-GL portion', and the symbol □ indicates the '1D11 portion'. The symbol "●" indicates the (negative) control 'R347 component'.

[0659] As can be seen from Figures 8A-8B, the number of stained cells in the "E02-GL section" is significantly higher than the number of stained cells in the "1D11 section". Example 8 In vitro cytotoxicity of Ad293 cells, with and without B7-H4 transfection (E02-GL-SG3932)

[0660] E02-GL was selected and conjugated to the topoisomerase I payload SG3932 at a mean drug-antibody ratio (DAR) of 8 (provided as E02-GL-SG3932), and its ability to target and kill human B7-H4 transfected and untransfected Ad293 cells was tested (e.g., the latter represents a negative control). The antibody NIP228 conjugated to SG3932 was used as a control. Transfected cells were readily targeted and killed by the E02-GL-SG3932 conjugate (with an IC50 of 53.3 ng / ml) (see Figure 9A). No significant killing effect was observed in untransfected cells after the addition of the E02-GL-SG3932 conjugate (see Figure 9B). In all experiments described herein involving E02-GL conjugated to a topoisomerase inhibitor, the E02-GL selected generally had the heavy chain sequence of SEQ ID NO.:51.

[0661] method:

[0662] Cell lines were isolated from tissue culture flasks using TrypLE Express (Gibco, Paisley, UK), precipitated by centrifugation, and resuspended in growth medium (RPMI-1640, 10% FBS). Viable cells were counted using the trypan blue exclusion method and a Vi-CELL XR cell viability analyzer (Beckman Coulter Life Sciences, Indianapolis, Indiana). The cell density was adjusted to 3.33 x 10⁴ cells / mL in growth medium. 75 μL / well of cell suspension (2.5 x 10³ cells) was added to 96-well clear-bottomed white-walled tissue culture plates and incubated overnight at 37°C in a humidified tissue culture incubator with 5% CO₂.

[0663] The ADC (e.g., NIP228-SG3932 and E02-GL-SG3932) was diluted in growth medium (RPMI-1640, 10% FBS) to a concentration of 240 μg / mL. Five-fold serial dilutions were prepared in growth medium (RPMI-1640, 10% FBS), and 25 μL was added to three wells of cultured cells, while nine-fold serial dilutions of the antibody (e.g., NIP228-SG3932 or E02-GL-SG3932) were added to control cells. 25 μL / well of growth medium (RPMI-1640, 10% FBS) was added to control cells that had undergone a similar treatment. Cells were cultured for six days in a humidified tissue culture incubator at 37°C and 5% CO2, during which time cell viability was assessed using the CellTiter-Glo® assay (Promega, Southampton, UK) according to the manufacturer's protocol. Efficacy was measured using an Envision multi-label microplate reader (PerkinElmer, Seer Green, UK). The half-maximal inhibitory concentration (IC50) values ​​of the test antibody (e.g., NIP228-SG3932 or E02-GL-SG3932) were determined by using a nonlinear regression model [log agonist vs. response - variable slope (four parameters)] in GraphPad Prism version 8 (GraphPad Software, La Jolla, CA) to generate the IC50 values, expressed as a percentage of cell viability relative to simulated control cells – ([(treated cells - background) / (simulated control cells - background)] x 100). Example 9 E02-GL-SG3932 induces bystander killing of tumor cells in vitro.

[0664] HT29-huB7-H4 selected 26 target-positive cells and GFP-labeled HT29 target-negative cells (plated individually or in a 1:1 ratio and cultured for 1 day) were treated with 200 ng / mL E02-GL-SG3932 or NIP228-SG3932 isotype control ADC for 6 days. At the end of treatment, the number of viable GFP-negative HT29-huB7-H4 selected 26 cells or GFP-positive HT29 cells was determined by flow cytometry. The results are shown in Figure 10: A) The dot plot shows representative images of the culture medium-treated samples from HT29-GFP cells and HT29+huB7-H4 selected 26 cells cultured individually or together at the end of treatment, analyzed by flow cytometry. The digits shown in the upper left and lower left quadrants reflect the percentages of HT29-GFP cells and HT29+huB7-H4 selected 26 cells, respectively. B) When plated alone, a decrease in cell count was observed in HT29+huB7H4 selected 26 cells after treatment with E02-GL-SG3932, which was not observed in target-negative HT29-GFP cells. When cells were plated in co-culture, cell counts decreased in both target-negative HT29-GFP cells and target-positive HT29+huB7H4 selected 26 cells, demonstrating bystander killing effect.

[0665] method:

[0666] Stable HT29-GFP cell lines expressing green fluorescent protein (GFP) were prepared using a lentiviral expression system. Cells were seeded at a total cell density of 15,000 cells / well in 24-well plates and cultured individually or in a 1:1 co-culture ratio for 1 day. The culture medium was then removed and replaced with fresh medium alone or with medium containing 200 ng / mL NIP228-SG3932 ADC or E02-GL-SG3932 ADC, and the cells were incubated for another 6 days. At the end of the treatment, the number of live GFP-negative HT29+huB7-H4 selected 26 cells or GFP-positive HT29 cells was determined using flow cytometry and FlowJo software. Example 10 E02-GL-SG3932 induces bystander killing of tumor cells in the body.

[0667] Tumor cells were subcutaneously implanted into 8- to 10-week-old female SCID mice. When the tumor reached an appropriate tumor volume range (typically 150-250 mm³), the animals were randomly assigned to treatment and control groups and administration was initiated. A single dose of the test substance was administered intravenously to the tumor-bearing mice. Animals were observed daily, and tumor size and body weight were measured and recorded two to three times weekly. The results are shown in Figure 11. Tumor volume was measured using calipers and calculated using the following formula: Tumor volume = length (mm) x width (mm)² / 2, where length and width are the longest and shortest diameters of the tumor, respectively. Example 11 E02-GL-SG3932 in patient-derived xenotransplantation It has effective in vivo activity in the (PDX) model.

[0668] Tumor tissue fragments were subcutaneously implanted into 6- to 8-week-old female athymic nude mice. When the tumor reached an appropriate tumor volume range (typically 150–300 mm³), the animals were randomly assigned to treatment and control groups and administration began. A single dose of the test substance was administered intravenously to the tumor-bearing mice. Animals were observed daily, and tumor size and body weight were measured and recorded twice weekly. The results are shown in Figure 12 and demonstrate that E02-GL-SG3932 has potent in vivo activity in a patient-derived xenograft (PDX) model. Tumor volume was measured using a digital caliper and calculated using the following formula: Tumor volume = length (mm) x width (mm)² x 0.5², where length and width are the longest and shortest diameters of the tumor, respectively.

[0669] Figure 13 shows the results of further evaluation of the in vivo antitumor response to E02-GL-SG3932 by determining the change in tumor volume after treatment, further demonstrating the antitumor activity of E02-GL-SG3932 in the PDX model after a single IV injection. The tumor volume at the start of treatment is called the initial tumor volume (ITV); the tumor volume at which the ADC treatment shows the maximum response is called the final tumor volume (ETV). If the ETV is less than the ITV, the antitumor response is calculated as follows: [(ETV-ITV) / ITV] x 100. Otherwise, the antitumor response is expressed as the percentage change in tumor volume in the treatment group relative to the medium control group: 100 - [1 - ((ETV-ITV) treatment / (ETV-ITV) medium) x 100]. Example 12 E02-GL-topo I inhibitor ADC It exhibits similar potency in in vitro and in vivo MX-1 cell models and HT29-derived models.

[0670] MX-1 model

[0671] The MX-1 xenograft model was conducted at Crown Biosciences (Taicang, China). The study was performed according to CRO and AstraZeneca IACUC guidelines. MX-1 tumor cells were cultured in vitro at 37°C in a 5% CO2 atmosphere in RPMI-1640 medium supplemented with 10% fetal bovine serum. Exponentially growing cells were harvested, and 5 × 10⁶ cells (in 0.1 ml of PBS:Matrix gel = 1:1) were implanted into the right abdomen of female BALB / c nude mice. Mice were immediately randomized to treatment groups (mean tumor volume = 153 mm³) using a matched overdistribution randomization method (Study Director™ software) before treatment. All antibody-drug conjugates were immediately diluted in buffer (25 mM histidine, 7% sucrose, 0.02% PS80, pH 6.0) before administration. ADCs were administered via a single intravenous dose. Tumor and body weight measurements were recorded twice weekly, and tumor volume was calculated using the equation length (mm) x width (mm)² / 2. Morbidity and mortality were monitored daily. Results are shown in Figure 14A.

[0672] HT29+huB7-H4 (selected for 4)

[0673] The HT29+huB7-H4 selected cell line was cultured in vitro at 37°C under a 5% CO2 atmosphere in McCoy's modified 5A medium supplemented with 10% fetal bovine serum. Exponentially growing HT29+B7-H4 selected cells were harvested, and 5 × 10⁶ cells (in 0.2 ml of PBS:Cultrex = 1:1) were implanted into the right abdomen of female CB-17 SCID mice. Mice were immediately randomized to treatment groups (mean tumor volume = 250 mm³) using a deterministic randomization method (Study Director™ software) prior to treatment. All antibody-drug conjugates were immediately diluted in buffer (25 mM histidine, 7% sucrose, 0.02% PS80, pH 6.0) before administration. A single dose of ADC was administered intravenously. Tumor and body weight measurements were recorded twice weekly, and tumor volume was calculated using the equation length (mm) x width (mm² / 2). Morbidity and mortality rates of the animals were monitored daily. The results are shown in Figure 14B. Example 13 In vitro cytotoxicity of cynomolgus monkey cells transfected with B7-H4 (E02-GL-SG3249)

[0674] E02-GL was selected and conjugated to the cytotoxin SG3249 (providing E02-GL-SG3249), and its ability to target and kill cynomolgus monkeys transfected with B7-H4 and untransfected cells was tested. For convenience, the antibody tested in this example containing the Maia heavy chain backbone (e.g., with a cysteine ​​insert, which provides an additional conjugable site for SG3249, see SEQ ID NO:48) was used. No loss of efficacy occurred when using other heavy chain backbones (e.g., lacking this cysteine ​​insert, see SEQ ID NO:52, for example). Antibody R347 conjugated to SG3249 was used as a control.

[0675] The "E02-GL" antibody has a CDR sequence of ZY0EQD-E02 (e.g., corresponding to it) ("GL" indicates that the antibody has been germinated). For example, E02-GL in such examples contains the VH chain sequence of SEQ ID NO:45, such as the germinated form of SEQ ID NO:43.

[0676] Transfected cells were readily targeted and killed by the E02-GL-SG3249 conjugate (IC50 of 0.6721 ng / ml). No significant killing effect was observed in untransfected cells after the addition of the E02-GL-SG3249 conjugate (see Figure 15). Example 14 E02-GL working mode

[0677] Monitoring the internalization kinetics of E02-GL in living cells

[0678] Live cells were treated with E02-GL conjugated to the fluorescent marker AF647. At 0 min post-treatment, fluorescent clusters were observed on the cell membrane (indicating binding to B7-H4 present on the membrane). Time-series analysis showed a steady increase in the number of intracellular fluorescent spots over time, indicating that the antibody was internalized along with the receptor antigen. Co-visualization with Lamp1-AF488 (a lysosomal marker) showed significant overlap, indicating that the bound antibody was internalized via endocytosis (see Figures 16A-16C).

[0679] The internalization of antibodies observed after binding to the target antigen is highly advantageous, as such internalization is generally considered a prerequisite for achieving the desired ADC effect.

[0680] The "E02-GL" antibody has a CDR sequence of ZY0EQD-E02 (e.g., corresponding to it) ("GL" indicates that the antibody has been germinated). For example, E02-GL in such examples contains the VH chain sequence of SEQ ID NO:45, such as the germinated form of SEQ ID NO:43.

[0681] E02-GL-SG3932 ADC: Mechanism of Action

[0682] After treating HT29+huB7-H4 selected 26 cells with NIP228, E02-GL, NIP228-SG3932, E02-GL-SG3932, or warhead SG3924 (as a control) (mAb or ADC 10 ug / ml, warhead 10 nM), lysates were prepared and Western blotted with antibodies against pATR, ATR, pChk1, Chk1, pATM, ATM, pChk2, ChK2, pH2AX, H2AX, and actin (as a loading control) – see Figure 17A. The results showed that the topoisomerase I poison warhead (SG3924) of E02-GL-SG3932 activated ATM and ATR signaling, indicating that E02-GL-SG3932 treatment led to double-stranded DNA breaks in vitro.

[0683] After treating MX-1 cells with NIP228, E02-GL, NIP228-SG3932, E02-GL-SG3932, or warhead SG3924 (as a control) (mAb or ADC 10 μg / ml, warhead 10 nM), lysates were prepared and analyzed by Western blot with antibodies against pATR, ATR, pATM, ATM, pH2AX, H2AX, and actin (as a control) – see Figure 17B. The results showed that the topoisomerase I poison warhead (SG3924) of E02-GL-SG3932 activated ATM and ATR communication, indicating that E02-GL-SG3932 treatment led to double-strand DNA breaks in vitro.

[0684] method:

[0685] HT29+huB7-H4 selected 26 and MX-1 cells were seeded at densities of 500,000 and 1,500,000 cells / well in 6-well plates containing 10% heat-inactivated FBS. The next day, the seeding medium was removed, and the cells were incubated together with HT29+huB7-H4 selected 26 cells, NIP228, E02-GL, NIP228-SG3932, and E02-GL-SG3932 (10 μg / mL) in complete medium. 10 nM warhead SG3924 was used as a control. After 72 hours, the cells were washed once with phosphate-buffered saline (PBS) and then lysed with Laemmil reduction buffer (loading buffer, Boston BioProducts). After a brief incubation, cell lysates were collected, and equal volumes of the cell lysates were loaded onto Bis NuPAGE Novex Bis-Tris gel (Ingenie), and proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Ingenie). The membrane was blocked with 5% skim milk powder and 0.1% Tween 20 (Sigma) (TBST) in Tris-buffered saline (pH 7.4) and incubated overnight at 4°C with antibodies from Cell Signaling against pATM-Ser1981 (#4526), ​​ATM (#2873), pATR-Thr1989 (#58014), ATR (#13934), pChk1-Ser345 (#2348), Chk1 (#2360), pChk2-Thr68 (#2197), Chk2 (#3440), pH2AX-Ser139 (#2577), and H2AX (#2595). An antibody against actin (A1978, Sigma) was used to ensure equal protein loading in all wells. The membrane was washed in 0.1% Tween 20 in TBS and then incubated for 1 hour with a streptavidin secondary antibody conjugated with wasabi peroxidase (HRP) (GE Healthcare). After washing, protein bands were detected using SuperSignal West Femto and SuperSignal West Pico chemiluminescent substrates (Pierce / Thermo Scientific). Images were captured and analyzed using an ImageQuant LAS4000 system (GE Healthcare).

[0686] E02-GL-SG3249 ADC: Mechanism of Action

[0687] After treating HCC1569 cells with E02-GL-SG3249 and SG3199 (as controls) (100 ng / ml and 100 pM, respectively), lysates were prepared and Western blotted against the following antibodies: pATR, ATR, pChk1, Chk1, pRPA32, RPA32, pATM, ATM (all involved in ATR communication), pChk2, ChK2, pKAP1, KAP1 (all involved in ATM communication), pDNA-PK, DNA-PK, pH2AX, H2AX, pBRCA1, BRCA1 (all involved in DNA double-strand breaks), pFANCD2, and GAPDH (loading control). The negative control was PBS treatment only.

[0688] The results showed that the PBD dimer warhead (SG3199) of E02-GL-SG3249 activated ATM and ATR communication, indicating that E02-GL-SG3249 treatment led to double-strand DNA breaks in vitro (see Figure 17C).

[0689] Caspase 3 / 7 activity

[0690] SKBR-3 cells were treated with E02-GL-SG3249, SG3199, and olaparib (control), and caspase 3 / 7 activity (e.g., apoptosis) was monitored using IncuCyte. A dose-dependent increase in caspase 3 / 7 activity levels was observed (see Figure 18). Example 15 In vitro activity of E02-GL-SG3249 and warhead SG3199 against tumor cells

[0691] Multiple cancer cell lines were treated with E02-GL-SG3249 (e.g., conjugated to cytotoxic agents), as shown in Table 15. The results are shown in Figure 19.

[0692] Example 16 E02-GL-SG3249 induces bystander killing of tumor cells in vitro.

[0693] HT29 B7-H4 expressing cells were treated with E02-GL-SG3249 for 4 days, after which the conditioned medium was removed and added to HT29 WT (i.e., non-B7-H4 expressing cells). A rapid decrease in cell viability was observed after the addition of conditioned medium, which was not observed in the untreated control (see Figure 20).

[0694] The "E02-GL" antibody has the CDR / VH sequence of ZY0EQD-E02 (e.g., corresponding to it) ("GL" indicates that the antibody has been germinated). Example 17 E02-GL-SG3249 inhibits the growth of tumor xenografts.

[0695] Tumor xenografts were prepared in mice using the following cancer cell lines:

[0696] - OVCAR4 (cisplatin-refractory ovarian cancer; high B7-H4 expression)

[0697] - HCC1569 (HER2-positive breast cancer; heterologous B7-H4 manifestation)

[0698] - MDA-MB-468 (Triple-negative breast cancer; low B7-H4 profile)

[0699] The "E02-GL" antibody has the CDR / VH sequence of ZY0EQD-E02 (e.g., corresponding to it) ("GL" indicates that the antibody has been germinated).

[0700] Compared with the control (mediator only), a significant reduction in tumor volume was observed, which was surprising, even for TNBC tumors with low levels of B7-H4 expression (see Figure 22), indicating that E02-GL-SG3249 has high efficacy in inhibiting tumor growth. Example 18 E02-GL-SG3249 induces bystander killing of tumor cells in the body.

[0701] B7-H4 xenografts were generated by co-transplanting HT29 cell lines expressing B7-H4 with those not expressing it (in a 1:1 ratio). The xenogeneity of these xenografts did not prevent the growth inhibition of E02-GL-SG3249 on them, which is evident (see Figure 23). This is highly advantageous because the inventors have already discovered that B7-H4 is xenogeneically expressed within tumors. Example 19 Compared to "1D11", E02-GL ADC exhibits superior in vitro cytotoxicity against cells expressing B7-H4.

[0702] Selected E02-GL (ZY0EQD_E02 germinated (GL)) was conjugated to cytotoxic SG3249 (providing E02-GL-SG3249 ADC) or AZ1508 (providing E02-GL-AZ1508) and compared with Genentech's "1D11" ADC (1D11 conjugated with (A114C-)MMAE) to compare their ability to target and kill cells expressing B7-H4.

[0703] The "E02-GL" antibody has the CDR / VH sequence of ZY0EQD-E02 (e.g., corresponding to it) ("GL" indicates that the antibody has been germinated).

[0704] Cytotoxicity assays (see Materials and Methods above) showed that E02-GL ADC (E02-GL-SG3249) had superior cytotoxicity compared to 1D11 ADC (1D11 conjugated with (A114C-)MMAE) – see Figure 21.

[0705] Perform ADC titration as follows: Example 20 E02-GL-SG3249 in patient-derived xenotransplantation It has effective in vivo activity in the (PDX) model.

[0706] Patient-derived xenograft models were generated using cancer cell lines with different B7-H4 expression levels, as described in Table 16. Tumor growth inhibition was observed in all models (see Figure 24). The "E02-GL" antibody has the CDR / VH sequence of ZY0EQD-E02 (e.g., its corresponding sequence) ("GL" indicates that the antibody has been germinated).

[0707] Example 21 E02-GL-SG3249 induces double-strand breaks in tumor xenografts.

[0708] A tumor xenograft model was generated (using HCC1954 cells) and treated with E02-GL-SG3249. The "E02-GL" antibody has the CDR / VH sequence of ZY0EQD-E02 (e.g., corresponding to it) ("GL" indicates that the antibody has been germinated).

[0709] Immunohistochemistry was then performed to determine the presence of γ H2AX (a marker of double-strand breaks) after treatment. An increase in the number of γ H2AX-positive tumor cells was observed up to 10 days after E02-GL-SG3249 treatment (see Figure 25). Example 22 Affinity analysis of E02_GL using KinExA 3200

[0710] The following experiments were performed using KinExA technology to obtain an estimate of the affinity (KD) between the selected E02_GL anti-B7H4 Fab and human B7H4. The "E02-GL" antibody has a CDR / VH sequence of ZY0EQD-E02 (e.g., corresponding to it) ("GL" indicates that the antibody has been germinated).

[0711] A range of human B7H4 concentrations were equilibrated overnight at 25°C in the presence of fixed concentrations of 5, 10, or 45 nM Fab. These concentrations were analyzed on a KinExA 3200, and the dataset was fitted globally (N-curve analysis). The results are summarized in Table 17.

[0712]

[0713] The 62% hB7H4 activity figure matches very well with the Rmax calculation and the Biacore-based affinity assessment of E02_GL. Example 23 Comparison of E02_GL combined with B7-H4 and mAb "1D11" combined with B7-H4

[0714] ELISA analysis was performed on selected E02_GL antibodies to determine their binding to human and mouse B7-H4. The binding was directly compared to mAb 1D11 (Genentech) and the R347 isotype control. The "E02-GL" antibody possesses the CDR / VH sequence of ZY0EQD-E02 (e.g., its corresponding sequence) ("GL" indicates that the antibody has been germinated).

[0715] Compared to mouse B7-H4, E02_GL showed a higher binding affinity for human B7-H4 (see, for example, Figure 2D). Advantageously, compared to Genentech's "1D11" mAb (which showed weaker binding), E02_GL showed significantly stronger binding affinity for human B7-H4, as demonstrated by ELISA and FACs analyses – see Figures 7 and 8, respectively. In fact, Genentech's "1D11" mAb bound human B7-H4 at a similar (low) level to mouse B7-H4 (indicating that "1D11" has lower specificity for human proteins compared to E02_GL). Example 24 Imaging studies showing tumor localization of E02_GL

[0716] In vivo imaging studies were performed as described in Materials and Methods (above), using an 800CW-labeled antibody (E02_GL). The "E02-GL" antibody has the CDR / VH sequence of ZY0EQD-E02 (e.g., corresponding to it) ("GL" indicates that the antibody has been germinated).

[0717] For HT29, p=0.06 was observed on days 3, 7, and 14; for CT26, p=0.06 was observed on days 3 and 7, and on day 10; and for 4T1, p=0.06 was observed on day 7. In all cases, B7-H4 Ab(E02_GL) was preferentially localized to tumors expressing B7-H4 compared to WT tumors - see Figures 26 and 27.

[0718] Total radiation efficiency yielded the clearest results. A similar trend was observed when normalized relative to tumor volume. Example 25 Toxicological studies were conducted using E02-GL-SG3249.

[0719] E02-GL-SG3249 was administered to male cynomolgus monkeys (N=2 / dose level). The "E02-GL" antibody has the CDR / VH sequence of ZY0EQD-E02 (e.g., corresponding to it) ("GL" indicates that the antibody has been germinated).

[0720] No abnormal toxicity was observed, and the toxicity was consistent with other similar PBD-ADCs (by monitoring standard target organs: kidney, bone marrow, skin; there was no evidence of target-related effects). Pharmacological studies

[0721] In vitro and in vivo pharmacological studies were conducted to further characterize the action and mechanism of action of the E02-GL antibody (the E02-GL antibody and its antigen-binding Fab intermediate, the latter referred to herein as "E02-INT"). The E02-GL-SG3932 ADC is an ADC targeting B7-H4 and comprises an anti-B7-H4 human IgG1κ monoclonal antibody (i.e., E02-GL) conjugated to a TOP1i warhead via a cleavable maleimide-PEG8-valine-alanine linker (a cleavable mal-PEG8-val-ala linker). The TOP1i warhead is referred to herein as SG3924. The TOP1i drug is covalently bound to the native cysteine ​​in the antibody via a thiosuccinimide linker, with each antibody binding approximately 8 drug molecules (i.e., DAR 8). A schematic diagram of the E02-GL-SG3932 ADC is shown in Figure 29A. The main differences between the E02-GL-SG3932 ADC and its competitors' ADCs are shown in Figure 29B.

[0722] The specific characteristics of E02-GL-SG3932 are described below and further elsewhere in this document:

[0723] mAb E02-INT: specifically binds to human and cynomolgus monkey B7-H4 (affinity 3.7 nM and 3.94 nM, respectively).

[0724] SG3932 warhead

[0725] Linker-warhead: Decomposable mal-PEG8-val-ala linker-SG3932 In vitro studies Example 26 B7-H4 phenotype analysis by immunohistochemistry

[0726] The expression profile of B7-H4 was evaluated using a validated IHC protocol to demonstrate its expression in FFPE normal cynomolgus monkey and human tissues, as well as human tumor tissues.

[0727] B7-H4 was observed in a limited number of normal human tissues, and when present, it was typically present in <10% of the total cells of the sample (except for the oviducts and pulmonary bronchi), limited to ductal or tubular epithelium, and primarily located on the apical membrane (Table 18). A similar spectrum of B7-H4 was detected in normal cynomolgus monkey tissues (Tables 2 and 19), where B7-H4 was observed in a limited number of tissues, with the spectrum limited to ductal or tubular epithelium and primarily located on the apical membrane and within the cytoplasm. Figure 30 shows representative images of immunohistochemical staining for B7-H4 in selected normal human and cynomolgus monkey tissues.

[0728]

[0729] c: cytoplasm; m: membrane.

[0730] All stained slides were reviewed and scored by a pathologist, who assessed the proportion of cells exhibiting B7-H4, staining intensity, and stained cell localization. Intensity was reported as weak (+), moderate (++), or strong (+++).

[0731] IHC was used to assess B7-H4 presentation in a range of human tumor tissues, and the results were scored by pathologists. Table 19 provides a summary of human tumor tissue presentation, and representative images are shown in Figure 31.

[0732]

[0733] a High B7-H4: Human tumor tissue samples containing 50% to 100% B7-H4 positive cells.

[0734] b. Low B7-H4: Human tumor tissue samples containing 5% to 49% B7-H4 positive cells.

[0735] c. Positive B7-H4: Human tumor tissue samples containing 1% to 4% B7-H4 positive cells.

[0736] d represents the proportion of human tumor tissue samples containing 1% to 100% B7-H4 positive cells.

[0737] This example demonstrates the presence of B7-H4 in various types of human cancer, including breast cancer, bile duct cancer, endometrial cancer, non-small cell lung cancer, squamous cell carcinoma, and ovarian serous carcinoma. Example 27 Comparison of sequence homology between human B7-H4 and heterologous homologs in cynomolgus monkeys, rhesus monkeys, mice, and rats

[0738] The amino acid sequence of human B7-H4 was identified and compared with heterologous homologs in cynomolgus monkeys, rhesus monkeys, mice, and rats. The percentage of identity was calculated based on these comparisons. B7-H4 is highly conserved in non-human primates; for the full-length domain and extracellular domain, human B7-H4 (hB7-H4) shares 98% and 99% sequence identity with cynomolgus monkeys (cyB7-H4) and rhesus monkeys (rhB7-H4), respectively (Figure 32). Rodent species show poor conservation; for the full-length B7-H4, mouse and rat B7-H4 share 87% and 86% sequence identity with hB7-H4, respectively, and 90% and 89% sequence identity with hB7-H4 in the extracellular domain, respectively.

[0739] This example demonstrates that the high amino acid sequence identity of B7-H4 in humans and non-human primates suggests that E02-GL-SG3932 may bind to B7-H4 in cynomolgus monkeys and rhesus monkeys. In contrast, the relatively low amino acid identity among B7-H4 in humans, mice, and rats suggests that binding of E02-GL-SG3932 to B7-H4 in mice or rats is unlikely. Example 28 Stability of E02-GL-SG3932 after 15 days of incubation in mouse, rat, cynomolgus monkey and human serum.

[0740] It is known that ADCs carrying drugs linked to cysteine ​​via thiosuccinimide exhibit some drug loss in the physiological environment due to the reverse Michael reaction. This process regenerates the cysteine ​​used for conjugation and the drug carrying maleimide, thereby reducing the ADC's DAR over time. This deconjugation process is a known characteristic of ADCs containing drugs linked to antibodies via thiosuccinimide.

[0741] The stability of E02-GL-SG3932 in sera from cynomolgus monkeys, mice, and rats was assessed by immunoprecipitation followed by reversible reduction mass spectrometry (rLCMS). Measurements showed that less than 20% of E02-GL-SG3932 was lost after incubation in mouse, rat, and cynomolgus monkey sera; at day 15, 84.5%, 83.5%, and 82.0% of SG3924 remained attached to E02-GL-SG3932 in mouse, rat, and cynomolgus monkey serum samples, respectively. The stability of E02-GL-SG3932 in human serum was assessed by immunocapture using human B7-H4-coated resin followed by rLCMS. Measurements showed minimal drug loss after incubation in human serum; at day 15, 81% of SG3924 remained attached to E02-GL-SG3932.

[0742] This example demonstrates that the drug release mechanism is via deconjugation through the reverse Michael reaction rather than linker cleavage, consistent with other ADCs prepared by conjugation of maleimide with cysteine ​​amino acids contained in interchain disulfides. Example 29 The binding affinity of antibody intermediate E02-INT Fab to recombinant B7-H4 antigen

[0743] The binding affinity of the anti-B7-H4 antibody E02-INT Fab to recombinant human, cynomolgus monkey, and mouse B7-H4 variants was determined by surface plasma resonance (SPR). The dissociation constant (KD) values ​​shown in Table 20 demonstrate that E02-INT Fab binds to immobilized human and cynomolgus monkey B7-H4 with similar affinity. In contrast, E02-INT Fab exhibits approximately 100-fold lower affinity for the mouse B7-H4 antigen compared to the human B7-H4 antigen.

[0744]

[0745] Fab: Antigen-binding fragment Example 30 E02-GL-SG3932 binding affinity to human FcRn and Fcγ receptors

[0746] The binding affinity of E02-GL-SG3932 to human FcRn and Fcγ receptors was assessed using SPR. The steady-state binding affinity (KD) of human FcRn to E02-GL-SG3932 was 4360 nM. The equilibrium KD of E02-GL-SG3932 to huFcγRI was 4.35 nM. The equilibrium KD of E02-GL-SG3932 to huFcγRIIa, huFcγRIIb, huFcγRIIIA-158V, and huFcγRIIIA-158F ranged from 3307 to 21640 nM. Example 31 Comparison of binding affinity of antibody intermediates E02-INT and E02-GL-SG3932 to recombinant human B7-H4 antigen

[0747] To assess whether the conjugation of the topoisomerase 1 linker-warhead affects the binding properties of the E02-INT antibody, the binding affinity of E02-INT and E02-GL-SG3932 was measured using DELFIA-ELISA and SPR methods. As shown in Figure 33, the DELFIA-ELISA results indicate that E02-INT and E02-GL-SG3932 bind similarly to immobilized recombinant human B7-H4, with EC50 values ​​of 1.98 nM and 1.71 nM, respectively. The kinetic rate constants (kon and koff) and equilibrium dissociation constants (KD) of E02-INT and E02-GL-SG3932 to human B7-H4 antigen were also determined using SPR and antibody capture methods. As shown in Table 21, E02-INT and E02-GL-SG3932 bind similarly to human B7-H4, with KD values ​​of 31.1 nM and 29.3 nM, respectively.

[0748]

[0749] RU: Resonance Unit

[0750] The results of Examples 30 and 31 show that the binding properties of the E02-INT antibody remain unchanged after conjugation with the topoisomerase 1 linker-warhead. Example 32 The antibody intermediate E02-INT binds to engineered HEK 293 cells expressing human, cynomolgus monkey, or mouse B7-H4.

[0751] The binding of antibody E02-INT to untransduced HEK 293 Jump In TREX cells and stable HEK 293 Jump In TREX cells expressing human, mouse, or cynomolgus monkey B7-H4 was measured using flow cytometry. Antibody E02-INT bound stable HEK 293 Jump In TREX cells expressing human, mouse, and cynomolgus monkey B7-H4, but not to untransduced HEK 293 Jump In TREX cells that were B7-H4 negative (Fig. 34). Binding to HEK 293 Jump In TREX cells expressing mouse B7-H4 was reduced compared to cells expressing human or cynomolgus monkey B7-H4. Example 33 Antibody intermediates E02-INT and E02-GL-SG3932 bind to human breast cancer cell lines and HT29 cells that stably express human B7-H4.

[0752] The binding of antibodies E02-INT and E02-GL-SG3932 to human breast cancer cell lines MX-1 and MDA-MB-468, as well as to engineered HT29 colorectal cancer cells stably expressing human B7-H4, was measured using flow cytometry. As shown in Figure 35, E02-INT and E02-GL-SG3932 similarly bound to HT29 cells stably expressing human B7-H4 (HT29-huB7-H4 selector 4 and HT29-huB7-H4 selector 26), but not to B7-H4-negative, untransduced HT29 cells. E02-INT and E02-GL-SG3932 also bound to MX-1 and MDA-MB-468 cells, indicating that the antibody intermediates and ADCs can recognize endogenously expressed B7-H4 in human cancer cell lines. The binding modulotropic properties of E02-INT and E02-GL-SG3932 were comparable.

[0753] Examples 32 and 33 demonstrate that the cell-binding properties of the parental antibody remain unchanged after conjugation with the topoisomerase 1 linker-warhead. Example 34 In vitro cytotoxicity of E02-GL-SG3932

[0754] The effect of E02-GL-SG3932 treatment on cell viability was determined using CellTiter-Glo assays on the target-negative human colorectal cancer cell line HT29, engineered human colorectal cancer cell line HT29-huB7-H4 Selected 26, and human breast cancer cell line MX-1. As shown in Figure 36, E02-GL-SG3932 exhibited cytotoxicity against B7-H4-expressing HT29-huB7-H4 Selected 26 and MX-1 cells, with IC50 values ​​of 0.036 μg / mL and 0.029 μg / mL, respectively. In contrast, no difference in activity was observed between E02-GL-SG3932 and the isotype-matched control ADC (NIP228-SG3932) in the B7-H4-negative HT29 cell line, indicating that E02-GL-SG3932 can specifically kill human B7-H4-expressing cancer cells. Example 35 In vitro cytotoxicity of E02-GL-SG3932

[0755] When IgG antibodies bind to cell surface antigens via their Fab domain, the Fc moiety of the antibody can bind to FcγRIIIa on natural killer cells. The interaction between the Fc domain and FcγRIIIa induces cross-linking of FcγR, thereby triggering the release of cytotoxic particles containing perforin and granzymes, leading to target cell death—a process known as antibody-dependent cytotoxicity (ADCC). The potential of antibodies E02-INT and E02-GL-SG3932 to activate ADCC activity was evaluated using isolated primary human NK cells and the human breast cancer SKBR3 cell line as target cells. In this assay, both E02-INT and E02-GL-SG3932 generated significantly increased ADCC activity compared to untreated co-cultured cells (Figures 36A-36C). The activity of E02-GL-SG3932 was slightly decreased compared to E02-INT, but this difference was not statistically significant, indicating that both E02-INT and E02-GL-SG3932 can induce ADCC activity in vitro.

[0756] Next, human breast cancer SK-BR-3 cells were co-cultured with NK cells isolated from six healthy donors in the presence of 1 μg / mL E02-GL-SG3932, antibody intermediate E02-INT, isotype-matched control antibody NIP228, and isotype-matched control ADC NIP228-SG3932 (Figure 37). ADCC activity was assessed by the bright and stable red fluorescence signal generated by Incycte® Annexin V dye binding to phosphatidylinosine exposed on the extracellular surface of apoptotic cells. Fold changes were based on the maximum cell death of untreated NK / SK-BR-3 co-cultured cells. Maximum cell death was calculated by dividing the average red target in the experimental wells by the average maximum cell death in stelloprene-treated SK-BR-3 cells. Example 36 Internalization and lysosomal transport of antibody E02-INT

[0757] The internalization and intracellular transport properties of antibody E02-INT were assessed using quantitative live-cell imaging in MX-1 human breast cancer cells and HT29-huB7-H4-selected 26 human colorectal cancer cells overexpressing B7-H4. Time-lapse images showed a strong E02-INT membrane signal persisting from 0 min to 120 min in both the human breast cancer (MX-1) and colorectal cancer (HT29-huB7-H4-selected 26) cell lines, with internalization of E02-INT continuously increasing by 240 min (Figs. 38, 39A, and 39B). Internalization kinetics measurements in both cell lines showed internalization half-lives of 127 (±35 SD) min and 102 (±18 SD) min in HT29-huB7-H4-selected 26 and MX-1 cells, respectively (Fig. 39C).

[0758] Intracellular transport of E02-INT was determined using confocal microscopy by measuring colocalization with early endosome markers, namely early endosome antigen 1 (EEA1) and lysosomal markers, namely lysosome-associated membrane protein 1 (LAMP1). As shown in Figure 40, E02-INT was enriched in LAMP1-modified subcellular compartments, while colocalization with EEA1 was limited, indicating that after internalization, E02-INT was transported into the lysosomal compartments of the cell. Example 37 In vitro activation of DNA damage response communication by E02-GL-SG3932 and SG3924

[0759] Following treatment with E02-GL-SG3932 or its TOP1i warhead SG3924, Western blotting was used to assess DDR pathway activation in the MX-1 human breast cancer cell line and the engineered colorectal cancer cell line HT29-huB7-H4. As shown in Figure 41, treatment of MX-1 cells with 10 μg / mL E02-GL-SG3932 or 10 nM SG3924 induced ATM signaling pathway activation. Notably, ATM (Ser 1981) phosphorylation increased as early as 24 hours and continued to increase throughout the 72-hour treatment period. Similarly, E02-GL-SG3932 and SG3924 induced ATR activation (Thr 1989 phosphorylation), observed at 48 and 72 hours. An increase in γH2AX was observed 24 hours after treatment and persisted into the 72-hour treatment period, indicating DNA damage.

[0760] The effect of E02-GL-SG3932 or its TOP1i warhead on DDR signaling was also examined in the colorectal cancer cell line HT29-huB7-H4 selected 26 (engineered to represent B7-H4). As shown in Figure 42, treatment with 10 μg / mL E02-GL-SG3932 or 10 nM SG3924 resulted in activation of the DDR signaling pathway, as evidenced by increased phosphorylation of ATR (Thr 1989), its downstream targets Chk1 (Ser 345), and Chk2 (Thr 68). This increase in phosphorylation persisted throughout the 72-hour treatment period. Similarly, activation of ATM (Ser 1981) and its downstream target KAP1 (Ser 824) was observed at 48 and 72 hours after treatment with E02-GL-SG3932 and the TOP1i warhead. An increase in γH2AX was observed 48 hours after treatment and persisted into the 72-hour treatment period, indicating DNA damage.

[0761] In summary, these results confirm that in two different cell lines, E02-GL-SG3932 activates the DDR pathway consistent with the mechanism of action of its TOP1i warhead. In vivo studies

[0762] In vitro studies demonstrated that E02-GL-SG3932 can bind to human and cynomolgus monkey B7-H4 with similar affinity. Specifically, E02-GL-SG3932 specifically binds to and is cytotoxic to tumor cells expressing B7-H4. Furthermore, it can induce moderate ADCC activity in co-culture assays of isolated primary NK cells, where the antibody intermediate of E02-GL-SG3932 (E02-INT) is internalized into tumor cells and transported to the lysosomal compartment. Treatment with E02-GL-SG3932 or its TOP1i warhead activated the DDR signaling pathway in B7-H4-expressing cell lines. To further elucidate the mechanism of action of E02-GL-SG3932 and determine whether these in vitro findings translate into antitumor activity, an in vivo mouse model was used. Example 38 Pharmacodynamic Study of E02-GL-SG3932 in HT29-huB7-H4 Selected Xenotransplantation Model 26

[0763] The pharmacodynamic effects of E02-GL-SG3932 treatment were evaluated in a human tumor xenograft mouse model using immunodeficient CB-17 SCID mice. Animals were subcutaneously (SC) inoculated with the engineered human colorectal cancer cell line HT29-huB7-H4 Select 26 to represent human B7-H4, and after tumor volume reached approximately 250–300 mm³, animals were randomly assigned to receive either E02-GL-SG3932 or a control via IV injection. Tumors were collected at designated time points, fixed in 10% neutral buffered formalin, subsequently processed, and embedded in paraffin blocks. Human IgG, γH2AX lesions, lysed caspase-3, and epithelial cell density over time were examined in tumor samples using IHC and image analysis techniques. Figures 43A-43F show representative IHC images of human IgG, γH2AX, and cleaved caspase-3 in tumors collected 168 hours after a single IV dose of 7 mg / kg E02-GL-SG3932 or the type-matched control ADC NIP228 SG3932.

[0764] As shown in Figures 44A-44D, a dose-dependent accumulation of E02-GL-SG3932 over time was observed in tumor cells, as seen by human IgG IHC assay. The accumulation of E02-GL-SG3932 was associated with increased positive staining of γH2AX lesions, predicting the induction of DNA damage. Compared to controls, elevated levels of cleaved caspase-3 and an overall decrease in epithelial cell density were observed over time in tumors treated with E02-GL-SG3932.

[0765] In summary, these data indicate that E02-GL-SG3932 binds to B7-H4 on tumor cells, causing DNA damage and apoptotic cell death. Example 39 Antitumor efficacy of E02-GL-SG3932 in subcutaneous human breast cancer and colorectal cancer xenograft models, in vivo efficacy, PDX

[0766] The antitumor activity of antibody intermediates E02-INT and E02-GL-SG3932 was investigated in a human tumor xenograft mouse model using immunodeficient CB-17 SCID mice.

[0767] Selected xenotransplantation models using HT29 or HT29-huB7-H4

[0768] For the first time, E02-GL-SG3932, a B7-H4-negative HT29 cell line, and HT29-huB7-H4 Selected 26 derived from the HT29 cell line and engineered to represent human B7-H4 were evaluated in two independent studies in a pair of colorectal cancer cell line xenograft models. In both studies, animals were injected with HT29 or HT29-huB7-H4 Selected 26 cells in sclerosis cells, and after the tumor volume grew to an average of 178 mm3 (HT29) or 194 mm3 (HT29-huB7-H4 Selected 26), the animals were randomized to receive an IV injection of either E02-GL-SG3932 or a control. As shown in Figures 45A and 45B, compared with the control group treated with the medium, neither E02-GL-SG3932 nor the allotype-matched control ADC NIP228-SG3932 significantly inhibited the growth of HT29 xenograft tumors when administered as a single IV dose of 10 mg / kg, showing 12% (p=0.7006) and 14% (p=0.6593) TGI, respectively. In contrast, compared with the control group treated with the medium or the type-matched control ADC NIP228-SG3932, E02-GL-SG3932 significantly inhibited the growth of HT29-huB7-H4 selected 26 xenograft tumors when administered in single IV doses of 5 mg / kg, 2.5 mg / kg and 1.25 mg / kg (Figures 46A-46C), with TGI of 42% (p<0.001), 37% (p=0.0005) and 31% (p=0.0039) relative to the medium, respectively.

[0769] MX-1 breast cancer xenograft model

[0770] The effects of E02-GL-SG3932, the allotype-matched control ADC NIP228-SG3932, and E02-INT (an antibody intermediate of E02-GL-SG3932) were evaluated in an MX-1 breast cancer xenograft model. Animals were injected with MX-1 cells (SCs), and after the tumor volume reached an average of 270 mm³, the animals were randomly assigned to either the test or control drug via intravenous injection. Compared to the control group treated with the drug, E02-GL-SG3932 significantly inhibited the growth of MX-1 xenograft tumors when administered via single intravenous doses of 5 mg / kg, 2.5 mg / kg, and 1.25 mg / kg (Figures 47A and 47B), with TGIs of 100% (p<0.001), 96% (p<0.001), and 98% (p<0.001), respectively.

[0771] Antibody intermediate E02-INT administered at 10 mg / kg did not significantly inhibit tumor growth (TGI = -1%, p > 0.9999). The lack of E02-INT activity at this high dose level in SCID mice suggests that ADCC may not be a significant contributor to the activity of E02-GL-SG3932 in vivo, and that the antitumor effect of E02-GL-SG3932 is driven by its TOP1i warhead.

[0772] MX-1 and MDA-MB-468 breast cancer xenograft models

[0773] To further elucidate the dose-dependent effect of E02-GL-SG3932 treatment in vivo, dose levels ranging from 0.125 mg / kg to 2 mg / kg were evaluated in MX-1 and MDA-MB-468 breast cancer xenograft models. In both studies, animals were injected with MX-1 or MDA-MB-468 cells (SCs), and after tumor volume grew to an average of approximately 138 mm3 (MX-1) or 120 mm3 (MDA-MB-468), the animals were randomly assigned to receive an IV injection of either E02-GL-SG3932 or a control. As shown in Figure 48, a single intravenous dose of E02-GL-SG3932 resulted in dose-dependent inhibition of the MX-1 xenograft. Treatment with E02-GL-SG3932 at dose levels of 2 mg / kg, 1.5 mg / kg, and 1 mg / kg all resulted in 100% TGI compared to the untreated group on day 40. E02-GL-SG3932 doses of 0.75 mg / kg, 0.5 mg / kg, 0.25 mg / kg, and 0.125 mg / kg resulted in dose-dependent %TGIs of 99.7%, 80.1%, 52.1%, and -5.5%, respectively. Similarly, a single intravenous dose of E02-GL-SG3932 resulted in dose-dependent inhibition of the MDA-MB-468 xenograft (Figure 49). Compared with the untreated group at day 55, E02-GL-SG3932 treatment at dose levels of 2 mg / kg, 1.5 mg / kg, and 1 mg / kg resulted in TGIs of 97.7%, 75.2%, and 66.6%, respectively. E02-GL-SG3932 doses of 0.75 mg / kg, 0.5 mg / kg, 0.25 mg / kg and 0.125 mg / kg resulted in dose-dependent %TGI of 52.8%, 58.4%, 25.5% and 31.8%, respectively. Example 40 Antitumor efficacy of E02-GL-SG3932 in a patient-derived triple-negative breast cancer xenograft model

[0774] The antitumor activity of E02-GL-SG3932 was investigated in a cohort of 26 human TNBC PDX models using immunocompromised athymic nude mice. These PDX models were established from human tumor samples without prior in vitro culture, and their histological, cytogenetic, genetic, and other biomarker profiles, as well as their response to standard treatment, have been investigated. Tumor fragments were subcutaneously transplanted into mice, and once the tumor volume reached approximately 94 to 189 mm³, the animals were randomly assigned to receive a single IV injection of either 1.25 mg / kg or 3.5 mg / kg of E02-GL-SG3932 or a control. To assess the relationship between B7-H4 performance and efficacy, fresh, untreated tumors with volumes of approximately 500 to 1183 mm³ were collected from three additional mice in each model, fixed in 10% neutral buffered formalin, subsequently processed, and embedded in paraffin blocks. Then, IHC and image analysis techniques were used to characterize the performance of B7-H4 on the tumor cell membrane in each model.

[0775] IHC analysis showed that these PDX models represented heterologous tumor manifestations of B7-H4, with varying levels of IHC staining intensity and tumor staining proportions, including a model of B7-H4 with almost undetectable levels (HBCx-15) (Figure 50).

[0776] Tumor growth inhibition was observed after a single dose of 1.25 mg / kg E02-GL-SG3932, with 46.2% of the models (12 out of 26) showing a reduction in tumor volume of 30% or more from baseline (Figure 51). Among these, 75% (9 out of 12) showed elevated levels of B7-H4, with an H score of 100 or higher (Figures 52A and 52B). For the 1.25 mg / kg dose level of E02-GL-SG3932, a significant correlation was identified between H score classification and responder status (Fischer exact test, p = 0.047), indicating that elevated B7-H4 levels were associated with a response to treatment with 1.25 mg / kg E02-GL-SG3932.

[0777] Compared to the 1.25 mg / kg dose level, a single dose of 3.5 mg / kg E02-GL-SG3932 showed greater antitumor activity, with 69.2% (18 out of 26) of the tested models exhibiting a 30% or greater reduction in tumor growth from baseline (Figure 53). Of these, 66.7% (12 out of 18) showed elevated levels of B7-H4, with an H score of 100 or higher (Figures 54A and 54B). At this dose level, the association between H score classification and responder status was not statistically significant (Fischer exact test, p = 0.073).

[0778] At this dose level, increased activity of the NIP228-SG3932 allotype ADC was also observed, with five models (T330, BCX-017-LOP, T168, HBCx-15, and HBCx-6) showing a 30% or greater reduction in tumor growth from baseline (Figure 53). These five models shared a common characteristic: defective homologous DNA repair, defined as the presence of BRCA1 mutations or a negative score in the RAD51 lesion formation assay, with the latter suggested as a functional measure of homologous DNA repair capacity.

[0779] This example demonstrates that administration of E02-GL-SG3932 to tumors with elevated B7-H4 expression levels and defective homologous DNA repair (defined as (1) the presence of BRCA1 mutations and (2) a negative score in the RAD51 lesion formation assay) can reduce tumor volume. Figures 53B-53E show that at low doses, the response of E02-GL-SG3932 is associated with B7-H4 expression levels and HR deficiency. In another study, a PDX model treated with 7 mg / kg showed high levels of allotype control ADC activity (Figures 60A-60B and 61).

[0780] This example also demonstrates that E02-GL-SG3932 exhibits potent activity in HR-deficient tumors and HR-proficient tumors with elevated B7-H4, and that HR-deficient tumors are more sensitive to TOP1i-induced damage and have a lower B7-H4 expression threshold for activity. Example 41 Antitumor efficacy of E02-GL-SG3932 in a patient-derived cholangiocarcinoma xenograft model

[0781] The antitumor activity of E02-GL-SG3932 was investigated in a cohort of 37 human cholangiocarcinoma PDX models. In the preliminary study phase, human cholangiocarcinoma PDX fragments were implanted into female mice from different backgrounds (athymic nude mice, Balb / c nude mice, or NOD / SCID mice) and allowed to grow to approximately 1000–1500 mm³. These tumors were then harvested and reimplanted into study mice. When the tumors reached an average tumor volume of 150–300 mm³, animals were matched to treatment or control groups based on tumor volume, and administration began on day 0. Each mouse received a single IV injection of E02-GL-SG3932 at a dose of 1.25 mg / kg or 3.5 mg / kg, or a control (untreated). Figures 55A–55G present the results for the first study group. Figures 56A–56K show the results for the second study group. Example 42 E02-GL-SG3932 TOP1i connector-bullet is associated with a wider therapeutic index (TI).

[0782] A head-to-head comparative study was conducted on the AZ'0133 linker-warhead selected for E02-GL-SG3932 to determine the efficacy, pharmacokinetics, and toxicity of the SG3932 warhead compared to four alternative linkers. The cleavable mal-PEG8-val-ala linker-SG3932 linker-warhead ADC offered the widest relative TI and showed advantages over the benchmark. Four comparative linker-warheads are presented below. Data collected from the comparative study are provided in Table 22.

[0783] Comparative linker-warhead

[0784] E02-INT-Dissociable mal-PEG8-val-ala linker-SG3932

[0785]

[0786] E02-INT-SG4010

[0787]

[0788] E02-INT-SG4057

[0789]

[0790] E02-INT-SG4052

[0791]

[0792]

[0793] The mp-PEG8-Val-Ala-SG3932 linker-warhead of E02-GL-SG3932 outperformed each of the comparative compounds. When conjugated to the E02-INT antibody, the ADC prepared with the cleavable mal-PEG8-val-ala linker-SG3932 linker-warhead exhibited the highest in vivo activity (Figures 57A and 57B). As shown in Figure 58, the cleavable mal-PEG8-val-ala linker-SG3932 linker-warhead ADC demonstrated the cleanest safety profile in rat toxicity studies. As shown in Figure 59, the cleavable mal-PEG8-val-ala linker-SG3932 linker-warhead ADC exhibited good PK characteristics and the widest relat...

Claims

1. An antibody or antigen-binding fragment thereof that binds to B7-H4, the antibody or antigen-binding fragment comprising: i. heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) respectively containing the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO:

12.

2. The antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the antibody or antigen-binding fragment thereof comprises: i. a variable heavy (VH) chain and a variable light (VL) chain respectively containing the amino acid sequences of SEQ ID NO: 45 and SEQ ID NO: 34; ii. a variable heavy (VH) chain and a variable light (VL) chain respectively containing the amino acid sequences of SEQ ID NO: 33 and SEQ ID NO: 34; or iii. a variable heavy (VH) chain and a variable light (VL) chain respectively containing the amino acid sequences of SEQ ID NO: 43 and SEQ ID NO:

34.

3. The antibody or antigen-binding fragment thereof as claimed in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises: i. a VH chain and a VL chain containing the amino acid sequences of SEQ ID NO: 45 and SEQ ID NO: 34, respectively.

4. The antibody or antigen-binding fragment thereof as described in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof binds to the OVCAR4 cell line.

5. An antibody or antigen-binding fragment thereof as claimed in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region containing the amino acid sequence of SEQ ID NO:

41.

6. An antibody or antigen-binding fragment thereof as claimed in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region containing the amino acid sequence of SEQ ID NO:

52.

7. An antibody or antigen-binding fragment thereof as claimed in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a light chain constant region containing the amino acid sequence of SEQ ID NO:

42.

8. An antibody or antigen-binding fragment thereof as claimed in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 51; and a light chain containing the amino acid sequence of SEQ ID NO:

44.

9. An antibody or antigen-binding fragment thereof as claimed in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 48; and a light chain containing the amino acid sequence of SEQ ID NO:

44.

10. An antibody or antigen-binding fragment thereof as described in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is conjugated with a heterologous drug agent.

11. An antibody or antigen-binding fragment thereof as claimed in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is conjugated to one or more heterologous agents selected from the group consisting of: topoisomerase I inhibitors, tubulolysin derivatives, pyrrolobenzodiazepines, antimicrobial agents, precursors, enzymes, lipids, biological response modifiers, pharmaceutical reagents, lymphatic mediators, heterologous antibodies, fragments of heterologous antibodies, polyethylene glycol (PEG), radioisotopes, or combinations thereof.

12. The antibody or antigen-binding fragment thereof as claimed in claim 11, wherein the antibody or antigen-binding fragment thereof is conjugated to one or more heterologous agents selected from topoisomerase I inhibitors, tubulolysin derivatives, pyrrolobenzodiazepines, or combinations thereof.

13. The antibody or antigen-binding fragment thereof as claimed in claim 12, wherein the antibody or antigen-binding fragment thereof is conjugated to a heterologous agent selected from the group consisting of: tubulolysin AZ1508, pyrrolobenzodiazepine SG3315, pyrrolobenzodiazepine SG3249, or combinations thereof.

14. An antibody or antigen-binding fragment thereof as described in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is conjugated to: ; and / or 15. The antibody or antigen-binding fragment thereof as described in claim 14, wherein the antibody or antigen-binding fragment thereof is conjugated to:

16. The antibody or antigen-binding fragment thereof as described in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody.

17. The antibody or antigen-binding fragment thereof as described in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is a humanized monoclonal antibody.

18. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-17.

19. A polynucleotide encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1-17.

20. A host cell comprising the polynucleotides as described in claim 19.

21. A method for generating an antibody or antigen-binding fragment thereof that binds to B7-H4, the method comprising expressing the polynucleotide as described in claim 19 in a host cell.

22. An antibody or an antigen-binding fragment thereof that can be obtained by the method described in claim 21.

23. Use of an antibody or antigen-binding fragment as described in claim 15 for the preparation of a medicament for treating cancer, wherein the cancer comprises cancer cells expressing B7-H4.

24. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in claim 15.

25. Use of a pharmaceutical composition as claimed in claim 24 for the preparation of a medicament for treating cancer, wherein the cancer comprises cancer cells expressing B7-H4.

26. The use as described in claim 23 or claim 25, wherein the cancer is selected from breast cancer, ovarian cancer, endometrial cancer, bile duct cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, and gastric cancer.

27. The use as described in claim 23 or claim 25, wherein the cancer is selected from breast cancer, ovarian cancer, endometrial cancer and bile duct cancer.

28. The use as described in claim 27, wherein the cancer is breast cancer selected from hormone receptor-positive (HR+) breast cancer, human epidermal growth factor receptor 2-positive (HER2+) breast cancer, and triple-negative breast cancer (TNBC).

29. An in vitro method for detecting the presence of B7-H4 peptide in a sample, the method comprising: i. Contact the sample with an antibody or an antigen-binding fragment thereof as described in any one of claims 1-18 to provide an antibody-antigen complex; ii. Detect the presence of the antibody-antigen complex; iii. Where the presence of the antibody-antigen complex confirms the presence of the B7-H4 peptide; iv. Where the absence of the antibody-antigen complex confirms the absence of the B7-H4 peptide.

30. The method as claimed in claim 29, wherein the presence of the antibody-antigen complex indicates the presence of cancer cells, and wherein the absence of the antibody-antigen complex indicates the absence of cancer cells.

31. The method as described in claim 29 or 30, wherein the B7-H4 polypeptide is a component of cancer cells.

32. An antibody-drug conjugate (ADC) comprising: (i) an antibody or antigen-binding fragment thereof bound to a B7-H4 polypeptide, the antibody or antigen-binding fragment comprising: HCDR1 containing the amino acid sequence of SEQ ID NO: 7; HCDR2 containing the amino acid sequence of SEQ ID NO: 8; HCDR3 containing the amino acid sequence of SEQ ID NO: 9; LCDR1 containing the amino acid sequence of SEQ ID NO: 10; LCDR2 containing the amino acid sequence of SEQ ID NO: 11; and LCDR3 containing the amino acid sequence of SEQ ID NO: 12; (ii) conjugated to SG3932, wherein the ADC has a drug-to-antibody ratio (DAR) of 8.

33. The ADC as claimed in claim 32, wherein the antibody or its antigen-binding fragment comprises a variable heavy (VH) chain containing the amino acid sequence of SEQ ID NO: 45 and a variable light (VL) chain containing the amino acid sequence of SEQ ID NO:

34.

34. The ADC as claimed in claim 32 or 33, the ADC comprising a heavy chain (HC) containing the amino acid sequence of SEQ ID NO: 51 and a light chain (LC) containing the amino acid sequence of SEQ ID NO:

44.

35. A pharmaceutical composition comprising an ADC as described in any one of claims 32-34.

36. Use of an ADC as described in any one of claims 32-34 for the preparation of a medicament for treating cancer, wherein the cancer comprises cancer cells expressing B7-H4.

37. Use of a pharmaceutical composition as described in claim 35 for the preparation of a medicament for treating cancer, wherein the cancer comprises cancer cells expressing B7-H4.

38. The use as described in claims 23, 25, 36 or 37, wherein the cancer cell has a homologous DNA repair defect.

39. The use as described in claim 38, wherein the homologous DNA repair defect is defined by the presence of a BRCA1 mutation.

40. The use as described in claim 38, wherein the homologous DNA repair defect is defined by a negative score in the RAD51 lesion formation assay.

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