TWI806247B - ANTI-HSP90α ANTIBODY AND USES THEREOF - Google Patents

Abstract

Discloased is an isolated antibody comprises: novel complementarity determining regions capable of specifically binding to the HSP90α epitope containing two EDK sites in the amino acid 235 to 244 and amino acid 251 to 260 regions, respectively. Also disclosed are nucleic acid molecules corresponding to the aforesaid antibody, a pharmaceutical composition comprising the aforesaid antibody or the corresponding nucleic acid molecules, and the methods for treating and monitoring a cancer using the aforesaid antibody.

Description

Anti-HSP90α antibody and use thereof

The present disclosure relates to a novel anti-HSP90α antibody and uses thereof. More specifically, the disclosure relates to a novel isolated anti-HSP90α antibody, pharmaceutical compositions comprising the antibody, and methods of using the antibody to treat cancer.

Cancer development and progression depend not only on genetic and epigenetic alterations in epithelial cells but also on key changes in their stromal microenvironment, which consists of the extracellular matrix (ECM) and stromal cells such as fibroblasts and immune cells )composition. See Pandol S, Edderkaoui M, Gukovsky I, Lugea A, Gukovskaya A. Desmoplasia of pancreatic ductal adenocarcinoma. Clin Gastroenterol Hepatol 2009;7(11):S44-7. Desmoplasia, a common feature of many malignancies such as pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC), is defined by abundant ECM as well as abundant expression of α-smooth actin (α-SMA) markers of myofibroblasts (myofibroblasts). Such myofibroblasts, also known as activated fibroblasts or cancer-associated fibroblasts (CAFs), contribute to tumor growth, immunosuppression and malignant progression. See the following literature: Orimo A, Gupta PB, Sgroi DC, Arenzana-Seisdedos F, Delaunay T, Naeem R, et al. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 2005; 121(3):335-48; Lakins MA, Ghorani E, Munir H, Carla P. Martins CP, Shields JD. Cancer-associated fibroblasts induce antigen-specific deletion of CD8 ⁺ T cells to protect tumor cells. Nat Commun 2018; 9(1): 948; Turley SJ, Cremasco V, Astarita JL. Immunological hallmarks of stromal cells in the tumor microenvironment. Nat Rev Immunol 2015; 15(11): 669-82; and Beacham DA, Cukierman E. Stromagenesis: the changing face of fibroblastic microenvironments during tumor progression. Semin Cancer Biol 2005;15(5):329-41. They constitute the stromal cells of most tumors and arise from a variety of cellular sources, such as tissue-resident fibroblasts, stellate cells, mesenchymal stem/precursor cells, and infiltrating fibroblasts. See Sugimoto H, Mundel TM, Kieran MW, Kalluri R. Identification of fibroblast heterogeneity in the tumor microenvironment. Cancer Biol Ther 2006;5(12):1640-6. In addition, CAFs can also be produced by endothelial-mesenchymal transition (EndoMT) of endothelial cells. See Zeisberg EM, Potenta S, Xie L, Zeisberg M, Kalluri R. Discovery of endothelial to mesenchymal transition as a source for carcinoma-associated fibroblasts. Cancer Res 2007;67(21):10123-8. In our previous study, EndoMT-derived CAFs with α-SMA ⁺ CD31 ⁺ cells were detected near osteopontin (OPN) expressing macrophages observed in CRC tissue samples. See Fan CS, Chen WS, Chen LL, Chen CC, Hsu YT, Chua KV, et al. Osteopontin-integrin engagement induces HIF-1α-TCF12-mediated endothelial-mesenchymal transition to exacerbate colorectal cancer. Oncotarget 2018; 9(4) : 4998-5015. OPN induces EndoMT in endothelial cells, and the resulting EndoMT-derived CAFs promote CRC cell stemness by secreting HSP90α, thereby exhibiting potent tumor-promoting effects. See Fan CS, Chen WS, Chen LL, Chen CC, Hsu YT, Chua KV, et al. Osteopontin-integrin engagement induces HIF-1α-TCF12-mediated endothelial-mesenchymal transition to exacerbate colorectal cancer. Oncotarget 2018; 9(4) : 4998-5015. Recently, we also found that endoMT-derived CAFs mixed with PDAC cells transplanted into mice can significantly recruit bone marrow-derived macrophages, block immune T cells and promote tumor growth. See Fan CS, Chen LL, Hsu TA, et al. Endothelial-mesenchymal transition harnesses HSP90α-secreting M2-macrophages to exacerbate pancreatic ductal adenocarcinoma. J Hematol Oncol 2019;12:138. HSP90α secreted by EndoMT-derived CAFs could further induce macrophage M2 polarization and more HSP90α secretion through cell surface receptors CD91 and TLR4 and the downstream MyD88-JAK2/TYK2-STAT-3 pathway. See Fan CS, Chen LL, Hsu TA, et al. Endothelial-mesenchymal transition harnesses HSP90α-secreting M2-macrophages to exacerbate pancreatic ductal adenocarcinoma. J Hematol Oncol 2019;12:138.

HSP90α is a well-known cell chaperone that helps fold, mature and transport many client proteins in cells, including cancer-related Bcr-Abl, ErbB2/Neu, Akt, HIF-1α, mutant Both p53 and Raf-1 are its auxiliary targets. See Trepel JB, Mollapour M, Giaccone G, Neckers L. Targeting the dynamic Hsp90 complex in cancer. Nat Rev Cancer 2010;10(8):537-49. In addition to being inside the cell, HSP90α can also be secreted from the keratinocytes and fibroblasts in the injured tissue to the outside of the cell. In addition, in an unfavorable tissue microenvironment, cancer cells will also express and secrete a large amount of HSP90α to accelerate the growth of the epithelial cells of cancer cells. Mesenchymal transition (EMT), migration, invasion and metastasis. See Li W, Li Y, Guan S, Fan J, Cheng CF, Bright AM, et al. Extracellular heat shock protein-90α: linking hypoxia to skin cell motility and wound healing. EMBO J 2007; 26(5): 1221- 33; Xu A, Tian T, Hao J, Liu J, Zhang Z, Hao J, et al. Elevation of serum HSP90α correlated with the clinical stage of non-small cell lung cancer. J Cancer Mol 2007; 3(4): 107-12; and Wang X, Song X, Zhuo W, Fu Y, Shi H, Liang Y, et al. The regulatory mechanism of HSP90α secretion and its function in tumor malignancy. Proc Natl Acad Sci USA 2009; 106(50) : 21288-93. Clinically, in patients with several malignant tumors, including CRC and PDAC, elevated levels of HSP90α in serum/plasma can be detected in the early stages of cancer. See Wang X, Song X, Zhuo W, Fu Y, Shi H, Liang Y, et al. The regulatory mechanism of HSP90α secretion and its function in tumor malignancy. Proc Natl Acad Sci USA 2009; 106(50): 21288-9 ; Wang X, Song X, Zhuo W, Fu Y, Shi H, Liang Y, et al. The regulatory mechanism of HSP90α secretion and its function in tumor malignancy. Proc Natl Acad Sci USA 2009; 106(50): 21288-93 ; Chen JS, Hsu YM, Chen CC, Chen LL, Lee CC, Huang TS. Secreted heat shock protein 90α induces colorectal cancer cell invasion through CD91/LRP-1 and NF-κB-mediated integrin α _V expression. J Biol Chem 2010 ;285(33):25458-66; and Chen CC, Chen LL, Li CP, Hsu YT, Jiang SS, Fan CS, et al. Myeloid-derived macrophages and secreted HSP90α induce pancreatic ductal adenocarcinoma development. OncoImmunology 2018; 7( 5): e1424612. Pancreatitis is known to be a high-risk factor for PDAC, and blood samples from patients with pancreatitis can also detect elevated levels of extracellular HSP90α (eHSP90α), which means that excess eHSP90α may be related to the formation of PDAC Relatedly, this hypothesis can be supported by an experimental mouse model in which eHSP90α can be obtained from bone marrow-derived macrophages infiltrating the pancreas and from all transgenic mice expressing activated K-Ras in pancreatic ductal epithelial cells before spontaneous development of PDAC. Stimulates pancreatic ductal epithelial cell production to promote PDAC formation. See Chen CC, Chen LL, Li CP, Hsu YT, Jiang SS, Fan CS, et al. Myeloid-derived macrophages and secreted HSP90α induce pancreatic ductal adenocarcinoma development. OncoImmunology 2018;7(5):e1424612. In addition, HSP90α or recombinant HSP90α (rHSP90α) secreted by EndoMT-derived CAFs can induce the expression of M2 markers in macrophages and the feedforward loop of massive HSP90α secretion, thus explaining why M2-polarized macrophages not only It will cause immunosuppression and promote angiogenesis, and also create a tumor microenvironment rich in eHSP90α to promote tumor growth and deterioration of PDAC cells. See Fan CS, Chen LL, Hsu TA, et al. Endothelial-mesenchymal transition harnesses HSP90α-secreting M2-macrophages to exacerbate pancreatic ductal adenocarcinoma. J Hematol Oncol 2019;12:138. In conclusion, eHSP90α plays a key role in both the initial stage of tumor formation and its subsequent progression, and can be considered as an important therapeutic target.

In our previous study, we synthesized a small-molecule HSP90α inhibitor that cannot cross the cell membrane to target eHSP90α. See Chen CC, Chen LL, Li CP, Hsu YT, Jiang SS, Fan CS, et al. Myeloid-derived macrophages and secreted HSP90α induce pancreatic ductal adenocarcinoma development. OncoImmunology 2018;7(5):e1424612. Although it has a certain inhibitory effect on the growth of PDAC cells into tumors and metastasis, but because it is easily excreted from mice, frequent administration has caused spleen enlargement in mice. On the other hand, we obtained encouraging results when using mouse monoclonal antibody to HSP90α, in our previously published study, HSP90α mouse monoclonal antibody against EndoMT-promoted and M2 macrophage Cell-involved tumor growth of PDAC cells has a significant therapeutic effect, and the antibody can reduce the connection of eHSP90α to its cell surface receptors, thereby preventing the eHSP90α-induced feed-forward loop of HSP90α expression and secretion. See Fan CS, Chen LL, Hsu TA, et al. Endothelial-mesenchymal transition harnesses HSP90α-secreting M2-macrophages to exacerbate pancreatic ductal adenocarcinoma. J Hematol Oncol 2019;12:138. This finding is also supported by another of our findings. Octyl gallate (OG), an antioxidant and preservative that is safely used in food and cosmetics, we found that octyl gallate can block eHSP90α and its cell surface Ligation of the receptor TLR4 inhibits EndoMT-derived CAF-induced macrophage M2 polarization and the associated eHSP90α feed-forward loop. See Chua KV, Fan CS, Chen LL, Chen CC, Hsieh SC, Huang TS. Octyl Gallate induces pancreatic ductal adenocarcinoma cell apoptosis and suppresses endothelial-mesenchymal transition-promoted M2-macrophages, HSP90α secretion, and tumor growth. Cells 2020;9(1):91.

In conclusion, eHSP90α is a potential therapeutic target for interstitial proliferation and M2 macrophage-associated tumor progression, and the development of effective anti-HSP90α antibodies is a valuable and promising strategy for the development of targeted therapies against eHSP90α.

In one aspect, described herein is an isolated antibody. The isolated antibody comprises: a novel complementarity determining region (CDR) capable of specifically binding to the HSP90α epitope containing the amino acid sequence EDK in the regions of amino acids 235-244 and amino acids 251-260.

In some embodiments, HSP90α can be eHSP90α.

In some embodiments, the isolated antibody may comprise: the heavy chain complementarity determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region sequence of SEQ ID NO: 2 or SEQ ID NO: 12; and SEQ ID NO: 7 or Light chain complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region sequence of SEQ ID NO: 17.

In some embodiments, the heavy chain CDR1, CDR2 and CDR3 are from SEQ ID NO:2, and the light chain CDR1, CDR2 and CDR3 may be from SEQ ID NO:7.

In some embodiments, the isolated antibody may comprise a polypeptide that is at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO: 2. chain variable region, and one light chain variable region that is at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the sequence of SEQ ID NO: 7 .

In some embodiments, the heavy chain CDR1 can have the sequence of SEQ ID NO: 3, the heavy chain CDR2 can have the sequence of SEQ ID NO: 4, the heavy chain CDR3 can have the sequence of SEQ ID NO: 5, and the light chain CDR1 can have the sequence of SEQ ID NO :8 sequence, the light chain CDR2 may have the sequence of SEQ ID NO:9, and the light chain CDR3 may have the sequence of SEQ ID NO:10.

In some embodiments, the heavy chain CDR1, CDR2, and CDR3 may be from SEQ ID NO:12, and the light chain CDR1, CDR2, and CDR3 are from SEQ ID NO:17.

In some embodiments, the isolated antibody may comprise a polypeptide that is at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO: 12. chain variable region, and one light chain variable region that is at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the sequence of SEQ ID NO: 17 .

In some embodiments, the heavy chain CDR1 can have the sequence of SEQ ID NO: 13, the heavy chain CDR2 can have the sequence of SEQ ID NO: 14, the heavy chain CDR3 can have the sequence of SEQ ID NO: 15, and the light chain CDR1 can have the sequence of SEQ ID NO :18 sequence, the light chain CDR2 may have the sequence of SEQ ID NO:19, and the light chain CDR3 may have the sequence of SEQ ID NO:20.

In some embodiments, the isolated antibody can be an antibody comprising an Fc region, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a single chain antibody, a scFv multimer, a monoclonal antibody, a monovalent antibody, a multispecific antibody, a humanized antibody or a chimeric antibody.

Also described herein are nucleic acid molecules comprising nucleic acid sequences encoding the antibodies disclosed herein.

In some embodiments, provided herein is a host cell comprising the nucleic acid molecule.

Also described herein is a pharmaceutical composition comprising an antibody described herein or a nucleic acid molecule comprising a nucleic acid sequence encoding an antibody disclosed herein. The pharmaceutical composition may further include pharmaceutically acceptable Received carrier and/or another therapeutic agent (such as another cancer drug, cytotoxic agent or immunomodulator). An example of a therapeutic agent may include, but is not limited to, gemcitabine.

eHSP90α can be used in the therapeutic strategy of tissue interstitial hyperplasia, especially cancerous tissue interstitial hyperplasia, therefore, also described herein is a method for treating cancerous tissue interstitial hyperplasia in a subject, comprising: administering to the desired subject a targeted A therapeutic agent for eHSP90α.

In some embodiments, the antibodies described herein are also useful for inhibiting cancer cell growth or cancer cell metastasis. Accordingly, described herein are methods of treating cancer in a subject comprising: administering to a subject in need thereof an effective amount of an antibody described herein or a nucleic acid molecule comprising a nucleic acid sequence encoding an antibody disclosed herein.

In some embodiments, the antibodies described herein are also used to reduce or prevent interstitial hyperplasia. Accordingly, described herein are methods for treating interstitial hyperplasia in a subject comprising: administering to a subject in need thereof an effective amount of an antibody described herein or a nucleic acid molecule comprising a nucleic acid sequence encoding an antibody disclosed herein. Here, interstitial hyperplasia may be interstitial hyperplasia of cancerous tissue.

In some embodiments, the method can also include administering to the subject another therapeutic agent. The time for administering the therapeutic agent is not particularly limited. In some embodiments, the therapeutic agent can be administered at the same time as the antibody or nucleic acid molecule is administered. In some embodiments, the therapeutic agent can be administered after the antibody or nucleic acid molecule is administered.

In some embodiments, blood HSP90α levels in a subject can be detected using the isolated antibodies described herein to monitor tumor shrinkage in the subject in a method of treating cancer or stromal hyperplasia. In some embodiments, the subject's blood HSP90α level can be the subject's blood HSP90α level in whole blood or serum.

In some embodiments, the cancer may be characterized by interstitial proliferation.

In some embodiments, the cancer may be characterized by M2 macrophage progression.

Examples of cancer may include, but are not limited to, pancreatic cancer, colon cancer, breast cancer, liver cancer, or lung cancer.

Also provided herein is a method of assessing tumor shrinkage in a subject, comprising: obtaining a blood sample from the subject; and determining the amount of HSP90α in the blood sample. In some embodiments, the blood sample can be whole blood or serum.

In some embodiments, the method for assessing tumor shrinkage in a subject may comprise: obtaining a blood sample from a subject administered IgG and administering an antibody as described herein or a nucleic acid molecule comprising a nucleic acid sequence encoding an antibody disclosed herein Another blood sample from another subject; determining the amount of HSP90α in the blood sample of the subject administered an IgG, antibody or nucleic acid molecule as described herein. When the HSP90α content in the blood sample of the subject administered the antibody or nucleic acid molecule disclosed herein is lower than the HSP90α content in the blood sample of the subject administered IgG, it means that the antibody or nucleic acid molecule described herein can effectively inhibit tumor growth or Reduce tumor volume.

The details of one or more implementations of the disclosure are set forth in the description below. Other features, objects and advantages of the present disclosure are apparent from the description and claims.

Figure 1 is a set of charts showing the analysis of the characteristics of mouse anti-HSP90α monoclonal antibody. (a) The HSP90α-binding ability of the culture supernatants of six fusion tumor strains derived from rHSP90α-immunized mice was analyzed by western blotting. (b) The HSP90α-binding activity of the culture supernatants of the above six fusion tumor strains was analyzed by ELISA. (c) Subtype characterization of Clone-2 and Clone-6 antibodies identified as IgG2b isotype immunoglobulins containing kappa light chains. (d) Analysis of Clone-2 and The effect of Clone-6 antibody on the molecular binding between HSP90α and CD91 and the molecular binding between CD91 and IKKα. The test condition is that the pancreatic ductal adenocarcinoma PANC-1 cell line is stimulated with rHSP90α (15 μg/ml) and there is a 10 μg/ml control IgG or Clone-2 or Clone-6 antibodies. (e) Cell invasion assay (Transwell invasion assay) showed that rHSP90α could induce the invasion ability of cancer cells. The test conditions were SW620, LoVo and BxPC-3 cancer cell lines were pretreated with PBS or rHSP90α (15 μg/ml) for 16 hours. (f) The effect of Clone-2 or Clone-6 antibody on rHSP90α-induced cancer cell invasion ability was analyzed by cell invasion ability test method. The test condition was that SW620 and PANC-1 cancer cell lines were treated with 15 μg/ml rHSP90α plus 10 μg/ml Control IgG or Clone-2 or Clone-6 antibodies were pretreated for 16 hours. (g) The effect of Clone-2 or Clone-6 antibody on the stemness of rHSP90α-induced cancer cells was analyzed by the cell sphere-forming ability test method. The test conditions were that SW620, PANC-1 and Panc 02 cancer cell lines were respectively implanted into cells containing 15 μg /ml of rHSP90α plus 10 μg/ml of control IgG or Clone-2 or Clone-6 antibody in the spheroid formation medium.

Fig. 2 is a chart of characteristic analysis of humanized anti-HSP90α antibody. (a) Binding kinetics of humanized HSP90α antibodies Clone-2-chimera, Clone-2-hA, Clone-2-hB and Clone-2-hC to HSP90α were analyzed using Biacore T200 assay. (b) Calculate the equilibrium dissociation of Clone-2-chimera, Clone-2-hA, Clone-2-hB and Clone-2-hC antibodies from the molecular binding sensorgram of (a) and apply a simple 1:1 interaction model Constant (K _D ) value. (c) Analyze the effects of Clone-2-chimera, Clone-2-hA, Clone-2-hB and Clone-2-hC antibodies on the invasion ability of rHSP90α-induced cancer cells by cell invasion ability test method, the test condition is PANC-1 Cancer cell lines were pretreated for 16 hours with 15 μg/ml rHSP90α plus 10 μg/ml control IgG, Clone-2, Clone-2-chimera, Clone-2-hA, Clone-2-hB or Clone-2-hC antibodies. (d) The effects of Clone-2-chimera, Clone-2-hA, Clone-2-hB and Clone-2-hC antibodies on rHSP90α-induced cancer cell stemness were analyzed by cell sphere-forming ability test method, and the test conditions were PANC -1 cells were seeded with 15 μg/ml rHSP90α plus 10 μg/ml control IgG, Clone-2, Clone-2-chimera, Clone-2-hA, Clone-2-hB or Clone-2-hC antibody class Spheroid Formation Medium. (e) To analyze the toxicity of 17-AAG and humanized HSP90α antibody HH01 (Clone-2-hA) to retinal pigment epithelial cells. AAG and HHO1 antibodies were treated for 72 hours and then their survival rates were assessed. (f) Pharmacokinetic study of humanized HSP90α antibody HH01, the test condition was a single dose of 10 mg/kg HH01 antibody was injected intravenously (IV) into 5 male mice.

Fig. 3 is a set of identification results showing the HSP90α epitope recognized by Clone-2-chimera and HH01 (namely Clone-2-hA) antibodies. (a) The region of HSP90α bound by the Clone-2 antibody was roughly outlined by protein domain scanning assay. (b) The amino acid sequence of the HSP90α site bound by the Clone-2-chimera antibody was further deduced by peptide scanning assay. (c) The amino acid sequence of the HSP90α site bound by the HH01 antibody was deduced by peptide scanning assay. (d) The key amino acids at the HSP90α site bound by Clone-2-chimera and HH01 antibodies were deduced by alanine scanning assay. (e) The effect of the HSP90α epitope on the invasion ability of rHSP90α-induced cancer cells was analyzed by the cell invasion ability test method. The test condition was that the PANC-1 cancer cell line was treated with 15 μg/ml rHSP90α plus 15 μg/ml control peptide (P-ctrl ) or epitope-containing peptide (P-46) pretreatment for 16 hours. (f) The effect of HSP90α epitope on rHSP90α-induced cancer cell stemness was analyzed by cell spheroid forming ability test method. The test condition was that PANC-1 cells were implanted with 15 μg/ml rHSP90α plus 15 μg/ml control peptide ( P-ctrl) or spheroid formation medium containing epitope peptide (P-46).

Figure 4 is a set of experimental mouse data showing the preventive efficacy of HH01 antibody in a mouse model of PDAC with interstitial hyperplasia. (a) Masson's trichrome stain revealed that in With the participation of EndoMT cells, the tumors grown from Panc 02 cancer cells have the phenomenon of interstitial hyperplasia. (b) The timing of administration of HH01 antibody to prevent the growth of stromal proliferative tumors in a mouse PDAC model in which EndoMT cells participate in the tumor growth of Panc 02 cancer cells. (c) The superficial tumor volume was measured from the body surface, showing that the tumor growth curve of the mice treated with HHO1 antibody was significantly suppressed compared with the IgG-treated control group. (d) Tumor weight of sacrificed mice on day 30 after inoculation. (e) Masson's trichrome staining of tumor tissue sections from mice treated with control IgG or HHO1 antibody. (f) Serum HSP90α content of mice treated with control IgG or HHO1 antibody.

Figure 5 is a set of experimental mouse data showing the therapeutic efficacy of HH01 antibody in a mouse model of PDAC with interstitial hyperplasia. (a) Timing of administration of HH01 antibody to treat tissue mesenchymal proliferative tumor growth in a mouse model in which EndoMT cells participate in Panc 02 cancer cell tumor growth. (b) The length and width of the body surface were measured and the subcutaneous tumor volume was estimated, showing that the tumor growth curve of mice treated with HH01 antibody was significantly suppressed compared with the control group using IgG. (c) Tumor weight of sacrificed mice on day 41 after inoculation. (d) Masson trichrome staining of tumor sections from mice treated with control IgG or HHOl antibody. (e) Serum HSP90α levels of mice treated with control IgG or HHO1 antibody.

Figure 6 is a set of experimental mouse data showing the therapeutic efficacy of HH01 antibody in a humanized mouse PDAC model with interstitial hyperplasia. (a) Timing of administration of HHO1 antibody to treat stromal proliferative tumor growth in a humanized mouse model in which EndoMT cells participate in tumor growth of PANC-1 cancer cells. (b) The length and width were measured from the body surface and the subcutaneous tumor volume was estimated to draw the tumor growth curves of mice treated with control IgG, control IgG plus gemcitabine, and HHO1 antibody plus gemcitabine, respectively. (c) Tumor weight of sacrificed mice on day 39 after inoculation. (d) Masson's trichrome staining of tumor sections from mice of different treatment groups. (e) Serum HSP90α levels in mice treated with control IgG, control IgG plus gemcitabine, or HHO1 antibody plus gemcitabine.

Figure 7 is a set of experimental mouse data showing the therapeutic efficacy of the HH01 antibody in the PDAC model of interstitial hyperplasia induced by the K-Ras ^G12D mutation in pancreatic duct cells. (a) In the transgenic mouse model of K-Ras ^G12D mutation in pancreatic duct cells (ie, LSL-KrasG12D/Pdx1-Cre mouse, hereinafter referred to as KC mouse), the administration of HH01 antibody for the treatment of interstitial hyperplastic PDAC time. (b) Kaplan-Meier survival curves of KC mice treated with control IgG and HHO1 antibodies, respectively. (c) Illustration of pancreas and liver appearance and interstitial hyperplasia in KC mice treated with control IgG or HHO1 antibody. (d) Serum HSP90α content of KC mice treated with control IgG or HHO1 antibody.

Figure 8 is a set of experimental mouse data showing that eHSP90α-induced M2 macrophages have a significant ability to promote tumor growth. (a) Macrophages treated with rHSP90α can promote the growth of Panc 02 cancer cells. C57BL/6 mice were injected subcutaneously with Panc 02 cells alone or with BMDM pretreated with PBS, 100 ng/ml LPS or 15 μg/ml rHSP90α for 24 hours (n=6 in each group). The size of the growing tumor was measured from the surface of the mouse body using a vernier caliper, and the volume of the tumor was estimated using the formula ½×length× ^width2 . (b) Continuing from (a), different groups of experimental mice were sacrificed on the 28th day after Panc 02 cancer cell inoculation, and the tumors were taken out and weighed. ^@p < 0.01 when compared to the "Panc 02" or "Panc 02+BMDM" groups. (c) Immunohistochemical staining analysis of F4/80, CD163, CD4 and CD8 in tumor tissues of experimental mice. H/E is hematoxylin and eosin staining. (d) Fluorescent analysis of tumor tissue stained with DAPI in experimental mice.

Figure 9 is a set of experimental mouse data showing the therapeutic efficacy of HH01 antibody in a mouse PDAC model in which M2 macrophages promote progression. (a) The administration time of HH01 antibody in the mouse model in which the treatment of M2 macrophages promotes the tumor growth of Panc 02 cancer cells. (b) The length and width were measured from the body surface and the subcutaneous tumor volume was estimated to draw the tumor growth curves of mice treated with control IgG and HHO1 antibody, respectively. (c) On day 42 after inoculation of Panc 02 cancer cells plus M2 macrophages, the mice were sacrificed to remove their tumors and weigh them. (d) Serum HSP90α content of mice treated with control IgG or HHO1 antibody. (e) with control IgG or HH01 Immunohistochemical staining analysis of CD163, CD204, CD4, and CD8 of antibody-treated mouse tumors. H/E is hematoxylin and eosin staining.

Figure 10 is a continuation of the data in Figure 9 showing the results of immunohistofluorescent staining analysis of F4/80, iNOS, Arginase 1, CD4, CD8 and TNF-α from tumors of mice treated with control IgG and HHO1 antibodies . (a and b) Increases of F4/80 ⁺ iNOS ⁺ M1 macrophages (as indicated by white arrows) and F4/80 ⁺ Arginase 1 ⁺ M2 macrophages were observed in tumor tissues of HH01-treated mice Visible disappearance of cells (as indicated by the yellow arrow in the control IgG group). (c) An increase in CD4 ⁺ TNF-α ⁺ cells (indicated by arrows) was observed in tumor tissues from HH01-treated mice. (d) An increase in CD8 ⁺ TNF-α ⁺ cells (indicated by arrows) was observed in tumor tissues from HH01-treated mice.

Described herein are novel antibodies that bind HSP90α, eg, eHSP90α.

The isolated antibody may comprise novel CDRs capable of specifically binding the HSP90α epitope comprising the amino acid sequence EDK in the region of ₂₃₅ AEEKEDKEEE ₂₄₄ and ₂₅₁ ESEDKPEIED ₂₆₀ .

The isolated antibody may comprise: the heavy chain complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region sequence of SEQ ID NO: 2 or SEQ ID NO: 12; and the light chain of SEQ ID NO: 7 or SEQ ID NO: 17. The light chain complementarity determining regions CDR1, CDR2 and CDR3 of the chain variable region sequence.

In one aspect, the heavy chain CDR1, CDR2 and CDR3 can be from SEQ ID NO:2 and the light chain CDR1, CDR2 and CDR3 can be from SEQ ID NO:7. In some embodiments, the isolated antibody may comprise a polypeptide that is at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO: 2. chain variable region, and one light chain variable region that is at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the sequence of SEQ ID NO: 7 . In some embodiments, the heavy chain CDR1 can have the sequence of SEQ ID NO: 3, the heavy chain CDR2 can have the sequence of SEQ ID NO: 4, the heavy chain CDR3 can have the sequence of SEQ ID NO: 5, and the light chain CDR1 can have the sequence of SEQ ID NO :8 sequence, the light chain CDR2 may have the sequence of SEQ ID NO:9, and the light chain CDR3 may have the sequence of SEQ ID NO:10. In some embodiments, there is also provided a nucleic acid molecule comprising a nucleic acid sequence that encodes a heavy chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 2, and a sequence that is at least 80% identical to the sequence of SEQ ID NO: 7 A light chain variable region with at least 80% sequence identity. In some embodiments, the nucleic acid molecule may comprise a sequence that is at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO: 1 . The sequence encodes a heavy chain variable region, and a sequence that is at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the sequence of SEQ ID NO: 6 to encode the light chain variable region.

The sequences of SEQ ID NO: 1 to SEQ ID NO: 10 are listed in Table 1 below.

Bold font: CDR

In one aspect, the heavy chain CDR1, CDR2 and CDR3 can be from SEQ ID NO:12 and the light chain CDR1, CDR2 and CDR3 can be from SEQ ID NO:17. In some embodiments, the isolated antibody may comprise a polypeptide that is at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO: 12. chain variable region, and one light chain variable region that is at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the sequence of SEQ ID NO: 17 . In some embodiments, the heavy chain CDR1 can have the sequence of SEQ ID NO: 13, the heavy chain CDR2 can have the sequence of SEQ ID NO: 14, the heavy chain CDR3 can have the sequence of SEQ ID NO: 15, and the light chain CDR1 can have the sequence of SEQ ID NO :18 sequence, the light chain CDR2 may have the sequence of SEQ ID NO:19, and the light chain CDR3 may have the sequence of SEQ ID NO:20. In some embodiments, there is also provided a nucleic acid molecule comprising a nucleic acid sequence that encodes a heavy chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 12, and a sequence that is at least 80% identical to the sequence of SEQ ID NO: 17 A light chain variable region with at least 80% sequence identity. In some embodiments, the nucleic acid molecule may comprise a sequence at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO: 11. The sequence encodes a heavy chain variable region, and a sequence that is at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the sequence of SEQ ID NO: 16 to encode the light chain variable region.

The sequences of SEQ ID NO: 11 to SEQ ID NO: 20 are listed in Table 2 below.

Bold font: CDR

The antibody can specifically bind to HSP90α. More specifically, the antibody binds with high affinity to HSP90α compared to other non-HSP90α proteins. In addition, the CDRs of the heavy or light chain variable regions can be determined by any method known in the art.

The antibodies described herein exhibit high binding affinity to eHSP90α. Therefore, the antibodies described herein can inhibit interstitial proliferation in tumors to further inhibit tumor growth or reduce tumor size.

According to the antibody sequences disclosed herein and their CDRs, skilled artisans can use any method known in the art to generate various forms of anti-HSP90α antibodies, and the generated anti-HSP90α antibodies can specifically bind to amino acids 235 to 244 and amine groups The HSP90α epitope contains two EDK sites in the acid 251 to 260 region.

According to the sequence of HSP90α epitope, the synthetic peptide can competitively inhibit the tumor-promoting function of eHSP90α.

The term "antibody" as used herein includes various antibody structures having antigen-binding activity. For example, antibodies may include, but are not limited to: an antibody comprising an Fc region, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a single chain antibody, a scFv multimer, a monoclonal antibody, a monovalent antibody, a multispecific antibody, a humanized antibody or a chimeric antibody. In some embodiments, the antibody is a humanized antibody.

Also described herein is a pharmaceutical composition comprising an antibody described herein. The pharmaceutical composition comprises: the isolated antibody as described herein and a pharmaceutically acceptable carrier.

Also described herein is a pharmaceutical composition comprising a nucleic acid molecule capable of encoding an antibody as described herein. The pharmaceutical composition includes: a nucleic acid molecule capable of encoding the antibody as described herein and a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable carrier" means that the carrier must be compatible with the active ingredient (eg, capable of stabilizing the antibody) and not deleterious to the subject to be treated. The carrier may be at least one selected from the group consisting of active agents, adjuvants, dispersants, wetting agents and suspending agents. Examples of carriers may be, but are not limited to, microcrystalline cellulose, mannitol, dextrose, skim milk powder, polyethylene, polyvinylpyrrolidone, starch, or combinations thereof.

Antibodies, nucleic acid molecules, or pharmaceutical compositions containing one or more thereof may be administered to a subject orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

Also described herein is the use of an antibody or nucleic acid molecule for the preparation of a medicament for the treatment of cancer.

Also described herein are methods for treating cancer in a subject comprising: administering to the subject an effective amount of an antibody or nucleic acid molecule as described herein.

Examples of cancers may include, but are not limited to, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, liver cancer, lymphoma, kidney cancer, osteosarcoma, ovarian cancer , pancreatic, prostate, skin (including basal and squamous cell carcinomas, and melanoma), stomach, thymus, and thyroid. In some embodiments, the cancer is characterized by interstitial hyperplasia symptoms, and specifically, interstitial hyperplasia can be found within or around the tumor. In some embodiments, the cancer may be pancreatic cancer, colorectal cancer, breast cancer, liver cancer, or lung cancer.

The antibodies described herein inhibit cancer tissue stromal proliferation, immunosuppression, growth and metastasis.

The term "subject" refers to a human or non-human animal.

The term "treating", "treat" or "treatment" refers to the administration of an antibody, nucleic acid molecule or pharmaceutical composition to a subject for the purpose of curing, alleviating, alleviating, altering, remedial, ameliorating or affecting Disease, symptom, or predisposition to disease. "Effective amount" refers to the amount of antibody or nucleic acid molecule required to give the desired effect to a subject. As known to those skilled in the art, effective amounts depend on the route of administration, the use of excipients and the possibility of co-administration with other methods of treatment, such as the use of other active agents.

The details of one or more implementations of the disclosure are set forth in the description below. Other features, objects and advantages of the present disclosure will be apparent from the description and claims.

Example

Without further elaboration, those skilled in the art can make full use of the present disclosure based on the above description. Accordingly, the following specific examples should be construed as illustrative only, and in no way limiting the remainder of the disclosure. All publications cited herein are hereby incorporated by reference in their entirety.

Materials and methods

cell culture

Human PDAC cell line PANC-1 was cultured in a humidified incubator at 37°C and 5% CO ₂ with the addition of 10% fetal bovine serum (FBS) and 100units/ml of penicillin, 100μg/ml of streptomycin and 2mM _L -glutamic acid (1×PSG) was cultured together in Dulbecco's Modified Eagle's Medium (DMEM). Human PDAC cell line BxPC-3, human CRC cell line SW620 and mouse PDAC cell line Panc ₀₂ were treated with RPMI- 1640 culture medium together. Under the same growth conditions, the human CRC cell line LoVo was maintained in Ham's F-12 medium containing 20% FBS and 1×PSG, and the human retinal pigment epithelial cell line ARPE-19 (ATCC CRL-2302 ^TM ; American Type Culture Collection , Manassas, VA, USA) were cultured in ATCC-prepared DMEM:F12 medium (model #30-2006, ATCC) supplemented with 10% FBS and 1×PSG. Human umbilical vein endothelial cells (HUVEC) were isolated and cultured in a humidified incubator at 37°C and 5% CO ₂ with 20% FBS, 100 units/ml of penicillin, 100 μg/ml of streptomycin, 30 μg/ml The M199 medium of endothelial cell growth supplement (EMD Millipore, Billerica, MA, USA) was cultivated together. See Fan CS, Chen LL, Hsu TA. Endothelial-mesenchymal transition harnesses HSP90α-secreting M2-macrophages to exacerbate pancreatic ductal adenocarcinoma. J Hematol Oncol 2019;12:138. For EndoMT induction, HUVECs were pre-cultured with M199 medium containing 2% FBS for 16 hours, then 0.3 μg/ml OPN was added and cultured for another 24 hours. Mouse endothelial cell line 3B-11 (ATCC CRL-2160 ^TM ) was grown in RPMI-1640 medium supplemented with 10% FBS and 1×PSG. For EndoMT induction, 3B-11 cells were treated in the same manner as HUVECs, except that RPMI-1640 medium containing 1% FBS was used. Mouse bone marrow-derived macrophages (BMDM) were prepared as previously published and cultured in DMEM supplemented with 10% FBS and 1×PSG. See Fan CS, Chen LL, Hsu TA, et al. Endothelial-mesenchymal transition harnesses HSP90α-secreting M2-macrophages to exacerbate pancreatic ductal adenocarcinoma. J Hematol Oncol 2019;12:138.

mouse model

Experiments with mice were performed with permission from the Institutional Animal Care and Use Committee of the National Institutes of Health (NHRI-IACUC-106031-A, 109022-M2-S02, and 109196-A). To establish a tissue-mesenchymal hyperplastic tumor xenograft model, 12-week-old C57BL/6 mice were inoculated subcutaneously with a mixture of Panc 02 cancer cells and EndoMT-derived CAFs (ie, OPN-treated 3B-11 cells). See Fan CS, Chen LL, Hsu TA, et al. Endothelial-mesenchymal transition harnesses HSP90α-secreting M2-macrophages to exacerbate pancreatic ductal adenocarcinoma. J Hematol Oncol 2019;12:138; and Chua KV, Fan CS, Chen LL, Chen CC, Hsieh SC, Huang TS. Octyl gallate induces pancreatic ductal adenocarcinoma cell apoptosis and suppresses endothelial-mesenchymal transition-promoted M2-macrophages, HSP90α secretion, and tumor growth. Cells 2020;9(1):91. Further illustrations of mouse treatment models are presented in the figures and their associated captions and text. The monitoring of therapeutic effects included measuring the size of subcutaneously growing tumors from the body surface with a vernier caliper, and estimating the tumor volume with the formula 1/2×length× ^width2 . Finally, the tumors were removed and weighed after the mice were sacrificed. In order to establish a tissue-mesenchymal proliferative tumor transplantation model in humanized mice, NOD-SCID IL2R ^null (ASID) mice implanted with human hematopoietic stem cells (hHSC) were subcutaneously inoculated with PANC- A mixture of 1 cells and OPN-treated HUVECs. The humanized mice used here (i.e. ASID mice transplanted with hHSC) were provided by the National Center for Experimental Animals (Taipei, Taiwan), and each mouse contained human CD45 ⁺ cells exceeding 38% of the total lymphocytes. To establish a mouse PDAC model in which M2 macrophages promote progression, C57BL/6 mice were subcutaneously inoculated with a mixture of Panc 02 cancer cells and rHSP90α-treated BMDM. Further illustrations of mouse treatment models are presented in the figures and their associated captions and text. In addition, LSL-KrasG12D/Pdx1-Cre gene transfected mice (KC mice for short) were used as a mouse model for spontaneous PDAC to evaluate the therapeutic effect of HH01 antibody. LSL-KrasG12D and Pdx1-Cre mice are mouse models from the Human Cancer Consortium Library of the National Cancer Institute (Frederick, Maryland, USA). LSL-KrasG12D mice were mated with Pdx1-Cre mice to generate LSL-KrasG12D mice. KrasG12D/Pdx1-Cre mice, and transgenic mice were further manipulated as indicated in the figure and associated text.

Western blot analysis

According to commonly used technical procedures, 732-amino acid (aa) full-length recombinant human HSP90α (rHSP90α; PeproTech Co., Cranbury, NJ, USA) was used to immunize mice to generate mouse fusion tumors. See Yokoyama WM. Production of monoclonal antibody supernatant and ascites fluid. Curr Protoc Mol Biol 2008;83:11.10.1-11.10.10. Next, fusion tumors were initially screened using the general procedure of western blotting. See Fan CS, Chen LL, Hsu TA, et al. Endothelial-mesenchymal transition harnesses HSP90α-secreting M2-macrophages to exacerbate pancreatic ductal adenocarcinoma. J Hematol Oncol 2019;12:138. Fusoma medium was collected for reaction with membrane strips containing HSP90α and molecular weight marker proteins blotted from electrophoretic gels. After washing three times with PBS supplemented with 0.05% Tween-20 (i.e., PBST), the membrane strips were incubated with horseradish catalase-conjugated secondary antibody for 1 hour. After washing 3 times with PBST, and 0.3 mg/ml of 3,3',5,5'-tetramethylbenzidine in 0.015% H ₂ O ₂ (Sigma-Aldrich, St.Louis, MO, USA) Immunoreactive protein bands can be seen after the reaction.

Enzyme-linked immunosorbent assay (ELISA)

To quantify the binding activity of the prepared antibodies to HSP90α, 96-well plates were coated with capture buffer (BioLegend, San Diego, CA, USA) containing 12.5 ng/ml rHSP90α, and incubated overnight at 4°C. The coated well plates were washed twice with PBST and then blocked with PBST plus 3% bovine serum albumin (BSA). The prepared antibody was added and incubated at room temperature for 2 hours, and after washing 3 times with PBST, a horseradish catalase-conjugated secondary antibody was added and incubated at 37°C for another 1 hour. After washing 3 times, 0.015% H ₂ O ₂ containing 0.3 mg/ml 3,3',5,5'-tetramethylbenzidine was added to each well and incubated at room temperature for 10 minutes in the dark. OD ₄₅₀ values were measured with an Infinite M200 microplate analyzer (TECAN, Männedorf, Switzerland) after stopping the reaction by adding H ₂ SO ₄ (0.5M). Additionally, immunoglobulin (IgG) isotype characterization of anti-HSP90α antibodies was performed using a mouse Ig isotype ELISA Ready-Set-Go! ^TM kit (eBioscience ^TM , Thermo Fisher Scientific, Waltham, MA, USA). As previously described, the ELISA assay protocol was also used to measure the amount of secreted HSP90α in mouse serum samples. See Chen JS, Hsu YM, Chen CC, Chen LL, Lee CC, Huang TS. Secreted heat shock protein 90α induces colorectal cancer cell invasion through CD91/LRP-1 and NF-κB-mediated integrin α _V expression. J Biol Chem 2010 ;285(33):25458-66.

Proximity Ligation Assay (PLA)

PANC-1 cells seeded on glass coverslips at a density of 2×10 ⁵ cells (per 22×22 mm coverslip) were incubated with medium containing 0.5% serum for 16 hours at 37°C and 5% CO ₂ in a humidified environment . Cells were then added to PBS or rHSP90α at 15 μg/ml in the absence or presence of 10 μg/ml control IgG or tested anti-HSP90α antibody for an additional 24 hours. Treated cells were then fixed with 3% paraformaldehyde and blocked with the blocking solution provided in the Duolink in situ PLA kit (Olink Bioscience, Uppsala, Sweden). In addition, cell samples were incubated with HSP90α antibody (1:80, cat. #AHP-1339, AbD Serotec, Raleigh, NC, USA) or IKKα antibody (1:80, cat. #3285, Epitomics Co. , Burlingame, CA, USA) was cultured together with mixed CD91 antibody (1:80, cat. #550495, BD Biosciences, San Jose, CA, USA). After washing three times with Tris buffer supplemented with Tween 20 (0.05%), cell samples were incubated with PLA probes for subsequent ligation and amplification procedures according to the manufacturer's instructions of the Duolink in situ PLA kit. Finally, nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Images were taken and analyzed using a Leica TCS SP5 II confocal microscope and LAS AF Lite 4.0 software (Leica, Wetzlar, Germany).

Cancer cell invasiveness assay

Cancer cells were serum-starved in medium containing 0.5% FBS for 16 h in _a humidified incubator at 37°C and 5% CO, and then treated in the absence or presence of 10 μg/ml of control IgG or the tested anti-HSP90α antibody. In case, PBS or rHSP90α at 15 μg/ml was added. After another 24 hours, the treated cells were collected and divided into aliquots (1 × ¹⁰⁵ cells per sample) in medium containing 0.5% FBS, and each aliquot was seeded onto pre-coated In the upper chamber of the Transwell ^™ with 5-fold dilutions of Matrigel (BD Biosciences). Cancer cells were allowed 16 hours to invade and migrate through the matrigel to the lower chamber containing medium and 10% FBS. Cancer cells remaining on the Transwell ^™ mesh plates were then fixed and stained with Giemsa. Use the Axiovert S100/AxioCam HR microscope system (Carl Zeiss, Oberkochen, Germany) to take pictures of the invasive cancer cells on the underside of the sieve plate, and use the Image-Pro Plus software (Media Cybernetics, Inc., Silver Spring, MD, USA ) for quantification.

Determination of Cell Spheroid Formation Ability (Cell Stemness Assay)

One thousand cancer cells were seeded into each well of a 24-well plate pre-coated with a 4 mm thick layer of 0.5% agarose. After 24 hours of incubation in serum-free medium at 37°C and 5% CO in _a humidified environment, PBS or 15 μg/ml of IgG was added to the cells in the absence or presence of 10 μg/ml of control IgG or the tested anti-HSP90α antibody. For rHSP90α, continue to culture for 10-14 days, and supplement some fresh serum-free medium every 3 days. Finally, under the Olympus IX 71 inverted microscope (Center Valley, PA, USA), the cell spheroids characterized by compactness, non-fixation, and diameter > 100 μm were photographed and counted, and the formula (number of cell spheroids/1000 )×100% to calculate the percentage of cell spheroid formation.

Cloning and sequencing of antibody genes in fusion tumor cells

Total RNA was isolated from mouse fusion tumor cells producing anti-HSP90α IgG and converted to cDNA by reverse transcriptase and random hexanucleotide primers. Mouse Ig-Primer Set (Sigma-Aldrich) was used to PCR amplify the variable domains (ie _VH and _VL domains) of IgG heavy (H) and light (L) chains. The PCR product was transferred into the pJET1.2/blunt transfer vector provided in the CloneJET PCR Colonization Kit (Thermo Fisher Scientific), followed by colony selection and DNA sequencing analysis.

Construction and expression of recombinant antibodies

The cDNA sequences of IgG _VH and _VL domains were synthesized by GeneDireX Inc. (Taoyuan, Taiwan) and inserted into pFUSEss-CHIg-hG1e1 and pFUSE2ss-CLIg-hk plasmids (InvivoGen, San Diego, CA, USA), respectively. The site preceding the region encoding the constant domains of the H and L chains in . In terms of cloning using pFUSEss-CHIg-hG1e1 as a vector, the _VH cDNA sequence was inserted into the EcoRI - NheI site following the human IL-2 signal sequence to express the presence of DNA at residues Q250T, E356D, M358L, and L428M. Recombinant protein with mutated Fc fragment. On the other hand, the cDNA of the _VL domain was inserted into the pFUSE2ss-CLIg-hk plasmid after cutting with EcoRI and BsiWI . The recombinant DNA sequences encoding recombinant IgG H and L chains were further amplified using hIgHG-F/hIgHG-R and CLIg-F/CLIg-R primers, and then inserted into pcDNA3.4-TOPO plasmids (Thermo Fisher Scientific), respectively. Finally, ExpiCHO cells were co-transfected with recombinant pcDNA3.4-TOPO plasmids encoding H and L chains at a ratio of 2:3. After culturing for 8-10 days, the culture medium of the transfected ExpiCHO cells was harvested, and the recombinant antibody was purified using a protein A column of Gibco ^™ ExpiCHO ^™ Expression System (Thermo Fisher Scientific).

Antibody Humanization and Optimization

The F(ab')2 region of the mouse monoclonal antibody was humanized with the help of computer simulation calculations, and all calculations were performed by Discovery Studio 2018 software (BIOVIA Inc., San Diego, CA, USA). The complementarity determining regions (CDRs) were identified by published methods. See Kabat EA, Wu TT, Perry HM, Gottesman KS, Foeller C. Sequences of proteins of immunological interest. USDepartment of Health and Human Services, NIH, Bethesda, MD, 1991. The three-dimensional (3D) structures of the VL and _VH domains were modeled through the X-ray templates of PdbID: _4X0K and PdbID: 4Y5X, respectively. See Johnson JL, Entzminger KC, Hyun J, Kalyoncu S, Heaner DP, Morales Jr IA, Sheppard A, Gumbart JC, Maynard JA, Lieberman RL. Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization. Acta Crystallogr D Biol Crystallogr 2015;71:896-906; and Moraga I, Wernig G, Wilmes S, Gryshkova V, Richter CP, Hong WJ, Sinha R, Guo F, Fabionar H, Wehrman TS, Krutzik P, Demharter S, Plo I, Weissman IL, Minary Majeti PR, Constantinescu SN, Piehler J, Garcia KC. Tuning cytokine receptor signaling by re-orienting dimer geometry with surrogate ligands. Cell 2015;160:1196–1208. Graft the CDRs of a mouse antibody into an in-house human template for further calculation of the 3D structure. Computes mutation proposals including aggregation, post-translational modifications, proteolytic cleavage, pharmacokinetics, and stability to improve recombinant antibodies.

HSP90α binding affinity assay

To measure the binding properties of anti-HSP90α antibodies to HSP90α, binding kinetic assays were performed using a Biacore T200 (Cytiva Co., Marlborough, MA, USA). will be interested in anti- The HSP90α antibody was applied as a moiety on the Protein A Series S sensor chip to achieve approximately 3000~5000 response units. HBS-EP+ buffer containing a range of concentrations of rHSP90α (1.5625-100 nM) was injected onto the wafer surface and the binding was monitored for 60 seconds with a final dissociation time of 1500 seconds.

Retinal Cytotoxicity Test

ARPE-19 cells were seeded on 96-well plates at a density of 4000 cells per well, and then treated with different concentrations of 17-AAG or anti-HSP90α antibody HH01 for 72 hours, and then added 3-(4,5-dimethyl Thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole (MTS; Sigma-Aldrich) was reacted, and finally the OD ₄₉₀ was measured to Assess cell viability.

Pharmacokinetic determination

The pharmacokinetics of anti-HSP90α antibody HHO1 was studied using Bltw:CD1 (ICR) male mice (BioLASCO Taiwan Co., Taipei, Taiwan). Experimental mice were kept in a state where food and water could be freely obtained at any time. First, 10 mg/kg of HH01 antibody was intravenously injected into mice (n=5) weighing 25-30 g, in order to determine the serum exposure of HH01 antibody amount, blood samples were collected after giving HH01 at 0.04, 0.08, 0.25, 1, 2, 3, 7, 9, 14, 16, 18, 21, 23, 25, 35, 42, 49, 56 and 63 days, using An ELISA kit (Cayman Chemical Co., Michigan, USA) measured the human IgG1 content of these samples to represent the remaining HHO1 content in mouse serum.

Cloning, expression and purification of full-length and truncated HSP90α

Cloning, expression and purification of full-length HSP90α (aa1-732) and its three truncated fragments, including N-terminal plus linker region (aa1-272), N-terminal to middle region (aa1-629) and C-terminal region (aa630-732). Briefly, each 8×His-tagged cDNA sequence was amplified by PCR from a human ORF colony (NM_005348) with primers containing Xho I and Bam HI sites. The PCR product was purified, cut with restriction enzymes, and inserted into pET-23a plasmid (Sigma-Aldrich). The recombinant plasmids were then transformed into E. coli ^ClearColi® recipient cells (competent cells, Lucigen Co., Middleton, WI, USA), and recombination was stimulated by the addition of isopropyl-D-thiogalactopyranoside DNA performance. Full-length and truncated HSP90α exhibiting 8×His tags were further purified using Ni-NTA columns (Qiagen, Germantown, MD, USA) according to the manufacturer's instructions. Finally, the purified protein was dialyzed against a storage buffer composed of 25 mM Tris-HCl (pH, 7.0), 50 mM NaCl, 0.1% Triton X-100, 50% glycerol, 1 mM EDTA and 1 mM DTT, and tested by Bradford protein assay ( Bio-Rad Laboratories, Hercules, USA) to determine the protein concentration.

Identification of HSP90α Epitope of Anti-HSP90α Antibody

First, a region-scanning strategy was used to initially determine the region of HSP90α responsible for binding to our prepared antibody. The above-mentioned full-length HSP90α and its three truncated fragments were used to coat a 96-well ELISA plate to determine the binding activity of the tested mouse monoclonal antibody. The results show that the tested mouse monoclonal antibody binds to the aa1-272 region of HSP90α. Therefore, in the peptide scanning strategy, a 132 peptide library was synthesized by Mimotopes Pty.Ltd. (Melbourne, Victoria, Australia). It consists of a series of 10-aa peptides shifted by 2 aa to span the sequence of the aa21~272 region of HSP90α. This series of peptides is used to coat 96-well ELISA plates to determine the binding activity of the tested antibodies. The results of peptide scanning analysis indicated that ₂₃₅ AEEKEDKEEE ₂₄₄ and ₂₅₁ ESEDKPEIED ₂₆₀ of HSP90α are the epitopes that bind to the tested antibody. Therefore, the alanine scanning strategy was further used to study which amino acid residue is critical for the binding of the tested antibody. Each amino acid was sequentially replaced with alanine, and 19 peptides were synthesized (Genozyme Biotech Inc., Taipei, Taiwan), and then ELISA was performed as described above to measure the binding activity of the tested antibodies.

Masson's trichrome staining

Mouse paraffin-embedded tissue sections were deparaffinized with xylene and rehydrated through a series of ethanol dilutions. Masson's trichrome staining was performed using the Trichrome Stain Kit (ScyTek Laboratories Inc., Utah, USA) according to the procedure provided by the manufacturer. After the sections were stained, they were dehydrated with a series of ethanol solutions and xylene with increasing concentrations, and finally mounted with mounting solution overnight at room temperature in the dark. Sections were observed and photographed using an Axiovert S100/AxioCam HR microscope system.

Immunohistochemical staining

Sections of 4-μm thick mouse tissue were deparaffinized with xylene and rehydrated in progressively diluted ethanol solutions. Conformational retrieval was performed by heating the sections in 10 mM citrate buffer, pH 6.0, under high pressure for 15 minutes. Endogenous peroxidase activity was inhibited by adding 0.3% H ₂ O ₂ . Before staining, sections were blocked with PBS plus 3% BSA for 30 min at room temperature. Subsequently, anti-F4/80 antibody (1:100, cat.#MCA497R, AbD Serotec), anti-CD163 antibody (1:80, cat.#sc-33560, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-CD204 Antibody (1:100, cat.#GTX51749, GeneTex Inc., Hsinchu City, Taiwan), anti-CD4 antibody (1:100, cat.#GTX44531, GeneTex Inc.), or anti-CD8 antibody (1:100, cat. #GTX16696, GeneTex Inc.) was added and reacted overnight at 4°C. After washing, the secondary antibody was added and reacted at room temperature for 30 minutes. Sections were detected using the DAKO REAL EnVision Detection System (Produktionsvej 42, DK-2600 Glostrup, Denmark) and counterstained with hematoxylin. The stained sections were dehydrated, sealed with mounting solution, and finally observed and photographed using the Axiovert S100/AxioCam HR microscope system.

Immunohistofluorescence staining

Mouse tissue sections were deparaffinized, rehydrated, and antigenic conformation restored as described above. Sections were blocked with PBS plus 5% BSA for 30 min at room temperature. Then, add 3 groups respectively Antibody and react overnight at 4°C. Antibody group 1 was composed of anti-F4/80 antibody (1:100, cat. #MCA497R, AbD Serotec), anti-iNOS antibody (1:100, cat. #GTX15323, GeneTex Inc.), and anti-Arginase 1 antibody (1:1: 50, cat.#sc-271430, Santa Cruz Biotechnology). Antibody panel 2 included anti-CD4 antibody (1:100, cat. #GTX44531, GeneTex Inc.) and anti-TNF-α antibody (1:75, cat. #sc-52746, Santa Cruz Biotechnology). Antibody group 3 consisted of anti-CD8 antibody (1:100, cat. #GTX16696, GeneTex Inc.) and anti-TNF-α antibody (1:75, cat. #sc-52746, Santa Cruz Biotechnology). After washing, fluorescently labeled secondary antibodies were added and reacted at room temperature for 1 hour, and the nuclei were counterstained with DAPI. Finally, the slices were observed using a Leica TCS SP5 II confocal microscope and LAS AF Lite 4.0 software. Take pictures and analyze.

result

Preparation and characteristic analysis of mouse anti-HSP90α monoclonal antibody

Six fusionoma strains were derived from mice immunized with a recombinant protein of human HSP90α (rHSP90α). Their culture supernatants were collected for determination of binding activity to HSP90α using standard Western blotting and ELISA protocols. Except for clone-5, the antibodies produced by these fusion tumors could all bind to the antigen rHSP90α (Fig. 1(a)). The binding activity was further quantified by ELISA. Clone-2 and Clone-6 exhibited the top two highest HSP90α binding contents (Fig. 1(b)), and thus were selected for the following further assays. Antibodies from both colonies were purified and both were identified as immunoglobulins of the IgG2b isotype containing kappa light chains (Fig. 1(c)). They could effectively block the binding of eHSP90α to its cell surface receptor CD91 and the binding of CD91 to downstream IKKα (Fig. 1(d)). Given that eHSP90α exerts its cancer-promoting effect through multiple mechanisms, such as inducing cancer cell EMT, migration, invasion, and gain-of-stemness, consistent with previous studies, our Transwell invasion assay showed that rHSP90α can act as a cancer cell. potent inducer of cell invasiveness (Fig. 1(e)), however the induction of rHSP90α can be induced by Clone-2 and Clone-6 anti-HSP90α antibodies were largely eliminated (Fig. 1(f)). In addition, rHSP90α can also induce cell stemness in cancer cells (reflected by increased sphere-forming ability), and this induction can also be significantly inhibited by Clone-2 and Clone-6 antibodies (Fig. 1(g)). Taken together, these data suggest that anti-HSP90α monoclonal antibodies can be developed and used to inhibit eHSP90α-promoted tumor cell progression.

Humanization of Clone-2 anti-HSP90α antibody

Next, we cloned and analyzed the cDNA sequences of the variable domains (ie, V _H and V _L ) of the IgG H and L chains of Clone-2 and Clone-6 anti-HSP90α antibodies, respectively. The cDNA sequences and encoded amino acid sequences are listed in Table 1 and Table 2. Clone-2 was further selected for humanization. The _VH and _VL DNA sequences were synthesized and inserted into plastid vectors, in front of the regions expressing the constant domains of the human IgG H and L chains (i.e., _CH and _CL ), respectively, in the expression "Clone-2- chimera" recombinant IgG1 antibody. In addition, according to the suggestion of computer-aided structural modeling and calculation, we constructed three mutants of Clone-2-chimera, named Clone-2-hA, Clone-2-hB and Clone-2-hC, aiming to improve Properties of antibodies in terms of aggregation, protease cleavage, post-translational modifications, pharmacokinetics, and stability. Mutation sites are listed in Table 3. These engineered humanized recombinant antibodies were expressed by ExpiCHO cells and analyzed for HSP90α binding affinity after purification. Kinetic analysis was performed using Biacore T200 to determine the binding properties of Clone-2-chimera, Clone-2-hA, Clone-2-hB and Clone-2-hC antibodies to rHSP90α (Fig. 2(a)). The equilibrium dissociation constant (K _D ) values of Clone-2-chimera, Clone-2-hA, Clone-2-hB and Clone-2-hC antibodies were further calculated, which were 0.94×10 ^-10 and 1.87×10 ^-10 respectively , 1.48×10 ^-10 , and 1.47×10 ^-10 M (Fig. 2(b)), indicating that the mutations in Clone-2-hA, Clone-2-hB and Clone-2-hC antibodies did not significantly reduce their response to HSP90α binding affinity.

To preliminarily evaluate the anticancer efficacy of these humanized anti-HSP90α antibodies, we assayed their inhibitory activity on rHSP90α-induced PDAC cell invasion and spheroid formation. The results of the Transwell invasion test showed that Clone-2-chimera and Clone-2-hA were superior to Clone-2-hB, Clone-2-hC and their mother line Clone- 2 (Fig. 2(c)). In the spheroid formation assay, Clone-2-hA still showed the most outstanding efficacy in inhibiting rHSP90α-induced spheroid formation in PANC-1 cells (Fig. 2(d)). Therefore, Clone-2-hA was finally selected and named HH01 antibody for further in vitro and in vivo assays. The solubility of the HH01 antibody in PBS is >10mg/ml, and the Dynamic Light Scattering assay (Dynamic Light Scattering assay) shows that the HH01 antibody is a molecule with a diameter in the range of 10-50nm, and the average diameter is about 19.33nm, indicating that HH01 Antibodies do not readily form aggregates. Considering clinical application, small molecule HSP90 inhibitors such as geldanamycin and resorcinol derivatives can cause damage to retinal photoreceptor cells when they accumulate to a certain extent in the retina. We studied the effect of HH01 antibody on ARPE- 19 The effect on the survival rate of retinal pigment epithelial cells was used to further evaluate whether the HH01 antibody showed any toxicity to non-cancerous retinal cells. As shown in Figure 2(e), 17-AAG exhibited cytotoxicity to ARPE-19 cells with an IC ₅₀ of 64.7nM, however, HH01 antibody had no effect on ARPE-19 cells even when its concentration reached 594.3nM (100μg/ml). 19 cells caused any appreciable cytotoxicity. In addition, the pharmacokinetics of the HH01 antibody was studied in male mice, and the results showed that in the mouse blood, the clearance rate of the HH01 antibody was 3×10 ^-3 ml/min/kg, and the volume at steady state was 80ml/kg , the half-life (T _1/2 ) of the HH01 antibody was measured to be about 18.4 days (Fig. 2(f)). It shows that the HH01 antibody has a long T _1/2 and a high serum exposure after intravenous administration, and is suitable for further development as a therapeutic drug.

Identification of the epitope recognized by the HH01 antibody

As listed in Table 3, the HH01 antibody can specifically bind human HSP90α, and its complementarity determining region (CDR) has a novel amino acid (aa) sequence. Therefore, we want to identify which region of HSP90α is bound by the HH01 antibody. In the preliminary identification work, we used the HSP90α region scanning strategy to define the HSP90α region responsible for binding to Clone-2 antibody. The full-length HSP90α (aa1~732) and its three truncated fragments, including the N-terminal plus linker region (aa1~272), the N-terminal to middle region (aa1~629) and the C-terminal region (aa630~732) were Colonization, expression and purification. Subsequently, the binding activity of Clone-2 antibody to the full-length and truncated three HSP90α fragments was determined by ELISA method. The results showed that the binding ability of the Clone-2 antibody to the aa1~272 region was similar to that of the full-length protein (Figure 3(a)), indicating that the binding epitope of the Clone-2 antibody may be between the N-terminal of HSP90α and the linker Within the area (aa1~272). Further, we used a peptide scanning strategy to identify the HSP90α epitopes bound by Clone-2-chimera and HH01 antibodies. A peptide library consisting of a series of 10-aa peptides with an 8-aa overlap between any two consecutive peptides was established to scan the sequence of the aa21~272 region of HSP90α. The binding activity of Clone-2-chimera and HH01 antibodies to this series of 10-aa peptides was determined by ELISA. The results showed that the possible epitope sites of the Clone-2-chimera antibody were ₂₃₅ AEEKEDKEEE ₂₄₄ and ₂₅₁ ESEDKPEIED ₂₆₀ of HSP90α (Fig. 3(b)). Consistently, these two sites could also serve as the binding epitopes of the HHO1 antibody (Fig. 3(c)). Based on the sequences of these two sites, we synthesized two series of 10-aa peptides, sequentially replacing amino acid residues with alanine. The alanine scanning strategy was used to identify the key amino acids that Clone-2-chimera and HH01 antibodies bind to the epitope site. The results showed that when E237, E239, K241, E253, and K255 were replaced with alanine, the binding activity of Clone-2-chimera antibody to the epitope site decreased sharply (Fig. 3(d)). Furthermore, another D240 was also responsible for the binding to the HHO1 antibody (Fig. 3(d)). According to the above epitope mapping results, we synthesized a peptide with the aa227~272 region sequence of HSP90α. The peptide competitively inhibited rHSP90α-induced PANC-1 cell invasion (Fig. 3(e)) and spheroid formation (Fig. 3(f)), confirming that the epitope-containing region of HSP90α may be a novel anticancer strategy target.

Evaluation of the preventive and therapeutic effects of HH01 antibody in PDAC transplantation model of histo-mesenchymal hyperplasia

The HH01 antibody was used for further in vivo anticancer tests. In our previous study, EndoMT-derived CAFs significantly promoted the tumor growth of pancreatic ductal adenocarcinoma Panc 02 cells in experimental mice. Our Masson trichrome staining results further revealed that EndoMT cell-involved Panc 02 tumors exhibited a significant degree of tissue-stromal proliferation, which was not observed in tumors derived from Panc 02 cells alone (Fig. 4(a)). To investigate the in vivo anticancer activity of the HH01 antibody, we followed the previous administration method using a mouse monoclonal antibody and measured its cancer prevention activity in a mouse PDAC model in which EndoMT participated. C57BL/6 mice were subcutaneously inoculated with Panc 02 cells+EndoMT cells. On day 4 after inoculation, the mice were further intravenously administered 5 mg/kg of control IgG or HHO1 antibody every 3 days for a total of 8 doses ( FIG. 4( b )). As shown in Figure 4(c), the HH01 antibody effectively inhibited the tumor growth of Panc 02 cancer cells in the subcutaneous area of mice in which EndoMT was involved. All mice were sacrificed on day 30 after inoculation, and their tumors were weighed. As expected, HH01 antibody significantly inhibited the tumor growth ability of Panc 02 plus EndoMT cells ( p <0.001, Fig. 4(d)). HH01 antibody also significantly prevented tumor interstitial proliferation (Fig. 4(e)). Furthermore, we detected increased eHSP90α content in the serum of a mouse PDAC model of interstitial hyperplasia, and the serum HSP90α content was significantly decreased in mice treated with HH01 antibody (Fig. 4(f)). These data indicate that the HH01 antibody is an effective agent that prevents tumor growth of interstitial proliferative PDAC cells.

To evaluate the therapeutic effect of HH01 antibody in a murine PDAC model of interstitial proliferation, C57BL/6 mice were subcutaneously inoculated with Panc 02 plus EndoMT cell grafts. On the 20th day after inoculation, the mice had tumors with an average size of ⁷⁵ mm, and the mice were injected intravenously with each dose of 5 mg/kg control IgG or HH01 antibody, once every 7 days, a total of three times ( Figure 5(a)). In vivo measurements showed that HH01 treatment completely inhibited the growth kinetics of subcutaneous tumors (Fig. 5(b)). Tumors were weighed on day 41, and the data showed that tumor growth was indeed inhibited in the HHOl -treated group of mice ( FIG. 5( c )). HH01 treatment also significantly reduced the degree of tumor interstitial proliferation (Fig. 5(d)) and serum HSP90α content (Fig. 5(e)). Taken together, these data suggest that the HH01 antibody can be developed as an effective therapeutic agent for interstitial proliferative PDAC.

Evaluation of the therapeutic effect of HH01 antibody in a humanized mouse PDAC transplantation model of interstitial hyperplasia

We also evaluated the therapeutic effect of the HH01 antibody in a humanized mouse PDAC model of interstitial proliferation. hHSC-transplanted ASID mice were inoculated subcutaneously with a mixture of human pancreatic ductal adenocarcinoma PANC-1 cells and EndoMT-derived cells. On day 20 after inoculation, mice were divided into 3 groups for further treatment (Fig. 6(a)). Group A mice (n=3) were intravenously injected with three doses of control IgG at a dose of 5 mg/kg at intervals of 7 days. Group B mice (n=5) were injected three times with control IgG plus two gemcitabine, while group C mice (n=5) were injected three times with HHO1 antibody plus two gemcitabine. The mice received the first dose of 5 mg/kg IgG (group B) or HH01 (group C) on the 20th day after inoculation of PANC-1+EndoMT cells, and 4 days later, the first dose of 100 mg/kg gemcitabine was further intravenously injected. The other two doses of control IgG or HH01 antibody with and another dose of gemcitabine were each administered at 7-day intervals (Figure 6(a)). Until the 39th day, body surface measurements were performed on subcutaneously growing tumors every 3 days, and the tumor growth curve was plotted as Fig. 6(b). Mice treated with IgG plus gemcitabine had significantly suppressed tumors after a second administration of gemcitabine compared to mice treated with IgG alone. However, in mice treated with HH01 plus gemcitabine, effective tumor growth cessation was observed only 4 days after the first dose of HH01 injection, with further tumor shrinkage following administration of a second dose of gemcitabine and a third dose of HH01 (Fig. 6 (b)). All mice were sacrificed on day 39, and the tumors of mice treated with HHO1 plus gemcitabine were significantly suppressed (Fig. 6(c)). The combination of HH01 antibody and gemcitabine also showed significant efficacy in inhibiting tumor interstitial proliferation ( FIG. 6( d )) and serum HSP90α content ( FIG. 6( e )). These data indicate that the HHO1 antibody may be more effective against interstitial proliferative PDAC in combination with other agents such as gemcitabine.

HH01 antibody in K-Ras ^G12D Evaluation of Therapeutic Effects of Induced Interstitial Hyperplasia in a Mouse PDAC Model

Next, we evaluated the therapeutic effect of the HH01 antibody in a mouse model of spontaneously developing PDAC. Clinically, more than 90% of PDAC patients harbor activating K-Ras mutants in their adenocarcinoma cells. In the transgenic mouse model, pancreatic ductal epithelial cells containing activated mutant K-Ras ^G12D can spontaneously develop PDAC through pancreatic ductal lesions (ADM) and pancreatic ductal intraepithelial neoplasia (PanIN). These transgenic mice expressing K-Ras ^G12D in pancreatic duct epithelial cells (LSL-KrasG12D/Pdx1-Cre mice, referred to as KC mice) developed ADM as early as 3 months after birth, and 3-6 months is the PanIN evolution period , and then developed signs of PDAC at 6 months of age. The results of Masson's trichrome staining showed that a small amount of interstitial hyperplasia could be detected in the pancreas of KC mice at the age of 3 months, and the whole pancreas showed extensive interstitial hyperplasia after the age of 6 months. This was not detected in LSL-KrasG12D control mice. In order to evaluate the therapeutic effect of HH01 antibody, KC mice were treated after developing signs of PDAC at 6 months old, and each dose of 5 mg/kg of control IgG or HH01 antibody was injected intravenously, starting once a week for 4 days. One dose, followed by one dose every two weeks for 4 doses, and finally one dose every month for 2 doses, for a total of 10 doses of treatment ( FIG. 7( a )). The end point of the whole experiment was day 450, and the survival curves of mice treated with IgG or HH01 were drawn by Kaplan-Meier method ( FIG. 7( b )). The results showed that 6 of 7 IgG-treated mice died within 450 days, but 7 of 9 HH01-treated mice were still alive at 450 days. Median survival times for IgG and HHOl -treated mice were 344 days and >450 days, respectively (analyzed by Log rank, p = 0.004). Unlike the smooth pancreas appearance of normal C57BL/6 mice or LSL-KrasG12D control mice, the pancreas of KC mice showed nodular, sclerotic appearance regardless of whether they received HH01 treatment (Fig. 7(c)). However, Masson's trichrome staining showed that the pancreas interstitial hyperplasia of KC mice treated with HH01 and still alive at day 450 was obviously restored ( FIG. 7( c )). It was observed that one IgG-treated KC mouse and one HH01-treated KC mouse had small tumor nodules on the appearance of the liver, but in number, the HH01-treated mice were significantly less than the IgG-treated mice (Fig. 7(c )). Masson's trichrome staining further showed that the interstitial hyperplasia of the liver tissues of mice treated with HH01 was significantly improved ( FIG. 7( c )). In addition, regardless of IgG or HH01-treated KC mice, the HSP90α content of the last serum sample collected before sacrifice was compared with the sample collected before IgG or HH01 treatment (i.e. post-treatment vs. pre-treatment comparison). Our data showed that the increase of HSP90α level in the serum of KC mice could be effectively suppressed by HH01 treatment ( FIG. 7( d )).

Evaluation of the Efficacy of HH01 Antibody Therapy in a M2 Macrophage-Facilitated Mouse PDAC Model

To establish a mouse PDAC model involving M2 macrophages, we mixed rHSP90α-treated BMDM with Panc 02 cancer cells and inoculated them subcutaneously in C57BL/6 mice. For comparison with M1 macrophages, Panc 02 cells and LPS-treated BMDM were inoculated into another group of mice. The "Panc 02+rHSP90α-treated BMDM" group started to grow on day 6 after inoculation, and tumors continued to grow faster than other groups (Fig. 8(a)). On day 30 after inoculation, all mice were sacrificed, and the tumors were weighed. As shown in Figure 8(b), rHSP90α-treated BMDM significantly promoted the growth of Panc 02 tumors, which was in contrast to the tumor suppressive effect produced by LPS-treated BMDM. The results of immunohistochemical staining analysis showed that compared with tumor blocks from other mouse groups, tumor tissues from the "Panc 02+rHSP90α-treated BMDM" group contained more CD163 ⁺ cells, but CD4 ⁺ and CD8 ⁺ The content of cells was significantly less (Fig. 8(c)). In the group of LPS-treated BMDM mice, the suppressed tumor tissues contained relatively high content of CD4 ⁺ and CD8 ⁺ cells. Nuclear staining with 4',6-diamidino-2-phenylindole (DAPI) also showed increased chromatin condensation and nuclear fragmentation (Fig. Phagocytes recruit CD4 ⁺ and CD8 ⁺ immune cells and cause more apoptosis to suppress the growth of Panc 02 tumors. Therefore, these results confirm that eHSP90α-induced M2 macrophages can exhibit effective immunosuppressive and tumor-promoting abilities. Further, we evaluated the therapeutic effect of HH01 antibody in this M2 macrophage-promoted mouse PDAC model. C57BL/6 mice were subcutaneously inoculated with BMDM cell grafts treated with Panc 02 plus rHSP90α. On day 27 after inoculation, each mouse developed approximately 0.1- ^cm3 tumors and started treatment with 5 mg/kg of control IgG or HHO1 antibody (Fig. 9(a)). Since the half-life of HH01 in mouse blood is about 18.4 days, a second dose was added after an interval of 7 days to strengthen the effect of HH01 antibody. As shown in Figure 9(b), a sharp tumor shrinkage after HH01 treatment was observed immediately after 3 days. All mice were sacrificed on day 42, and the results showed that the tumors of HHOl-treated mice were significantly shrunk compared to the persistently growing tumors of mice in the control group ( p = 0.001, Figure 9(c)). Consistently, the increase in serum HSP90α content was also significantly suppressed after HH01 treatment (Fig. 9(d)). Immunohistochemical analysis of tumor samples also showed that the amount of CD4 ⁺ and CD8 ⁺ cells suppressed by M2 macrophages was effectively increased due to the reduction of M2 macrophages after HH01 treatment (Fig. 9(e)). Finally, immunohistofluorescent staining results confirmed these results. An increase in F4/80 ⁺ iNOS ⁺ cells (M1 macrophages) and a decrease in F4/80 ⁺ Arginasel ⁺ cells (M2 macrophages) were observed in tumor tissues from HH01-treated mice (Fig. 10(a) and 10 (b)). Consistently, CD4 ⁺ TNF-α ⁺ cells (effector T cells) and CD8 ⁺ TNF-α ⁺ cells (cytotoxic T cells) were increased in response to HH01 antibody treatment (Fig. 10(c) and 10(d )). These data suggest that the HH01 antibody is an effective agent for inhibiting M2 macrophages from promoting PDAC progression and enhancing immunity in the tumor microenvironment.

in conclusion

Interstitial hyperplasia is a hallmark of many malignant tumors and is closely related to rapid tumor growth, metastasis, and poor treatment outcomes. Reagents and strategies that can inhibit the proliferation of interstitial tissue to alleviate the malignant progression of tumors and improve the efficacy of treatment have always been an urgent medical need for cancer patients. The proliferation of cancer-associated fibroblasts (CAFs) is one of the main causes of tissue-mesenchymal hyperplasia, and the endothelial-mesenchymal transition (EndoMT) of endothelial cells provides a rich source of CAFs. EndoMT-derived CAFs recruit large numbers of bone marrow-derived macrophages to tumors and promote the polarization of these macrophages towards the M2 type. M2 macrophages not only secrete IL-10 and TGF-β to effectively suppress cytotoxic T cells, but also produce VEGF, bFGF and PDGF to stimulate tumor angiogenesis. In addition, these M2 macrophages express and secrete large amounts of eHSP90α, creating an eHSP90α-rich tumor microenvironment that promotes cancer cell proliferation and acquisition of cell stemness. Therefore, eHSP90α and the eHSP90α-enriched tumor microenvironment may be considered as targets for novel cancer therapies. In our previously published mouse tumor model results, EndoMT-derived CAFs significantly promoted tumor growth in pancreatic ductal adenocarcinoma cell xenografts. However, EndoMT-promoting tumor growth was significantly inhibited when mice received intravenous injection of mouse anti-HSP90α monoclonal antibody from day 4 after cancer cell inoculation. We have sequenced the gene of this mouse anti-HSP90α monoclonal antibody. After computer-aided analysis and suggestions, we further constructed humanized and improved antibody genes. The recombinant gene has been introduced into ExpiCHO cells to express the humanized anti-HSP90α antibody HH01. Through a series of analysis, we know that the CDR of the HH01 antibody has a novel amino acid sequence, which shows a high binding affinity to eHSP90α, with a KD value of 1.87×10 ^-10 M. The HH01 antibody does not readily form aggregates. It has good water solubility, but it is not easily excreted from mice, and its half-life in blood is >18.4 days. It has no cytotoxicity to retinal pigment epithelial cells and does not cause spleen enlargement in mice. The HH01 antibody also showed superiority in anticancer function. It effectively inhibits the invasiveness and spheroid-forming ability of pancreatic ductal adenocarcinoma and colorectal cancer cell lines. Results from three of our mouse cancer models demonstrate that the HH01 antibody abolishes EndoMT-promoted stromal-proliferative tumor growth alone and synergistically in combination with gemcitabine. In the gene transfer mouse model of spontaneous PDAC formation, it can effectively inhibit the development of interstitial hyperplastic and pancreatic duct adenocarcinoma and its liver metastasis caused by K-Ras mutation, thereby prolonging the survival time of experimental mice . Furthermore, in a mouse model of PDAC with exacerbated M2 macrophages, the HH01 antibody effectively inhibited tumor growth and enhanced the immunity of the tumor microenvironment.

All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification may be replaced by another feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only one example of a generic series of equivalent or similar features.

Furthermore, from the above description, one skilled in the art can easily ascertain the essential characteristics described, and without departing from the spirit and scope thereof, can make various changes and modifications of the present disclosure to adapt it to various usages and conditions. Accordingly, other embodiments are within the scope of the claims.

<110> National Institutes of Health

<120> Anti- HSP90α antibody and use thereof

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Claims (13)

An isolated antibody comprising: heavy chain complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region sequence of SEQ ID NO: 2, wherein the heavy chain CDR1 has the sequence of SEQ ID NO: 3, and the heavy chain CDR2 has the sequence of SEQ ID NO: 3 ID NO: 4 sequence, the heavy chain CDR3 has the sequence of SEQ ID NO: 5; and the light chain complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region sequence of SEQ ID NO: 7, wherein the light chain CDR1 has the sequence of SEQ ID NO: 7 ID NO:8 sequence, the light chain CDR2 has the sequence of SEQ ID NO:9, and the light chain CDR3 has the sequence of SEQ ID NO:10. An isolated antibody comprising: heavy chain complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region sequence of SEQ ID NO: 12, wherein the heavy chain CDR1 has the sequence of SEQ ID NO: 13, and the heavy chain CDR2 has the sequence of SEQ ID NO: 13 ID NO: 14 sequence, the heavy chain CDR3 has the sequence of SEQ ID NO: 15; and the light chain complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region sequence of SEQ ID NO: 17, wherein the light chain CDR1 has the sequence of SEQ ID NO: 17 ID NO: 18 sequence, the light chain CDR2 has the sequence of SEQ ID NO: 19, and the light chain CDR3 has the sequence of SEQ ID NO: 20. The antibody according to claim 1 or 2, wherein the isolated antibody is an antibody comprising an Fc region, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a single-chain antibody, and a scFV Multimer, a monoclonal antibody, a monovalent antibody, a multispecific antibody, a humanized antibody or a chimeric antibody. A nucleic acid molecule comprising a nucleic acid sequence encoding the isolated antibody of any one of claims 1-3. A pharmaceutical composition, comprising the isolated antibody described in any one of Claims 1 to 3 or the nucleic acid molecule described in Claim 4; and a pharmaceutically acceptable carrier. The use of a pharmaceutical composition for preparing a drug for treating cancer, wherein the pharmaceutical composition includes: the isolated antibody as described in any one of claims 1 to 3 or the nucleic acid molecule as described in claim 4, and the cancer Features of interstitial hyperplasia or deterioration of M2 macrophages. The use as described in claim 6, wherein the cancer has a characteristic of interstitial proliferation. The use according to claim 6, wherein the cancer has a characteristic of being exacerbated by M2 macrophages. The use as described in claim 6, wherein the cancer is pancreatic cancer, colorectal cancer, breast cancer, liver cancer or lung cancer. The use as described in claim 6, wherein the pharmaceutical composition further includes a therapeutic agent. The use as described in claim 10, wherein the therapeutic agent is gemcitabine. A use of the isolated antibody according to any one of claims 1 to 3, for detecting the blood HSP90α content of a subject to monitor tumor shrinkage of the subject. A use of a pharmaceutical composition for preparing a drug for treating tissue interstitial hyperplasia, wherein the pharmaceutical composition includes: the isolated antibody as described in any one of Claims 1 to 3 or the nucleic acid molecule as described in Claim 4.

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