Treatment methods for bone cancer

Abstract

This disclosure provides compositions and methods for treating bone cancer, e.g., osteosarcoma, in patients who have previously failed at at least one alternative therapy for the treatment of osteosarcoma. The method includes promoting the opening of Cx43 hemichannels in osteocytes by using, for example, a composition comprising an anti-connexin 43 antibody. This disclosure provides methods and compositions for treating cancer, e.g., bone cancer, in subjects or patients where needed, and includes administering at least one dose of an anti-Cx43 antibody to the subject or patient. In some embodiments, the anti-Cx43 antibody promotes the opening of Cx43 hemichannels. In some embodiments, the anti-Cx43 antibody comprises a specific CDR amino acid sequence.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Application 63 / 504,162, filed on 24 May 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Incorporation of sequence lists This application includes a sequence listing submitted via the Patent Center. The sequence listing, named 172628-202002_PCT_SL.xml, created on 8 May 2024 and having a size of 14,456 bytes, is incorporated herein by reference in its entirety. [Background technology]

[0003] Osteosarcoma is the most common primary malignant bone tumor, originating from primitive osteogenic mesenchymal stem cells. Osteosarcoma has a bimodal age distribution, with a first peak in adolescents aged 10-14 years and a second peak in adults over 65 years. It is generally more common in men than in women, with a rate of 5.4 / million / year in men compared to 4.0 / million / year in women, and more common in Black (6.8 / million) and Hispanic (6.5 / million) populations than in White (4.6 / million).

[0004] Osteosarcoma typically occurs in the long bones of the limbs near the epiphysis of the metaphyseal growth plate. Of all cancer deaths in childhood and adolescence, 8.9% were due to malignancies of bone and joints. The 5-year survival rate for osteosarcoma was 68%, with no significant difference between sexes. The expected 5-year survival rate for patients with metastasis is 15%–30% (Lin et al., Trends in molecular medicine, 23(8):737-755 (2017)), and patient survival is age-related, with older patients having lower survival rates. The prognosis for osteosarcoma is very poor. Statistically, the 5-year and 10-year overall survival rates are 0.23 and 0.18, respectively (Kempf-Bielacket al., J Clin Oncol., 23(3):559-568 (2005)). Tumor stage, metastasis, local recurrence, chemotherapy plan, anatomical location, tumor size, and tumor cell destruction rate after neoadjuvant chemotherapy are all related to treatment outcomes (Ottaviani et al., Cancer Treat Res., 152:3-13 (2009)).

[0005] For high-grade osteosarcoma (including intramedullary and surface morphology), neoadjuvant chemotherapy is recommended (grade 1A), followed by re-evaluation and re-staging using chest X-ray, local X-ray, PET, or bone scan. For resectable tumors, extensive resection should be performed. If the resection margin is negative and the response to chemotherapy is good, chemotherapy should be continued. If the response to chemotherapy is poor, the chemotherapy plan can be modified. If the resection margin is positive and the response to chemotherapy is good, chemotherapy should be continued, and other local treatments (surgery, radiotherapy, etc.) can be performed concurrently. If the response to chemotherapy is poor, the chemotherapy plan may be changed, and other local treatments (surgery, radiotherapy, etc.) should be performed concurrently. For unresectable tumors, only radiotherapy and chemotherapy should be considered. Patients should be continuously monitored after treatment.

[0006] Osteocytes exhibit high expression of connexin Cx43 in intercellular junction channels and hemichannels. Gap junction channels mediate cell communication between two adjacent cells, maintaining normal cellular metabolic function, while hemichannels enable molecular exchange between the cell and the extracellular environment. Hemichannels are normally closed but are activated and open under certain conditions such as mechanical stress or ischemia, cytokines, or ion stimulation. Open hemichannels can pass small molecules (<1.2 kDa) such as ATP, NAD+, glutamate, and inositol triphosphate (IP3) (Goodenough et al., Nature reviews Molecular cell biology, 4(4):285-294 (2003)). Opening of Cx43 hemichannels in osteocytes has been shown to have a strong inhibitory effect on the growth, migration, and bone metastasis of breast cancer tumors (Zhou et al., Oncogene, 35(43):5597-5607 (2016)). The current first-line chemotherapy drugs for osteosarcoma are methotrexate, doxorubicin, cisplatin, and ifosfamide. As recommended by CSCO in 2020, there is no second-line treatment plan demonstrating an overall survival benefit, so participation in clinical trials is an opportunity for osteosarcoma patients who have failed first-line treatment to achieve better efficacy or to resume treatment. The literature has reported that a drug is considered effective in clinical trials if it provides a response rate of more than 5% or a 4-month PFS rate of more than 40%. Systemic chemotherapy is the first-line treatment for advanced osteosarcoma, and there have been no major breakthroughs in the treatment of advanced osteosarcoma in the past few decades. Currently, the need for drug therapy in advanced osteosarcoma is great and unmet. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Lin et al.,Trends in molecular medicine,23(8):737-755(2017)

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

[0008] One aspect of the present disclosure is a method for treating bone cancer in a subject in need thereof, comprising administering to the subject at least one dose of an anti-connexin 43 (Cx43) antibody or an antigen-binding fragment thereof, wherein the anti-Cx43 antibody comprises a heavy-chain variable region comprising an HCDR1 amino acid sequence comprising SEQ ID NO: 1, an HCDR2 amino acid sequence comprising SEQ ID NO: 2, and an HCDR3 amino acid sequence comprising SEQ ID NO: 3, and a light-chain variable region comprising an LCDR1 amino acid sequence comprising SEQ ID NO: 4, an LCDR2 amino acid sequence comprising SEQ ID NO: 5, and an LCDR3 amino acid sequence comprising SEQ ID NO: 6.

[0009] In some embodiments, the at least one dose of the anti-Cx43 antibody or an antigen-binding fragment thereof is from about 0.001 mg / kg to about 300 mg / kg. In some particular embodiments, the at least one dose of the anti-Cx43 antibody or an antigen-binding fragment thereof is about 1 mg / kg, about 3 mg / kg, about 6 mg / kg, about 12 mg / kg, about 18 mg / kg, about 24 mg / kg, about 30 mg / kg, or about 36 mg / kg.

[0010] In some embodiments, the anti-Cx43 antibody or its antigen-binding fragment is administered approximately once a day to approximately once every 12 months. In some specific embodiments, the anti-Cx43 antibody or its antigen-binding fragment is administered approximately once every two weeks, approximately once every three weeks, or approximately once every four weeks. In one embodiment, the anti-Cx43 antibody or its antigen-binding fragment is administered approximately once every three weeks.

[0011] In some embodiments, the anti-Cx43 antibody or its antigen-binding fragment is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, or subcutaneously. In specific embodiments, the anti-Cx43 antibody or its antigen-binding fragment is administered intravenously.

[0012] In some embodiments, the anti-Cx43 antibody or its antigen-binding fragment is administered within approximately 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, 150 minutes, 180 minutes, or longer.

[0013] In some embodiments, the anti-Cx43 antibody or its antigen-binding fragment is administered in a pharmaceutically acceptable composition. In some embodiments, the composition includes a pharmaceutically acceptable carrier.

[0014] In some embodiments, the anti-Cx43 antibody or its antigen-binding fragment includes a heavy chain variable region containing an amino acid sequence according to SEQ ID NO: 7, and / or a light chain variable region containing an amino acid sequence according to SEQ ID NO: 8.

[0015] In some embodiments, the anti-Cx43 antibody comprises a heavy chain containing an amino acid sequence according to any one of SEQ ID NOs: 9 and 11-13, and / or a light chain containing an amino acid sequence according to SEQ ID NO: 10.

[0016] In some embodiments, the anti-Cx43 antibody is a humanized antibody.

[0017] In some embodiments, an anti-Cx43 antibody or its antigen-binding fragment enhances Cx43 hemichannel opening in the subject.

[0018] In some embodiments, bone cancer is osteosarcoma.

[0019] In some embodiments, osteosarcoma is resectable, while in other embodiments, it is not.

[0020] In some embodiments, the osteosarcoma is stage IA, stage IB, stage II-A, stage II-B, stage III, stage IV-A, or stage IV-B.

[0021] In some embodiments, osteosarcoma is low-grade, while in other embodiments, osteosarcoma is high-grade.

[0022] In some embodiments, osteosarcoma is primary osteosarcoma, osteoblastic osteosarcoma, chondroblastic osteosarcoma, fibroblastic osteosarcoma, small cell osteosarcoma, telangiectatic osteosarcoma, paraosteal osteosarcoma, periosteal osteosarcoma, typical osteosarcoma, osteoblastic osteosarcoma sclerosing osteosarcoma, chondroblastomatous osteosarcoma, chondromyxoid fibrous osteosarcoma, clear cell osteosarcoma, malignant fibrous histiocytomatous osteosarcoma, giant cell osteosarcoma, and / or epithelioid osteosarcoma.

[0023] In some embodiments, the subjects have failed to receive treatment with at least one standard osteosarcoma care, including chemotherapy, surgery, immunotherapy, or radiotherapy, or a combination thereof.

[0024] In some embodiments, the subjects have failed to receive treatment with methotrexate, doxorubicin, cisplatin, and ifosfamide, apatinib, analotinib, vindesine, vincristine, docetaxel, paclitaxel, irinotecan, bortezomib, albumin-conjugated paclitaxel, nedaplatin (Aqupla), pemetrexed, etoposide, gemcitabine, lovaplatin, recombinant human endostatin, eribulin, dacarbazine, pazopanib, immune checkpoint inhibitors, surgery, or radiotherapy, or a combination thereof.

[0025] In some embodiments, after treatment with an anti-Cx43 antibody or its antigen-binding fragment, subjects achieve (i) progression-free survival of at least 1 month, 2 months, 3 months, at least 4 months, at least 5 months, or at least 6 months; (ii) improvement in overall survival of at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 6 months; (iii) improvement in EQ-5D score; (iv) improvement in disease progression as assessed by PERCIST criteria, RECIST criteria, and / or ICDS criteria; (v) improvement in pain score as assessed by NRS; and / or (vi) reduction of at least 1%, at least 2%, at least 3%, at least 5%, at least 8%, at least 10%, at least 15%, at least 20%, or at least 30% in tumor size.

[0026] In some embodiments, subjects achieve a complete or partial response to treatment with an anti-Cx43 antibody.

[0027] Further aspects of the present disclosure provide a method for treating bone cancer in a subject requiring it, comprising administering at least one dose of an anti-connexin 43 (Cx43) antibody or its antigen-binding fragment to the subject, wherein the at least one dose of the anti-Cx43 antibody is between approximately 0.01 mg / kg and approximately 300 mg / kg.

[0028] In some embodiments, at least one dose of the anti-Cx43 antibody or its antigen-binding fragment is about 1 mg / kg, about 3 mg / kg, about 6 mg / kg, about 12 mg / kg, about 18 mg / kg, about 24 mg / kg, about 30 mg / kg, or about 36 mg / kg.

[0029] In some embodiments, the anti-Cx43 antibody or its antigen-binding fragment is administered approximately once a day to approximately once every 12 months. In some specific embodiments, the anti-Cx43 antibody or its antigen-binding fragment is administered approximately once every two weeks, approximately once every three weeks, or approximately once every four weeks. In one embodiment, the anti-Cx43 antibody or its antigen-binding fragment is administered approximately once every three weeks.

[0030] In some embodiments, the anti-Cx43 antibody or its antigen-binding fragment is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically. In one embodiment, the anti-Cx43 antibody or its antigen-binding fragment is administered intravenously.

[0031] In some embodiments, the anti-Cx43 antibody or its antigen-binding fragment is administered within approximately 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, 150 minutes, 180 minutes, or longer.

[0032] In some embodiments, the anti-Cx43 antibody or its antigen-binding fragment is administered in a pharmaceutically acceptable composition. In some embodiments, the pharmaceutically acceptable composition includes a pharmaceutically acceptable carrier.

[0033] In some embodiments, the anti-Cx43 antibody or its antigen-binding fragment comprises a heavy chain variable region including an HCDR1 amino acid sequence including SEQ ID NO: 1, an HCDR2 amino acid sequence including SEQ ID NO: 2, and an HCDR3 amino acid sequence including SEQ ID NO: 3, and a light chain variable region including an LCDR1 amino acid sequence including SEQ ID NO: 4, an LCDR2 amino acid sequence including SEQ ID NO: 5, and an LCDR3 amino acid sequence including SEQ ID NO: 6.

[0034] In some embodiments, the anti-Cx43 antibody or its antigen-binding fragment includes a heavy chain variable region containing an amino acid sequence according to SEQ ID NO: 7, and / or a light chain variable region containing an amino acid sequence according to SEQ ID NO: 8.

[0035] In some embodiments, the anti-Cx43 antibody comprises a heavy chain containing an amino acid sequence according to any one of SEQ ID NOs: 9 and 11-13, and / or a light chain containing an amino acid sequence according to SEQ ID NO: 10.

[0036] In some embodiments, the anti-Cx43 antibody is a humanized antibody.

[0037] In some embodiments, an anti-Cx43 antibody or its antigen-binding fragment enhances Cx43 hemichannel opening in the subject.

[0038] In some embodiments, bone cancer is osteosarcoma.

[0039] In some embodiments, osteosarcoma is resectable, but in some other embodiments, osteosarcoma is not resectable.

[0040] In some embodiments, the osteosarcoma is stage IA, stage IB, stage II-A, stage II-B, stage III, stage IV-A, or stage IV-B.

[0041] In some embodiments, osteosarcoma is low-grade, while in some other embodiments, osteosarcoma is high-grade.

[0042] In some embodiments, osteosarcoma is primary osteosarcoma, osteoblastic osteosarcoma, chondroblastic osteosarcoma, fibroblastic osteosarcoma, small cell osteosarcoma, telangiectatic osteosarcoma, paraosteal osteosarcoma, periosteal osteosarcoma, typical osteosarcoma, osteoblastic osteosarcoma sclerosing osteosarcoma, chondroblastomatous osteosarcoma, chondromyxoid fibrous osteosarcoma, clear cell osteosarcoma, malignant fibrous histiocytomatous osteosarcoma, giant cell osteosarcoma, and / or epithelioid osteosarcoma.

[0043] In some embodiments, the subjects have failed to receive treatment with at least one standard treatment for osteosarcoma, including chemotherapy, surgery, radiotherapy, immunotherapy, or a combination thereof.

[0044] In some embodiments, the subjects have failed to receive treatment with methotrexate, doxorubicin, cisplatin, and ifosfamide, apatinib, analotinib, vindesine, vincristine, docetaxel, paclitaxel, irinotecan, bortezomib, albumin-conjugated paclitaxel, nedaplatin (Aqupla), pemetrexed, etoposide, gemcitabine, lovaplatin, recombinant human endostatin, eribulin, dacarbazine, pazopanib, immune checkpoint inhibitors, surgery, or radiotherapy, or a combination thereof.

[0045] In some embodiments, after treatment with an anti-Cx43 antibody or its antigen-binding fragment, subjects achieve (i) progression-free survival of at least 1 month, 2 months, 3 months, at least 4 months, at least 5 months, or at least 6 months; (ii) improvement in overall survival of at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 6 months; (iii) improvement in EQ-5D score; (iv) improvement in disease progression as assessed by PERCIST criteria, RECIST criteria, and / or ICDS criteria; (v) improvement in pain score as assessed by NRS; and / or (vi) reduction of at least 1%, at least 2%, at least 3%, at least 5%, at least 8%, at least 10%, at least 15%, at least 20%, or at least 30% in tumor size.

[0046] In some embodiments, subjects achieve a complete or partial response to treatment with an anti-Cx43 antibody or its antigen-binding fragment. [Brief explanation of the drawing]

[0047] [Figure 1] This shows the pain severity rating on a numerical evaluation scale. [Figure 2]The VAS scale for the EQ-5D questionnaire is shown. [Figure 3] This is a swimlane plot showing the overall efficacy determined by the principal investigators for 24 enrolled participants. Participant S02001 received ALMB-0168 at a dose of 1 mg / kg per dose; Participant S02002 received ALMB-0168 at a dose of 3 mg / kg per dose; Participants S01001, S01002, and S02003 received ALMB-0168 at a dose of 6 mg / kg per dose; Participants S02006 and S06001 received ALMB-0168 at a dose of 12 mg / kg per dose; Participants S02007, S06002, and S06008 received ALMB-0168 at a dose of 18 mg / kg per dose; Participants S02010, S09001, and S1100 Participant 3 received ALMB-0168 at a dose of 24 mg / kg; participants S02012, S06004, S01003, and S03001 received ALMB-0168 at a dose of 30 mg / kg; participants S01004 and S06005 received ALMB-0168 at a dose of 36 mg / kg; an upward-pointing triangle indicates a partial response (PR), a circle indicates stable disease (SD), a diamond indicates progressive disease (PD), a right-pointing triangle indicates the trial is ongoing, an "X" indicates the end of the trial (EOS), a pentagon indicates the initiation of a new anticancer therapy, and a plus sign indicates death. [Figure 4] This is a waterfall plot showing the best-case change in the target lesion determined by the principal investigator compared to baseline (efficacy evaluable analysis set). [Figure 5] This is the left knee prosthesis visible at baseline in participant S01001. Recurrent metastasis was observed in the right upper femur. [Figure 6A]These are cross-sectional images of participant S01001 along its longest axis: a baseline image collected on October 28, 2021 (Figure 6A), and the first five tumor evaluation images collected on December 20, 2021 (Figure 6B), February 23, 2022 (Figure 6C), April 19, 2022 (Figure 6D), June 27, 2022 (Figure 6E), and August 29, 2022 (Figure 6F), respectively. The participant achieved a partial response and increased bone mineral density according to RECIST 1.1. [Figure 6B] These are cross-sectional images of participant S01001 along its longest axis: a baseline image collected on October 28, 2021 (Figure 6A), and the first five tumor evaluation images collected on December 20, 2021 (Figure 6B), February 23, 2022 (Figure 6C), April 19, 2022 (Figure 6D), June 27, 2022 (Figure 6E), and August 29, 2022 (Figure 6F), respectively. The participant achieved a partial response and increased bone mineral density according to RECIST 1.1. [Figure 6C] These are cross-sectional images of participant S01001 along its longest axis: a baseline image collected on October 28, 2021 (Figure 6A), and the first five tumor evaluation images collected on December 20, 2021 (Figure 6B), February 23, 2022 (Figure 6C), April 19, 2022 (Figure 6D), June 27, 2022 (Figure 6E), and August 29, 2022 (Figure 6F), respectively. The participant achieved a partial response and increased bone mineral density according to RECIST 1.1. [Figure 6D] These are cross-sectional images of participant S01001 along its longest axis: a baseline image collected on October 28, 2021 (Figure 6A), and the first five tumor evaluation images collected on December 20, 2021 (Figure 6B), February 23, 2022 (Figure 6C), April 19, 2022 (Figure 6D), June 27, 2022 (Figure 6E), and August 29, 2022 (Figure 6F), respectively. The participant achieved a partial response and increased bone mineral density according to RECIST 1.1. [Figure 6E]These are cross-sectional images of participant S01001 along its longest axis: a baseline image collected on October 28, 2021 (Figure 6A), and the first five tumor evaluation images collected on December 20, 2021 (Figure 6B), February 23, 2022 (Figure 6C), April 19, 2022 (Figure 6D), June 27, 2022 (Figure 6E), and August 29, 2022 (Figure 6F), respectively. The participant achieved a partial response and increased bone mineral density according to RECIST 1.1. [Figure 6F] These are cross-sectional images of participant S01001 along its longest axis: a baseline image collected on October 28, 2021 (Figure 6A), and the first five tumor evaluation images collected on December 20, 2021 (Figure 6B), February 23, 2022 (Figure 6C), April 19, 2022 (Figure 6D), June 27, 2022 (Figure 6E), and August 29, 2022 (Figure 6F), respectively. The participant achieved a partial response and increased bone mineral density according to RECIST 1.1. [Figure 7]Images of participant S01001 are shown: baseline image (Panel 1) collected on October 28, 2021, and four tumor evaluation images collected on December 20, 2021 (Panel 2), February 23, 2022 (Panel 3), April 19, 2022 (Panel 4), and August 29, 2022 (Panel 5), respectively. Panel 1: mean lesion size is approximately 187.45, standard deviation is approximately 128.3, area is 11.02 cm2, maximum length ≥ 706 units, minimum length ≤ -252 units. Panel 2: mean lesion size is approximately 476.07 units, standard deviation is approximately 292.39 units, area is 6.9 cm2, maximum length ≥ 1391 units, minimum length ≤ -252 units. Third panel: The mean lesion size is approximately 796.28 units, the standard deviation is approximately 457 units, the area is 5.621 cm², the maximum length is ≥ 1954 units, and the minimum length is ≤ -344 units. Fourth panel: The mean lesion size is approximately 1009.3 units, the standard deviation is approximately 398.84 units, the area is 5.533 cm², the maximum length is ≥ 1697 units, and the minimum length is ≤ -115 units. Fifth panel: The mean lesion size is approximately 970.93 units, the standard deviation is approximately 23 units, the area is 4.594 cm², the maximum length is ≥ 1320 units, and the minimum length is ≤ 49 units. [Figure 8A] These images show lesion 1 (Figure 8A) and lesion 2 (Figure 8B) of participant S02007, collected at baseline (left panel) and after treatment (right panel). As shown in the left panel of Figure 8A, the length of lesion 1 at baseline is 3.19 cm. As shown in the right panel of Figure 8A, the length of lesion 2 at baseline is 2.09 cm. As shown in the left panel of Figure 8B, the length of lesion 1 after treatment is 1.94 cm. As shown in the right panel of Figure 8B, the length of lesion 2 after treatment is 1.41 cm. [Figure 8B]These images show lesion 1 (Figure 8A) and lesion 2 (Figure 8B) of participant S02007, collected at baseline (left panel) and after treatment (right panel). As shown in the left panel of Figure 8A, the length of lesion 1 at baseline is 3.19 cm. As shown in the right panel of Figure 8A, the length of lesion 2 at baseline is 2.09 cm. As shown in the left panel of Figure 8B, the length of lesion 1 after treatment is 1.94 cm. As shown in the right panel of Figure 8B, the length of lesion 2 after treatment is 1.41 cm. [Modes for carrying out the invention]

[0048] This disclosure provides methods and compositions for treating cancer, such as bone cancer, in subjects or patients where required, comprising administering at least one dose of an anti-Cx43 antibody to the subject or patient. In some embodiments, the anti-Cx43 antibody promotes the opening of Cx43 hemichannels. In some embodiments, the anti-Cx43 antibody comprises a specific CDR amino acid sequence. In some embodiments, the anti-Cx43 antibody is administered according to a drug regimen.

[0049] definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings that are ordinarily understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms.

[0050] It should be understood that the present invention is not limited to, and is therefore subject to change, the specific methodologies, protocols, and reagents described herein. The terms used herein are intended solely to describe specific embodiments and are not intended to limit the scope of the present invention as defined solely by the claims.

[0051] As used herein, the articles “a,” “an,” and “the” are used to refer to one or more (i.e., at least one) grammatical objects of the articles. For example, “element” means one or more elements.

[0052] The use of alternatives (e.g., "or") should be understood to mean one, both, or any combination thereof of the alternatives.

[0053] The term "and / or" should be understood to mean either one or both of the alternatives.

[0054] As used herein, the terms “about” or “approximately” refer to a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to the quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of reference. In one embodiment, the terms “about” or “approximately” refer to a range of a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of reference.

[0055] As used herein, the terms “substantially” or “essentially” refer to a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that is approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of reference. In one embodiment, the terms “essentially the same” or “substantially the same” refer to a range of quantities, levels, values, numbers, frequencies, percentages, dimensions, size, volume, weight, or length that is approximately the same as the quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of reference.

[0056] Throughout this specification, unless otherwise required by context, the terms “comprise,” “comprises,” and “comprising” are understood to mean including the described process or element or group of processes or elements, but not to mean excluding any other process or element or group of processes or elements. In certain embodiments, the terms “include,” “have,” “contain,” and “comprise” are used as synonyms.

[0057] The phrase "consists of" means that what follows it is included and limited to it. Therefore, the phrase "consists of" indicates that the listed elements are necessary or essential, and that other elements may not be present.

[0058] "Essentially consisting of" means including any elements listed after the phrase, but limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure of the listed elements. Thus, the phrase "essentially consisting of" indicates that the listed elements are necessary or essential, but the other elements are not optional and may or may not be present depending on whether or not they affect the activity or action of the listed elements.

[0059] The term “to provide” is used in accordance with its usual meaning of supplying or providing for use. In some embodiments, a protein (e.g., an antibody) is provided directly by administering the protein, and in other embodiments, a protein (e.g., an antibody) is provided effectively by administering the nucleic acid encoding the protein. In certain embodiments, the present invention envisions compositions comprising various combinations of nucleic acids, antigens, peptides, and / or epitopes.

[0060] Throughout this specification, references to “one embodiment,” “embodiment,” “specific embodiment,” “related embodiment,” “specific embodiment,” “additional embodiment,” or “further embodiment,” or any combination thereof, mean that any particular feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment of the present invention. Therefore, occurrences of the aforementioned phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, specific features, structures, or characteristics can be combined in any preferred manner in one or more embodiments.

[0061] As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to molecules having amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids in a polypeptide. As used herein, these terms refer to both short chains, also commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, also commonly referred to in the art as polypeptides or proteins. A “polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0062] As used herein, the terms “percent sequence identity” or “sequence identity” refer to the degree of identity between any given query sequence and a target sequence. The percentage of identity of any query nucleic acid or amino acid sequence to another target nucleic acid or amino acid sequence can be determined using tools and techniques known in the art, such as NCBI BLAST.

[0063] The term "pharmaceutical preparation" or "pharmaceutical composition" refers to a preparation containing a therapeutic agent (e.g., an anti-Cx43 antibody). It is in a form that enables the biological activity of the antibody to be effective and does not contain additional components that are unacceptably toxic to the subject to whom the preparation is administered.

[0064] As used herein, the term "antigen" is a molecule that can be bound by an antibody or a T cell receptor. In certain embodiments, binding moieties other than antibodies are engineered to specifically bind to an antigen, such as an aptamer, an avimer, etc.

[0065] As used herein, the term "specifically binds" is not intended to indicate that an antibody binds exclusively to its intended target. Rather, an antibody "specifically binds" when its affinity for its intended target is about 5-fold greater when compared to its affinity for non-target molecules. It is appropriate that there is no significant cross-reaction or cross-linking with unwanted substances. The affinity of the antibody is, for example, at least about 5-fold, such as 10-fold, such as 25-fold, particularly 50-fold, particularly 100-fold or more greater for the target molecule than for non-target molecules. In some embodiments, specific binding between an antibody or other binding agent and an antigen is at least 10 6 M -1 of binding affinity. An antibody is, for example, at least about 10 7 M -1 , for example, about 10 8 M -1 ~ about 10 9 M -1 , about 10 9 M -1 ~ about 10 10 M -1 , or about 10 10 M -1 ~ about 10 11 M -1Antibodies can bind with affinity. Antibodies can bind with EC of, for example, 50 nM or less, 10 nM or less, 1 nM or less, 100 pM or less, or more preferably 10 pM or less. As is known in the art, antibodies that are specifically immunoreactive to an antigen can be selected using various immunoassay formats. For example, solid-phase ELISA immunoassays are conventionally used to select monoclonal antibodies that are specifically immunoreactive to a protein. For a description of immunoassay formats and conditions that may be used to determine specific immunoreactivity, see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1988.

[0066] Affinity-matured antibodies have one or more modifications in one or more hypervariable regions, which results in improved antibody affinity to an antigen compared to parent antibodies without these modifications. In one embodiment, affinity-matured antibodies have nanomolar or picomolar affinity to a target antigen. Affinity-matured antibodies are produced by procedures known in the art. Marks et al. Bio / Technology 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDRs and / or framework residues has been described by Barbas et al., Proc Nat. Acad. Sci USA, 91:3809-3813 (1994); Schier et al., Gene, 169:147-155 (1995); Yelton et al., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol., 154(7):3310-9 (1995); and Hawkins et al., J. Mo / . Biol., 226:889-896 (1992).

[0067] "Cancer" or "tumor" is intended to include any malignant or neoplastic growth in a patient, including initial tumors and any metastases. Cancer can be hematological or solid tumor types. Hematological malignancies include tumors of hematological origin, such as myeloma (e.g., multiple myeloma), leukemia (e.g., Waldenström syndrome), chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, granulocytic leukemia, monocytic leukemia, acute lymphoblastic leukemia, and other leukemias), lymphoma (e.g., B-cell lymphomas such as diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, plasmacytoma, or reticular sarcoma), and myeloproliferative neoplasms such as myelodysplastic syndromes, thrombocythemia, polycythemia vera, or myelofibrosis. Solid tumors may originate from organs and include cancers of bone, skin, lung, brain, breast, prostate, ovaries, colon, kidney, pancreas, liver, esophagus, stomach, intestines, bladder, uterus, cervix, testes, and adrenal glands. As used herein, cancer cells, including tumor cells, refer to cells that divide at an abnormal (increased) rate, or cells whose control of proliferation or survival differs from that of cells in the same tissue from which the cancer cells originate or survive. Cancer cells include, but are not limited to, cells in carcinomas, sarcomas, myelomas, leukemias, lymphomas, and tumors of the nervous system, including gliomas, meningiomas, medulloblastomas, schwannomas, or epididymatomas. In a more preferred embodiment, bone is bone cancer. In a more preferred embodiment, bone cancer is osteosarcoma.

[0068] The term "affinity" refers to the strength of the binding reaction between the antibody's binding domain and its epitope. It is the sum of the attractive and repulsive forces acting between the binding domain and the epitope. As used herein, the term affinity refers to the dissociation constant KD.

[0069] The term “epitope” includes any determinant, preferably polypeptide determinant, that can specifically bind to an immunoglobulin or T cell receptor. In certain embodiments, an epitope determinant includes a chemically active surface group of a molecule such as an amino acid, sugar side chain, phosphoryl, or sulfonyl, and in certain embodiments, may have specific three-dimensional structural properties and / or specific charge properties. In one embodiment, an epitope is a region of an antigen to which an antibody binds. In certain embodiments, an antibody is said to bind specifically to an antigen if it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. Methods for epitope mapping are well known in the art, including X-ray cocrystallization, array-based oligopeptide scanning, site-directed mutagenesis, high-throughput mutagenesis mapping, and hydrogen-deuterium exchange. Epitopes can be formed from both continuous amino acids or discontinuous amino acids juxtaposed by the tertiary folding of proteins. Epitopes formed from sequential amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three, more commonly at least five or eight to ten, amino acids in a unique spatial conformation.

[0070] The terms “promote,” “enhance,” and “induce,” as used interchangeably herein, refer to any statistically significant increase in biological activity (e.g., hemichannel opening). For example, “promote” may refer to an increase of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in biological activity.

[0071] As used herein, the terms “bone cancer” or “bone tumor” refer to any cancer related to bone, including cancer originating from bone, or cancer originating from other organs or tissues (such as the breast, prostate, lungs, kidneys, and thyroid) and metastasizing to the bone. Therefore, non-limiting examples of bone cancer include osteosarcoma, multiple myeloma, Ewing's sarcoma, osteoporosis, osteopenia, osteoblastoma, chondroma, chondromyxofibroma, mesenchymal chondrosarcoma, chordoma, enameloma, or metastasis of osteosarcoma.

[0072] Osteosarcoma is the most common primary malignant bone tumor. By definition, osteosarcoma is an osteoid spindle cell neoplasm with several histological subtypes exhibiting variable radiographic appearance, intrinsic cellular heterogeneity, and overall prognosis. Subtypes of osteosarcoma include, but are not limited to, osteoblastic osteosarcoma, chondroblastic osteosarcoma, fibroblastic osteosarcoma, small cell osteosarcoma, telangiectatic osteosarcoma, paraosteal osteosarcoma, periosteal osteosarcoma, typical osteosarcoma, osteoblastic osteosarcoma sclerosing osteosarcoma, chondroblastomatous osteosarcoma, chondromyxofibromatoid osteosarcoma, clear cell osteosarcoma, malignant fibrous histiocytomatous osteosarcoma, giant cell osteosarcoma, or epithelioid osteosarcoma. The World Health Organization considers the osteoblastic, chondroblastic, and fibroblastic subtypes of conventional intramedullary osteosarcoma, which is the most common type of osteosarcoma.

[0073] Osteoblastic osteosarcoma arises from the abnormal proliferation of osteoblasts. Osteoblastic osteosarcoma typically occurs in the metaphysical regions of long bones such as the distal femur, proximal tibia, and proximal humerus, but can also occur in other bones. Osteoblastic osteosarcoma is characterized by the setting of abundant extracellular osteoid production. The matrix, consisting of immature bone, is deposited in a lace-like pattern along with malignant tumor cells incorporated throughout the extracellular matrix.

[0074] Chondroblastic osteosarcoma, as defined by the WHO, is a histological entity characterized by a tendency to exhibit a high degree of hyaline cartilage-likeness and by the predominance of a cartilage-like matrix closely associated with non-cartilaginous elements (osteoid or bone matrix). Chondroblastic osteosarcoma typically occurs in long bones such as the femur and tibia. It can also affect other bones, including the humerus and pelvis. Chondroblastic osteosarcoma is more common in adolescents and young adults, but can occur at any age.

[0075] Fibroblastic osteosarcoma is characterized by the presence of spindle-shaped cells similar to fibroblasts. Fibroblastic osteosarcoma is often highly vascularized and can resemble hemangiopericytoma. Histopathologically, osteoid production is minimal and localized.

[0076] Small cell osteosarcoma, characterized by the presence of small, round, and uniform cells within the tumor, is a rare subtype of osteosarcoma. Histopathologically, small cell osteosarcoma cells have large, hyperpigmented nuclei with poor cytoplasm. Small cell osteosarcoma primarily occurs in the metaphysical region of long bones.

[0077] Benign giant cells, which resemble osteoclasts, are found in approximately 25% of osteosarcomas. In rare osteosarcomas, the abundance of benign giant cells can mask the background malignant cells, potentially leading to a misdiagnosis of giant cell tumor. This is more likely to occur in the sacrum, a frequent site for giant cell tumors. The radiographic differences between giant cell tumors and osteosarcomas are less pronounced in the sacrum than in the long bones. It is worth reminiscing that typical giant cell tumors occur in skeletally mature patients. Tumors that appear to be giant cell tumors but occur in skeletally immature individuals should be thoroughly and carefully sampled to rule out giant cell-rich osteosarcomas.

[0078] There are many classification systems for grading osteosarcoma. One widely used system is the four-stage (Bloder) system, based on the percentage of anaplasia in the tumor. Most conventional central osteosarcomas fall into the Grade 3 or Grade 4 category. Tumors assigned to Grade 1 are low-grade central osteosarcomas and typical paraosteal osteosarcomas. Osteosarcoma of the jaw often tends to show a Grade 2 histological structure.

[0079] The general staging systems for malignant bone tumors are as follows: the Enneking system for staging malignant musculoskeletal tumors and the American Joint Committee on Cancer (AJCC) system for staging osteosarcoma. The Enneking system is based on the histological grade of the tumor, its local extent, and the presence or absence of metastasis. The AJCC system for osteosarcoma is based on the malignancy of the tumor, its size, presence, and the location of metastasis. The different stages of the AJCC system are shown below.

[0080] [Table 1]

[0081] II. Antibodies The term “antibody” as used herein is used in its broadest sense and specifically includes full-length antibodies, antibody peptides or immunoglobulins, monoclonal antibodies, chimeric antibodies, polyclonal antibodies, human antibodies, humanized antibodies, and antibodies derived from non-human species, such as human antibodies derived from human germline immunoglobulin sequences transduced into mice, sheep, chickens, or goats; recombinant antigen-binding forms such as monobodies and diabodies; multispecific antibodies formed from at least two full-length antibodies (e.g., bispecific antibodies) (e.g., each part includes an antigen-binding region of the antibody against a different antigen or epitope); and any of the above, including dAb, Fv, scFv, Fab, F(ab)'2, Fab', for example, antibodies or individual antigen-binding fragments of antibodies from which they are derived.

[0082] Antibody-like conjugated peptide mimes are also intended in the methods described herein. Liu et al. (2003) described “antibody-like conjugated peptide mimes” (ABIPs), which are peptides that act as truncated antibodies and have the particular advantages of a longer serum half-life and a less complicated synthesis method.

[0083] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope. The modifier “monoclonal” indicates a characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of the antibody by any particular method.

[0084] Antibody "antigen-binding fragments" preferably include variable regions of at least the heavy and / or light chains of an anti-Cx43 antibody. For example, an anti-Cx43 antibody antigen-binding fragment may include the amino acid sequences of SEQ ID NOs. 7 and 8. Examples of such antigen-binding fragments include the Fab fragment, Fab' fragment, Fv fragment, scFv, and F(ab')2 fragment. Antibody antigen-binding fragments can be produced by enzymatic cleavage or recombination techniques. For example, papain or pepsin cleavage can be used to produce the Fab or F(ab')2 fragment, respectively. Antibodies can also be produced in various truncated forms using antibody genes in which one or more stop codons are introduced upstream of the native stop site. For example, a recombinant construct encoding the heavy chain of the F(ab')2 fragment may be designed to include DNA sequences encoding the CH1 domain and hinge region of the heavy chain. In one embodiment, the antigen-binding fragment facilitates the opening of the Cx43 hemichannel in a target and the effects associated with the opening of the Cx43 hemichannel.

[0085] A "therapeutic monoclonal antibody" is an antibody used for the treatment of human subjects. The therapeutic monoclonal antibodies disclosed herein include anti-Cx43 antibodies. Antibody "effector function" refers to the biological activity resulting from the Fc region of the antibody (natural sequence Fc region or amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and cell surface receptors (e.g., B cell receptor downregulation; BCR). To evaluate the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as those described in U.S. Patent No. 5,500,362 or No. 5,821,337, can be performed.

[0086] Depending on the amino acid sequence of the constant domains of their heavy chains, full-length antibodies can be assigned to different "classes." There are five major classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further classified into "subclass" isotypes, e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of antibodies are well known. The "light chains" of antibodies from any vertebrate species can be assigned to one of two distinct types called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0087] A portion of the present invention, such as a polypeptide, peptide, antigen, or immunogen, may be conjugated or linked covalently or noncovalently to another portion, such as an adjuvant, protein, peptide, support, fluorescent moiety, or label. The terms “conjugate” or “immunoconjugate” are used broadly to define an operative association of one portion with another drug and are not intended to refer only to any type of operative association, and are not particularly limited to chemical “conjugation.”

[0088] As used herein, the term “hypervariable region” refers to an amino acid residue of an antibody that is involved in antigen binding. The hypervariable region generally consists of amino acid residues derived from the "complementarity-determining region" or "CDR" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain, and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or residues derived from the "hypervariable loop" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain, and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk This includes J.Mal.Biol.196:901-917(1987). The "framework region" or "FR" residues are variable domain residues other than the hypervariable region residues as defined herein. The hypervariable region or its CDR can be transferred from one antibody chain to another antibody chain or another protein to confer antigen-binding specificity to the resulting (complex) antibody or binding protein.

[0089] As those skilled in the art will understand, the CDRs disclosed herein may also include variants. Generally, the amino acid identity between individual variant CDRs is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Thus, a “variant CDR” is one that has a specific identity with respect to the parent CDR of the present invention and shares biological functions that include, but are not limited to, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and / or activity of the parent CDR.

[0090] Amino acid substitutions are typically single-residue substitutions. Insertions are usually on the order of approximately 1 to 20 amino acid residues, but larger insertions can also be tolerated. Deletions range from approximately 1 to 20 amino acid residues, but in some cases, deletions may be larger.

[0091] Substitutions, deletions, insertions, or any combination thereof can be used to arrive at the final derivative or variant. Generally, these changes are made to a few amino acids to minimize molecular alterations, particularly to the immunogenicity and specificity of antigen-binding proteins. However, in certain circumstances, larger changes may be permissible.

[0092] As used herein, the terms “Fab” or “Fab region” mean a polypeptide comprising the VH, CH1, VL, and CL immunoglobulin domains. Fab may refer to this isolated region, or to this region in the context of a full-length antibody, antibody fragment, or Fab fusion protein, or any other antibody embodiment outlined herein.

[0093] As used herein, the terms “Fv,” “Fv fragment,” or “Fv region” refer to a polypeptide comprising the VL and VH domains of a single antibody.

[0094] As used herein, the term "framework" refers to the region of the antibody variable domain excluding the region defined as the CDR. Each antibody variable domain framework can be further subdivided into contiguous regions separated by the CDR (FR1, FR2, FR3, and FR4).

[0095] The "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody containing the minimal sequence derived from the non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the hypervariable region of the recipient are replaced by residues from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, that possess the desired specificity, affinity, and capabilities. In some cases, framework region (FR) residues of the human antibody are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in the recipient antibody or donor antibody. These modifications are made to further improve the performance of the antibody. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0096] An “isolated” antibody is an antibody identified, separated, and / or recovered from its natural environment. In certain embodiments, the antibody is purified to (1) more than 95% or 99% by weight of the protein as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a spinning cup sequencer, or (3) to homogenize by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. An isolated antibody contains an in situ antibody in a recombinant cell because at least one component of the antibody’s natural environment is absent. However, typically, an isolated antibody is prepared by at least one purification step.

[0097] Anti-Cx43 antibody Certain aspects of this disclosure provide compositions and methods for treating bone cancer, such as osteosarcoma. The methods and compositions include an anti-Cx43 antibody that specifically binds to the Cx43 hemichannel and promotes its opening in osteocytes.

[0098] The anti-Cx43 antibody may be any antibody known in the art that specifically binds to Cx43. In various embodiments, the anti-Cx43 antibody used in the method includes an HCDR1 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1, an HCDR2 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 2, an HCDR3 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 3, and / or an LCDR1 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 4, an LCDR2 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 5, and an LCDR3 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 6. In some specific embodiments, the anti-Cx43 antibody includes the same HCDR1 amino acid sequence as SEQ ID NO: 1, the same HCDR2 amino acid sequence as SEQ ID NO: 2, the same HCDR3 amino acid sequence as SEQ ID NO: 3, and / or the same LCDR1 amino acid sequence as SEQ ID NO: 4, the same LCDR1 amino acid sequence as SEQ ID NO: 5, and the same LCDR3 amino acid sequence as SEQ ID NO: 6.

[0099] In various embodiments, the anti-Cx43 antibody used in this method includes a heavy chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 7; and / or a light chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 8. In some specific embodiments, the anti-Cx43 antibody includes the same heavy chain variable region as SEQ ID NO: 7; and / or the same light chain variable region as SEQ ID NO: 8.

[0100] In various embodiments, the anti-Cx43 antibody used in this method comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NOs: 9 and 11-13, and / or a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NOs: 10. In some specific embodiments, the anti-Cx43 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NOs: 9; and a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NOs: 10. In some other specific embodiments, the anti-Cx43 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 11; and a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID NO: 10. In some other specific embodiments, the anti-Cx43 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 12; and a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID NO: 10. In some further specific embodiments, the anti-Cx43 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 13; and a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID NOs: 10.

[0101] In some embodiments, the anti-Cx43 antibody contains the same heavy chain as SEQ ID NOs. 9 and any one of SEQ ID NOs. 11-13, and / or the same light chain as SEQ ID NOs. 10. In preferred embodiments, the anti-Cx43 antibody contains the same heavy chain as SEQ ID NOs. 9 and the same light chain as SEQ ID NOs. 10. In one embodiment, the anti-Cx43 antibody contains the same heavy chain as SEQ ID NOs. 11 and the same light chain as SEQ ID NOs. 10. In another embodiment, the anti-Cx43 antibody contains the same heavy chain as SEQ ID NOs. 12 and the same light chain as SEQ ID NOs. 10. In yet another embodiment, the anti-Cx43 antibody contains the same heavy chain as SEQ ID NOs. 13 and the same light chain as SEQ ID NOs. 10.

[0102] In various embodiments, the anti-Cx43 antibody may comprise a mixture or cocktail of two or more anti-Cx43 antibodies, each of which binds to the same or different epitopes on Cx43.

[0103] In various embodiments, antibodies that bind to epitopes partially or completely located within the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 14) are provided herein. In some embodiments, the epitope may comprise one or more amino acids selected from the group consisting of F1, S3, R4, P5, T6, E7, K8, T9, and I10 of SEQ ID NO: 14. In one embodiment, the epitope comprises F1, S3, R4, P5, T6, E7, K8, T9, and I10 of SEQ ID NO: 14. In some embodiments, the epitope may comprise all 10 amino acids of SEQ ID NO: 14. In a particular embodiment, the epitope comprises all 10 amino acids of SEQ ID NO: 14.

[0104] The anti-Cx43 antibody is substantially pure and preferably substantially homogeneous (i.e., free from contaminating proteins, etc.). A “substantially pure” antibody means a composition containing at least about 90% by weight, at least about 95% by weight, or 97% by weight of the antibody, based on the total weight of the protein in the composition. A “substantially homogeneous” antibody means a composition containing a protein in which at least about 99% by weight of the protein is a specific antibody, e.g., an anti-Cx43 antibody, based on the total weight of the protein.

[0105] In some embodiments, the anti-Cx43 antibody is a humanized antibody. In some embodiments, the anti-Cx43 antibody is a monoclonal antibody. In some embodiments, a humanized monoclonal antibody.

[0106] Pharmaceutical preparations containing anti-Cx43 antibodies One aspect of this disclosure provides a pharmaceutical formulation for treating cancer, such as bone cancer including osteosarcoma, comprising an anti-Cx43 antibody. In some embodiments, the anti-Cx43 antibody promotes the opening of Cx43 hemichannels in osteocytes. The anti-Cx43 antibody may be a complete antibody or an antigen-binding fragment thereof.

[0107] As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic and absorption retardants, buffers, and other physiologically compatible excipients. Preferably, the carrier is suitable for parenteral, oral, or topical administration. Depending on the route of administration, the active compound, e.g., small molecule or bioagent, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0108] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injection solutions or dispersions, as well as conventional excipients for the preparation of tablets, pills, capsules, and the like. The use of such media and agents for the formulation of pharmaceutically active substances is known in the art. Unless any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions provided herein is intended. Auxiliary active compounds may also be incorporated into the compositions.

[0109] A pharmaceutically acceptable carrier may contain a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0110] Suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions provided herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, as well as injectable organic esters, such as ethyl oleate. Appropriate fluidity can be maintained where necessary, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it may be useful to include isotonic agents in the composition, such as sugars, polyalcohols such as mannitol and sorbitol, or sodium chloride. Long-term absorption of the injectable composition can be achieved by including absorption-delaying agents, such as monostearate and gelatin, in the composition.

[0111] These compositions may also contain functional excipients such as preservatives, wetting agents, emulsifiers, and dispersants.

[0112] Therapeutic compositions typically must be sterile, non-systematic, and stable under manufacturing and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. Sterile injectable solutions may be prepared by incorporating the required amount of the active compound in a suitable solvent, along with one or a combination of the components listed above, as needed, and then by sterilization, e.g., microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method includes vacuum drying and freeze-drying (lyophilization) of the powder of the active ingredient and any additional desired components obtained from its pre-sterilized filtered solution. The active agent may be mixed under sterile conditions with additional pharmaceutically acceptable carriers and any preservatives, buffers, or propellants as needed.

[0113] The prevention of microbial presence can be ensured by both the sterilization procedures described above and the inclusion of various antimicrobial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. Furthermore, long-term absorption of the injectable pharmaceutical form can be achieved by the inclusion of absorption-delaying agents such as aluminum monostearate and gelatin.

[0114] For ease of administration and uniformity of dosage, it may be advantageous to formulate parenteral compositions in unit dosage forms. As used herein, unit dosage forms refer to physically distinct units suitable as unit doses for a patient being treated, each unit containing a predetermined amount of the active agent calculated to produce the desired therapeutic effect, along with any necessary pharmaceutical carriers. The specifications of a unit dosage form are determined and directly depend on (a) the distinctive characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the field in which such an active compound is formulated for the treatment of susceptibility in an individual.

[0115] The actual dose levels of the active ingredients in the pharmaceutical compositions disclosed herein can be varied to obtain an amount of the active ingredient effective in achieving a desired therapeutic response for a particular patient, composition, and mode of administration without causing toxicity to the patient. As used herein in the context of administration, “parenteral” means a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injections and infusions.

[0116] As used herein, the phrases “parenteral administration” and “administered parenterally” refer to modes of administration other than enteral (i.e., via the gastrointestinal tract) and topical administration, typically by injection or infusion, and include, but are not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injections and infusions. Intravenous injections and infusions are frequently (but not exclusively) used for antibody administration.

[0117] The formulations described herein are administered to mammals, preferably humans, requiring treatment with anti-Cx43 antibodies, by known methods such as intravenous administration as a bolus or by continuous infusion over a period of time, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intra-sacral, intrathecal, oral, topical, or inhalation routes.

[0118] In some embodiments, the formulation is administered to mammals by intravenous or subcutaneous (i.e., under the skin) administration. For such purposes, the formulation may be injected using a syringe. However, other devices for administering the formulation are available, such as injection devices (e.g., I, NJECT-EASEc and GENJECT(trademark) devices, injection pens (GENPEN(trademark)), etc.); automated injection devices, needleless devices (e.g., MEDIJECTOR(trademark) and BIOJECTOR(trademark); and subcutaneous patch delivery systems.

[0119] In certain embodiments, this disclosure relates to kits for single-dose units. Such kits include containers for aqueous formulations of therapeutic proteins or antibodies, which include pre-filled syringes, either single-chamber or multi-chamber. Exemplary pre-filled syringes are available from Vetter GmbH (Ravensburg, Germany).

[0120] III. Treatment methods for bone cancer One aspect of this disclosure provides a method for treating cancer, including bone cancer, such as osteosarcoma. The method may include promoting / enhancing the opening of Cx43 hemichannels in osteocytes in a target area, for example, by using an anti-Cx43 antibody. Currently, there is no second-line therapy for osteosarcoma, and therefore, Cx43 modulation (e.g., promoting the opening of Cx43 hemichannels using an anti-Cx43 antibody) can be used as a second-line therapy for patients who have failed standard treatment for osteosarcoma, or as at least one alternative therapy for treating osteosarcoma. Cx43 modulation may be a monotherapy for bone cancer, including osteosarcoma, or may be in combination with other cancer therapies described herein or known in the art.

[0121] In some embodiments, anti-Cx43 antibodies are used as a second-line therapy in subjects who have failed at least one alternative therapy (standard treatment) for bone cancer (e.g., osteosarcoma). In some other embodiments, anti-Cx43 antibodies are used as a first-line therapy in subjects for the treatment of bone cancer (e.g., osteosarcoma). In some embodiments, anti-Cx43 antibodies are used as neoadjuvant therapy for bone cancer (e.g., osteosarcoma) in subjects.

[0122] Various cells can communicate with each other and with the extracellular environment via hemichannels and gap junctions formed by the protein connexin. Connexin proteins are ubiquitously expressed throughout the body. Six connexin proteins constitute one hemichannel, and two hemichannels constitute one gap junction channel. Gap junctions are clusters of channels located in the plasma membrane between adjacent cells and mediate intercellular communication. Hemichannels are distinct entities from gap junction channels. Hemichannels enable the exchange of molecules between intracellular compartments and the extracellular environment.

[0123] Normal bone is composed of three main cell types: osteoblasts, resorbative osteoclasts, and osteocytes. Osteocytes make up about 95% of osteocytes and maintain the bone remodeling process by coordinating osteolytic and osteoblastic activity. When cancer cells invade bone, much of normal bone function is affected. Cancer cells interact with the local microenvironment to promote their survival through bone destruction and angiogenesis.

[0124] Osteocytes express hemichannels known as connexin (Cx) 43 hemichannels. These osteocyte hemichannels are normally closed and can open when exposed to mechanical stimuli, resulting in the release of various factors into the bone microenvironment. Factors released by hemichannel opening may mediate other processes that can reduce tumor cell migration and bone metastases. Alendronate (AD), an effective and commonly used bisphosphonate drug, has been shown to open Cx43 hemichannels in osteocytes. Bisphosphonates are a class of drugs known to treat many bone disorders, including bone metastases. Bisphosphonate administration has been shown to be associated with a reduced incidence of bone metastases and a reduction in mortality in breast cancer patients. AD is associated with reduced tumor growth as well as reduced bone destruction and pain. AD inhibits osteoclast activity and induces the opening of Cx43 hemichannels in osteocytes. However, AD administration is associated with several severe side effects.

[0125] Cx43, also known as gap junction alpha-1 protein (GJA1), is a 43.0 kDa protein composed of 382 amino acids (NCBI reference sequence: NP_000156.1). GJA1 contains a long C-terminal tail, an N-terminal domain, and multiple transmembrane domains. The protein passes through the phospholipid bilayer four times, leaving its C-terminus and N-terminus exposed to the cytoplasm. The C-terminal tail consists of 50 amino acids and contains post-translational modification sites, as well as binding sites for transcription factors, cytoskeletal elements, and other proteins. As a result, the C-terminal tail is central to functions such as pH gating and channel assembly regulation. In particular, the DNA region of the GJA1 gene encoding this tail (NCBI gene ID: 2697) is highly conserved, indicating that it is either resistant to mutations or becomes lethal if mutated. The N-terminal domain, on the other hand, is involved in channel gating and oligomerization and can therefore control the switching between the open and closed states of channels. The transmembrane domain forms a gap junction channel, and the extracellular loop facilitates proper channel docking. Furthermore, the two extracellular loops form disulfide bonds that interact with the two hexamers to form a complete gap junction channel.

[0126] "Treatment" refers to therapeutic treatment. Those requiring treatment include those who already have a disease. Thus, a patient being treated herein, for example, a human, may have been diagnosed with a disease such as bone cancer. A disease, for example, bone cancer, is "inhibited" or "treated" if at least one symptom of the condition (such as responsive / unresponsive, or as determined by indicators known in the art and described herein) is alleviated, terminated, slowed, minimized, or prevented. The terms "patient" and "subject" are used interchangeably herein.

[0127] The terms "subject" or "patient" refer to either human or non-human animals, such as primates, mammals, and vertebrates. In certain embodiments, the subject is human.

[0128] Treatment can be appropriately administered to subjects, particularly humans, who have, are susceptible to, or are at risk of developing such cancers, including bone cancers such as osteosarcoma. The determination of subjects at "risk" can be made by any objective or subjective determination based on diagnostic tests or the opinion of the subject or healthcare provider (e.g., genetic testing, enzyme or protein markers, family history, etc.). Identifying subjects who require such treatment may be at the discretion of the subject or healthcare professional and may be subjective (e.g., opinion) or objective (e.g., measurable by tests or diagnostic methods).

[0129] Anti-Cx43 antibodies can be stored as lyophilized solids, aqueous formulations, or any other form known in the art. In the case of anti-Cx43 antibodies stored as lyophilized solids, the antibody is reconstituted in a solution such as sterile water for injection before administration. When prepared for injection from either the lyophilized form or an aqueous formulation, for example, the final concentration of the reconstituted anti-Cx43 antibody after dilution (e.g., in saline, Ringer's solution, or a 5% dextrose infusion system) may be approximately 0.1 mg / ml to approximately 80 mg / ml for administration. The final concentrations may be approximately 0.1 mg / ml to approximately 80 mg / ml, approximately 0.5 mg / ml to approximately 70 mg / ml, approximately 1 mg / ml to approximately 60 mg / ml, approximately 5 mg / ml to approximately 50 mg / ml, approximately 10 mg / ml to approximately 40 mg / ml, approximately 15 mg / ml to approximately 30 mg / ml, or approximately 20 mg / ml to approximately 25 mg / ml. In some embodiments, the final dosage form may have a concentration of less than 0.1 mg / ml, about 0.1 mg / ml, about 0.5 mg / ml, about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 10 mg / ml, about 15 mg / ml, about 20 mg / ml, about 25 mg / ml, about 30 mg / ml, about 35 mg / ml, about 40 mg / ml, about 45 mg / ml, about 50 mg / ml, about 55 mg / ml, about 60 mg / ml, about 65 mg / ml, about 70 mg / ml, about 75 mg / ml, about 80 mg / ml, or greater than 80 mg / ml.

[0130] As used herein, the term “effective dose” refers to the amount of a drug, such as an anti-Cx43 antibody, that, when administered to a patient or subject, results in the treatment, prognosis, or diagnosis of cancer (e.g., bone cancer). The administration plan may be adjusted to provide an optimal therapeutic response. The effective dose is also the amount in which any toxic or adverse effects (side effects) of the drug are minimized and / or the beneficial effects outweigh them. The therapeutic effective dose varies depending on the patient being treated and their disease state, the patient’s weight and age, the severity of the disease state, the mode of administration, the course of the condition, the patient’s medical history, and their response to the anti-Cx43 antibody, and can be readily determined by those skilled in the art. For example, the effective dose or amount of anti-Cx43 antibody ranges from about 0.001 mg / kg to about 300 mg / kg. In some embodiments, the dose of anti-Cx43 antibody is approximately 0.01 mg / kg to approximately 200 mg / kg, approximately 0.1 mg / kg to approximately 100 mg / kg, approximately 0.2 mg / kg to approximately 90 mg / kg, approximately 0.5 mg / kg to approximately 80 mg / kg, approximately 0.8 mg / kg to approximately 70 mg / kg, approximately 1 mg / kg to approximately 60 mg / kg, approximately 3 mg / kg to approximately 50 mg / kg, approximately 6 mg / kg to approximately 40 mg / kg, approximately 9 mg / kg to approximately 36 mg / kg, approximately 12 mg / kg to approximately 33 mg / kg, approximately 15 mg / kg to approximately 30 mg / kg, approximately 18 mg / kg to approximately 27 mg / kg, or approximately 21 mg / kg to approximately 24 mg / kg. In some embodiments, the dose of anti-Cx43 antibody is approximately 0.001 mg / kg, approximately 0.01 mg / kg, approximately 0.1 mg / kg, approximately 1 mg / kg, approximately 3 mg / kg, approximately 6 mg / kg, approximately 9 mg / kg, approximately 12 mg / kg, approximately 15 mg / kg, approximately 18 mg / kg, approximately 21 mg / kg, approximately 24 mg / kg, approximately 27 mg / kg, approximately 30 mg / kg, approximately 33 mg / kg, approximately 36 mg / kg, and approximately 40 mg / kg. The levels are approximately g / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 120 mg / kg, 140 mg / kg, 160 mg / kg, 180 mg / kg, 200 mg / kg, 220 mg / kg, 240 mg / kg, 260 mg / kg, 280 mg / kg, 300 mg / kg, or more than 300 mg / kg.

[0131] In some specific embodiments, the effective amount or dose of anti-Cx43 antibody is 1 mg / kg, 3 mg / kg, 6 mg / kg, 9 mg / kg, 12 mg / kg, 15 mg / kg, 18 mg / kg, 21 mg / kg, 24 mg / kg, 27 mg / kg, 30 mg / kg, 33 mg / kg, or 36 mg / kg.

[0132] In some embodiments, the effective amount or dose of anti-Cx43 antibody may be in the range of about 1 mg to about 25,000 mg. In some embodiments, the dose of anti-Cx43 antibody is about 2 mg to 20,000 mg, about 5 mg to 15,000 mg, about 10 mg to 10,000 mg, about 15 mg to 10,000 mg, about 20 mg to 5,000 mg, about 25 mg to 4,000 mg, about 30 mg to 3,000 mg, about 40 mg to 2,000 mg, about 50 mg to 1,000 mg, about 60 mg to 900 mg, about 70 mg to 800 mg, about 80 mg to 700 mg, about 90 mg to 600 mg, about 100 mg to 500 mg, about 150 mg to 450 mg, about 200 mg to 400 mg, about 250 mg to 350 mg, or about 275 mg to 325 mg. In some embodiments, the dose of anti-Cx43 antibody is less than 1 mg, approximately 1 mg, approximately 2 mg, approximately 3 mg, approximately 4 mg, approximately 5 mg, approximately 10 mg, approximately 15 mg, approximately 20 mg, approximately 25 mg, approximately 30 mg, approximately 35 mg, approximately 40 mg, approximately 45 mg, approximately 50 mg, approximately 75 mg, approximately 100 mg, approximately 150 mg, approximately 200 mg, approximately 250 mg, approximately 300 mg, approximately 350 mg, approximately 400 mg, approximately 450 mg, approximately 500 mg, approximately 550 mg, approximately 600 mg, approximately 650 mg, approximately 700 mg, approximately 750 mg, approximately 800 mg, approximately 85 mg The amounts are 0 mg, approximately 900 mg, approximately 950 mg, approximately 1000 mg, approximately 1250 mg, approximately 1500 mg, approximately 1750 mg, approximately 2000 mg, approximately 2250 mg, approximately 2500 mg, approximately 2750 mg, approximately 3000 mg, approximately 4000 mg, approximately 5000 mg, approximately 6000 mg, approximately 7000 mg, approximately 8000 mg, approximately 9000 mg, approximately 10,000 mg, approximately 12,500 mg, approximately 15,000 mg, approximately 17,500 mg, approximately 20,000 mg, approximately 22,500 mg, approximately 25,000 mg, or more than 25,000 mg.

[0133] In the methods described herein, the effective amount or dose of anti-Cx43 antibody is approximately once a day, approximately once every two days, approximately once every three days, approximately once every four days, approximately once every five days, approximately once every six days, approximately once a week, approximately once every eight days, approximately once every nine days, approximately once every ten days, approximately once every eleven days, approximately once every twelve days, approximately once every thirteen days, approximately once every two weeks, approximately once every fifteen days, approximately once every sixteen days, approximately once every seventeen days, approximately once every eighteen days, approximately once every nineteen days, approximately once every twenty days, approximately once every three weeks, approximately once every twenty-two days, approximately once every twenty-three days, approximately once every twenty-four days, approximately once every twenty-five days, approximately once every twenty-six days, approximately once every twenty-seven days, approximately once every four weeks, approximately once every twenty-nine days, approximately once every thirty days. It is administered approximately once every 31 days, 32 days, 33 days, 34 days, every 5 weeks, 36 days, 37 days, 38 days, 39 days, 40 days, or every 41 days, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, every 13 weeks, every 15 weeks, every 16 weeks, every 17 weeks, every 18 weeks, every 19 weeks, every 20 weeks, every 21 weeks, every 22 weeks, every 23 weeks, every 24 weeks, every 6 months, or in cycles longer than 24 weeks or 6 months.

[0134] In some specific embodiments, the dose of anti-Cx43 antibody is administered approximately once every three weeks. In one specific embodiment, the dose of anti-Cx43 antibody is administered once every three weeks.

[0135] According to certain embodiments of the present disclosure, multiple doses of an anti-Cx43 antibody (or a pharmaceutical composition comprising a combination of an anti-Cx43 antibody and any additional therapeutic activator described herein) may be administered to a subject over a defined time course. A method according to this aspect of the present disclosure includes administering multiple doses of the anti-Cx43 antibody of the present disclosure to a subject in succession.

[0136] As used herein, “sequential administration” means that each dose of anti-Cx43 antibody is administered to a subject at different time points, for example, on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). This disclosure includes a method comprising administering to a patient sequentially a single initial dose of anti-Cx43 antibody, followed by one or more secondary doses of anti-Cx43 antibody, and optionally followed by one or more tertiary doses of anti-Cx43 antibody. Anti-Cx43 antibody may be administered in doses ranging from 0.001 mg / kg to approximately 300 mg / kg.

[0137] The terms “initial dose,” “secondary dose,” and “tertiary dose” refer to the chronological order of administration of the anti-Cx43 antibody in this disclosure. Thus, the “initial dose” is the dose administered at the start of the treatment plan (also called the “baseline dose”). The “secondary dose” is the dose administered after the initial dose, and the “tertiary dose” is the dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of anti-Cx43 antibody, but generally may differ from one another in terms of administration frequency. However, in certain embodiments, the amounts of anti-Cx43 antibody contained in the initial, secondary, tertiary, and / or subsequent doses may vary from one another during the course of treatment (e.g., adjusted upward or downward as appropriate).

[0138] Anti-Cx43 antibodies may be administered to the patient as a single treatment or over a series of treatments, or at any point prior to diagnosis. Anti-Cx43 antibodies may be administered as a monotherapy or in combination with other drugs or therapies useful in treating the condition in question.

[0139] In the methods described herein, doses of anti-Cx43 antibody may be administered to a patient over a period of less than 5 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 110 minutes, about 120 minutes, about 150 minutes, about 180 minutes, or more than 180 minutes. In some specific embodiments, doses of anti-Cx43 antibody may be administered to a patient over a period of about 30 minutes. In one specific embodiment, doses of anti-Cx43 antibody may be administered intravenously to a patient over a period of 30 minutes.

[0140] In the methods described herein, an anti-Cx43 antibody is administered to a patient. If the anti-Cx43 antibody is in a solid formulation, for example, in a dry state, the administration process may include a step of converting the formulation into a liquid state. In one embodiment, the dry formulation can be reconstituted with the above-mentioned liquid, for example, for use by injection, for example, intravenous, intradermal, intramuscular, intratumoral, intraperitoneal, or subcutaneous injection. In one particular embodiment, and in another embodiment, the solid or dry formulation can be administered topically, for example, in the form of a patch, cream, aerosol, or suppository.

[0141] In the methods described herein, the anti-Cx43 antibody may be administered to a subject alone or in combination with another agent. The anti-Cx43 antibody may be administered before, with, or after the administration of the additional agent. In one embodiment, the dose of the co-administered agent may be gradually reduced over time or completely tapered off during the treatment period with the anti-Cx43 antibody. Non-limiting examples of co-administered agents include chemotherapy agents, radiotherapy, immunotherapy, and surgery.

[0142] Non-exclusive examples of such chemotherapeutic agents include platinum-based chemotherapeutic agents (e.g., cisplatin, carboplatin), taxanes (e.g., paclitaxel (Taxol®), docetaxel (Taxotere®), EndoTAG-PM (a formulation of paclitaxel encapsulated in a positively charged lipid complex; MediGene), Abraxane® (a formulation of paclitaxel bound to albumin)), tyrosine kinase inhibitors (e.g., imatinib / Gleevec®, sunitinib / Sutent®, dasatinib / Sprycel®), and combinations thereof.

[0143] In another embodiment, at least one additional anticancer agent includes an EGFR inhibitor, such as an anti-EGFR antibody or a small molecule inhibitor of EGFR signaling. An example of an anti-EGFR is cetuximab (Erbituxl®), which is commercially available from ImClone Systems Incorporated. Other examples of anti-EGFR antibodies include matsuzumab (EMD72000), panitumumab (Vectibix®; Amgen), nimotuzumab (TheraCIM®), and mAb 806. An exemplary small molecule inhibitor of the EGFR signaling pathway is gefitinib (Iressa®), which is marketed by AstraZeneca and Teva. Other examples of small molecule inhibitors of the EGFR signaling pathway include erlotinib (OSI-774: Tarceva®), OSI Pharma; lapatinib (Tykerb®, GlaxoSmithKline); canertinib (canertinib dihydrochloride, Pfizer); peritinib (Pfizer); PKI-166 (Novartis); PD158780; and AG 1478(4-(3-chloroanilino)-6,7-dimethoxyquinazoline) is one example.

[0144] In yet another embodiment, at least one additional anticancer agent comprises a VEGF inhibitor. Exemplary VEGF inhibitors include anti-VEGF antibodies such as bevacizumab (Avastatin®: Genentech). In yet another embodiment, at least one additional anticancer agent comprises an anti-ErbB 2 antibody. Suitable anti-ErbB 2 antibodies include trastuzumab and pertuzumab.

[0145] In one embodiment, the improved efficacy of the combination according to this disclosure can be demonstrated by achieving a therapeutic synergy. The terms “therapeutic synergy” or “synergistic effect” are used when a combination of two products at a given dose is more effective than the best of each of the two products individually at the same dose. In one example, a therapeutic synergy can be evaluated by comparing the combination to the best monotherapy using estimates obtained from a two-way analysis of variance with repeated measures (e.g., a time factor) of the parameter tumor volume.

[0146] In some embodiments, the patient or subject has previously failed at at least one alternative therapy or standard care for bone cancer (e.g., osteosarcoma). Non-limiting examples of failed therapies or standard care for bone cancer (e.g., osteosarcoma) are any therapies or agents described elsewhere in this disclosure that can be administered concurrently with an anti-Cx43 antibody.

[0147] In some embodiments, the patient or subject has previously failed at at least one therapy for osteosarcoma. In some specific embodiments, at least one therapy for osteosarcoma includes chemotherapy, surgery, radiotherapy, or immunotherapy, or a combination thereof. In some specific embodiments, the patient or subject has previously failed at at least one therapy including methotrexate, doxorubicin, cisplatin, and ifosfamide, apatinib, analotinib, vindesine, vincristine, docetaxel, paclitaxel, irinotecan, bortezomib, albumin-conjugated paclitaxel, nedaplatin (Aqupla), pemetrexed, etoposide, gemcitabine, lovaplatin, recombinant human endostatin, eribulin, dacarbazine, pazopanib, immune checkpoint inhibitors, surgery, or radiotherapy, or a combination thereof.

[0148] Patients or subjects are examined for cancer-related parameters using techniques and methods known in the art before, during, or after treatment with anti-Cx43 antibodies. Non-limiting examples of common medical techniques and methods used to examine and diagnose osteosarcoma include radiography, computed tomography (CT) scans, magnetic resonance imaging (MRI), positron emission tomography (PET), bone scintigraphy (bone scans), biopsies, the EQ-5D questionnaire to assess quality of life, the PERCIST criteria to assess disease progression, and numerical rating scales to assess pain levels. The selection of techniques and methods, as well as the frequency of examinations, may be determined and / or adjusted by those skilled in the art based on the specific condition of the patient or subject.

[0149] In some embodiments, treatment with anti-Cx43 antibodies against osteosarcoma improves disease progression as assessed by PERCIST, RECIST, and / or ICDS criteria compared to a control population that may, but is not limited to, received the same elective therapy as the subject, or failed but did not receive anti-Cx43 antibodies against osteosarcoma.

[0150] In some embodiments, treatment with an anti-Cx43 antibody for osteosarcoma improves overall survival by 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 6 months compared to a control value that may include, but is not limited to, the mean or median overall survival of an osteosarcoma population that received or failed the same elective therapy as the subjects but did not receive an anti-Cx43 antibody for osteosarcoma.

[0151] In some embodiments, treatment with anti-Cx43 antibodies against osteosarcoma improves 6-month progression-free survival by 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 6 months compared to a control value that may include, but is not limited to, the mean or median overall survival of an osteosarcoma population that received or failed the same elective therapy as the subjects but did not receive anti-Cx43 antibodies against osteosarcoma.

[0152] In some embodiments, subjects treated with an anti-Cx43 antibody for osteosarcoma have a response period of at least 10 days, at least 15 days, at least 20 days, at least 25 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or longer.

[0153] In some embodiments, subjects treated with anti-Cx43 antibodies for osteosarcoma have response times of less than 2 years, less than 11 months, less than 10 months, less than 9 months, less than 8 months, less than 7 months, less than 6 months, less than 5 months, less than 4 months, less than 3 months, less than 2 months, less than 1 month, less than 25 days, less than 20 days, less than 15 days, less than 10 days, or less than 5 days.

[0154] In some embodiments, treatment with an anti-Cx43 antibody against osteosarcoma reduces tumor size by at least 1%, at least 2%, at least 3%, at least 5%, at least 8%, at least 10%, at least 15%, at least 20%, or at least 30%.

[0155] In some embodiments, treatment with an anti-Cx43 antibody for osteosarcoma improves the European Health-Related Quality of Life 5-Dimensional (EQ-5D) score. In some embodiments, treatment with an anti-Cx43 antibody for osteosarcoma improves the EQ-5D Health Index by at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, or 1 compared to baseline. In some embodiments, treatment with an anti-Cx43 antibody for osteosarcoma improves the Visual Analog Scale (VAS) score of the EQ-5D questionnaire by at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or 100.

[0156] In some embodiments, treatment with an anti-Cx43 antibody for osteosarcoma reduces the numerical rating scale (NRS) score by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or 10.

[0157] In some embodiments, treatment with anti-Cx43 antibodies against osteosarcoma improves bone mineral density (BMD) of the lumbar spine, hip joint, and femur and neck. In some embodiments, treatment with anti-Cx43 antibodies against osteosarcoma improves bone mineral density (BMD) of the lumbar spine, hip joint, and femur and neck by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more.

[0158] Treatment with anti-Cx43 antibodies for osteosarcoma reduces the dose and / or frequency of morphine use by the subject or patient. In some embodiments, treatment with anti-Cx43 antibodies for osteosarcoma reduces the dose and / or frequency of morphine use by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%. In certain embodiments, treatment with anti-Cx43 antibodies for osteosarcoma completely phasing out morphine use.

[0159] As used herein, “response” or “responsiveness” to treatment means that the subject has an improvement in at least one parameter of disease progression. The subject may have a “partial response” or a “full response” to treatment. The response, partial response, or full response to treatment can be determined based on methods known in the art. Those skilled in the art can determine an appropriate method based on the type of disease being evaluated. Non-limiting examples of methods include the RECIST criteria, the ICDS criteria, and the PERCIST criteria. In some embodiments, the subject has a full response to treatment with an anti-Cx43 antibody. In some other embodiments, the subject has a partial response to treatment with an anti-Cx43 antibody.

[0160] In some embodiments, after treatment with an anti-Cx43 antibody, subjects do not experience (i) adverse events, serious adverse events, or adverse events of grade 3 or higher; (ii) adverse events occurring under treatment (TEAE); (iii) adverse drug reactions (ADR); (iv) Cx43-related cardiac events or severe hepatic events; and / or (v) dose-limiting toxicity.

[0161] An "adverse event" or "AE" refers to any adverse medical event in a participant who has been administered a drug, which may manifest as a symptom, sign, illness, or abnormal test result, but is not necessarily causally related to the drug.

[0162] "Adverse drug reaction" or "ADR" refers to any reaction that is harmful to the human body, or an unintended reaction that is thought to be potentially related to a drug in a clinical trial. A drug and an AE have at least one likely reasonable causal relationship; that is, a correlation cannot be ruled out.

[0163] "Adverse events occurring during treatment" or "TEAEs" refer to events that occur during treatment but do not occur or worsen compared to the pre-treatment period.

[0164] A “serious adverse event” or “SAE” refers to one of the following situations in which a participant receives a drug: (ii) life-threatening: referring to the risk of death of the participant at the time of the event, rather than the risk of death if the event worsens; (iii) requiring hospitalization or extended hospitalization. It will be understood by those skilled in the art that SAEs include, but are not limited to, the above list.

[0165] Non-limiting examples of "dose-limiting toxicity" include grade 4 neutropenia lasting ≥7 days, or grade ≥3 neutropenia with fever (ANC < 1000 / mm²). 3Grade ≥3 neutropenia with a single temperature > 38°C or a sustained temperature ≥ 38°C for more than 1 hour, or with infection; Grade 4 thrombocytopenia or Grade 3 thrombocytopenia with bleeding; Grade ≥3 hepatotoxicity: ALT or AST ≥ 5 × upper limit of normal (ULN) (for participants with normal baseline) or ≥ 5 × baseline value (for participants with a combination of liver metastasis and abnormal baseline); or bilirubin ≥ 3 × ULN (for participants with normal baseline) or ≥ 3 × baseline value (for participants with abnormal baseline); Grade ≥3 nephrotoxicity: serum creatinine ≥ 3 × ULN (for participants with normal baseline) or ≥ 3 × baseline value (for participants with abnormal baseline); Grade ≥3 cardiotoxicity: QTc > 500 ms or QTc increase ≥ 60 ms. [Examples]

[0166] The following embodiments are included to demonstrate preferred embodiments of the present invention. Those skilled in the art will understand that the techniques disclosed in the following embodiments are techniques that the inventors have found to work well in carrying out the present invention and can therefore be considered to constitute a preferred mode for its implementation. However, those skilled in the art will understand that many modifications can be made in light of this disclosure to the particular embodiments disclosed, and that similar or comparable results can still be obtained without departing from the spirit and scope of the present invention.

[0167] Example 1. Overview and preclinical studies of ALMB-0168 ALMB-0168 is a humanized monoclonal antibody prepared from recombinant CHO cell lines under GMP conditions. Like typical humanized antibodies, ALMB-0168 consists of two identical heavy chains (IgG4 variant) and two identical light chains (κ chains) covalently linked by four pairs of disulfide bonds. The heavy and light chains consist of 446 and 219 amino acid residues, respectively. Based on the total amino acid composition, the molecular weight is 146.2 kDa. As with most antibodies, the heavy chain is glycosylated at a conserved glycosylation site in the constant region.

[0168] ALMB-0168 is a novel Cx43 humanized IgG4 monoclonal antibody that targets the Cx43 hemichannel in osteocytes. The initial antibody is obtained by immunizing mice with human Cx43. The mouse antibody is then humanized using common humanization approaches, namely complementarity-determining region (CDR) transplantation (Bowers et al., J Biol Chem., 288(11):7688-7696(2013)) (also known as modification) and germline sequence modeling.

[0169] ALMB-0168 is the first drug designed to inhibit the growth of primary and metastatic bone tumors by activating the Cx43 hemichannel, which releases essential substances such as ATP into the extracellular environment.

[0170] Based on preclinical studies, ALMB-0168 can effectively activate osteocyte Cx43 hemichannel opening both in vivo and in vitro. In several mouse models of breast cancer bone metastases and in situ osteosarcoma, administration of ALMB-0168 inhibited tumor growth in a dose-dependent manner and extended the lifespan of tumor-bearing animals, suggesting its potential as a treatment for malignant bone tumors. Furthermore, ALMB-0168 has favorable nonclinical safety attributes supporting FIH doses. The physicochemical properties of ALMB-0168 are shown in Table 1.

[0171] [Table 2]

[0172] Pharmacological effects and mechanism of action Osteocytes exhibit high expression of connexin Cx43 in intercellular junction channels and hemichannels. Gap junction channels mediate cell communication between two adjacent cells, maintaining normal cellular metabolic function, while hemichannels enable molecular exchange between the cell and the extracellular environment. Hemichannels are normally closed but are activated and open under certain conditions such as mechanical stress or ischemia, cytokines, or ion stimulation. Open hemichannels can pass small molecules (<1.2 kDa) such as ATP, NAD+, glutamate, and inositol triphosphate (IP3) (Goodenough et al., Nature reviews Molecular cell biology, 4:285-294 (2003)). Opening of Cx43 hemichannels in osteocytes has been shown to have a strong inhibitory effect on bone metastases from breast cancer tumors (Zhou et al., Oncogene, 35:5597-5607 (2016)).

[0173] ALMB-0168 is a humanized IgG4 monoclonal antibody that targets osteocyte hemichannels. The initial antibody is obtained by immunizing mice with human Cx43. The mouse antibody is then humanized using common humanization approaches, namely complementarity-determining region (CDR) transplantation (Bowers et al., J Biol Chem., 288(11):7688-7696(2013)) (also known as modification) and germline sequence modeling.

[0174] ALMB-0168 is designed to activate the Cx43 hemichannel, thereby releasing key anticancer agents (e.g., ATP) into the extracellular environment. ATP can activate killer CD8+ T cells and CD4+ T cells, while significantly inhibiting Treg cells with a negative tumor immune state. Furthermore, ATP binds to the P2Y11 receptor on cancer cells, reducing the positive regulation of P2Y11 on CXCR4, and thus inhibiting the growth and metastasis of primary and metastatic bone tumors.

[0175] Based on preclinical studies, ALMB-0168 can effectively activate osteocyte Cx43 hemichannel opening both in vivo and in vitro. In several mouse models of breast cancer bone metastases and in situ osteosarcoma, ALMB-0168 inhibited tumor growth in a dose-dependent manner and extended the lifespan of tumor-bearing animals, suggesting its potential as a treatment for malignant bone tumors. Furthermore, ALMB-0168 possesses favorable nonclinical safety attributes that support first-in-the-heart (FIH).

[0176] Summary of Preclinical Trials Summary of Preclinical Pharmacodynamic Studies In vitro testing In vitro studies showed that ALMB-0168 does not affect gap junctions. ALMB-0168 did not affect the normal function of gap junctions. After 16 hours of treatment of mouse bone MOL-Y4 cells with ALMB-0168, gap junctions were unaffected, but the positive control glycyrrhetinic acid (a known gap junction blocker) significantly inhibited gap junction function (p<0.001).

[0177] ALMB-0168 specifically bound to and activated the Cx43 hemichannel in osteocytes. The binding activity of ALMB-0168 to mouse osteocytes (MOL-Y4) and primary human osteocytes was determined by ELISA (enzyme-linked immunosorbent assay). The results showed that the antibody concentrations corresponding to 50% of the maximum effect (EC50) for MOL-Y4 cells and human osteocytes were 37.3±0.6 μg / ml and 41.0±6.5 μg / ml, respectively. Furthermore, the binding of ALMB-0168 to its target effectively activated the Cx43 hemichannel opening, and as determined by EB staining, the EC50 values ​​reached 2.33±0.15 μg / ml and 3.97±0.90 μg / ml for MOL-Y4 cells and human osteocytes, respectively.

[0178] ALMB-0168 treatment inhibited the migration of breast cancer and osteosarcoma cells. A conditioned medium (MOL-Y4) recovered from ALMB-0168-treated osteocytes significantly inhibited the migration of breast cancer cells (MDA-MB-231) and osteosarcoma cells (OS17) in vitro (p<0.001). Cell migration was determined by a 24-well tissue culture plate migration test with a membrane filter inserted. The results showed that the conditioned medium from ALMB-0168-treated osteocytes inhibited cancer cell migration by 50% compared to the conditioned medium from osteocytes treated with a vehicle. Treatment of the conditioned medium with ATPase (an enzyme that hydrolyzes ATP) eliminated the inhibitory effect, and extracellular ATP levels increased after ALMB-0168 treatment.

[0179] In vivo testing Tissue distribution and activation of Cx43 hemichannels by ALMB 0168 in mice. Four hours after intraperitoneal injection of ALMB-0168 (25 mg / kg), the antibody was detected only in mouse osteocytes and not in other organs expressing connexin (liver, heart, and spleen). Furthermore, ALMB-0168 treatment significantly increased Cx43 hemichannel opening in mouse osteocytes, as shown by EB staining assay.

[0180] ALMB 0168 inhibited bone metastases from breast cancer in mice. Once-weekly injections of ALMB-0168 at 25 mg / kg significantly inhibited human breast cancer growth in a mouse xenograft model (p<0.05).

[0181] ALMB-0168 inhibited osteosarcoma growth and extended the survival of tumor-bearing mice in a mouse in-situ transplantation model. In a mouse model of in-situ osteosarcoma, weekly administration of 15-25 mg / kg of ALMB-0168 significantly inhibited tumor growth in the tibia and extended lifespan.

[0182] ALMB-0168 treatment increased plasma ATP levels. To evaluate the changes in plasma ATP levels due to activation of Cx43 hemichannels by ALMB-0168 administration, mice were injected with ALMB-0168, and then plasma ATP levels were measured by a luciferin / luciferase assay. The results showed that ALMB-0168-treated mice had significantly higher ATP levels (p<0.001), more than 5 times higher, than saline-treated mice.

[0183] Summary of Preclinical Pharmacokinetic Studies Pharmacokinetic study of ALMB-0168 in cynomolgus monkeys after a single intravenous infusion. A total of 18 cynomolgus monkeys (9 monkeys / sex) were randomized to three dose groups: a low-dose group, a medium-dose group, and a high-dose group, each receiving an IV dose of 5, 15, and 50 mg / kg, respectively. Blood samples were collected from each group before administration, at the end of administration, 10 minutes after administration, and at 2, 8, 24, 48, 72 hours, 5, 7, 10, 14, 17, 21, and 28 days after administration. Serum concentrations of ALMB-0168 were determined using a valid ELISA method with a limit of quantification of 0.01 μg / ml. PK parameters were determined using non-compartmental analysis (NCA) with WinNonlin software.

[0184] Serum PK parameters can be summarized as follows: 1. Except for a slightly higher AUC in women than in men in the medium-dose group (p < 0.05, ratio = 1.32), there were no significant sex differences in PK parameters (p ≥ 0.05). 2. Following a single intravenous infusion of ALMB-0168 in cynomolgus monkeys, the maximum serum concentration and systemic exposure of ALMB-0168 were proportional to the dose. The dosing ratio for the low-dose, medium-dose, and high-dose groups was 1:3:10. The mean Cmax and AUClast ratios for the low-dose, medium-dose, and high-dose groups were 1:3.04:10.92 and 1:3.10:10.95, respectively. 3. Regression analysis was performed for Cmaxdose and AUClastdose. The results showed that the slopes of the Cmaxdose and AUClastdose curves were 1.04 and 1.04, respectively, with 95% confidence intervals of [1.11, 0.98] and [1.15, 0.94], respectively, and R² values ​​of 0.99 and 0.96, respectively. The results showed that exposure to ALMB-0168 in cynomolgus monkeys was linearly associated with single intravenous infusion doses of 5, 15, and 50 mg / kg.

[0185] Overview of Preclinical Toxicology Research Single-dose toxicity study of ALMB-0168 120 mice (60 mice / sex) were randomly assigned to groups 1, 2, 3, and 4, with 15 mice / sex in each group. Mice in group 1 (0 mg / kg) were treated with ALMB-0168 buffer as a vehicle control, while mice in groups 2, 3, and 4 were treated with ALMB-0168 at doses of 50 mg / kg, 150 mg / kg, and 500 mg / kg, respectively.

[0186] Under the test conditions, no significant test substance-related toxicity was observed in mice that received a single IV dose of ALMB-0168 at doses of 50, 150, and 500 mg / kg.

[0187] A total of eight cynomolgus monkeys (4 of each sex) were randomly assigned to groups 1-4, with one monkey of each sex in each group. Group 1 received placebo as a control, while groups 2, 3, and 4 were administered ALMB-0168 at doses of 50, 150, or 500 mg / kg, respectively. On day 1, the monkeys received a single intravenous infusion. Animals were observed for approximately 4 hours after administration, followed by periodic clinical monitoring. Body weight, food consumption, and body temperature were measured, and electrocardiograms, hematological tests, coagulation tests, clinical chemistry tests, urinalysis, and serum anti-drug antibody and toxicology analyses were performed. On day 15, all animals were sacrificed and thoroughly examined.

[0188] During the study, no deaths or illnesses were observed in any of the animals in groups 1-4. No abnormal clinical findings related to the test substance were observed in any of the animals.

[0189] Multiple-dose toxicity study of ALMB-0168 1. Repeated-dose toxicity study in monkeys Forty-eating monkeys were randomized into groups 1-4, 24 monkeys / sex, and 6 monkeys / sex / group. ALMB-0168 buffer was used as a vehicle control in group 1, and ALMB-0168 was administered at doses of 25, 75, and 250 mg / kg in groups 2, 3, and 4, respectively. The monkeys received the ALMB-0168 once a week for six consecutive weeks on days 1, 8, 15, 22, 29, 36, and 43 (a total of seven doses).

[0190] Throughout the study, no signs of death or near-death were observed in animals in groups 1-4.

[0191] Throughout the study, no significant abnormalities were observed in analytes-related or toxicological aspects of body weight, food consumption, body temperature, cardiovascular function, respiratory function, electrocardiogram, blood pressure, ophthalmology, hematology, coagulation, clinical chemistry, urinalysis, lymphocyte subsets, cytokines, serum immunoglobulins, serum complement, and functional observation batteries. Neurobehavioral studies were evaluated in animals treated with ALMB-0168 at doses of 25, 75, and 250 mg / kg compared to same-sex animals in the vehicle control group.

[0192] Serum anti-drug antibody (ADA) analysis: In the ALMB-0168 25 mg / kg dose group, 2 out of 12 monkeys tested positive for ADA on day 15 post-administration, with a maximum titer of 1:4. In the ALMB-0168 75 mg / kg dose group, 1 / 12 monkeys tested positive for ADA pre-2 days and on day 15 post-administration, with titers less than 1. No increase in titer was observed throughout the study. Therefore, the positive results were considered unrelated to ALMB-0168. In the ALMB-0168 250 mg / kg dose group, 2 / 12 monkeys tested positive for ADA on day 15 post-administration, with a maximum titer of 1:8. The TK results were C in ADA-positive animals of the same group. 最大 and AUC last This showed that it was not significantly different from that of other animals.

[0193] Toxicology: Table 2 shows the mean TK parameters of the test substance in cynomolgus monkey serum. All reported values ​​are median TK values. 最大 Except for that, it was average.

[0194] [Table 3-1] [Table 3-2] Abbreviation: AF = Accumulation coefficient (AUC) 22日目 / AUC 1日目 );AUC=area under the concentration-time curve;C 最大 =Maximum concentration; N = Number of animals per sex per group; T 最大 = Time to reach maximum concentration; t 1,2 = Half-life; SD = Standard Deviation

[0195] Under the study conditions, ALMB-0168 was administered intravenously to cynomolgus monkeys at doses of 25, 75, and 250 mg / kg once weekly for 6 consecutive weeks (a total of 7 doses), followed by a 4-week recovery period. Local injection site reactions related to the test substance were observed, and a dose-dependent decrease in lymphocyte counts in the spleens of the animals was observed in all dose groups. After a 4-week recovery period, chronic active and hemorrhagic injection site inflammation completely resolved in all animals except one animal in the low-dose group. The findings observed in the spleen (dose-dependent decrease in lymphocyte counts in germinal centers and border regions) completely resolved in the 25 and 75 mg / kg dose groups after the 4-week recovery period, and tended to resolve in the 250 mg / kg dose group (minimal in one male, mild in one female). Therefore, these findings were not considered to have any adverse effects on the spleen. Thus, 250 mg / kg was a NOAEL (No Observed Adverse Event) in the study.

[0196] 2. Repeated-dose toxicity study in mice 980 mice (490 mice / sex) were randomized into eight groups (Groups 1-8): Groups 1, 2, 3, and 4 had 40 mice / sex / group, Group 5 had 45 mice / sex / group, and Groups 6, 7, and 8 had 95 mice / sex / group. Mice were administered intravenously once a week for six consecutive weeks (a total of seven doses) at the following doses: Groups 1 and 5 (vehicle control groups) (ALMB-0168 buffer, 0 mg / kg), Groups 2 and 6 (ALMB-0168), 75 mg / kg (ALMB-0168), and 250 mg / kg (ALMB-0168). The administration day was defined as Day 1.

[0197] The parameters evaluated in this study included clinical observation, body weight, food consumption, fundus examination, hematology, coagulation, clinical chemistry, urinalysis, T lymphocyte subset determination (CD3+, CD3+CD4+, CD3+CD8+, CD4+CD8+), and cytokine testing (IL-2, IL-4, IL-5, IL-6, TNF-α, IFN-γ). Six weeks after administration (day 44), the first 20 animals / sex / groups of groups 1, 2, 3, and 4 were euthanized, and the remaining 20 animals / sex / groups were euthanized after a subsequent 4-week recovery period (day 72). Gross dissection was performed on all animals in groups 1-4, organ weights were collected, and gross dissection observation and microscopic examination were completed. Histopathological evaluation was performed on all standard tissues in groups 1 and 4, tissues of animals that died unexpectedly before the scheduled time, and all abnormal tissues in groups 2 and 3.

[0198] Throughout the experiment, no deaths or near-death experiences related to the test substance occurred in any of the animals in groups 1 through 8.

[0199] Throughout the trial, no abnormal changes related to the test product were observed in clinical observations or injection site observations.

[0200] No abnormal changes in test substance-related parameters in body weight, weight gain, food consumption, body temperature, ophthalmological examination, hematology, coagulation, T lymphocyte subsets, and cytokines were observed throughout the study in animals administered 25, 75, and 250 mg / kg of ALMB-0168.

[0201] Animals administered ALMB-0168 at doses of 25, 75, and 250 mg / kg (excluding females administered at 25 mg / kg) showed increased LDH levels on day 44. After a 4-week recovery period, LDH levels decreased to those of the vehicle control group. No microscopic findings were observed. Therefore, it was not considered an adverse reaction.

[0202] At the end of administration and the end of the 4-week recovery period, no abnormalities related to the test substance were observed in organ weight, organ / body weight ratio, organ / brain weight ratio, gross anatomical observation, or microscopic examination in any of the animals. No local irritation related to the test substance was observed at the injection site in any of the animals during the study.

[0203] In conclusion, under the study conditions, mice were administered ALMB0168 at doses of 25, 75, and 250 mg / kg intravenously once a week for six consecutive weeks (a total of seven doses), followed by a four-week recovery period. The results showed no adverse systemic toxicity or target organ toxicity. Therefore, the NOAEL (No Observed Adverse Emission Limit) of ALMB-0168 in this study was 250 mg / kg.

[0204] Other related toxicity studies 1. Study of tissue cross-reactivity in normal cynomolgus monkey tissues The tissue cross-reactivity of ALMB-0168 was evaluated using streptavidin-biotin immunohistochemistry in 34 different types of normal cynomolgus monkey tissue (each tissue type was derived from three different animals).

[0205] Positive staining was observed in the cell membranes of positive control sections of MLO-Y4 cells and mouse osteocyte cell lines highly expressing Cx43 hemichannels after staining with the test substance at 5 μg / ml and 20 μg / ml, respectively. Negative control sections of the test substance group did not show positive staining on frozen human skeletal muscle tissue. No positive staining was observed in the positive and negative control photographs of the isotype control group and the negative control group.

[0206] In the test substance groups (ALMB-0168-biotin, 5 μg / ml and 20 μg / ml), nonspecific staining of round cells was observed in bone marrow, ileum, and gastric tissue in the isotype control and negative control groups, as well as in tubular epithelial cells and hepatocytes. No nonspecific positive staining was observed in other tissues in each test group.

[0207] Under the conditions of this study, no specific staining for ALMB-0168 was observed in normal cynomolgus monkey tissue.

[0208] 2. Tissue cross-reactivity test in normal human tissue Using the streptavidin-biotin method of IHC staining, we evaluated whether ALMB-0168 exhibited tissue cross-reactivity with 34 frozen tissues from normal humans (each tissue originating from three different individuals).

[0209] In the test substance groups (ALMB-0168-biotin, 5 μg / ml and 20 μg / ml), nonspecific staining of round cells was observed in bone marrow, colon, ileum, and gastric tissue in the isotype control and negative control groups. Nonspecific staining was also observed in tubular epithelial cells and hepatocytes. No nonspecific positive staining was observed in other tissues in each test group.

[0210] Under the test conditions, no specific staining for ALMB-0168 was observed in frozen normal human tissue.

[0211] 3. Tissue response tests in mouse tissues Using the streptavidin-biotin method of IHC staining, we evaluated whether ALMB-0168 exhibited cross-reactivity with 30 frozen tissue samples from normal mice (each tissue sample from three different individuals).

[0212] In the isotype control and negative control groups of the test substance groups (ALMB-0168-biotin, 5 μg / ml and 20 μg / ml), nonspecific staining was observed in adrenal cortical cells and bone marrow cells, gastric round cells, renal tubular epithelial cells, and hepatocytes. No nonspecific positive staining was observed in other tissues in each test group.

[0213] Under the test conditions, no specific staining for ALMB-0168 was observed in normal mouse tissue.

[0214] 4. In vitro human blood cell hemolysis test This provides a reference for clinical trials of the test substance. The test substance at a concentration of 10 mg / ml was analyzed on the day of the in vitro test. The precision of the upper, middle, and lower layers of the test sample was 99.93%, 101.20%, and 101.63%, respectively, with a coefficient of variation of 0.87%. The test substance was not detected in the negative and positive controls. The analytical results met the requirements.

[0215] After incubation at 37±0.5℃ for 3 hours, the positive control tube was completely hemolyzed, the solution was clear red, and there were no RBCs at the bottom of the tube. In both the test material tube and the negative control tube, red blood cells were observed to settle at the bottom of the tube, the upper solution was colorless and clear, and there was no hemolysis or agglutination.

[0216] Under the conditions of this study, ALMB-0168 at a concentration of 10 mg / ml did not have a hemolytic effect on human RBCs in vitro and did not cause RBC agglutination.

[0217] 5. Cytokine release study in human peripheral blood mononuclear cells (PBMCs) In vitro cytokine release from stimulated PBMC samples was tested after 24-hour incubation with ALMB-0168. For technical controls, research-grade anti-CD3 antibody and research-grade human IgG isotype control were used as positive and negative controls, respectively, while untreated PBMC samples were used as a baseline control.

[0218] All donor-derived PBMCs reacted with CD3 antibodies in both stimulation modes, demonstrating that the PBMC samples had the ability to stimulate and induce the release of IL-2, IL-6, IL-10, IFN-γ, and TNF-α. Elevated IL-6 was detected in several PBMC samples, and wet coating stimulation was used for most of these samples.

[0219] Human IgG controls under both stimulation forms did not induce significant release responses for IL-2, IL-10, IFN-γ, and TNF-α. Under soluble stimulation, IL-6 responses differed among multiple donors. No dose-response trend was observed, and responses did not reach the level of positive control biological CD3 antibodies achieved by the majority of donors.

[0220] Under both forms of stimulation, ALMB-0168 induced little to no response to IL-2, IL-10, IFN-γ, and TNF-α cytokine release. Under soluble stimulation, IL-6 responses differed among multiple donors. The response did not reach the level of positive control biological CD3 antibody achieved by the majority of donors.

[0221] First human safety study in Australia As of May 2021, a total of two participants (one at 1 mg / kg and the other at 3 mg / kg) were enrolled in the first-in-human trial in Australia and received five cycles of administration (once every three weeks). There were no serious adverse events, fatal adverse events, or adverse events leading to participant withdrawal. One participant in the 1 mg / kg group experienced a drug-related adverse event of abdominal pain, which fluctuated between grade 1 and grade 2 and resolved after symptomatic treatment with oral medication. One participant in the 3 mg / kg group experienced a drug-related adverse event of grade 1 fatigue, which resolved spontaneously without drug treatment. Other adverse events that occurred in both participants during the trial included diarrhea, abdominal discomfort, nausea, herpes zoster on the left side of the upper lip, hyperglycemia, hypercalcemia, and redness and itching at the site of the peripherally inserted central catheter, all of which were grade 1 and judged to be unrelated to the drug.

[0222] Known potential benefits Prior to the commencement of this study, a first-in-human clinical study of ALMB-0168-AU-101 was conducted in Australia to evaluate the safety, tolerability, PK, PD, preliminary efficacy, and immunogenicity of ALMB-0168 in patients with incurable osteosarcoma and bone metastases for which there were no better treatment options. This study guided the starting dose to reduce exposure to ineffective doses and further ensure safety. In this first-in-human clinical trial, the administration of the first and second dose groups (1 mg / kg and 3 mg / kg, respectively) to the first and second participants demonstrated good safety and tolerability. As described in the study protocol, all participants were closely monitored throughout the study, and any adverse reactions experienced by participants were closely monitored and addressed in a timely manner until they returned to normal or became clinically stable.

[0223] In conclusion, ALMB-0168 is a humanized monoclonal IgG4 antibody that targets the Cx43 hemichannel and has a clear mechanism of action. Preclinical studies have shown that ALMB-0168 has good efficacy, safety, and controllability, and may provide efficacy benefits to patients with osteosarcoma and bone metastatic tumors.

[0224] Example 2. A multicenter, single-arm, open-label Phase I / II clinical trial to evaluate the safety and efficacy of ALMB-0168 in patients with osteosarcoma - Trial protocol This study is registered on ClinicalTrials.gov as NCT04886765.

[0225] Research objectives and endpoints Part I of the Research The primary objectives of Part I are to evaluate the safety and tolerability of ALMB-0168 in patients with osteosarcoma who have failed standard treatment, and to investigate the recommended Phase II dose (RP2D).

[0226] The secondary objectives of Part I are as follows: • To evaluate the pharmacokinetic (PK) profile of ALMB-0168; • To evaluate the efficacy of ALMB-0168 in the treatment of patients with osteosarcoma; • To evaluate the immunogenicity of ALMB-0168.

[0227] The primary endpoints for Part I are as follows: Physical examination, vital signs, pulse oxygen saturation, safety testing, electrocardiogram, and the occurrence of AEs, SAEs, and DLTs; • MTD (if any).

[0228] The secondary endpoints for Part I are as follows: • Pharmacokinetic indicators include, but are not limited to, AUC0-t, AUC0-inf, C-max, C-trough, T-max, CL and λz, and t1 / 2. • Efficacy indicators: Overall response rate (ORR), progression-free survival (PFS), duration of response (DoR), time to response (TTR), disease control rate (DCR), 6-month progression-free survival rate (6m-PFSR), overall survival (OS); percentage change from baseline in alkaline phosphatase (ALP) or lactate dehydrogenase (LDH); skeletal-related events (SRE); changes from baseline in numerical rating scales (NRS) and the European Quality of Life 5-Dimensional (EQ-5D) score; bone mineral density of the lumbar spine, hip, femur, and neck; dosage and frequency of morphine use; • Incidence of anti-drug antibodies (ADA).

[0229] Part II of the Research The primary objective of Part II is to further evaluate the safety and efficacy of ALMB-0168 in patients with high-grade osteosarcoma who were unable to further determine RP2D after standard treatment.

[0230] The second objective of Part II is as follows: • Further evaluation of the pharmacokinetic (PK) profile of ALMB-0168; • To evaluate the effectiveness of ALMB-0168; • Further evaluation of the immunogenicity of ALMB-0168.

[0231] The primary endpoints of Part II are as follows: Physical examination, vital signs, pulse oxygen saturation, safety testing, electrocardiogram, and occurrence of AEs and SAEs; • Efficacy indicator: 6-month progression-free survival rate (6m-PFSR).

[0232] The secondary endpoints for Part II are as follows: • Efficacy indicators: Overall response rate (ORR), duration of response (DoR), time to response (TTR), disease control rate (DCR), overall survival (OS); percentage change from baseline in alkaline phosphatase (ALP) or lactate dehydrogenase (LDH); skeletal-related events (SRE); changes from baseline in numerical rating scales (NRS) and the European Quality of Life 5-Dimensional (EQ-5D) score; bone mineral density of the lumbar spine, hip, femur, and neck; dosage and frequency of morphine use; • PK index: Includes, but is not limited to, C-maximum and C-trough. • Incidence of anti-drug antibodies (ADA).

[0233] research design Overall design This is a multicenter, open-label, single-arm phase I / II clinical trial to evaluate the safety and preliminary efficacy of ALMB-0168 in patients with osteosarcoma who have failed standard treatment.

[0234] The trial will be conducted in two phases (Part I: dose escalation phase and Part II: dose expansion phase), with all participants receiving multiple cycles of intravenous injection of ALMB-0168 every three weeks. Part I will enroll patients with high-grade osteosarcoma who have failed standard treatment, and Part II will enroll patients with high-grade osteosarcoma from the Part I trial population. Accelerated titration and a standard "3+3" escalation design will be used in the dose escalation phase. The accelerated titration escalation method will be used for the first two dose groups (1 mg / kg, 3 mg / kg). One participant will be enrolled in each group, and if the participant does not experience a drug-related grade ≥2 TEAE within three weeks after the initial dose, the dose will be escalated to the next group. If the participant experiences a drug-related grade ≥2 TEAE, the 3+3 dose escalation method will be used at the dose level. The 3+3 dose escalation method will be used starting from the third dose group (6 mg / kg). Dose escalation for each subsequent cohort will be determined according to the incidence of ALMB-0168-related adverse events (AEs) assessed by NCI CTCAE v5.0 within three weeks after the first dose (DLT observation period). Dose escalation will continue until at least one-third of participants (if six or more participants are assessed) experience a DLT at that dose level. The acceptable level prior to the dose level is considered the MTD, and safety can be reassessed by selecting an appropriate dose between the two doses after discussion and communication between the sponsor and the investigator.

[0235] The selection of the RP2D from the acceptable dose range is based on short-term and long-term safety information, as well as findings from preclinical dose determination studies, PK, PD, and antitumor activity studies. For dose groups that have completed the DLT observation period, after a comprehensive analysis of participant benefits by the principal investigator and sponsor based on an evaluation of previous clinical data, they may be expanded to a maximum of eight participants (excluding the MTD expansion cohort) to provide further information on safety, PK profile, and antitumor activity. Safety data from the expanded participants will not be used as the basis for determining the MTD.

[0236] In the dose escalation phase, expansion studies will be conducted in 1 to 3 dose groups to investigate initial efficacy in patients with high-grade osteosarcoma who have failed standard treatment, and to further document the safety and pharmacokinetic properties of ALMB-0168. Each dose group is planned to have a maximum of 60 participants, and a decision will be made on whether to increase the number of participants after efficacy and safety data are obtained.

[0237] All participants will sign an ICF prior to any specific research activity. Eligible participants will be treated at the dose level determined during the dose escalation phase after enrollment. Theoretically, one treatment cycle in this study is defined as 21 days (3 weeks). All participants will receive intravenous injections of ALMB-0168 every 3 weeks. Participants will receive multiple cycles of treatment until they require treatment for progressive disease or antitumor therapy. All participants will be closely monitored for safety.

[0238] All participants will be followed up individually 28 days after their last treatment, followed by a one-year follow-up to assess long-term outcomes.

[0239] Dose escalation design Dose-limiting toxicity Dose-limiting toxicity (DLT) is defined as the following toxic responses associated with ALMB-0168 that occur during the first treatment cycle (21 days): • Grade 4 neutropenia lasting ≥ 7 days; or fever (ANC < 1000 / mm²) 3 neutropenia of grade ≥3 accompanied by infection (single temperature >38.3°C or sustained temperature ≥38°C for more than 1 hour); • Grade 4 thrombocytopenia or grade 3 thrombocytopenia accompanied by bleeding; • Grade ≥3 hepatotoxicity: ALT or AST ≥ 5 × upper limit of normal (ULN) (for participants with normal baseline) or ≥ 5 × baseline value (for participants with liver metastases and abnormal baseline); or bilirubin ≥ 3 × ULN (for participants with normal baseline) or ≥ 3 × baseline value (for participants with abnormal baseline); · Grade ≥ 3 nephrotoxicity: Serum creatinine ≥ 3 × ULN (for participants with normal baseline values) or ≥ 3 × baseline value (for participants with abnormal baseline values); · Grade ≥ 3 cardiotoxicity: QTc > 500 ms or QTc increase ≥ 60 ms; · Other Grade ≥ 3 non-hematological toxicities, excluding the following: □ Grade 3 nausea and / or vomiting lasting less than 72 hours without prophylactic and effective antiemetic treatment; □ Fatigue or asthenia lasting less than 7 days. □ Transient and asymptomatic laboratory test abnormalities lasting less than 3 days; □ Alopecia, rash, and diarrhea controllable under standard supportive care. · Toxicity responses for which the principal investigator of the clinical trial determines that ALMB-0168 should be permanently discontinued.

[0240] The above AEs are graded according to the National Cancer Institute - Common Terminology Criteria for Adverse Events (NCI-CTCAE) Version 5.0.

[0241] Maximum tolerated dose MTD Definition: Use of accelerated titration and standard "3+3" dose escalation design in dose escalation stages: Use of accelerated titration for the first two dose groups (1 mg / kg, 3 mg / kg). One participant is enrolled in each group, and if the participant does not experience grade 2 or higher toxicity related to ALMB-0168, the dose is escalated to the next dose group. If the participant experiences grade 2 or higher toxicity related to ALMB-0168, the 3+3 dose escalation method is used at the dose level. The 3+3 dose escalation method is used starting from the third dose group (6 mg / kg). Dose escalation for each subsequent cohort is determined according to the incidence of ALMB-0168-related adverse events (AEs) assessed by NCI CTCAE v5.0 within 3 weeks after the first dose (DLT observation period). Dose escalation continues until more than one-third of the participants experience DLT at that dose level (if more than 6 participants are assessed). The tolerable dose level preceding the dose level is considered the MTD (Most Tolerable Dose).

[0242] After Cycle 1, all Grade ≥ 4 adverse events during the extension period are also factors in determining dose escalation and MTD.

[0243] Estimation of initial dose The safe clinical starting dose for this study will be calculated using the results of toxicity studies in non-human primates and mice. The specific methodology will be consistent with the guidance provided in ICH S9. The non-observed adverse effect level (NOAEL) for ALMB-0168 is 250 mg / kg in both non-human primates and rodents. Given the novel target of ALMB-0168, a safety factor greater than 100 × NOAEL will be used to ensure that the first dose does not cause adverse effects in humans. The final starting dose of the drug in this first-in-human study is 1 mg / kg.

[0244] Determining the Maximum Dose Increase In a non-GLP dose-ranging study in non-human primates (NHPs), ALMB-0168 was administered intravenously to cynomolgus monkeys at weekly doses of 20, 60, and 200 mg / kg, and no adverse reactions were observed in any dose group over the following two weeks. Subsequently, in a GLP repeated-dose toxicity study, cynomolgus monkeys were administered intravenously once weekly at doses of 25, 75, and 250 mg / kg for six weeks, followed by a four-week recovery period. No significant adverse reactions were found. Furthermore, in a GLP repeated-dose toxicity study in mice, mice were repeatedly administered ALMB-0168 at weekly doses of 25, 75, and 250 mg / kg for six weeks, and no significant adverse reactions were observed after four weeks of observation and recovery. Therefore, the NOAEL (No Observed Adverse Effect Level) of ALMB-0168 is 250 mg / kg in both NHPs and rodents.

[0245] In preclinical efficacy studies, weekly injections of 25 mg / kg of ALMB-0168 significantly inhibited the growth of human breast cancer in a mouse xenograft model. In a mouse model of in situ osteosarcoma, weekly administration of 15–25 mg / kg of ALMB-0168 significantly inhibited tumor growth in the tibia and extended lifespan. The maximum escalation dose in clinical trials is tentatively set at 30 mg / kg, which will also be adjusted as needed based on PK, PD, and safety data obtained for each dose group of participants in Australia and China.

[0246] Method and frequency of administration A 21-day (3-week) treatment cycle is defined as one treatment cycle in this study. All participants will receive intravenous injections of ALMB-0168 every 3 weeks. Participants will receive multiple treatment cycles until progressive disease or unacceptable toxicity occurs. All participants will be closely monitored for safety.

[0247] All participants will be followed up individually 28 days after their last treatment, followed by a one-year follow-up to assess long-term outcomes.

[0248] Dose escalation group setting The dose escalation level is based on the ALMB-0168-related adverse events observed during the first cycle of medication and the dose escalation gradient, as specified in the dose escalation setting table. If two or more participants experience an ALMB-0168-related grade ≥ 2 TEAE in the first cycle of medication, or if one participant experiences an ALMB-0168-related grade ≥ 3 TEAE, the scale of the inter-cohort dose escalation may be reduced from a 100% dose escalation to a dose increase of 50% or less from the previous dose level, as necessary and as discussed between the investigator and the sponsor. ALMB-0168 is escalated at dose levels of 18 mg / kg or higher with an increase of 33% or less. If there are problems with safety assessment, the dose escalation can be appropriately reduced using a similar procedure. It is expected that there will be no more than seven dose groups evaluated during the study. The actual number of dose groups and dose levels will depend on the maximum tolerated dose (MTD) and the safety profile observed during the study. If necessary, the principal investigator and the sponsor may discuss intermediate dose escalation levels. Refer to Table 3 below for dose group settings at specific dose escalation stages.

[0249] [Table 4] a: Accelerated titration with dose escalation is used for the first two dose groups (1 mg / kg, 3 mg / kg). One participant is enrolled in each group, and if the participant does not have a Grade 2 or higher TEAE related to ALMB-0168, the dose is escalated to the next dose group. If the participant experiences a Grade ≥2 TEAE related to ALMB-0168, a 3+3 dose escalation method is used at the dose level. * The MTD cohort will include up to 10 participants, including 3–6 participants in the dose escalation phase, to determine safety. The dose levels and percentage increases from previous dose levels for each dose group will be adjusted based on trial data from the First in Human Clinical Trial in Australia (ALMB-0168-AU-101). After completing the DLT observation period, the dose group may be expanded to a maximum of eight participants (excluding the MTD expansion cohort) after the principal investigator and sponsor have evaluated previous clinical data and analyzed the participant benefits.

[0250] If the safety and tolerability of the dose group remain good after gradually increasing the dose to the pre-specified maximum dose group, the principal investigator and sponsor will jointly decide whether to investigate higher doses.

[0251] Dosage escalation principle Use accelerated titration and the standard "3+3" dose escalation design during dose escalation: Use the accelerated titration escalation method for the first two dose groups (1 mg / kg, 3 mg / kg), and the 3+3 design dose escalation method starting with the third dose group (6 mg / kg). At least three participants will be enrolled in each dose group, and a decision will be made within three weeks after the three participants complete the first dose at their respective dose levels (DLT observation period), based on ALMB-0168-related adverse events (AEs) assessed according to NCI CTCAE version 5.0. If none of the three participants experience a DLT, the next group will be treated at the next dose level according to the dose escalation rules. If one participant experiences a DLT at each dose level, enroll three additional participants. Dose escalation will continue until more than one-third of the participants experience a DLT at that dose level (if more than six participants are evaluated). The tolerable dose level prior to the dose level is considered the MTD. The selection of the RP2D from the acceptable dose range is based on short-term and long-term safety information, as well as findings from preclinical dose determination studies, PK, and antitumor activity studies. For dose groups that have completed the DLT observation period, after a comprehensive analysis of participant benefits by the principal investigator and sponsor based on an evaluation of previous clinical data, they may be expanded to a maximum of eight participants (excluding the MTD expansion cohort) to provide further information on safety, PK profile, and antitumor activity. Safety data from the expanded participants will not be used as the basis for determining the MTD.

[0252] Expansion stage In the expansion phase, expansion trials will be conducted in 1 - 3 dose groups, registering up to 60 osteosarcoma patients in each dose group to preliminarily investigate efficacy, further characterize the safety and PK properties of ALMB - 0168, investigate efficacy, and clarify the PR2D, etc.

[0253] Definition of End of Study A participant is considered to have completed the study if he / she has completed all stages of dosing and the study - end visit (28 days after the last dose). The end - of - study date is defined as the last visit date of the last patient in the study. The maximum follow - up period is 12 months. Thus, if the last - enrolled patient dies or is followed for 12 months, whichever is earlier, the trial is considered to have ended.

[0254] Study Population Inclusion Criteria 1. Patients with high - grade osteosarcoma confirmed by histopathology. 2. Defined according to different stages. a) Part I: Osteosarcoma patients with bone lesions (primary or metastatic) that have failed standard treatment, for example, but not limited to, the following. i) Osteosarcoma of stage III with or without lung metastasis or with bone metastasis according to the American Joint Committee on Cancer (AJCC) bone tumor staging system; ii) Locally advanced inoperable osteosarcoma; iii) Multifocal osteosarcoma. b) Part II: Patients with high - grade osteosarcoma that have failed standard treatment.

[0255] Standard treatment failure is defined as progressive disease during or within 6 months after chemotherapy with first - line or more chemotherapy drugs (including high - dose methotrexate, doxorubicin [cumulative dose > 350 mg], and cisplatin). For patients with progressive disease beyond 6 months, the principal investigator of the trial should conduct a risk - benefit assessment and obtain consent from the participant or his / her guardian.

[0256] 3. Patients were 16 years of age or older, regardless of gender. 4. Patients with an ECOG (Eastern Cooperative Oncology Group) PS score of 0, 1, or 2. 5. Patients with measurable or unmeasurable lesions according to RECIST v1.1. Unmeasurable lesions should be confirmed by conventional imaging techniques, including isotope bone scans, CT, or MRI. Participants included in Part II should have at least one measurable lesion confirmed by CT or MRI at baseline. 6. Patients with the following major systemic functions are as follows: a) Bone marrow reserve: Absolute neutrophil count (ANC) ≥ 1.5 × 10⁹ / L; Platelet count ≥ 75 × 10⁹ 9 / L; Hemoglobin ≥ 9 g / dL, no blood transfusions in the past 14 days (participant requiring a blood transfusion); b) Liver function: Total bilirubin ≤ 1.5 × upper limit of normal (ULN) (unless the patient has experienced grade 1 bilirubin elevation due to Gilbert's disease or a similar syndrome with delayed bilirubin binding). Transaminases (AST / SGOT and / or ALT / SGPT) ≤ 3 × ULN (liver metastases < 5 × ULN); c) Renal function: Normal serum creatinine ≤ 1.5 mg / dL (133 μmol / L) or calculated creatinine clearance ≥ 50 ml / min (Cockroft-Gault formula); d) Coagulation: Defined as having an International Rate of Standardization (INR) ≤ 2. 7. Female participants of childbearing potential must have a negative serum or urine pregnancy test within 7 days prior to the first dose and must not be lactating. Male participants with a female partner of childbearing potential and female participants of childbearing potential must use two acceptable methods of contraception, including one barrier method, during the study and within 3 months after the last dose. Male participants must avoid donating sperm during the study. Female participants must avoid donating eggs during the study. 8. Expected survival period ≥ 3 months. 9. Participants (or their guardians) understand the entire process of this study, can voluntarily participate in the study, and can sign the ICF.

[0257] intervention ALAM-0168 ALMB-0168 is a sterile, pyrogen-free injectable monoclonal antimicrobial agent for human connexin 43, provided by the clinical trial sponsor, Shanghai Therapeutics (AlaMab) Inc. The strength is as follows: Each vial contains 6 ml (150 mg) of ALMB-0168 monoclonal antimicrobial agent at a strength of 25 mg / ml. Storage conditions are -20±5°C, protected from light, and shaken.

[0258] ALMB-0168 is administered by intravenous injection over at least 30 minutes, and all participants receive intravenous infusion of ALMB-0168 alone every three weeks. Participants will receive multiple cycles of treatment until progressive disease or unacceptable toxicity occurs.

[0259] Currently, there are no drugs in the same class as ALMB-0168. In preclinical animal studies, chronic active hemorrhagic inflammation and lymphopenia were observed in the central and marginal zones of the spleen. Therefore, participants should be closely monitored during the study for infusion reactions, infections, flu-like symptoms, rashes, muscle and arthralgia, fever and chills, as well as toxicity to the heart, liver, kidneys, and other organ systems. Participants receiving their first ALMB-0168 infusion must be monitored for at least 48 hours post-infusion in an area equipped with resuscitation devices and emergency medications.

[0260] If a participant experiences an adverse reaction during the study, the principal investigator should provide necessary supportive care according to clinical needs and allow for discontinuation and / or dose adjustment of ALMB-0168.

[0261] If all ALMB-0168-related adverse events (AEs) that occurred during the previous treatment cycle do not revert to baseline levels or Grade 1 on the day of the subsequent scheduled treatment, treatment should be delayed and the toxicity response should be graded weekly. The day on which a participant receives the next cycle of ALMB-0168 treatment is recorded as Day 1 of the next treatment cycle. If all ALMB-0168-related AEs revert to baseline levels or NCI CTCAE Grade 1 within 3 weeks, medication may be continued.

[0262] Participants requiring a treatment delay of more than 21 days due to ALMB-0168-related toxicity experienced in the previous treatment cycle will permanently discontinue the study treatment and complete the study by making a treatment completion visit four weeks after the last treatment.

[0263] Preparation / Handling / Storage / Responsibility for ALMB-0168 The clinical trial sponsor should be responsible for delivering ALMB-0168 to the research facility, and the principal investigator or their designated representative should sign to confirm receipt.

[0264] ALMB-0168 may only be used in this study and may only be handled by a designated person authorized by the Principal Investigator. The Principal Investigator / Pharmacist responsible for administering the drug should distribute, recycle, and destroy ALMB-0168 and maintain accurate records in accordance with the study procedures. Used ALMB-0168 and its packaging should be disposed of and destroyed as medical waste at the study site, and unused or expired drugs should be destroyed by the sponsor after collection.

[0265] Instructions for reheating, diluting, mixing, and preparing ALMB-0168 should be recorded in the corresponding accompanying documentation.

[0266] This study will employ a non-randomized, open-label design.

[0267] The use of ALMB-0168 must strictly adhere to clinical protocol regulations, and the dose applied to each participant should be recorded in the eCRF.

[0268] The principal investigator at the research site should check and count the drugs during distribution, retrieval, and destruction, and sign documentation for the movement, transfer, and destruction of the drugs for confirmation.

[0269] Dosage form, appearance, packaging, and labeling The sign should include the following: Protocol number: ALMB-0168-CN-101 Drug number: Drug: ALMB-0168 injection Strength: 150 mg, 6 ml / vial Manufacturing batch number: Dosage form: IV injection Shelf life: Indications: High-grade osteosarcoma following failure of standard treatment Instructions for use: Please refer to the enclosed document for details.

[0270] Storage conditions: -20°C ± 5°C. Protect from light and shaking before use. Infusion solutions should be used within 28 hours of preparation, including 24 hours if refrigerated at 2°C to 8°C, and 4 hours if administered and temporarily stored at room temperature of 18°C ​​to 26°C (including storage and infusion time of the infusion solution in the infusion bag). If refrigerated, the diluted solution for infusion should be returned to room temperature before administration.

[0271] The label should also include the statement, "The drug is provided by Shanghai Therapeutics (AlaMab) Inc. and is for use only in clinical trials."

[0272] Product preservation and stability ALMB-0168 should be stored at -20°C ± 5°C, protected from light and shaking before use. The infusion solution should be used within 28 hours of preparation, including a maximum of 4 hours if stored at room temperature between 18°C ​​and 26°C, and a maximum of 24 hours if refrigerated between 2°C and 8°C (including storage and infusion time of the infusion solution in the infusion bag).

[0273] The diluted solution for injection should not be frozen or shaken.

[0274] ALMB-0168 should be ensured to remain stable within its proposed shelf life under the proposed packaging and storage conditions.

[0275] Concomitant medication and treatment Given that ALMB-0168 is a therapeutic monoclonal antibody, the likelihood of drug-drug interactions between ALMB-0168 and small molecule formulations is low. Since ALMB-0168 is expected to break down into amino acids and circulate to other proteins, it is unlikely to affect drug-metabolizing enzymes or transporters. Medication to treat conditions present before the study or new conditions arising during the study may be administered throughout the study. Blood products, analgesics, hypnotics, and other medications for symptom management may be administered at the discretion of the principal investigator. In addition to the drugs listed in the exclusion criteria (Section 5.2) and Section 6.5.1, other medications necessary for the safety and health of the participants may be administered at the discretion of the principal investigator. The principal investigator may administer any concomitant medications or treatments necessary for appropriate supportive care.

[0276] All concomitant medications and treatments under study should be documented in detail in the eCRF. For concomitant or supportive therapies for osteosarcoma, the name of the treatment, indication (reason for administration), dose, unit, frequency, start date, and end date should be collected. For all other concomitant therapies, the name of the treatment, indication, start date, and end date should be collected.

[0277] Concomitant medication should include not only prescription drugs but also all over-the-counter (OTC) medications, herbal remedies, and vitamins.

[0278] Prohibited / Permitted Foods, Drugs and Treatments During the study, any antitumor treatment (including cytotoxic chemotherapy, targeted drugs, radiotherapy, endocrine therapy, and traditional Chinese medicine with antitumor indications) and all other investigational drugs are prohibited. If a participant requires such a drug, they must first withdraw from the study.

[0279] The following drugs are permitted for use as appropriate during the trial. (1) Long-term medication necessary for co-existing conditions (such as hypertension and diabetes); (2) Hormone replacement therapy or oral contraceptives not intended for antitumor treatment; (3) With the understanding that the interests and safety of the participants are protected, the principal investigator should provide symptomatic and supportive care for drug toxicity responses or to control tumor symptoms.

[0280] Research evaluation and procedures Screening should be completed within 28 days prior to C1D1. Participants who meet all inclusion criteria and do not meet exclusion criteria will be randomized.

[0281] Demographic data, including date of birth, sex, age, and nationality, will be collected from day -28 to day -1.

[0282] Height will be measured only at the time of screening. Weight will be measured during the screening period and on CXD1 (if weight measurement at screening is completed within 72 hours prior to administration, weight measurement is not required on C1D1).

[0283] Medical history and other past medical history Medical history refers to clinically significant information (or signs and symptoms if a diagnosis cannot be confirmed) regarding previous illnesses and associated conditions that occurred before signing the ICF, including past and present medical history. Of this, tumor history includes the time of diagnosis, pathological classification, staging, and screening stage. All medical history related to a participant's tumor diagnosis must be traceable back to the original diagnosis. In addition, a history of smoking, alcoholism, substance abuse, surgery, or clinically significant abnormalities found during relevant screening tests should also be recorded as past medical history in the original medical record and eCRF.

[0284] Pre- / conjunctional medication, treatment, and pre-tumor therapy Premedication refers to any medication taken within 28 days prior to the day of administration of the investigational drug.

[0285] Concomitant medication and treatment refers to the simultaneous administration of two or more drugs / treatments, and in this study, it is defined as any drug / treatment administered at any point in time from the first concurrent / simultaneous dose of ALMB-0168 up to 28 days after the last dose.

[0286] At each visit from the initial screening to the end of the study, the principal investigator should inquire about any medications / treatments the participant has taken since the last visit. Any previous medications and concomitant treatments should be recorded in the original medical record and eCRF, and all concomitant medications should be recorded with the following information: drug / non-drug name, cause of administration, dosage, units, frequency of administration, route of administration, start date and end date; concomitant non-drug treatments should include the name of the non-drug treatment, description of the treatment, cause of treatment, start date and end date of treatment. If the reason for using concomitant medications and treatments conforms to the definition of AE, relevant information should be recorded in the participant's original medical record and eCRF.

[0287] Treatment may include surgery, chemotherapy, radiation therapy, hormone therapy, or immunotherapy. The following data should be collected: start and end dates of treatment, name of the treatment plan, dosage, units, frequency, best response, and reason for discontinuing treatment.

[0288] Safety and other evaluations The following items will be tested in the examination. (1) Hematology: Red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), white blood cell count (WBC), platelet count (PLT), number and percentage of neutrophils, lymphocytes, eosinophils, basophils and monocytes, reticulocyte count, etc. (2) Blood chemistry: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), total bilirubin (TBIL), direct bilirubin (DBIL), total protein (TP), albumin (ALB), urea or blood urea nitrogen (BUN), creatinine (Cr), creatinine clearance (Ccr), glucose (Glu), potassium (K+), sodium (Na+), chloride (Cl-), calcium (Ca2+), magnesium (Mg2+), phosphorus (P), and carbon dioxide; (3) Cardiac zymogram: Creatine kinase (CK) and its isozyme (CK-MB). (4) Urinalysis: pH, specific gravity (SG), glucose (GLU), protein (PRO), red blood cells (RBC), white blood cells (WBC), ketones (KET), occult blood, bilirubin, nitrite, urobilinogen. (5) Coagulation function: Prothrombin time (PT), activated partial thromboplastin time (APTT), international normalized ratio (INR), thrombin time (TT), plasma fibrinogen (Fbg). (6) Serum or urine pregnancy test: Only for women who may be pregnant. (7) Viral serological tests: Hepatitis B test (hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (HBsAb), e antigen (HBeAg), e antibody (HBeAb), core antibody (HBcAb), additional HBV-DNA test in patients positive for hepatitis B surface antigen), hepatitis C test (hepatitis C antibody, additional HCV-RNA test in patients positive for HCV antibody), AIDS test (anti-HIV antibody), syphilis test (anti-Treponema pallidum specific antibody).

[0289] Hematology (hematology and reticulocyte count), blood chemistry, cardiac zymogram, and urinalysis: performed at baseline (-7 to -1 day), C1D1, C1D8, C1D15, C2D1, C2D8, C2D15, D1 of each subsequent cycle, and at the final visit of the trial. If the C1D1 physical examination is expected to be performed within 72 hours of the baseline physical examination, it may be omitted.

[0290] Coagulation function: This was performed at screening, before the administration of D1 in each cycle, and at the final visit of the study. It may be omitted if the C1D1 physical examination is expected to be performed within 72 hours of the baseline physical examination.

[0291] Serum or urine pregnancy tests were performed at screening, before the D1 dose of each cycle, and at the final visit of the study.

[0292] Virology / serology: The test is performed during screening.

[0293] The principal investigator will examine all laboratory findings, assess the participant's change from baseline in laboratory findings based on reference ranges, determine the clinical significance of each abnormal laboratory value, and record it as “Not Clinically Significant (NCS)” or “Clinically Significant (CS)” (unmarked as “Normal”). Any abnormal laboratory tests that occur during the study and are accompanied by the following symptoms should be listed as adverse events and recorded in the original medical record and eCRF. • Directly removing participants from the study; • To cause serious harmful consequences. • To produce clear clinical symptoms. • Those deemed clinically significant by the principal investigator.

[0294] The reviewed test report will be signed and dated by the principal investigator.

[0295] A physical examination including skin, mucous membranes, lymph nodes, head (including skull, ears, eyes, nose, mouth, and throat), neck, chest (including rib cage, breasts, lungs, and heart), abdomen (including liver, gallbladder, spleen, kidneys, bladder, stomach, and intestines), spine / limbs, nervous system, and other areas.

[0296] Examinations are performed at baseline (-7 to -1 day), C1D1, C1D8, C1D15, C2D1, C2D8, C2D15, D1 of each subsequent cycle, and at the final visit of the study. If the C1D1 physical examination is expected to be performed within 72 hours of the baseline physical examination, it may be omitted. Clinically significant abnormalities compared to baseline should be recorded as adverse events (AEs).

[0297] Vital signs include sitting blood pressure, respiration, pulse, and body temperature. Vital signs are measured at baseline (-7 to -1 day), C1D1, C1D8, C1D15, C2D1, C2D8, C2D15, D1 of each subsequent cycle, and at the final visit of the study. The C1D1 physical examination may be omitted if it is expected to be performed within 72 hours of the baseline physical examination. Clinically significant abnormal values ​​compared to baseline should be reported as adverse events (AEs).

[0298] Sitting systolic and diastolic blood pressure should be measured on the same arm each time, and the measurement should be taken after the participant has rested in a seated position for at least 5 minutes. All records should be made using standard equipment, and automated or manual measurement is acceptable. However, the principal investigator should continue to perform all assessments of individual participants using the same method used for the first measurement.

[0299] Participant performance status will be assessed according to the ECOG Performance Status Scale (see Appendix I for details). Examinations will be performed at baseline (day -7 to day -1), C1D1, C2D1, D1 of each subsequent cycle, and at the final visit of the study. The C1D1 physical examination may be omitted if it is expected to be performed within 72 hours of the baseline physical examination.

[0300] The tests will be performed at baseline (-7 to -1 day), 30 minutes (±10 minutes) before and 30 minutes after administration on C1D1, C1D8, and C1D15, 30 minutes (±10 minutes) before and 30 minutes (±10 minutes) after administration on D1 of each subsequent cycle, and upon the final visit at the end of the study. The principal investigator may increase the frequency of the tests depending on the participant's condition. Participants must rest in a supine position for at least 5 minutes before receiving three 12-lead electrocardiograms.

[0301] The procedure was performed at baseline (from day -14 to day -1). During the study, the principal investigator may perform examinations as needed based on clinical needs. Participants must rest in a supine position for at least 5 minutes before the UCG.

[0302] The examination was performed at baseline (from day -28 to day -1). During the study, the principal investigator will perform the examination as needed.

[0303] The activity schedule is summarized in Table 4 below.

[0304] [Table 5-1]

[0305] [Table 5-2] Abbreviations: ADA = Anti-drug antibody; BP = Blood pressure; C = Cycle; CBC = Complete blood count; CMP = Comprehensive metabolic panel; D = Day; diff = Difference; ECG = Electrocardiogram; ECHO = Echocardiography; ECOG = Eastern Cooperative Oncology Group; PK = Pharmacokinetics; PS = Performance status; RETICS = Reticulocyte count; EOS = End of study * Instructions for arranging procedures / evaluations: Day 1 (D1) of any cycle is defined as the day on which ALMB-0168 is administered within a given cycle. The procedure / evaluation can be adjusted within + / - 3 days. The -7 day screening / baseline test and related evaluations can be completed within + / -7 days (from -14 day to -1 day). All pre-administration tests and other evaluations listed for D1 can be completed within 3 days prior to administration, with the exception that the pre-administration electrocardiogram for C1D1 should be completed on C1D1 at the time of the actual administration visit. • The listed visits, laboratory tests, and other evaluations for D2 and D1 of the subsequent cycle can be scheduled within + / - 3 days. • For days 8 and 15 of cycles 1 and 2, complete hospital visits and / or laboratory or other evaluations within a + / - 2 day timeframe. All imaging procedures, tumor assessments, and bone mineral density tests conducted in accordance with the SoA may be completed within a 5-day window from the specified date. • The final follow-up visit after the trial should be an in-person visit 28 days after the last treatment, and can be completed within a 3-day window of + / - from the designated date. a. Informed consent must be obtained ≤ 28 days before the start of the investigational treatment. b. Vital signs, including resting heart rate, respiration, blood pressure, and body temperature, will be measured at screening, C1D1, C1D8, C1D15, C2D1, C2D8, and C2D15, D1 of each subsequent cycle, and at the final visit of the study. The C1D1 physical examination may be omitted if it is expected to be performed within 72 hours of the baseline physical examination. c. A physical examination including the skin, mucous membranes, lymph nodes, head (including skull, ears, eyes, nose, mouth, and throat), neck, chest (including rib cage, breasts, lungs, and heart), abdomen (including liver, gallbladder, spleen, kidneys, bladder, stomach, and intestines), spine / limbs, and nervous system shall be performed at screening, C1D1, C1D8, C1D15, C2D1, C2D8, C2D15, D1 of each subsequent cycle, and at the final visit of the study. The C1D1 physical examination may be omitted if it is expected to be performed within 72 hours of the baseline physical examination. d. Height: Height will be measured only during screening. e. Weight: Weight will be measured during the screening period and on CXD1 (if weight measurement at screening is completed within 72 hours prior to administration, weight measurement is not required on C1D1). f. Echocardiography (ECHO): ECHO will be performed within 14 days prior to the start of the study treatment and as needed during the study. g. 12-lead electrocardiogram: Three 12-lead electrocardiograms should be performed: at screening, 30 minutes (±10 minutes) before and 30 minutes (±10 minutes) after administration of C1D1, at any point in time on C1D8 and C1D15, 30 minutes (±10 minutes) before and 30 minutes (±10 minutes) after administration of D1 in each subsequent cycle, upon arrival at the end of the study, and whenever clinical symptoms are observed. h. All medical adverse events (AEs) that occur from the time of signing the ICF until 28 days after the last dose will be collected for evaluation. Specific DLT evaluations will be performed from the start of medication until the end of C1 (C1D21), or before the scheduled C2D1 dose (i.e., C1D22). i. Complete blood count includes hematology and reticulocyte count. Hematology: Red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), white blood cell count (WBC), platelet count (PLT), and the number and percentage of neutrophils, lymphocytes, eosinophils, basophils, and monocytes. This is performed at screening, C1D1, C1D8, C1D15, C2D1, C2D8, C2D15, D1 of each subsequent cycle, and at the visit at the end of the study. If the C1D1 physical examination is expected to be performed within 72 hours after the baseline physical examination, it may be omitted. Samples for hematology are collected before each administration. j. Blood chemistry: Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), total bilirubin (TBIL), direct bilirubin (DBIL), total protein (TP), albumin (ALB), urea or blood urea nitrogen (BUN), creatinine (Cr), creatinine clearance (Ccr), glucose (Glu), potassium (K+), sodium (Na+), chloride (Cl-), calcium (Ca2+), magnesium (Mg2+), phosphorus (P), and carbon dioxide. Cardiac zymograms include creatine kinase (CK) and its isoenzyme (CK-MB). These are performed at screening, on C1D1, C1D8, C1D15, C2D1, C2D8, C2D15, D1 of each subsequent cycle, and at the end of the study visit. If the C1D1 physical examination is expected to take place within 72 hours of the baseline physical examination, it may be omitted. k. Coagulation markers (PT / aPTT / INR): Prothrombin time (PT), activated partial thromboplastin time (APTT), international normalized ratio (INR), thrombin time (TT), and plasma fibrinogen (Fbg) are tested at screening, before the D1 dose of each cycle, and at the end-of-study visit. If the C1D1 test time is within 72 hours after the baseline test, the C1D1 test is not required. The thrombin activator time / prothrombin time test at screening is performed at a local laboratory using standard methods. 1. Urinalysis: pH, specific gravity (SG), glucose (GLU), protein (PRO), red blood cells (RBC), white blood cells (WBC), ketones (KET), occult blood, bilirubin, nitrite, and urobilinogen. This is performed at screening, C1D1, C1D8, C1D15, C2D1, C2D8, and C2D15, D1 of each subsequent cycle, and at the final visit of the study. If the C1D1 physical examination is expected to be performed within 72 hours of the baseline physical examination, it may be omitted. m. Serum or urine pregnancy test: A serum pregnancy test is performed at baseline (completed within 7 days of the first dose, and if the baseline test is performed within 72 hours, a C1D1 test is not required), and a urine pregnancy test can be performed at other times. For women of potential pregnancy, a pregnancy test should be performed before the D1 dose of each cycle and at the end of the study visit. n. Viral serology: Tested at baseline. Hepatitis B testing (hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (HBsAb), e antigen (HBeAg), e antibody (HBeAb), core antibody (HBcAb), additional HBV-DNA testing in patients positive for hepatitis B surface antigen), hepatitis C (hepatitis C antibody, additional HCV-RNA testing in patients positive for HCV antibody), AIDS (anti-HIV antibody), syphilis (anti-Treponema pallidum specific antibody). o. PK blood sample collection time: 1. All participants in the escalation phase and the first 10 participants in the expansion phase will be sampled intensively. Intensive sampling times: Before administration (within 30 minutes) and immediately after administration (within 5 minutes), 2 hours (±15 minutes), 4 hours (±30 minutes), and 8 hours (±1 hour) after administration on C1D1; 24 hours (±2 hours) after administration on C1D2 and 48 hours (±2 hours) after administration on C1D3; 168 hours (±6 hours) after administration on C1D8 and 336 hours (±12 hours) after administration on C1D15; on D1 of each subsequent cycle, before administration (within 30 minutes) and immediately after administration (within 5 minutes); at the end of the study. 2. The remaining participants in the expansion phase will be sampled sparsely. Sparse sampling times: On D1 of each cycle, before administration (within 30 minutes) and immediately after administration (within 5 minutes); at the end of the study visit. 3. The number and timing of PK sampling may vary depending on the actual conditions of the study. If an infusion reaction or serious adverse event occurs, the principal investigator may, in cooperation with the laboratory on the same visit, test the collected blood and perform unscheduled PK sampling. p. Blood sample collection time for immunogenicity: D1 of cycles 1, 2, 3, 5, and 7, and before administration (within 30 minutes) at the end of the study (4 weeks after the last treatment). Samples will be used for anti-drug antibody (ADA) testing. q. Both RECIST and ICDS criteria will be used for response assessment in the study. Radiologists should be reminded that, in addition to size assessment, ICDS criteria will also be used for density assessment. If the principal investigator deems it necessary, imaging procedures other than those specified (such as PET-CT) may be performed and the response may be assessed according to RECIST criteria. r. All participants will visit the hospital every three months until 12 months after the last dose to follow up on their disease status, subsequent antitumor treatment, and survival status. These visits will have a time frame of ±7 days.

[0306] Response evaluation Tumor evaluation Appropriate imaging procedures in tumor-related anatomical regions include CT, MRI contrast-enhanced scans and / or PET-CT of the tumor-related site, CT or MRI of the brain, and technetium-99 isotope bone scans.

[0307] Results from participants within four weeks prior to drug administration are acceptable in tumor assessments performed during the screening period. During the screening period, the principal investigator should select an appropriate tumor assessment method according to the participant's condition, and tumor assessments during the follow-up period should be performed using the same method as the baseline assessment. During the study, the first tumor assessment will be performed at week 6 (end of C2) and every nine weeks thereafter (3 cycles). If clinical symptoms are found, additional scans may be performed during the study.

[0308] Response assessments are performed using both RECIST v1.1 (Appendix III) and ICDS criteria (Appendix VI). Radiologists should be reminded that, in addition to size assessment, ICDS criteria are also used for density assessment.

[0309] Research endpoints Overall response rate (ORR): Defined as the proportion of patients with the best response (complete response (CR) or partial response (PR) (i.e., CR+PR) from the start of ALMB-0168 treatment to the discontinuation of the study, as assessed according to the response evaluation criteria for solid tumors (RECIST v1.1, Appendix III; ICDS, detailed in Appendix VI). Participants initially assessed as CR or PR should be re-evaluated at least 4 weeks later.

[0310] Progression-free survival (PFS): The time from the start of ALMB-0168 to the first recorded date of progressive disease (PD) or death, whichever occurs first.

[0311] The 6-month progression-free survival rate (6m-PFSR) is defined as the percentage of participants who survive without PD within 6 months of the start of the ALMB-0168 study.

[0312] Time to Response (DoR): Defined as the time from the first assessment of complete response (CR) or partial response (PR) to the first day the participant shows evidence of tumor progression or dies.

[0313] Time to response (TTR): Defined as the time from the first dose to the first occurrence of an objective response (PR or CR).

[0314] Disease control rate (DCR): Defined as the percentage of evaluable participants who have a partial response (PR) + complete response (CR) after treatment and have stable disease (SD).

[0315] Overall survival (OS) is defined as the time from the start of the experimental treatment to death from any cause.

[0316] The rate of change from baseline in alkaline phosphatase (ALP) or lactate dehydrogenase (LDH).

[0317] Skeletal-related events (SREs) include changes from baseline in the number of pathological fractures, spinal cord compression, malignant hypercalcemia, disease-modifying radiotherapy, or surgeries for bone lesions.

[0318] Change from baseline in NRS score (see Appendix IV for details).

[0319] Change from baseline in the EQ-5D score (see Appendix V for details).

[0320] Bone mineral density of the lumbar spine, hip joint, femur, and neck was measured by dual-energy X-ray absorptiometry (DXA), and changes from baseline were collected.

[0321] Changes from baseline in the dose and frequency of morphine use.

[0322] The timing of evaluations of efficacy indicators, including ALP, LDH, SRE, NRS, EQ-5D, bone mineral density, and morphine use, is detailed in Table SoA.

[0323] Response criteria The principal investigator will perform imaging assessments during the screening period and each treatment cycle according to the Response Assessment Criteria for Solid Tumors (RECIST v1.1) and ICDS criteria. The same imaging techniques used at baseline should be used throughout the study, and lesions can be divided into measurable and unmeasurable lesions. If a participant's tumor-related symptoms worsen during treatment, tumor assessments may be performed in advance at the discretion of the principal investigator. For participants who discontinue treatment before progressive disease, tumor assessments will be performed every three months after the last dose.

[0324] Participants showing progressive disease on imaging tests should be removed from the study.

[0325] In rare cases, if the principal investigator believes the treatment will benefit the participant, a participant with radiographically confirmed disease progression (PD) may also continue with the study treatment. This should be discussed with the sponsor first.

[0326] If deemed necessary by the principal investigator, imaging procedures other than those specified (e.g., PET-CT) may be performed, response assessments may be conducted according to the PERCIST criteria, and the above data may be summarized for efficacy assessment.

[0327] Skeletal-related events (SREs), dosage and frequency of morphine use, NRS scores, and EQ-5D scores should be collected at each visit (see SOA for details).

[0328] Pharmacokinetics, immunogenicity, and biomarker evaluation PK index Blood samples for PK will be collected from the contralateral arm at the following times as specified in the State of Analysis (SoA). The number and timing of PK sampling may vary depending on the actual conditions of the study. In the event of an infusion reaction or serious adverse event, the principal investigator may, in cooperation with the same-visit laboratory, test the collected blood and perform unscheduled PK sampling.

[0329] All participants in the escalation phase and the first 10 participants in the expansion phase will be sampled intensively. Intensive sampling points: Before administration (within 30 minutes), immediately after administration (within 5 minutes), 2 hours (±15 minutes), 4 hours (±30 minutes), and 8 hours (±1 hour) after administration in C1D1; 24 hours (±2 hours) after administration in C1D2 and 48 hours (±2 hours) after administration in C1D3; 168 hours (±6 hours) after administration in C1D8, and 336 hours (±12 hours) after administration in C1D15. On D1 of each subsequent cycle, before administration (within 30 minutes) and immediately after administration (within 5 minutes); at the end of the study visit. 2. The remaining participants in the expansion phase will be sampled sparsely. Sparse sampling points: On D1 of each cycle, before administration (within 30 minutes) and immediately after administration (within 5 minutes); at the end of the study visit.

[0330] PK index:C 最大 , t 最大 AUC 0-t , C トラフ , and CL, V z , λ z , t 1 / 2 AUC0-inf (Regarding participants who received only concentrated sampling).

[0331] Immunogenicity index To assess the immunogenicity of ALMB-0168, blood samples should be tested for anti-drug antibodies. Samples from participants for immunogenicity / drug concentration analysis may also be used to assist the sponsor or its nominees in assessing safety or efficacy, addressing issues arising during or after the trial, or evaluating relevant biomarkers.

[0332] Blood sample collection timing: Pre-administration (within 30 minutes) on Day 1 of cycles 1, 2, 3, 5, and 7, and at the end of the test (4 weeks after the last treatment). The sample will be used for the anti-drug antibody (ADA) test.

[0333] A blood sample for determining the anti-ALMB-0168 antibody (ADA) is collected from the contralateral arm at the time specified in the State of Analysis (SoA).

[0334] Statistical considerations This study lacks a formal statistical hypothesis, and the results of the statistical analysis are preliminary.

[0335] Sample size estimation The Phase I portion of the study consists of accelerated titration and a standard "3+3" dose escalation. 1–6 participants are enrolled in the first two dose groups, and 3–6 participants are enrolled in the third and subsequent dose groups, with up to 7 dose groups including 10 participants receiving MTD levels. Dose groups that complete the DLT observation period may be expanded to a maximum of 8 participants (excluding the MTD expansion cohort) after the principal investigator and sponsor evaluate previous clinical data and analyze participant benefits. Therefore, the maximum number of participants should not exceed 58.

[0336] Up to 180 participants will be enrolled in the Phase II dose expansion, according to the final number of participants.

[0337] Analysis group Full Analysis Set (FAS): Includes all participants who received at least one dose of ALMB-0168 after successful enrollment according to the Basic Intention to Treat (ITT) principle. The FAS will be used for demographic and baseline characterization analyses.

[0338] Protocol Set (PPS): Defined as a subset of participants in the Full Analysis Set (FAS). Participants who meet the inclusion and exclusion criteria have at least one post-baseline tumor assessment, have 80%–120% medication compliance, and have no material violations of the clinical trial protocol.

[0339] Pharmacokinetic analysis set (PKS): Participants who have received at least one dose of ALMB-0168 and have at least one measurable plasma concentration. Reasons for excluding a participant or a participant's plasma concentration from the PKS include, but are not limited to, the following factors that affect plasma concentration (e.g., incorrect treatment, concomitant medication), inappropriate treatment of blood samples, and inaccurate blood sample testing.

[0340] Safety Analysis Set (SS): The SS includes all participants who receive at least one dose of ALMB-0168 and undergo safety evaluation. Unless otherwise specified, all analyses are based on the SS. The SS set is defined as being equivalent to the FAS set.

[0341] Anti-drug antibody set (ADA set, ADAS): Participants receiving at least one dose of ALMB-0168 for which ADA data is available.

[0342] Efficacy evaluable analysis set (EES): Includes all participants who received at least one dose of the study drug and underwent efficacy evaluation at least once after administration.

[0343] Pharmacokinetic concentration set (PKCS): This includes all participants who have received at least one dose of the study drug and have at least one evaluable plasma concentration data.

[0344] Pharmacokinetic parameter set (PKPS): This includes all participants who receive at least one dose of the study drug and have at least one evaluable PK parameter.

[0345] Immunogenicity analysis set (IS): This includes all participants who have received at least one dose of the study drug, have evaluable immunogenicity data at baseline, and have at least one evaluable immunogenicity data after baseline.

[0346] Biomarker analysis set: This includes all enrolled participants who have received at least one dose of the investigational drug and have at least one biomarker data.

[0347] statistical analysis A detailed summary of the data collected from this study and the methods for statistical analysis are included in the Statistical Analysis Plan (SAP). The SAP is developed after the protocol and CRF are completed and completed before the database lock. The SAP specifies and describes in detail all planned statistical analyses based on the main features of the protocol. Any changes to the protocol that have a significant impact on the Statistical Analysis Plan, at the discretion of the sponsor or principal investigator, will require a new revision of the SAP to align with the study protocol.

[0348] For statistical descriptions, categorical variables are represented by the number of participants and their percentages (%). Continuous variables are represented by the number of participants, missing values, mean, standard deviation, median, quartiles, minimum, and maximum values.

[0349] Regarding participant placement, the registration, medication administration, dropout or exclusion of participants in each group, and dataset splitting will be summarized and presented along with a participant placement flowchart.

[0350] Statistically describe demographic data and baseline characteristics. For continuous variables in baseline data, calculate the number of participants, mean, standard deviation, median, minimum, and maximum. For count and ranking data, calculate the number of participants and percentage. Detailed participant demographic data, baseline characteristics, past medical history, and treatment history are presented in tables.

[0351] Pharmacokinetics and immunogenicity 1. Pharmacokinetic analysis The mean (mean ± SD) dose-time curves for ALMB-0168 are plotted for each dose group. Individual PK parameters of participants are listed, and descriptive statistical analysis (mean, median, range, standard deviation, coefficient of variation, etc.) is performed on the PK parameters.

[0352] PK parameter: C 最大 , t 最大 , C トラフ , and CL, Vz, λz, t 1 / 2 AUC 0-t AUC 0-inf (For participants who underwent concentrated sampling only)

[0353] 2. Immunogenicity analysis: Summarize and present the number and percentage of ADA-positive participants.

[0354] Safety analysis 1. AEs and drug-related AEs. For the analysis of adverse events (AEs) and drug-related AEs, summarize the number of participants in each group, the number of AEs, and the percentages of AEs, drug-related AEs, AEs leading to study termination, AEs leading to death, and SAEs.

[0355] The organ-specific broad categories (SOCs) and priority terms (PTs) of AEs and drug-related AEs are coded according to the MedDRA dictionary, and the number of participants, the number of AEs, and the percentage of AEs and drug-related AEs are summarized by SOC / PT.

[0356] The number of participants, the number of AEs, and the percentage of AEs and drug-related AEs are classified by SOC / PT and severity. Each participant's AE class is counted only once under the highest severity level during the same period (SOC or PT).

[0357] All adverse events (AEs), including those occurring during non-medication periods, drug-related AEs, SAEs, AEs leading to trial termination, AEs leading to death, and adverse events of special interest (AESIs) are listed separately.

[0358] 2. Laboratory Tests Baseline and time points after the last visit. Measured values ​​at each time point, minimum and maximum values ​​of measurements after baseline, observed values ​​at the last visit, and changes from baseline should be statistically analyzed according to dose group and total.

[0359] The normal and abnormal changes in each indicator before and after treatment will be compared through cross-tabulation of clinical judgments before and after treatment. The laboratory test indicators will be listed in detail for each group.

[0360] 3. Vital signs The statistical time points for vital sign testing include baseline, each follow-up visit after baseline, and the follow-up visit for early discontinuation. Descriptive statistical results at baseline, follow-up visits after baseline, and the end of the study are statistically analyzed by group and total, and then normal and abnormal changes in each indicator before and after treatment are compared using pre- and post-treatment clinical judgment cross-tabulations (where applicable).

[0361] 4. Physical examination and electrocardiogram Physical examination items: general condition, skin, head and neck, chest, abdomen, back, limbs, nerves, and mental state. Normal and abnormal changes in each dose group before and after treatment will be compared through cross-tabulation of pre- and post-treatment clinical judgments.

[0362] Electrocardiogram (ECG) testing includes heart rate, PR, QRS complex, and QTc. Statistical analysis of baseline, each visit after baseline, minimum and maximum values ​​of measurements after baseline, observations at the last visit, and changes from baseline is performed by dose group and total. Normal and abnormal changes in each dose group before and after treatment are compared through cross-tabulation of pre- and post-treatment clinical judgments.

[0363] A detailed list of physical examinations and electrocardiogram tests will be compiled for each group.

[0364] 5. ECOG score ECOG score statistics are compiled at baseline, each visit after baseline, and the final visit. Descriptive statistics are performed at baseline, at visits after baseline, and at the end of treatment for the cohort, and changes in ECOG scores before and after treatment are compared by cross-tabulation.

[0365] Effectiveness analysis The response can be evaluated as PD, SD, CR, and PR according to RECIST V1.1 and ICDS criteria. Specific analytical indicators include, but are not limited to, the following. • Overall Response Rate (ORR): Summarizes the number and percentage of participants who had an objective tumor response (CR or PR). • Overall Survival (OS): This refers to the time from randomization to death from any cause, or to the last follow-up visit for patients who failed to be followed up; or, for participants who are still alive at the end of the study, to the end of follow-up. The last contact date is the most recent date on which a participant is known to be alive. • Progressive disease-free survival (PFS): Defined as the time from the first dose to the onset of progressive disease or death, whichever comes first. For participants without progressive disease or death, the date of the last tumor assessment is used as the endpoint, and their data are treated as censored values. • 6-month progression-free survival rate (6m-PFSR): The percentage of participants who survived without PD within 6 months of the start of the ALMB-0168 program. • Time to response (TTR): Defined as the time from the first dose to the first occurrence of an objective tumor response (CR or PR). • DoR (Time to Response): The time from the first assessment of complete response (CR) or partial response (PR) to the first day the participant shows evidence of tumor progression or dies. • Disease control rate (DCR): Defined as the percentage of evaluable participants who have a partial response (PR) + complete response (CR) after treatment and have stable disease (SD). Participant responses are evaluated according to RECIST 1.1 and ICDS criteria, ORR and DCR are calculated, and 95% confidence intervals (CIs) are calculated for ORR and DCR using the Clopper-Pearson exact method. DoR, PFS and TTR are estimated using the Kaplan-Meier method, and the median DoR, PFS, TTR, censorship, quartiles, and their 95% CIs are listed, and Kaplan-Meier curves are plotted. • Analysis of Numerical Rating Scale (NRS) scores: Record the NRS score and the change from baseline at each visit. • European Quality of Life Scale 5-Dimensional (EQ-5D) Score: Record the EQ-5D score and the change from baseline at each visit. If deemed necessary by the principal investigator, imaging procedures other than those specified in the protocol (e.g., PET-CT) may be performed, response assessments may be conducted according to the PERCIST criteria, and these data may be included and summarized for efficacy assessment.

[0366] Note Appendix I: ECOG Performance Status Scale The ECOG performance status scale is shown in Table 5.

[0367] [Table 6]

[0368] Appendix II: New York Heart Association (NYHA) Functional Classification Table 6 shows the New York Heart Association's functional cardiac classification.

[0369] [Table 7]

[0370] Excerpt from Appendix III of the response evaluation criteria for solid tumors (RECIST 1.1) RECIST criteria history Assessing changes in tumor burden is a crucial feature of the clinical evaluation of malignant tumor treatment. Both tumor reduction (objective response) and time to progression are important endpoints for decision-making in clinical research on malignant tumors. Years of research evidence supporting tumor reduction as an endpoint in phase II trials for screening new antitumor agents have been found. These studies suggest that, for various solid tumors, drugs that contribute to tumor reduction in some patients are likely (though imperfect) to improve overall patient survival or have other opportunities to enter event evaluation in randomized phase III studies. Currently, objective response is more reliable than any other biomarker in evaluating efficacy in phase II screening studies. Furthermore, in phase II and III clinical trials in patients with advanced tumors, time to progression (or progression-free survival) is increasingly being used as an endpoint for determining efficacy, and this is also based on anatomical measurements of tumor size.

[0371] However, the two endpoints for determining tumor size—objective response and time to progression—are only valuable if they are supported by criteria that are widely accepted and readily usable based on the anatomical structure of tumor volume. Tumor response criteria were first published by the World Health Organization (WHO) in 1981 and are primarily used in trials where tumor response is the primary endpoint. The WHO criteria introduce the concept of evaluating the response to treatment by performing a holistic assessment of tumor volume by calculating the sum of the two-dimensional sizes of the lesion and the change from baseline during treatment. However, for more than a decade after the publication of the criteria, oncology groups and pharmaceutical companies using the criteria often modified them to adapt to new technologies or to suggest ambiguities in the original literature, which led to confusion in the interpretation of study results. In fact, the application of various response criteria resulted in widely varying treatment outcomes for the same treatment method. In response to these problems, an international working group was established in the mid-19th century to standardize and simplify the response criteria. The new criteria, also known as RECIST (Response Evaluation Criteria in Solid Tumors), were published in 2000. Key features of the early version of RECIST included the definition of the smallest measurable lesion size, a description of the number of lesions to be followed (up to 10; up to 5 per organ), and an overall assessment of tumor volume using one-dimensional (instead of two-dimensional) measurements. These criteria have been widely adopted by academic groups, oncology groups, and the pharmaceutical industry, with the primary endpoint of the criteria being objective response or progressive disease. Furthermore, the NMPA has accepted RECIST as an appropriate standard for these assessments.

[0372] Purpose of the guidelines This guideline describes standard measurement methods for solid tumors in clinical studies of malignancies in adults and children and defines objective criteria for determining changes in tumor size. Since all measures of effectiveness are based on the assessment of anatomical tumor volume and its changes in the study, these criteria are expected to be used in all studies with objective response as the primary study endpoint, and in studies that take stable disease, tumor progression, or analysis progression time as indicators. The proportion of patients meeting the criteria for endpoints predicting the effectiveness of a drug or treatment plan is not assumed in this paper. These definitions depend on the type of malignancy in the ongoing study and the specific drug being studied. The study protocol must include an appropriate statistical section defining the number of samples for the study and the effectiveness parameters on which the inclusion criteria are based. This guideline not only provides definitions and criteria for determining tumor response but also presents proposals for criteria reporting of clinical study outcomes where tumor response is considered the study endpoint.

[0373] These guidelines can be used in research on malignant brain tumors, but criteria for evaluating responses in this field are also published separately. International guidelines for evaluating responses in lymphoma are also published separately, so these guidelines are not used in research on malignant lymphoma.

[0374] Finally, many oncologists rely on repeated imaging studies to track patients' tumor lesions in their daily clinical practice and determine further treatment plans based on objective indicators and symptoms. These RECIST guidelines should not generally be used for clinical decision-making unless the oncologist deems them appropriate for treatment decisions.

[0375] Baseline tumor measurement At baseline, tumor lesions / lymph nodes are classified as measurable and unmeasurable, as follows:

[0376] 1. Measurable lesions Neoplastic lesion: A tumorous lesion has at least one accurately measurable diameter (recorded as the longest diameter of the measuring surface), and its minimum length is as follows: • For computed tomography (CT), the slice thickness is 10 mm (CT slice thickness is 5 mm or less). • 10 mm measured with a caliper for clinical testing (lesions that cannot be accurately measured with a caliper should be recorded as unmeasurable). • For chest X-rays, 20 mm.

[0377] Malignant lymph nodes: When evaluated by CT (CT slice thickness of 5 mm or less is recommended), the short axis of the lymph node must reach 15 mm in order to be considered pathologically measurable with magnification. Only the short axis length should be measured and tracked preoperatively and for follow-up.

[0378] 2. Unmeasurable lesions All other lesions include small lesions (maximum diameter less than 10 mm, or pathological lymph node short axis less than 10 mm to 15 mm) and truly unmeasurable lesions. Lesions considered truly measurable include leptomeningeal lesions, ascites, pleural or pericardial effusion, inflammatory chest disease, lymphatic infiltration of skin / pulmonary tissue, peritoneal dissemination, and intra-abdominal masses / abdominal organ enlargements that cannot be confirmed by repeated imaging techniques and physical examination.

[0379] 3. Special considerations regarding measurable lesions Bone lesions, cystic lesions, and locally treated lesions require particular attention, as outlined below. Bone lesions: Bone scans, positron emission tomography (PET) scans, or simple radiographs are not considered appropriate imaging techniques for measuring bone lesions. However, these techniques can be used to confirm the presence or absence of bone lesions. Regarding osteolytic lesions or mixtures of osteolytic and osteogenic lesions that have defined soft tissue components, if the soft tissue components meet the above definition of measurability, they can be evaluated as measurable lesions using tomographic imaging techniques such as CT or MRI. • Osteogenesis lesions are unmeasurable. Cystic lesions: • Lesions that meet the criteria for a simple cyst on radiographic imaging are defined as simple cysts and should not be considered malignant (neither measurable nor unmeasurable). If cystic lesions represent cystic metastatic lesions and satisfy the measurability defined above, they can be considered measurable. However, if non-cystic lesions are present in the same patient, the non-cystic lesions should be preferred as target lesions.

[0380] Locally treated lesions: Tumor lesions located in previously irradiated areas or areas treated with other local therapies are generally considered unmeasurable lesions unless they show obvious progression. Study protocols should detail the conditions under which such lesions are considered measurable.

[0381] Measurement method specifications 1. Measurement of lesions When clinically evaluated, all measurements should be recorded in metric units using a caliper. All baseline evaluations should be performed as close to the start of treatment as possible, no more than four weeks prior.

[0382] 2.Measurement method Each lesion identified and reported at baseline and follow-up should be documented using the same decision-making methods and techniques. Imaging tests (instead of clinical tests) should generally be performed unless the lesion is found to be unsuitable for imaging during follow-up.

[0383] Clinical lesions: Only superficial lesions (e.g., subcutaneous nodules) with caliper measurements of 10 mm or more in diameter are considered measurable. In the case of skin lesions, it is recommended to use color photographs that measure the percentage of lesion size. As mentioned above, when a lesion can be examined both clinically and by imaging, imaging should be used because the imaging evaluation is more objective and can be used for the final review of the clinical study.

[0384] Chest X-ray: Compared to chest X-ray, chest CT is preferred, especially when progressive disease is used as an important endpoint, because CT scans are more sensitive than X-rays in identifying new lesions. However, if X-rays reveal a clear lesion boundary surrounded by an inflated lung, it can be considered measurable.

[0385] CT and MRI: CT is currently the most effective and repeatable testing method for evaluating lesion response. As defined by guidelines, the CT slice thickness should be 5 mm or less relative to the measurable lesion. If the CT slice thickness exceeds 5 mm, the smallest measurable lesion should be twice the slice thickness. MRI can also be used in certain situations (such as whole-body scans).

[0386] Ultrasonography: Ultrasonography is not suitable for evaluating lesion size and should not be applied as a measurement method. Ultrasonography cannot be perfectly reproduced between two adjacent observations. Furthermore, the results depend on the examiner. Technique and measurement results cannot be guaranteed to be the same in one test and the next. If a new lesion is detected by ultrasound during the course of the study, verification by CT or MRI is recommended. If CT radiation exposure is a concern, MRI can be used instead to detect the lesion being examined.

[0387] Endoscopy and laparoscopy: These techniques are not recommended for objective tumor evaluation. However, they are useful in confirming a complete pathological response by biopsy or in determining a complete response or recurrence after surgical resection.

[0388] Tumor Markers: Tumor markers cannot be used alone to assess the objective response of a tumor. However, if a tumor marker begins to rise above the upper limit of normal, it must be reduced to the normal range when used to determine the patient's full response. Since tumor markers are disease-specific, indicators for testing for specific diseases should be included in the protocol. Specific guidelines have been published regarding changes in CA-125 (ovarian cancer recurrence) and changes in PSA (prostate cancer recurrence). Furthermore, the Gynecologic Cancer Intergroup has developed the CA-125 progression criterion for clinical trials using objective tumor assessment indicators for first-line treatment of ovarian cancer.

[0389] Cytology, Histology: These techniques may often be used to distinguish between partial and complete responses in individual cases when required by clinical research protocols (e.g., residual benign tumor lesions in germ cell tumors). If exudation is known to be a potential adverse consequence of treatment (e.g., certain paclitaxel-based chemotherapy agents or angiogenesis inhibitors), measurable tumor lesions meeting the criteria for response or stable disease require cytological confirmation of tumor exudation that occurs or worsens during treatment to distinguish between response (or stable disease) and progressive disease.

[0390] Tumor response assessment 1. Evaluation of all tumors and measurable lesions. To assess objective response or potential future progression, baseline assessment of total tumor burden in all tumor lesions is necessary as a reference for subsequent measurements. In clinical protocols with objective response as the primary treatment endpoint, only patients with measurable lesions at baseline are enrolled. Measurable lesions are defined as the presence of at least one measurable lesion. For studies using progressive disease (time to progression or degree of progression at a fixed date) as the primary treatment endpoint, the protocol inclusion criteria must clarify whether enrollment is limited to patients with measurable lesions or whether patients without measurable lesions may also be enrolled.

[0391] 2. Baseline recording of target and non-target lesions If more than one measurable lesion is present at baseline assessment, all lesions should be recorded and measured, with a total of no more than five target lesions (no more than two per organ) representing all involved organs (i.e., patients with only one or two cumulative organs should have a maximum of two or four target lesions as baseline measurable lesions).

[0392] The target lesion must be selected based on lesion size (maximum diameter), represent all involved organs, and the measurement must be repeatable. If the largest lesion cannot be repeatedly measured, another largest lesion that can be repeatedly measured should be selected.

[0393] Lymph nodes can still be imaged in a normal anatomical structure without tumor metastasis and therefore require special attention. Pathological lymph nodes, defined as measurable nodules or target lesions, must meet the following criteria: Short diameter ≥ 15 mm as measured by CT. At baseline, only the short diameter should be detected. Radiologists typically use the short diameter of the nodule to determine whether tumor metastasis is present. Nodule size is generally represented by two-dimensional data from imaging studies (axial plane is common for CT scans, while axial, sagittal, and coronal planes are selected for MRI). The short diameter is determined by the minimum value adopted. For example, a 20 mm × 30 mm abdominal nodule with a short-axis diameter of 20 mm can be considered a malignant measurable nodule. In this example, 20 mm is the measured value of the nodule. Nodules with a diameter of 10 mm or more but less than 15 mm should not be considered target lesions. Nodules smaller than 10 mm are not considered pathological and should not be recorded or further observed.

[0394] The calculated sum of the diameters of all target lesions (including the maximum diameter of non-nodular lesions and the short-axis diameter of nodular lesions) is the sum of baseline diameters. If lymph node diameters are included, only the short axis is included, as described above. The sum of baseline diameters is used as the baseline disease criterion.

[0395] All remaining lesions, including pathological lymph nodes, should be considered non-target lesions without measurement, but should be recorded at baseline assessment as follows: “Present,” “Absent,” or, in the case of a small number, “Obvious Progression.” Extensive non-target lesions in the same organ, such as numerous enlarged pelvic lymph nodes or extensive liver metastases, may be recorded as a single item in the case report.

[0396] 3. Response evaluation criteria 3.1. Evaluation of response to target lesions Complete response (CR): All target lesions are absent, and the short-axis diameter of any pathological lymph node (whether it is a target lesion or not) must be <10 mm.

[0397] Partial response (PR): The sum of the diameters of all target lesions decreases by at least 30% compared to the sum of baseline diameters.

[0398] Progressive disease (PD): The sum of the diameters of all target lesions increases by at least 20% compared to the sum of the diameters of the smallest lesions studied (including the sum of baseline lesion diameters, if small). In addition to a 20% relative increase, the sum of diameters must also show an absolute increase of at least 5 mm (Note: The presence of one or more new lesions may also be considered progressive).

[0399] Stable disease (SD): Based on the sum of the diameters of the smallest lesions in the study, a decrease in lesions is not a partial response (PR), and an increase in lesions is not a progressive disease (PD).

[0400] 3.2. Considerations for evaluating the response to target lesions If the target lesion is located in a lymph node: Even if lymph nodes resolve to less than 10 mm in the study, the measured short-axis diameter should be recorded (on the same anatomical surface as the baseline measurement). This means that if a target lesion is present in a lymph node, a lymph node with a short-axis diameter < 10 mm is considered normal, and therefore the sum of the target lesion diameters will not be zero even if the full response criteria are met. Case report forms or other data collection methods may be designed to record nodular target lesions separately. For a full response, each node must have a short-axis diameter of less than 10 mm. For PR, SD, and PD, the sum of the target lesion diameters includes the actual short-axis diameter of the nodules.

[0401] If the target lesion is too small to be measured: For all lesions (nodular and non-nodular) recorded at baseline in the study, their actual measurements must be recorded in subsequent evaluations, even if the lesions are very small (e.g., 2 mm). However, some lesions or lymph nodes recorded as target lesions at baseline may have signals too weak on the CT scan to be accurately measured by the radiologist, and they may be reported as too small to be measured. In this case, it is important to record one measurement in the case report. If the radiologist determines that the lesion may disappear, the measurement may be recorded as 0 mm. If a lesion is present with a weak signal, a default value of 5 mm should be assigned (Note: This rule does not apply to normal lymph nodes, as normal lymph nodes usually have a definable size and are often surrounded by fat, such as in the retroperitoneum; however, if a lymph node is present with a signal too weak to be measured, a default value of 5 mm should also be assigned). The default value of 5 mm is based on the thickness of the CT tomography scan (the default value of 5 mm does not change with this thickness). Measurements of such lesions (too small to measure) may lack reproducibility, and default values ​​should be assigned to prevent erroneous responses or misdiagnosis due to measurement errors. Even in these cases, if a radiologist can provide actual measurements, even if they are less than 5 mm, these should be recorded.

[0402] When the lesions split or merge during treatment If non-nodular lesions are separated, the maximum diameters of all fragments must be summed for the total diameter of the target lesion. Similarly, when lesions merge, the long axis diameter can be retained, which helps to obtain the maximum diameter of the individual lesions before merging. If the lesions have merged completely to the point where they can no longer be separated, the vector of the maximum diameter in this case should be the maximum diameter of the merged lesion.

[0403] 3.3. Evaluation of non-target lesions This section defines the criteria for the response to non-target lesion tumors. While some non-target lesions are actually measurable, they do not need to be measured; they only need to be qualitatively assessed at the points specified in the protocol.

[0404] Complete response (CR): All non-target lesions resolve, and tumor markers return to normal. All lymph nodes are non-pathological in size (short diameter < 10 mm).

[0405] Incomplete response / non-progressive disease (non-CR or non-PD): One or more non-target lesions are present and / or tumor markers are consistently above normal levels.

[0406] Progressive disease (PD): Existing non-target lesions show clear progression. Note: The presence of one or more new lesions is also considered a progressive disease.

[0407] 3.4. Considerations for evaluating the progression of non-target lesions The definition of progression of non-target lesions is explained further below.

[0408] If the patient has measurable lesions: In this case, due to the apparent progression of non-target lesions, the non-target lesions worsen to the extent that treatment must be discontinued. Even if the target lesion is assessed as stable disease or partial response, the total tumor volume increases sufficiently to support discontinuation of treatment. Mild increases in one or more non-target lesions are usually not sufficient to be considered apparent progression. In cases of stable disease or partial response of the target lesion, it is extremely rare for total tumor progression to be defined solely by changes in non-target lesions.

[0409] If none of the patient's non-target lesions are measurable: This occurs when the inclusion criteria do not specify that measurable lesions must be present in some Phase III trials. The overall assessment is still based on the above criteria, but in this case there is no measurable data for the lesions. Worsening of non-target lesions is not easy to assess (by definition: all lesions are truly unmeasurable). Therefore, if changes in non-target lesions result in an increase in overall disease burden equivalent to the development of progressive disease in target lesions, clear progression should be defined based on non-target lesions, and effective trials for assessment should be established. For example, an increase in tumor volume represents a further 73% increase in volume (this corresponds to a 20% increase in the diameter of a measurable lesion). Another example is peritoneal exudation ranging from "minimal" to "large." Lymphoid lesions ranged from "local" to "extensive." Or, "evidence-based changes in treatment" as described in the protocol. In cases of clear progression, the patient should generally be considered to have had progressive disease at that point. Ideally, there would be unmeasurable lesions that can be evaluated by objective criteria, but there may be lesions that are inherently not objectively evaluable and therefore must be significantly enlarged.

[0410] 3.5. New lesions The presence of new malignant lesions indicates a progressive disease. Therefore, several comments regarding new lesions are important. There are no specific criteria for identifying new lesions through imaging. However, the discovery of new lesions should be obvious. For example, progression should not be attributed to differences in imaging techniques, changes in imaging morphology, or other lesions other than tumors (e.g., some so-called new bone lesions are simply healed original lesions or recurrent original lesions). It is important that the patient's baseline lesions show a partial or complete response. For example, necrosis of a liver lesion may be recorded on a CT report as a new cystic lesion, even though it is not actually necrotic.

[0411] Lesions detected during follow-up and not identified at baseline are considered new and suggest progressive disease. For example, in a patient with visceral lesions identified at baseline, if metastases are found on a head CT or MRI, intracranial metastases are considered the basis for progressive disease, even if the patient did not undergo a head examination at baseline.

[0412] If a new lesion is suspected due to its small size, continued treatment and follow-up evaluation are necessary to confirm whether it is indeed a new lesion. If repeated examinations confirm that it is clearly a new lesion, the time to progression should be calculated from the time it was first discovered.

[0413] Since FDG-PET evaluation generally requires additional tests for confirmation, it is appropriate to combine FDG-PET findings with CT findings to assess progression (especially for newly suspected diseases). New lesions can be identified by FDG-PET examination following the procedure below.

[0414] Negative FDG-PET results at baseline and positive FDG-PET results during follow-up suggest a progressive disease.

[0415] There was no FDG-PET at baseline, but a positive FDG-PET result was obtained during follow-up.

[0416] If a new lesion identified by positive FDG-PET during follow-up is confirmed by CT, it indicates the presence of a progressive disease.

[0417] If a new lesion identified by positive FDG-PET during follow-up is not confirmed by CT, an additional CT scan is required for confirmation (if confirmed, the time to progression should be calculated from the time the abnormality was detected in the early FDG-PET scan).

[0418] If a positive FDG-PET scan during follow-up confirms an existing lesion identified by CT, and the lesion has not progressed at the time of imaging, then there is no progressive disease.

[0419] 4. Evaluation of the best overall response The assessment of the best overall response is a record of the best response from the start to the end of the trial, taking into account any necessary conditions for confirmation. In some cases, the response may not be confirmed until the end of treatment, and therefore the protocol should specify whether the response assessment at the end of treatment is included in the assessment of the best overall response. The protocol must specify how any new treatment preceding progression will affect the best response. The patient's best response depends primarily on the outcomes of targeted and non-targeted lesions, as well as the presence or absence of new lesions, and the nature of the study, protocol requirements, and outcome measures. Specifically, in non-randomized trials where response is the primary endpoint, confirmation of PR or CR is necessary to determine which is the best overall response.

[0420] 4.1. Response assessments are assumed to be performed at each specific time point of the protocol. Table 1 provides a summary of the overall response at each time point for patients with measurable disease at baseline.

[0421] If the patient does not have a measurable lesion (i.e., no target lesion), the evaluation can be found in Table 2.

[0422] 4.2. (Explanation of cases where evaluation is omitted or impossible) If lesions are not imaged / measured at a specific point in time, the patient is unassessable at that time. If only partial lesions can be assessed at one time, the case is usually unassessable at that time unless a convincing argument can be made that the missing lesions do not affect the response assessment at that point in time. This is more likely to occur in the context of progressive disease. For example, if a patient has three lesions with a total diameter of 50 mm at baseline, and later only two lesions with a total diameter of 80 mm are evaluable, the patient will be assessed as having progressive disease, regardless of the impact of the missing lesions.

[0423] 4.3. Best Overall Response: All Points When all information about the patient is available, the best overall response can be determined.

[0424] Assessment of the best overall response when confirmation of a complete or partial response is not required in the study: The best response in the study is the best response at all time points. For example, if a patient has SD in the first cycle, PR in the second cycle, and PD in the final cycle, the best overall response is PR. If the best overall response is SD, it must also meet the protocol-specified minimum time from baseline. If the minimum time criterion is not met, the best overall response of SD is not accepted and will be determined by subsequent assessment. For example, if a patient has SD in the first cycle and PD in the second cycle, but the minimum time for SD is not met, the best overall response is PD. Similarly, a patient who was assessed as SD in the first cycle and then became unfollowed is considered unassessable.

[0425] Assessment of the best overall response when confirmation of a complete or partial response is required in the study: A complete or partial response may only be confirmed if each participant meets the criteria for a partial or complete response specified in the study, and the response is confirmed at a subsequent point in time (generally 4 weeks later) as specified in the protocol. In this case, a description of the best overall response is provided in Table 3.

[0426] 4.4. Special notes regarding response evaluation If nodular lesions are included in the overall assessment of the target lesions, and the nodules decrease to a "normal" size (<10 mm), scan reports regarding lesion size still exist. To avoid overestimation, measurements are recorded even if the nodules are normal, as reflected by an increase in nodule size. As mentioned earlier, this means that no 0s are recorded in the CRF for participants with a complete response.

[0427] When response confirmation is required during a study, repeated "unmeasurable" time points complicate the best response assessment. The analytical design for the study must address how responses are judged based on these missing data. For example, in most studies, a participant's PR-NE-PR response can be considered confirmed.

[0428] Symptom progression should be reported when a participant experiences an overall worsening that necessitates discontinuation of treatment without objective evidence. Objective progression should be assessed as much as possible, even after discontinuation of treatment. Symptom worsening is not an objective responder, but it is a reason for discontinuing treatment. The objective responder in such participants is assessed by the description of target and non-target lesions, as shown in Tables 1-3.

[0429] Cases defined as early progression, early death, or unassessable are exceptions to the trial and should be clearly stated in each protocol (depending on the treatment interval and treatment cycle).

[0430] In some cases, it can be difficult to distinguish local lesions from normal tissue. If the assessment of complete response is based on such a definition, it is recommended that biopsy be performed before assessing the complete response of the local lesion. If abnormal imaging results of local lesions in some participants are thought to indicate fibrosis or scar formation of the lesion, FDG-PET should be used as an evaluation criterion, as with biopsy, to confirm the complete response. In this case, the use of FDG-PET should be pre-documented in a protocol supported by reports from the relevant medical literature. However, it must be recognized that both FDG-PET and biopsy can produce false positive results in the assessment of complete response due to their own limitations (including resolution / sensitivity).

[0431] [Table 8]

[0432] In the case of SD, at least one post-registration follow-up measurement must meet the SD criteria, and follow-up and registration should be separated by at least 6-8 weeks.

[0433] [Table 9] Note: For non-target lesions, "non-CR / non-PD" refers to a response superior to SD. Since SD is increasingly used as an endpoint for response assessment, a non-CR / non-PD response is established when the lesion cannot be measured.

[0434] In cases of ambiguous findings regarding progression (e.g., very small, uncertain new lesions; cystic changes or necrosis in existing lesions), treatment may be continued until the next evaluation. If progressive disease is confirmed at the next evaluation, the date of progression should be the date of previously suspected progression.

[0435] [Table 10] Note: CR = complete response, PR = partial response, SD = stable disease, PD = progressive disease, NE = unassessable. Superscript "a": If CR actually occurs at the first time point and any disease occurs at a subsequent time point, the response is still PD at the subsequent time point (because the disease reappears after CR), even if the participant meets the PR criteria relative to baseline. The best response depends on whether SD occurs within the shortest treatment interval. However, sometimes the response is assessed as CR at the first time point, but a scan at a subsequent time point suggests a small lesion, and therefore, in reality, the participant's response should be PR rather than CR at the first time point. In this case, the CR assessed at the first time point should be changed to PR, and the best response should be PR.

[0436] 5. Frequency of tumor re-evaluation The frequency of tumor reassessment during treatment should depend on the treatment plan and be consistent with the type and schedule of treatment. However, in Phase II trials where the benefit of treatment is unknown, follow-up every 6-8 weeks (timed to coincide with the end of the cycle) is reasonable, and the length of the interval may be adjusted in special plans or cases. The protocol should specify which tissue sites should be evaluated at baseline (usually those most likely to be involved in metastases of the tumor type under study) and how often the evaluation should be repeated. Typically, target and non-target lesions are evaluated at each time point. In some arbitrary cases, certain non-target lesions may be evaluated less frequently. For example, bone scans may only need to be repeated if complete response (CR) is identified in the target disease or if progression of bone lesions is suspected.

[0437] At the end of treatment, tumor reassessment depends on whether response rate or time to event (progression / death) is used as the endpoint of the clinical study. For time to event (e.g., TTP / DFS / PFS), routine repeated assessments as defined in the protocol are required. In randomized controlled studies, in particular, planned assessments should be outlined in the schedule (e.g., 6-8 weeks after treatment or 3-4 months post-treatment) and should not be affected by any other events that may cause disparities in treatment groups in terms of treatment delays, dosing intervals, and timing of disease assessment.

[0438] 6. Response evaluation / Confirmation of response period 6.1. Confirmation In non-randomized clinical trials where response is the primary endpoint, confirmation of PR and CR is necessary to ensure that the response is not a result of evaluation error. This also allows for a sound interpretation of the results in the context of historical data, but responses documented in the historical data of these studies should also be confirmed. However, in all other cases, such as randomized trials (Phase II or Phase III), or in trials where a stable or progressive disease is the primary endpoint, response confirmation is no longer necessary as it has no value in interpreting the trial results. However, eliminating the need for response confirmation makes central reviews to prevent offsets even more important, especially in open-label studies.

[0439] For SD (Stable Decline), at least one measurement should meet the SD criteria specified in the protocol within the shortest possible time interval after the start of the study (generally 6-8 weeks or more).

[0440] 6.2. The total response period is calculated from the time when the CR or PR criterion is first met (whichever was measured first) until when relapse or progressive disease is objectively recorded (the smallest measured value recorded in the study is used as the criterion for progressive disease). The total complete response period is calculated from the time when the CR criterion is first met until when relapse or progressive disease is objectively recorded.

[0441] 6.3. Period of stable illness This is calculated from the start of treatment (or from the time of randomization in a randomized trial) to the progression of the disease by referring to the sum of the minimum measurements in the trial (if the baseline sum is the minimum, this is used as the reference for the PD calculation). The clinical relevance of the duration of stable disease varies by study and disease. If the proportion of patients who achieve stable disease over the minimum duration is the study endpoint in a particular study, the protocol should specify the minimum time interval between two measurements, as defined in the SD.

[0442] Note: Response time, stable disease duration, and PFS are influenced by the frequency of follow-up after baseline assessment. Defining a standard frequency of follow-up is not within the scope of these guidelines. The frequency of follow-up should be determined based on disease classification and staging, treatment cycle, standard practice, etc. When inter-study comparisons are necessary, the limitations of the precision of measured endpoints should be taken into consideration.

[0443] 7. Progression-free survival (PFS) / progression-free survival (PPF) rate This guideline focuses on the use of objective response as a trial endpoint in Phase II clinical trials. In some cases, response rates may not be optimal for evaluating the potential anticancer activity of a new drug / program. In these cases, PFS / PPF at a delimited time point may be considered a suitable substitute for providing an early signal of the biological activity of the new drug. However, in uncontrolled studies, these evaluations are clearly questionable, as seemingly valuable observations may be related to biological factors such as patient screening rather than the effect of the drug intervention. Therefore, Phase II clinical trials using these study endpoints are best designed with randomized controls. However, since the clinical manifestations of some tumors are always consistent (generally always poor), non-randomized studies are reasonable. However, in these cases, because there is no positive control, evidence of efficacy should be carefully documented when evaluating expected PFS or PPF.

[0444] Note: Age is in years, and weight is in kilograms (kg).

[0445] Appendix IV Numerical Rating Scale (NRS) Scores A score of 0-10 represents different degrees of pain. Ask the patient to rate their pain level by marking a number or circling the diagram that best represents their pain level.

[0446] 0: No pain. 1-3: Mild pain (does not affect sleep). 4-6: Moderate pain. 7-9: Severe pain (inability to fall asleep or waking up during sleep due to pain). 10: Smart.

[0447] Different degrees of pain are also shown in Figure 1.

[0448] Appendix V: European Health-Related Quality of Life Questionnaire 5-Dimensional (EQ-5D) The EQ-5D format is shown below. Record the patient's name, randomization number, date of visit, and corresponding medication prescribed at the time of visit. The relevant scales are shown in Figure 2.

[0449] [Table 11]

[0450] Appendix VI: ICDS (Modified Reverse Choy Density / Size Criteria) Evaluation Criteria

[0451] [Table 12] This should be evaluated by contrast-enhanced spiral CT or MRI. CT attenuation of each tumor should be measured in Hunsfield units (HU) during the portal venous phase. SLD represents the sum of the longest diameters.

[0452] Example 3. A multicenter, single-arm, open-label Phase I / II clinical trial to evaluate the safety and efficacy of ALMB-0168 in patients with osteosarcoma - Results Twenty-four participants were enrolled to receive the humanized monoclonal antibody (ALMB-0168) involved at doses of 1 mg / kg, 3 mg / kg, 6 mg / kg, 12 mg / kg, 18 mg / kg, 24 mg / kg, 30 mg / kg, or 36 mg / kg every three weeks. Baseline characteristics are shown in Table 12.

[0453] Of the 24 participants, 3 were not evaluated and dropped out of the study, 2 experienced symptom progression, and 19 were evaluable (2 PR, 8 SD, and 9 PD). The disease progression of the 19 participants is shown in Figure 3. The best change in the determined target lesion diameter for the 19 participants is shown in Figure 4.

[0454] Regarding EES, the overall response rate was 10.5% (2 / 19), and the DCR was 52.6% ((2+8) / 19). Table 13 shows the efficacy data for the dose groups: 1 mg / kg (1 PD), 3 mg / kg (1 PD), 6 mg / kg (1 PR, 2 SD), 12 mg / kg (1 SD, 1 PD, 1 NE), 18 mg / kg (1 PR, 1 SD, 1 PD), 24 mg / kg (1 SD, 2 PD), 30 mg / kg (3 PD, 1 SD, 1 NE), and 36 mg / kg (2 PD, 1 SD, 1 NE).

[0455] None of the 24 participants experienced DLT events. A total of 80 drug-related adverse events were observed, of which anemia (9 participants, 11.25%) and proteinuria (8 participants, 10.00%) had an incidence of ≥10%. There was only one treatment-related adverse event of grade ≥3, and there were no serious drug-related adverse events. Adverse events that occurred during treatment are summarized in Table 14.

[0456] According to pharmacokinetic data, exposure to ALMB-0168 in participants after a single infusion increased proportionally with the dose within the dose range of 1–36 mg / kg, with a mean peak concentration (C 最大The effective dose ranges from approximately 19.7 to 691 μg / mL, and the mean area under the drug-time curve (AUC) is approximately 19.7-691 μg / mL. 0-t ) is approximately 4365-123795h * The concentration was μg / mL. ALMB-0168 was slowly excreted in vivo, with a mean excretion half-life of approximately 8.54–14.8 days and a mean excretion rate of approximately 8.75–13.0 ml / h. This resulted in a limited tissue distribution with a mean apparent distribution volume of approximately 3.55–4.88 L.

[0457] [Table 13]

[0458] [Table 14]

[0459] [Table 15]

[0460] Participant S01001 received chemotherapy after wide resection of osteosarcoma in the left lower femur and for lung metastases. Following progression, Participant S01001 received systemic chemotherapy with a different drug and palliative resection of lung metastases. Participant S01001 received third-line chemotherapy and palliative resection of metastases in the right lower femur. Following fourth-line chemotherapy, osteosarcoma recurred and progressed in the right femur. Participant met the protocol inclusion criteria and participated in the clinical trial of ALMB-0168. Participant S01001 was enrolled in September 2021 and treated with ALMB-0168. The right femoral lesion continued to decrease (Figures 5 and 6B-6F), Hu levels continued to increase (Figure 7), and the PR response was the best. This participant was treated with ALMB-0168 for more than 18 months in the study.

[0461] Regarding participant S02007, she had undergone surgery and chemotherapy for right distal femoral osteosarcoma; one year later, she underwent surgical resection of a tibial metastasis and progressed after chemotherapy for a pulmonary lesion. This participant met the protocol inclusion criteria, participated in the clinical trial of this drug, and achieved the best partial response (PR). At baseline, participant S02007 had lesion 1 with a major diameter of 31.9 mm (Figure 8A, left panel) and lesion 2 with a major diameter of 19.4 mm (Figure 8B, left panel), totaling 51.3 mm. After treatment with ALMB-0168 in this study, lesion 1 decreased to 20.9 mm (Figure 8A, right panel) and lesion 2 decreased to 14.1 mm (Figure 8B, left panel), totaling 35.0 mm, which is a 31.8% reduction from baseline and represents the best partial response.

[0462] These data demonstrate that ALMB-0168 has remarkably good efficacy and acceptable safety in patients with metastatic or unresectable osteosarcoma after failure to standard treatment in a Phase I / II dose-escalation trial.

[0463] [Table 16-1]

[0464] [Table 16-2]

[0465] [Table 17-1]

[0466] [Table 17-2]

Claims

1. A method for treating bone cancer in a subject requiring treatment, comprising administering at least one dose of an anti-connexin 43 (Cx43) antibody or its antigen-binding fragment to the subject, wherein the anti-Cx43 antibody is HCDR amino acid sequence: HCDR1: Sequence ID 1; HCDR2: Sequence ID No. 2; HCDR3: Sequence ID 3 Heavy chain variable region including, LCDR amino acid sequence: LCDR1: Sequence ID 4; LCDR2: Sequence ID 5; LCDR3: Sequence ID 6 A method comprising a light chain variable region including a light chain.

2. The method according to claim 1, wherein the at least one dose of the anti-Cx43 antibody or its antigen-binding fragment is about 0.001 mg / kg to about 300 mg / kg.

3. The method according to claim 1 or 2, wherein the at least one dose of the anti-Cx43 antibody or its antigen-binding fragment is about 1 mg / kg, about 3 mg / kg, about 6 mg / kg, about 12 mg / kg, about 18 mg / kg, about 24 mg / kg, about 30 mg / kg, or about 36 mg / kg.

4. The method according to any one of claims 1 to 3, wherein the anti-Cx43 antibody or its antigen-binding fragment is administered approximately once a day to approximately once every 12 months.

5. The method according to any one of claims 1 to 4, wherein the anti-Cx43 antibody or its antigen-binding fragment is administered approximately once every two weeks, approximately once every three weeks, or approximately once every four weeks.

6. The method according to any one of claims 1 to 5, wherein the anti-Cx43 antibody or its antigen-binding fragment is administered once every three weeks.

7. The method according to any one of claims 1 to 6, wherein the anti-Cx43 antibody or its antigen-binding fragment is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, or subcutaneously.

8. The method according to any one of claims 1 to 7, wherein the anti-Cx43 antibody or its antigen-binding fragment is administered intravenously.

9. The method according to claim 8, wherein the anti-Cx43 antibody or its antigen-binding fragment is administered within approximately 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, 150 minutes, 180 minutes or longer.

10. The method according to any one of claims 1 to 9, wherein the anti-Cx43 antibody or its antigen-binding fragment is administered in a pharmaceutically acceptable composition.

11. The method according to any one of claims 1 to 10, wherein the anti-Cx43 antibody or its antigen-binding fragment comprises a heavy chain variable region containing an amino acid sequence according to SEQ ID NO: 7, and / or a light chain variable region containing an amino acid sequence according to SEQ ID NO:

8.

12. The method according to any one of claims 1 to 11, wherein the anti-Cx43 antibody comprises a heavy chain having an amino acid sequence according to any one of SEQ ID NOs: 9 and 11 to 13, and / or a light chain having an amino acid sequence according to SEQ ID NO:

10.

13. The method according to any one of claims 1 to 12, wherein the anti-Cx43 antibody is a humanized antibody.

14. The method according to any one of claims 1 to 13, wherein the anti-Cx43 antibody or its antigen-binding fragment enhances the opening of the Cx43 hemichannel in the target.

15. The method according to any one of claims 1 to 14, wherein the bone cancer is osteosarcoma.

16. The aforementioned osteosarcoma, (i) Resectable or unresectable; (ii) Stage I-A, Stage I-B, Stage II-A, Stage II-B, Stage III, Stage IV-A, or Stage IV-B; (iii) Low grade or high grade; and / or The method according to claim 15, wherein (iv) primary osteosarcoma, osteoblastic osteosarcoma, chondroblastic osteosarcoma, fibroblastic osteosarcoma, small cell osteosarcoma, telangiectatic osteosarcoma, paraosteal osteosarcoma, periosteal osteosarcoma, typical osteosarcoma, osteoblastic osteosarcoma sclerosing osteosarcoma, chondroblastomatous osteosarcoma, chondromyxoid fibrous osteosarcoma, clear cell osteosarcoma, malignant fibrous histiocytomatous osteosarcoma, giant cell osteosarcoma, and / or epithelioid osteosarcoma.

17. The method according to any one of claims 1 to 16, wherein the subject has failed to treat with at least one standard treatment for osteosarcoma, including chemotherapy, surgery, immunotherapy, or radiotherapy, or a combination thereof.

18. The method according to any one of claims 1 to 17, wherein the subject has failed to receive treatment with methotrexate, doxorubicin, cisplatin, and ifosfamide, apatinib, analotinib, vindesine, vincristine, docetaxel, paclitaxel, irinotecan, bortezomib, albumin-conjugated paclitaxel, nedaplatin (Aqpla), pemetrexed, etoposide, gemcitabine, lovaplatin, recombinant human endostatin, eribulin, dacarbazine, pazopanib, immune checkpoint inhibitors, surgery, or radiotherapy, or a combination thereof.

19. After treatment with the anti-Cx43 antibody or its antigen-binding fragment, the subject, (i) progression-free survival of at least one month, two months, three months, at least four months, at least five months, or at least six months; (ii) Improvement in overall survival of at least one month, at least two months, at least three months, at least four months, at least five months, or at least six months; (iii) Improvement of EQ-5D score; (iv) Improvement of disease progression as assessed by the PERCIST criteria, RECIST criteria, and / or ICDS criteria; (v) Improvement in pain score as assessed by NRS; and / or (vi) A reduction of at least 1%, at least 2%, at least 3%, at least 5%, at least 8%, at least 10%, at least 15%, at least 20%, or at least 30% in tumor size. A method according to any one of claims 1 to 18, which achieves the objective.

20. The method according to any one of claims 1 to 19, wherein the subject achieves a complete or partial response to the treatment with the anti-Cx43 antibody or its antigen-binding fragment.

21. A method for treating bone cancer in a subject requiring treatment, comprising administering to the subject at least one dose of an anti-connexin 43 (Cx43) antibody or an antigen-binding fragment thereof, wherein the at least one dose of the anti-Cx43 antibody is approximately 0.01 mg / kg to approximately 300 mg / kg.

22. The method according to claim 21, wherein the at least one dose of the anti-Cx43 antibody or its antigen-binding fragment is about 1 mg / kg, about 3 mg / kg, about 6 mg / kg, about 12 mg / kg, about 18 mg / kg, about 24 mg / kg, about 30 mg / kg, or about 36 mg / kg.

23. The method according to claim 21 or 22, wherein the anti-Cx43 antibody or its antigen-binding fragment is administered approximately once a day to approximately once every 12 months.

24. The method according to any one of claims 21 to 23, wherein the anti-Cx43 antibody or its antigen-binding fragment is administered approximately once every two weeks, approximately once every three weeks, or approximately once every four weeks.

25. The method according to any one of claims 21 to 24, wherein the anti-Cx43 antibody or its antigen-binding fragment is administered once every three weeks.

26. The method according to any one of claims 21 to 25, wherein the anti-Cx43 antibody or its antigen-binding fragment is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally.

27. The method according to any one of claims 21 to 26, wherein the anti-Cx43 antibody or its antigen-binding fragment is administered intravenously.

28. The method according to claim 27, wherein the anti-Cx43 antibody or its antigen-binding fragment is administered within approximately 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, 150 minutes, 180 minutes, or longer.

29. The method according to any one of claims 21 to 28, wherein the anti-Cx43 antibody or its antigen-binding fragment is administered in a pharmaceutically acceptable composition.

30. The anti-Cx43 antibody or its antigen-binding fragment, HCDR amino acid sequence: HCDR1: Sequence ID 1; HCDR2: Sequence ID No. 2; HCDR3: Sequence ID 3 Heavy chain variable region including; and LCDR amino acid sequence: LCDR1: Sequence ID 4; LCDR2: Sequence ID 5; LCDR3: Sequence ID 6 The method according to any one of claims 21 to 29, comprising a light chain variable region including a light chain variable region.

31. The method according to any one of claims 21 to 30, wherein the anti-Cx43 antibody or its antigen-binding fragment comprises a heavy chain variable region containing an amino acid sequence according to SEQ ID NO: 7, and / or a light chain variable region containing an amino acid sequence according to SEQ ID NO:

8.

32. The method according to any one of claims 21 to 31, wherein the anti-Cx43 antibody comprises a heavy chain having an amino acid sequence according to any one of SEQ ID NOs: 9 and 11 to 13, and / or a light chain having an amino acid sequence according to SEQ ID NO:

10.

33. The method according to any one of claims 21 to 32, wherein the anti-Cx43 antibody is a humanized antibody.

34. The method according to any one of claims 21 to 33, wherein the anti-Cx43 antibody or its antigen-binding fragment enhances the opening of the Cx43 hemichannel in the target.

35. The method according to any one of claims 21 to 34, wherein the bone cancer is osteosarcoma.

36. The aforementioned osteosarcoma is (i) Resectable or unresectable; (ii) Stage I-A, Stage I-B, Stage II-A, Stage II-B, Stage III, Stage IV-A, or Stage IV-B; (iii) Low grade or high grade; and / or The method according to claim 35, wherein (iv) primary osteosarcoma, osteoblastic osteosarcoma, chondroblastic osteosarcoma, fibroblastic osteosarcoma, small cell osteosarcoma, telangiectatic osteosarcoma, paraosteal osteosarcoma, periosteal osteosarcoma, typical osteosarcoma, osteoblastic osteosarcoma sclerosing osteosarcoma, chondroblastomatous osteosarcoma, chondromyxoid fibrous osteosarcoma, clear cell osteosarcoma, malignant fibrous histiocytomatous osteosarcoma, giant cell osteosarcoma, and / or epithelioid osteosarcoma.

37. The method according to any one of claims 21 to 36, wherein the subject has failed to treat with at least one standard treatment for osteosarcoma, including chemotherapy, surgery, radiotherapy, or immunotherapy, or a combination thereof.

38. The method according to any one of claims 21 to 37, wherein the subject has failed to receive treatment with methotrexate, doxorubicin, cisplatin, and ifosfamide, apatinib, analotinib, vindesine, vincristine, docetaxel, paclitaxel, irinotecan, bortezomib, albumin-conjugated paclitaxel, nedaplatin (Aqpla), pemetrexed, etoposide, gemcitabine, lovaplatin, recombinant human endostatin, eribulin, dacarbazine, pazopanib, immune checkpoint inhibitors, surgery, or radiotherapy, or a combination thereof.

39. After treatment with the anti-Cx43 antibody or its antigen-binding fragment, the subject: (i) progression-free survival of at least one month, two months, three months, at least four months, at least five months, or at least six months; (ii) Improvement in overall survival of at least one month, at least two months, at least three months, at least four months, at least five months, or at least six months; (iii) Improvement of EQ-5D score; (iv) Improvement of disease progression as assessed by the PERCIST criteria, RECIST criteria, and / or ICDS criteria; (v) Improvement in pain score as assessed by NRS; and / or (vi) A reduction in tumor size of at least 1%, at least 2%, at least 3%, at least 5%, at least 8%, at least 10%, at least 15%, at least 20%, or at least 30% A method according to any one of claims 21 to 38, which achieves the objective.

40. The method according to any one of claims 21 to 39, wherein the subject achieves a complete or partial response to the treatment with the anti-Cx43 antibody or its antigen-binding fragment.

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