Methods of treating bone cancer

Abstract

The present disclosure provides compositions and methods for treating osteosarcoma, such as osteosarcoma in a patient who has previously received at least one first-tier osteosarcoma treatment but has failed treatment. The methods comprise promoting the opening of Cx43 hemichannels in bone cells by, for example, using a composition comprising an anticonnexin 43 antibody.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 504,162, filed May 24, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Merging of sequence lists

[0004] This application contains a sequence list, which has been submitted through the Patent Center. The sequence list, named 172628-202002_PCT_SL.xml, was created on May 8, 2024, and is 14,456 bytes in size, and is incorporated herein by reference in its entirety. Background Technology

[0005] Osteosarcoma is the most common primary malignant bone tumor, originating from primitive bone-forming mesenchymal stem cells. Osteosarcoma exhibits a bimodal age distribution, with the first peak in adolescents aged 10-14 years and the second peak in adults over 65 years of age. Its incidence is generally higher in men than women, at 5.4 per million per year for men and 4.0 per million per year for women, and higher in Black (6.8 per million) and Hispanic (6.5 per million) individuals than in White (4.6 per million).

[0006] Osteosarcoma typically occurs near the metaphyseal growth plates of the long bones of the limbs. 8.9% of all childhood and adolescent cancer deaths are due to malignant tumors of the bone and joints. The 5-year survival rate for osteosarcoma is 68%, with no significant gender difference. The expected 5-year survival rate for metastatic patients is 15% to 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-Bielack et al., J Clin Oncol., 23(3): 559-568 (2005)). Tumor stage, metastasis, local recurrence, chemotherapy regimen, anatomical location, tumor size, and tumor cell destruction rate after neoadjuvant chemotherapy are all associated with treatment outcomes (Ottaviani et al., Cancer Treat Res., 152: 3-13 (2009)).

[0007] For high-grade osteosarcoma (including intramedullary and superficial types), preoperative chemotherapy (Grade 1A) is recommended, followed by reassessment and restaging via chest X-ray, local X-ray, PET, or bone scan. For resectable tumors, wide resection should be performed. Chemotherapy should continue when the resection margins are negative and the response to chemotherapy is good; the chemotherapy regimen may be modified when the response is poor. When the resection margins are positive and the response to chemotherapy is good, chemotherapy should continue, and other local treatments (surgery, radiotherapy, etc.) may be administered concurrently; the chemotherapy regimen may be modified when the response is poor, and other local treatments (surgery, radiotherapy, etc.) should be administered concurrently. For unresectable tumors, only radiotherapy and chemotherapy are considered. Continuous monitoring of patients is necessary after treatment.

[0008] Osteocytes highly express the connectant protein Cx43 in gap junction channels and hemichannels. Gap junction channels mediate cell communication between two adjacent cells to maintain normal cellular metabolic function, while hemichannels allow molecular exchange between the cell and its extracellular environment. Hemichannels are normally closed, but under certain conditions, such as mechanical stress or local ischemia, cytokine or ion stimulation, they can be activated and opened. Open hemichannels allow small molecules (<1.2 kDa) such as ATP, NAD+, glutamate and inositol triphosphate (IP3) to pass through (Goodenough et al., Nature reviews Molecular cellbiology, 4(4): 285-294 (2003)). The opening of osteocyte Cx43 hemichannels has been shown to strongly inhibit the growth, migration and bone metastasis of breast cancer tumors (Zhou et al., Oncogene, 35(43): 5597-5607 (2016)).

[0009] Currently, first-line chemotherapy drugs for osteosarcoma include methotrexate, doxorubicin, cisplatin, and ifosfamide. According to the CSCO (Chinese Society of Clinical Oncology) recommendations in 2020, since there are no second-line treatment regimens demonstrating overall survival benefit, participating in clinical trials is an opportunity for osteosarcoma patients who have failed first-line treatment to achieve better efficacy or be retreated. Literature has reported that in clinical trials, a drug is considered effective if it provides a response rate >5% or a 4-month progression-free survival rate >40%. Systemic chemotherapy is the primary treatment for advanced osteosarcoma, and no major breakthroughs have been achieved in its treatment over the past few decades. To date, there remains a significant and unmet need for drug treatment of advanced osteosarcoma. Summary of the Invention

[0010] One aspect of this disclosure provides a method of treating bone cancer in a subject of need, comprising administering to the subject at least one dose of an anti-connector protein 43 (Cx43) antibody or an antigen-binding fragment thereof, the anti-Cx43 antibody comprising: a heavy chain variable region comprising the following amino acid sequences: an HCDR1 amino acid sequence having SEQ ID NO: 1, an HCDR2 amino acid sequence having SEQ ID NO: 2, and an HCDR3 amino acid sequence having SEQ ID NO: 3; and a light chain variable region comprising the following amino acid sequences: an LCDR1 amino acid sequence having SEQ ID NO: 4, an LCDR2 amino acid sequence having SEQ ID NO: 5, and an LCDR3 amino acid sequence having SEQ ID NO: 6.

[0011] In some embodiments, 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. In some specific 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.

[0012] In some embodiments, the anti-Cx43 antibody or its antigen-binding fragment is administered at a frequency of approximately once daily to approximately once every 12 months. In some specific embodiments, the anti-Cx43 antibody or its antigen-binding fragment is administered at a frequency of approximately once every 2 weeks, approximately once every 3 weeks, or approximately once every 4 weeks. In one embodiment, the anti-Cx43 antibody or its antigen-binding fragment is administered at a frequency of approximately once every 3 weeks.

[0013] In some embodiments, the anti-Cx43 antibody or its antigen-binding fragment is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, or subcutaneously. In one specific embodiment, the anti-Cx43 antibody or its antigen-binding fragment is administered intravenously.

[0014] In some implementations, the anti-Cx43 antibody or its antigen-binding fragment is administered over a period of approximately 5 minutes, approximately 10 minutes, approximately 15 minutes, approximately 20 minutes, approximately 25 minutes, approximately 30 minutes, approximately 40 minutes, approximately 50 minutes, approximately 60 minutes, approximately 70 minutes, approximately 80 minutes, approximately 90 minutes, approximately 100 minutes, approximately 110 minutes, approximately 120 minutes, approximately 150 minutes, approximately 180 minutes, or longer.

[0015] In some embodiments, the anti-Cx43 antibody or its antigen-binding fragment is administered in a pharmaceutically acceptable composition. In some embodiments, the composition comprises a pharmaceutically acceptable carrier.

[0016] In some embodiments, the anti-Cx43 antibody or its antigen-binding fragment comprises a heavy chain variable region having the amino acid sequence according to SEQ ID NO: 7, and / or a light chain variable region having the amino acid sequence according to SEQ ID NO: 8.

[0017] In some embodiments, the anti-Cx43 antibody comprises a heavy chain having an amino acid sequence according to any one of SEQ ID NO: 9 and 11-13, and / or a light chain having an amino acid sequence according to SEQ ID NO: 10.

[0018] In some implementations, the anti-Cx43 antibody is a humanized antibody.

[0019] In some implementations, anti-Cx43 antibodies or their antigen-binding fragments enhance the opening of Cx43 hemichannels in subjects.

[0020] In some implementations, bone cancer is osteosarcoma.

[0021] In some implementations, osteosarcoma is resectable, while in others it is unresectable.

[0022] In some implementations, osteosarcoma is classified as stage IA, stage IB, stage II-A, stage II-B, stage III, stage IV-A, or stage IV-B.

[0023] In some implementations, osteosarcoma is low-grade, while in others it is high-grade.

[0024] In some implementation schemes, osteosarcoma is defined as primary osteosarcoma, osteoblastic osteosarcoma, chondrogenic osteosarcoma, fibroblastic osteosarcoma, small cell osteosarcoma, capillary dilatational osteosarcoma, periosteal osteosarcoma, periosteal osteosarcoma, classic osteosarcoma, osteoblastic osteosarcoma-sclerosing osteosarcoma, chondroblastoma-like osteosarcoma, chondromycinoid fibrosarcoma-like osteosarcoma, clear cell osteosarcoma, malignant fibrous histiocytoma-like osteosarcoma, giant cell rich osteosarcoma, and / or epithelioid osteosarcoma.

[0025] In some implementation schemes, the subjects had previously received at least one standard treatment for osteosarcoma, including chemotherapy, surgery, immunotherapy, radiation therapy, or a combination thereof, but the treatment failed.

[0026] In some implementation schemes, the subjects had previously failed treatment with methotrexate, doxorubicin, cisplatin and ifosfamide, apatinib, anlotinib, vindesin, vincristine, docetaxel, paclitaxel, irinotecan, bortezomib, albumin-bound paclitaxel, nedaplatin (Aqupla), pemetrexed, etoposide, gemcitabine, lobaplatin, recombinant human endostatin, eribulin, dacarbazine, pazopanib, immune checkpoint inhibitors, surgery, radiotherapy, or combinations thereof.

[0027] In some implementations, after treatment with anti-Cx43 antibody or its antigen-binding fragment, the subject achieves: (i) progression-free survival of at least 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months; (ii) improvement in overall survival of at least 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months; (iii) improvement in EQ-5D score; (iv) improvement in disease progression as assessed by PERCIST, RECIST, 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%, 2%, 3%, 5%, 8%, 10%, 15%, 20%, or 30%.

[0028] In some implementation schemes, the subjects achieved complete or partial remission after treatment with the anti-Cx43 antibody.

[0029] Another aspect of this disclosure provides a method for treating bone cancer in a subject in need, comprising administering to the subject at least one dose of an anti-connector protein 43 (Cx43) antibody or an antigen-binding fragment thereof, wherein the at least one dose of the anti-Cx43 antibody is from about 0.01 mg / kg to about 300 mg / kg.

[0030] In some implementations, 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.

[0031] In some embodiments, the anti-Cx43 antibody or its antigen-binding fragment is administered at a frequency of approximately once daily to approximately once every 12 months. In some specific embodiments, the anti-Cx43 antibody or its antigen-binding fragment is administered at a frequency of approximately once every 2 weeks, approximately once every 3 weeks, or approximately once every 4 weeks. In one embodiment, the anti-Cx43 antibody or its antigen-binding fragment is administered at a frequency of approximately once every 3 weeks.

[0032] In some embodiments, the anti-Cx43 antibody or its antigen-binding fragment is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally. In one embodiment, the anti-Cx43 antibody or its antigen-binding fragment is administered intravenously.

[0033] In some implementations, the anti-Cx43 antibody or its antigen-binding fragment is administered over a period of approximately 5 minutes, approximately 10 minutes, approximately 15 minutes, approximately 20 minutes, approximately 25 minutes, approximately 30 minutes, approximately 40 minutes, approximately 50 minutes, approximately 60 minutes, approximately 70 minutes, approximately 80 minutes, approximately 90 minutes, approximately 100 minutes, approximately 110 minutes, approximately 120 minutes, approximately 150 minutes, approximately 180 minutes, or longer.

[0034] 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 comprises a pharmaceutically acceptable carrier.

[0035] In some embodiments, the anti-Cx43 antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising: an HCDR1 amino acid sequence having SEQ ID NO: 1, an HCDR2 amino acid sequence having SEQ ID NO: 2, and an HCDR3 amino acid sequence having SEQ ID NO: 3; and a light chain variable region comprising: an LCDR1 amino acid sequence having SEQ ID NO: 4, an LCDR2 amino acid sequence having SEQ ID NO: 5, and an LCDR3 amino acid sequence having SEQ ID NO: 6.

[0036] In some embodiments, the anti-Cx43 antibody or its antigen-binding fragment comprises a heavy chain variable region having the amino acid sequence according to SEQ ID NO: 7, and / or a light chain variable region having the amino acid sequence according to SEQ ID NO: 8.

[0037] In some embodiments, the anti-Cx43 antibody comprises a heavy chain having an amino acid sequence according to any one of SEQ ID NO: 9 and 11-13, and / or a light chain having an amino acid sequence according to SEQ ID NO: 10.

[0038] In some implementations, the anti-Cx43 antibody is a humanized antibody.

[0039] In some implementations, anti-Cx43 antibodies or their antigen-binding fragments enhance the opening of Cx43 hemichannels in subjects.

[0040] In some implementations, bone cancer is osteosarcoma.

[0041] In some implementations, osteosarcoma is resectable, while in others it is unresectable.

[0042] In some implementations, osteosarcoma is classified as stage IA, stage IB, stage II-A, stage II-B, stage III, stage IV-A, or stage IV-B.

[0043] In some implementations, osteosarcoma is low-grade; while in others, it is high-grade.

[0044] In some implementation schemes, osteosarcoma is defined as primary osteosarcoma, osteoblastic osteosarcoma, chondrogenic osteosarcoma, fibroblastic osteosarcoma, small cell osteosarcoma, capillary dilatational osteosarcoma, periosteal osteosarcoma, periosteal osteosarcoma, classic osteosarcoma, osteoblastic osteosarcoma-sclerosing osteosarcoma, chondroblastoma-like osteosarcoma, chondromycinoid fibrosarcoma-like osteosarcoma, clear cell osteosarcoma, malignant fibrous histiocytoma-like osteosarcoma, giant cell rich osteosarcoma, and / or epithelioid osteosarcoma.

[0045] In some implementation schemes, the subjects had previously received at least one standard treatment for osteosarcoma, including chemotherapy, surgery, radiation therapy, immunotherapy, or a combination thereof, but the treatment failed.

[0046] In some implementation schemes, the subjects had previously failed treatment with methotrexate, doxorubicin, cisplatin and ifosfamide, apatinib, anlotinib, vindesin, vincristine, docetaxel, paclitaxel, irinotecan, bortezomib, albumin-bound paclitaxel, nedaplatin (Aqupla), pemetrexed, etoposide, gemcitabine, lobaplatin, recombinant human endostatin, eribulin, dacarbazine, pazopanib, immune checkpoint inhibitors, surgery, radiotherapy, or combinations thereof.

[0047] In some implementations, after treatment with anti-Cx43 antibody or its antigen-binding fragment, the subject achieves: (i) progression-free survival of at least 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months; (ii) improvement in overall survival of at least 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months; (iii) improvement in EQ-5D score; (iv) improvement in disease progression as assessed by PERCIST, RECIST, 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%, 2%, 3%, 5%, 8%, 10%, 15%, 20%, or 30%.

[0048] In some implementations, treatment with the anti-Cx43 antibody or its antigen-binding fragment resulted in complete or partial remission in the subject. Attached Figure Description

[0049] Figure 1 The pain level is shown on a digital rating scale.

[0050] Figure 2 The VAS scale of the EQ-5D questionnaire is shown.

[0051] Figure 3 This is a lane diagram showing the investigator-determined overall efficacy for the 24 enrolled participants. Participant S02001 received 1 mg / kg ALMB-0168 per dose; participant S02002 received 3 mg / kg ALMB-0168 per dose; participants S01001, S01002, and S02003 received 6 mg / kg ALMB-0168 per dose; participants S02006 and S06001 received 12 mg / kg ALMB-0168 per dose; participants S02007, S06002, and S06008 received 18 mg / kg ALMB-0168 per dose; participants S02010, S09001, and S11003 received 24 mg / kg ALMB-0168 per dose; and participants S02012, S06004, S01003, and S03001 received 30 mg / kg ALMB-0168 per dose. ALMB-0168; Participants S01004 and S06005 received 36 mg / kg ALMB-0168 per dose; an equilateral triangle indicates partial remission (PR), a circle indicates stable disease (SD); a diamond indicates disease progression (PD); a right-pointing triangle indicates the trial is in progress; a cross indicates the study has ended (EOS); a pentagon indicates the start of a new anticancer treatment; a plus sign indicates death.

[0052] Figure 4 It is a waterfall plot, showing the optimal change of the target lesion relative to the baseline as determined by the researchers (evaluable efficacy analysis set).

[0053] Figure 5 The left knee prosthesis was visible at baseline in participant S01001; recurrent metastases were observed in the proximal right femur.

[0054] Figures 6A to 6F This is a cross-sectional image of participant S01001 along its major axis: the baseline image acquired on October 28, 2021. Figure 6A ), and 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 6FThe first five tumor evaluation images were collected. The participant achieved partial remission according to RECIST 1.1 and had increased bone mineral density.

[0055] Figure 7 Images of participant S01001 are shown: the baseline image acquired on October 28, 2021 (sub-image 1), and four tumor evaluation images acquired on December 20, 2021 (sub-image 2), February 23, 2022 (sub-image 3), April 19, 2022 (sub-image 4), and August 29, 2022 (sub-image 5). Sub-image 1: Lesion mean size approximately 187.45 cm², standard deviation approximately 128.3, area 11.02 cm². 2 The maximum length is ≥706 units, and the minimum length is ≤-252 units; the second subplot shows that the average lesion size is approximately 476.07 units, the standard deviation is approximately 292.39 units, and the area is 6.9 cm². 2 Maximum length ≥ 1391 units, minimum length ≤ -252 units; Third subplot: lesion average size approximately 796.28 units, standard deviation approximately 457 units, area 5.621 cm². 2 The maximum length is ≥1954 units, and the minimum length is ≤-344 units; Fourth subfigure: The average size of the lesion is approximately 1009.3 units, the standard deviation is approximately 398.84 units, and the area is 5.533 cm². 2 Maximum length ≥ 1697 units, minimum length ≤ -115 units; Fifth subfigure: Lesion average size approximately 970.93 units, standard deviation approximately 23 units, area 4.594 cm². 2 The maximum length is ≥1320 units, and the minimum length is ≤49 units.

[0056] Figures 8A to 8B This shows lesion 1 in participant S02007 ( Figure 8A ) and lesion 2 ( Figure 8B Images of [the patient] were captured at baseline (left subplot) and after treatment (right subplot). Figure 8A As shown in the left sub-image, the length of lesion 1 at baseline is 3.19 cm; Figure 8A As shown in the right-hand sub-figure, the length of lesion 2 at baseline is 2.09 cm; Figure 8B As shown in the left sub-image, the length of lesion 1 after treatment is 1.94 cm; Figure 8B As shown in the right-hand sub-figure, the length of lesion 2 after treatment is 1.41 cm. Detailed Implementation

[0057] This disclosure provides methods and compositions for treating cancer (e.g., bone cancer) in a subject or patient of need, including 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 the Cx43 hemichannel. 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 dosing regimen.

[0058] I. Definition

[0059] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural forms and plural terms shall include singular forms.

[0060] It should be understood that the present invention is not limited to the specific methodologies, schemes, and reagents described herein, and therefore variations are possible. The terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is defined only by the claims.

[0061] As used in this text, the articles “a,” “an,” and “the” are used to refer to one or more (i.e., at least one) of the grammatical objects in this text. For example, “an element” means one element or more than one element.

[0062] The use of alternatives (e.g., "or") should be understood to mean any one, both, or any combination of the alternatives.

[0063] The term “and / or” should be understood to mean one or both of the alternatives.

[0064] As used herein, the term "about" or "approximately" means a quantity, level, value, number, frequency, percentage, size, volume, weight, or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference quantity, level, value, number, frequency, percentage, size, volume, weight, or length. In one embodiment, the term "about" or "approximately" means a series of quantities, levels, values, numbers, frequencies, percentages, sizes, volumes, weights, or lengths that are ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%.

[0065] As used herein, the terms “substantially” or “largely” mean approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher of a reference quantity, level, value, number, frequency, percentage, size, weight, or length. In one embodiment, the terms “substantially the same” or “largely the same” mean a range of quantities, levels, values, numbers, frequencies, percentages, sizes, weights, or lengths that are approximately the same as a reference quantity, level, value, number, frequency, percentage, size, weight, or length.

[0066] Throughout this specification, unless the context otherwise requires, the word "comprise / comprises / comprising" should be understood to imply that it includes the stated steps or elements or a group of steps or elements, but does not exclude any other steps or elements or a group of steps or elements. In certain embodiments, the terms "comprising," "having," "containing," and "including" are used synonymously.

[0067] The phrase "composed of" means including but not limited to anything that follows the phrase "composed of". Therefore, the phrase "composed of" indicates that the listed elements are necessary or mandatory, and no other elements may exist.

[0068] "consistently composed of..." means including any of the elements listed after the phrase, and is limited to other elements that do not interfere with or affect the activity or function of the listed elements as specified in this disclosure. Thus, the phrase "consistently composed of..." indicates that the listed elements are necessary or required, but other elements are not optional and may be present or absent depending on whether they affect the activity or function of the listed elements.

[0069] The term "provide" is used in its usual sense, meaning to supply or provide for use. In some embodiments, a protein (e.g., an antibody) is provided by direct administration of the protein, while in other embodiments, a protein (e.g., an antibody) is efficiently provided by administration of a nucleic acid encoding the protein. In some aspects, the invention contemplates compositions comprising various combinations of nucleic acids, antigens, peptides, and / or epitopes.

[0070] Throughout this specification, references to "an embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a specific embodiment," "an additional embodiment," or "another embodiment," or combinations thereof, indicate that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Therefore, the aforementioned phrases appearing throughout this specification do not necessarily all refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic may be combined in any suitable manner in one or more embodiments.

[0071] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to a molecule 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, etc.) and long chains (also commonly referred to in the art as polypeptides or proteins). “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0072] As used herein, the term "sequence identity percentage" or "sequence identity" refers 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 relative to another target nucleic acid or amino acid sequence can be determined using tools and techniques known in the art, such as NCBI BLAST.

[0073] The terms "pharmaceutical formulation" or "pharmaceutical composition" refer to a formulation containing a therapeutic agent (e.g., an anti-Cx43 antibody). Its form allows the antibody's biological activity to be effective and does not contain any additional components that would have unacceptable toxicity to the subject to which the formulation will be administered.

[0074] As used herein, the term "antigen" is a molecule that can be bound by an antibody or a T-cell receptor. In some embodiments, the binding portion other than the antibody is engineered to specifically bind to the antigen, such as an aptamer, affinity, etc.

[0075] As used herein, the term "specific binding" is not intended to mean that an antibody binds only to its intended target. Rather, an antibody is considered to have "specific binding" if its affinity for its intended target is approximately five times greater than its affinity for a non-target molecule. Appropriately, there is no significant cross-reactivity or cross-binding with the undesired substance. For example, the antibody's affinity for the target molecule will be at least about five times its affinity for a non-target molecule, such as 10 times, such as 25 times, especially 50 times, and particularly 100 times or more. In some embodiments, specific binding between an antibody or other binding agent and an antigen means at least 10... 6 M -1 The binding affinity. Antibodies can, for example, bind with at least about 10... 7 M -1 The affinity combination, such as between about 10 8 M -1 To about 10 9 M -1 Between, between about 10 9 M -1 To about 10 10 M -1 Between, or between about 10 10 M -1 To about 10 11 M -1 The affinity between them. Antibodies can bind, for example, at EC values ​​of 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, a variety of immunoassay formats can be used to select antibodies that specifically react with the antigen. For example, solid-phase ELISA is commonly used to select monoclonal antibodies that specifically react with the protein. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Press, 1988, which describes immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0076] Affinity-matured antibodies possess one or more alterations in one or more hypervariable regions that result in increased affinity for the antigen compared to parent antibodies without these alterations. In one respect, affinity-matured antibodies will have nanomolar or even picomolar affinity for the target antigen. Affinity-matured antibodies are produced using procedures known in the art. Marks et al., Bio / Technology 10:779-783 (1992), describe affinity maturation via VH and VL domain shuffling. Random mutagenesis of CDRs and / or framework residues is described in: 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).

[0077] The term "cancer" or "tumor" is intended to encompass any malignant or neoplastic growth in a patient, including the initial tumor and any metastatic lesions. Cancer can be hematologic or solid tumor type. Hematologic tumors include tumors of hematologic origin, including, for example, myeloma (e.g., multiple myeloma), leukemia (e.g., Waldenström macroglobulinemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, granulocytic leukemia, monocytic leukemia, acute lymphoblastic leukemia, 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 reticulum cell sarcoma), and myeloproliferative tumors, such as myelodysplastic syndromes, thrombocythemia, polycythemia vera, or myelofibrosis. Solid tumors can originate from organs and include cancers of organs such as bone, skin, lung, brain, breast, prostate, ovary, colon, kidney, pancreas, liver, esophagus, stomach, intestine, bladder, uterus, cervix, testis, and adrenal glands. As used herein, cancer cells, including tumor cells, are cells that divide at an abnormal (increased) rate or whose growth or survival is controlled differently from cells in the same tissue from which cancer cells originate or are present. Cancer cells include, but are not limited to, cells in carcinomas, sarcomas, myelomas, leukemias, lymphomas, and nervous system tumors, including gliomas, meningiomas, medulloblastomas, schwannomas, or ependymomas. In a preferred embodiment, the cancer is bone cancer. In a more preferred embodiment, the bone cancer is osteosarcoma.

[0078] The term "affinity" refers to the strength of the binding reaction between the binding domain of an antibody and the 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.

[0079] The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specifically binding to immunoglobulins or T-cell receptors. In some embodiments, the epitope determinant includes chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in some embodiments, may have specific three-dimensional structural features and / or specific charge features. In one embodiment, an epitope is an antigenic region that is bound by an antibody. In some embodiments, when an antibody preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules, it is referred to as specifically binding antigen. Methods for epitope mapping are well known in the art, such as X-ray co-crystallization, array-based oligopeptide scanning, site-directed mutagenesis, high-throughput mutagenesis mapping, and hydrogen-deuterium exchange. Epitopes can be formed from either consecutive amino acids or non-consecutive amino acids arranged side-by-side through the ternary folding of a protein. Epitopes formed from consecutive amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed from ternary folds are generally lost upon treatment with denaturing solvents. Epitopes typically comprise at least three, more commonly at least five, or eight to ten amino acids with a unique spatial conformation.

[0080] The terms “promote,” “enhance,” and “induce,” which are used interchangeably in this article, refer to any statistically significant increase in biological activity (e.g., half-channel opening). For example, “promote” can refer to an increase in biological activity of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0081] As used herein, the term “bone cancer” or “bone tumor” refers to any cancer associated with bone, including cancers originating from bone or originating from other organs or tissues (such as the breast, prostate, lung, kidney, and thyroid) and metastasizing to bone. Therefore, non-limiting examples of bone cancer include osteosarcoma, multiple myeloma, Ewing's sarcoma, osteoporosis, osteopenia, osteoblastoma, chondroma, chondromycinoid fibroma, mesenchymal chondrosarcoma, chordoma, ameloblastoma, or metastatic bone cancer.

[0082] Osteosarcoma is the most common primary malignant bone tumor. By definition, osteosarcoma is a spindle cell tumor that forms osteoid tissue and has several histological subtypes, exhibiting variable radiographic features, inherent cellular heterogeneity, and overall prognosis. Subtypes of osteosarcoma include, but are not limited to: osteoblastic osteosarcoma, chondrogenic osteosarcoma, fibroblastic osteosarcoma, small cell osteosarcoma, capillary osteosarcoma, periosteal osteosarcoma, classic osteosarcoma, osteoblastic osteosarcoma-sclerosing osteosarcoma, chondroblastoma-like osteosarcoma, chondromyxoid fibromatosis-like osteosarcoma, clear cell osteosarcoma, malignant fibrous histiocytoma-like osteosarcoma, giant cell-rich osteosarcoma, or epithelioid osteosarcoma. The World Health Organization considers osteoblastic, chondrogenic, and fibroblastic subtypes to be the common subtypes of intramedullary osteosarcoma (the most common type of osteosarcoma).

[0083] Osteoblastic osteosarcoma originates from the abnormal proliferation of osteoblasts. It typically occurs in the metaphysis 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 a rich background of extracellular osteoid formation. The matrix, composed of immature bone, is deposited in a lace-like pattern, and malignant tumor cells are distributed throughout the extracellular matrix.

[0084] The World Health Organization defines chondrogenic osteosarcoma as a histological entity characterized by the presence of a predominantly chondrogenic matrix, which often exhibits high hyaline cartilage density and is closely associated with non-chondrogenic components (osteoid or bone matrix). Chondrogenic osteosarcoma commonly occurs in long bones, such as the femur and tibia. It can also affect other bones, including the humerus and pelvis. Chondrogenic osteosarcoma is more common in adolescents and young adults, but it can occur at any age.

[0085] Fibroblastic osteosarcoma is characterized by the presence of spindle cells resembling fibroblasts. It is highly vascularized and may resemble hemangiopericytoma. Histopathologically, bone production is minimal and focal.

[0086] Small cell osteosarcoma is a rare subtype of osteosarcoma characterized by the presence of small, round, and homogeneous cells within the tumor. Histopathologically, small cell osteosarcoma cells have sparse cytoplasm and large, deeply stained nuclei. Small cell osteosarcoma primarily occurs in the metaphysis of long bones.

[0087] Benign giant cells (similar to osteoclasts) are found in approximately 25% of osteosarcomas. In rare osteosarcomas, the number of benign giant cells is so high that they may mask malignant cells in the background, leading to misdiagnosis as a giant cell tumor. This is more likely to occur in the sacrum (a common site for giant cell tumors). The radiographic differences between giant cell tumors and osteosarcomas are less pronounced in the sacrum than in long bones. It is important to remember that giant cell tumors typically occur in patients with mature bones. Tumors that appear to be giant cell tumors but occur in individuals with immature bones should be thoroughly and carefully sampled to rule out osteosarcomas rich in giant cells.

[0088] There are many classification systems for grading osteosarcoma. One widely used system is the Broder system, which grades tumors based on the percentage of intermediate changes. Most common central osteosarcomas fall into grade 3 or 4. Tumors designated as grade 1 are low-grade central osteosarcomas and common paraosteal osteosarcomas. Jawbone osteosarcomas typically tend to exhibit grade 2 histological features.

[0089] The commonly used staging systems for malignant bone tumors are the Enneking Malignant Musculoskeletal Tumor Staging System and the American Joint Committee on Cancer (AJCC) Osteosarcoma Staging System. The Enneking system is based on the tumor's histological grade, its local extent, and the presence or absence of metastases. The AJCC system for osteosarcoma is based on tumor grade, size, and the presence and location of metastases. The different stages of the AJCC system are shown below.

[0090]

[0091] II. Antibodies

[0092] The term “antibody” is used in the broadest sense and specifically encompasses full-length antibodies, antibody peptides or immunoglobulins, monoclonal antibodies, chimeric antibodies, polyclonal antibodies, human antibodies, humanized antibodies, and antibodies derived from non-human species, including human antibodies derived from human immunoglobulin sequences introduced into non-human species (e.g., mice, sheep, chickens, or goats), recombinant antigen-binding forms such as monomers and dimers, multispecific antibodies formed from at least two full-length antibodies (e.g., bispecific antibodies, each containing an antigen-binding region of an antibody against a different antigen or epitope), and individual antigen-binding fragments of any of the foregoing, such as fragments of antibodies or antibodies derived from them, including dAb, Fv, scFv, Fab, F(ab)'2, Fab'.

[0093] The methods described in this paper also consider antibody-like binding peptide mimics. Liu et al. (2003) described “antibody-like binding peptide mimics” (ABiP), which are peptides that function as simplified antibodies and have certain advantages such as longer serum half-life and less cumbersome synthesis methods.

[0094] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, meaning that the individual antibodies constituting the group are identical and / or bind to the same epitopes. The modifier "monoclonal" indicates that the antibody is derived from a substantially homogeneous group of antibodies and is not to be construed as requiring the antibody to be produced by any particular method.

[0095] The "antigen-binding fragment" of the antibody preferably comprises at least the variable region of the heavy chain and / or light chain of the anti-Cx43 antibody. For example, the antigen-binding fragment of the anti-Cx43 antibody may contain the amino acid sequences SEQ ID NO: 7 and 8. Examples of such antigen-binding fragments include the Fab fragment, Fab' fragment, Fv fragment, scFv, and F(ab')2 fragment. The antigen-binding fragment of the antibody can be generated by enzymatic digestion or recombinant techniques. For example, the Fab or F(ab')2 fragment can be generated by digestion with papain or pepsin, respectively. Antibodies can also be generated in various truncated forms by using antibody genes with one or more stop codons introduced upstream of the natural termination site. For example, a recombinant construct encoding the heavy chain of the F(ab')2 fragment can be designed as a DNA sequence including the CH1 domain and hinge region encoding the heavy chain. In one aspect, the antigen-binding fragment promotes the opening of the Cx43 hemichannel in the subject and the effects associated with the opening of the Cx43 hemichannel.

[0096] "Therapeutic monoclonal antibodies" are antibodies intended for the treatment of human subjects. The therapeutic monoclonal antibodies disclosed herein include anti-Cx43 antibodies. Antibody "effective functions" refer to the biological activities attributable to the Fc region of the antibody (the native sequence Fc region or the Fc region of an amino acid sequence variant). Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. To assess the ADCC activity of the molecule of interest, in vitro ADCC assays can be performed, such as those described in U.S. Patent Nos. 5,500,362 or 5,821,337.

[0097] Full-length antibodies can be classified into different "classes" based on the amino acid sequence of their heavy chain constant domains. There are five main classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM. Several of these classes can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different antibody classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different antibody classes are well-known. The "light chain" of antibodies from any vertebrate species can be classified into one of two distinct types based on the amino acid sequence of its constant domain, called kappa (κ) and lambda (λ).

[0098] Parts of this invention, such as polypeptides, peptides, antigens, or immunogens, may be covalently or non-covalently conjugated or linked to other parts, such as adjuvants, proteins, peptides, supports, fluorescent portions, or tags. The terms “conjugate” or “immunoconjugate” are used broadly to define the operative binding of one part to another agent and are not intended to refer only to any particular type of operative binding, and are not particularly limited to chemical “conjugation.”

[0099] When used in this article, the term "hypervariate region" refers to the antibody amino acid residues responsible for antigen binding. Hypervariable regions typically contain amino acid residues from the complementarity-determining region (CDR) or the chain variable domain (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 edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), and / or amino acid residues from the hypervariable ring (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 J. Mal. Biol.). 196:901-917 (1987)). “Frame region” or “FR” residues are variable domain residues other than hypervariable region residues as defined herein. Hypervariable regions or their CDRs 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.

[0100] As those skilled in the art will understand, the CDRs disclosed herein may also include variants. Generally, the amino acid identity among the variant CDRs is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Thus, a “variant CDR” is a CDR that has specified identity with the parental CDR of the present invention and shares biological functions (including, but 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 parental CDR).

[0101] Amino acid substitutions are typically single residues; insertions are usually on the order of about one to about twenty amino acid residues, but rather large insertions may be tolerated. Deletions range from about one to about twenty amino acid residues, but in some cases deletions can be much larger.

[0102] Substitution, deletion, insertion, or any combination thereof can be used to obtain the final derivative or variant. Generally, these changes are made on a few amino acids to minimize alterations to the molecule, particularly the immunogenicity and specificity of antigen-binding proteins. However, larger changes may be tolerated in certain cases.

[0103] As used herein, the term "Fab" or "Fab region" refers to a polypeptide that includes the VH, CH1, VL, and CL immunoglobulin domains. Fab may refer to the region existing alone, or the region in the context of a full-length antibody, antibody fragment, or Fab fusion protein, or any other antibody implementation as outlined herein.

[0104] As used herein, the terms “Fv,” “Fv fragment,” or “Fv region” refer to a polypeptide that includes the VL and VH domains of a single antibody.

[0105] As used herein, the term "framework" refers to the antibody variable domain region other than those regions defined as CDRs. Each antibody variable domain frame can be further subdivided into consecutive regions separated by CDRs (FR1, FR2, FR3, and FR4).

[0106] The “humanized” form of non-human (e.g., rodent) antibodies is a chimeric antibody containing a minimal sequence derived from a non-human antibody. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies) in which residues from the receptor hypervariable region are replaced by residues from the hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate, possessing the desired specificity, affinity, and capability. In some instances, the frame region (FR) residues of a human antibody are replaced by corresponding non-human residues. Furthermore, humanized antibodies may include residues not found in the receptor or donor antibody. These modifications are made to further refine antibody performance. For more 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).

[0107] "Isolated" antibodies are antibodies that have been identified, isolated, and / or recovered from components of their native environment. In some embodiments, the antibody will be purified to the following criteria: (1) a protein content greater than 95% by weight, or alternatively, greater than 99% by weight, as determined by the Lowry method; (2) sufficient to obtain at least 15 N-terminal or internal amino acid sequence residues using a rotary cup sequencer; or (3) homogeneity achieved by SDS-PAGE under reducing or non-reducing conditions, detected using Coomassie blue or silver staining. Isolated antibodies include recombinant antibodies present in situ within cells, as at least one component of the antibody's native environment will be absent. However, typically isolated antibodies will be prepared through at least one purification step.

[0108] Anti-Cx43 antibody

[0109] Certain aspects of this disclosure provide compositions and methods for treating bone cancers, such as osteosarcoma. These methods and compositions comprise an anti-Cx43 antibody that specifically binds to the Cx43 hemichannel and promotes its opening in bone cells.

[0110] The anti-Cx43 antibody can be any antibody known in the art that specifically binds to Cx43. In various embodiments, the anti-Cx43 antibody used in these methods comprises an HCDR1 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 1, an HCDR2 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 2, and an HCDR3 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 3; and / or an LCDR1 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 4, an LCDR2 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 5, and an LCDR3 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 6. In some specific embodiments, the anti-Cx43 antibody comprises the same HCDR1 amino acid sequence as SEQ ID NO: 1, the same HCDR2 amino acid sequence as SEQ ID NO: 2, and 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.

[0111] In various embodiments, the anti-Cx43 antibody used in this method comprises 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 comprises the same heavy chain variable region as SEQ ID NO: 7; and / or the same light chain variable region as SEQ ID NO: 8.

[0112] 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 NO: 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 NO: 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 any one of SEQ ID NO: 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 NO: 10. In some other 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 any one of 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 with any one of SEQ ID NO: 10. In some other 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 any one of 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 with 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 with any one of 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 with any one of SEQ ID NO: 10.

[0113] In some embodiments, the anti-Cx43 antibody comprises the same heavy chain as any one of SEQ ID NO: 9 and 11-13, and / or the same light chain as any one of SEQ ID NO: 10. In a preferred embodiment, the anti-Cx43 antibody comprises the same heavy chain as SEQ ID NO: 9, and the same light chain as any one of SEQ ID NO: 10. In one embodiment, the anti-Cx43 antibody comprises the same heavy chain as SEQ ID NO: 11, and the same light chain as any one of SEQ ID NO: 10. In another embodiment, the anti-Cx43 antibody comprises the same heavy chain as SEQ ID NO: 12, and the same light chain as any one of SEQ ID NO: 10. In yet another embodiment, the anti-Cx43 antibody comprises the same heavy chain as SEQ ID NO: 13, and the same light chain as any one of SEQ ID NO: 10.

[0114] In various implementations, the anti-Cx43 antibody may comprise a mixture or combination of two or more anti-Cx43 antibodies, wherein each antibody binds to the same or different epitopes on Cx43.

[0115] In various embodiments, this document provides an antibody that binds to an epitope located partially or entirely within the amino acid sequence FLSRPTEKTI (SEQ ID NO: 14). 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 consists of F1, S3, R4, P5, T6, E7, K8, T9, and I10 of SEQ ID NO: 14. In some embodiments, the epitope may comprise all ten amino acids of SEQ ID NO: 14. In some embodiments, the epitope consists of all ten amino acids of SEQ ID NO: 14.

[0116] Anti-Cx43 antibodies are substantially pure and are expected to be substantially homogeneous (i.e., free from contaminating proteins, etc.). "Substantially pure" means a composition containing at least about 90%, 95%, or 97% antibody by weight, based on the total weight of the protein in the composition. "Substantially homogeneous" means a composition containing protein in which at least about 99% of the protein by weight is a specific antibody, such as an anti-Cx43 antibody.

[0117] 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, it is a humanized monoclonal antibody.

[0118] Pharmaceutical formulations containing anti-Cx43 antibodies

[0119] One aspect of this disclosure provides a pharmaceutical formulation for treating cancer (e.g., bone cancer including osteosarcoma), the pharmaceutical formulation comprising an anti-Cx43 antibody. In some embodiments, the anti-Cx43 antibody promotes the opening of Cx43 hemichannels in bone cells. The anti-Cx43 antibody may be a complete antibody or an antigen-binding fragment thereof.

[0120] As used herein, a “pharmaceuticalally acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption-delaying agents, 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., a small molecule or biopharmaceutical) may be encapsulated in the material to protect the compound from acids and other natural conditions that could inactivate it.

[0121] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders used for the provisional preparation of sterile injectable solutions or dispersions, as well as conventional excipients used for the preparation of tablets, pills, capsules, etc. The use of such media and reagents in the formulation of pharmaceutically active substances is known in the art. Unless to some extent there is incompatibility between any conventional media or reagent and the active compound, its use in the pharmaceutical compositions provided herein is considered. Complementary active compounds may also be incorporated into the composition.

[0122] Pharmaceutically acceptable carriers may include pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0123] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions provided herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, as well as injectable organic esters, such as ethyl oleate. Appropriate flowability can be maintained, for example, by using coating materials (such as lecithin), by maintaining the desired particle size in the case of dispersions, and by using surfactants. In many cases, it may be useful to include isotonic agents, such as sugars, polyols (such as mannitol, sorbitol), or sodium chloride, in the composition. Prolonged absorption of the injectable composition can be achieved by including agents that delay absorption (e.g., monostearate and gelatin) in the composition.

[0124] These compositions may also contain functional excipients, such as preservatives, wetting agents, emulsifiers, and dispersants.

[0125] Therapeutic compositions must generally be sterile, pyrogen-free, and stable under manufacturing and storage conditions. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentrations. Sterile solutions for injection can be prepared by incorporating the desired amount of the active compound with one or more of the ingredients listed above (as needed) into a suitable solvent, followed by sterilization, for example, by microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile solvent containing a basic dispersion medium and the other desired ingredients listed above. For sterile powders used to prepare sterile solutions for injection, preparation methods include vacuum drying and lyophilization (freeze-drying), which produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile filtered solution. The active agent can be mixed under sterile conditions with additional pharmaceutically acceptable carriers and any necessary preservatives, buffers, or propellants.

[0126] The presence of microorganisms can be prevented through the sterilization procedures described above, as well as by including various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). It may also be necessary to include isotonic agents, such as sugars and sodium chloride, in the composition. Additionally, prolonged absorption of injectable drugs can be achieved by including agents that delay absorption (such as aluminum monostearate and gelatin).

[0127] Formulating parenteral compositions in unit dosage forms may be advantageous for ease of administration and dosage uniformity. As used herein, a unit dosage form refers to a physically discrete unit suitable for use as a single dose for a patient to be treated: each unit contains a predetermined amount of active agent, the amount of which is calculated to produce the desired therapeutic effect when combined with any desired drug carrier. The specifications of a unit dosage form depend on and are directly dependent on (a) the unique properties of the active compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations in the art of formulating such active compounds to treat individual sensitivities.

[0128] The actual dose level of the active ingredient in the pharmaceutical compositions disclosed herein can be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and route of administration without being toxic to the patient. As used herein in the context of administration, "parenteral" means a route of administration other than enteral and local administration, typically by injection, and including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, intradermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0129] As used herein, the phrases “parenteral administration” and “extragastric administration” refer to administration methods other than intravenous (i.e., via the digestive tract) and local administration, typically by injection or infusion, and including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intrasacral, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, intradermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. Intravenous injection and infusion are commonly (but not exclusively) used for antibody administration.

[0130] The formulation described herein is administered to mammals, preferably humans, requiring treatment with anti-Cx43 antibodies, according to known methods, such as intravenous administration by bolus injection or continuous infusion over a period of time, via intramuscular, intraperitoneal, intracerebrospinal fluid, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, local, or inhalation routes.

[0131] In some embodiments, these preparations are administered to mammals via intravenous or subcutaneous (i.e., subcutaneous) administration. For such purposes, a syringe can be used to inject the preparation. However, other devices for administering the preparation are also available, such as injection devices (e.g., INJECT-EASE). ™ and GENJECT ™ Devices); Injection pens (such as GENPEN) ™ ); auto-injector devices, needle-free devices (e.g., MEDIJECTOR) ™ and BIOJECTOR ™ ); and subcutaneous patch delivery systems.

[0132] In one specific embodiment, this disclosure relates to a kit for a single-dose administration unit. Such kits include aqueous formulation containers for therapeutic proteins or antibodies, including single-compartment or multi-compartment pre-filled syringes. Exemplary pre-filled syringes are available from Vetter GmbH, Ravensburg, Germany.

[0133] III. Methods of treating bone cancer

[0134] One aspect of this disclosure provides methods for treating cancers (e.g., bone cancer including osteosarcoma). These methods may include promoting / enhancing the opening of Cx43 hemichannels in the bone cells of a subject in need by using, for example, an anti-Cx43 antibody. Currently, there is a lack of second-line treatment for osteosarcoma; therefore, Cx43 modulation (e.g., promoting the opening of Cx43 hemichannels using an anti-Cx43 antibody) may be used as second-line treatment for patients who have failed standard treatment for osteosarcoma, or as at least first-line treatment for osteosarcoma. Cx43 modulation may be a standalone therapy for bone cancer including osteosarcoma, or used in combination with other cancer therapies described herein or known in the art.

[0135] In some implementations, the anti-Cx43 antibody is used as second-line treatment for subjects who have failed at least first-line therapy (standard treatment) for bone cancer (e.g., osteosarcoma). In some other implementations, the anti-Cx43 antibody is used as first-line treatment for treating a subject with bone cancer (e.g., osteosarcoma). In some implementations, the anti-Cx43 antibody is used as neoadjuvant therapy for a subject with bone cancer (e.g., osteosarcoma).

[0136] Cells communicate with each other and with the extracellular environment through hemichannels and gap junction channels formed by connexins. Connexins are universally expressed throughout the body. Six connexins form a hemichannel, and two hemichannels form a gap junction channel. Gap junction channels are clusters of channels located in the plasma membrane between adjacent cells, mediating intercellular communication. Hemichannels are separate entities from gap junction channels. Hemichannels allow molecular exchange between intracellular compartments and the extracellular environment.

[0137] Normal bone is composed of three main cell types: osteoblasts, osteoclasts, and osteocytes. Osteocytes make up about 95% of bone cells and maintain bone remodeling by coordinating osteoclast and osteogenic activities. When cancer cells invade bone, many normal bone functions are affected. Cancer cells interact with the local microenvironment, promoting cancer cell survival through bone destruction and vascularization.

[0138] Bone cells express hemichannels called connexin (Cx)43 hemichannels. These bone cell hemichannels are normally closed but can open upon exposure to mechanical stimuli, leading to the release of various factors into the bone microenvironment. Factors released from these open hemichannels can mediate other processes that can reduce tumor cell migration and bone metastasis. Alendronate (AD), an effective and commonly used bisphosphonate drug, has been shown to open Cx43 hemichannels in bone cells. Bisphosphonates are a class of drugs known for treating many bone diseases, including bone metastases. Bisphosphonate administration has been shown to be associated with reduced incidence of bone metastases and mortality in breast cancer patients. AD has been associated with reduced tumor growth, bone destruction, and pain relief. AD inhibits osteoclast activity and induces the opening of Cx43 hemichannels in bone cells. However, AD administration is accompanied by a variety of serious side effects.

[0139] Cx43, also known as gap junction α-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 crosses the phospholipid bilayer four times, exposing its C-terminus and N-terminus to the cytoplasm. The C-terminal tail, consisting of 50 amino acids, includes post-translational modification sites and binding sites for transcription factors, cytoskeletal elements, and other proteins. Therefore, the C-terminal tail is crucial for regulating functions such as pH gating and channel assembly. Notably, the DNA region encoding this tail in the GJA1 gene (NCBI gene ID: 2697) is highly conserved, indicating it is either mutagenic or lethal upon mutation. Meanwhile, the N-terminal domain is involved in channel gating and oligomerization, thus controlling the switching between open and closed states of channels. The transmembrane domains form gap junction channels, while the extracellular loop facilitates proper channel docking. In addition, the two extracellular rings form disulfide bonds, which interact with the two hexamers to form complete gap junction channels.

[0140] “Treatment” refers to therapeutic treatment. People requiring treatment include those who already have a disease. Therefore, the patient(s) to be treated in this document (e.g., a person) may have been diagnosed with a disease such as bone cancer. A disease (e.g., bone cancer) is “suppressed” or “treated” if at least one symptom of the disease (determined based on responsiveness / non-responsiveness, or indicators known in the art and described herein) is relieved, terminated, slowed, minimized, or prevented. The terms “patient” and “subject” are used interchangeably herein.

[0141] The terms "subject" or "patient" refer to humans or non-humans, such as primates, mammals, and vertebrates. In a particular implementation, the subject is a human.

[0142] Treatment may be appropriately administered to subjects who have, possess, are susceptible to, or are at risk of developing this type of cancer (e.g., bone cancer, including osteosarcoma), particularly humans. Identification of subjects “at risk” can be made by any objective or subjective determination by the subject or healthcare provider, through diagnostic tests or opinions (e.g., genetic testing, enzyme or protein biomarkers, family history, etc.). Identifying subjects requiring this treatment can be a judgment of the subject or healthcare professional and can be subjective (e.g., opinion) or objective (e.g., measured by tests or diagnostic methods).

[0143] Anti-Cx43 antibodies can be stored as lyophilized solids or aqueous formulations, or in any other form known in the art. When anti-Cx43 antibodies are stored as lyophilized solids, the antibody is reconstituted in a solution (such as water for injection) prior to administration. If prepared from lyophilized or aqueous formulations for infusion, the final administration concentration of the anti-Cx43 antibody (e.g., after reconstitution and dilution in a saline, Ringer's solution, or 5% glucose infusion system) can be from about 0.1 mg / ml to about 80 mg / ml. Final concentrations can be from about 0.1 mg / ml to about 80 mg / ml, from about 0.5 mg / ml to about 70 mg / ml, from about 1 mg / ml to about 60 mg / ml, from about 5 mg / ml to about 50 mg / ml, from about 10 mg / ml to about 40 mg / ml, from about 15 mg / ml to about 30 mg / ml, or from about 20 mg / ml to about 25 mg / ml. In some implementations, the final dosage form concentration may be 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.

[0144] As used herein, the term "effective amount" refers to the amount of a drug (such as an anti-Cx43 antibody) that, when administered to a patient or subject, is sufficient to affect the treatment, prognosis, or diagnosis of cancer (e.g., bone cancer). Dosing regimens can be adjusted to provide an optimal therapeutic response. An effective amount also refers to the amount in which any toxic or harmful effects (side effects) of the drug are minimized and / or outweighed by beneficial effects. Therapeutic effective amounts will vary depending on the patient being treated and their disease condition, the patient's weight and age, the severity of the disease condition, the route of administration, the course of the disease, the patient's clinical history, and the response to the anti-Cx43 antibody, which can be readily determined by those skilled in the art. For example, the effective amount or dose range for anti-Cx43 antibodies is from about 0.001 mg / kg to about 300 mg / kg. In some embodiments, the dosage of the anti-Cx43 antibody is about 0.01 mg / kg to about 200 mg / kg, about 0.1 mg / kg to about 100 mg / kg, about 0.2 mg / kg to about 90 mg / kg, about 0.5 mg / kg to about 80 mg / kg, about 0.8 mg / kg to about 70 mg / kg, about 1 mg / kg to about 60 mg / kg, about 3 mg / kg to about 50 mg / kg, about 6 mg / kg to about 40 mg / kg, about 9 mg / kg to about 36 mg / kg, about 12 mg / kg to about 33 mg / kg, about 15 mg / kg to about 30 mg / kg, about 18 mg / kg to about 27 mg / kg, or about 21 mg / kg to about 24 mg / kg. In some implementations, the dosage of the 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. / kg, approximately 50mg / kg, approximately 60mg / kg, approximately 70mg / kg, approximately 80mg / kg, approximately 90mg / kg, approximately 100mg / kg, approximately 120mg / kg, approximately 140mg / kg, approximately 160mg / kg, approximately 180mg / kg, approximately 200mg / kg, approximately 220mg / kg, approximately 240mg / kg, approximately 260mg / kg, approximately 280mg / kg, approximately 300mg / kg, or greater than 300mg / kg.

[0145] In some specific implementations, 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.

[0146] In some embodiments, the effective amount or dose range of the anti-Cx43 antibody can be from about 1 mg to about 25,000 mg. In some embodiments, the dose of the 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 5000 mg, about 25 mg to 4000 mg, about 30 mg to 3000 mg, about 40 mg to 2000 mg, about 50 mg to 1000 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 implementations, the dose of anti-Cx43 antibody is less than 1 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg. mg, about 900 mg, about 950 mg, about 1000 mg, about 1250 mg, about 1500 mg, about 1750 mg, about 2000 mg, about 2250 mg, about 2500 mg, about 2750 mg, about 3000 mg, about 4000 mg, about 5000 mg, about 6000 mg, about 7000 mg, about 8000 mg, about 9000 mg, about 10,000 mg, about 12,500 mg, about 15,000 mg, about 17,500 mg, about 20,000 mg, about 22,500 mg, about 25,000 mg, or more than 25,000 mg.

[0147] In the method described herein, the effective amount or dose of anti-Cx43 antibody is administered approximately once daily, approximately every 2 days, approximately every 3 days, approximately every 4 days, approximately every 5 days, approximately every 6 days, approximately once a week, approximately every 8 days, approximately every 9 days, approximately every 10 days, approximately every 11 days, approximately every 12 days, approximately every 13 days, approximately every 2 weeks, approximately every 15 days, approximately every 16 days, approximately every 17 days, approximately every 18 days, approximately every 19 days, approximately every 20 days, approximately every 3 weeks, approximately every 22 days, approximately every 23 days, approximately every 24 days, approximately every 25 days, approximately every 26 days, approximately every 27 days, approximately every 4 weeks, approximately every 29 days, approximately every 3 weeks ... Once every 0 days, approximately once every 31 days, approximately once every 32 days, approximately once every 33 days, approximately once every 34 days, approximately once every 5 weeks, approximately once every 36 days, approximately once every 37 days, approximately once every 38 days, approximately once every 39 days, approximately once every 40 days, or approximately once every 41 days, approximately once every 6 weeks, approximately once every 7 weeks, approximately once every 8 weeks, approximately once every 9 weeks, approximately once every 10 weeks, approximately once every 11 weeks, approximately once every 12 weeks, approximately once every 13 weeks, approximately once every 15 weeks, approximately once every 16 weeks, approximately once every 17 weeks, approximately once every 18 weeks, approximately once every 19 weeks, approximately once every 20 weeks, approximately once every 21 weeks, approximately once every 22 weeks, approximately once every 23 weeks, approximately once every 24 weeks or 6 months, or approximately once every 24 weeks or 6 months.

[0148] In some specific implementations, the anti-Cx43 antibody dose is administered approximately every 3 weeks. In one particular implementation, the anti-Cx43 antibody dose is administered every 3 weeks.

[0149] According to certain embodiments of this disclosure, multiple doses of anti-Cx43 antibody (or a pharmaceutical composition comprising an anti-Cx43 antibody and any additional therapeutically active agent mentioned herein) may be administered to a subject over a predetermined time period. A method according to this disclosure includes sequentially administering multiple doses of the disclosed anti-Cx43 antibody to a subject.

[0150] As used herein, “sequential administration” means administering each dose of anti-Cx43 antibody to a subject at different time points, for example, on different days separated by predetermined intervals (e.g., hours, days, weeks, or months). This disclosure includes a method comprising sequentially administering to a patient a single initial dose of anti-Cx43 antibody, followed by one or more second doses of anti-Cx43 antibody, and optionally subsequently, one or more third doses of anti-Cx43 antibody. The anti-Cx43 antibody can be administered at doses ranging from 0.001 mg / kg to about 300 mg / kg.

[0151] The terms "initial dose," "second dose," and "third dose" refer to the order in which the anti-Cx43 antibody of this disclosure is administered. Thus, the "initial dose" is the dose administered at the start of the treatment regimen (also known as the "baseline dose"); the "second dose" is the dose administered after the initial dose; and the "third dose" is the dose administered after the second dose. The initial, second, and third doses may all contain the same amount of anti-Cx43 antibody, but may typically differ from each other in terms of administration frequency. However, in some embodiments, the amount of anti-Cx43 antibody contained in the initial, second, third, and / or subsequent doses differs from each other during treatment (e.g., adjusted upwards or downwards as appropriate).

[0152] Anti-Cx43 antibodies are suitable for administration to patients in a single or series of treatments and can be administered at any time from the date of diagnosis. Anti-Cx43 antibodies can be administered as the sole treatment or in combination with other medications or therapies available for the treatment of the condition in question.

[0153] In the methods described herein, a dose of the anti-Cx43 antibody may be administered to the patient within 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, a dose of the anti-Cx43 antibody may be administered to the patient within about 30 minutes. In one specific embodiment, a dose of the anti-Cx43 antibody may be administered intravenously to the patient within 30 minutes.

[0154] In the methods described herein, an anti-Cx43 antibody is administered to a patient. If the anti-Cx43 antibody is in a solid (e.g., dry) formulation, the administration process may include the step of converting the formulation into a liquid state. In one aspect, the dry formulation may be reconstituted (e.g., by means of a liquid as described above) for injection, such as intravenous, intradermal, intramuscular, intratumoral, intraperitoneal, or subcutaneous injection. In one specific embodiment, on the other hand, the solid or dry formulation may be applied topically, such as in a patch, cream, aerosol, or suppository.

[0155] In the methods described herein, the anti-Cx43 antibody may be administered to the subject alone or in combination with another agent. The anti-Cx43 antibody may be administered before, simultaneously with, or after the administration of an additional agent. In one embodiment, during anti-Cx43 antibody treatment, the dose of the co-administered agent may be reduced or completely withdrawn over time. Non-limiting examples of co-administered agents include chemotherapy drugs, radiation therapy, immunotherapy, and surgery.

[0156]

[0157]

[0158] In yet another embodiment, at least one additional anticancer agent includes a VEGF inhibitor. Exemplary VEGF inhibitors include antiVEGF antibodies, such as bevacizumab (Avastatin@Genentech). In yet another embodiment, at least one additional anticancer agent includes an anti-ErbB2 antibody. Suitable anti-ErbB2 antibodies include trastuzumab and pertuzumab.

[0159] In one aspect, the improved effectiveness of the combination according to this disclosure can be demonstrated by achieving a therapeutic synergy. The term "therapeutic synergy" or "synergistic effect" is used when the combination of two products at a given dose is more effective than the optimal effect of either product used alone at the same dose. In one example, the therapeutic synergy can be evaluated by comparing the effect of the combination with the optimal single agent on the parameter tumor volume using estimates obtained from a two-way analysis of variance with repeated measures (e.g., time factor).

[0160] In some implementations, the patient or subject has failed at least one first-line or standard treatment for bone cancer (e.g., osteosarcoma). Non-limiting examples of failed treatment or standard treatment for bone cancer (e.g., osteosarcoma) are any treatments or agents described elsewhere in this disclosure that can be administered in combination with an anti-Cx43 antibody.

[0161] In some implementations, the patient or subject has failed at least one prior treatment for osteosarcoma. In some specific implementations, at least one osteosarcoma treatment includes chemotherapy, surgery, radiation therapy, immunotherapy, or a combination thereof. In some specific implementations, the patient or subject has failed at least one prior treatment, including methotrexate, doxorubicin, cisplatin and ifosfamide, apatinib, anlotinib, vinblastine, vincristine, docetaxel, paclitaxel, irinotecan, bortezomib, albumin-bound paclitaxel, nedaplatin (Aqupla), pemetrexed, etoposide, gemcitabine, lobaplatin, recombinant human endostatin, eribulin, dacarbazine, pazopanib, immune checkpoint inhibitors, surgery, radiation therapy, or a combination thereof.

[0162] Before, during, or after treatment with anti-Cx43 antibodies, examine cancer-related parameters of the patient or subject using techniques and methods known in the art. Non-limiting examples of commonly used medical techniques and methods for examining and diagnosing osteosarcoma include: X-rays, computed tomography (CT) scans, magnetic resonance imaging (MRI), positron emission tomography (PET), bone scintillation scans (bone scans), biopsies, the EQ-5D questionnaire for assessing quality of life, the PERCIST criteria for assessing disease progression, and digital rating scales for assessing pain intensity. The selection of techniques and methods, as well as the frequency of examinations, can be determined and / or adjusted by those skilled in the art based on the specific circumstances of the patient or subject.

[0163] In some implementations, treatment of osteosarcoma with anti-Cx43 antibodies improves disease progression as assessed by PERCIST, RECIST, and / or ICDS criteria compared to a control. This control may include, but is not limited to, osteosarcoma patients who have received the same number of lines of treatment as the subject or have had the same number of lines of treatment failure as the subject, but have not received anti-Cx43 antibody treatment for osteosarcoma.

[0164] In some implementations, treatment with anti-Cx43 antibodies 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. This control may include, but is not limited to, the mean or median overall survival of an osteosarcoma population that has received the same number of lines of treatment as the subject or has had the same number of lines of treatment failure as the subject, but has not received anti-Cx43 antibody treatment for osteosarcoma.

[0165] In some implementations, treatment with anti-Cx43 antibodies for 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. The control may include, but is not limited to, the mean or median overall survival of an osteosarcoma population that has received the same number of lines of treatment as the subject or has had the same number of lines of treatment failure as the subject, but has not received anti-Cx43 antibody treatment for osteosarcoma.

[0166] In some implementations, the duration of response in subjects treated with anti-Cx43 antibodies for osteosarcoma is 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.

[0167] In some implementation schemes, the time to remission for subjects treated with anti-Cx43 antibodies is 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.

[0168] In some implementations, treatment of osteosarcoma with anti-Cx43 antibodies 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%.

[0169] In some embodiments, treatment of osteosarcoma with anti-Cx43 antibodies improved the European Five-Dimensional Health Inventory (EQ-5D) score. In some embodiments, compared to baseline, treatment of osteosarcoma with anti-Cx43 antibodies improved 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. In some embodiments, treatment of osteosarcoma with anti-Cx43 antibodies improved the Visual Analogue Scale (VAS) score on 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.

[0170] In some implementations, treatment of osteosarcoma with anti-Cx43 antibodies reduces the Digital 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.

[0171] In some embodiments, treatment of osteosarcoma with anti-Cx43 antibodies improves bone mineral density (BMD) in the lumbar spine, hip, femur, and neck. In some embodiments, treatment of osteosarcoma with anti-Cx43 antibodies improves BMD in the lumbar spine, hip, 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%, at least 100%, or more.

[0172] Treatment of osteosarcoma with anti-Cx43 antibodies reduces the dose and / or frequency of morphine use in subjects or patients. In some embodiments, treatment of osteosarcoma with anti-Cx43 antibodies 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 some embodiments, treatment of osteosarcoma with anti-Cx43 antibodies completely and gradually discontinues morphine use.

[0173] As used herein, "response" or "having a response" to treatment means that at least one disease progression parameter of the subject has improved. A subject may have a "partial response" or a "complete response" to treatment. Response, partial response, or complete response to treatment can be determined based on methods known in the art. Those skilled in the art can determine appropriate methods based on the type of disease being evaluated. Non-limiting examples of such methods include RECIST criteria, ICDS criteria, and PERCIST criteria. In some embodiments, the subject has a complete response to anti-Cx43 antibody treatment. In some other embodiments, the subject has a partial response to anti-Cx43 antibody treatment.

[0174] In some implementations, after treatment with anti-Cx43 antibodies, the subject did not experience: (i) an adverse event, a serious adverse event, or a grade 3 or higher adverse event; (ii) a treatment-induced adverse event (TEAE); (iii) an adverse drug reaction (ADR); (iv) a Cx43-related cardiac event or a serious liver event; and / or (v) a dose-limiting toxicity.

[0175] "Adverse events" or "AEs" refer to any adverse medical event that occurs after a participant has taken a drug. These events may manifest as symptoms, signs, illnesses, or abnormal laboratory test results, but are not necessarily causally related to the drug.

[0176] "Adverse drug reaction" or "ADR" refers to any harmful or unexpected reaction that is considered to be related to a drug in clinical studies. There must be at least one plausible causal relationship between the drug and the ADR, meaning the correlation cannot be ruled out.

[0177] "Treating adverse events during treatment" or "TEAEs" refers to events that occur during treatment but are worse than events that did not occur or occurred before treatment.

[0178] A “serious adverse event” or “SAE” means any of the following situations that occur after a participant has received the drug: (i) resulting in death; (ii) life-threatening: referring to the participant’s risk of death at the time the event occurs, not the risk of death in the event’s progression; (iii) requiring hospitalization or prolonged hospitalization. Those skilled in the art will understand that SAEs include, but are not limited to, the foregoing list.

[0179] Non-limiting examples of "dose-limiting toxicities" include: grade 4 neutropenia lasting ≥7 days; or grade 3 neutropenia with fever (ANC < 1000 / mm³). 3 And a single body temperature >38.3℃ or a sustained body temperature ≥38℃ for more than 1 hour) or accompanied by 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 (for participants with liver metastases and abnormal baseline); or bilirubin ≥3 × ULN (for participants with normal baseline) or ≥3 × baseline (for participants with abnormal baseline); grade 3 nephrotoxicity: serum creatinine ≥3 × ULN (for participants with normal baseline) or ≥3 × baseline (for participants with abnormal baseline); grade 3 cardiotoxicity: QTc > 500ms or QTc increase ≥60ms.

[0180] Example

[0181] The following embodiments illustrate preferred embodiments of the present invention. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques discovered by the inventors that work well in practicing the present invention, and therefore can be considered as constituting preferred practices of the present invention. However, based on this disclosure, those skilled in the art will understand that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the present invention, and similar or related results can still be obtained.

[0182] Example 1. Overview and Preclinical Studies of ALMB-0168

[0183] ALMB-0168 is a humanized monoclonal antibody prepared under GMP conditions from a recombinant CHO cell line. Similar to 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. Like most antibodies, the heavy chain is glycosylated at conserved glycosylation sites in the constant region.

[0184] ALMB-0168 is a novel humanized IgG4 monoclonal antibody targeting the Cx43 hemichannel of bone cells. The initial antibody was obtained by immunizing mice with human Cx43. The mouse antibody was then humanized using common humanization methods (i.e., complementarity-determining region, CDR) transplantation (Bowers et al., J Biol Chem., 288(11): 7688-7696 (2013)) (also known as modification) and germline sequence modeling.

[0185] ALMB-0168 is the first drug designed to inhibit the growth of primary and metastatic bone tumors by activating the Cx43 hemichannel and releasing key substances such as ATP into the extracellular environment.

[0186] Based on preclinical evaluations, ALMB-0168 effectively activated the opening of Cx43 hemichannels in osteocytes both in vivo and in vitro. In several mouse models of breast cancer bone metastases and osteosarcoma in situ, administration of ALMB-0168 inhibited tumor growth in a dose-dependent manner and prolonged the lifespan of tumor-bearing animals, demonstrating its potential as a treatment for malignant bone tumors. Furthermore, ALMB-0168 exhibited a favorable nonclinical safety profile, supporting a first-in-human dose. The physicochemical properties of ALMB-0168 are shown in Table 1.

[0187] Table 1. Physicochemical properties of ALMB-0168

[0188]

[0189] Pharmacological effects and mechanisms of action

[0190] Osteocytes highly express the connectant protein Cx43 in gap junction channels and hemichannels. Gap junction channels mediate cell-cell communication between two adjacent cells to maintain normal cellular metabolic function, while hemichannels allow molecular exchange between the cell and its extracellular environment. Hemichannels are normally closed, but under certain conditions, such as mechanical stress or local ischemia, cytokine or ion stimulation, they can be activated and opened. Open hemichannels allow small molecules (<1.2 kDa) such as ATP, NAD+, glutamate, and inositol triphosphate (IP3) to pass through (Goodenough et al., Nature reviews Molecular cellbiology, 4:285-294 (2003)). The opening of osteocyte Cx43 hemichannels has been shown to strongly inhibit bone metastasis of breast cancer tumors (Zhou et al., Oncogene, 35: 5597-5607 (2016)).

[0191] ALMB-0168 is a humanized IgG4 monoclonal antibody targeting the osteocyte hemichannel. The initial antibody was obtained by immunizing mice with human Cx43. The mouse antibody was then humanized using common humanization methods (i.e., complementarity-determining region, CDR) transplantation (Bowers et al., J Biol Chem., 288(11): 7688-7696 (2013)) (also known as modification) and germline sequence modeling.

[0192] ALMB-0168 is designed to activate the Cx43 hemichannel, allowing key anticancer agents such as ATP to be released into the extracellular environment. On one hand, ATP can activate cytotoxic CD8+ T cells and CD4+ T cells while significantly inhibiting Treg cells with negative tumor immune conditions. On the other hand, ATP can bind to the P2Y11 receptor on cancer cells, reducing the positive regulation of CXCR4 by P2Y11, thereby inhibiting the growth and metastasis of primary and metastatic bone tumors.

[0193] Based on preclinical evaluations, ALMB-0168 effectively activated the opening of the Cx43 hemichannel in osteocytes both in vivo and in vitro. In several mouse models of breast cancer bone metastases and osteosarcoma in situ, ALMB-0168 inhibited tumor growth in a dose-dependent manner and prolonged the lifespan of tumor-bearing animals, demonstrating its potential as a treatment for malignant bone tumors. Furthermore, ALMB-0168 exhibited a favorable nonclinical safety profile, supporting a first-in-human trial.

[0194] Overview of preclinical research

[0195] Overview of Preclinical Pharmacodynamic Studies

[0196] In vitro test

[0197] In vitro experiments showed that ALMB-0168 had no effect on gap junctions. ALMB-0168 does not affect the normal function of gap junctions. Treatment of mouse osteoblasts MOL-Y4 with ALMB-0168 for 16 hours had no effect on gap junctions, while the positive control glycyrrhetinic acid (a known gap junction inhibitor) significantly inhibited gap junction function (p < 0.001).

[0198] ALMB-0168 specifically binds to and activates the Cx43 hemichannel in osteocytes. The binding activity of ALMB-0168 with mouse osteocytes (MOL-Y4) and human primary osteocytes was determined by ELISA (enzyme-linked immunosorbent assay). The results showed that the antibody concentrations corresponding to the maximum 50% effective concentration (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 opening of the Cx43 hemichannel; EB staining showed EC50 values ​​of 2.33 ± 0.15 µg / ml for MOL-Y4 cells and 3.97 ± 0.90 µg / ml for human osteocytes.

[0199] ALMB-0168 treatment inhibited the migration of breast cancer and osteosarcoma cells. Conditioned medium collected from ALMB-0168-treated osteocytes (MOL-Y4) significantly inhibited the migration of breast cancer cells (MDA-MB-231) and osteosarcoma cells (OS17) in vitro (p < 0.001). Cell migration was measured using a 24-well tissue culture plate migration assay with an inserted membrane filter. The results showed that ALMB-0168-treated osteocyte conditioned medium inhibited cancer cell migration by 50% compared to solvent-treated osteocyte conditioned medium. The inhibitory effect disappeared after treatment with ATPase (an enzyme used to hydrolyze ATP), indicating that extracellular ATP levels increased after ALMB-0168 treatment.

[0200] In vivo testing

[0201] Tissue distribution of ALMB 0168 in mice and activation of Cx43 hemichannels. Four hours after intraperitoneal injection of ALMB-0168 (25 mg / kg), antibodies were detected only in mouse osteocytes and not in other organs expressing connexins (liver, heart, spleen). Furthermore, EB staining assays showed that ALMB-0168 treatment significantly increased the opening of Cx43 hemichannels in mouse osteocytes.

[0202] ALMB 0168 inhibits bone metastasis of breast cancer in mice. In a mouse xenograft model, weekly injection of 25 mg / kg ALMB-0168 significantly inhibited the growth of human breast cancer (p < 0.05).

[0203] In a mouse orthotopic transplantation model, ALMB-0168 inhibited osteosarcoma growth and prolonged the survival of tumor-bearing mice. In a mouse orthotopic osteosarcoma model, weekly administration of 15-25 mg / kg ALMB-0168 significantly inhibited tibial tumor growth and prolonged lifespan.

[0204] ALMB-0168 treatment increased plasma ATP levels. To assess the changes in plasma ATP levels induced by ALMB-0168 administration activating the Cx43 hemichannel, plasma ATP levels in mice after ALMB-0168 injection were measured using a luciferase / luciferase assay. The results showed that the plasma ATP levels in ALMB-0168-treated mice were significantly higher (p < 0.001) than those in saline-treated mice, exceeding the ATP levels by more than 5 times.

[0205] Overview of Preclinical Pharmacokinetic Studies

[0206] Pharmacokinetic study of ALMB-0168 in cynomolgus monkeys after a single intravenous infusion

[0207] A total of 18 cynomolgus macaques (9 per sex) were randomly assigned to three dosage groups: low, medium, and high dose groups, with intravenous doses of 5 mg / kg, 15 mg / kg, and 50 mg / kg, respectively. Blood samples were collected from each group at time points including before administration, at the end of administration, 10 minutes after administration, and at 2 hours, 8 hours, 24 hours, 48 ​​hours, 72 hours, 5 days, 7 days, 10 days, 14 days, 17 days, 21 days, and 28 days after administration. Serum ALMB-0168 concentrations were determined using a validated ELISA method with a limit of quantification of 0.01 μg / ml. PK parameters were determined using non-compartmental analysis (NCA) in WinNonlin software.

[0208] Serum PK parameters are summarized as follows: :

[0209] 1. Except for a slightly higher AUC in females than in males in the medium-dose group (p < 0.05, ratio = 1.32), there were no significant sex differences in PK parameters (p ≥ 0.05).

[0210] 2. Following a single intravenous infusion of ALMB-0168 in cynomolgus monkeys, the maximum serum concentration and systemic exposure of ALMB-0168 were dose-proportionate. The administration ratio of the low, medium, and high dose groups was 1:3:10. The mean Cmax and AUClast ratios for the low, medium, and high dose groups were 1:3.04:10.92 and 1:3.10:10.95, respectively.

[0211] 3. Regression analysis was performed on Cmax-dose and AUClast-dose. The results showed that the slopes of the Cmax-dose and AUClast-dose curves were 1.04 and 1.04, respectively, with 95% confidence intervals of [1.11, 0.98] and [1.15, 0.94], and R² values ​​of 0.99 and 0.96, respectively. The results indicate that the exposure to ALMB-0168 in cynomolgus monkeys was linearly correlated with single intravenous infusion doses of 5 mg / kg, 15 mg / kg, and 50 mg / kg.

[0212] Overview of Preclinical Toxicology Studies

[0213] Single-dose toxicity study of ALMB-0168

[0214] 120 mice (60 mice / sex) were randomly divided into groups 1, 2, 3, and 4, with 15 mice / sex in each group. Group 1 mice (0 mg / kg) were treated with ALMB-0168 buffer as a solvent control, while groups 2, 3, and 4 were treated with ALMB-0168 at 50 mg / kg, 150 mg / kg, and 500 mg / kg, respectively.

[0215] Under the conditions of this study, no significant test-drug-related toxicities were observed in mice that received a single intravenous injection of ALMB-0168 at doses of 50 mg / kg, 150 mg / kg, and 500 mg / kg.

[0216] A total of eight cynomolgus macaques (4 per sex) were randomly assigned to groups 1-4, with one macaque per sex in each group. Group 1 served as a placebo control, while groups 2, 3, and 4 were administered ALMB-0168 at 50 mg / kg, 150 mg / kg, or 500 mg / kg, respectively. On day 1, the macaques received a single intravenous infusion. Animals were observed for approximately 4 hours post-administration, followed by routine clinical observation. Body weight, food intake, and body temperature were measured, and ECG, hematology, coagulation, clinical chemistry, urinalysis, serum anti-drug antibody testing, and toxicokinetics were performed. On day 15, all animals were euthanized and subjected to a comprehensive gross examination.

[0217] No deaths or illnesses were observed in any of the animals in groups 1–4 during the study period. No abnormal clinical findings related to the test substance were found in any of the animals.

[0218] ALMB-0168 Repeated-Dose Toxicity Study

[0219] 1. Repeated-dose toxicity study in monkeys

[0220] Forty-eight cynomolgus macaques were randomly divided into groups 1-4, with 24 macaques of each sex and 6 macaques of each sex per group. Group 1 used ALMB-0168 buffer as a solvent control, while groups 2, 3, and 4 used ALMB-0168 at 25 mg / kg, 75 mg / kg, and 250 mg / kg, respectively. The macaques were administered the drug once a week on days 1, 8, 15, 22, 29, 36, and 43 (a total of 7 doses) for 6 consecutive weeks.

[0221] No signs of death or near death were observed in any of the animals in groups 1-4 throughout the study.

[0222] Throughout the study, compared with the same-sex animals in the solvent control group, animals treated with ALMB-0168 at 25 mg / kg, 75 mg / kg, and 250 mg / kg showed no analyte-related or toxicologically significant abnormalities in body weight, food intake, body temperature, cardiovascular function, respiratory function, ECG, blood pressure, ophthalmology, hematology, coagulation, clinical chemistry, urinalysis, lymphocyte subsets, cytokines, serum immunoglobulins, serum complement, and the combination of functional observations used to assess neurobehavioral function.

[0223] Serum antidrug antibody (ADA) analysis: In the 25 mg / kg ALMB-0168 dose group, 2 / 12 monkeys tested positive for ADA on day 15 post-administration, with the highest titer being 1:4. In the 75 mg / kg ALMB-0168 dose group, 1 / 12 monkeys tested positive for ADA on day-2 before administration and day 15 post-administration, with titers less than 1; no increase in titer was observed throughout the study period. Therefore, the positive results were considered unrelated to ALMB-0168. In the 250 mg / kg ALMB-0168 dose group, 2 / 12 monkeys tested positive for ADA on day 15 post-administration, with the highest titer being 1:8. TK results showed that the C... max and AUC last It is not significantly different from other animals.

[0224] Toxicokinetics: The mean TK parameters of the test sample in cynomolgus monkey serum are shown in Table 2. Except for T... max Except for the median, all reported values ​​are averages.

[0225] Table 2. Mean TK parameters of ALMB-0168 in cynomolgus monkey serum (repeated dosing study)

[0226]

[0227]

[0228]

[0229] Under the conditions of this study, ALMB-0168 was administered intravenously to cynomolgus monkeys weekly at doses of 25 mg / kg, 75 mg / kg, and 250 mg / kg for 6 weeks (7 doses in total), followed by a 4-week recovery period. Local injection site reactions associated with the test substance were observed, and a dose-dependent decrease in lymphocyte counts in the spleen was observed in all dose groups. After the 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 the germinal centers and marginal zones) completely resolved after the 4-week recovery period in the 25 mg / kg and 75 mg / kg dose groups, and showed a tendency to recover in the 250 mg / kg dose group (very slight in one male and mild in one female). Therefore, this finding was considered to have no adverse effect on the spleen. Thus, 250 mg / kg was the no-observed-adverse-effect level (NOAEL) in this study.

[0230] 2. Repeated-dose toxicity study in mice

[0231] Nine hundred and eighty mice (490 mice / sex) were randomly divided into eight groups (groups 1-8). Groups 1, 2, 3, and 4 each contained 40 mice / sex; group 5 contained 45 mice / sex; and groups 6, 7, and 8 each contained 95 mice / sex. Mice were administered the drug intravenously once weekly for six weeks (a total of seven doses) at the following dosages: Groups 1 and 5 (solvent control group) (ALMB-0168 buffer, 0 mg / kg); Groups 2 and 6 (ALMB-0168) 25 mg / kg; Groups 3 and 7 (ALMB-0168) 75 mg / kg; and Groups 4 and 8 (ALMB-0168) 250 mg / kg. Day 1 was defined as the day of administration.

[0232] The parameters evaluated in this study included clinical observation, body weight, food intake, fundus examination, hematology, coagulation, clinical chemistry, urinalysis, T lymphocyte subset assays (CD3+, CD3+CD4+, CD3+CD8+, CD4+CD8+), and cytokine assays (IL-2, IL-4, IL-5, IL-6, TNF-α, IFN-γ). Six weeks after drug administration (day 44), the first 20 animals / sex / groups in 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 dissections were performed on all animals in groups 1 through 4, organ weights were collected, and gross anatomical observations and microscopic examinations were completed. Histopathological evaluations were performed on all standard tissues from groups 1 and 4, tissues from animals that died unexpectedly before the predetermined time point, and all abnormal tissues from groups 2 and 3.

[0233] Throughout the study, no animals in groups 1-8 experienced any test-substance-related deaths or near-death experiences.

[0234] No abnormal changes related to the test substance were observed during clinical and injection site observations throughout the study.

[0235] Throughout the study, no abnormal changes in body weight, weight gain, food intake, body temperature, ophthalmological examination, hematology, coagulation, T lymphocyte subsets, and cytokines related to the test substance were observed in animals administered ALMB-0168 at doses of 25 mg / kg, 75 mg / kg, and 250 mg / kg.

[0236] Animals administered ALMB-0168 at doses of 25 mg / kg, 75 mg / kg, and 250 mg / kg (except for female animals administered 25 mg / kg) showed elevated LDH levels on day 44. After a 4-week recovery period, LDH levels returned to the solvent control group levels. No microscopic findings were observed. Therefore, this is not considered an adverse reaction.

[0237] At the end of administration and the 4-week recovery period, no abnormalities related to the test product were observed in any animal in terms of organ weight, organ-to-body weight ratio, organ-to-brain weight ratio, gross anatomical examination, or microscopic examination. No local irritation related to the test product was observed at the injection site in any animal during this study.

[0238] In summary, under the conditions of this study, mice were administered ALMB-0168 intravenously once weekly at doses of 25 mg / kg, 75 mg / kg, and 250 mg / kg for 6 consecutive weeks (7 doses in total), followed by a 4-week recovery period. The results showed no adverse systemic or target organ toxicity. Therefore, the NOAEL for ALMB-0168 in this study was 250 mg / kg.

[0239] Other relevant toxicity studies

[0240] 1. Study on tissue cross-reactivity in normal cynomolgus monkey tissues

[0241] The tissue cross-reactivity of ALMB-0168 was evaluated in 34 different normal cynomolgus monkey tissues (each tissue from three different animals) using streptavidin-biotin immunohistochemistry.

[0242] Positive staining was observed in the cell membranes of MLO-Y4 cells (a mouse bone cell line highly expressing the Cx43 hemichannel) in the positive control section after staining with 5 μg / ml and 20 μg / ml ALMB-0168-biotin, respectively. No positive staining was observed in the negative control section of the test sample group on frozen human skeletal muscle tissue. No positive staining was observed in either the positive or negative control photographs of the isotype control and negative control groups.

[0243] In the test groups (ALMB-0168-Biotin, 5 μg / ml and 20 μg / ml), nonspecific staining of round cells was observed in the bone marrow, ileum, and stomach tissues of the isotype control and negative control groups, and nonspecific staining was also observed in renal tubular epithelial cells and hepatocytes. No nonspecific positive staining was observed in other tissues of each test group.

[0244] Under the conditions of this study, no specific staining of ALMB-0168 was observed in normal cynomolgus monkey tissues.

[0245] 2. Tissue cross-reactivity test in normal human tissues

[0246] The streptavidin-biotin method with IHC staining was used to evaluate whether ALMB-0168 had tissue cross-reactivity with 34 frozen tissues from normal individuals (each tissue was derived from 3 different individuals).

[0247] In the test groups (ALMB-0168-Biotin, 5 μg / ml and 20 μg / ml), nonspecific staining of round cells was observed in the bone marrow, colon, ileum, and stomach tissues of the isotype control and negative control groups, and nonspecific staining was also observed in renal tubular epithelial cells and hepatocytes. No nonspecific positive staining was observed in other tissues of each test group.

[0248] Under the conditions of this experiment, no specific staining of ALMB-0168 was observed in frozen normal human tissue.

[0249] 3. Tissue reaction test in mouse tissues

[0250] The cross-reactivity of ALMB-0168 with 30 frozen tissues from normal mice (each tissue derived from 3 different individuals) was evaluated using the streptavidin-biotin method with IHC staining.

[0251] Nonspecific staining was observed in adrenal cortical cells and bone marrow cells, gastric round cells, and renal tubular epithelial cells and hepatocytes in the isotype control and negative control groups of the test sample groups (ALMB-0168-Biotin, 5 μg / ml and 20 μg / ml). No nonspecific positive staining was observed in other tissues of each test group.

[0252] Under the conditions of this experiment, no specific staining of ALMB-0168 was observed in normal mouse tissues.

[0253] 4. Human blood cell in vitro hemolysis test

[0254] This study provides a reference for the clinical research of the test sample. The test sample was analyzed at a concentration of 10 mg / ml on the day of the in vitro test. The accuracies for the upper, middle, and lower layers of the test sample were 99.93%, 101.20%, and 101.63%, respectively, with a coefficient of variation of 0.87%. The test sample was not detected in the negative and positive controls. The analytical results met the requirements.

[0255] After incubation at 37 ± 0.5℃ for 3 hours, the positive control tube showed complete hemolysis, with a clear red solution and no red blood cells at the bottom. For the test tube and negative control tube, red blood cells were observed to settle to the bottom, while the upper layer of solution was colorless and clear, showing no hemolysis or aggregation.

[0256] Under the conditions of this study, ALMB-0168 at a concentration of 10 mg / ml did not cause hemolysis of human erythrocytes in vitro and did not induce erythrocyte aggregation.

[0257] 5. Cytokine release assay in human peripheral blood mononuclear cells (PBMCs)

[0258] After 24 hours of incubation with ALMB-0168, the in vitro cytokine release from the stimulated PBMC samples was tested. 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 served as baseline controls.

[0259] All donor-derived PBMCs reacted with CD3 antibodies under both stimulation modalities, indicating that the PBMC samples possess the ability to stimulate and induce the release of IL-2, IL-6, IL-10, IFN-γ, and TNF-α. Elevated IL-6 levels were detected in some PBMC samples, and most of these samples underwent wet-coating stimulation.

[0260] Human IgG controls under both stimulation modalities showed almost no induction of IL-2, IL-10, IFN-γ, and TNF-α release. Under the soluble stimulation modalities, IL-6 responses varied across multiple donors. No dose-response trend was observed, and the response did not reach the level of positive control biological CD3 antibodies achieved by most donors.

[0261] Under both stimulation modalities, ALMB-0168 induced minimal responses to the release of IL-2, IL-10, IFN-γ, and TNF-α cytokines. Under the soluble stimulation modality, IL-6 responses varied across multiple donors. The response did not reach the level of positive control biological CD3 antibodies achieved by most donors.

[0262] Australia's first human safety study

[0263] As of May 2021, the first human study in Australia had enrolled a total of two participants (one at 1 mg / kg and the other at 3 mg / kg), and had received five cycles of dosing (every three weeks). No serious adverse events, fatal adverse events, or adverse events leading to participant withdrawal occurred. One participant in the 1 mg / kg group experienced a drug-related adverse event of abdominal pain, fluctuating between grade 1 and 2, which resolved after symptomatic treatment with oral medication. One participant in the 3 mg / kg group experienced a grade 1 drug-related adverse event of fatigue, which resolved spontaneously without medication. Other adverse events occurring in both participants during the study included diarrhea, abdominal discomfort, nausea, herpes zoster on the left upper lip, hyperglycemia, hypercalcemia, and redness and itching at the peripherally inserted central venous catheter site, all grade 1 and determined to be drug-independent.

[0264] Known potential benefits

[0265] Prior to the initiation of this study, the first-in-human clinical trial of ALMB-0168-AU-101 was conducted in Australia to evaluate the safety, tolerability, pharmacokinetic (PK), disease progression (PD), preliminary efficacy, and immunogenicity of ALMB-0168 in patients with incurable osteosarcoma and bone metastases for whom no better alternative treatment options were available, and to guide the starting dose for this study to minimize ineffective dose exposure and further ensure safety. For this first-in-human clinical trial, dosing of the first and second participants in the first and second dose groups (1 mg / kg and 3 mg / kg, respectively) has been completed and demonstrated good safety and tolerability. As described in the study protocol, all participants were closely monitored throughout the study, and any adverse events experienced by participants were promptly and closely monitored and managed until they returned to normal or became clinically stable.

[0266] In summary, ALMB-0168 is a humanized monoclonal IgG4 antibody targeting the Cx43 hemichannel with a well-defined mechanism of action. Preclinical studies have shown that ALMB-0168 has good efficacy, safety, and controllability, and may bring therapeutic benefits to patients with osteosarcoma and bone metastases.

[0267] Example 2. A multicenter, single-arm, open-label study evaluating the safety and efficacy of ALMB-0168 in patients with osteosarcoma. Phase I / II Clinical Trial – Study Protocol

[0268] This study is registered on ClinicalTrials.gov under number NCT04886765.

[0269] Research objectives and endpoints

[0270] Part I of the study

[0271] The primary objective of Part I is to evaluate the safety and tolerability of ALMB-0168 in osteosarcoma patients who have failed standard therapy and to explore the recommended phase II dose (RP2D).

[0272] The secondary objective of Part I is:

[0273] • Evaluate the pharmacokinetic (PK) characteristics of ALMB-0168;

[0274] • Evaluate the efficacy of ALMB-0168 in treating patients with osteosarcoma;

[0275] • Evaluate the immunogenicity of ALMB-0168.

[0276] The primary endpoint of Part I is:

[0277] • Physical examination, vital signs, pulse oximetry, safety laboratory tests, ECG, and the occurrence of AE, SAE, and DLT;

[0278] • MTD (if applicable).

[0279] The secondary endpoint of Part I is:

[0280] • Pharmacokinetic parameters: including but not limited to AUC0-t, AUC0-inf, Cmax, Ctrough, Tmax, CL, λz and t1 / 2;

[0281] • Efficacy endpoints: Objective 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); rate of change of alkaline phosphatase (ALP) or lactate dehydrogenase (LDH) relative to baseline; skeletal-related events (SREs); changes of Numerical Rating Scale (NRS) and European Five-Dimensional Health Scale (EQ-5D) scores relative to baseline; bone mineral density of the lumbar spine, hip, femur, and neck; morphine dosage and frequency;

[0282] • Incidence of anti-drug antibodies (ADA).

[0283] Part II of the study

[0284] The primary objective of Part II is to further evaluate the safety and efficacy of ALMB-0168 in patients with high-grade osteosarcoma who have failed standard therapy, in order to further determine RP2D.

[0285] The secondary objective of Part II is:

[0286] • Further evaluate the pharmacokinetic (PK) characteristics of ALMB-0168;

[0287] • Evaluate the efficacy of ALMB-0168;

[0288] • Further evaluate the immunogenicity of ALMB-0168.

[0289] The principal endpoint of Part II is:

[0290] • Physical examination, vital signs, pulse oximetry, safety laboratory tests, ECG, and the occurrence of adverse events (AEs) and sarcastic anemias (SAEs);

[0291] • Efficacy endpoint: 6-month progression-free survival rate (6m-PFSR).

[0292] The secondary endpoint of Part II is:

[0293] • Efficacy endpoints: Objective response rate (ORR), duration of response (DoR), time to response (TTR), disease control rate (DCR), overall survival (OS); rate of change of alkaline phosphatase (ALP) or lactate dehydrogenase (LDH) relative to baseline; skeletal-related events (SREs); changes of Numerical Rating Scale (NRS) and European Five-Dimensional Health Scale (EQ-5D) scores relative to baseline; bone mineral density of the lumbar spine, hip, femur, and neck; morphine dosage and frequency;

[0294] •PK metrics: including but not limited to Cmax and Ctrough;

[0295] • Incidence of anti-drug antibodies (ADA).

[0296] Research Design

[0297] Overall Design

[0298] This is a multicenter, open-label, single-arm phase I / II clinical trial to evaluate the safety and preliminary efficacy of ALMB-0168 in osteosarcoma patients who have failed standard therapy.

[0299] This study will be conducted in two phases (Part I: dose escalation phase; and Part II: dose expansion phase). All participants will receive multiple cycles of intravenous ALMB-0168 every 3 weeks. Part I will enroll patients with high-grade osteosarcoma who have failed standard therapy, and Part II will enroll high-grade osteosarcoma patients from the Part I population. The dose escalation phase will employ accelerated titration and a standard “3+3” escalation design. The first two dose groups (1 mg / kg, 3 mg / kg) will use an accelerated titration escalation method. One participant will be enrolled in each group, and the dose will be escalated to the next group if the participant does not experience a drug-related grade ≥2 TEAE within 3 weeks of the first dose. If the participant experiences a drug-related grade ≥2 TEAE, the dose level will be escalated using a 3+3 dose escalation method. 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 based on the incidence of ALMB-0168-related adverse events (AEs) assessed by NCI CTCAE v5.0 within 3 weeks of the first dose (DLT observation period). Dose escalation will continue until a dose level in which ≥1 / 3 of the participants (when ≥6 participants are assessed) experience a DLT. The tolerable level prior to this dose level will be 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 investigator.

[0300] The selection of RP2D from the tolerable dose range will be based on short-term and long-term safety information, as well as findings from preclinical dose-finding studies, pharmacokinetic (PK), PD, and antitumor activity studies. For dose groups that have completed the DLT observation period, investigators and sponsors may extend the study to up to eight participants (excluding the MTD extension cohort) after a comprehensive analysis of the participants' benefits, based on an assessment of previous clinical data. This extension will provide further information on safety, PK characteristics, and antitumor activity. Safety data from the extended participants will not be used to determine the MTD.

[0301] In the dose expansion phase, an expansion study will be conducted in 1-3 dose groups to explore the preliminary efficacy of ALMB-0168 in patients with high-grade osteosarcoma who have failed standard therapy, and to further describe its safety, pharmacokinetic characteristics, etc. The plan is to enroll a maximum of 60 participants per dose group; whether to increase the number of participants will be decided after obtaining efficacy and safety data.

[0302] All participants will sign an ICF prior to any specific study activity. Eligible participants will receive treatment at the dose level determined by the dose escalation phase after enrollment. Theoretically, this study defines one treatment cycle 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 disease progression or when it is necessary to initiate anti-tumor therapy. The safety of all participants will be closely monitored.

[0303] All participants will be followed up in person 28 days after the last treatment, followed by a one-year follow-up to assess long-term outcomes.

[0304] Dosage escalation design

[0305] Dose-limiting toxicity

[0306] Dose-limiting toxicities (DLTs) are defined as the following toxicities associated with ALMB-0168 that occur during the first treatment cycle (21 days):

[0307] • Grade 4 neutropenia lasting ≥7 days; or grade 3 neutropenia with fever (ANC < 1000 / mm³) 3 And a single body temperature >38.3℃ or a sustained body temperature ≥38℃ for more than 1 hour) or accompanied by infection;

[0308] • Grade 4 thrombocytopenia or Grade 3 thrombocytopenia with bleeding;

[0309] • Grade 3 hepatotoxicity: ALT or AST ≥ 5 × upper limit of normal (ULN) (for participants with normal baseline) or ≥ 5 × baseline (for participants with liver metastases and abnormal baseline); or bilirubin ≥ 3 × ULN (for participants with normal baseline) or ≥ 3 × baseline (for participants with abnormal baseline).

[0310] • Grade 3 nephrotoxicity: Serum creatinine ≥ 3 × ULN (for participants with normal baseline values) or ≥ 3 × baseline value (for participants with abnormal baseline values);

[0311] • Grade 3 cardiotoxicity: QTc > 500ms or QTc increase ≥ 60ms;

[0312] • Other non-hematologic toxicities of grade ≥3, excluding:

[0313] □ Grade 3 nausea and / or vomiting lasting less than 72 hours without preventative and effective antiemetic treatment;

[0314] □ Fatigue or weakness lasting less than 7 days;

[0315] □ Transient asymptomatic laboratory test abnormalities lasting less than 3 days;

[0316] □ Hair loss, rash, and diarrhea that are manageable with standard supportive care.

[0317] • Researchers determined that the toxic effects of ALMB-0168 should be permanently discontinued.

[0318] The aforementioned adverse events (AEs) will be classified according to the National Cancer Institute Common Terminology Standard for Adverse Events (NCI-CTCAE) version 5.0.

[0319] Maximum tolerated dose

[0320] MTD Definition: The dose escalation phase will employ an accelerated titration and standard “3+3” escalation design: the first two dose groups (1 mg / kg, 3 mg / kg) will use an accelerated titration escalation method. One participant will be enrolled in each group, and the dose will be escalated to the next dose group if the participant does not experience ≥ Grade 2 toxicity related to ALMB-0168. If the participant experiences ≥ Grade 2 toxicity related to ALMB-0168, a 3+3 dose escalation method will be used for that dose level. A 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 based on the incidence of ALMB-0168-related adverse events (AEs) assessed according to NCI CTCAE v5.0 within 3 weeks after the first dose (DLT observation period). Dose escalation will continue until a dose level in which ≥1 / 3 of the participants (when ≥6 participants are assessed) experience DLT. The tolerable dose level prior to this dose level will be considered the MTD.

[0321] After the first cycle, all adverse events of grade 4 or higher during the extension period will also be one of the factors determining dose escalation and MTD.

[0322] Initial dose estimation

[0323] Results from toxicity studies in non-human primates and mice will be used to calculate the safe starting clinical dose for this study. The methodology will be consistent with the guidelines provided in ICH S9. The no-observed-adverse-effect level (NOAEL) for ALMB-0168 in both non-human primates and rodents is 250 mg / kg. Considering the novel target of ALMB-0168, a safety factor exceeding 100 × NOAEL will be used to ensure that the first dose will not cause adverse effects in humans. The final starting dose for this drug in this first-in-human study is 1 mg / kg.

[0324] Determination of maximum incremental dose

[0325] In a non-GLP dose-range exploration study in non-human primates (NHP), ALMB-0168 was administered intravenously once weekly to cynomolgus monkeys at doses of 20 mg / kg, 60 mg / kg, and 200 mg / kg. No adverse reactions were observed in any dose group during the subsequent 2 weeks. Subsequently, in a GLP repeated-dose toxicology study, cynomolgus monkeys were administered intravenously once weekly at doses of 25 mg / kg, 75 mg / kg, and 250 mg / kg for 6 weeks, followed by a 4-week recovery period. No significant adverse reactions were observed. Furthermore, in a GLP repeated-dose toxicology study in mice, mice were repeatedly administered weekly at doses of 25 mg / kg, 75 mg / kg, and 250 mg / kg for 6 weeks. No significant adverse reactions were observed after a 4-week observation and recovery period. Therefore, the NOAEL for ALMB-0168 in both NHP and rodents is 250 mg / kg.

[0326] In preclinical efficacy studies, weekly injections of 25 mg / kg ALMB-0168 significantly inhibited the growth of human breast cancer in a mouse xenograft model. In a mouse orthotopic osteosarcoma model, weekly administration of 15–25 mg / kg ALMB-0168 significantly inhibited tibial tumor growth and prolonged lifespan. The maximum escalation dose in clinical studies is tentatively set at 30 mg / kg, and this dose will be adjusted as necessary based on pharmacokinetic (PK), PD, and safety data obtained from participants in each dose group in Australia and China.

[0327] Application methods and frequency

[0328] In this study, 21 days (3 weeks) is defined as one treatment cycle. All participants will receive an intravenous injection of ALMB-0168 every 3 weeks. Participants will receive multiple cycles of treatment until disease progression or intolerable toxicity occurs. The safety of all participants will be closely monitored.

[0329] All participants will be followed up in person 28 days after the last treatment, followed by a one-year follow-up to assess long-term outcomes.

[0330] Dose escalation group settings

[0331] Dose escalation levels will be based on ALMB-0168-related adverse events observed during the first dosing cycle and the dose escalation gradients specified in the dose escalation settings table. If two or more participants experience ≥ Grade 2 TEAEs related to ALMB-0168 during the first dosing cycle, or one participant experiences ≥ Grade 3 TEAEs related to ALMB-0168, the dose escalation between cohorts may be reduced as needed, for example, from 100% dose escalation to no more than 50% of the previous dose level, as determined jointly by the investigator and sponsor. ALMB-0168 dose levels at 18 mg / kg and above will be escalated in increments not exceeding 33%. If any issues arise during the safety assessment, the dose escalation may be appropriately reduced following the same procedure. It is expected that no more than seven dose groups will be evaluated during this study. The actual number of dose groups and dose levels will depend on the maximum tolerated dose (MTD) and the safety profiles observed during this study. If necessary, the investigator and sponsor may discuss intermediate dose escalation levels. See Table 3 below for the specific dose group settings for each dose escalation phase.

[0332] Table 3. Part I: Specific Dosage Increment Settings

[0333]

[0334] a: The first two dose groups (1 mg / kg, 3 mg / kg) will be administered using an accelerated titration escalation method. One participant will be enrolled in each group, and if the participant does not experience a grade ≥2 TEAE related to ALMB-0168, the dose will be escalated to the next dose group. If the participant experiences a grade ≥2 TEAE related to ALMB-0168, a 3+3 dose escalation method will be used for that dose level.

[0335] * The MTD cohort will include up to 10 participants, including 3-6 participants in the dose escalation phase, to determine safety.

[0336] The dose level for each dose group and the percentage increase relative to the previous dose level will be adjusted based on data from the first human clinical study in Australia (ALMB-0168-AU-101).

[0337] For dose groups that have completed the DLT observation period, the number of participants can be expanded to a maximum of 8 (excluding the MTD extension cohort) after the investigator and sponsor have evaluated the previous clinical data and analyzed the benefits to the participants.

[0338] When the dose is increased to the pre-specified highest dose group, if the safety and tolerability of that dose group remain good, the investigator and the sponsor will jointly decide whether to explore higher doses.

[0339] Dosage escalation principle

[0340] The dose escalation phase will employ accelerated titration and a standard “3+3” escalation design: the first two dose groups (1 mg / kg, 3 mg / kg) will use accelerated titration escalation, and from the third dose group (6 mg / kg) onwards, a 3+3 escalation design will be used. At least three participants will be enrolled in each dose group, and the decision will be based on adverse events (AEs) related to ALMB-0168 evaluated according to NCI CTCAE version 5.0 within three weeks after the first dose is completed at that dose level (DLT observation period). If no DLT occurs in these three participants, the next group will be treated at the next lower dose level according to the dose escalation rules. At each dose level, if one participant experiences DLT, three more participants will be enrolled. Dose escalation will continue until at a dose level ≥1 / 3 of the participants (when ≥6 participants are evaluated) experience DLT. The tolerable dose levels prior to this dose level will be considered the mean time to treatment (MTD). The selection of RP2D from the tolerable dose range will be based on short-term and long-term safety information, as well as findings from preclinical dose-finding studies, pharmacokinetic (PK) studies, and antitumor activity studies. For dose groups that have completed the DLT observation period, investigators and sponsors may extend the study to up to eight participants (excluding the MTD extension cohort) after a comprehensive analysis of the benefits received from previous clinical data, based on an assessment of previous clinical data. This extension will provide further information on safety, PK characteristics, and antitumor activity. Safety data from the extended participants will not be used to determine the MTD.

[0341] Expansion phase

[0342] In the expansion phase, an expansion study will be conducted in 1-3 dose groups, with a maximum of 60 osteosarcoma patients enrolled in each dose group, to initially explore efficacy, further describe the safety and PK characteristics of ALMB-0168, explore efficacy, clarify PR2D, etc.

[0343] End of study definition

[0344] Participants are considered to have completed the study if they have completed all phases of dosing and the end-of-study visit (28 days after the last dose). The study end date is defined as the date of the last visit of the last patient in the study. The maximum follow-up period is 12 months. Therefore, the study will be considered closed when the last enrolled patient dies or when 12 months of follow-up is completed (whichever occurs first).

[0345] Study population

[0346] Selection criteria

[0347] 1. Patients with high-grade osteosarcoma confirmed by histopathology.

[0348] 2. Defined according to different stages:

[0349] a) Part I: Patients with osteosarcoma with bone lesions (primary or metastatic) who have failed standard treatment, including but not limited to:

[0350] i) Stage III osteosarcoma with bone metastases, with or without lung metastases, according to the American Joint Committee on Cancer (AJCC) bone tumor staging system;

[0351] ii) Unresectable locally advanced osteosarcoma;

[0352] iii) Multifocal osteosarcoma.

[0353] b) Part II: Patients with high-grade osteosarcoma who have failed standard treatment.

[0354] Standard treatment failure is defined as disease progression within 6 months during or after first-line or higher chemotherapy (including high-dose methotrexate, doxorubicin [cumulative dose > 350 mg], and cisplatin); for patients with disease progression more than 6 months later, investigators should conduct a risk-benefit assessment and obtain consent from the participant or their guardian.

[0355] 3. Patients aged ≥16 years, regardless of gender.

[0356] 4. Patients with an ECOG (Eastern Cooperative Oncology Group) PS score of 0, 1, or 2.

[0357] 5. Patients with measurable or non-measurable lesions according to RECIST v1.1 criteria. Non-measurable lesions should be confirmed by routine 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.

[0358] 6. The main system functions are as follows for patients:

[0359] a) Bone marrow reserve: Absolute neutrophil count (ANC) ≥ 1.5 × 10⁻⁶ 9 / L; Platelet count ≥75×10 9 / L; Hemoglobin ≥9g / dL, no blood transfusion in the past 14 days (participants who need blood transfusion);

[0360] b) Liver function: Total bilirubin ≤1.5 × upper limit of normal (ULN) (unless the patient has grade 1 bilirubin elevation due to Gilbert's disease or a syndrome similar to slow bilirubin conjugation). Transaminases (AST / SGOT and / or ALT / SGPT) ≤3 × ULN (<5 × ULN for patients with liver metastases);

[0361] c) Renal function: Serum creatinine is normal ≤1.5 mg / dL (133 μmol / L) or creatinine clearance is calculated to be ≥50 ml / min (Cockroft-Gault formula);

[0362] d) Coagulation: defined as International Normalized Ratio (INR) ≤ 2.

[0363] 7. Female participants of reproductive potential must have a negative serum or urine pregnancy test within 7 days prior to the first dose and must not be breastfeeding. Male participants with female partners of reproductive potential and female participants of reproductive potential must use two acceptable methods of contraception, including one barrier method, during the study period and for 3 months after the last dose. Male participants must avoid donating sperm during the study period; female participants must avoid donating eggs during the study period.

[0364] 8. Expected survival ≥ 3 months.

[0365] 9. Participants (or their guardians) must understand the entire research process, voluntarily participate in the research, and sign the ICF.

[0366] Intervention measures

[0367] ALAM-0168

[0368] ALMB-0168 is a sterile, pyrogen-free monoclonal antibody injection against human connexin 43, provided by the sponsor, AlaMab Therapeutics (Shanghai) Inc. Specifications: Each vial contains 6 ml (150 mg) of 25 mg / ml ALMB-0168 monoclonal antibody. Storage conditions: -20±5℃, protected from light, and avoid shaking.

[0369] ALMB-0168 will be administered via intravenous injection over at least 30 minutes. All participants will receive a single intravenous infusion of ALMB-0168 every 3 weeks. Participants will receive multiple cycles of treatment until disease progression or intolerable toxicity occurs.

[0370] Currently, there are no comparable drugs to ALMB-0168. Chronic active hemorrhagic inflammation and lymphopenia were observed in the central and peripheral regions of the spleen in preclinical animal studies. Therefore, participants should be closely monitored during the study for infusion reactions, infections, flu-like symptoms, rash, myalgia 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 observed for at least 48 hours post-infusion in an area equipped with resuscitation equipment and emergency medications.

[0371] During the study, if a participant experiences an adverse reaction, the investigator should provide necessary supportive care as needed and allow for the suspension of ALMB-0168 administration and / or dose adjustment.

[0372] If all ALMB-0168-related adverse events (AEs) occurring in a previous treatment cycle do not return to baseline or Grade 1 by the next scheduled treatment date, treatment should be delayed, and toxicity should be graded weekly. The date the participant receives the next cycle of ALMB-0168 treatment will be recorded as Day 1 of the next treatment cycle. If all ALMB-0168-related AEs return to baseline or NCI CTCAE Grade 1 within 3 weeks, dosing can continue.

[0373] If a participant requires a treatment delay of more than 21 days due to ALMB-0168-related toxicity occurring in a previous treatment cycle, the study treatment will be permanently discontinued, and the study will end upon completion of the treatment end visit 4 weeks after the last treatment.

[0374] Preparation / processing / storage / responsibility of ALMB-0168

[0375] The sponsor shall be responsible for delivering ALMB-0168 to the research center, and the researcher or a person designated by the researcher shall sign for receipt.

[0376] ALMB-0168 may only be used in this study and may only be managed by a designated person authorized by the investigator. The investigator / pharmacist responsible for administering the medication shall distribute, collect, and dispose of ALMB-0168 in accordance with the study procedures and maintain accurate records. Used ALMB-0168 and its packaging shall be disposed of and destroyed as medical waste at the research center, and unused or expired medication shall be collected and destroyed by the sponsor.

[0377] Instructions for rewarming, dilution, mixing, and preparation of ALMB-0168 should be recorded in the relevant instruction manual.

[0378] This study employed a non-random, open-label design.

[0379] The use of ALMB-0168 must strictly follow the clinical protocol, and the dose applied to each participant should be recorded in the eCRF.

[0380] Researchers at the research center should inspect and count the drugs during distribution, recall, and destruction, and sign off on the drug transfer, handover, and destruction documents.

[0381] Dosage form, appearance, packaging and labeling

[0382] The label should include the following:

[0383] Scheme Number: ALMB-0168-CN-101

[0384] Drug Number:

[0385] Medication: ALMB-0168 injection

[0386] Specifications: 150mg, 6ml / bottle

[0387] Production batch number:

[0388] Dosage form: Intravenous injection

[0389] Validity period:

[0390] Indication: High-grade osteosarcoma after failure of standard treatment

[0391] Instructions for use: See the instruction manual for details.

[0392] Storage conditions: -20℃±5℃. Protect from light and avoid shaking before use. The infusion solution should be used within 28 hours of preparation, including 24 hours at 2℃-8℃ and 4 hours at room temperature (18℃-26℃) (including storage and infusion time in the infusion bag). If refrigerated, the diluted infusion solution should be warmed to room temperature before administration.

[0393] The label should also include a statement: "This drug is provided by AlaMab Therapeutics (Shanghai) Inc. and is for clinical research purposes only."

[0394] Product storage and stability

[0395] ALMB-0168 should be stored at -20℃±5℃ and protected from light and shaking before use. The infusion solution should be used within 28 hours after preparation, including up to 4 hours of storage at room temperature (18℃-26℃) and up to 24 hours of refrigeration at 2℃-8℃ (including storage and infusion time of the infusion solution in the infusion bag).

[0396] The diluted infusion solution should not be frozen or shaken.

[0397] It should be ensured that ALMB-0168 remains stable within the intended shelf life under the intended packaging and storage conditions.

[0398] Combination therapy and treatment

[0399] Given that ALMB-0168 is a therapeutic monoclonal antibody, the likelihood of drug interactions between ALMB-0168 and small molecule formulations is low. Since ALMB-0168 is expected to degrade into amino acids and cycle to other proteins, it is unlikely to affect drug-metabolizing enzymes or transporters. Medications for treating pre-existing conditions or new conditions that develop during the study may be administered throughout the study. Blood products, analgesics, hypnotics, and other symptomatic medications may be administered at the investigator's discretion. Other medications necessary for the safety and health of participants, in addition to those listed in the exclusion criteria (Section 5.2) and Section 6.5.1, may be administered at the investigator's discretion. Investigators may administer any necessary concomitant medications or treatments to provide adequate supportive care.

[0400] All concomitant medications and treatments during the study period should be documented in detail in the eCRF. For concomitant or supportive treatments for osteosarcoma, the treatment name, indication (reason for use), dose, unit, frequency, start date, and stop date should be collected. For all other concomitant treatments, the treatment name, indication, start date, and stop date should be collected.

[0401] Combined medication use should include not only prescription drugs, but also all OTC drugs, traditional Chinese medicine, and vitamins.

[0402] Prohibited / Permitted Foods, Drugs and Treatments

[0403] During the study, all anti-tumor treatments (including cytotoxic chemotherapy, targeted therapies, radiotherapy, endocrine therapy, and traditional Chinese medicine with anti-tumor indications) and all other investigational drugs are prohibited. If a participant needs to use such drugs, they must first withdraw from the study.

[0404] The following medications may be used at the discretion of the researchers during the study period:

[0405] (1) Long-term medication required to treat comorbidities (such as hypertension and diabetes);

[0406] (2) Hormone replacement therapy or oral contraceptives not intended for anti-tumor treatment;

[0407] (3) Under the premise of protecting the interests and safety of the participants, the researchers should provide symptomatic and supportive treatment for the toxic reactions of the drug or for controlling tumor symptoms.

[0408] Research evaluation and procedures

[0409] Screening should be completed within 28 days prior to C1D1. Participants who meet all inclusion criteria and no exclusion criteria will be randomly assigned to groups.

[0410] Demographic data, including date of birth, sex, age, and nationality, will be collected from day -28 to day -1.

[0411] Height is measured only at screening. Weight will be measured during screening and at CXD1 (if the weight check at screening is completed within 72 hours before administration, there is no need to check weight at C1D1).

[0412] Medical history and other past medical history

[0413] Medical history refers to significant clinical information (or signs and symptoms, if a definitive diagnosis cannot be made) about past and comorbid diseases that occurred prior to signing the ICF, including past medical history and present medical history. For oncology, this includes the date of diagnosis, pathological classification, staging diagnosis, and screening stage. All medical history related to a participant's oncology diagnosis must be traced back to the original diagnosis. In addition, smoking history, alcohol consumption history, drug abuse history, surgical history, or significant clinical abnormalities found in relevant examinations at the time of screening will also be recorded as past medical history in the original medical record and eCRF.

[0414] Previous / concomitant medications, treatments, and previous anti-tumor therapies

[0415] Previous medication refers to any medication taken within 28 days prior to the date of administration of the study drug.

[0416] Concomitant use of medications and treatments refers to the simultaneous administration of two or more drugs / treatments, which in this study is defined as any drug / treatment administered at any time from the first administration of ALMB-0168 concurrently / concurrently to 28 days after the last administration.

[0417] During each visit from initial screening to the end of the study, researchers should inquire about any medications / treatments the participant has taken since the last visit. Any prior medications and concomitant treatments will be recorded in the original medical record and eCRF. For all concomitant medications, the following information will be recorded: drug / non-drug name, reason for use, dosage, unit, frequency of administration, route of administration, start date, and end date. For concomitant non-drug treatments, the name of the non-drug treatment, description of the treatment, reason for treatment, start date, and end date should be included. If the reason for using concomitant medications and treatments meets the definition of an AE, the relevant information should be recorded in the participant's original medical record and eCRF.

[0418] Treatment may include surgery, chemotherapy, radiation therapy, hormone therapy, or immunotherapy. The following data should be collected: treatment start and end dates, treatment regimen name, dosage, units, frequency, best response, and reason for discontinuation of treatment.

[0419] Security and other assessments

[0420] The following items are tested in the laboratory.

[0421] (1) Hematology: Red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), white blood cell count (WBC), platelet count (PLT), count and percentage of neutrophils, lymphocytes, eosinophils, basophils and monocytes, reticulocyte count, etc.;

[0422] (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;

[0423] (3) Myocardial enzyme profile: creatine kinase (CK) and its isoenzyme (CK-MB);

[0424] (4) Urine analysis: pH, specific gravity (SG), glucose (GLU), protein (PRO), red blood cells (RBC), white blood cells (WBC), ketone bodies (KET), occult blood, bilirubin, nitrite and urobilinogen;

[0425] (5) Coagulation function: prothrombin time (PT), activated partial thromboplastin time (APTT), international normalized ratio (INR), thrombin time (TT) and plasma fibrinogen (Fbg);

[0426] (6) Serum or urine pregnancy test: only applicable to women of childbearing potential;

[0427] (7) Viral serology: Hepatitis B testing (Hepatitis B surface antigen (HBsAg), Hepatitis B surface antibody (HBsAb), e antigen (HBeAg), e antibody (HBeAb) and core antibody (HBcAb), with further testing for HBV-DNA in patients with positive Hepatitis B surface antigen), Hepatitis C (Hepatitis C antibody, with further testing for HCV-RNA in patients with positive HCV antibody), AIDS (anti-HIV antibody) and syphilis (anti-Treponema pallidum specific antibody).

[0428] Hematologic tests (hematology and reticulocyte count), blood chemistry, myocardial enzyme profile, and urinalysis: performed at baseline (days -7 to -1), C1D1, C1D8, C1D15, C2D1, C2D8, C2D15, day 1 of each subsequent cycle, and at the end-of-study visit. If the C1D1 physical examination is expected to be performed within 72 hours of the baseline physical examination, it can be omitted.

[0429] Coagulation function: Performed at screening, before administration of D1 in each cycle, and at the end-of-study visit. This step can be omitted if the C1D1 physical examination is expected to be performed within 72 hours of the baseline physical examination.

[0430] Serum or urine pregnancy tests: performed at screening, before administration of D1 in each cycle, and at the end-of-study visit.

[0431] Viral serology: This will be tested during screening.

[0432] Researchers will examine all laboratory test results, assess changes in participants' laboratory test results relative to baseline based on laboratory reference ranges, determine the clinical significance of each abnormal laboratory value, and record them as "No Clinical Significance (NCS)" or "Clinically Significant (CS)" ("Normal Status" does not require labeling). Abnormal laboratory test results occurring during the study period, accompanied by the following symptoms, should be classified as adverse events and recorded in the original medical records and eCRF:

[0433] • Directly leads to participants withdrawing from the study;

[0434] • Leading to serious adverse consequences;

[0435] • Leads to obvious clinical symptoms;

[0436] • Researchers believe it has clinical significance.

[0437] The reviewed laboratory report will be signed and dated by the researcher.

[0438] Physical examination, including skin, mucous membranes, lymph nodes, head (including skull, ears, eyes, nose, mouth, and throat), neck, chest (including thorax, breasts, lungs, and heart), abdomen (including liver, gallbladder, spleen, kidneys, bladder, stomach, and intestines), spine / limbs, nervous system, etc.

[0439] Physical examinations will be performed at baseline (days -7 to -1), C1D1, C1D8, C1D15, C2D1, C2D8, C2D15, D1 of each subsequent cycle, and at the end-of-study visit. If the C1D1 physical examination is expected to be performed within 72 hours of the baseline physical examination, it can be omitted. Any clinically significant abnormalities compared to baseline should be recorded as adverse events (AEs).

[0440] Vital signs include sitting blood pressure, respiration, pulse, and temperature. Vital signs checks will be performed at baseline (days -7 to -1), C1D1, C1D8, C1D15, C2D1, C2D8, C2D15, D1 of each subsequent cycle, and at the end-of-study visit. If the C1D1 physical examination is expected to be performed within 72 hours of the baseline physical examination, it can be omitted. Any clinically significant abnormalities compared to baseline should be reported as adverse events (AEs).

[0441] Sit-and-reach systolic and diastolic blood pressure should be measured with the same arm each time, and should be taken at least 5 minutes after the participant has rested in a seated position. All records will be taken using standard equipment; automated or manual measurements are acceptable. However, researchers should continue to use the same methods employed for the first measurement for all assessments of individual participants.

[0442] Participants' physical condition will be assessed using the ECOG Performance Status Scale (see Appendix I). Examinations will be conducted at baseline (days -7 to -1), C1D1, C2D1, D1 of each subsequent cycle, and at the end-of-study visit. If the C1D1 physical examination is expected to be performed within 72 hours of the baseline physical examination, it can be omitted.

[0443] The examinations will be performed at baseline (days -7 to -1), 30 minutes (±10 minutes) before and 30 minutes after administration of C1D1, C1D8, and C1D15, 30 minutes (±10 minutes) before and 30 minutes (±10 minutes) after administration of D1 in each subsequent cycle, and at the end-of-study visit. Investigators may increase the frequency of examinations based on participant condition. Participants should rest in a supine position for at least 5 minutes before undergoing any of the three 12-lead ECG examinations.

[0444] The examination was conducted at baseline (day -14 to day -1). Additionally, researchers may perform examinations as clinically necessary during the study. Participants should rest in a supine position for at least 5 minutes before undergoing a UCG examination.

[0445] Examinations will be conducted at baseline (day -28 to day -1). During the study, researchers will conduct examinations as needed clinically.

[0446] The event schedule is summarized in Table 4 below.

[0447]

[0448]

[0449]

[0450]

[0451]

[0452] Relief Evaluation

[0453] Tumor assessment

[0454] Imaging procedures for appropriate tumor-related anatomical regions include: CT, MRI with contrast enhancement and / or PET-CT of the tumor-related site, CT or MRI of the head, and technetium-99 isotope bone scan.

[0455] For tumor assessments conducted during screening, participants within 4 weeks prior to drug administration had acceptable outcomes. During screening, investigators should select appropriate tumor assessment methods based on the participant's condition, and tumor assessments during follow-up should be performed using the same methods as baseline assessments. The first tumor assessment during the study will be conducted at week 6 (at the end of C2), followed by assessments every 9 weeks (3 cycles). Additional scans may be performed during the study if clinical symptoms are detected.

[0456] Relief assessment will be conducted using both RECIST v1.1 (Appendix III) and ICDS criteria (Appendix VI). Radiologists should be emphasized that, in addition to size assessment, density assessment will also be performed using ICDS criteria.

[0457] Study endpoints

[0458] Objective response rate (ORR): defined as the proportion of patients who achieved a best response of complete response (CR) or partial response (PR) (i.e., CR + PR) from the start of ALMB-0168 treatment to withdrawal from the study, as assessed according to the RECIST v1.1 criteria for evaluating efficacy in solid tumors (see Appendix III; ICDS, Appendix VI). Participants initially assessed as CR or PR should be confirmed at least 4 weeks later.

[0459] Progression-free survival (PFS): refers to the time from the start of ALMB-0168 treatment to the date of first recorded disease progression (PD) or death (whichever comes first).

[0460] The 6-month progression-free survival rate (6m-PFSR) is defined as the percentage of participants who survive 6 months without PD starting ALMB-0168.

[0461] Duration of Response (DoR): defined as the time from the initial evaluation of CR or PR (best response) until the date the participant first shows evidence of tumor progression or death.

[0462] Time to Response (TTR): Defined as the time from the first dose to the first objective response (PR or CR).

[0463] Disease control rate (DCR): defined as the percentage of evaluable participants who achieved PR+CR remission or disease stability (SD) after treatment.

[0464] Overall survival (OS): defined as the time from the start of research treatment to death from any cause.

[0465] The rate of change of alkaline phosphatase (ALP) or lactate dehydrogenase (LDH) relative to baseline.

[0466] Skeletal events (SREs): These include changes in the number of pathological fractures, spinal cord compressions, malignant tumor-related hypercalcemia, and disease-reducing radiotherapy or surgery targeting bone lesions relative to baseline.

[0467] Changes in NRS scores relative to baseline (see Appendix IV for details).

[0468] Changes in EQ-5D scores relative to baseline (see Appendix V for details).

[0469] Bone mineral density of the lumbar spine, hip, femur and neck: measured by dual-energy X-ray absorptiometry (DXA), collecting changes relative to baseline.

[0470] Variations in morphine dosage and frequency relative to baseline.

[0471] For the time points of efficacy assessment indicators including ALP, LDH, SRE, NRS, EQ-5D, bone mineral density, and morphine use, please refer to Table SoA.

[0472] Relief Standard

[0473] Researchers will conduct imaging assessments during the screening period and each treatment cycle according to the RECIST v1.1 criteria and ICDS criteria. The same imaging techniques as at baseline should be used throughout the study, and lesions can be categorized as measurable or non-measurable. If a participant's tumor-related symptoms worsen during treatment, an earlier tumor assessment may be performed at the researcher's discretion. For participants who discontinue treatment before disease progression, tumor assessments will be performed every 3 months after the last dose.

[0474] Participants whose imaging studies show disease progression should withdraw from the study.

[0475] In rare cases, if researchers believe the treatment is beneficial to the participants, those with radiographically confirmed PD may continue the study treatment. Consultation with the sponsor is required beforehand.

[0476] If researchers deem it necessary, they may perform imaging procedures other than those specified in the protocol (such as PET-CT), and may evaluate remission according to the PERCIST criteria. The above data may be included and aggregated for efficacy assessment.

[0477] Skeletal-related events (SREs), morphine dosage and frequency, NRS and EQ-5D scores should be collected at their respective visits (see SOA for details).

[0478] Pharmacokinetics, Immunogenicity and Biomarker Evaluation

[0479] PK index

[0480] Blood samples for PK will be collected from the contralateral arm at the following time points specified by the SoA. The number and timing of PK samples may vary depending on the specific circumstances of the study. In the event of a transfusion reaction or serious adverse event, the investigator may cooperate with the laboratory on the same visit to test the collected blood and perform unplanned PK sampling.

[0481] Intensive sampling will be performed on all participants in the incremental phase and the first 10 participants in the expansion phase. Intensive sampling time points: before (within 30 minutes) of C1D1 administration, and immediately after (within 5 minutes), 2 hours (±15 minutes), 4 hours (±30 minutes), and 8 hours (±1 hour) after administration; 24 hours (±2 hours) after C1D2 administration and 48 hours (±2 hours) after C1D3 administration; 168 hours (±6 hours) after C1D8 administration and 336 hours (±12 hours) after C1D15 administration. Sparse sampling will also be performed before (within 30 minutes) and immediately after (within 5 minutes) of administration on D1 of each subsequent cycle; and at the end-of-study visit. 2. The remaining participants in the expansion phase will be sparsely sampled. Sparse sampling time points: before (within 30 minutes) and immediately after (within 5 minutes) of administration on D1 of each cycle; and at the end-of-study visit.

[0482] PK Index: C max t max AUC 0-t C trough , and CL, V z , λ z t 1 / 2 AUC 0-inf (For participants in intensive sampling only).

[0483] Immunogenicity indicators

[0484] To evaluate the immunogenicity of ALMB-0168, anti-drug antibodies in blood samples should be detected. Participant samples used to analyze immunogenicity / drug concentration can also be used to assess safety or efficacy, resolve issues arising during or after the study, or assist the sponsor or its designated personnel in evaluating relevant biomarkers.

[0485] Blood samples will be collected before D1 administration (within 30 minutes) in cycles 1, 2, 3, 5, and 7, and at the end of the study (4 weeks after the last treatment). Samples will be used for anti-drug antibody (ADA) testing.

[0486] Blood samples used to determine anti-ALMB-0168 antibody (ADA) will be collected from the contralateral arm at the time points specified by SoA.

[0487] Statistical considerations

[0488] This study did not have formal statistical hypotheses, and the statistical analysis results are exploratory.

[0489] Sample size estimation

[0490] The Phase I portion of this study includes accelerated titration and a standard “3+3” dose escalation. The first two dose groups enrolled 1 to 6 participants, and the third and subsequent dose groups enrolled 3 to 6 participants, for a maximum of seven dose groups, including 10 participants administered at the MTD level. For dose groups that have completed the DLT observation period, expansion to a maximum of eight participants (excluding the MTD expansion cohort) may be considered after the investigator and sponsor have evaluated previous clinical data and analyzed participant benefits. Therefore, the maximum number of participants should not exceed 58.

[0491] The Phase II dose expansion will enroll up to 180 participants, depending on the final number of participants.

[0492] Analyzing the population

[0493] Full Analysis Set (FAS): Includes all participants who received at least one dose of ALMB-0168 after successful enrollment according to the intention-to-treat (ITT) principle. FAS was used for demographic and baseline characteristic analyses.

[0494] The Protocol Compliance Set (PPS) is defined as a subset of participants in the full analysis set (FAS). Participants who meet the inclusion and exclusion criteria, have undergone at least one post-baseline tumor assessment, have medication adherence between 80% and 120%, and have not committed any serious violations of the clinical study protocol.

[0495] Pharmacokinetic 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 their plasma concentration from the PKS include, but are not limited to, factors affecting plasma concentrations such as incorrect treatment, concomitant medication, improper blood sample handling, and incorrect blood sample testing.

[0496] Safety Analysis Set (SS): The SS will include all participants who have received at least one dose of ALMB-0168 and underwent a safety evaluation. Unless otherwise specified, all analyses will be based on the SS. The definition of the SS set is consistent with that of the FAS set.

[0497] Anti-drug antibody set (ADA set, ADAS): Participants who have received at least one dose of ALMB-0168 and have available ADA data.

[0498] Evaluable efficacy analysis set (EES): includes all participants who have received at least one dose of the study drug and have received at least one post-dose efficacy evaluation.

[0499] Pharmacokinetic concentration set (PKCS): includes all participants who have received at least one dose of the study drug and have at least one evaluable plasma concentration data.

[0500] Pharmacokinetic parameter set (PKPS): Includes all participants who have received at least one dose of the study drug and have at least one evaluable PK parameter.

[0501] Immunogenicity analysis set (IS): 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.

[0502] Biomarker analysis set: includes all enrolled participants who have received at least one dose of the study drug and have at least one biomarker data.

[0503] Statistical analysis

[0504] The detailed summary and statistical analysis methods for the data collected in this study will be included in the Statistical Analysis Plan (SAP). The SAP will be developed after the protocol and CRF are finalized and will be finalized before the database is locked. The SAP will detail and describe the statistical analysis for all plans based on the key features of the protocol. Any changes to the protocol that, in the opinion of the sponsor or principal investigator, have a significant impact on the Statistical Analysis Plan will require a new SAP revision to ensure consistency with the research protocol.

[0505] For statistical descriptions, categorical variables will be described using the number of participants and percentage (%); continuous variables will be described using the number of participants, missing values, mean, standard deviation, median, quartiles, minimum, and maximum values.

[0506] For participant management, the process will be summarized and presented, including the enrollment, medication administration, withdrawal or exclusion of participants in each group, as well as the data set division, and a flowchart of the participant management process will be provided.

[0507] Statistical descriptions of demographic data and baseline characteristics: For continuous variables in the baseline data, the number of participants, mean, standard deviation, median, minimum, and maximum values ​​will be calculated; for count and tiered data, the number of participants and percentage will be calculated. Participant entry statistics, baseline characteristics, medical history, and treatment history will be presented in a detailed list.

[0508] Pharmacokinetics and Immunogenicity

[0509] 1. Pharmacokinetic Analysis

[0510] Mean (mean ± SD) dose-time curves for ALMB-0168 will be plotted for each dose group. Individual pharmacokinetic (PK) parameters for each participant will be listed, and descriptive statistical analyses (mean, median, range, standard deviation, coefficient of variation, etc.) will be performed on the PK parameters.

[0511] PK parameter: C max t max C trough , and CL, Vz, λz, t 1 / 2 AUC 0-t AUC 0-inf (For participants in intensive sampling only)

[0512] 2. Immunogenicity analysis :

[0513] The number and percentage of ADA-positive participants will be summarized and presented.

[0514] Security Analysis

[0515] 1. AEs and drug-related AEs .

[0516] The analysis of adverse events (AEs) and drug-related AEs will summarize the following data for each group of participants: number of participants, number and percentage of AEs, drug-related AEs, AEs leading to study termination, AEs leading to death, and SAEs.

[0517] The systemic organ classification (SOC) and preferred term (PT) for adverse events (AEs) and drug-related AEs will be coded according to the MedDRA dictionary, and the number of participants, the number of AEs, and the percentage of AEs and drug-related AEs will be summarized by SOC / PT:

[0518] The number of participants, the number of adverse events (AEs), and the percentage of AEs and drug-related AEs will be summarized by SOC / PT and severity. Under the same term (SOC or PT), a participant's AE of a certain category will be counted only once at the highest severity.

[0519] All adverse events (including those occurring outside of medication use), drug-related adverse events, subacute adverse events (SAEs), adverse events leading to study termination, adverse events leading to death, and adverse events of particular concern (AESIs) will be listed separately.

[0520] 2. Laboratory tests

[0521] Each time point after baseline and the time of the last visit. The measurements at each time point, the minimum and maximum values ​​of the measurements after baseline, the observations at the last visit, and the changes relative to baseline should be statistically analyzed according to the dose group and the total.

[0522] We will compare the normal and abnormal changes of various indicators before and after treatment using a contingency table of clinical assessments. Laboratory test indicators will be listed in detail by group.

[0523] 3. Vital signs

[0524] The statistical time points for vital sign examinations include baseline, each visit after baseline, and the visit time point for early withdrawal. Descriptive statistics at baseline, post-baseline visit time points, and at the end of the study will be statistically analyzed by group and total. Then, a clinical judgment contingency table will be used to compare normal and abnormal changes in various indicators before and after treatment (if applicable).

[0525] 4. Physical examination and ECG

[0526] Physical examination items: general condition, skin, head and neck, chest, abdomen, back, limbs, nerves, and mental status. Normal and abnormal changes before and after treatment are compared in a clinical assessment contingency table for each dosage group.

[0527] Electrocardiogram (ECG) examination included heart rate, PR, QRS complex, and QTc. Statistical analysis was performed on baseline, post-baseline visits, minimum and maximum values ​​of post-baseline measurements, last visit observations, and changes relative to baseline, categorized by dose group and total. Normal and abnormal changes before and after treatment were compared in each dose group using a pre- and post-treatment clinical judgment contingency table.

[0528] List the physical examinations and ECG examinations in detail, grouped by group.

[0529] 5. ECOG rating

[0530] The statistical time points for ECOG scores include baseline, each visit after baseline, and the last visit. Descriptive statistics will be performed by cohort at baseline, each visit after baseline, and at the end of treatment, and changes in ECOG scores before and after treatment will be compared using a contingency table.

[0531] Therapeutic effect analysis

[0532] According to RECIST V1.1 and ICDS standards, remission can be evaluated as PD, SD, CR, and PR. Specific analytical indicators include, but are not limited to, the following:

[0533] • Objective Response Rate (ORR): This will summarize the number and percentage of participants who achieved an objective tumor response (CR or PR).

[0534] • Overall survival (OS): refers to the time from randomization to death from any cause. Alternatively, for patients lost to follow-up, it is the time to the last follow-up; or for participants still alive at the end of the study, it is the time to the end of follow-up. The last contact date is the most recent date on which a participant's survival was known.

[0535] • Progression-free survival (PFS): Defined as the period from first dose to disease progression or death (whichever occurs first). For participants who did not experience disease progression or death, the date of their last tumor assessment will be used as the endpoint, and their data will be treated as censored values.

[0536] • 6-month progression-free survival (6m-PFSR): Defined as the percentage of participants who survive 6 months without PD starting ALMB-0168.

[0537] • Time to Response (TTR): Defined as the time from the first dose to the first objective tumor response (CR or PR).

[0538] • Duration of Response (DoR): The time from the initial assessment of CR or PR (best response) until the date the participant first shows evidence of tumor progression or dies.

[0539] • Disease control rate (DCR): Defined as the percentage of evaluable participants who achieve PR+CR remission or disease stability (SD) after treatment.

[0540] • Participant remission will be evaluated according to RECIST 1.1 and ICDS criteria. ORR and DCR will be calculated, and 95% confidence intervals (CIs) for ORR and DCR will be calculated using the Clopper-Pearson exact method. DoR, PFS, and TTR will be estimated using the Kaplan-Meier method, and the median DoR, PFS, TTR, truncation rate, quartiles, and their 95% CIs will be listed. Kaplan-Meier curves will also be plotted.

[0541] • Numerical Rating Scale (NRS) score analysis: describes the NRS score at each visit and the change relative to baseline.

[0542] • European Five-Dimensional Health Scale (EQ-5D) score: describes the EQ-5D score at each visit and the change relative to baseline.

[0543] • If the investigators deem it necessary, imaging procedures other than those specified in the protocol (such as PET-CT) may be performed, and remission can be evaluated according to the PERCIST criteria. These data can be included and aggregated for efficacy assessment.

[0544] appendix

[0545] Appendix I ECOG Performance Status Scale

[0546] The ECOG performance status scale is shown in Table 5.

[0547] Table 5. ECOG Performance Status Scale

[0548]

[0549] Appendix II New York Heart Association (NYHA) Functional Classification

[0550] The New York Heart Association functional classification is shown in Table 6.

[0551] Table 6. NYHA Classification of Cardiac Function

[0552]

[0553] Appendix III: Excerpt from the Evaluation Criteria for Treatment of Solid Tumors (RECIST 1.1)

[0554] RECIST Standard History

[0555] Assessing changes in tumor burden is a crucial characteristic of clinical evaluation in the treatment of malignant tumors. Tumor shrinkage (objective response) and time to progression are both important endpoints in clinical research on malignant tumors. Years of research evidence support the use of tumor shrinkage as an endpoint in phase II studies for screening new anti-tumor drugs. These studies demonstrate that for various solid tumors, drugs that induce tumor shrinkage in certain patients are likely (though not perfectly) to improve overall survival or have other opportunities for inclusion in event evaluation in randomized phase III studies. Currently, objective response is more reliable than any other biomarker when evaluating the efficacy of phase II screening studies. Furthermore, in phase II and III clinical studies of advanced patients, time to progression (or progression-free survival) is increasingly being used as an endpoint to determine efficacy, also based on anatomical measurements of tumor size.

[0556] However, the two endpoints for tumor assessment—objective response and time to progression—are only valuable when supported by widely accepted and easily usable standards based on tumor burden anatomy. The criteria for tumor response were initially published by the World Health Organization (WHO) in 1981, primarily for trials with tumor response as the primary endpoint. The WHO criteria introduced the concept of a comprehensive assessment of tumor burden by calculating the sum of the two-dimensional dimensions of the lesions, evaluating treatment response by changes relative to baseline during treatment. However, in the decade following its publication, oncology groups and pharmaceutical companies using these criteria frequently modified them to accommodate new technologies or address unclear points in the original literature, leading to confusion in the interpretation of research results. In fact, the application of various response criteria resulted in significant differences in treatment outcomes for the same treatment method. To address these issues, 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 (Responsiveness Evaluation Criteria for Solid Tumors), were published in 2000. Key features of the initial version of RECIST include: defining a minimum measurable lesion size, describing the number of lesions to be followed up (up to 10; up to 5 per organ), and using one-dimensional (rather than two-dimensional) measurements to assess the overall tumor burden. These criteria are widely adopted by the academic community, oncology community, and pharmaceutical industry, with objective response or disease progression as the initial endpoint. Furthermore, the NMPA accepts RECIST as the appropriate standard for these assessments.

[0557] Purpose of the Guide

[0558] This guideline describes standard measurement methods for solid tumors in clinical studies of malignant tumors in adults and children, and defines objective criteria for determining changes in tumor size. These criteria are expected to be used in all studies with objective response as the primary endpoint, as well as studies using disease stabilization, tumor progression, or time to progression as indicators, because all efficacy measurements are based on an assessment of anatomical tumor burden and its changes in the study. This document does not presuppose the proportion of patients meeting the endpoint criteria for predicting the efficacy of a drug or treatment regimen: these definitions depend on the type of malignancy in the ongoing study and the specific drug being investigated. Study protocols must include an appropriate statistical section defining the sample size and the efficacy parameters upon which inclusion criteria are based. This guideline not only provides definitions and criteria for determining tumor response but also offers recommendations for standard reporting of clinical study results with tumor response as the endpoint.

[0559] While these guidelines can be used for research on malignant brain tumors, response evaluation criteria in this field have been published separately. Because international guidelines for evaluating response in lymphoma have also been published separately, these guidelines are not applicable to research on malignant lymphomas.

[0560] Finally, many oncologists rely on repeated imaging studies in their daily clinical practice to follow up on patients' tumor lesions and to determine further treatment based on objective indicators and symptoms. These RECIST guidelines should generally not be used for clinical decision-making unless the oncologist deems them suitable for treatment decisions.

[0561] Baseline tumor measurement

[0562] At baseline, tumor lesions / lymph nodes were classified as measurable and non-measurable, as described below.

[0563] 1. Measurable lesions

[0564] Tumor lesions: Tumor lesions. A tumor lesion has at least one accurately measurable diameter (recorded as the longest diameter in the measuring plane), and its minimum length is as follows:

[0565] • For computed tomography (CT) scans (CT slice thickness not exceeding 5 mm), the value is 10 mm.

[0566] • Clinical examination using calipers, with a measurement of 10mm (lesions that cannot be accurately measured with calipers should be recorded as unmeasurable).

[0567] • For chest X-rays, the value is 20mm.

[0568] Malignant lymph nodes: When evaluated by CT (CT slice thickness is recommended not to exceed 5 mm), the short axis of the lymph node must reach 15 mm to be considered pathologically enlarged and measurable. Only the short axis length is measured and followed up preoperatively and during follow-up.

[0569] 2. Unmeasurable lesions

[0570] All other lesions, including small lesions (maximum diameter less than 10 mm or short axis of pathological lymph nodes of 10 mm to less than 15 mm) and truly unmeasurable lesions. Lesions considered truly unmeasurable include leptomeningeal lesions, ascites, pleural or pericardial effusions, inflammatory mastopathy, lymphatic vessels infiltrating the skin / lungs, peritoneal dissemination, and abdominal masses / enlarged abdominal organs that cannot be confirmed by repeat imaging techniques and physical examination.

[0571] 3. Special considerations regarding measurable lesions

[0572] Special attention should be paid to bone lesions, cystic lesions, and lesions that have undergone local treatment. A summary is as follows: Bone lesions:

[0573] • Bone scans, positron emission tomography (PET), or conventional X-rays are not considered appropriate imaging techniques for measuring bone lesions. However, these techniques can be used to confirm the presence or disappearance of bone lesions.

[0574] • For osteolytic lesions or mixed osteolytic / osteoblastic lesions with a clear soft tissue component, if the soft tissue component meets the definition of measurability above, they can be evaluated as measurable lesions using tomographic imaging techniques (such as CT or MRI).

[0575] • Osteoblastic lesions are not measurable. Cystic lesions:

[0576] • Lesions that meet the definition criteria for simple cysts on radiographic imaging should not be considered malignant (neither measurable nor unmeasurable) because they are defined as simple cysts.

[0577] • A cystic lesion can be considered a measurable lesion if it represents a cystic metastatic lesion and meets the measurability definition above. However, if non-cystic lesions are present in the same patient, non-cystic lesions should be preferred as target lesions.

[0578] Lesions treated locally :

[0579] • Tumor lesions located in previously irradiated areas or sites treated with other localized areas are generally considered unmeasurable lesions unless they show clear progression. The study protocol should specify under what conditions such lesions will be considered measurable.

[0580] Measurement Method Specifications

[0581] 1. Lesion measurement

[0582] All measurements should be recorded using metric units, or calipers if clinically evaluated. All baseline assessments must be performed as close as possible to the start of treatment, but no earlier than four weeks.

[0583] 2. Measurement Method

[0584] The same assays and techniques should be used when describing each identified and reported lesion at baseline and during follow-up. Imaging examination (rather than clinical examination) should generally be performed unless the lesion is found to be unsuitable for imaging at follow-up.

[0585] Clinical lesions: Only superficial lesions (such as subcutaneous nodules) with a diameter greater than or equal to 10 mm, as measured by calipers, are considered measurable. For cases of skin lesions, it is recommended to use color photographs with the lesion size proportions as measured. As mentioned earlier, when a lesion can be examined by both clinical and imaging methods, imaging should be used because imaging evaluation is more objective and can be used for final review in clinical studies.

[0586] Chest X-ray: Chest CT is preferred over chest X-ray, especially when disease progression is used as an important endpoint, as CT scans are more sensitive than X-rays in identifying new lesions. However, if an X-ray shows a well-defined lesion surrounded by inflated lung tissue, it is considered measurable.

[0587] CT and MRI: CT is currently the most effective and reproducible method for evaluating lesion response. As defined in the guidelines, the CT slice thickness should not exceed 5 mm for measurable lesions. When the CT slice thickness exceeds 5 mm, the minimum measurable lesion should be twice the slice thickness. In certain situations (such as whole-body scans), MRI can also be used.

[0588] Ultrasound examination: Ultrasound is not suitable for evaluating lesion size and should not be used as a measurement method. Ultrasound examinations cannot be perfectly reproduced between two adjacent observations. Furthermore, results depend on the examiner. Technical and measurement results cannot be guaranteed to be identical between one examination and its subsequent examination. If a new lesion is detected by ultrasound during the study, CT or MRI verification is recommended. If there are concerns about CT radiation exposure, MRI can be used instead of ultrasound to examine the lesion.

[0589] Endoscopy and laparoscopy: These techniques are not recommended for objective tumor evaluation. However, they can help confirm complete pathological remission through biopsy, or determine complete remission or recurrence after surgical resection.

[0590] Tumor markers: Tumor markers cannot be used alone to evaluate objective response to tumors. However, when tumor markers begin to rise above the upper limit of normal, they must be brought down to the normal range if used to determine complete response in a patient. Because tumor markers are disease-specific, indications for disease-specific testing should be included in the protocol. Specific guidelines have been published regarding changes in CA-125 (ovarian cancer recurrence) and PSA changes (prostate cancer recurrence). Furthermore, the Gynecologic Cancer Collaboration has developed CA-125 progression criteria for clinical studies using objective tumor markers as first-line treatment for ovarian cancer.

[0591] Cytology and histology: These techniques can often be used to differentiate between partial and complete remission in individual cases, if required by the clinical trial protocol (e.g., residual benign tumor lesions in a tumor type of germ cell tumor). When 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 remission or disease stability require cytological confirmation of neoplastic exudation that occurred or worsened during treatment in order to differentiate between remission (or disease stability) and disease progression.

[0592] Tumor remission evaluation

[0593] 1. Evaluation of all tumors and measurable lesions

[0594] To evaluate objective response or potential future progression, it is necessary to perform a baseline assessment of the total tumor burden of all tumor lesions as a reference for subsequent measurements. In clinical protocols with objective response as the primary treatment endpoint, only patients with measurable lesions at baseline will be included. A measurable lesion is defined as having at least one measurable lesion. For studies with disease progression (time to progression or degree of progression at a fixed date) as the primary treatment endpoint, the inclusion criteria must clearly define whether the study is limited to patients with measurable lesions or whether patients without measurable lesions can also be included.

[0595] 2. Baseline records of target lesions and non-target lesions

[0596] When there is more than one measurable lesion at baseline, all lesions should be recorded and measured, totaling no more than 5 (no more than 2 per organ), as target lesions representing all affected organs (i.e., patients with only one or two affected organs should select a maximum of two or four target lesions as baseline measurement lesions).

[0597] Target lesions must be selected based on lesion size (maximum diameter), representing all affected organs, and measurements must be repeatable. If the largest lesion cannot be measured repeatedly, another largest lesion that can be measured repeatedly should be selected.

[0598] Lymph nodes can still be imaged within normal anatomical structures without tumor metastasis, therefore special care is required. Pathological lymph nodes, defined as measurable nodules or even target lesions, must meet the following criteria: a short diameter ≥15 mm as measured by CT. Only the short diameter needs to be measured at baseline. Radiologists typically use the short diameter of a nodule to determine the presence of tumor metastasis. Nodule size is usually represented by two-dimensional data from imaging examinations (axial plane is commonly used for CT scans, while axial, sagittal, or coronal planes are selected for MRI). The short diameter is determined by the minimum value taken. For example, an abdominal nodule measuring 20 mm × 30 mm with a short axis diameter of 20 mm can be considered a malignant, measurable nodule. In this example, 20 mm is the measurement of the nodule. Nodules with a diameter ≥10 mm but <15 mm should not be considered target lesions. Nodules with a diameter <10 mm are not considered pathological and should not be recorded or further observed.

[0599] The sum of all target lesion diameters (including the maximum diameter of non-nodular lesions and the short axis diameter of nodular lesions) will be the sum of baseline diameters. If lymph node diameters are involved, only the short axis will be included, as described above. The sum of baseline diameters will be used as a reference for baseline disease.

[0600] All remaining lesions, including pathological lymph nodes, should be considered non-target lesions and do not require measurement, but should be recorded at baseline as follows: "Present", "Absent", or, in rare cases, "Clear Progression". Extensive non-target lesions in the same organ may be recorded as a single item on the case report form, such as numerous enlarged pelvic lymph nodes or extensive liver metastases.

[0601] 3. Mitigation Evaluation Criteria

[0602] 3.1 Evaluation of Target Lesion Response

[0603] Complete remission (CR): All target lesions disappear and the short axis diameter of any pathological lymph node (whether or not a target lesion) must be <10 mm.

[0604] Partial remission (PR): The sum of the diameters of all target lesions is reduced by at least 30% relative to the sum of the diameters at baseline.

[0605] Disease progression (PD): The sum of the diameters of all target lesions increases by at least 20% relative to the sum of the diameters of the smallest lesion during the study period (including the sum of the baseline diameters of the lesions, if it is the smallest). In addition to a relative increase of 20%, the sum of diameters must also demonstrate an absolute increase of at least 5 mm (Note: the appearance of one or more new lesions can also be considered as progression).

[0606] Disease stability (SD): Based on the total diameter of the smallest lesions during the study period, lesion shrinkage did not reach PR, and lesion enlargement did not reach PD.

[0607] 3.2. Precautions for evaluating the response of target lesions

[0608] When the target lesion is located in a lymph node :

[0609] The measured short-axis diameter (in the same anatomical plane as the baseline measurement) should be recorded, even if the lymph nodes shrink to less than 10 mm during the study. This means that when target lesions are present in lymph nodes, the sum of the diameters of the target lesions will not be zero, even if the criteria for complete remission are met, because lymph nodes with a short-axis diameter <10 mm are considered normal. Case report forms or other data collection methods can be designed to record nodular target lesions separately. For complete remission, the short-axis diameter of each lymph node must be <10 mm. For PR, SD, and PD, the sum of the diameters of the target lesions will include the actual short-axis diameter of the lymph nodes.

[0610] When the target lesion is too small to be measured :

[0611] 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 too weak a signal on CT scans for the radiologist to measure accurately and may be reported as too small to measure. In such cases, it is important to record a measurement in the case report form. If the radiologist believes the lesion may have disappeared, the measurement can be recorded as 0 mm. If the lesion is present but has a weak signal, a default value of 5 mm should be specified (Note: This rule does not apply to normal lymph nodes, as they typically have a definite size and are often surrounded by fat, such as in the retroperitoneum; however, a default value of 5 mm should also be specified if the lymph node is present but has a weak signal that prevents measurement). The default value of 5 mm is based on the thickness of the CT scan (the default value of 5 mm will not change with this thickness). Measurements of such lesions (too small to measure) may lack reproducibility, so a default value should be specified to prevent evaluation as false remission or false progression due to measurement error. To reiterate, if the radiologist can provide the actual measurement, even if it is less than 5 mm, it should be recorded.

[0612] When the lesion splits or merges during treatment

[0613] When separating non-nodular lesions, the maximum diameters of all fragments must be added together to calculate the total diameter of the target lesion. Similarly, when lesions fuse, the major axis diameter can be preserved, which helps to obtain the maximum diameter of each individual lesion before fusion. If the lesions have completely fused to the point that they can no longer be separated, then the maximum diameter vector in this case should be the maximum diameter of the fused lesion.

[0614] 3.3. Evaluation of non-target lesions

[0615] This section defines the response criteria for non-target tumors. Although some non-target lesions are actually measurable, measurement is unnecessary; only qualitative evaluation at the time points specified in the protocol is required.

[0616] Complete remission (CR): All non-target lesions disappeared, and tumor markers returned to normal. All lymph nodes were of non-pathological size (short diameter <10mm).

[0617] Incomplete remission / non-disease progression (non-CR or non-PD): The presence of one or more non-target lesions and / or tumor markers that are persistently elevated above normal levels.

[0618] Disease progression (PD): Existing non-target lesions show clear progression. Note: The appearance of one or more new lesions is also considered disease progression.

[0619] 3.4. Precautions for evaluating the progression of non-target lesions

[0620] The definition of non-target lesion progression is further explained as follows: :

[0621] When a patient has measurable lesions: In this case, for clear progression of non-target lesions, the non-target lesions have worsened to the point where treatment must be discontinued. Even if target lesions are evaluated as stable or in partial remission, the overall tumor burden has increased sufficiently to justify discontinuing treatment. A slight increase in one or more non-target lesions is generally insufficient to be considered clear progression. It is extremely rare to define overall tumor progression solely by changes in non-target lesions when target lesions are stable or in partial remission.

[0622] When a patient has no measurable non-target lesions: This occurs when the inclusion criteria for certain phase III studies do not require the presence of measurable lesions. The overall assessment remains based on the aforementioned criteria, but in this case, there is no measurable data for lesions. Progression in non-target lesions is difficult to assess (by definition: all lesions are truly unmeasurable). Therefore, when changes in non-target lesions lead to an increase in overall disease burden, equivalent to disease progression in target lesions, progression should be clearly defined based on the definition of non-target lesions, and an effective diagnostic method should be established for evaluation. For example, an increase in tumor burden represents an additional 73% increase in volume (equivalent to a 20% increase in the diameter of a measurable lesion). Another example is peritoneal effusion changing from “small” to “large”; lymphatic lesions changing from “local” to “extensive”; or “modification of treatment based on sufficient evidence” as described in the protocol. In cases of clear progression, the patient should be considered to have experienced overall disease progression at that point in time. While ideally, unmeasurable lesions should be evaluated using objective criteria, there may be lesions that are inherently impossible to objectively assess and therefore must be significantly enlarged.

[0623] 3.5. New lesions

[0624] The appearance of new malignant lesions indicates disease progression; therefore, some commentary on new lesions is important. There are no specific criteria for identifying new lesions through imaging; however, the discovery of new lesions should be definitive. For example, progression cannot be attributed to differences in imaging techniques, changes in imaging morphology, or lesions other than the tumor (e.g., some so-called new bone lesions are simply healed or recurrent primary lesions). This is important when a patient's baseline lesions show partial or complete remission. For example, necrosis of liver lesions may be recorded as a new cystic lesion on a CT report when it is not actually present.

[0625] Lesions detected during follow-up that were not identified at baseline will be considered new lesions, indicating disease progression. For example, in patients with visceral lesions identified at baseline, intracranial metastasis will be considered evidence of disease progression when metastases are found on head CT or MRI, even if no head examination was performed at baseline.

[0626] If a new lesion is ambiguous 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 indeed a new lesion, the progression time should be calculated from the time it was first discovered.

[0627] FDG-PET evaluation often requires additional testing for confirmation; therefore, combining FDG-PET and CT results to assess progression is reasonable (especially for new suspected lesions). New lesions can be identified using FDG-PET examination according to the following procedure:

[0628] A negative FDG-PET result at baseline but a positive FDG-PET result at follow-up indicates disease progression.

[0629] No FDG-PET at baseline but positive FDG-PET result at follow-up:

[0630] If new lesions identified by a positive FDG-PET scan are confirmed by CT during follow-up, then disease progression has occurred.

[0631] If new lesions identified by positive FDG-PET are not confirmed by CT during follow-up, additional CT will be required for confirmation (if confirmed, the progression time should be calculated from the time when the abnormality was detected by early FDG-PET).

[0632] If a positive FDG-PET scan at follow-up confirms the existing lesion identified by CT, and the lesion does not progress on imaging, then disease progression will not occur.

[0633] 4. Best overall remission evaluation

[0634] The best overall response assessment records the best response from the start to the end of the study, taking into account any necessary confirmatory conditions. Sometimes, a response cannot be confirmed until the end of treatment; therefore, the protocol should specify whether the response assessment at the end of treatment is included in the best overall response assessment. The protocol must specify how any new treatments prior to progression affect the best response. A patient's best response depends primarily on the outcome of target and non-target lesions and the presence of new lesions, as well as on the nature of the study, protocol requirements, and outcome measures. Specifically, in non-randomized studies where response is the primary endpoint, either a partial response (PR) or a complete remission (CR) must be confirmed to determine which is the best overall response.

[0635] 4.1. It is assumed that remission will be evaluated at each specific time point in the protocol. Table 1 summarizes the overall remission status of patients with measurable disease at baseline at each time point.

[0636] If the patient has no measurable lesions (no target lesions), the evaluation can be found in Table 2.

[0637] 4.2. Explanation of cases with missing evaluations and those that cannot be evaluated.

[0638] If lesion imaging / measurement is not performed at a specific time point, the patient will be unevaluable at that time point. If only part of the lesion can be evaluated at a time, the case will generally be unevaluable at that time point unless a compelling argument can be made that the missing lesion will not affect the remission evaluation at the specified time point. This is more likely to occur in cases of disease progression. For example, if a patient has 3 lesions at baseline with a total diameter of 50 mm, and later only 2 lesions with a total diameter of 80 mm are evaluable, the patient will be evaluated as having disease progression, regardless of the impact of the missing lesions.

[0639] 4.3. Optimal Overall Remission: All Time Points

[0640] If all information about the patient is available, the best overall response can be determined.

[0641] The study does not require confirmation of the best overall response at the time of complete or partial remission: 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 last cycle, the best overall response will be PR. When the best overall response is SD, the minimum time from baseline specified in the protocol must also be met. If the minimum time criterion is not met, the best overall response of SD will not be accepted, and it will be determined by subsequent evaluation. For example, if a patient has SD in the first cycle and PD in the second cycle but does not meet the minimum time for SD, the best overall response will be PD. Similarly, patients who are assessed as SD in the first cycle and lost to follow-up will be considered unevaluable.

[0642] The study requires confirmation of the optimal overall response assessment for complete or partial remission: a complete or partial remission can only be confirmed when each participant meets the criteria for partial or complete remission as specified in the study, and the remission is confirmed at a subsequent time point specified in the protocol (usually 4 weeks later). In this case, a description of the optimal overall remission is provided in Table 3.

[0643] 4.4. Special Notes Regarding Mitigation Evaluation

[0644] Even when nodular lesions are included in the overall assessment of target lesions, and the nodules shrink to a “normal” size (<10 mm), scan reports regarding lesion size will still be provided. To avoid overestimation, measurements will be recorded even if the nodules appear normal but show an increase in size. As previously mentioned, this means that a value of 0 will not be recorded on the CRF for participants with complete remission.

[0645] If remission confirmation is required during the study, repeated "unmeasurable" time points complicate the evaluation of optimal remission. The study's analytical plan must address how to determine remission using these missing data. For example, in most studies, a participant's remission as PR-NE-PR can be considered confirmed.

[0646] When a participant experiences a general deterioration requiring discontinuation of treatment without objective evidence, symptomatic progression should be reported. Objective progression should be evaluated as far as possible, even after treatment cessation. Symptom exacerbation is not a descriptive indicator of objective remission, but rather a reason for discontinuation of treatment. Objective remission in such participants will be evaluated through descriptions of target and non-target lesions, as shown in Tables 1 through 3.

[0647] Cases defined as early progression, early death, and non-evaluable are exceptions to the study and should be clearly described in each protocol (depending on the treatment interval and treatment duration).

[0648] In some cases, it may be difficult to distinguish between localized lesions and normal tissue. When the evaluation of complete remission is based on such a definition, we recommend performing a biopsy before evaluating complete remission of localized lesions. When abnormal imaging results of localized lesions in some participants are considered to represent fibrosis or scarring of the lesions, FDG-PET is used as a biopsy-like evaluation criterion to confirm complete remission. In such cases, the use of FDG-PET should be prospectively described in the protocol and supported by reports from the specialist medical literature on this case. However, it must be acknowledged that both FDG-PET and biopsy, due to their inherent limitations (including resolution / sensitivity), can lead to false positive results when evaluating complete remission.

[0649] Table 7. Time-point remission: Participants with target lesions (with or without non-target lesions)

[0650]

[0651] In SD cases, at least one follow-up measurement after enrollment must meet the SD criteria, and the follow-up should be at least 6-8 weeks apart from enrollment.

[0652] Table 8. Time-point remission - Participants with only non-target lesions

[0653]

[0654] Note: For non-target lesions, "non-CR / non-PD" refers to a response superior to SD. Because SD is increasingly used as an endpoint for response evaluation, a non-CR / non-PD response standard has been established for cases without measurable lesions.

[0655] For suspected progression (e.g., very small and indeterminate new lesions; cystic changes or necrosis of existing lesions), treatment may continue until the next evaluation. If disease progression is confirmed at the next evaluation, the progression date should be the date of the previously suspected progression.

[0656] Table 9. Optimal Overall Remission Requires Confirmation of CR and PR

[0657]

[0658] Note: CR = Complete Remission, PR = Partial Remission, SD = Stable Disease, PD = Progressive Disease, NE = Not Evaluable. Superscript "a": If CR actually occurs at the initial time point, and any disease recurs at a subsequent time point, then even if the participant meets the PR criteria relative to baseline, the response at the subsequent time point will still be PD (because the disease will recur after CR). Optimal response depends on whether SD occurs within the shortest treatment interval. However, sometimes a response is evaluated as CR at the initial time point, but subsequent scans reveal small lesions; therefore, the participant's actual response at the initial time point should be PR, not CR. In this case, the CR assessed at the initial time point should be changed to PR, and the optimal response should be PR.

[0659] 5. Frequency of tumor re-evaluation

[0660] The frequency of tumor re-evaluation during treatment depends on the treatment regimen and should be consistent with the type and schedule of treatment. However, in phase II studies where the treatment benefit is unclear, follow-up every 6 to 8 weeks (consistent with the end of a cycle) is reasonable, although the length of the interval may be adjusted for specific protocols or cases. The protocol should specify which tissue sites to evaluate at baseline (typically the sites most likely to metastasize for the tumor type under study) and the frequency of re-evaluation. Generally, target and non-target lesions are evaluated at each step. In some optional cases, certain non-target lesions may be evaluated less frequently. For example, bone scans may only need to be repeated when the target disease is identified as CR or bone lesion progression is suspected.

[0661] At the end of treatment, tumor re-evaluation depends on whether the clinical study used response rate or time to event (progression / death) as the endpoint. For time to event (e.g., TTP / DFS / PFS), routine repeat evaluations as specified in the protocol are required. Especially in randomized comparative studies, the planned evaluations should be outlined in the timeline (e.g., every 6 to 8 weeks during treatment or every 3 to 4 months after treatment) and should not be affected by treatment delays, dosing intervals, or any other events that may cause an imbalance in the timing of disease evaluation between treatment groups.

[0662] 6. Relief Evaluation / Relief Confirmation Period

[0663] 6.1. Confirmation

[0664] In non-randomized clinical studies with remission as the primary endpoint, partial response (PR) and complete remission (CR) must be confirmed to ensure that remission is not an outcome of misjudgment. This also allows for the reasonable interpretation of results in the context of historical data, but remissions described in the historical data of these studies should also be confirmed. However, in all other cases, such as randomized studies (phase II or III) or studies with disease stability or disease progression as the primary endpoint, confirmation of remission is no longer required, as it has no value for the interpretation of the study results. Nevertheless, eliminating the need for remission confirmation will make central review to prevent bias even more important, especially in non-blinded studies.

[0665] For SD, at least one measurement should meet the SD criteria specified in the protocol within the shortest time interval after the start of the study (usually no less than 6–8 weeks).

[0666] 6.2. Overall remission is calculated from the date of first achieving CR or PR (based on the date of first measurement) until the date of objectively recorded relapse or disease progression (the minimum measurement recorded in the study is used as a reference for disease progression). Overall complete remission is calculated from the date of first achieving CR until the date of objectively recorded relapse or disease progression.

[0667] 6.3. Disease Stability Period

[0668] The duration of disease stability is calculated from the start of treatment (or, in randomized studies, from the randomization time) until disease progression, using the minimum sum of measurements from the reference study (if the baseline sum is minimum, it is used as a reference for PD calculation). The clinical relevance of disease stability varies by study and disease. If the proportion of patients achieving and maintaining disease stability for the shortest possible time is the study endpoint for a particular study, the protocol should specify the minimum time interval between two measurements, as defined by SD.

[0669] Note: Remission, disease stability, and PFS are affected by the follow-up frequency after baseline assessment. Defined standard follow-up frequencies are not covered in this guideline. Follow-up frequencies should be determined based on disease subtype and stage, treatment duration, standard practices, etc. If comparisons between different studies are necessary, limitations in the accuracy of the measured endpoints should be considered.

[0670] 7. Progression-free survival (PFS) / Percentage of progression-free survival (PPF)

[0671] This guideline focuses on using objective response as the endpoint in Phase II clinical trials. In some cases, response rate may not be the optimal indicator for evaluating the potential anticancer activity of a new drug / regimen. In these cases, PFS / PPF at specific time points can be considered appropriate surrogate indicators providing initial signals of the drug's biological activity. However, it is clear that these evaluations are questionable in uncontrolled studies, as seemingly valuable observations may be related to biological factors such as patient selection rather than the effect of the drug intervention. Therefore, Phase II clinical trials with these endpoints are best designed as randomized controlled trials. However, non-randomized studies are reasonable because some tumors have consistently poor clinical presentations (often, always poor). However, in these cases, due to the lack of a positive control, efficacy evidence should be carefully documented when evaluating the expected PFS or PPF.

[0672] Note: Age is in years, and weight is in kilograms (kg).

[0673] Appendix IV: Numerical Rating Scale (NRS) Scoring

[0674] A scale of 0-10 represents different levels of pain. Ask the patient about their pain level and mark it, or ask the patient to circle the number that best represents their pain level.

[0675] 0: Painless.

[0676] 1-3: Mild pain (does not affect sleep).

[0677] 4-6: Moderate pain.

[0678] 7-9: Severe pain (inability to fall asleep or waking up in pain during sleep).

[0679] 10: Severe pain.

[0680] Pain of varying degrees is also present Figure 1 As shown in the image.

[0681] Appendix V: European Five-Dimensional Health Scale (EQ-5D)

[0682] The EQ-5D form is shown below. Record the patient's name, random number, visit date, and corresponding medication administered during the visit. Relevant scales are available in... Figure 2 As shown in the image.

[0683] Table 10. EQ-5D Questionnaire

[0684]

[0685] Appendix VI ICDS (Modified Inverse Choi Density / Size Standard) Evaluation Criteria

[0686] Table 11. ICDS Evaluation Criteria

[0687]

[0688] Evaluation should be performed using contrast-enhanced spiral CT or MRI.

[0689] The CT attenuation value of each tumor should be measured in Henle units (HU) during the portal vein period.

[0690] SLD represents the sum of the longest diameters.

[0691] Example 3. A multicenter, single-arm, open-label study evaluating the safety and efficacy of ALMB-0168 in patients with osteosarcoma. Phase I / II Clinical Trial – Results

[0692] Twenty-four participants were enrolled and treated with the humanized monoclonal antibody (ALMB-0168) 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 3 weeks. Baseline characteristics are shown in Table 12.

[0693] Of the 24 participants, 3 were unevaluated and withdrew from the study, 2 experienced symptomatic progression, and 19 were evaluable (2 partial response, 8 stable disease, and 9 progressive disease). The disease progression of these 19 participants was... Figure 3 The optimal variation in target lesion diameter determined by the researchers for these 19 participants is shown in the figure. Figure 4 As shown in the image.

[0694] For EES, the objective response rate was 10.5% (2 / 19), and the disease control rate (DCR) was 52.6% ((2+8) / 19). Efficacy data for the dosage 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) are shown in Table 13.

[0695] No treatment-related adverse events (DLT) occurred in any of the 24 participants. A total of 80 drug-related adverse events were observed, of which: anemia (9 participants, 11.25%) and proteinuria (8 participants, 10.00%) occurred in ≥10% of cases. Only one treatment-related adverse event of grade ≥3 occurred, and there were no drug-related serious adverse events. Adverse events occurring during treatment are summarized in Table 14.

[0696] According to pharmacokinetic data, following a single intravenous infusion, participants' ALMB-0168 exposure increased proportionally with dose within the dose range of 1 mg / kg–36 mg / kg, with the mean peak concentration (C0) being [missing data]. max The effective concentration ranged from approximately 19.7 μg / mL to 691 μg / mL, with a mean area under the drug-time curve (AUC). 0-t The effective rate is approximately 4365 h*μg / ml–123795 h*μg / ml. ALMB-0168 is slowly eliminated in vivo, with a mean elimination half-life of approximately 8.54 days–14.8 days and a mean elimination rate of approximately 8.75 ml / h–13.0 ml / h. It exhibits limited tissue distribution, with a mean apparent volume of distribution of approximately 3.55 L–4.88 L.

[0697]

[0698]

[0699]

[0700]

[0701] Participant S01001 received chemotherapy after radical resection of osteosarcoma in the left distal femur and subsequent lung metastasis. Following progression, Participant S01001 received systemic chemotherapy with another drug and palliative resection of the lung metastases. Participant S01001 also received third-line chemotherapy and palliative resection of the right distal femur metastases. After fourth-line chemotherapy, the right femoral osteosarcoma recurred and progressed. This participant met the inclusion criteria and enrolled in the ALMB-0168 clinical trial. Participant S01001 was enrolled in September 2021 and received ALMB-0168 treatment. The right femoral lesion continued to shrink (…). Figure 5 and Figures 6B-6F The Hu value continued to increase. Figure 7 The best response was PR. This participant received ALMB-0168 treatment for more than 18 months in the study.

[0702] Participant S02007 underwent surgery and chemotherapy for osteosarcoma of the distal right femur; one year later, surgical resection of tibial metastases and chemotherapy for lung lesions progressed. This participant met the inclusion criteria for the protocol and participated in the clinical trial of this drug, achieving a best response of PR. At baseline, the long diameter of lesion 1 in participant S02007 was 31.9 mm (…). Figure 8A (Left side sub-image) The long diameter of lesion 2 is 19.4 mm ( Figure 8B (Left sub-image), totaling 51.3 mm. After treatment with ALMB-0168 in this study, the long axis of lesion 1 shrank to 20.9 mm (…). Figure 8A (See right sub-image), the long diameter of lesion 2 has shrunk to 14.1 mm. Figure 8B (Left sub-figure), total 35.0 mm, a 31.8% reduction relative to the baseline, with PR being the best mitigation.

[0703] These data demonstrate that, in a phase I / II dose-escalation trial, ALMB-0168 exhibits unexpectedly good efficacy and tolerable safety in patients with metastatic or unresectable osteosarcoma who have failed standard therapy.

[0704] Table 15. List of Abbreviations

[0705]

[0706]

[0707]

[0708]

[0709]

Claims

1. A method of treating bone cancer in a subject of need, comprising administering to the subject at least one dose of an anti-connector protein 43 (Cx43) antibody or an antigen-binding fragment thereof, wherein the anti-Cx43 antibody comprises The heavy chain variable region comprises the following HCDR amino acid sequence: HCDR1: SEQ ID NO: 1; HCDR2: SEQ ID NO: 2; HCDR3: SEQ ID NO: 3; and The light chain variable region comprises the following LCDR amino acid sequence: LCDR1: SEQ ID NO: 4; LCDR2: SEQ ID NO: 5; LCDR3: SEQ ID NO:

6.

2. The method according to claim 1, wherein the at least one dose of 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 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 at a frequency of about once daily to about 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 at a frequency of about once every 2 weeks, about once every 3 weeks, or about once every 4 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 at a frequency of approximately once every 3 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 of claim 8, wherein the anti-Cx43 antibody or its antigen-binding fragment is administered over a period of 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 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 having an amino acid sequence according to SEQ ID NO: 7, and / or a light chain variable region having 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 NO: 9 and 11-13, 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 subject.

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

16. The method of claim 15, wherein the osteosarcoma is (i) Resectable or unresectable; (ii) Stage IA, Stage IB, Stage II-A, Stage II-B, Stage III, Stage IV-A, or Stage IV-B; (iii) Low-level or high-level; and / or (iv) Primary osteosarcoma, osteoblastic osteosarcoma, chondrogenic osteosarcoma, fibroblastic osteosarcoma, small cell osteosarcoma, capillary dilatational osteosarcoma, periosteal osteosarcoma, classical osteosarcoma, osteoblastic osteosarcoma-sclerosing osteosarcoma, chondroblastoma-like osteosarcoma, chondromycinoid osteosarcoma, clear cell osteosarcoma, malignant fibrous histiocytoma-like osteosarcoma, giant cell rich osteosarcoma and / or epithelioid osteosarcoma.

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

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

19. The method according to any one of claims 1 to 18, wherein after treatment with the anti-Cx43 antibody or its antigen-binding fragment, the subject achieves: (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) Overall survival improved by 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, RECIST and / or ICDS criteria; (v) Improvement in pain score as assessed by the NRS; and / or (vi) The tumor size is reduced 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%.

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

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

22. The method of 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 of claim 21 or 22, wherein the anti-Cx43 antibody or its antigen-binding fragment is administered at a frequency of about once daily to about 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 at a frequency of about once every 2 weeks, about once every 3 weeks, or about once every 4 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 at a frequency of about once every 3 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 of claim 27, wherein the anti-Cx43 antibody or its antigen-binding fragment is administered over a period of 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 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 method according to any one of claims 21 to 29, wherein the anti-Cx43 antibody or its antigen-binding fragment comprises The heavy chain variable region comprises the following HCDR amino acid sequence: HCDR1: SEQ ID NO: 1; HCDR2: SEQ ID NO: 2; HCDR3: SEQ ID NO: 3; and The light chain variable region comprises the following LCDR amino acid sequence: LCDR1: SEQ ID NO: 4; LCDR2: SEQ ID NO: 5; LCDR3: SEQ ID NO:

6.

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 having an amino acid sequence according to SEQ ID NO: 7, and / or a light chain variable region having 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 NO: 9 and 11-13, 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 subject.

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

36. The method of claim 35, wherein the osteosarcoma is (i) Resectable or unresectable; (ii) Stage IA, Stage IB, Stage II-A, Stage II-B, Stage III, Stage IV-A, or Stage IV-B; (iii) Low-level or high-level; and / or (iv) Primary osteosarcoma, osteoblastic osteosarcoma, chondrogenic osteosarcoma, fibroblastic osteosarcoma, small cell osteosarcoma, capillary dilatational osteosarcoma, periosteal osteosarcoma, classical osteosarcoma, osteoblastic osteosarcoma-sclerosing osteosarcoma, chondroblastoma-like osteosarcoma, chondromycinoid osteosarcoma, clear cell osteosarcoma, malignant fibrous histiocytoma-like osteosarcoma, giant cell rich osteosarcoma and / or epithelioid osteosarcoma.

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

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

39. The method according to any one of claims 21 to 38, wherein after treatment with the anti-Cx43 antibody or its antigen-binding fragment, the subject achieves: (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) Overall survival improved by 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, RECIST and / or ICDS criteria; (v) Improvement in pain score as assessed by the NRS; and / or (vi) The tumor size is reduced 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%.

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

Images