Pharmaceutical compositions comprising mixed antibodies for Anti-CTLA4 and Anti-PD1

Abstract

The present disclosure relates to a mixed antibody comprising anti-CTLA4 and anti-PD1, or a use of the mixed antibody in combination with a chemotherapeutic drug and / or an anti-angiogenic agent in the treatment of hepatocellular carcinoma.

Description

Pharmaceutical composition comprising mixed antibodies of anti-CTLA4 and anti-PD1 and therapeutic use thereof Technical Field

[0001] The present disclosure relates to the treatment of cancer, particularly hepatocellular carcinoma, including immunotherapy and combination therapy. More specifically, the present disclosure relates to the use of a pharmaceutical composition comprising a mixed antibody of anti-CTLA4 and anti-PD1 for the treatment of hepatocellular carcinoma. Background Art

[0002] In China, primary liver cancer is the fifth most common malignant tumor and the second leading cause of cancer-related death. Approximately 400,000 new cases of liver cancer are diagnosed, and 391,000 deaths are attributed to the disease, representing 44% and 47% of the global incidence, respectively. This represents a serious threat to people's lives and health. Hepatocellular carcinoma (HCC) is the predominant pathological type of primary liver cancer, accounting for 85% to 90% of cases. HCC treatment is characterized by multidisciplinary involvement and the coexistence of multiple treatment approaches. Common treatments include hepatectomy, liver transplantation, ablation, transarterial chemoembolization, radiotherapy, and systemic anti-tumor therapy. Choosing the right treatment approach for patients with different liver cancer stages can maximize efficacy. Due to the insidious onset of HCC, less than 30% of patients are eligible for radical treatment at the time of initial diagnosis. More than two-thirds of HCC patients are already at an advanced stage at diagnosis, making them ineligible for curative treatment.

[0003] Surgical resection, local ablation, and liver transplantation are potentially curative, but tumor recurrence is very common, with a 5-year recurrence rate of up to 70% after surgical resection or local ablation. Currently, there is no globally recognized adjuvant treatment for HCC after radical treatment. Patients with high-risk recurrence factors are given high attention clinically, and intervention measures are often actively taken in the hope of preventing or delaying recurrence, including antiviral drugs, hepatic artery intervention, systemic chemotherapy containing oxaliplatin, molecular targeted therapy drugs, and traditional Chinese medicine. These therapies may have some efficacy, but with the exception of antiviral drug treatment, other treatments lack strong evidence-based medical support. Therefore, there is a huge unmet clinical need, and there is an urgent need to develop adjuvant treatment options after radical treatment to prevent tumor recurrence.

[0004] For unresectable or metastatic HCC, systemic anti-tumor therapy can control disease progression and prolong patient survival, including molecular targeted drug therapy, immunotherapy, chemotherapy, and traditional Chinese medicine treatment. The efficacy of molecular targeted drugs such as sorafenib is relatively limited; HCC is poorly sensitive to chemotherapy, such as oxaliplatin, and chemotherapy alone has limited effectiveness; the development of first-line treatment drugs has been in a bottleneck period for a long time. With the advancement of immune research, immune checkpoint inhibitors (ICIs) have the advantages of killing residual cancer cells, improving chemotherapy sensitivity, reducing side effects, alleviating late symptoms, and prolonging survival. They have produced significant and lasting therapeutic effects in many tumors, including HCC. Although existing ICIs combined with anti-vascular drugs have achieved great breakthroughs in the treatment of advanced HCC, the 5-year survival rate of patients with advanced HCC using existing combination therapy regimens remains low, and there is still unmet clinical need. Further exploration of new combination therapy regimens is needed to further improve the prognosis of patients with advanced HCC.

[0005] ZPML265 is a hybrid antibody drug formulation consisting of a recombinant humanized IgG1 monoclonal antibody targeting human CTLA4 and a recombinant humanized IgG4 monoclonal antibody targeting human PD1. These two different antibodies are produced by a single host cell. This hybrid antibody can simultaneously and specifically bind to CTLA4 and PD1, thereby blocking the immune checkpoint signaling pathways between CTLA4 and B7-1 / B7-2, and between PD1 and PDL1. This relieves the inhibitory effects of these two pathways on T lymphocytes, restoring their functional activity and anti-tumor immune responses, thereby enabling the body to fight and kill tumors.

[0006] ZPML264 injection is a pharmaceutical formulation containing only the anti-PD-1 antibody from ZPML265, whose sequence is identical to that of the anti-PD-1 antibody component of the mixed antibody ZPML265. ZPML264's early clinical trial results demonstrated good safety and tolerability, with no significant uncontrolled adverse event safety signals compared to marketed drugs such as pembrolizumab, nivolumab, toripalimab, sintilimab, and camrelizumab. Furthermore, its efficacy has been demonstrated across multiple tumor types.

[0007] Bevacizumab is an anti-angiogenic drug that can normalize abnormal vascular structure and function. Vascular normalization can enhance the infiltration of immune cells into tumors, allowing the tumor microenvironment to have more CD8+ T cells and B lymphocytes, thereby improving the tumor immune microenvironment.

[0008] Therefore, there is an unmet clinical need for patients with unresectable or metastatic hepatocellular carcinoma who have not received previous systemic treatment, and the development of more effective drugs is urgently needed.

[0009] SUMMARY OF THE INVENTION

[0010] The technical problem addressed by this disclosure is to provide an innovative immunotherapy that outperforms the currently approved and recognized best-in-class standard of care for hepatocellular carcinoma in China, or to fill the clinical gap in adjuvant therapy for HCC following surgical resection or local ablation. This disclosure is centered on patient needs and driven by clinical value. It is expected to further enhance patient survival while maintaining safety, meeting the growing and diverse clinical needs.

[0011] The present disclosure provides a pharmaceutical composition for treating liver cancer, particularly hepatocellular carcinoma, comprising an effective amount of mixed antibodies of anti-CTLA4 and anti-PD1.

[0012] At the same time, the present disclosure also provides a method for treating liver cancer, especially hepatocellular carcinoma, using a pharmaceutical composition comprising an effective amount of mixed antibodies of anti-CTLA4 and anti-PD1.

[0013] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0014] In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent, including but not limited to chemotherapeutic agents (chemotherapeutic drugs), cytotoxic agents, radiotherapeutic agents, cancer vaccines, tumor reducing agents, targeted anti-cancer agents, anti-angiogenic agents, biological response modifiers, cytokines, hormones, anti-metastatic agents, and immunotherapeutic agents.

[0015] In some embodiments, the additional therapeutic agent is a chemotherapeutic drug and / or an anti-angiogenic agent, preferably a chemotherapeutic drug is oxaliplatin-based systemic chemotherapy, and preferably an anti-angiogenic agent is bevacizumab.

[0016] More preferably, the oxaliplatin-based systemic chemotherapy is XELOX or FOLFOX4.

[0017] Preferably, the hepatocellular carcinoma is unresectable or metastatic hepatocellular carcinoma that has not been previously treated systematically, or is hepatocellular carcinoma with high-risk recurrence factors after surgical resection or local ablation therapy.

[0018] In some embodiments, the hybrid antibody is produced by a single host cell that contains nucleic acids encoding two different antibodies, anti-CTLA4 and anti-PD1, wherein the sequences of the anti-CTLA4 antibody heavy chain HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 1, 2, and 3, respectively, the sequences of the anti-CTLA4 antibody light chain LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 4, 5, and 6, respectively, the sequences of the anti-PD1 antibody heavy chain HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 9, 10, and 11, respectively, and the sequences of the anti-PD1 antibody light chain LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 12, 13, and 14, respectively.

[0019] In some embodiments, the heavy chain variable region sequence of the anti-CTLA4 antibody is shown in SEQ ID NO: 7, the light chain variable region sequence of the anti-CTLA4 antibody is shown in SEQ ID NO: 8, the heavy chain variable region sequence of the anti-PD1 antibody is shown in SEQ ID NO: 15, and the light chain variable region sequence of the anti-PD1 antibody is shown in SEQ ID NO: 16.

[0020] In some embodiments, the heavy chain sequence of the anti-CTLA4 antibody is shown in SEQ ID NO: 17, the light chain sequence of the anti-CTLA4 antibody is shown in SEQ ID NO: 18, the heavy chain sequence of the anti-PD1 antibody is shown in SEQ ID NO: 19, and the light chain sequence of the anti-PD1 antibody is shown in SEQ ID NO: 20.

[0021] The dosage of the mixed antibody is 5 mg / kg or 7.5 mg / kg, administered once every three weeks by intravenous infusion on the first day, and a treatment cycle of 21 days.

[0022] In some more specific embodiments, the mixed antibody is administered at a dose of 5 mg / kg or 7.5 mg / kg, administered on day 1 of each three-week dosing cycle; in addition, chemotherapy drugs and / or anti-angiogenic agents may be additionally administered. Preferably, the bevacizumab injection is administered at a dose of 7.5 mg / kg or 15 mg / kg, administered on day 1 of each three-week dosing cycle; the XELOX chemotherapy regimen includes oxaliplatin combined with capecitabine, with an oxaliplatin dose of 85 mg / m2, administered on day 1 of each three-week dosing cycle, and a capecitabine dose of 1000 mg / m2, administered on day 1 to day 14 of each three-week dosing cycle. The mixed antibody, bevacizumab, and oxaliplatin are all administered by intravenous infusion, and capecitabine is administered orally.

[0023] At the same time, the present disclosure also provides the use of a mixed antibody comprising an effective amount of anti-CTLA4 and anti-PD1 in the preparation of a pharmaceutical composition for treating unresectable or metastatic hepatocellular carcinoma that has not been previously treated systemically, wherein the pharmaceutical composition may additionally include a chemotherapeutic drug and / or an anti-angiogenic agent.

[0024] Furthermore, the present disclosure provides the use of a mixed antibody comprising an effective amount of anti-CTLA4 and anti-PD1 in the preparation of a pharmaceutical composition for treating hepatocellular carcinoma with high-risk recurrence factors after surgical resection or local ablation therapy.

[0025] The results of a phase Ib clinical study of the hybrid antibody disclosed herein as a single drug showed that 25 patients with hepatocellular carcinoma enrolled in the 5 mg / kg dose group showed a certain therapeutic effect; the results of a phase Ib / II clinical study of the hybrid antibody combined with bevacizumab as the first-line treatment for advanced HCC showed that both the 5 mg / kg dose group and the 7.5 mg / kg dose group showed efficacy superior to the existing standard treatment. At the same time, based on the published data on the effectiveness of ICIs combined with oxaliplatin-based chemotherapy as the first-line treatment for HCC, it was shown that ICIs combined with chemotherapy can exert a synergistic anti-tumor effect in advanced HCC. Therefore, the combination of the hybrid antibody provided by the present disclosure with bevacizumab and / or oxaliplatin-based chemotherapy is a new drug development strategy centered on patient needs and guided by clinical value for the first-line treatment of hepatocellular carcinoma in internal medicine, which can largely address unmet clinical needs. DETAILED DESCRIPTION

[0026] the term

[0027] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0028] Before describing the present disclosure in detail below, it should be understood that the present disclosure is not limited to the specific methodologies, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs.

[0029] Certain embodiments disclosed herein include numerical ranges, and certain aspects of the present disclosure may be described in terms of ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions in terms of ranges are intended for simplicity and convenience and should not be considered as strict limitations on the scope of the present disclosure. Therefore, descriptions in terms of ranges should be considered to specifically disclose all possible subranges and all possible specific numerical points within the range, as these subranges and numerical points have been clearly stated herein. Regardless of the width of the numerical value, the above principles apply equally. When describing in terms of ranges, the range includes the endpoints of the range.

[0030] When referring to a measurable value such as an amount, a temporal duration, etc., the term "about" is meant to include variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0031] As used herein, the three-letter and one-letter codes for amino acids are as described in J. Biol. Chem, 243, p3558 (1968).

[0032] The term "antibody" as used herein typically refers to a Y-shaped tetrameric protein comprising two heavy (H) polypeptide chains (HC) and two light (L) polypeptide chains (LC) held together by covalent disulfide bonds and non-covalent interactions. Natural IgG antibodies have this structure. Each light chain comprises a light chain variable domain (VL) and a light chain constant domain (CL). Each heavy chain comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH) (or heavy chain constant region (CH)).

[0033] Five major classes of antibodies are known in the art: IgA, IgD, IgE, IgG, and IgM. The corresponding heavy chain constant domains are called α, δ, ε, γ, and μ, respectively. IgG and IgA can be further divided into different subclasses, for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called κ and λ, based on the amino acid sequence of their constant domains.

[0034] The term "variable region" or "variable domain" shows significant changes in the amino acid composition from one antibody to another, and is primarily responsible for antigen recognition and binding. The variable region of each light chain / heavy chain pair forms an antigen binding site, so that a complete IgG antibody has two binding sites (i.e., it is bivalent). The variable region of the heavy chain (VH) and the variable region of the light chain (VL) each contain three regions with extreme variability, referred to as hypervariable regions (HVRs), or more generally, referred to as complementary determining regions (CDRs). VH and VL each have four skeleton regions FR (or framework regions), represented by FR1, FR2, FR3, and FR4, respectively. Therefore, CDR and FR sequences typically appear in the following sequence of the heavy chain variable domain (VH) (or light chain variable domain (VL)): FR1-HCDR1 (LCDR1)-FR2-HCDR2 (LCDR2)-FR3-HCDR3 (LCDR3)-FR4.

[0035] The types of "antibodies" in a broad sense may include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primatized antibodies, CDR-grafted antibodies, human antibodies (including recombinantly produced human antibodies), recombinantly produced antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies (including muteins and variants thereof), etc.

[0036] The term "monoclonal antibody" (or "mAb") refers to a substantially homogeneous antibody produced by a single cell clone that is directed against a specific antigenic epitope. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, synthetic technology, or a combination of these techniques.

[0037] It should be noted that the division of the CDRs and FRs in the antibody variable regions of the present disclosure is determined according to the Kabat definition. Other naming and numbering systems, such as Chothia, IMGT, or AHo, are also known to those skilled in the art. Therefore, humanized antibodies containing one or more CDRs derived from any naming system based on the antibody sequences of the present disclosure are clearly within the scope of the present disclosure.

[0038] The term "antigen" refers to a substance that is recognized and specifically bound by an antibody or antibody binding fragment. In a broad sense, an antigen can include any immunogenic fragment or determinant of a selected target, including a single epitope, multiple epitopes, a single domain, multiple domains, or a complete extracellular domain (ECD) or protein.

[0039] The term "epitope" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope can be formed by adjacent amino acids or non-adjacent amino acids juxtaposed by tertiary folding of a protein.

[0040] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear, cyclic, or branched, it may contain modified amino acids, particularly conservatively modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified, for example, by glycosylation, lipidation, acetylation, phosphorylation, methylation, and the like.

[0041] As used herein, the term "hybrid antibody" refers to an antibody containing a limited number of primary antibody species, optionally no more than two, three, four, five, six, seven, eight, nine, or ten, produced from host cells (optionally cells from a single host cell line) that have been transfected with DNA encoding at least two different antibodies (optionally full-length primate IgG antibodies) with different binding specificities. In some embodiments, DNA encoding at least two different heavy chains (HCs) and at least two different light chains (LCs) can be introduced into the same host cell. For example, a host cell can be transfected with DNA encoding at least two but no more than four different antibodies with different binding specificities. In some embodiments, the sequences of all transfected DNA encoding HCs and LCs can be mutated to alter the amino acid sequence of the antibody, thereby disfavoring non-homologous HC / LC pairing and strongly favoring homologous HC / LC pairing. In the case of introducing two different HCs into the host cell, one or both of the two different HCs can be optionally altered to disfavor heterodimer formation. In some embodiments, only one heavy chain is altered to prevent heterodimer formation. In some embodiments, when DNA encoding only two different antibodies is introduced into a host cell, only one of the antibodies encoded by the DNA comprises one or more partner-directed changes that favor cognate HC / LC pairing, while the other antibody does not comprise such changes. In some embodiments, the host cell produces only two major antibody species, wherein each HC predominantly pairs with its cognate LC, and most antibodies are tetramers containing two heavy chains having the same amino acid sequence and two light chains having the same amino acid sequence (see PCT / US2017 / 030676).

[0042] The term "pharmaceutical composition" refers to a formulation or combination of formulations containing one, two, or more active ingredients, which allows the active ingredients contained therein to exist in a biologically effective form and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. When a "pharmaceutical composition" is present as a combination of separate formulations containing two or more different active ingredients, they can be administered simultaneously, sequentially, separately, or at intervals, with the goal of exerting the biological activities of the multiple active ingredients for combined treatment of a disease.

[0043] The term "pharmaceutical carrier" or "pharmaceutically acceptable carrier" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic agent is administered.

[0044] The term "effective amount" refers to a dose of a pharmaceutical formulation comprising an active ingredient of the present disclosure that produces the desired effect in a treated patient after administration to the patient in a single or multiple doses. The effective amount can be readily determined by the attending physician, who is skilled in the art, by considering a variety of factors such as: ethnic differences; weight, age, and health status; the specific disease involved; the severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.

[0045] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0046] The term "transfection" as used herein refers to the introduction of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.

[0047] The term "isolated polynucleotide" or "isolated nucleic acid" refers to a nucleic acid molecule, DNA or RNA, that has been removed from its natural environment. For example, for the purposes of this disclosure, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in a heterologous host cell or a purified polynucleotide (partially or substantially) in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that normally contain the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present disclosure, as well as positive and negative strand forms and double-stranded forms. Isolated polynucleotides or nucleic acids of the present disclosure also include such molecules produced synthetically. In addition, the polynucleotide or nucleic acid may or may not include regulatory elements, such as promoters, ribosome binding sites, or transcription terminators.

[0048] The terms "nucleic acid molecule encoding," "coding DNA sequence," and "coding DNA" refer to the order of deoxyribonucleotides along a deoxyribonucleic acid chain. The order of these deoxyribonucleotides determines the order of amino acids along a polypeptide (protein) chain. Thus, a nucleic acid sequence encodes an amino acid sequence.

[0049] Methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as the Cold Spring Harbor Manual of Antibody Laboratory Techniques, Chapters 5-8 and 15. The engineered antibodies or antigen-binding fragments thereof disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes the antibodies can be purified and collected using conventional techniques. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange.

[0050] As used herein, the term "individual" or "subject" refers to any animal, such as a mammal or marsupial. Individuals of the present disclosure include, but are not limited to, humans, non-human primates (e.g., cynomolgus or rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any type of poultry.

[0051] As used herein, the terms "disease," "condition," or "disorder" refer to any change or disorder that damages or interferes with the normal function of a cell, tissue, or organ. For example, "disease" includes, but is not limited to, tumors, pathogen infection, autoimmune diseases, T-cell dysfunction, or immune tolerance deficiency (e.g., transplant rejection).

[0052] As used herein, the term "tumor" refers to a disease characterized by the pathological proliferation of cells or tissues, and their subsequent migration or invasion of other tissues or organs. Tumor growth is typically uncontrolled and progressive, without inducing or inhibiting normal cell proliferation. Tumor includes "cancer" and refers broadly to all malignant tumors.

[0053] As used herein, the term "treatment" refers to clinical intervention aimed at altering the course of a disease in an individual or cell, and can be either preventative or interventional in the clinical pathological process. Therapeutic effects include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, slowing the progression of a disease, improving or relieving the condition, and alleviating or improving the prognosis.

[0054] The term "combination" as used herein refers to a treatment regimen that provides at least two or more different therapies to achieve a specified therapeutic effect. The therapies can be physical, such as radiotherapy, or chemical, such as administering a drug to a subject, and the drug also includes a combination drug. "Combination drug" refers to a combination of two or more pharmaceutical preparations, each of which has an active ingredient, that need to be used in combination when administered to a subject. The active ingredients can be mixed together to form a single dosing unit, or they can be independently administered as dosing units and used separately; when administered, different pharmaceutical preparations can be administered essentially synchronously, simultaneously or sequentially.

[0055] Relapse-free survival (RFS) was defined as the time from randomization to the first documented disease recurrence (local, regional, or distant) or death from any cause, whichever occurred first.

[0056] Overall survival (OS) is the time from the start of study treatment to death from any cause. Patients who were still in follow-up at the cut-off date were censored based on the cut-off date. Subjects lost to follow-up were censored based on the date of last contact.

[0057] Progression-free survival (PFS) refers to the time from the start of study treatment to the first imaging assessment of disease progression or death from any cause (whichever occurs first).

[0058] The objective response rate (ORR) was defined as the best confirmed ORR during the study period, including both complete responses (CR) and partial responses (PR). According to RECIST v1.1, additional imaging was required to confirm the first occurrence of a PR or CR at ≥4 weeks.

[0059] Disease control rate (DCR) refers to the percentage of cases with the best efficacy evaluation as "CR+PR+SD".

[0060] Complete remission (CR): All target lesions disappear and the short diameter of all pathological lymph nodes (including target nodules and non-target nodules) must be reduced to <10mm.

[0061] Partial response (PR): The sum of the target lesion diameters decreased by at least 30% compared with the baseline level.

[0062] Disease progression (PD): The minimum value of the sum of all target lesion diameters measured during the entire experimental study is used as a reference, and the sum of diameters increases by at least 20% (if the baseline measurement value is the minimum, the baseline value is used as a reference); in addition, the absolute value of the sum of diameters must increase by at least 5 mm (the appearance of one or more new lesions is also considered as disease progression).

[0063] Stable disease (SD): The target lesion has not decreased to the level of PR, nor increased to the level of PD, but is somewhere in between. The minimum sum of the diameters can be used as a reference for research.

[0064] Duration of response (DOR) was defined as the time from the first tumor assessment as CR or PR (whichever was recorded first) to the first assessment as PD or death.

[0065] Treatment-emergent adverse events (TEAEs) refer to adverse events that occur during treatment.

[0066] Treatment-related adverse events (TRAEs) are adverse events related to study drugs that occur during treatment.

[0067] A serious adverse event (SAE) refers to an adverse medical event (AE) such as death, life-threatening, permanent or severe disability or loss of function, the need for hospitalization or prolonged hospitalization, and congenital abnormalities or birth defects that occurs after the subject receives the investigational drug.

[0068] Dose-limiting toxicity (DLT) was defined as grade ≥3 non-hematologic toxicity or grade ≥4 hematologic toxicity occurring within 21 days (1 cycle) after the first dose.

[0069] The maximum tolerated dose (MTD) was defined as the highest dose level of the drug at which no more than one of six subjects experienced DLT during the 21-day treatment period (one cycle) after the first dose.

[0070] Example

[0071] The present disclosure is further described below with reference to specific examples. It should be understood that these examples are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure.

[0072] Example 1 Obtaining the hybrid antibody ZPML265

[0073] A hybrid antibody produced by a single host cell line was prepared using the method disclosed in PCT / US2017 / 030676. The hybrid antibody contains two active ingredients: a recombinant humanized IgG1 monoclonal antibody targeting human CTLA4 and a recombinant humanized IgG4 monoclonal antibody targeting human PD1. This hybrid antibody can specifically bind to both human CTLA4 and PD1. After the two antibodies are simultaneously expressed in a single host cell line, they are collected, purified, and combined with a pharmaceutically acceptable carrier to form a single hybrid antibody pharmaceutical formulation, ZPML265. The amino acid sequences of the anti-CTLA4 and anti-PD1 antibodies are shown in Table 1 below.

[0074] Table 1 Amino acid sequences of the components of the mixed antibody ZPML265

[0075] Example 2 Obtaining ZPML264

[0076] ZPML264 injection contains only one active ingredient: the recombinant humanized IgG4 monoclonal antibody targeting human PD-1, contained in ZPML265. The sequence of this antibody is shown in Table 1. After expression in a host cell line, this antibody is harvested, purified, and combined with a pharmaceutically acceptable carrier to form the pharmaceutical formulation ZPML264.

[0077] Example 3: A multicenter, open-label, phase Ib study evaluating the efficacy and safety of ZPML265 in patients with advanced malignant solid tumors

[0078] Dosage:

[0079] In a Phase Ib clinical study conducted in China, a total of 419 subjects were enrolled, including 25 patients with hepatocellular carcinoma. Patients were aged 18 years or older, had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, had an expected survival of 3 months or more, and had the following vital organ function levels before the first use of the investigational drug:

[0080] 1) Absolute neutrophil count ≥1.5×10 9 / L;

[0081] 2) Platelets ≥75×10 9 / L;

[0082] 3) Hemoglobin ≥90 g / L;

[0083] 4) serum albumin ≥30 g / L;

[0084] 5) Alanine aminotransferase (AST) and aspartate aminotransferase (ALT) ≤ 2.5 × upper limit of normal reference value (ULN) (for patients with liver cancer or liver metastasis, ≤ 5 × ULN is allowed);

[0085] 6) Total bilirubin ≤1.5×ULN (≤3×ULN is allowed for patients with Gilbert syndrome);

[0086] 7) International normalized ratio (INR) ≤ 1.5 [INR ≤ 2.3 or prothrombin time (PT) prolonged ≤ 6 seconds in subjects with liver cancer]; activated partial thromboplastin time (APTT) ≤ 1.5 × ULN;

[0087] 8) Serum creatinine ≤ 1.5 × ULN. If the subject's creatinine level is > 1.5 × ULN, creatinine clearance (CrCl) ≥ 50 mL / min (calculated according to the Cockcroft-Gault formula);

[0088] 9) Left ventricular ejection fraction (LVEF)>50%.

[0089] Dosage regimen: ZPML265 is administered at a dose of 5 mg / kg once every three weeks (Q3W) via intravenous infusion.

[0090] Effectiveness Results:

[0091] A total of 25 subjects with advanced hepatocellular carcinoma were enrolled in this study and treated with the 5 mg / kg dose group. None of the 25 subjects with hepatocellular carcinoma achieved a CR or PR, and 10 subjects (40.0%) had a best response evaluation result of SD. The confirmed ORR was 0% (95% CI: 0.000, 13.719), and the confirmed DCR was 40.0% (95% CI: 21.125, 61.335). Among the three subjects with hepatocellular carcinoma who had not previously received systemic treatment, one had a best response evaluation result of SD, and two had PD. Among the subjects who had previously received systemic therapy, 5 had not received immunotherapy, 3 of whom had a best efficacy evaluation result of SD, with a confirmed DCR of 60.0% (95% CI: 14.663, 94.726). Among the 17 subjects who had previously received immunotherapy, 6 had a best efficacy evaluation result of SD, with a confirmed DCR of 35.3% (95% CI: 14.210, 61.672). It is worth mentioning that among the subjects who had previously received immunotherapy, 2 subjects were found to have a PD response based on the RECISTv1.1 criteria at the first response evaluation, but continued to receive study treatment and achieved a PR tumor shrinkage, which continued to maintain the PR status.

[0092] Efficacy summary: ZPML265 alone showed certain efficacy in hepatocellular carcinoma.

[0093] Safety results:

[0094] Safety data were compiled from 609 subjects treated with ZPML265. As of May 31, 2022, 546 (89.7%) patients experienced TEAEs and 451 (74.1%) patients experienced TEAEs related to ZPML265. Grade ≥3 TEAEs and SAEs were reported in 187 (30.7%) and 156 (25.6%) patients, respectively, and Grade ≥3 TEAEs related to ZPML265 and SAEs related to ZPML265 were reported in 102 (16.7%) and 75 (12.3%) patients, respectively.

[0095] In contrast to ZPML265 monotherapy, another meta-analysis that summarized 2,664 cases of Nivolumab combined with Ipilimumab showed that the overall incidence of treatment-related serious adverse events was 29.6%, and the incidence of grade ≥3 TRAEs was 39.9%, which were higher than the relevant data for ZPML265 monotherapy.

[0096] Existing data show that the treatment safety is acceptable and no new safety signals have been found compared with other immunotherapy drugs.

[0097] Example 4 A Phase Ib / II Clinical Study to Evaluate the Safety, Pharmacokinetics, and Preliminary Efficacy of ZPML265 or ZPML264 Injection Combined with Bevacizumab Injection in Patients with Advanced Liver Cancer

[0098] ZPML265 or ZPML264 injection combined with bevacizumab injection is being evaluated in an open, multicenter Phase Ib / II clinical study of patients with unresectable or metastatic hepatocellular carcinoma who have not received systemic treatment. The study is divided into three cohorts: Cohort A, Cohort B, and Cohort C. Cohort A includes 50 subjects, Cohort B includes 26 subjects, and Cohort C includes 40 subjects. Patients must be over 18 years old, have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, have an expected survival of 3 months or more, and have the following vital organ function levels before the first use of the investigational drug:

[0099] 1) Absolute neutrophil count ≥1.5×10 9 / L;

[0100] 2) Platelets ≥75×10 9 / L;

[0101] 3) Hemoglobin ≥90 g / L;

[0102] 4) serum albumin ≥30 g / L;

[0103] 5) AST and ALT ≤ 5 × upper limit of normal reference value (ULN);

[0104] 6) Total bilirubin ≤1.5×ULN (≤3×ULN is allowed for patients with Gilbert syndrome);

[0105] 7) serum creatinine ≤1.5×ULN. If the patient's creatinine level is >1.5×ULN, the creatinine clearance (CLcr) calculated by the Cockcroft-Gault equation is ≥50 mL / min;

[0106] 8) Left ventricular ejection fraction (LVEF)>50%;

[0107] 9) Proteinuria <2+ (when urine protein ≥2+, 24-hour urine protein quantification should be performed, and patients ≤1g can be selected);

[0108] 10) For patients not receiving anticoagulant therapy, the international normalized ratio (INR) or activated partial thromboplastin time (APTT) should be ≤ 2×ULN.

[0109] Dosage regimen:

[0110] Cohort A: ZPML265 was administered at a dose of 5 mg / kg every 3 weeks via intravenous infusion, while bevacizumab injection was administered at a dose of 7.5 mg / kg or 15 mg / kg every 3 weeks via intravenous infusion.

[0111] Cohort B: ZPML264 was administered at a fixed dose of 200 mg every 3 weeks via intravenous infusion, while bevacizumab injection was administered at a dose of 7.5 mg / kg or 15 mg / kg every 3 weeks via intravenous infusion.

[0112] Cohort C: ZPML265 was administered at a dose of 7.5 mg / kg every 3 weeks via intravenous infusion, while bevacizumab injection was administered at a dose of 7.5 mg / kg or 15 mg / kg every 3 weeks via intravenous infusion.

[0113] Safety results:

[0114] As of the data analysis cutoff of February 20, 2023, the safety analysis set for Cohort A included 50 subjects, and the safety analysis set for Cohort C included 40 subjects. This study is currently ongoing, and the preliminary safety results of Cohorts A and C are summarized as follows:

[0115] Queue A:

[0116] Among the 50 subjects in Cohort A, 49 (98.0%) experienced TEAEs and 43 (86.0%) experienced TRAEs. Of these, 42 (84.0%) experienced ZPML265-related TEAEs and 40 (80.0%) experienced bevacizumab-related TEAEs. Sixteen (32.0%) experienced ZPML265-related TEAEs of grade ≥3 and 14 (28.0%) experienced bevacizumab-related TEAEs of grade ≥3. Six (12.0%) experienced ZPML265-related SAEs and six (12.0%) experienced bevacizumab-related SAEs. Twenty-six (52.0%) experienced immune-related TEAEs and seven (14.0%) experienced immune-related TEAEs of grade ≥3. TEAEs related to ZPML265 that led to discontinuation of treatment occurred in 18 patients (36.0%), and 1 patient (2.0%) experienced permanent discontinuation of treatment related to ZPML265. One patient (2.0%) experienced an adverse event related to ZPML265 that led to withdrawal from the trial. No adverse events related to ZPML265 that resulted in death were observed.

[0117] Queue C:

[0118] Among the 40 subjects in Cohort C (ZPML265 7.5 mg / kg + bevacizumab 15 mg / kg), a total of 40 (100.0%) experienced TEAEs and 35 (87.5%) experienced TRAEs. Of these, 32 (80.0%) experienced ZPML265-related TEAEs and 29 (72.5%) experienced bevacizumab-related TEAEs. Six (15.0%) experienced ZPML265-related TEAEs of grade ≥3 and 6 (15.0%) experienced bevacizumab-related TEAEs of grade ≥3. Three (7.5%) experienced ZPML265-related SAEs and two (5.0%) experienced bevacizumab-related SAEs. Eleven (27.5%) experienced immune-related TEAEs, and no immune-related TEAEs of grade ≥3 were observed. Nine patients (22.5%) experienced TEAEs leading to discontinuation of ZPML265-related treatment, but no patients permanently discontinued ZPML265-related treatment. No ZPML265-related adverse events led to trial withdrawal, and no ZPML265-related adverse events resulted in death.

[0119] The above data show that ZPML2655mg / kg or 7.5mg / kg combined with bevacizumab has good safety and does not show a significant increase in toxicity compared with PD-1 monoclonal antibody combined with bevacizumab.

[0120] Effectiveness Results:

[0121] As of February 20, 2023, a total of 108 subjects were included in the efficacy evaluable set, including 47 subjects in cohort A, 26 subjects in cohort B, and 35 subjects in cohort C.

[0122] In Cohort A, the overall objective response rate (ORR) was 38.3% (95% CI: 24.5%, 53.6%), with one subject (2.1%) achieving a complete response (CR) and 17 subjects (36.2%) achieving a partial response (PR) as their best outcome. Seventeen subjects (36.2%) had a sustained outcome as their best outcome, resulting in a sustained response (DCR) of 74.5% (95% CI: 59.7%, 86.1%). Twelve subjects (25.5%) had a partial response as their best outcome. Median progression-free survival (mPFS) was 7.0 (95% CI: 3.1-9.9) months.

[0123] In Cohort B, the overall ORR was 23.1% (95% CI: 9.0%, 43.6%), with one subject (3.8%) achieving a complete response (CR) and five subjects (19.2%) achieving a partial response (PR). Twelve subjects (46.2%) had a sustained outcome as their best response, resulting in a sustained response (DCR) of 69.2% (95% CI: 48.2%, 85.7%). Seven subjects (26.9%) achieved a partial response as their best response. The mean progression-free survival (mPFS) was 5.4 months (95% CI: 2.4-8.5).

[0124] In Cohort C, the overall ORR was 34.3% (95% CI: 19.1%, 52.2%). No subject achieved a CR, and 12 subjects (34.3%) had a PR as their best response outcome. Seventeen subjects (48.6%) had a SD as their best response outcome, resulting in a DCR of 82.9% (95% CI: 66.4%, 93.4%). Six subjects (17.1%) had a PD as their best response outcome.

[0125] As of the data analysis deadline, ZPML265 5mg / kg plus bevacizumab and ZPML265 7.5mg / kg plus bevacizumab demonstrated comparable efficacy, both demonstrating superior efficacy compared to ZPML264 plus bevacizumab. Follow-up for the ZPML265 7.5mg / kg plus bevacizumab group is shorter, and ORR data are not yet fully mature, but there is a trend toward further improvement.

[0126] Example 5: A randomized, controlled, open-label, multicenter phase II / III clinical study evaluating ZPML265 combination therapy versus sintilimab combined with bevacizumab as first-line treatment for patients with advanced hepatocellular carcinoma

[0127] In a Phase II / III clinical study conducted in China, approximately 668 patients with unresectable or metastatic hepatocellular carcinoma who had not received systemic treatment were planned to be enrolled. Patients were required to be between 18 and 75 years old; Barcelona Clinic Liver Cancer (BCLC) stage C, or stage B not suitable for radical surgery and / or local treatment; Eastern Cooperative Oncology Group (ECOG) performance status score of 0 or 1; Child-Pugh liver function class A or class B ≤7; and the functional level of important organs before the first use of the investigational drug:

[0128] 1) Absolute neutrophil count ≥1.5×10 9 / L;

[0129] 2) Platelets ≥75×10 9 / L;

[0130] 3) Hemoglobin ≥90 g / L;

[0131] 4) Albumin ≥29 g / L;

[0132] 5) Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (AKP) are all ≤5×ULN;

[0133] 6) total bilirubin (TBIL) ≤ 2 × ULN;

[0134] 7) Creatinine clearance (CrCl) calculated by the Cockcroft-Gault equation ≥50 mL / min;

[0135] 8) International normalized ratio (INR) ≤ 2 or prothrombin time (PT) exceeds the upper limit of the normal range ≤ 6 seconds;

[0136] 9) Urine protein <2+ (if urine protein ≥2+, 24-hour urine protein quantification is required, and patients with 24-hour urine protein quantification <1.0g can be included in the group).

[0137] Dosage regimen:

[0138] Phase II clinical study

[0139] Group 1 received ZPML265 combined with bevacizumab and chemotherapy, administered in the following order: ZPML265 at a dose of 7.5 mg / kg, intravenously on Day 1, every 3 weeks. Bevacizumab at a dose of 15 mg / kg, intravenously on Day 1, every 3 weeks. Chemotherapy was the XELOX regimen, with oxaliplatin at a dose of 85 mg / m 2 , intravenous drip, D1, Q3W; capecitabine dosage is 1000mg / m 2 , oral, twice a day, D1-14, then rest for 7 days, Q3W.

[0140] Group 2 received ZPML265 combined with bevacizumab, administered sequentially as follows: ZPML265 at a dose of 7.5 mg / kg, intravenously on Day 1, every 3 weeks; bevacizumab at a dose of 15 mg / kg, intravenously on Day 1, every 3 weeks.

[0141] Group 3 received ZPML265 combined with chemotherapy, which was administered in the following order: ZPML265 at a dose of 7.5 mg / kg, intravenous drip, D1, Q3W. Chemotherapy was the XELOX regimen, and oxaliplatin at a dose of 85 mg / m 2 , intravenous drip, D1, Q3W; capecitabine dosage is 1000mg / m 2 , oral, twice a day, D1-14, then rest for 7 days, Q3W.

[0142] The control group received sintilimab combined with bevacizumab, and the drugs were administered in the following order: sintilimab was administered at a dose of 200 mg, intravenous drip, D1, Q3W; bevacizumab was administered at a dose of 15 mg / kg, intravenous drip, D1, Q3W.

[0143] Phase III clinical study

[0144] The dosing regimens for the experimental and control groups were the same as those for the Phase II clinical study.

Claims

1. A pharmaceutical composition for treating hepatocellular carcinoma, comprising an effective amount of a mixed antibody of anti-CTLA4 and anti-PD1, wherein the mixed antibody is produced by a single host cell containing nucleic acids encoding two different antibodies, anti-CTLA4 and anti-PD1, wherein: The sequences of the anti-CTLA4 antibody heavy chain HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 1, 2 and 3, respectively; the sequences of the anti-CTLA4 antibody light chain LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 4, 5 and 6, respectively; the sequences of the anti-PD1 antibody heavy chain HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 9, 10 and 11, respectively; and the sequences of the anti-PD1 antibody light chain LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 12, 13 and 14, respectively.

2. The pharmaceutical composition according to claim 1, wherein The heavy chain variable region sequence of the anti-CTLA4 antibody is shown in SEQ ID NO: 7, the light chain variable region sequence of the anti-CTLA4 antibody is shown in SEQ ID NO: 8, the heavy chain variable region sequence of the anti-PD1 antibody is shown in SEQ ID NO: 15, and the light chain variable region sequence of the anti-PD1 antibody is shown in SEQ ID NO:

16.

3. The pharmaceutical composition according to claim 1, wherein The heavy chain sequence of the anti-CTLA4 antibody is shown in SEQ ID NO: 17, the light chain sequence of the anti-CTLA4 antibody is shown in SEQ ID NO: 18, the heavy chain sequence of the anti-PD1 antibody is shown in SEQ ID NO: 19, and the light chain sequence of the anti-PD1 antibody is shown in SEQ ID NO:

20.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the hepatocellular carcinoma is unresectable or metastatic hepatocellular carcinoma that has not been previously treated with systemic therapy.

5. The pharmaceutical composition according to claim 4, wherein the dosage of the mixed antibody is 5 mg / kg or 7.5 mg / kg, administered once every three weeks by intravenous infusion on day 1, and a treatment cycle of 21 days.

6. The pharmaceutical composition according to claim 4, further comprising a chemotherapeutic drug and / or an anti-angiogenic agent.

7. The pharmaceutical composition according to claim 6, wherein the chemotherapy drugs are oxaliplatin and capecitabine, and / or the anti-angiogenic agent is bevacizumab.

8. The pharmaceutical composition according to claim 7, wherein the dosage of bevacizumab is 7.5 mg / kg or 15 mg / kg, and each three-week administration cycle is administered by intravenous infusion on the first day of each administration cycle.

9. The pharmaceutical composition according to claim 7, wherein the dosage of oxaliplatin is 85 mg / m 2 The administration cycle was three weeks, and the drug was administered by intravenous infusion on the first day of each administration cycle; the dose of capecitabine was 1000 mg / m 2 , one dosing cycle is three weeks, and the drug is administered orally from day 1 to day 14 of each cycle.

10. The pharmaceutical composition according to any one of claims 1 to 3, wherein the hepatocellular carcinoma is hepatocellular carcinoma with high-risk recurrence factors after surgical resection or local ablation therapy. The pharmaceutical composition according to claim 10 , wherein the mixed antibody is administered at a dosage of 5 mg / kg or 7.5 mg / kg, with one administration cycle consisting of three weeks, and is administered by intravenous infusion on the first day of each administration cycle.

12. A method for treating hepatocellular carcinoma, comprising administering the pharmaceutical composition of any one of claims 1 to 11 to a subject.

13. Use of a mixed antibody comprising an effective amount of anti-CTLA4 and anti-PD1 in the preparation of a pharmaceutical composition for treating hepatocellular carcinoma, wherein the mixed antibody is produced by a single host cell containing nucleic acids encoding two different antibodies, anti-CTLA4 and anti-PD1, wherein: The heavy chain sequence of the anti-CTLA4 antibody is shown in SEQ ID NO: 17, the light chain sequence of the anti-CTLA4 antibody is shown in SEQ ID NO: 18, the heavy chain sequence of the anti-PD1 antibody is shown in SEQ ID NO: 19, and the light chain sequence of the anti-PD1 antibody is shown in SEQ ID NO: 20; the hepatocellular carcinoma is unresectable or metastatic hepatocellular carcinoma that has not been previously treated systemically; or, the hepatocellular carcinoma is hepatocellular carcinoma with high-risk factors for recurrence after surgical resection or local ablation therapy.

14. The use according to claim 13, wherein the pharmaceutical composition further comprises a chemotherapeutic drug and / or an anti-angiogenic agent.

15. The use according to claim 14, wherein the chemotherapy drugs are oxaliplatin and capecitabine, and / or the anti-angiogenic agent is bevacizumab.