WO2023198089A1 - Pharmaceutical composition comprising anti-ctla4 and anti-pd1 antibody mixture and therapeutic use thereof - Google Patents

Abstract

Provided are an anti-CTLA4 and anti-PD1 antibody mixture, a combination of the antibody mixture, a chemotherapeutic drug and an anti-angiogenic agent, and use thereof for treating colorectal cancer.

Description

Pharmaceutical compositions containing mixed antibodies against CTLA4 and anti-PD1 and their therapeutic uses Technical field

The present disclosure relates to the treatment of cancer, particularly colorectal cancer, including immunotherapy and combination therapy. More specifically, the present disclosure relates to the use of a pharmaceutical composition comprising a mixed antibody against CTLA4 and anti-PD1 to treat colorectal cancer.

Background technique

The 2020 version of the GLOBOCAN statistical report shows that in 2020, there will be approximately 19.3 million new cases of cancer and nearly 10 million cancer deaths around the world. Among them, the proportion of new cases of colorectal cancer (CRC) is about 10.0%, ranking third; the proportion of deaths is about 9.4%, ranking second. In February 2022, the latest cancer statistics from the National Cancer Center of China showed that the incidence rate of colorectal cancer in China ranked third, with 408,000 cases; the mortality rate ranked fourth, with 195,600 deaths. The incidence and mortality of colorectal cancer remain on the rise, and it is an important disease that threatens human health worldwide. Since the early symptoms of CRC are not obvious, about 20% of patients have metastasized or progressed to an advanced stage when diagnosed, and more than 50% of patients with early-stage CRC will eventually relapse or metastasize.

The treatment of CRC is mainly surgery, combined with endoscopic treatment, chemotherapy and radiotherapy. Palliative care for advanced colorectal cancer is stratified based on molecular marker testing results. dMMR (mismatch repair protein deficiency)/MSI-H (microsatellite instability high) type CRC accounts for about 12 to 15% of CRC in the entire population, and the late stage accounts for about 5%. KRAS and NRAS are GTPase proteins encoded by RAS family member genes. They participate in signal transduction of epidermal growth factor receptors and regulate cell growth, differentiation, proliferation, and survival. 40% to 50% of colorectal cancer patients have KRAS point mutations; 3.8% of colorectal cancer patients have NRAS gene point mutations. And, as a member of the RAF proto-oncogene family, the BRAF gene is located downstream of the RAS gene and is a key member of the RAS-RAF-MEK kinase pathway. Among Asian colorectal cancer patients, the BRAF mutation rate is 5.4% to 6.7%. Among patients with metastatic colorectal cancer with BRAF gene mutations, 90% have BRAF V600E mutations.

For patients with dMMR/MSI-H CRC, immune checkpoint inhibitors (ICIs) have become the standard treatment option for first-line and later-line palliative care, and are superior to chemotherapy in perioperative neoadjuvant treatment of CRC. Efficacy. Currently, a variety of anti-PD-1/PD-L1 monoclonal antibodies have been approved globally for the second-line and subsequent treatment of dMMR/MSI-H metastatic colorectal cancer (mCRC), such as pembrolizumab, nivolumab Uzumab, envolizumab, tislelizumab, slutizumab, putelizumab, etc.; based on the KEYNOTE-177 study, pembrolizumab has been approved for dMMR/MSI- H mCRC first-line treatment. However, no anti-PD-1/PD-L1 mAb combined with anti-CTLA-4 mAb has been approved for this indication.

For patients with MSS (microsatellite stable) CRC, ICIs, as a late-line systemic treatment option, have poor efficacy. Anti-PD-1 monoclonal antibodies combined with standard treatment regimens have certain efficacy in the first-line treatment of mCRC, but still need to be further improved. In first-line treatment of mCRC, RAS/RAF gene mutations suggest that patients receiving anti-EGFR targeted therapy have a poorer prognosis compared with anti-VEGF monoclonal antibodies (such as bevacizumab). However, this prognostic indicator has not yet been confirmed in immunotherapy.

ZPML265 is a mixed antibody pharmaceutical preparation 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 mixed antibody can specifically bind to CTLA4 and PD1 at the same time, thereby blocking the two immune checkpoint signaling pathways of CTLA-4 and B7-1/B7-2, as well as PD-1 and PD-L1, and releasing the impact of the two pathways on T lymphocytes. The inhibitory effect of cells restores their functional activity and anti-tumor immune response, thereby enabling the body to achieve the purpose of fighting and killing tumors. Different from the combined use of nivolumab and ipilimumab, ZPML265 can achieve continuous administration of CTLA-4 monoclonal antibody during the combined treatment, thereby further improving its therapeutic effect.

However, the proportion of dMMR/MSI-H CRC is small, and the vast majority of CRC does not respond well to immune checkpoint inhibitors. There are currently no immunotherapy products for the entire colorectal cancer population or other biomarker-enriched populations. Approved, there are unmet clinical needs, and the field urgently needs to solve this problem. Therefore, immunological and/or combination therapy regimens are of great interest as potential treatment options.

Summary of the invention

The technical problem to be solved by this disclosure is to provide an immunotherapy to fill the clinical gap that there is no dual-target immune (double-immune) therapeutic drug for colorectal cancer in the world, thereby solving such unmet clinical needs. need.

The present disclosure provides a pharmaceutical composition for treating colorectal cancer, comprising an effective amount of a mixed antibody against CTLA4 and anti-PD1.

In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

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

In some embodiments, the additional therapeutic agents are chemotherapeutic agents and anti-angiogenic agents, with preferred chemotherapeutic agents being oxaliplatin and capecitabine, and the preferred anti-angiogenic agent being bevacizumab.

In some embodiments, the colorectal cancer of the present disclosure is unresectable or metastatic colorectal cancer.

Preferably, the unresectable or metastatic colorectal cancer is mismatch repair protein deficient (dMMR) or microsatellite unstable high (MSI-H) colorectal cancer; another preferred solution is that the unresectable or metastatic colorectal cancer Metastatic colorectal cancer is microsatellite stable (MSS) colorectal cancer.

Therefore, the present disclosure also preferably provides a pharmaceutical composition for the treatment of unresectable or metastatic mismatch repair protein-deficient (dMMR) or microsatellite instability-high (MSI-H) colorectal cancer, which contains an effective amount Mixed anti-CTLA4 and anti-PD1 antibodies.

At the same time, the present disclosure also preferably provides a pharmaceutical composition for treating unresectable or metastatic microsatellite stable (MSS) colorectal cancer, which contains an effective amount of anti-CTLA4 and anti-PD1 mixed antibodies, chemotherapy Drugs and anti-angiogenic agents.

More preferably, the unresectable or metastatic microsatellite stable (MSS) colorectal cancer is the case where the KRAS, NRAS and BRAF genes are all wild type; alternatively, the unresectable or metastatic microsatellite stable (MSS) colorectal cancer can also be This is a case where any one of the KRAS, NRAS, and BRAF genes is mutated.

In some embodiments, the mixed antibodies are produced from 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 chains HCDR1, HCDR2 and HCDR3 are respectively SEQ. ID NO: 1, 2, and 3, the sequences of the anti-CTLA4 antibody light chain LCDR1, LCDR2, and LCDR3 are respectively SEQ ID NO: 4, 5, and 6, and the sequences of the anti-PD1 antibody heavy chain HCDR1, HCDR2, and HCDR3 are respectively They are shown in SEQ ID NO: 9, 10, and 11, and the sequences of the anti-PD1 antibody light chain LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO: 12, 13, and 14 respectively.

In some embodiments, the heavy chain variable region sequence of the anti-CTLA4 antibody is set forth in SEQ ID NO:7, the light chain variable region sequence of the anti-CTLA4 antibody is set forth in SEQ ID NO:8, and the heavy chain sequence of the anti-PD1 antibody is set forth in SEQ ID NO:7. The variable region sequence 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.

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

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

The dosage of the chemotherapy drug oxaliplatin injection is 130 mg/m ² , which is administered by intravenous infusion on the first day, with a treatment cycle of 21 days; the dosage of the chemotherapy drug capecitabine tablets is 1000 mg/m ² , taken orally, twice a day, starting on the first day of every 3 weeks, stopping for 1 week after every 2 weeks, and every 3 weeks (21 days) is a treatment cycle; dosage of bevacizumab injection It is 7.5mg/kg, intravenous infusion, administered once on the first day of every 3 weeks, and every 3 weeks is a treatment cycle.

Among them, oxaliplatin can be used for a maximum of 8 treatment cycles, and capecitabine tablets can be used for a maximum of 2 years.

And, the present disclosure also provides a method for treating colorectal cancer, which method includes administering a pharmaceutical composition to a subject, and the pharmaceutical composition is the aforementioned mixed antibody containing an effective amount of anti-CTLA4 and anti-PD1.

The present disclosure also preferably provides a method of treating unresectable or metastatic mismatch repair protein deficient (dMMR) or microsatellite unstable high (MSI-H) colorectal cancer, the method comprising administering to a subject a drug combination The pharmaceutical composition is the aforementioned mixed antibody containing an effective amount of anti-CTLA4 and anti-PD1.

The present disclosure also preferably provides a method of treating unresectable or metastatic microsatellite stable (MSS) colorectal cancer, the method comprising administering to a subject a pharmaceutical composition, the pharmaceutical composition comprising an effective amount of A mix of anti-CTLA4 and anti-PD1 Antibodies, chemotherapeutic drugs, and anti-angiogenic agents.

The results of a Phase I clinical study of the disclosed mixed antibody as a single agent showed that it had certain efficacy and good safety in 27 patients with advanced CRC. At the same time, clinical studies based on the hybrid antibody of the present disclosure combined with bevacizumab have also shown that immune checkpoint inhibitors combined with anti-angiogenic drugs can exert a synergistic anti-tumor effect in advanced colorectal cancer. Therefore, the mixed antibody single drug or the combination regimen of mixed antibodies, chemotherapy drugs and bevacizumab provided by the present disclosure is expected to have controllable safety, good patient compliance, and can be used in the medical treatment of advanced colorectal cancer. To a large extent, it fills the gap where dual-immune combination therapy drugs have not yet been approved and solves the urgent unmet clinical needs.

Detailed ways

the term

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. Enter the general.

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

Certain embodiments disclosed herein include numerical ranges, and certain aspects of the disclosure may be described in terms of ranges. Unless otherwise stated, it should be understood that numerical ranges or range descriptions are only for the purpose of simplicity and convenience and should not be considered to strictly limit the scope of the present disclosure. Accordingly, descriptions expressed in range terms should be considered to specifically disclose all possible subranges and all possible specific numerical points within such ranges, as if such subranges and numerical points were expressly written herein. The above principles apply equally regardless of the width of the values stated. When a range description is used, the range includes the endpoints of the range.

When referring to measurable values such as amounts, temporary durations, etc., the term "about" is meant to include ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±20% of the specified value. A variation of ±1% in some cases, or ±0.1% in some cases.

The three-letter and single-letter codes for amino acids used herein are as described in J. Biol. Chem, 243, p3558 (1968).

As used herein, the term "antibody" typically refers to an antibody containing two heavy (H) polypeptide chains (HC) and two light (L) polypeptide chains (LC) held together by covalent disulfide bonds and non-covalent interactions. ) Y-type tetrameric protein. Natural IgG antibodies have such a structure. Each light chain consists of a variable domain (VL) and a constant domain (CL). Each heavy chain contains a variable domain (VH) and a constant region (CH).

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, such as IgG can be divided into IgG1, IgG2, IgG3, 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 distinct types, termed kappa and lambda, based on the amino acid sequence of their constant domains.

The term "variable region" or "variable domain" shows significant variation in the amino acid composition from one antibody to another and is primarily responsible for antigen recognition and binding. The variable regions of each light/heavy chain pair form the antibody binding site, such that a complete IgG antibody has two binding sites (i.e. it is bivalent). The variable region (VH) of the heavy chain and the variable region (VL) of the light chain each contain three regions of extreme variability known as the hypervariable region (HVR), or more commonly, as Complementarity determining region (CDR), VH and VL each have 4 framework regions FR (or framework regions), represented by FR1, FR2, FR3, and FR4 respectively. Therefore, CDR and FR sequences typically occur 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.

Types of "antibodies" in a broad sense can 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, intrabodies, multispecific antibodies, bispecific antibodies, single chain antibodies, monovalent antibodies, multivalent antibodies, single domain antibodies, Nanobodies, synthetic antibodies (including Mutated proteins and their variants), etc.

The term "monoclonal antibody" (or "monoclonal antibody") refers to a substantially homogeneous antibody produced by a single cell clone and directed only 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 the above technologies.

It should be noted that the division of CDRs and FRs in the antibody variable region of the present disclosure is determined based on 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 nomenclature system based on the antibody sequences of the disclosure are expressly within the scope of the disclosure.

The term "antigen" refers to a substance 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 the selected target, including single epitopes, multiple epitopes, and single structures. domain, multi-domain, or complete extracellular domain (ECD) or protein.

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear, cyclic or branched, it may contain modified amino acids, especially 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.

As used herein, the term "mixed antibodies" refers to cells containing DNA from a host that has been transfected with DNA encoding at least two different antibodies with different binding specificities, optionally full-length primate IgG antibodies A limited number of major antibody species, optionally no more than two, three, four, five, six, seven, eight, optionally produced by cells from a single host cell line) One species, nine species, or ten species. In some embodiments, DNA encoding at least two different heavy chains (HC) and at least two different light chains (LC) can be introduced into the same host cell. For example, the host cell can be genetically modified with DNA encoding at least two but no more than four different light chains (LC). DNA transfection of different antibodies with different binding specificities. In some embodiments, the sequences of all transfected DNA encoding HC and LC can be mutated, thereby changing the amino acid sequence of the antibody to disfavor non-homologous HC/LC pairing and strongly favor homologous HC/LC pairing. Where two different HCs are introduced into a host cell, one or both of the two different HCs can optionally be altered such that formation of heterodimers is not favored. In some embodiments, only one heavy chain is altered to prevent heterodimer formation. In some embodiments, where DNA encoding only two different antibodies is introduced into a host cell, only one of the antibodies encoded by the DNA contains one or more partner orientation changes such that homology is favored. HC/LC pairing, whereas the other antibody did not contain such changes. In some embodiments, the host cell produces only two major antibody species, where each HC primarily pairs with its cognate LC, and most antibodies are those containing two heavy chains with the same amino acid sequence and two heavy chains with the same amino acid sequence. Tetramer of light chains (see PCT/US2017/030676).

The term "pharmaceutical composition" refers to a preparation or a combination of preparations containing one, two or more active ingredients, which allows the active ingredients contained therein to be present in a biologically active form and which does not contain any chemicals necessary for administration. Additional components of the formulation have unacceptable toxicity to subjects. When a "pharmaceutical composition" exists in the form of a combination of separate preparations containing two or more active ingredients, it can be administered simultaneously, sequentially, separately or at intervals. The purpose is to exert the biological activity of the multiple active ingredients and use them together. For treating diseases.

The term "pharmaceutically acceptable 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.

The term "effective amount" refers to that dose of a pharmaceutical formulation containing an active ingredient of the present disclosure that produces the desired effect in the patient being treated when administered to the patient in single or multiple doses. The effective amount can be readily determined by the attending physician, who is one of skill in the art, by considering 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 dosing regimen selected; and the use of any concomitant therapy.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid is introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be identical in nucleic acid content to the parent cells but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cells are included herein.

The term "transfection" as used herein refers to the introduction of exogenous nucleic acid into a eukaryotic cell. Transfection can be achieved by various means known in the art, including electroporation, microinjection, liposome fusion, etc.

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

Methods for producing and purifying antibodies and antigen-binding fragments are well known and available in the art, such as Cold Spring Harbor's Antibody Experimentation Technical Guide, Chapters 5-8 and 15. Engineered antibodies of the present disclosure, or antigen-binding fragments thereof, may 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 stably transfect CHO cells. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones were expanded in serum-free medium in bioreactors to produce antibodies. The culture medium secreting antibodies can be purified and collected using conventional techniques. Antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange.

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

As used herein, the terms "disease" or "disorder" or "disorder" or the like refer to any change or disorder that impairs or interferes with the normal function of a cell, tissue or organ. For example, the "disease" includes but is not limited to: tumors, pathogenic infections, autoimmune diseases, T cell dysfunction diseases, or immune tolerance defects (such as transplant rejection), etc.

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

The term "treatment" as used herein refers to clinical intervention in an attempt to modify an individual or to treat a cell-induced disease process, either prophylactically or in the clinical pathological process. Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing the direct or indirect pathological consequences of any disease, preventing metastasis, slowing down the progression of the disease, improving or alleviating the condition, alleviating or improving the prognosis, etc.

As used herein, the term "combination" refers to a treatment regimen that provides at least two or more different therapies to achieve a specified therapeutic effect, and the therapies may be either physical, such as radiation therapy, or chemical, such as administration of Subject drugs, the drugs also include combination drugs. "Combination drug" refers to a combination of two or more pharmaceutical preparations each having active ingredients, which need to be used together when administered to a subject. The active ingredients can be mixed together to form a single dosing unit, or they can be formed into separate dosing units and used separately; during administration, different pharmaceutical preparations can be administered substantially simultaneously, simultaneously or sequentially.

Overall survival (OS) refers to the time between the start of study treatment and death from any cause. For patients who are still followed up as of the analysis cut-off date (Cut-off), the cut-off date will be used as the censoring time, and for subjects who are lost to follow-up, the date of the last contact with them will be used as the censoring time.

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

Objective response rate (ORR) was defined as the best confirmed ORR during the study period, including complete response (CR) and partial response (PR) cases. According to RECIST v1.1, the first occurrence of PR or CR requires additional imaging to confirm the lesion at ≥4 weeks.

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

Complete response (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 <10 mm.

Partial response (PR): The sum of target lesion diameters is reduced by at least 30% from baseline.

Disease progression (PD): Taking the minimum value of the sum of the diameters of all measured target lesions during the entire experimental study as a reference, the relative increase in the diameter sum is at least 20% (if the baseline measurement value is the smallest, the baseline value is used as the reference); otherwise In addition, the absolute value of the diameter and sum must be increased by at least 5 mm (the appearance of one or more new lesions is also considered disease progression).

Stable disease (SD): The reduction of the target lesion does not reach the PR level, and the increase does not reach the PD level. It is somewhere in between. The minimum value of the sum of diameters can be used as a reference during research.

Duration of response (DOR) was defined as the time from when the tumor was first assessed as CR or PR (whichever was recorded first) to when the tumor was first assessed as PD or death.

Treatment-emergent adverse events (TEAE) refer to adverse events that occur during treatment.

Treatment-related adverse events (TRAE) refer to adverse events related to the study drug that occur during treatment.

Serious adverse events (SAE) refer to death, life-threatening, permanent or severe disability or loss of function after the subject receives the investigational drug, the subject needs hospitalization or prolonged hospitalization, and adverse events such as congenital anomalies or birth defects. Medical events.

Example

The present disclosure will be further described below with reference to specific embodiments. 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.

Example 1 Obtaining mixed antibody ZPML265

Prepare a mixed antibody produced by a single host cell line (see PCT/US2017/030676). The mixed antibody contains two active ingredients, namely a recombinant humanized IgG1 monoclonal antibody targeting human CTLA4 and an antibody targeting human PD1. Recombinant humanized IgG4 monoclonal antibody. This mixed antibody can specifically bind to human CTLA4 and PD1 at the same time. After the two antibodies are simultaneously expressed through a single host cell line, they are collected, purified, and combined with a pharmaceutically acceptable carrier to form a single mixed antibody drug preparation ZPML265. anti- The respective amino acid sequences of CTLA4 antibodies and anti-PD1 antibodies are shown in Table 1 below.

Table 1 Amino acid sequence of each component of mixed antibody ZPML265

Example 2 Effectiveness and safety of ZPML265 monotherapy in the treatment of advanced colorectal cancer

Administration:

In a phase I clinical study conducted in China, 27 patients with pathologically confirmed metastatic or recurrent colorectal cancer were enrolled, 26 of whom were colorectal adenocarcinoma, and the patients were over 18 years old, according to the Eastern Cooperative Oncology Group (ECOG) performance status score of 0 or 1, expected survival ≥3 months, and functional level of vital organs before the first use of the investigational drug:

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

2) Platelets ≥75×10 ⁹ /L;

3) Hemoglobin ≥90g/L;

4) Serum albumin ≥30g/L;

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

6) Total bilirubin ≤1.5×ULN (patients with Gilbert syndrome are allowed ≤3×ULN);

7) International normalized ratio (INR) ≤ 1.5; activated partial thromboplastin time (APTT) ≤ 1.5 × ULN;

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

9) Cardiac left ventricular ejection fraction (LVEF) >50%.

Dosing schedule: The dose of ZPML265 is 5 mg/kg, administered by intravenous infusion once every three weeks.

Effectiveness results:

ZPML265 monotherapy treated CRC. Among 27 subjects, the confirmed ORR was 7.4% (95% CI: 0.910, 24.290), and the DCR was 25.9% (95% CI: 11.114, 46.285), of which 2 patients had PR. 5 cases SD. ZPML265 single agent has shown certain effectiveness in advanced CRC.

Safety results:

Safety data summarized data from 609 subjects treated with ZPML265. As of May 31, 2022, 546 (89.7%) patients had experienced TEAEs, and 451 (74.1%) patients had 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 and SAEs related to ZPML265 were reported in 102 (16.7%) and 75 (12.3%) patients. SAE related to ZPML265.

Among them, TRAEs with an incidence of ≥10% include: rash (17.6%), pruritus (12.3%), hypothyroidism (11.3%), hyperthyroidism (10.3%), anemia (10%), and aspartate Acid aminotransferase was elevated (10%). The incidence of grade ≥3 TRAEs was 16.7%, and TRAEs with an incidence ≥1% included anemia (2.3%), increased aspartate aminotransferase (1.1%), and increased alanine aminotransferase (1.0% ).

Compared with ZPML265 monotherapy, another meta-analysis that summarized 2664 cases of nivolumab combined with ipilimumab showed that the overall incidence of treatment-related serious adverse events The rate was 29.6%, and the incidence rate of grade ≥3 TRAE was 39.9%, which was higher than the relevant data of ZPML265 monotherapy.

It can be seen from the existing data that the safety of the treatment is acceptable, and no new safety signals were found compared with other immunotherapy drugs.

in conclusion:

Overall, ZPML265 monotherapy shows promising therapeutic effects in the treatment of advanced CRC. In terms of safety, the incidence of TRAEs is similar to that of anti-PD-(L)1 monotherapy and lower than that of Nivolumab+Ipilimumab dual-immune drug combination therapy, that is, , while ensuring the effectiveness of the drug, the safety is better than the existing clinical solutions in the existing technology.

Example 3: An evaluation of ZPML265 alone or in combination with bevacizumab and XELOX regimen in the first-line treatment of unresectable locally advanced disease or phase II clinical studies in metastatic CRC

In the phase II clinical study of ZPML265 single agent combined with bevacizumab and XELOX regimen as first-line treatment of unresectable locally advanced or metastatic CRC, patients with unresectable locally advanced or metastatic colorectal adenocarcinoma confirmed by histological examination were enrolled. , divided into 2 cohorts: Cohort 1 enrolled the MSI-H population, and Cohort 2 enrolled the low-grade microsatellite instability (MSI-L) or MSS and RAS and BRAF wild-type populations. Patients are aged ≥18 years old, regardless of gender. Eastern Cooperative Oncology Group (ECOG) performance status score 0-1. Expected survival ≥3 months. The functional level of vital organs must meet the following requirements before using the investigational drug for the first time:

1) Neutrophil count (ANC) ≥1.5×10 ⁹ /L;

2) Platelets (PLT) ≥90×10 ⁹ /L;

3) Hemoglobin (Hb) ≥90g/L;

4) Albumin (ALB) ≥ 30g/L;

5) Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 2.5 × upper limit of normal range (ULN). For subjects with liver metastases, AST and ALT are required to be ≤ 5 × ULN;

6) Serum total bilirubin (TBIL) ≤ 1.5 × ULN (subjects with Gilbert syndrome require TBIL < 3 × ULN);

7) Serum creatinine (Cr) ≤ 1.5 × ULN or creatinine clearance (CCr) ≥ 50 mL/min (apply the standard Cockcroft-Gault formula);

8) Cardiac left ventricular ejection fraction (LVEF)>50%;

9) Qualitative urine protein ≤1+ (if ≥2+, a 24-hour urine protein test is required. If the result is <1g, inclusion is allowed);

10) Activated partial thromboplastin time (APTT) ≤ 1.5 × ULN, international normalized ratio (INR) ≤ 1.5, prothrombin time (PT) ≤ 1.5 × ULN.

Dosing regimen:

Queue 1:

The dosage of ZPML265 is 5mg/kg, intravenous infusion, once every 3 weeks on the first day, and every 3 weeks is a treatment cycle;

Queue 2:

The dosage of ZPML265 is 5mg/kg, intravenous infusion, once every 3 weeks on the first day, and every 3 weeks is a treatment cycle;

The dosage of bevacizumab injection is 7.5mg/kg, intravenous infusion, once every 3 weeks on the first day, and every 3 weeks is a treatment cycle;

Oxaliplatin injection: The dosage is 130 mg/m ² , intravenous infusion, once every 3 weeks on the first day, and every 3 weeks is a treatment cycle; a maximum of 8 treatment cycles can be used;

Capecitabine tablets: The dosage is 1000 mg/m ² , taken orally, twice a day, starting on the first day of every 3 weeks, stopping for 1 week after every 2 weeks, and every 3 weeks is a treatment cycle; The maximum duration of medication is 2 years.

Phased effectiveness results:

This study is still in the enrollment stage. As of March 13, 2023, 3 subjects have been enrolled in Cohort 1, and 3 subjects have at least 1 post-treatment tumor evaluation, including 2 PR and 1 SD. The ORR was 66.7% and the DCR was 100%. Cohort 2 has enrolled 14 subjects, 8 of whom have at least 1 post-treatment tumor assessment, 4 of whom have PR, 4 have SD, ORR is 50%, and DCR is 100%; among them, the best efficacy is Among the 4 subjects with SD, 3 had only the first tumor evaluation, and 1 had 2 tumor evaluations. The tumor reduction ratios were 23%, 26%, 29% and 29% respectively, which are very close to the PR standard ( shrink by at least 30%), it is expected that the probability of conversion to PR after continuing to receive study treatment is greater.

It can be seen that ZPML265 monotherapy for the treatment of MSI-H CRC, or ZPML265 combined with bevacizumab and chemotherapy, has shown outstanding effectiveness data.

Safety results:

As of March 13, 2023, among the 3 subjects in Cohort 1, 1 (33.3%) had TEAE, and no adverse events related to ZPML265 have occurred. In Cohort 2, 11 subjects entered the safety analysis set, and 7 subjects (63.6%) experienced TEAEs, all of which were AEs related to combined medication (TRAEs). Major TRAEs (≥ 2 subjects) occurred. ) had decreased neutrophil count (3, 27.3%), decreased platelet count (3, 27.3%), decreased white blood cell count (2, 18.2%), vomiting (2, 18.2%), nausea (2, 18.2%) , diarrhea (2, 18.2%); 1 subject (9.1%) experienced grade ≥3 TRAEs, which were decreased platelet count (9.1%) and hypertriglyceridemia (9.1%); 1 subject (9.1%) A SAE occurred in 9.1%) subjects, which was a decrease in platelet count (9.1%). No TEAEs resulting in death occurred.

Safety data suggest that ZPML265 is safe when used as a single agent; when combined with bevacizumab and chemotherapy, it mainly shows chemotherapy-related hematological toxicity and gastrointestinal reactions, and is well tolerated.

in conclusion:

In summary, ZPML265 monotherapy is initially more effective than PD-(L)1 monoclonal antibody monotherapy in the first-line treatment of patients with MSI-H advanced CRC. treatment, and the safety profile is good, with no new safety signals emerging. ZPML265 is combined with bevacizumab and chemotherapy as a first-line treatment for patients with advanced CRC in MSS. The preliminary efficacy is good. The tumor size of all subjects shows a shrinking trend, and the combination of drugs does not increase safety risks.

Claims (27)

1. A pharmaceutical composition for the treatment of colorectal cancer, which contains an effective amount of a mixed antibody against CTLA4 and anti-PD1. The mixed antibody is produced by a single host containing nucleic acids encoding two different antibodies against CTLA4 and anti-PD1. Produced by cells, among which, the sequences of anti-CTLA4 antibody heavy chain HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 1, 2 and 3 respectively, and the sequences of anti-CTLA4 antibody light chain LCDR1, LCDR2 and LCDR3 are respectively SEQ ID NO: As shown in 4, 5 and 6, the sequences of anti-PD1 antibody heavy chain HCDR1, HCDR2 and HCDR3 are respectively SEQ ID NO: 9, 10 and 11. The sequences of anti-PD1 antibody light chain LCDR1, LCDR2 and LCDR3 are respectively SEQ ID NO. NO: shown in 12, 13 and 14.
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, and 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 of 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, and the heavy chain sequence of the anti-PD1 antibody is shown in SEQ ID NO: 17. The chain sequence 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 colorectal cancer is unresectable or metastatic colorectal cancer.
5. The pharmaceutical composition of claim 4, wherein the unresectable or metastatic colorectal cancer is mismatch repair protein-deficient (dMMR) or microsatellite instability-high (MSI-H) colorectal cancer.
6. The pharmaceutical composition according to claim 5, the dosage of the mixed antibody is 5 mg/kg, administered once every three weeks, administered by intravenous infusion on the first day, and 21 days is a treatment cycle.
7. The pharmaceutical composition of claim 4, wherein the unresectable or metastatic colorectal cancer is microsatellite stable (MSS) colorectal cancer.
8. The pharmaceutical composition according to claim 7, wherein the KRAS, NRAS and BRAF genes of the microsatellite stable (MSS) colorectal cancer are all wild type.
9. The pharmaceutical composition according to claim 7, wherein any one of the KRAS, NRAS and BRAF genes of the microsatellite stable (MSS) colorectal cancer is a mutant type.
10. As for the pharmaceutical composition according to any one of claims 7 to 9, the dosage of the mixed antibody is 5 mg/kg, administered once every three weeks, administered by intravenous infusion on the first day, and 21 days is a treatment cycle.
11. The pharmaceutical composition according to any one of claims 7 to 9, further comprising a chemotherapeutic drug and an anti-angiogenic agent.
12. The pharmaceutical composition according to claim 11, wherein the chemotherapy drugs are oxaliplatin and capecitabine, and the anti-angiogenic agent is bevacizumab.
13. The pharmaceutical composition according to claim 12, the dosage of oxaliplatin is 130 mg/m ² and is administered by intravenous infusion on the first day. A treatment cycle is 21 days; the dosage of capecitabine is 1000 mg/m ² , taken orally, twice a day, starting on the first day of every 3 weeks, stopping for 1 week after every 2 weeks, every 3 weeks It is a treatment cycle; the dosage of bevacizumab is 7.5 mg/kg, administered by intravenous infusion on the first day, and 21 days is a treatment cycle.
14. A method of treating colorectal cancer, the method comprising administering to a subject a pharmaceutical composition containing an effective amount of a mixed antibody encoding anti-CTLA4 and anti-PD1, the mixed antibody being composed of a mixture encoding both anti-CTLA4 and anti-PD1 The nucleic acids of different antibodies are produced by a single host cell. Among them, the sequences of anti-CTLA4 antibody heavy chains HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 1, 2 and 3 respectively, and the sequences of anti-CTLA4 antibody light chains LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 1, 2 and 3 respectively. The sequences are shown in SEQ ID NO: 4, 5 and 6 respectively. The sequences of anti-PD1 antibody heavy chains HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 9, 10 and 11 respectively. The sequences of anti-PD1 antibody light chains LCDR1, LCDR2 and The sequences of LCDR3 are shown in SEQ ID NO: 12, 13, and 14 respectively.
15. The method of claim 14, 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, and the anti-CTLA4 antibody is shown in SEQ ID NO: 8. The heavy chain variable region sequence of the 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.
16. The method of claim 14, 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, and the heavy chain sequence of the anti-PD1 antibody It is shown in SEQ ID NO: 19, and the light chain sequence of the anti-PD1 antibody is shown in SEQ ID NO: 20.
17. The method of any one of claims 14-16, wherein the colorectal cancer is unresectable or metastatic colorectal cancer.
18. The method of claim 17, wherein the unresectable or metastatic colorectal cancer is mismatch repair protein-deficient (dMMR) or microsatellite instability-high (MSI-H) colorectal cancer.
19. According to the method of claim 18, the dosage of the mixed antibody is 5 mg/kg, administered once every three weeks, administered by intravenous infusion on the first day, and 21 days is a treatment cycle.
20. The method of claim 17, wherein the unresectable or metastatic colorectal cancer is microsatellite stable (MSS) colorectal cancer.
21. The method of claim 20, wherein the KRAS, NRAS and BRAF genes of the microsatellite stable (MSS) colorectal cancer are all wild type.
22. The method of claim 20, wherein any one of the KRAS, NRAS and BRAF genes of the microsatellite stable (MSS) colorectal cancer is mutated.
23. According to the method according to any one of claims 20 to 22, the dosage of the mixed antibody is 5 mg/kg, administered once every three weeks, administered by intravenous infusion on the first day, and 21 days is a treatment cycle.
24. The method of any one of claims 20-22, further comprising a chemotherapeutic agent and an anti-angiogenic agent.
25. The method of claim 24, wherein the chemotherapy drugs are oxaliplatin and capecitabine, and the anti-angiogenic agent is bevacizumab.
26. The method according to claim 25, the dosage of oxaliplatin is 130 mg/m ² , administered by intravenous infusion on the first day, and 21 days is a treatment cycle; the dosage of capecitabine is 1000 mg/m 2 m ² , taken orally, twice a day, starting on the first day of every 3 weeks, stopping for 1 week after every 2 weeks, and every 3 weeks is a treatment cycle; the dosage of bevacizumab is 7.5mg/ kg, administered by intravenous infusion on the first day, with a treatment cycle of 21 days.
27. A pharmaceutical composition for treating unresectable or metastatic colorectal cancer, which contains an effective amount of a mixed antibody against CTLA4 and anti-PD1. The mixed antibody is composed of two different types of antibodies encoding anti-CTLA4 and anti-PD1. The nucleic acid of the antibody is produced by a single host cell, 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, and the heavy chain sequence of the anti-PD1 antibody The sequence is shown in SEQ ID NO: 19, and the light chain sequence of the anti-PD1 antibody is shown in SEQ ID NO: 20.