CN115531530A

CN115531530A - Application of anti-human VEGF antibody in preparation of drugs for treating solid tumors

Info

Publication number: CN115531530A
Application number: CN202210759542.1A
Authority: CN
Inventors: 薛黎婷; 杨文清; 石明亮; 刘恋; 任晋生
Original assignee: Jiangsu Simcere Pharmaceutical Co Ltd
Current assignee: Shandong Simcere Bio Pharmaceutical Co ltd
Priority date: 2021-06-30
Filing date: 2022-06-30
Publication date: 2022-12-30

Abstract

The invention relates to an application of an anti-human VEGF antibody in preparing a medicament for treating solid tumors, in particular to an application of the anti-human VEGF antibody in preparing a medicament for treating advanced or metastatic solid tumors, and an application of a combination of the anti-human VEGF antibody, paclitaxel and carboplatin in preparing a medicament for treating lung cancer.

Description

Application of anti-human VEGF antibody in preparation of drugs for treating solid tumors

The invention is claimed in 2021, 6/30, and filed by the Chinese patent office, entitled "application of anti-human VEGF antibody in preparing medicine for treating solid tumor", priority of Chinese patent application No. 202110740378.5, the entire content of which is incorporated by reference into the present invention.

Technical Field

The invention relates to the field of medicines, in particular to application of an anti-human VEGF antibody in preparation of a medicine for treating solid tumors.

Background

Tumors are a major public health problem in China as well as globally. Tumor death accounts for 1/4 of all causes of death in China. In 2016, month 1, the national Cancer center director Chen Moqing brought the lead team to release the Cancer present-suffering data of residents in China for the first time on the Cancer communication (Cancer Letter) in the international famous Cancer professional journal. The result shows that the number of cases diagnosed as cancer and still alive within 5 years in China is about 749 ten thousand, the incidence rate of cancer is 250.28/10 ten thousand in 5 years in total, the incidence rate of the cancer is 556/10 ten thousand in 5 years, and the cumulative incidence rate (0-74 years) is 21.20 percent. The breast cancer, the colorectal cancer, the lung cancer, the gastric cancer and the esophagus cancer are the first five cancers with the highest prevalence rate in China, and account for 56.1 percent of the burden of the cancers in China.

Vascular Endothelial Growth Factor (VEGF) is a disulfide-linked heparin-binding dimeric glycoprotein that specifically binds to receptors on endothelial cell surfaces and exerts biological effects. Studies have shown that VEGF plays a key role in tumor growth and metastasis. How to effectively treat solid tumors, especially advanced or metastatic solid tumors, by the anti-human VEGF antibody has important significance.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention provides an application of an anti-human VEGF antibody in preparing a medicament for treating a patient with advanced or metastatic solid tumor or an application of an anti-human VEGF antibody, paclitaxel and carboplatin in preparing a medicament for treating lung cancer, which is used for improving the treatment effect on advanced or metastatic solid tumor.

In a first aspect, the invention provides the use of an anti-human VEGF antibody, the heavy chain variable region of which comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region of which comprises LCDR1, LCDR2 and LCDR3, in the manufacture of a medicament for the treatment of patients with advanced or metastatic solid tumors, wherein:

the HCDR1 is selected from a sequence shown in any one of SEQ ID NO 5-7;

the HCDR2 is selected from a sequence shown in any one of SEQ ID NO 8-10;

the HCDR3 is selected from a sequence shown in any one of SEQ ID NO 11-12;

the LCDR1 is selected from a sequence shown in any one of SEQ ID NO 13-14;

the LCDR2 is selected from a sequence shown in any one of SEQ ID NO 15-16; and the combination of (a) and (b),

the LCDR3 is selected from a sequence shown in SEQ ID NO. 17.

In some specific embodiments, the heavy chain variable region has the amino acid sequence as set forth in SEQ ID NO:1, or a sequence corresponding to SEQ ID NO:1, and a sequence having at least 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region has the sequence set forth in SEQ ID NO:3, or a sequence corresponding to SEQ ID NO:3 compared to a sequence having at least 95%, 96%, 97%, 98% or 99% identity.

In some embodiments, the heavy chain constant region of the antibody has the amino acid sequence as set forth in SEQ ID NO:2, or a sequence corresponding to SEQ ID NO:2, and a light chain constant region having a sequence as set forth in SEQ ID NO:4, or a sequence corresponding to SEQ ID NO:4 compared to a sequence having at least 95%, 96%, 97%, 98% or 99% identity.

In some embodiments, the solid tumor is a histologically, cytologically, or imagewise confirmed advanced solid tumor or metastatic malignant solid tumor.

In some specific embodiments, the solid tumor is colorectal cancer, breast cancer, gastric cancer, lung cancer, ovarian cancer, cervical cancer, liver cancer, pancreatic tail cancer, or duodenal papillary cancer.

In some specific embodiments, the patient is a patient lacking or ineffective standard therapy.

In some embodiments, the patient has previously received a chemotherapy, radiation therapy, and/or surgical treatment regimen.

In some specific embodiments, the patient has or has prior to surgical resection a metastatic lesion, optionally 1, 2, 3 or greater than 3 metastatic lesions.

In some specific embodiments, the anti-human VEGF antibody is administered to the patient in an effective amount of 1.5 to 8mg/kg, preferably 2mg/kg, 4mg/kg, 5mg/kg, 6mg/kg or 7.5mg/kg;

preferably, the dosage form of the anti-human VEGF antibody is a single dose dosage form, each dose containing an amount of antibody capable of being administered to a patient in the effective administration amount.

In some specific embodiments, the active ingredient of the medicament comprises only the anti-human VEGF antibody, or the medicament further comprises an active ingredient other than the anti-human VEGF antibody (other therapeutic agent other than the anti-human VEGF antibody).

In a second aspect, the invention also provides a method of treating a patient with advanced or metastatic solid tumor, the method comprising administering to the patient an effective amount of an anti-human VEGF antibody whose heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 and whose light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein:

the HCDR1 is selected from SEQ ID NO: 5-7;

the HCDR2 is selected from SEQ ID NO: 8-10;

the HCDR3 is selected from SEQ ID NO: 11-12;

the LCDR1 is selected from SEQ ID NO: 13-14;

the LCDR2 is selected from SEQ ID NO: 15-16; and the combination of (a) and (b),

the LCDR3 is selected from SEQ ID NO:17, and (b) is shown in the specification.

In some specific embodiments, the heavy chain variable region has the amino acid sequence as set forth in SEQ ID NO:1, or a sequence corresponding to SEQ ID NO:1, and a light chain variable region having a sequence as set forth in SEQ ID NO:3, or a sequence corresponding to SEQ ID NO:3 compared to a sequence having at least 95%, 96%, 97%, 98% or 99% identity.

In some embodiments, the heavy chain constant region of the antibody has the amino acid sequence as set forth in SEQ ID NO:2, or a sequence corresponding to SEQ ID NO:2, and a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:4, or a sequence corresponding to SEQ ID NO:4 compared to a sequence having at least 95%, 96%, 97%, 98% or 99% identity.

In some specific embodiments, the solid tumor is a histologically, cytologically, or imagewise confirmed advanced solid tumor or metastatic malignant solid tumor.

In some specific embodiments, the patient has one or more metastatic lesions present, optionally 2, 3 or more than 3 metastatic lesions present, or prior to surgical resection.

In some specific embodiments, the anti-human VEGF antibody is administered to the patient in an effective amount of 1.5 to 8mg/kg, preferably 2mg/kg, 4mg/kg, 5mg/kg, 6mg/kg or 7.5mg/kg.

In some specific embodiments, the anti-human VEGF antibody is administered 1 time every 2 weeks.

In a third aspect, the invention also provides an anti-human VEGF antibody, the heavy chain variable region of which comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region of which comprises LCDR1, LCDR2 and LCDR3, wherein:

the HCDR1 is selected from SEQ ID NO: 5-7;

the HCDR2 is selected from SEQ ID NO: 8-10;

the HCDR3 is selected from SEQ ID NO: 11-12;

the LCDR1 is selected from SEQ ID NO: 13-14;

the LCDR3 is selected from SEQ ID NO:17, and (b) is shown in the specification.

In some specific embodiments, the heavy chain variable region has the amino acid sequence as set forth in SEQ ID NO:1, or a sequence corresponding to SEQ ID NO:1, and a light chain variable region having a sequence as set forth in SEQ ID NO:3, or a sequence corresponding to SEQ ID NO:3 to a sequence having at least 95%, 96%, 97%, 98% or 99% identity.

In some specific embodiments, the heavy chain constant region of the antibody has the amino acid sequence as set forth in SEQ ID NO:2, or a sequence corresponding to SEQ ID NO:2, and a light chain constant region having a sequence as set forth in SEQ ID NO:4, or a sequence corresponding to SEQ ID NO:4 compared to a sequence having at least 95%, 96%, 97%, 98% or 99% identity.

In some specific embodiments, the patient has at least 1, 2, 3, or more than 3 metastatic lesions.

In some specific embodiments, the patient has or has prior to surgical resection a metastatic lesion, optionally, the metastatic lesion is 1, 2, 3 or more than 3 lesions.

In some specific embodiments, the effective amount of the antibody administered is 1.5 to 8mg/kg, preferably 2mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, or 7.5mg/kg;

In a fourth aspect, the present invention also provides the use of an anti-human VEGF antibody, paclitaxel and carboplatin for the manufacture of a medicament for the treatment of lung cancer, the heavy chain variable region of the antibody comprising HCDR1, HCDR2 and HCDR3 and the light chain variable region of the antibody comprising LCDR1, LCDR2 and LCDR3, wherein:

the HCDR1 is selected from SEQ ID NO: 5-7;

the HCDR2 is selected from SEQ ID NO: 8-10;

the HCDR3 is selected from SEQ ID NO: 11-12;

the LCDR1 is selected from SEQ ID NO: 13-14;

the LCDR3 is selected from SEQ ID NO:17, and (b) is shown in the specification.

In some embodiments, the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:1, or a sequence corresponding to SEQ ID NO:1, and a light chain variable region having a sequence as set forth in SEQ ID NO:3, or a sequence substantially identical to SEQ ID NO:3 compared to a sequence having at least 95%, 96%, 97%, 98% or 99% identity.

In some specific embodiments, the heavy chain has the amino acid sequence as set forth in SEQ ID NO:2, or a sequence corresponding to SEQ ID NO:2, and a sequence having at least 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain having the sequence set forth in SEQ ID NO:4, or a sequence corresponding to SEQ ID NO:4 compared to a sequence having at least 95%, 96%, 97%, 98% or 99% identity.

In some specific embodiments, the lung cancer is non-small cell lung cancer.

In some specific embodiments, the effective amount of the anti-human VEGF antibody is 0.8-7.5 mg/kg, preferably 0.8mg/kg,2.5mg/kg or 7.5mg/kg, the effective amount of paclitaxel is 10mg/kg, and the effective amount of carboplatin is 80mg/kg.

In some embodiments, the anti-human VEGF antibody is administered in a single dosage form, each containing an amount of the antibody effective for administration to a patient.

In some specific embodiments, the medicament further comprises an additional active ingredient (an additional therapeutic agent) other than the VEGF antibody, paclitaxel, and carboplatin.

In a fifth aspect, the invention also provides a method of treating lung cancer, the method comprising administering to a patient an effective amount of an anti-human VEGF antibody, paclitaxel and carboplatin, the antibody having a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein:

the HCDR1 is selected from SEQ ID NO: 5-7;

the HCDR2 is selected from SEQ ID NO: 8-10;

the HCDR3 is selected from SEQ ID NO: 11-12;

the LCDR1 is selected from SEQ ID NO: 13-14;

the LCDR3 is selected from SEQ ID NO:17, and (b) is shown in the specification.

In some specific embodiments, the lung cancer is non-small cell lung cancer.

In some embodiments, the effective amount of the anti-human VEGF antibody administered is 0.8-7.5 mg/kg, preferably 0.8mg/kg,2.5mg/kg or 7.5mg/kg, the effective amount of paclitaxel administered is 10mg/kg, and the effective amount of carboplatin administered is 80mg/kg.

In a sixth aspect, the invention also provides a combination of an anti-human VEGF antibody, paclitaxel and carboplatin for use in the treatment of lung cancer, the heavy chain variable region of said antibody comprising HCDR1, HCDR2 and HCDR3 and the light chain variable region of said antibody comprising LCDR1, LCDR2 and LCDR3, wherein:

the HCDR1 is selected from SEQ ID NO: 5-7;

the HCDR2 is selected from SEQ ID NO: 8-10;

the HCDR3 is selected from SEQ ID NO: 11-12;

the LCDR1 is selected from SEQ ID NO: 13-14;

the LCDR3 is selected from SEQ ID NO:17, and (b) is shown in the specification.

In some specific embodiments, the heavy chain variable region has the amino acid sequence as set forth in SEQ ID NO:1, or a sequence substantially identical to SEQ ID NO:1, and a light chain variable region having a sequence as set forth in SEQ ID NO:3, or a sequence corresponding to SEQ ID NO:3 compared to a sequence having at least 95%, 96%, 97%, 98% or 99% identity.

In some embodiments, the heavy chain constant region of the antibody has the amino acid sequence as set forth in SEQ ID NO:2, or a sequence corresponding to SEQ ID NO:2, and a light chain constant region having a sequence as set forth in SEQ ID NO:4, or a sequence substantially identical to SEQ ID NO:4 compared to a sequence having at least 95%, 96%, 97%, 98% or 99% identity.

In some specific embodiments, the lung cancer is non-small cell lung cancer.

In some specific embodiments, the dosage form of the anti-human VEGF antibody is a single dosage form comprising the anti-human VEGF antibody in an amount effective to administer the effective amount to the patient.

The VEGF antibody is a humanized rabbit anti-human VEGF antibody, and is used for treating advanced stage or metastatic fructification tumor. Based on the results of clinical experiments, the treatment scheme of the invention can more effectively realize the treatment of advanced or metastatic solid tumors, is beneficial to increasing the selection of patients with advanced or metastatic solid tumors and benefits more patients.

Definition and description of terms

Unless defined otherwise herein, scientific and technical terms related to the present invention shall have the meanings that are understood by those of ordinary skill in the art.

Furthermore, unless otherwise indicated herein, singular terms herein shall include the plural and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

The terms "comprising," "including," and "having," as used herein, are used interchangeably and are intended to mean the inclusion of a solution, meaning that there may be additional elements of the solution other than the listed elements. It should also be understood that the use of "including", "comprising" and "having" in this context also provides a "consisting of … …" solution.

The term "and/or" as used herein includes the meanings of "and", "or" and "all or any other combination of elements linked by the term.

The term "antibody" is used herein in the broadest sense and refers to a polypeptide or combination of polypeptides that comprises sufficient sequence from an immunoglobulin heavy chain variable region and/or sufficient sequence from an immunoglobulin light chain variable region to be capable of specifically binding to an antigen. Herein, "antibody" encompasses various forms and various structures as long as they exhibit the desired antigen binding activity, illustratively, including whole antibodies and antigen binding fragments. The term "desired antigen binding activity" as used herein means, for example, that an antibody typically binds specifically to an antigen and to substantially the same antigen with high affinity, but does not bind to an unrelated antigen with high affinity. Affinity is usually reflected in an equilibrium dissociation constant (KD), where a lower KD indicates a higher affinity. Illustratively, high affinity generally means having about 10 ^-7 M or less, about 10 ^-8 M or less, about 1X 10 ^-9 M or less, about 1X 10 ^-10 M or less, 1X 10 ^-11 M or less or 1X 10 ^-12 M or lower KD. KD is calculated as follows: KD = KD/Ka, where KD represents the dissociation rate and Ka represents the association rate. The equilibrium dissociation constant KD can be measured using methods well known in the art, such as surface plasmon resonance (e.g., biacore) or equilibrium dialysis assay.

The term "antibody" herein includes a typical "four-chain antibody" belonging to an immunoglobulin composed of two Heavy Chains (HC) and two Light Chains (LC); heavy chain refers to a polypeptide chain consisting of, in the direction from N-terminus to C-terminus, a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a Hinge Region (HR), a heavy chain constant region CH2 domain, a heavy chain constant region CH3 domain; and, when the full length antibody is of IgE isotype, optionally further comprising a heavy chain constant region CH4 domain; a light chain is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction; the heavy chains are connected with each other and the heavy chains are connected with each other through disulfide bonds to form a Y-shaped structure. Because of the differences in the amino acid composition and arrangement of the constant regions of immunoglobulin heavy chains, their antigenicity also differs. Accordingly, the term "immunoglobulin" is used herein to refer to five classes, or isotypes called immunoglobulins, namely IgM, igD, igG, igA and IgE, with their corresponding heavy chains being the μ, δ, γ, α and ε chains, respectively. The same class of Ig can be divided into different subclasses according to the differences of amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds, for example, igG can be divided into IgG1, igG2, igG3 and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are classified as either kappa or lambda chains by differences in the constant regions. Each of the five classes of Ig may have either a kappa chain or a lambda chain.

The "antibody" herein may be derived from any animal, including but not limited to humans and non-human animals which may be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, ostriches, alpacas, sheep, rabbits, mice, rats or chondroiidaes (e.g. shark).

"antibody" herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

The terms "full-length antibody," "intact antibody," and "intact antibody" are used interchangeably herein and refer to a antibody having a structure that is substantially similar to a native antibody structure.

The term "humanized antibody" herein refers to a non-human antibody that has been genetically engineered to have its amino acid sequence modified to increase homology to the sequence of a human antibody. Generally, all or a portion of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., variable region FR and/or constant regions) are derived from a human immunoglobulin (acceptor antibody). Humanized antibodies typically retain or partially retain the desired properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, the ability to increase immune cell activity, the ability to enhance an immune response, and the like.

The term "variable region" herein refers to the region of an antibody heavy chain or light chain involved in binding an antibody to an antigen, the "heavy chain variable region" being used interchangeably with "VH", "HCVR" and the "light chain variable region" being used interchangeably with "VL", "LCVR". The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, each domain comprising four conserved Framework Regions (FR) and three hypervariable regions (HVRs). See, e.g., kindt et al, kuby Immunology,6th ed., w.h.freeman and co., p.91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity.

The terms "complementarity determining region" and "CDR" are used interchangeably herein and generally refer to the hypervariable region (HVR) of a heavy chain variable region (VH), which may be abbreviated as HCDR, or a light chain variable region (VL), which may be abbreviated as LCDR, because of its spatial structure to allow precise complementarity to an epitope. The terms "framework region" or "FR region" are used interchangeably herein to refer to those amino acid residues in an antibody heavy chain variable region or light chain variable region other than the CDRs. Generally, a typical antibody variable region consists of 4 FR regions and 3 CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

For further description of the CDRs, reference is made to Kabat et al, j.biol.chem., 252; kabat et al, united states department of health and public service, "Sequences of proteins of immunological interest" (1991); chothia et al, J.mol.biol.196:901-917 (1987); al-Lazikani b. Et Al, j.mol.biol., 273; macCallum et al, J.mol.biol.262:732-745 (1996); abhinandan and Martin, mol.immunol., 45; lefranc m.p. et al, dev.comp.immunol., 27; and honeyger and pluckthun, j.mol.biol.,309 (2001). The "CDRs" herein may be labeled and defined by means known in the art, including but not limited to Kabat numbering system, chothia numbering system, or IMGT numbering system, using tool sites including but not limited to AbRSA sites (http:// cao. Lab share. Cn/AbRSA/cds. Php), abYsis sites (www.abysis.org/analysis/sequence _ input/key _ indication. Cgi), and IMGT sites (http:// www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign. Cgi # results).

Illustratively, SEQ ID NO:1 or 3 are as shown in the following table: wherein Kabat and Chothia use the abYsis website and IMGT uses the IMGT website.

TABLE 1 amino acid sequence listing of CDR regions of heavy and light chains of anti-VEGF antibody

The term "heavy chain constant region" herein refers to the carboxy-terminal portion of an antibody heavy chain that is not directly involved in binding of the antibody to an antigen, but exhibits effector functions, such as interaction with an Fc receptor, which has a more conserved amino acid sequence relative to the variable domain of the antibody. The "heavy chain constant region" comprises at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or a variant or fragment thereof. The "heavy chain constant region" includes a "full-length heavy chain constant region" having a structure substantially similar to a natural antibody constant region, and a "heavy chain constant region fragment" including only a portion of the full-length heavy chain constant region. Illustratively, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH 2 domain-CH 3 domain; when the antibody is IgE, it further comprises a CH4 domain; when the antibody is a heavy chain antibody, it does not include a CH1 domain. Illustratively, a typical "heavy chain constant region fragment" may be selected from the CH1, fc or CH3 domains.

The term "light chain constant region" herein refers to the carboxy-terminal portion of an antibody light chain that is not directly involved in binding of the antibody to an antigen, which light chain constant region may be selected from a constant kappa domain or a constant lambda domain.

The term "Fc" herein refers to the carboxy-terminal portion of an antibody that is papain-hydrolyzed from an intact antibody, typically comprising the CH3 and CH2 domains of the antibody. Fc regions include, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus. The C-terminal lysine of the Fc region (residue 447 according to the Kabat numbering system) may be removed, for example, during production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody, and thus, the Fc region may or may not include Lys447.

The term "identity" herein can be calculated by: to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

The percent identity between two sequences varies with the same position shared by the sequences, taking into account the number of gaps that need to be introduced and the length of each gap for optimal alignment of the two sequences.

The term "drug" herein refers to a substance used for the prevention, treatment and diagnosis of a disease, including "pharmaceutical compositions" or "drug combinations", illustratively, the drug also includes a cell therapeutic agent, such as a therapeutic CAR-T cell or CAR-NK cell. By "pharmaceutical composition" is meant a formulation that is present in a form that allows the biological activity of the active ingredients contained therein to be effective, and that does not contain additional ingredients, which may be one or more, that have unacceptable toxicity to the subject to whom the pharmaceutical composition is administered. The term "combination" or "pharmaceutical combination" as used herein refers to an unfixed combination, wherein the active agent and the at least one further active agent may be administered separately, either simultaneously or within time intervals, in particular where these time intervals allow the combination partners to show a cooperative (e.g. synergistic) effect. The term "non-fixed combination" means that the active ingredients (e.g., one active agent and at least one additional active agent) are both administered to a patient as separate entities either simultaneously or sequentially without specific time constraints, wherein such administration provides therapeutically effective levels of both compounds in the patient.

The term "treatment" herein refers to surgery or drug treatment (therapeutic or therapeutic treatment) with the purpose of preventing, slowing (reducing) the progression of an undesired physiological change or pathology, such as a cancer or tumor, in a subject being treated. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. Subjects in need of treatment include subjects already suffering from a condition or disease as well as subjects susceptible to a condition or disease or subjects for whom prevention of a condition or disease is intended. When terms such as slow, moderate, slow and the like are mentioned, the meaning also includes elimination, disappearance, non-occurrence and the like.

The term "patient" or "subject" herein refers to an organism that is receiving treatment for a particular disease or disorder as described herein. Examples of "patients" or "subjects" include mammals, such as humans, primates (e.g., monkeys), or non-primate mammals, that are being treated for a disease or disorder.

The term "effective amount" as used herein refers to an amount of a therapeutic agent that is effective to prevent or ameliorate a disease condition or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject. An "effective amount" also refers to an amount of a compound or cell having a therapeutic effect (e.g., CAR-T cells, CAR-NK cells) sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to treat, cure, prevent, or alleviate an increased rate of such a condition.

The term "single dose dosage form" herein refers to a dosage form comprising an effective administered amount of a drug to a subject or patient in a single administration. Illustratively, the amount of drug contained in a single dosage form may be converted by an effective dose. For example, an effective amount to be administered in mg/m2 can be converted to the drug content per dose in a single dosage form based on the body surface area of a human. For example, an effective amount in mg/kg can be converted to the drug content per dose in a single dosage form based on the body weight of a human.

Herein, the term "therapeutic agent" refers to a pharmaceutically active ingredient which includes not only anti-human VEGF antibody, carboplatin and paclitaxel, but also may further comprise other pharmaceutically active ingredients, illustratively, the therapeutic agent of the present invention includes immune checkpoint inhibitors, such as antibodies targeting PD-1 or PD-L1.

The term "cancer" herein refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers. The term "tumor" or "neoplasm" herein refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous (pre-cancerous) and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when referred to herein.

The term "advanced or metastatic solid tumor" refers to an advanced, unresectable and/or metastatic malignant solid tumor that has been histologically, cytologically or imagewise confirmed diagnosed. Throughout, the term "advanced or metastatic solid tumor" is a subset of solid tumors and is the underlying indication for solid tumors.

The term "Maximum Tolerated Dose (MTD)" is defined herein as the Dose level above which excessive toxicity occurs.

The term "Adverse Event (AE)" herein refers to any Adverse medical Event that occurs when a subject is treated with a study drug, but is not necessarily causally related to the treatment. An adverse event can be any adverse and unintended sign (including abnormal laboratory findings), symptom, or disease that is temporally linked to, but not necessarily related to, medication.

The term "Severe Adverse Event (SAE)" herein refers to an Adverse Event that meets at least one criterion that results in death, life-threatening, prolonged or permanent hospitalization, permanent or Significant disability or loss of function, and congenital malformations or birth defects in a subject.

The term "Dose Limiting Toxicity (DLT)" herein is defined as an adverse event that is positively associated with, may be associated with, or is not determinable with a test drug, of any of the following:

(1) The 4-grade neutrophilic granulocytopenia lasts for more than or equal to 3 days;

(2) Grade 4 anemia;

(3) The grade 3 or more neutrophilic granulocytes reduce fever;

(4) Grade 4 thrombocytopenia or grade 3 thrombocytopenia with bleeding;

(5) Grade 3 or more non-hematologic toxicity (except grade 3 nausea, vomiting, fever, anorexia, alkaline phosphatase abnormality, and 3 grade blood pressure can be well controlled after the blood pressure is reduced by using antihypertensive drug (systolic pressure <140mgHg and diastolic pressure <90 mgHg), or grade 3 proteinuria can be recovered to <2g/24h within 2 weeks);

(6) Arterial embolization of any grade;

the above adverse events were ranked according to the standard for toxicity assessment (NCI-CTC) version 4.0 of the american cancer institute.

Infusion-related responses at grade > 3 were not DLT, but required discontinuation of medication and replenishment of new subjects in the group.

The term "Objective Response Rate (ORR)" as used herein is defined as the incidence of confirmed CR or PR, and the target and non-target lesions are evaluated by confirmed radiological methods, and the evaluation criteria for efficacy of solid tumors are judged by reference to RECIST criteria 1.1.

Herein, the term "Disease Control Rate (DCR)": the incidence of CR, PR and SD was confirmed and evaluated by confirmed radiology methods for target and non-target lesions, and the evaluation criteria for the efficacy of solid tumors were judged according to RECIST criteria 1.1.

The term "Progression Free Survival (PFS)" herein: the time from the day of first dose to the time when disease progression is first observed (on an image basis), and if a patient died for other reasons before disease progression was observed, the number of days from the day of first dose to death was calculated. The actual time of tumor assessment will be used in the calculation. For patients who have not yet developed disease progression or death at the time of analysis, progression-free survival will have the time of last tumor assessment as the cutoff date. Progression free survival patients who have not been evaluated for tumors since the baseline period will have 1 day as the cutoff date. The investigator's judgment will be used to determine the time of progression.

The term "CR" herein refers to Complete Response, i.e. Complete remission; "PR" refers to Partial Response, i.e., partial remission; SD refers to Stable Disease, namely Stable Disease; "PD" refers to Progress Disease.

The term "C _max "is the plasma concentration of the active agent measured at the point of maximum or peak concentration.

The term "t _1/2 "also known as biological half-life or biological half-life" refers to the time it takes for the concentration of a drug in the blood or the amount of a drug in the body to be reduced by one-half.

AUC _0-∞ : area under the plasma concentration-time curve from zero to infinity.

AUC _0-t : area under the plasma concentration-time curve from zero to the last measurable concentration.

Safety Analysis Set (SS, safe Analysis Set): all patients who had been administered at least one trial.

Full Analysis Set (FAS, full Analysis Set): efficacy analysis was performed on all cases using at least one test drug according to the principle of intent test (ITT). The main efficacy index deficiency value is not carried out.

The term "Q2W" refers to administration once every two weeks.

Drawings

FIG. 1 PFS (days) after treatment of the subjects in each dose group;

FIG. 2 PFS (days) after treatment for all subjects;

FIG. 3 tumor inhibitory effect of VEGF antibody on lung cancer model;

FIG. 4. Tumor suppression effect of VEGF antibodies in combination with Paclitaxel and Carboplatin on a lung cancer model.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The examples are exemplary only and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and substitutions are intended to be within the scope of the invention.

The VEGF antibody (hereinafter, the VEGF antibody of the present invention) used in the following examples was produced and supplied by Shandong Mitsumadzu biopharmaceutical Co., ltd. And stored at 2-8 ℃. The sequence information of the VEGF antibody of the present invention is shown below.

Heavy chain variable region (SEQ ID NO: 1):

EVQLVESGGGLVKPGGSLRLSCAASGFSFSNNDVMCWVRQAPGKGLEWIGCIMTTDVVTEYANWAKSRFTVSRDSAKNSVYLQMNSLRAEDTAVYFCARDSVGSPLMSFDLWGPGTLVTVSS；

heavy chain constant region (SEQ ID NO: 2):

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；

light chain variable region (SEQ ID NO: 3):

DIQMTQSPSSLSASVGDRVTINCQASQSIYNNNELSWYQQKPGKPPKLLIYRASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCGGYKSYSNDGNGFGGGTKVEIK；

light chain constant region (SEQ ID NO: 4):

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

example 1 clinical trials of VEGF antibodies in patients with advanced or metastatic solid tumors

Purpose of study

Standard regimens are investigated for the safety and tolerability of single and multiple intravenous infusions of VEGF antibodies of the invention, as well as pharmacokinetic parameters, in patients with advanced or metastatic solid tumors who are refractory to standard regimens or have no standard treatment regimens. The anti-tumor curative effect of the VEGF antibody single medicine is observed through indexes such as Objective Remission Rate (ORR) and Disease Control Rate (DCR).

Design of research

Patients with advanced or metastatic solid tumors were selected as subjects using a multicenter, open, dose escalation design. The dose groups were 2mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, 7.5mg/kg, and the tolerance of single and multiple administrations was examined. Dose escalation dose-limiting toxicity (DLT) occurred within 28 days (d 1-d 28) after administration of the previous dose group as a basis during which blood samples were collected for drug concentration determination, as well as determination of VEGF and antibody titers. All subjects continued dosing until disease progression or intolerable adverse events occurred.

The dose escalation overall adopted the 3+3 design principle. Dose escalation should be terminated during the trial as if a dose group with a maximum trial dose and a Maximum Tolerated Dose (MTD) in the previous dose group had a DLT of > 1/3.

Population of subjects

Patients with advanced or metastatic solid tumors.

Inclusion criteria include：

1) Age: 18-74 years old;

2) Histologically or cytologically confirmed advanced or metastatic malignant solid tumors;

3) Those who fail standard anti-tumor therapy or have no standard treatment regimen at the present stage;

4) At least one measurable tumor lesion;

5) ECOG PS score 0-1;

6) Fertile eligible patients (male and female) must agree to use a reliable method of contraception (hormonal or barrier or abstinence) during the trial period and for at least 12 weeks after the last dose. Female patients in the fertile age must be negative in a blood pregnancy test within 7 days prior to enrollment;

7) The subject must give informed consent to the study before testing and voluntarily signs a written informed consent form;

exclusion criteria include：

1) The anti-VEGF protein drugs such as bevacizumab and the like are used in the past;

2) Histologically confirmed squamous cell lung or head and neck cancer;

3) Active phase of hepatitis B;

4) Symptomatic brain metastases;

5) Patients with proteinuria are examined before group entry, and in two examinations (on different days) in a screening period, urine protein is more than or equal to 1+ in any one examination;

6) Large surgical procedures (not including needle biopsies) or significant trauma occurred within 4 weeks prior to group entry;

7) Active bleeding occurred within 3 months before group entry;

8) Myocardial infarction or cerebral apoplexy appear in 6 months before the group;

9) Unstable angina, congestive heart failure or, new york cardiology society (NYHA) grade ii heart failure, uncontrolled arrhythmias, single-drug uncontrollable hypertension (systolic >140mmHg and/or diastolic >90 mmHg);

10 Pregnant or lactating women;

11 Known to be allergic to the therapeutic drugs or adjuvants used.

The exit criteria include：

All subjects had the right to withdraw from the trial at any stage of the study during the trial period according to the GCP and ICH guidelines, and in addition, subjects would withdraw from the trial if the following occurred during the trial.

1) Evidence of disease Progression (PD);

2) Infusion-related reactions at a level of not less than 3;

3) Dropping off;

4) The test drug is not used after taking part in the test;

5) Investigators considered subjects unsuitable for continued use of the test drug (e.g., intolerant adverse effects);

in addition to the above, other serious deviations or protocol violations during the trial have had an impact on drug resistance, safety or pharmacokinetic assessments.

Route of administration

Administration was by intravenous drip.

Test procedure

The test is divided into three phases, including a screening phase, a testing phase and a follow-up phase.

And (3) screening period:

the screening period was 14 days, baseline assessments were performed, confirming that the subjects met all entry conditions and did not have any of the conditions in the exclusion criteria.

And (3) test period:

in the single administration stage, the VEGF antibody is subjected to intravenous drip on the same day of administration and is observed for 28 days, the safety is evaluated by the laboratory examination of vital signs, physical examination, hematuria routine, hematobiochemistry, blood coagulation function and the like, the occurrence condition of adverse events is strictly monitored, and a blood sample is collected for pharmacokinetic study. Tumor assessments were performed 29 ± 3 days after dosing.

In the multiple administration stage, a subject receives 3 times of intravenous administration, the interval time of each administration is 2 weeks, the safety of laboratory examination such as vital signs, physical examination, hematuria routine, hematobiochemistry and blood coagulation function is evaluated within 29 +/-2 days after the 1 st administration to the 3 rd administration, adverse events are strictly monitored, and blood samples are collected for pharmacokinetic study. Safety and efficacy assessments will be made 29 ± 3 days after the 3 rd dose.

A follow-up period:

if the subjects complete 4 times of administration and 28 days after the last administration, and the treatment effect evaluation is SD or above SD, and the tolerance is good, under the premise of agreement between the subjects and researchers, the subjects can continue to receive the test drug treatment according to the original treatment scheme, and the treatment effect evaluation is performed every 6 weeks until the disease progresses or intolerant adverse reactions occur or other withdrawal criteria are met.

Research index

The safety index is as follows: including adverse events (graded according to CTCAE 4.0), vital signs, physical examination, ECOG physical performance scores, laboratory and auxiliary examination values (including blood routine, urine routine, liver function, kidney function, blood lipids, fasting plasma glucose, blood electrolytes, 12-lead electrocardiogram, etc.).

The effectiveness index is as follows: including Objective Remission Rate (ORR), disease Control Rate (DCR), and Progression Free Survival (PFS).

Pharmacokinetic/pharmacodynamic and immunogenicity indices: all plasma samples were assayed for VEGF antibody concentration and VEGF concentration of the present invention. anti-VEGF antibody titers were determined for plasma samples before each dose and at days 15 and 29 after the last dose. PK parameters include Kel, t1/2, tmax, cmax, AUC, vd, CL, MRT, AR, etc.

Statistics and analysis

The security analysis uses the SS set. All safety and tolerance evaluation indexes are subjected to descriptive statistics. Safety analyses included adverse events and severe adverse events, vital signs, physical examinations, laboratory examinations, electrocardiographic examinations, ECOG scores, drug exposures.

The FAS set was used for efficacy analysis. Efficacy indices include ORR, DCR and PFS. And (4) carrying out descriptive statistics on ORR and DCR, and calculating the number of cases and the percentage. PFS is depicted by a Kaplan-Meier survival plot.

Medicine powerAnd (3) chemical analysis: and performing non-atrioventricular model analysis on the blood concentration data. Pharmacokinetic parameters include C _max 、t _1/2 、AUC _0-∞ 、AUC _0-t Descriptive statistics are employed.

The statistical tests all used a two-sided test, and those with P less than or equal to 0.05 were considered statistically significant.

Results

1. Population of subjects

The group is divided into 25 subjects, and the group is divided into different subjects, which are shown in table 2. The SS and FAS sets were included in 25 subjects. Wherein 15 cases are male, 10 cases are female; the median age was 55 years, the main tumor types were colorectal cancer (12 cases), breast cancer (4 cases), and gastric cancer (3 cases), and other pathological types were lung cancer, ovarian cancer, cervical cancer, liver cancer, pancreatic tail cancer, and duodenal papillary carcinoma in 1 case each. All subjects had metastases, with 1 metastasis in 3 (12%), 2 in 11 (44%), 3 in 9 (36%), and > 3 metastases in 2 (8%).

Treatment and prior history, subjects received chemotherapy, with 23 subjects receiving more than 3 chemotherapy regimens; 23 subjects received surgical treatment of primary foci, 8 subjects received surgery of metastatic foci; 15 subjects received radiation therapy.

TABLE 2 grouping of subjects in different groups

2. Safety feature

(1)MTD

The study finally determined an MTD of 5mg/kg.

(2) Adverse reaction occurrence

Common adverse reactions in the test process include hypertension (17 cases, 68%), proteinuria (16 cases, 64%), asthenia (8 cases, 32%) and rash (6 cases, 24%); laboratory tests were mainly positive for hematuria (9 cases, 36%), decreased white blood cell count (6 cases, 24%), decreased platelet count (5 cases, 20%), increased alanine aminotransferase (5 cases, 20%) and ecg changes (5 cases, 20%). Most adverse reactions are well documented.

(3) Adverse events leading to discontinuation of test drug

In the test process, 12 subjects stop using the test drugs due to adverse events; adverse events in 9 cases were associated with the test drugs, including 4 cases of proteinuria, 1 case of proteinuria aggravated to grade 3, 1 case of hypertension aggravated, 1 case of hyperbilirubinemia; in 1 case of intestinal bleeding, perforation of the intestinal tract is possible. Other adverse events leading to withdrawal were judged by the investigator to be unrelated to the test drug.

(4)SAE

4 subjects developed 4 SAEs, femoral fracture, perforation of the intestinal tract, possible bleeding of the intestinal tract, proteinuria and ileus. Adverse events associated with study drug observed throughout the study were reported in the study of the same class of drugs. The most common adverse reactions occurring in the test process are hypertension, proteinuria, hypodynamia and rash; aspects of laboratory test abnormalities include positive urine blood, decreased white blood cell count, decreased platelet count, increased alanine aminotransferase, and changes in electrocardiogram; the mechanism of occurrence is mostly related to VEGF pathway inhibition. Within the MTD range, the adverse reactions of the test drugs can be effectively controlled, and the influence on the administration intensity is small.

3. Pharmacokinetics/pharmacodynamics

The pharmacokinetic parameters at different doses in this test are shown in the table below.

TABLE 3 pharmacokinetic parameters

4. Effectiveness of

The efficacy analysis population of the test was FAS set. All 25 subjects in the group were enrolled into the FAS panel, 24 subjects had at least 1 tumor assessment, and 1 subject had no tumor assessment; there were 11 subjects evaluated for tumors 1 day 85 (12 weeks) after completion of the first dose.

Based on the results of tumor assessment on day 29, 1 case was ORR (4%), 20 cases was DCR (80%), with 1 case being PR (4%), 19 cases being SD (76%), and 4 cases being PD (16%). 1 case (4%) was not evaluated for tumors. Based on the results of tumor assessment at day 85, 0 cases were ORR (0%), 8 cases were DCR (32%), 8 cases were SD (32%), 3 cases (12%) were PD, and 14 cases (56%) were not tumor assessed.

Median PFS was 122 days, 65 days for 25% quantile and 126 days for 75% quantile for all subjects in this trial. The PFS profile of the different dose groups is detailed in Table 4, FIGS. 1-2.

TABLE 4 PFS (day) after treatment for each dose group

And (4) conclusion:

the MTD of the VEGF antibody is 5mg/kg, and the adverse reaction of the test drugs can be effectively controlled within the MTD range. The therapeutic results show that the VEGF antibodies of the invention are effective in patients with advanced malignancies who fail standard therapy, with an ORR of 4%, a DCR of 80%, 0% and a DCR of 32% for all subjects on day 29. In conclusion, the VEGF antibody has good safety and tolerance, and shows an anti-tumor curative effect at first.

Example 2 evaluation of therapeutic Effect of VEGF antibody on human Lung cancer nude mouse transplanted tumor model

2.1 test materials and methods for tumor assessment (this example uses the test materials and methods for tumor assessment described in 2.1)

The tested drugs are:

the VEGF antibody is produced and provided by Shandong Xianshuidejin biopharmaceutical company Limited; bevacizumab (Avastin) was purchased from Roche (Roche); paclitaxel (Paclitaxel) was purchased from Hainan Quanxing pharmaceutical Co., ltd; carboplatin (Carboplatin) was purchased from zilu pharmaceutical limited. The preparation method of the medicine comprises the following steps: carboplatin was formulated and diluted with a 5% glucose solution (purchased from Guizhou Tiandi pharmaceutical Co., ltd.) and other drugs were formulated and diluted with sterile normal saline (purchased from Jiangsu Huai' an Shuanghe pharmaceutical Co., ltd.).

Cell line:

NCI-H460 cells: american model culture Collection (ATCC).

Reagent:

RPMI1640: gibco, cat number: 22400-089;

fetal bovine serum: hyclone, cat # s: SV30087.03;

antibacterial-antifungal agents (dual-action): gibco, cat number: 15240-062.

Experimental animals:

BALB/c Nude mice, female, 6-8 weeks, and the body weight ranges of 17.7-24.9g (example 2.2) and 17.3-23.6g (example 2.3) in the two experiments in this order, purchased from Shanghai Chang Biotech, inc., production license number: SCXK 2018-0003, and the animal certification numbers of the two experiments are 20180003008266 (example 2.2) and 20180003008991 (example 2.3) in sequence. A breeding environment: SPF grade.

The tumor evaluation method comprises the following steps:

tumor volume V =0.5 × a × b ² Wherein a is the major diameter of the tumor and b is the minor diameter of the tumor;

relative tumor volume RTV = V _t /V ₀ In which V is ₀ Tumor volume of the animal at the time of grouping; v _t Is the tumor volume of the animal after treatment.

Relative tumor proliferation rate T/C (%) = T _RTV /C _RTV X 100% where T _RTV Mean RTV for treatment groups; c _RTV The negative control group mean RTV.

Tumor growth inhibition ratio TGI (%) = (1-T/C) × 100%.

2.2 Evaluation of therapeutic Effect of VEGF antibody on human Lung cancer NCI-H460 nude mouse transplantation tumor model-Single drug

NCI-H460 cells cultured in RPMI16 containing 10% fetal bovine serum40 in culture medium (containing double antibody). When the saturation degree of the cells is 80% -90%, and the quantity reaches the requirement, the cells are collected, and the cell saturation degree is 1 multiplied by 10 ⁶ NCI-H460 cells were inoculated subcutaneously into experimental mice. The tumor is grown to the average volume of 147.62mm ³ The mice were divided randomly into 8 groups (n = 8), each group of mice was given saline (vehicle) via the tail vein, 0.8mg/kg,2.5mg/kg or 7.5mg/kg of the test agent VEGF antibody of the invention, 0.8mg/kg,2.5mg/kg or 7.5mg/kg of the control agent Avastin, or a combination of 10mg/kg of Paclitaxel and 80mg/kg of Carboplatin via intraperitoneal administration. The VEGF antibody of the invention and Avastin were administered 2 times per week for 3 weeks, and Paclitaxel and Carboplatin were administered 1 time per week for 2 times. Tumor volume was measured 3 times per week, mice were weighed, data were recorded, and tumor size change and mouse weight change were counted over 3 weeks.

On day 20 after administration, the VEGF antibodies of the invention inhibited tumor growth significantly and exhibited better dose dependence than the solvent control group (saline), 0.8mg/kg,2.5mg/kg and 7.5mg/kg. The 0.8mg/kg Avastin group showed no effect of inhibiting tumor growth relative to the solvent control group. The 2.5mg/kg and 7.5mg/kg Avastin groups showed significant tumor growth inhibition effect relative to the solvent control group, but did not show dose dependence. The VEGF antibody has obviously better tumor inhibition effect than Avastin under the same dosage. Neither the VEGF antibody nor Avastin of the invention seen weight loss or other significant toxic responses in animals at different doses. The 10mg/kg Paclixel +80mg/kg Carboplatin combination group also produced a certain anti-tumor effect, but the weight loss of the mice was obvious. See table 5 and figure 3 for details.

The result shows that the VEGF antibody has obvious tumor inhibition effect on a NCI-H460 mouse lung cancer model under the dosage of 0.8mg/kg,2.5mg/kg and 7.5mg/kg, and presents better dose dependence, and the tumor inhibition effect of the VEGF antibody is obviously superior to Avastin.

TABLE 5 antitumor Effect of the test substances on the NCI-H460 xenograft tumor model

Note: a. data are shown as mean ± Standard Deviation (SD).

b.P values the relative tumor volumes were analyzed using two-way ANOVA, showing P values for each group compared to vehicle controls.

2.3 Evaluation of therapeutic Effect of VEGF antibody on human Lung cancer NCI-H460 nude mouse transplantable tumor-combination chemotherapy

NCI-H460 cells were cultured in RPMI1640 medium (containing double antibody) containing 10% fetal bovine serum. When the saturation degree of the cells is 80% -90%, and the quantity reaches the requirement, the cells are collected, and the cell saturation degree is 1 multiplied by 10 ⁶ NCI-H460 cells were inoculated subcutaneously into experimental mice. The tumor is grown to the average volume of 147.79mm ³ Group administration, the day of group administration being defined as day 0 (D0), the mice were randomly divided into 8 groups (n = 8), and each group of mice was administered with physiological saline (Vehicle) via the tail vein, 2.5mg/kg or 7.5mg/kg of the test drug (i.e., the VEGF antibody of the present invention), 2.5mg/kg or 7.5mg/kg of the control Avastin, or administered with 10mg/kg of Paclitaxel and 80mg/kg of Carboplatin in combination via the abdominal cavity, or administered with 2.5mg/kg of the VEGF antibody of the present invention via the tail vein and administered with 10mg/kg of Paclitaxel and 80mg/kg of Carboplatin intraperitoneally, or administered with 2.5mg/kg of Avastin via the tail vein and administered with 10mg/kg of Paclitaxel and 80mg/kg of Carboplatin. The VEGF antibody of the invention and Avastin were administered 2 times per week for 3 weeks, and Paclitaxel and Carboplatin were administered 1 time per week for 2 times. Tumor volume was measured 3 times per week, mice were weighed, data were recorded, and tumor size change and mouse weight change were counted over 3 weeks.

On day 20 post-dose, 2.5mg/kg and 7.5mg/kg Avastin, both showed significant tumor growth inhibition effect relative to the solvent control (saline), but did not show dose dependence. The VEGF antibodies of the invention, 2.5mg/kg and 7.5mg/kg, both showed significant tumor growth inhibition effects relative to the solvent control group and were dose-dependent. Under the same dosage, the anti-tumor effect of the VEGF antibody is obviously superior to that of Avastin. The 2.5mg/kg Avastin +10mg/kg Paclitaxel +80mg/kg Carboplatin combination group showed significant tumor growth inhibition effect compared with the solvent control group, and the tumor inhibition effect was significantly enhanced compared with the 10mg/kg Paclitaxel +80mg/kg Carboplatin combination group and the 2.5mg/kg Avastin single drug group. The VEGF antibody of 2.5mg/kg +10mg/kg Paclitaxel +80mg/kg Carboplatin combined administration group shows a significant tumor growth inhibition effect relative to a solvent control group, the tumor inhibition effect is significantly enhanced compared with the combined group of 10mg/kg Paclitaxel +80mg/kg Carboplatin, but the tumor inhibition effect is slightly enhanced compared with a single drug group of the VEGF antibody of 2.5mg/kg, but no statistical difference exists. Compared with the combined administration group of 2.5mg/kg of Avastin, 10mg/kg of Paclixel and 80mg/kg of Carboplatin, the combined administration group of the VEGF antibody, 10mg/kg of Paclixel and 80mg/kg of Carboplatin has a better trend of tumor inhibition effect but has no statistical difference. In the administration process, the mice in the 10mg/kg Paclitaxel +80mg/kg Carboplatin combination group, the 2.5mg/kg VEGF antibody of the invention +10mg/kg Paclitaxel +80mg/kg Carboplatin combination group, the 2.5mg/kg Avastin +10mg/kg Paclitaxel +80mg/kg Carboplatin combination group have obvious body weight reduction, the mice recover after the 10mg/kg Paclitaxel +80mg/kg Carboplatin stop taking the drugs, and the tumor bearing mice in other groups show better tolerance to the tested drugs under all doses without death or morbidity. The VEGF antibody or Avastin combined group of the invention and 10mg/kg of Paclitaxel +80mg/kg of Carboplatin has no obvious toxicity increase compared with the 10mg/kg of Paclitaxel +80mg/kg of Carboplatin group. See table 6 and fig. 4 for details.

The result shows that the VEGF antibody has dose-dependent significant tumor inhibition effect on NCI-H460 mouse lung cancer models at the doses of 2.5mg/kg and 7.5m/kg, and the tumor inhibition effect is obviously superior to Avastin. The combination treatment of the VEGF antibody of 2.5mg/kg and the paclitaxel of 10mg/kg and the carboplatin of 80mg/kg has obvious tumor inhibition effect on a NCI-H460 mouse lung cancer model, and the tumor inhibition effect is obviously stronger than that of the group consisting of the paclitaxel of 10mg/kg and the carboplatin of 80mg/kg. The drug effect of the combination of the VEGF antibody of 2.5mg/kg, the paclitaxel of 10mg/kg and the carboplatin of 80mg/kg is better than that of the combination group of the Avastin of 2.5mg/kg, the paclitaxel of 10mg/kg and the carboplatin of 80mg/kg, which indicates that the VEGF antibody of the invention can enhance the effect of chemotherapy combination better than the Avastin.

TABLE 6 tumor-inhibiting Effect of the test substances on the NCI-H460 xenograft tumor model

Note: a. data are shown as mean ± SD.

b.P values are obtained from two-way ANOVA analysis of relative tumor volumes and the P values shown are values for each group compared to vehicle controls.

Sequence listing

<110> Jiangsu Xiansui pharmaceutical Co., ltd

Application of <120> anti-human VEGF antibody in preparation of drugs for treating solid tumors

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<141> 2022-06-30

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<213> Artificial Sequence (Artificial Sequence)

<400> 8

Cys Ile Met Thr Thr Asp Val Val Thr Glu Tyr Ala Asn Trp Ala Lys

1 5 10 15

Ser

<210> 9

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Thr Thr Asp Val Val

1 5

<210> 10

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Ile Met Thr Thr Asp Val Val Thr

1 5

<210> 11

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Asp Ser Val Gly Ser Pro Leu Met Ser Phe Asp Leu

1 5 10

<210> 12

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Ala Arg Asp Ser Val Gly Ser Pro Leu Met Ser Phe Asp Leu

1 5 10

<210> 13

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Gln Ala Ser Gln Ser Ile Tyr Asn Asn Asn Glu Leu Ser

1 5 10

<210> 14

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Gln Ser Ile Tyr Asn Asn Asn Glu

1 5

<210> 15

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Arg Ala Ser Thr Leu Ala Ser

1 5

<210> 16

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Arg Ala Ser

1

<210> 17

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Gly Gly Tyr Lys Ser Tyr Ser Asn Asp Gly Asn Gly

1 5 10

Claims

1. Use of a therapeutic agent comprising an anti-human VEGF antibody in the manufacture of a medicament for treating a patient having advanced or metastatic solid tumor, wherein the heavy chain variable region of said antibody comprises HCDR1, HCDR2 and HCDR3 and the light chain variable region of said antibody comprises LCDR1, LCDR2 and LCDR3, wherein:

the HCDR1 is selected from SEQ ID NO: 5-7;

the HCDR2 is selected from SEQ ID NO: 8-10;

the HCDR3 is selected from SEQ ID NO: 11-12;

the LCDR1 is selected from SEQ ID NO: 13-14;

the LCDR3 is selected from SEQ ID NO:17, and (b) is shown in the specification.

2. The use of claim 1, wherein the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:1, or a sequence corresponding to SEQ ID NO:1, and a light chain variable region having a sequence as set forth in SEQ ID NO:3, or a sequence corresponding to SEQ ID NO:3 compared to a sequence having at least 95%, 96%, 97%, 98% or 99% identity.

3. The use of any one of claims 1 to 2, wherein the heavy chain constant region of said antibody has the amino acid sequence as set forth in SEQ ID NO:2, or a sequence corresponding to SEQ ID NO:2, and a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:4, or a sequence corresponding to SEQ ID NO:4 compared to a sequence having at least 95%, 96%, 97%, 98% or 99% identity.

4. The use according to any one of claims 1 to 3, wherein the solid tumor is a solid tumor of advanced stage or metastatic malignant solid tumor as confirmed histologically, cytology or imaging.

5. The use according to any one of claims 1 to 4, wherein the solid tumor is colorectal cancer, breast cancer, gastric cancer, lung cancer, ovarian cancer, cervical cancer, liver cancer, pancreatic tail cancer, or duodenal papillary cancer.

6. The use of any one of claims 1 to 5, wherein the patient is one lacking or otherwise ineffective standard therapy.

7. The use according to any one of claims 1 to 6, wherein the patient has previously received a chemotherapy, radiation therapy and/or surgery regimen.

8. The use of any one of claims 1 to 7, wherein the patient has or has prior to surgical resection a metastatic lesion, optionally 1, 2, 3 or more than 3.

9. Use according to any one of claims 1 to 8, characterized in that the effective amount of anti-human VEGF antibody administered is between 1.5 and 8mg/kg, preferably 2mg/kg, 4mg/kg, 5mg/kg, 6mg/kg or 7.5mg/kg;

10. The use according to any one of claims 1 to 9, wherein the active ingredient of the medicament comprises only said anti-human VEGF antibody, or wherein the medicament further comprises an active ingredient other than said anti-human VEGF antibody.

11. Use of a therapeutic agent comprising an anti-human VEGF antibody, paclitaxel and carboplatin for the manufacture of a medicament for the treatment of lung cancer, wherein the heavy chain variable region of said antibody comprises HCDR1, HCDR2 and HCDR3 and the light chain variable region of said antibody comprises LCDR1, LCDR2 and LCDR3, wherein:

the HCDR1 is selected from SEQ ID NO: 5-7;

the HCDR2 is selected from SEQ ID NO: 8-10;

the HCDR3 is selected from SEQ ID NO: 11-12;

the LCDR1 is selected from SEQ ID NO: 13-14;

the LCDR3 is selected from SEQ ID NO:17, and (b) is shown in the specification.

12. The use of claim 11, wherein the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:1, or a sequence substantially identical to SEQ ID NO:1, and a light chain variable region having a sequence as set forth in SEQ ID NO:3, or a sequence corresponding to SEQ ID NO:3 compared to a sequence having at least 95%, 96%, 97%, 98% or 99% identity.

13. The use of claim 11 or 12, wherein the heavy chain constant region of the antibody has the amino acid sequence as set forth in SEQ ID NO:2, or a sequence corresponding to SEQ ID NO:2, and a light chain constant region having a sequence as set forth in SEQ ID NO:4, or a sequence corresponding to SEQ ID NO:4 compared to a sequence having at least 95%, 96%, 97%, 98% or 99% identity.

14. The use according to any one of claims 11 to 13, wherein the lung cancer is non-small cell lung cancer.

15. The use according to any one of claims 11 to 14, wherein said anti-human VEGF antibody is administered in an effective amount of 0.8 to 7.5mg/kg, preferably 0.8mg/kg,2.5mg/kg or 7.5mg/kg, said paclitaxel is administered in an effective amount of 10mg/kg, and said carboplatin is administered in an effective amount of 80mg/kg.

16. The use of claim 15, wherein said anti-human VEGF antibody is in a single dosage form, each containing an amount of antibody effective for administration to a patient.