CN117098770A - Polypeptide compound for SORT1 and drug conjugate thereof - Google Patents

Polypeptide compound for SORT1 and drug conjugate thereof Download PDF

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CN117098770A
CN117098770A CN202380010846.0A CN202380010846A CN117098770A CN 117098770 A CN117098770 A CN 117098770A CN 202380010846 A CN202380010846 A CN 202380010846A CN 117098770 A CN117098770 A CN 117098770A
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董志超
陈昌发
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Shanghai Zhipeptide Biotechnology Co ltd
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    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
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    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

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Abstract

A targeting peptide specifically targeting Sortilin/SORT1 protein, and a targeting peptide-drug conjugate comprising the targeting peptide. The targeting peptide is a linear peptide or cyclic peptide containing non-natural chemical modification, and can improve the half life and the stability of the targeting peptide-drug conjugate. The targeting peptide-drug conjugate can specifically bind with high affinity to a target protein Sortilin/SORT1, so that tumors expressing the Sortilin/SORT1 can be killed efficiently, and a novel anti-tumor targeting drug is provided for the field of tumor treatment.

Description

Polypeptide compound for SORT1 and drug conjugate thereof Technical Field
The invention relates to the technical field of biological medicine, in particular to a polypeptide compound aiming at a Sort1 target point, a medicine coupling medicine thereof, a preparation method and application thereof.
Background
Cancer is the first killer threatening human health. The data published by the world health organization International cancer research Commission (IARC) shows that in 2020, cancer is diagnosed in 1930 ten thousand cases worldwide, with 457 ten thousand new cancer in China, the first place worldwide. Although surgery can ablate localized tumors early in the day, it has little effect on advanced or systemic cancers; and the radiotherapy and the chemotherapy can bring side effects such as alopecia, vomiting and the like.
The targeting agent is "tomorrow star" in the antineoplastic market. One of the main characteristics of the targeting drug is that the targeting drug plays a role aiming at specific targets, the conditions of each patient are different, and the targeting drugs which can be selected are different, so that the individual treatment of tumors is realized to a certain extent. From the trend of medicine demand, obvious curative effect and small side effect are main demand directions of future product development, and under the drive of the market demand, the research and development and clinical application of the antitumor targeted medicine are one of the main development directions of the antitumor medicine industry in the future.
Sortilin (NP-002950.3), also known as neurotensin receptor 3 (NTSR 3), is a 100kDa type 1 membrane glycoprotein encoded by the SORT1 Gene (Gene ID: 6272), which is located on human chromosome 1P13.3. The SORT1 gene encodes variants of 5 Sortilin, including a full length sequence of 831 amino acids (aa), 4 truncated splice variants 694, 118, 60, and 20aa (Fatemeh Ghaemimanesh et al, 2021). Sortilin belongs to the family of vacuolated protein Sortilin 10 (VPS 10) receptors. The Vps10 protein family includes heterologous type 1 transmembrane receptors, including Sortilin (100 kDa), sorLA, sorCS1, sorCS2 and SorCS3, mature Sortilin being differentiated from the precursor prepro-Sortilin (Munck Petersen et al, 1999). Sortilin can be localized not only on golgi membranes as Sortilin, but also on cell membranes as a clearance receptor.
Sortilin consists of a pre-peptide propeptide (common signal sequence of proteins inserted into the cell membrane; 1-33 aa), pro-peptide propeptide (34-77 aa), large lumen/extracellular region (Vps 10p domain; 78-755 aa), transmembrane fragment of 756-778aa and intracellular domain of 779-831aa (FIG. 1) (Fatemeh Ghaemimanesh et al, 2021). Both signal peptidase and furin protein converting enzyme are responsible for cleavage of the propeptide and pro-peptide propeptide from the pre-peptide propeptide cleavage site to produce the mature form of the protein (Munck Petersen et al, 1999).
Sortilin was first discovered for its ability to bind neurotensin and then discovered to bind a range of ligands such as neurotrophic factor precursors. More and more studies confirm that Sortilin is able to interact with many molecules and affect their intracellular distribution. Sortilin regulates intracellular transport of neurotrophic factors, as in neurons and B lymphocytes. In particular, when the precursor form of Sortilin is cleaved to remove the N-terminal precursor moiety, it becomes mature and is then continuously secreted into the cell membrane, which exposes Sortilin to the cell membrane and binds its ligand, thereby facilitating its ligand binding and transducing its intracellular signal or directly mediating the intracellular internalization of the ligand. Current studies indicate that Sortilin has dual functions in cells: not only involved in the regulation of cellular transport of substances, but also as receptors to act intracellular to transduce signals of extracellular ligands (Fatemeh Ghaemimanesh et al, 2021).
The overexpression of Sortilin and its clinical pathological significance in oncology has been reported in succession in various types of human solid cancers (e.g., neuroendocrine, kim et al, 2018; breast cancer, demont et al, 2012; rhost et al, 2018; roselli et al, 2015; colorectal cancer, aerol et al, 2011; ovarian cancer, ghaaemimanesh et al, 2014; heat et al, 2009) and hematological malignancies (chronic lymphocytic leukemia, lia Farahi et al, 2019).
Antibody Drug Conjugates (ADCs) are mainly used in the field of targeted tumor therapy. ADC drugs are a class of targeted biological agents consisting of antibodies, linkers, and cytotoxic drugs.
The antibody used by the ADC has extremely high affinity with the surface antigen of the tumor cells, and normal cells containing the same target point can be continuously killed in the in vivo time due to long half-life period (1-3 weeks), so that the toxic and side effects of the drug are greatly increased, in addition, the antibody is a relatively clumsy transport tool and is difficult to pass through a tumor by a small beard, only 0.1% of the drug can reach tumor tissues, and the chemical linkage between the warhead and the antibody is required to be stable enough in order to ensure that other 99% of highly toxic warheads do not bring about systemic toxicity. But this obviously presents a hassle to the drug design in order to be able to release the warhead inside the cell, which is not too stable. Due to the large difference in molecular weight, for the same number of moles of payload (payload), the mass used for ADC drugs is around 300 times the payload and therefore the side effects are relatively large.
ADCs are also prone to aggregation, which can lead to modifications that reduce their ability to bind antigen. Protein aggregation is a major obstacle to ADC development, which can occur at every stage and during transport and long-term storage. Aggregates are immunogenic. In addition, protein aggregation can lead to product loss. In general, any chemical or physical degradation may result in structural changes to the ADC and excessive aggregation of the protein. There are various other factors that can lead to aggregation, such as frequent freezing/thawing, high protein and salt concentrations, elevated temperatures, or low pH. Furthermore, most payloads are hydrophobic, and binding the payload at high DAR on the protein surface can result in excessive aggregation of the protein, thus impeding successful development of ADCs.
In addition, antibody-conjugated drugs (ADCs) also present immunogenicity as antibody drugs, and the risk of immunogenicity affects the safety and efficacy of the drug in patients, even with fatal new diseases caused by the intersection of ADA and endogenous proteins.
PDC (Peptide-Drug Conjugate), a polypeptide conjugated Drug, consists of a linker (linker), a homing Peptide (homing Peptide) and a cytotoxic payload (payload), wherein the homing Peptide can specifically target a protein receptor overexpressed on the surface of tumor cells so as to transmit cytotoxin to induce apoptosis of the tumor cells. Compared with the prior ADC drugs, the PDC drugs have the characteristics of small molecular weight, strong tumor penetrability, low immunogenicity, large-scale synthesis by utilizing a solid phase synthesis method, low production cost, relatively good pharmacokinetics and the like, and become next-generation targeted antitumor drugs after small-molecule targeted drugs, monoclonal antibodies and ADC.
Disclosure of Invention
The invention aims to provide a targeting peptide with unnatural amino acid or cyclic peptide for targeting tumor to highly express a target protein Sortilin, and a targeting peptide-drug conjugate containing the targeting peptide.
In a first aspect of the invention, there is provided an isolated polypeptide comprising modified or unmodified unnatural amino acids, and/or two cysteine residues that are linked by intra-chain linker (intro-chain linker) cyclization, or a pharmaceutically acceptable salt thereof, and which polypeptide specifically targets a SORT1 protein;
wherein the intrachain linker isOr disulfide bonds.
In another preferred embodiment, the polypeptide is a linear peptide or a cyclic peptide.
In another preferred embodiment, the polypeptide is a cyclic peptide formed by cyclizing two cysteine residues separated in the polypeptide sequence, wherein the cysteine residues are located in the range of 1-6 from the N-terminus of the polypeptide sequence and in the range of 1-6 from the C-terminus of the polypeptide sequence, and the separation between the two cysteine residues is not less than 3 amino acids. .
In another preferred embodiment, the amino acid sequence structure of the polypeptide is as shown in formula I: x is X 0 -X 1 -X 2 -X 3 -X 4 -X 5 -Ala-X 7 -Val-Arg-X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 (I)
Wherein X is 0 Modified or unmodified Cys or none, or Z for acetylation (Ac) 0 -Cys, wherein Z 0 1-3 amino acid residues;
X 1 gly, D-Ala, val, asn, arg, gln or none, modified or unmodified for acetylation (Ac);
X 2 val, 1Nal, 2Nal, D-2-Nal, or Tyr;
X 3 arg or hArg;
X 4 is Ala, arg or none;
X 5 lys, hArg, arg or Cys;
X 7 gly, arg, D-Ala, leu, phe, or Cys;
X 10 is Asp or Asn
X 11 Is Val or Nle;
X 12 is Phe, 4-Cl-Phe, 4-F-Phe, aib or 1Nal;
X 13 lys, hArg, arg or Cys;
X 14 ser, aib, asn or Cys;
X 15 glu, arg, lys or Aib;
X 16 is Ser, arg or Aib;
X 17 is Tyr or Aib;
X 18 is Cys or none, or Cys-Z 1 Wherein Z is 1 1-3 amino acid residues.
In another preferred embodiment, when X 0 X is when it is absent 1 Gly, D-Ala, val, asn, arg or Gln modified for acetylation (Ac); when X is 0 X is not time-free 1 Is unmodified Gly, D-Ala, val, asn, arg or Gln.
In another preferred embodiment, the polypeptide is a cyclic peptide comprising two cysteine residues linked by intra-chain linker (intra-chain linker), wherein the cysteine residues are located in each of X of the polypeptide sequence of formula (I) 0 -X 5 Bit range and X 13 -X 18 Within a bit range.
In another preferred embodiment, the intrachain linker is
In another preferred embodiment, the polypeptide is selected from the group consisting of:
table A
In another preferred embodiment, the polypeptide is a synthetic polypeptide.
In a second aspect of the present invention, there is provided a drug conjugate having the structure of formula II: (D) n -L-P (II)
Wherein D is the payload;
l is a linker;
p is a targeting peptide which is a polypeptide according to claim 1;
n is a positive integer not less than 1, preferably n=1 to 4, most preferably n=1 or 2.
In another preferred embodiment, the payload is selected from the group consisting of: docetaxel, paclitaxel, or cabazitaxel, or derivatives thereof.
In another preferred embodiment, the linker is selected from the group consisting of: succinic acid or dimethyl glutaric acid.
In another preferred embodiment, the linker is succinic acid.
In another preferred embodiment, the targeting peptide is selected from the group consisting of the polypeptides shown in Table A.
In another preferred embodiment, the drug conjugate is selected from the group consisting of:
(Z1) (docetaxel) n-succinic acid-SMTB 05;
(Z2) (docetaxel) n-succinic acid-SMTB 01;
(Z3) (docetaxel) n-succinic acid-SMTB 19;
(Z4) (docetaxel) n-succinic acid-SMTB 46;
(Z5) (paclitaxel) n-succinic acid-SMTB 05;
(Z6) (paclitaxel) n-succinic acid-SMTB 01;
(Z7) (paclitaxel) n-succinic acid-SMTB 19;
(Z8) (paclitaxel) n-succinic acid-SMTB 46;
(Z9) (cabazitaxel) n-succinic acid-SMTB 05;
(Z10) (cabazitaxel) n-succinic acid-SMTB 01;
(Z11) (cabazitaxel) n-succinic acid-SMTB 19; or (b)
(Z12) (cabazitaxel) n-succinic acid-SMTB 46.
In another preferred embodiment, the payload is attached to a lysine residue of the targeting peptide via a linker.
In a third aspect of the present invention, there is provided a pharmaceutical composition comprising: (a) the drug conjugate of claim 2; and (b) a pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition further comprises other drugs for treating tumors.
In a fourth aspect of the invention there is provided the use of a polypeptide according to the first aspect of the invention, or a drug conjugate according to the second aspect of the invention, in the manufacture of a medicament for the treatment of a tumour or cancer.
In another preferred embodiment, the tumor or cancer comprises a solid tumor and a hematological tumor.
In another preferred embodiment, the tumor or cancer highly expresses the SORT1 protein.
In another preferred embodiment, the tumor or cancer includes, but is not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, multiple myelopathy, non-hodgkin's lymphoma, colorectal cancer, breast cancer, colorectal cancer, gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, cervical cancer, lymphatic cancer, adrenal tumor, or bladder tumor.
In a fifth aspect of the invention there is provided a method of preparing a polypeptide according to the first aspect of the invention, the method comprising the steps of:
(1) Synthesizing linear polypeptide by adopting a solid-phase synthesis method; and
(2) Alternatively, the linear polypeptide obtained in step (1) is cyclized with a cyclizing reagent to obtain a cyclic peptide.
In a sixth aspect of the invention there is provided a method of preparing a drug conjugate according to the second aspect of the invention, the method comprising the steps of:
(A) Providing a linker and a payload, reacting the linker with the payload in an organic solvent to obtain an intermediate;
(B) Activating the intermediate with an activating reagent;
(C) Subjecting the polypeptide of claim 1 to an activated ester reaction with the intermediate of step (a), thereby obtaining the drug conjugate;
(D) Purifying the drug conjugate of step (C).
In a seventh aspect of the invention there is provided the use of a polypeptide according to the first aspect of the invention in the manufacture of a detection reagent or kit for detecting a SORT1 protein.
In another preferred embodiment, the kit is for detecting SORT1 protein in a sample.
In another preferred embodiment, the sample comprises a blood sample, a body fluid sample, or a tissue sample.
In another preferred embodiment, the kit further comprises instructions describing the use of the kit for non-invasively detecting SORT1 protein expression in a subject.
In an eighth aspect of the present invention, there is provided a method for detecting a SORT1 protein in a sample, the method comprising the steps of: (i) Contacting a sample with a polypeptide according to the first aspect of the invention; (ii) Detecting whether a polypeptide-SORT 1 protein complex is formed, wherein the formation of the complex indicates the presence of SORT1 protein in the sample.
In another preferred embodiment, the detection is non-diagnostic and non-therapeutic.
In another preferred embodiment, the detection is used for diagnosis or prognosis of a tumor or cancer.
In a ninth aspect of the invention there is provided a kit comprising a polypeptide according to the first aspect of the invention.
In another preferred embodiment, the kit is for detecting SORT1 protein in a sample.
In another preferred embodiment, the sample comprises a blood sample, a body fluid sample, or a tissue sample.
In another preferred embodiment, the kit further comprises instructions describing the use of the kit for non-invasively detecting SORT1 protein expression in a subject.
In a tenth aspect of the invention there is provided a method of treating a tumour or cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical conjugate according to the second aspect of the invention or a pharmaceutical composition according to the third aspect of the invention.
In another preferred embodiment, the tumor or cancer comprises a solid tumor and a hematological tumor.
In another preferred embodiment, the tumor or cancer highly expresses the SORT1 protein.
In another preferred embodiment, the tumor or cancer includes, but is not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, multiple myelopathy, non-hodgkin's lymphoma, colorectal cancer, breast cancer, colorectal cancer, gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, cervical cancer, lymphatic cancer, adrenal tumor, or bladder tumor.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
Fig. 1 shows a schematic structural diagram of Sortilin.
FIG. 2 shows MS diagrams of the SMTB01 linear peptide.
Fig. 3 shows an MS diagram of SMTB 01.
Fig. 4 shows the high resolution mass spectrum results of SMTB 01-docetaxel conjugates.
FIG. 5 shows the relative body weight change (%) of MDA-MB-231 tumor-bearing female BALB/c nude mice following treatment with the test agents. The relative weight change was calculated based on the weight of the animals at the beginning of the administration. Data points represent the percent mean weight change for each group, error bars represent Standard Error (SEM).
FIG. 6 shows neutrophil content of MDA-MB-231 tumor-bearing female BALB/c nude mice on day 4 after administration of the subject treatment. Data represent mean values for each group, error bars represent Standard Error (SEM).
FIG. 7 shows neutrophil content of MDA-MB-231 tumor-bearing female BALB/c nude mice at day 18 after administration of the subject treatment. Data represent mean values for each group, error bars represent Standard Error (SEM).
FIG. 8 shows the tumor growth curve of MDA-MB-231 tumor-bearing female BALB/c nude mice after administration of the subject treatment. Data points represent mean tumor volumes for each group, error bars represent Standard Error (SEM).
Detailed Description
Through extensive and intensive studies, the present inventors have unexpectedly developed a class of targeting peptides that specifically target Sortilin, including linear or cyclic peptides containing unnatural amino acids, through extensive experimental screening, and prepared targeting peptide-drug conjugates using the same. The targeting peptide prepared by the invention is a linear peptide or cyclic peptide containing non-natural chemical modification, and can improve the half life of the targeting peptide-drug conjugate and improve the stability of the targeting peptide-drug conjugate. The targeting peptide-drug conjugate provided by the invention can be specifically combined with a target protein Sortilin with high affinity, so that tumors expressing the Sortilin can be effectively killed, and a novel anti-tumor targeting drug is provided for the field of tumor treatment.
In order that the present disclosure may be more readily understood, certain terms are first defined. As used in the present application, each of the following terms shall have the meanings given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.
As used herein, the terms "SORT1 protein", "Sortilin" are used interchangeably and refer to the protein encoded by the SORT1 Gene (Gene ID: 6272).
As used herein, the terms "isolated polypeptide", "targeting peptide", "peptide compound" are used interchangeably and refer to a synthetic polypeptide of the first aspect of the application that targets a SORT1 protein.
As used herein, the terms "drug conjugate," "targeting peptide-drug conjugate," "conjugate compound," are used interchangeably and refer to a conjugate formed by conjugation of a polypeptide of the first aspect of the application with one or more therapeutic agent molecules (e.g., small molecule compounds such as docetaxel, paclitaxel, or cabazitaxel) via a linker.
The targeting peptides of the application
In one aspect of the application, an isolated polypeptide is provided that is a targeting peptide that targets Sortilin. The polypeptide may be a linear peptide or a cyclic peptide comprising modified or unmodified unnatural amino acids, and/or two cysteine residues that are connected by intra-chain linker (intro-chain linker) cyclization. As used herein, the term "intra-chain linker" refers to a linker for cyclizing two cysteine residues within a linked polypeptide chain, which in a preferred embodiment of the application is Or disulfide bonds, preferably
The invention also includes active fragments, derivatives and analogues of the polypeptides of the first aspect of the invention. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that substantially retains the function or activity of binding Sortilin. The polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a polypeptide of the first aspect of the invention with another compound, such as a compound which increases the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to a sequence of such a polypeptide, for example a leader sequence, secretory sequence or a tag sequence of 6 His. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.
A preferred class of reactive derivatives refers to polypeptides having up to 6, preferably up to 3, more preferably up to 2, most preferably 1 amino acid replaced by an amino acid of similar or similar nature, as compared to the amino acid sequence of formula I. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.
Table A
The present invention also provides analogues of the polypeptides of the first aspect of the invention. These analogues may differ from the polypeptides of the first aspect of the invention by differences in amino acid sequence, by differences in modified forms that do not affect the sequence, or by both. Analogs also include analogs having residues other than the natural L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.
Some of the commonly used unnatural amino acids are listed in Table B below.
Table B
Modified (typically without altering the primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the polypeptide or during further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to improve their proteolytic resistance or to optimize solubility.
The polypeptides of the invention may also be used in the form of salts derived from pharmaceutically or physiologically acceptable acids or bases. These salts include, but are not limited to, salts formed with acids: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid or isethionic acid. Other salts include: salts with alkali or alkaline earth metals (such as sodium, potassium, calcium or magnesium), and in the form of esters, carbamates or other conventional "prodrugs".
The targeting peptide-drug conjugates of the invention
In one aspect of the present invention, there is provided a targeting peptide-drug conjugate comprising the targeting peptide of the first aspect of the present invention, having the structure shown in formula II: (D) n -L-P (II)
Wherein D is the payload;
l is a linker;
p is a targeting peptide according to the first aspect of the invention;
n is a positive integer not less than 1, preferably n=1 to 4, most preferably n=1 or 2.
In some embodiments, the payload is a small molecule compound, including but not limited to docetaxel, paclitaxel, or cabazitaxel.
Wherein the chemical structure of docetaxel is as follows:
The chemical structure of paclitaxel is shown below:
the chemical structure of cabazitaxel is as follows:
in some embodiments, the linker is selected from succinic acid or dimethylglutaric acid, preferably succinic acid.
In some embodiments, the targeted peptide-drug conjugates of the application are composed as shown in the following table:
the preparation method of the targeting peptide
In another aspect of the application, there is provided a method of preparing an isolated polypeptide (targeting peptide) of the first aspect of the application, in particular the method comprising the steps of:
(1) Synthesizing an initial polypeptide by adopting a solid-phase synthesis method;
(2) Cracking the product of the step (1) by using strong acid; adding a side chain protecting group scavenger, filtering, adding a proper amount of organic solvent for precipitation, centrifuging, washing the precipitate with the organic solvent, and drying to obtain crude peptide;
(3) Optionally, cyclizing the crude peptide obtained in step (2) with a cyclizing reagent to obtain a cyclized polypeptide.
In some embodiments, the resin support used in the solid phase synthesis in step (1) is selected from Wang resin or 2-CTC resin.
In some embodiments, the step (1) comprises the sub-steps of:
(a) Resin swelling-feeding (first amino acid/condensation reagent) -measuring resin substitution value-removing amino protecting group-solvent washing-monitoring-coupling amino acid-monitoring-solvent washing-removing amino protecting group-sequentially coupling residual amino acid-removing amino protecting group of last amino acid and washing;
(b) Adding acetic anhydride/DIEA to perform an acetylation reaction, and washing;
wherein, the amino protecting group refers to a chemical group introduced for protecting an amino group participating in a condensation reaction.
The amino protecting group may be selected from t-butoxycarbonyl (Boc), benzyloxycarbonyl (Z) or 9-fluorenyl-methoxycarbonyl (Fmoc), preferably 9-fluorenyl-methoxycarbonyl (Fmoc).
The solvent used in step (a) is selected from the group consisting of: dimethylformamide (DMF), dichloromethane (DCM) or N-methylpyrrolidone (NMP), preferably DMF or DCM.
The removing agent for removing amino protecting groups in the step (a) is selected from piperidine/DMF (PIP) with the concentration of 10-40%, preferably piperidine/DMF (PIP) with the concentration of 20-25%; the removal time is 20-50min, preferably 25-35min.
The step of condensing the amino acid in step (a) requires the addition of a condensing reagent selected from the group consisting of a carbodiimide type reagent, a benzotriazole salt type reagent or 1-hydroxybenzotriazole (HOBt), or a combination thereof.
The carbodiimide type reagent is selected from one of Dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) or N-diaminopropyl-N-Ethylcarbodiimide (EDC).
The benzotriazole onium salt reagent is selected from one of 2- (1H-benzotriazole L-1-yl) -1, 3-tetramethylurea tetrafluoroborate (TBTU), O-benzotriazole-N, N, N ', N' -tetramethylurea Hexafluorophosphate (HBTU), benzotriazole-1-oxy tris (dimethylamino) phosphonium hexafluorophosphate (BOP) or benzotriazol-1-yl-oxy-tripyrrolidinyl phosphonium hexafluorophosphate (PyBOP).
The coupling reagent is selected from the group consisting of Diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt), or 2- (1H-benzotrisazo L-1-yl) -1, 3-tetramethylurea tetrafluoroborate (TBTU) and 1-hydroxybenzotriazole (HOBt), with DIC (diisopropylcarbodiimide) and 1-hydroxybenzotriazole (HOBt) being further preferred. In some embodiments, the "monitoring" described in step (a) employs ninhydrin detection to monitor the condensation reaction of the polypeptide.
The sequential coupling of the remaining amino acids in step (a) refers to the sequential attachment of the amino acids from the C-terminus to the N-terminus according to the amino acid sequence of the polypeptide.
In some embodiments, the side chain protecting group scavenger described in step (2) is selected from one, two or a combination of several of anisole, triisopropylsilane, phenol, water, 1, 2-ethanedithiol or m-cresol.
In some specific embodiments, the side chain protecting group scavenger described in step (2) is selected from trifluoroacetic acid (TFA): triisopropylsilane: water=95:2.5:2.5 (V/V).
In some embodiments, the cyclizing reaction described in step (3) requires the addition of a cyclizing reagent selected from one or a combination of 1,2- (dibromomethyl) benzene, 1,3- (dibromomethyl) benzene, 1,4- (dibromomethyl) benzene, or other bromoalkyl substituted benzene reagents, preferably 1,2- (dibromomethyl) benzene, 1,3- (dibromomethyl) benzene, or 1,4- (dibromomethyl) benzene, and more preferably 1,3- (dibromomethyl) benzene, and ammonium bicarbonate (NH 4HCO 3).
The method for preparing the polypeptide provided by the invention can further comprise a purification step after the crude peptide is obtained from the step (2). The purification method employed includes, but is not limited to, reverse phase chromatography or ion exchange chromatography, preferably reverse phase chromatography.
Preparation method of targeting peptide-drug conjugate of the invention
In another aspect, the invention also provides a method for preparing the targeting peptide-drug conjugate of the invention, the method comprising:
(A) Providing a linker and a payload, reacting the linker with the payload in an organic solvent comprising an organic base to obtain an intermediate;
(B) Activating the intermediate with an activating reagent;
(C) Carrying out an activated ester reaction on the targeting peptide of the invention and the intermediate of the step (A), thereby obtaining the targeting peptide-drug conjugate;
(D) Purifying the targeting peptide-drug conjugate of step (C).
In some embodiments, the linker used in step (a) is selected from succinic acid or dimethyl glutaric acid.
In some embodiments, the payload in step (a) is a small molecule compound, including but not limited to docetaxel, paclitaxel, or cabazitaxel.
In some embodiments, the organic solvent in step (a) is selected from DMSO, DCM, DMF, or THF, preferably DMSO.
In some embodiments, the organic base in step (a) is selected from one or a combination of DIEA, TEA, DMAP; DMAP/triethylamine is preferred.
In some embodiments, the activating reagent in step (B) is selected from one of 2- (1H-benzotrisazo L-1-yl) -1, 3-tetramethyluronium tetrafluoroborate (TBTU), O-benzotriazole-N, N' -tetramethyluronium Hexafluorophosphate (HBTU), benzotriazol-1-oxy tris (dimethylamino) phosphonium hexafluorophosphate (BOP), or benzotriazol-1-yl-oxy-tripyrrolidinyl phosphonium hexafluorophosphate (PyBOP).
In some embodiments, the therapeutic agent in step (C) is linked to the targeting peptide at a lysine residue, and wherein the targeting peptide comprises 1, 2, 3, or 4 therapeutic agent molecules linked thereto.
In some embodiments, the purification method employed in step (D) includes, but is not limited to, reverse phase chromatography or ion exchange chromatography, preferably reverse phase chromatography.
Pharmaceutical compositions and methods of administration
In another aspect, the invention provides a pharmaceutical composition comprising (a) a safe and effective amount of a drug conjugate according to the second aspect of the invention; and (b) a pharmaceutically acceptable carrier. The amount of the drug conjugate of the invention in the pharmaceutical composition is generally 10. Mu.g to 100 mg per dose, preferably 100 to 1000. Mu.g per dose.
For the purposes of the present invention, an effective dose is about 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg of body weight of the drug conjugate of the invention administered to an individual. In addition, the drug conjugates of the invention may be used alone or in combination with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).
The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. The term refers to such agent carriers: they do not themselves induce the production of antibodies harmful to the individual receiving the composition and do not have excessive toxicity after administration. Such vectors are well known to those of ordinary skill in the art. A sufficient discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub.Co., N.J.1991). Such vectors include (but are not limited to): saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.
The pharmaceutically acceptable carrier in the therapeutic composition may contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
In general, the therapeutic compositions may be formulated as an injectable, such as a liquid solution or suspension; it can also be made into a solid form suitable for incorporation into a solution or suspension, and a liquid carrier prior to injection.
Once formulated into the compositions of the present invention, they may be administered by conventional routes including, but not limited to: intramuscular, intravenous, subcutaneous, intradermal or topical administration. The subject to be prevented or treated may be an animal; especially humans.
When the pharmaceutical composition of the present invention is used for actual treatment, various different dosage forms of the pharmaceutical composition can be employed according to the use condition. Preferably, there may be exemplified a freeze-dried powder injection, an oral preparation, etc.
These pharmaceutical compositions may be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents (isotonides), preservatives, wetting agents, emulsifying agents, dispersing agents, stabilizers and cosolvents, and the formulation process may be carried out in a conventional manner according to dosage forms.
Therapeutic applications
The invention also provides application of the polypeptide and the drug conjugate in preparing a drug for treating tumors or cancers. The present invention also provides a method of treating a tumor or cancer comprising administering to a subject in need thereof a therapeutically effective amount of a drug conjugate of the second aspect of the invention.
Wherein the tumor or cancer comprises a solid tumor and a hematological tumor, and the tumor or cancer highly expresses SORT1 protein. Tumors or cancers that may be treated using the drug conjugates of the application include, but are not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, multiple myelopathy, non-hodgkin's lymphoma, colorectal cancer, breast cancer, colorectal cancer, gastric cancer, liver cancer, leukemia, renal tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, cervical cancer, lymphatic cancer, adrenal tumor, or bladder tumor.
The main advantages of the application include:
(1) The targeting peptide in the drug conjugate is a linear peptide or cyclic peptide containing unnatural amino acid, the targeting peptide can improve half life of the conjugate and stability, and in addition, the drug coupled with the targeting peptide can bypass a drug resistance path, so that drug resistance in the treatment process is avoided.
(2) The plasma stability of the drug conjugate is obviously improved.
(3) The polypeptide conjugates are equally effective against payload-resistant cell lines.
The compounds of the present application may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments set forth below, embodiments formed by combining with other chemical synthetic methods, and equivalent alternatives well known to those skilled in the art, preferred embodiments including but not limited to the examples of the present application.
The chemical reactions of the embodiments of the present application are accomplished in a suitable solvent that is compatible with the chemical changes of the present application and the reagents and materials required therefor. In order to obtain the compounds of the present application, it is sometimes necessary for a person skilled in the art to modify or select the synthesis steps or reaction schemes on the basis of the embodiments already present.
The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
Example 1: preparation and purification of SMTB01
The SMTB01 targeting peptide was prepared as follows:
SMTB01 formula:
1.1 materials and reagents
Wang resin, substitution value 0.31mmol/g.
The amino acid is: fmoc-Cys (Trt) -OH, fmoc-Tyt (tbu) -OH, fmoc-Ser (tbu) -OH, fmoc-Glu (otbu) -OH, fmoc-Lys (boc) -OH, fmoc-Phe-OH, fmoc-Nle-OH, fmoc-Asp (tbu) -OH, fmoc-Arg (pbf) -OH, fmoc-Val-OH, fmoc-Gly-OH, fmoc-Ala-OH.
The synthesis reagent comprises the following steps: HOBt, DIC, DMF, DCM piperidine
1.2 instruments
CS-BIO type polypeptide synthesizer, waters600 semi-preparative high performance liquid chromatograph, beckman centrifuge, buchi rotary evaporator.
1.3 working up procedure (taking 0.15mmol as an example)
a. Solid phase chemical synthesis of polypeptides
Weighing 0.5g of Fmoc-Cys (Trt) -Wang resin, placing the Fmoc-Cys (Trt) -Wang resin in a reactor of a polypeptide synthesizer, adding 10ml of DCM, soaking for 2h, adding 15ml of 20% PIP/DMF solution, mixing for 25min to remove amino protecting groups, and washing the resin with DCM for 6 times; three times the amount of Fmoc-Tyt (tbu) -OH, DIC, HOBT was weighed into 10ml of DMF and dissolved, and the reaction was carried out in a reactor at room temperature with ninhydrin reaction monitoring the progress of the reaction. Monitoring blue-violet indicates incomplete condensation reaction, colorless indicates complete reaction, and then washing the resin with DCM for 6 times; 15ml of 20% PIP/DMF solution was then added and the amino protecting group was removed by mixing for 20min, and the resin was washed 6 times with DMF at which time ninhydrin was detected as bluish violet.
The coupling reaction of the next amino acid was continued as described above, and the cycle was repeated until all amino acids were coupled, the last amino acid was deprotected, washed, and a solution of acetic anhydride (1.2 eq) and DIEA (3.6 eq) in DMF was added to react for 30 minutes, and the resin was washed 6 times with DCM.
b. Cleavage and precipitation
The resin peptide was dried in vacuo and weighed. Preparing a cleavage reagent according to the proportion of adding 10ml of the cleavage reagent into 1g of resin, wherein the reagent proportion is TFA, triisopropylsilane, water=95:2.5:2.5 (V: V), adding the cleavage reagent into the resin, stirring at room temperature for reaction for 3 hours, filtering, removing part of TFA by rotary evaporation, adding 10 times of volume of glacial ethyl ether into the filtrate to precipitate polypeptide, centrifuging, repeatedly washing the precipitate with the glacial ethyl ether for 4-5 times, drying in vacuum, and weighing.
c. Preparation of cyclic peptides by reaction in liquid phase
Weighing 0.05mmol of dried crude peptide, adding 1ml of water for dissolution, adjusting the pH value to 8.0 by using ammonium bicarbonate, weighing 0.05mmol of cyclizing reagent 1, 3-di (bromomethyl) benzene for dissolution in acetonitrile, adding acetonitrile solution of the cyclizing reagent into the crude peptide solution for summarizing, and reacting for 0.5-1 h at room temperature.
d. Separation and purification
The crude peptide was purified by semi-preparative RP-HPLC.
(1) Purification
Chromatographic column: YMC-pack ODS-A-HG C18 preparation column (10 mm. Times.250 mm,10 μm)
Flow rate: 5ml/min
Detection wavelength: 215nm, 280nm
Mobile phase: phase A: 0.1% hac/water solution; and B phase: 0.1% HAc/acetonitrile
Gradient elution procedure is as follows table 1:
TABLE 1
(2) Analysis
Analysis of the collected product by Thermo U3000 type HPLC
Chromatographic column: kromasil C18 analytical column (4.6 mm. Times.250 mm,5 μm)
Flow rate: 1ml/min
Detection wavelength: 215nm
Mobile phase: phase A: 0.05% tfa/water; and B phase: 0.05% TFA/acetonitrile
Gradient elution procedure is as follows table 2:
TABLE 2
Collecting target components with purity of more than 90%, removing acetonitrile by rotary evaporation, and vacuum freeze drying. Molecular weight confirmation by ESI-MS, M/Z= 1117.52 (M+H) 2+ Consistent with theoretical molecular weight.
Example 2: preparation and purification of the remaining targeting peptides
The remaining targeted peptide compounds were prepared as described in example 1, molecular weight confirmation via ESI-MS:
TABLE 3 Table 3
Example 3: surface Plasmon Resonance (SPR) affinity assay
The affinity of the targeting peptides prepared in example 1 and example 2 to the SORT1 protein was studied using SPR techniques to determine the kinetic parameter Ka (M -1 s -1 )、Kd(s -1 ) KD (M). The affinity of the targeting peptides SMTB53 (SEQ ID NO: 53) and SMTB54 (SEQ ID NO: 54) for SORT1 proteins in the prior art was also determined.
The SORT1 protein is fixed on a CM5 chip by a Biacore 8K instrument through a standard amino coupling mode, and the coupling level is 1200-3000 RU. A concentration gradient sample was prepared by 2-fold dilution of PBST (0.02% Tween) in which the peptides were dissolved to give a 100nM stock solution. At 25 ℃, PBST is used as an operation buffer, 10mM GLy (PH 4.0) is used as a regeneration buffer, SPR is operated, the combination time is 60S, the dissociation time is 60-100S, and corresponding software is adopted to process and dynamically fit data.
The affinity assay results for the targeting peptides are shown in table 4 below:
TABLE 4 Table 4
Wherein NA represents no measurement or no binding.
Example 4: preparation of targeting peptide-drug conjugates
Targeting peptide-drug conjugates were prepared using SMTB01 as an example.
4.1 preparation of docetaxel succinate
DMAP (2.5 eq) was added to a DMSO solution (100 mg/ml) of docetaxel under stirring, and after stirring for 30 minutes, 5ml of a DMSO solution of succinic anhydride (1.2 eq) was added dropwise to the above solution, and the reaction was stirred for 12 hours after the addition within 30 minutes; after the reaction was completed, an equal volume of sodium bicarbonate solution was added, stirred for 30 minutes, HCl was added dropwise to the solution until a large amount of white precipitate was generated, centrifuged, and the precipitate was lyophilized after washing twice with water.
4.2 preparation of docetaxel succinate activated ester
TBTU (2 eq) was added to a DMSO solution of docetaxel succinate with stirring, DIEA (6 eq) was added to the solution and activated for 30 minutes.
4.3 Synthesis of SMTB01 conjugated with docetaxel
The SMTB01 was dissolved in 1% tea DMSO, to which an activated ester solution was added dropwise, the reaction was stirred at room temperature, the reaction was checked by HPLC until completion, and the reaction was terminated by adding an appropriate amount of acetic acid.
4.4 Purification of SMTB011 (SMTB 01-docetaxel conjugation)
(1) Purification
Chromatographic column: YMC-pack C4 preparation column (10 mm. Times.250 mm,10 μm)
Flow rate: 5ml/min
Detection wavelength: 215nm, 280nm
Mobile phase: phase A: 0.1% tfa/water; and B phase: 0.1% TFA/acetonitrile
Gradient elution procedure is as follows table 5:
TABLE 5
(2) Analysis
The collected product was analyzed by Thermo U3000 type HPLC.
Chromatographic column: kromasil C4 analytical column (4.6 mm. Times.250 mm,5 μm)
Flow rate: 1ml/min
Detection wavelength: 215nm
Mobile phase: phase A: 0.05% tfa/water; and B phase: 0.05% TFA/acetonitrile
Gradient elution procedure is as follows table 6:
TABLE 6
Collecting target components with purity of more than 90%, removing acetonitrile by rotary evaporation, and vacuum freeze drying.
The structure of the prepared SMTB011 is shown as follows:
the paclitaxel and cabazitaxel drug conjugates were prepared using similar methods.
Conjugates of other targeting peptides with docetaxel, paclitaxel or cabazitaxel are prepared by similar methods and are not described in detail herein.
The resulting partial conjugates were prepared as follows:
SMTB051 (SMTB 05-docetaxel conjugate)
SMTB191 (SMTB 19-docetaxel conjugate)
Smob 461 (smob 46-docetaxel conjugate)
Example 5: cytotoxicity assay of SMTB011
Test sample: SMTB011
Control sample: docetaxel (docetaxel)
The experimental method comprises the following steps:
test cell line: human ovarian clear cell carcinoma cell ES-2, breast duct carcinoma cell HCC-70, human ovarian carcinoma cell OV90, and human breast carcinoma cell MDA-MB-231.
Tumor cell lines were incubated at 37℃with 5% CO 2 Is cultured in an incubator of (a). Periodically passaging, taking the obtained extractLong term cells were used for plating.
1. Cell plating
(1) Cell staining was performed with trypan blue and living cells were counted.
(2) The cell concentration was adjusted to the appropriate concentration.
(3) mu.L of cell suspension was added to each well of the culture plate, and cell-free culture medium was added to the blank wells.
(4) The plates were incubated at 37℃with 5% CO 2 And 100% relative humidity overnight.
SMTB011 memory board preparation
1000x smtb011 storage plate was prepared: the SMTB011 was diluted with DMSO from the highest concentration gradient to the lowest concentration.
3.10 preparation of working solution of X conjugate and PDC treatment of cells
(1) Preparing 10X conjugate working solution: 990. Mu.L of cell culture medium was added to a 96-well plate with a V-bottom, and 10. Mu.L of SMTB011 was pipetted from a 1000 XSMTB011 storage plate into the cell culture medium of the 96-well plate. And adding 10 mu L of corresponding solvent into the solvent control and the blank control, and blowing and uniformly mixing by a gun.
(2) Adding the medicine: 10. Mu.L of 10 XSMTB011 work was added to the cell culture plate. To the vehicle control and the blank, 10 μl of the corresponding solvent was added.
(3) The 96-well cell plates were returned to the incubator for 72 hours.
Cell Activity detection by CellTiter-Glo luminescence method
The following steps were performed according to the instructions of the Promega CellTiter-Glo luminescence cell activity assay kit.
(1) CellTiter-Glo buffer was thawed and left to stand to room temperature.
(2) The CellTiter-Glo substrate was left to stand to room temperature.
(3) CellTiter-Glo working solution was prepared by adding CellTiter-Glo buffer to a bottle of CellTiter-Glo substrate to dissolve the substrate.
(4) The slow vortex shaking allowed for adequate dissolution.
(5) The cell culture plates were removed and allowed to stand for 30 minutes to equilibrate to room temperature.
(6) To each well 50. Mu.L (equal to half the volume of cell culture broth in each well) of CellTiter-Glo working fluid was added. The cell plates were wrapped with aluminum foil paper to protect from light. The rest CellTiter-Glo working solution is subpackaged into 50mL centrifuge tubes, stored at-20 ℃ in a dark place and used within one month.
(7) The plates were shaken on an orbital shaker for 2 minutes to induce cell lysis.
(8) The plates were left at room temperature for 10 minutes to stabilize the luminescence signal.
(9) The luminescence signal is detected at 2104 EnVision reader.
Meanwhile, the cytotoxicity was determined according to the above method using targeting peptide-drug conjugate (SMTB 531) targeting the SORT1 protein in the prior art as a positive control. The structure of the SMTB531 is as follows:
the data were analyzed according to the detection results as shown in table 7 below.
Table 7 cytotoxicity test results of SMTB011
Results and discussion: SMTB011 has a killing or growth inhibiting effect on ES-2, HCC-70, OV90, MDA-MB-231 tumor cells, with IC50 values lower than that of Docetaxel in some cell lines (e.g., HCC70, MDA-MB-231) and lower than that of SMTB531 in all 4 cell lines.
Example 6: cytotoxicity assay of smob 051, smob 191, smob 461
Test sample: SMTB051, SMTB191, SMTB461
Control sample: docetaxel (docetaxel)
The experimental method comprises the following steps:
test cell line: human ovarian clear cell carcinoma cell ES-2, breast duct carcinoma cell HCC-70, human ovarian carcinoma cell OV90, and human breast carcinoma cell MDA-MB-231.
Tumor cell linesAt 37 ℃,5% CO 2 Is cultured in an incubator of (a). Cells in the logarithmic growth phase were taken for plating at regular passages.
1. Cell plating
(1) Cell staining was performed with trypan blue and living cells were counted.
(2) The cell concentration was adjusted to the appropriate concentration.
(3) mu.L of cell suspension was added to each well of the culture plate, and cell-free culture medium was added to the blank wells.
(4) The plates were incubated at 37℃with 5% CO 2 And 100% relative humidity overnight.
SMTB051, SMTB191, SMTB461 memory board preparation
1000x SMTBs 051, SMTB191, SMTB461 storage plates were prepared: the SMTB051, SMTB191, SMTB461 were diluted with DMSO from the highest concentration gradient to the lowest concentration.
3.10 preparation of working solution of X conjugate and PDC treatment of cells
(1) Preparing 10X conjugate working solution: 990. Mu.L of cell culture medium was added to a 96-well plate with V-shaped bottom, and 10. Mu.L of SMTB051, SMTB191, SMTB461 were pipetted from 1000 XSMTB051, SMTB191, SMTB461, respectively, into the cell culture medium of the 96-well plate. And adding 10 mu L of corresponding solvent into the solvent control and the blank control, and blowing and uniformly mixing by a gun.
(2) Adding the medicine: mu.L of 10 XSMTB051, SMTB191, SMTB461 were operatively added to the cell culture plates. To the vehicle control and the blank, 10 μl of the corresponding solvent was added.
(3) The 96-well cell plates were returned to the incubator for 72 hours.
Cell Activity detection by CellTiter-Glo luminescence method
The following steps were performed according to the instructions of the Promega CellTiter-Glo luminescence cell activity assay kit.
(1) CellTiter-Glo buffer was thawed and left to stand to room temperature.
(2) The CellTiter-Glo substrate was left to stand to room temperature.
(3) CellTiter-Glo working solution was prepared by adding CellTiter-Glo buffer to a bottle of CellTiter-Glo substrate to dissolve the substrate.
(4) The slow vortex shaking allowed for adequate dissolution.
(5) The cell culture plates were removed and allowed to stand for 30 minutes to equilibrate to room temperature.
(6) To each well 50. Mu.L (equal to half the volume of cell culture broth in each well) of CellTiter-Glo working fluid was added. The cell plates were wrapped with aluminum foil paper to protect from light. The rest CellTiter-Glo working solution is subpackaged into 50mL centrifuge tubes, stored at-20 ℃ in a dark place and used within one month.
(7) The plates were shaken on an orbital shaker for 2 minutes to induce cell lysis.
(8) The plates were left at room temperature for 10 minutes to stabilize the luminescence signal.
(9) The luminescence signal is detected at 2104 EnVision reader.
Meanwhile, the cytotoxicity was measured as described above using the aforementioned SMTB531 as a positive control.
The data were analyzed according to the detection results as shown in table 8 below.
Table 8 cytotoxicity test results of SMTB051, SMTB191 and SMTB461
Results and discussion: SMTB051, SMTB191, SMTB461 have tumor cell killing or growth inhibiting effects on ES-2, HCC-70, OV90, and MDA-MB-231 tumor cells. Of these, SMTB191 has an IC50 value lower than that of SMTB531 in all of the 4 cell lines described above, and has an IC50 value lower than that of Docetaxel in HCC70 and MDA-MB-231 cell lines.
Example 7: animal model efficacy research of targeting peptide-drug conjugate
1. Experimental purposes the in vivo efficacy of the test subjects in a BALB/c nude mouse MDA-MB-231 subcutaneous engraftment model was evaluated.
2. Experiment design: as shown in Table 9 below
Table 9 in vivo efficacy experimental animal grouping and dosing regimenNote that: a.N number of mice per group b. dosing volume: based on the weight of the mice, 10. Mu.l/g. If body weight decreases by more than 15%, the dosing regimen should be adjusted accordingly. c. If the weight is reduced by more than 15 percent (compared with D0), the drug is stopped temporarily, and the weight of the mice is restored to be reduced<Administration was continued after 10%.
3. Experimental materials
3.1 laboratory animals
Species: a mouse
Strain: BALB/c nude
Week-old: 6-8 weeks of age
Gender: female
Weight of: 18-22 g
Quantity: 65, not including the remaining mice
3.2 raising Environment
Animals were housed in the experimental environment 7 days after arrival and the experiment was started. Animals were housed in SPF class animal houses in IVC (independent air supply system) cages (5 animals per cage). The number, sex, strain, date of receipt, dosing regimen, number, group and date of start of experiment of animals in the cages are noted per cage of animal information card. All cages, pads and drinking water are sterilized before use. The cages, feed and drinking water are replaced twice a week. The feeding environment and the illumination conditions are as follows:
Temperature: 20-26 DEG C
Humidity: 40 to 70 percent
Illumination period: 12 hours of illumination, 12 hours of no illumination (8 am on lamp-8 pm off lamp)
Cage utensil: made of polycarbonate, volume 300mm x 180mm x 150mm. The padding is corncob, and is replaced twice a week.
Food: the experimental animals were free to eat (irradiation sterilized, dry granular food) throughout the experimental period.
And (3) drinking water: the experimental animal can drink the sterilized water freely.
Cage identification: the number, sex, strain, date of receipt, dosing regimen, number, group and date of start of experiment of animals in the cage should be noted per cage of animal information card.
4. Experimental methods and procedures
4.1 cell culture
MDA-MB-231 cells are cultivated in an in vitro adherence way, and the cultivation condition is that 10 percent of fetal bovine serum and 1 percent of PS are added into an L-15 culture medium, and the culture is carried out at 37 ℃ and 0 percent of CO 2. Passaging is routinely performed 2 times a week. Cells were harvested, counted and inoculated while the cells remained in the exponentially growing phase.
4.2 tumor cell seeding
Each BALB/c nude mouse was inoculated with 0.2mL (10X 10) 6 Individual + Matrigel) MDA-MB-231 cells. The inoculation simultaneously marks the ear marks of the experimental animals as the only confirmation marks of the subsequent experiments. Waiting for tumor growth, and the average tumor volume reaches 132mm on the 27 th day after inoculation 3 Random group dosing was started at this time. The information after specific grouping is shown in table 9.
4.3 daily observations of laboratory animals
The development of the experimental protocol and any modification passed the evaluation approval of the Institutional Animal Care and Use Committee (IACUC) of the south-access drug Ming Kangde New drug development Co. The use and welfare of experimental animals was performed in compliance with the international committee for laboratory animal assessment and approval (AAALAC) specifications. Animals were monitored daily for health and mortality, and routine examinations included observation of tumor growth and the effects of drug treatment on daily performance of the animals such as behavioral activity, intake of water (visual inspection only), weight changes (weight measured twice a week), physical signs of appearance, or other abnormalities.
4.4 tumor measurement and Experimental index
The experimental index is to examine whether tumor growth is inhibited, retarded or cured. Tumor diameters were measured twice weekly with vernier calipers. The calculation formula of the tumor volume is: v=0.5a×b2, a and b represent the long and short diameters of the tumor, respectively.
The tumor-inhibiting effect of the compound was evaluated by TGI (%) or relative tumor proliferation rate T/C (%). TGI (%) reflects the tumor growth inhibition rate. Calculation of TGI (%): TGI (%) = [1- (mean tumor volume at the end of dosing of a treatment group-mean tumor volume at the beginning of dosing of a treatment group)/(mean tumor volume at the end of treatment of isotype control group-mean tumor volume at the beginning of treatment of isotype control group) ]x100.
Calculation of relative tumor proliferation rate T/C (%): T/C% = mean tumor volume at the end of treatment group dosing/mean tumor volume at the end of treatment of solvent control group x 100.
4.5 statistical analysis
Statistical analysis was based on mean and Standard Error (SEM) of tumor volumes per group at day 35 post-start dosing. One-way ANOVA was used for comparison among three or more groups. All data analysis was performed using GraphPad Prism software, with p <0.05 considered significant differences.
5. Experimental results
5.1 weight change conditions
Docetaxel 15mg/kg high dose mice began to drop off on day 25 after group dosing, and mice in this group were discontinued on day 28 after group dosing (4 doses) and the other groups were discontinued after 5 doses. The results are shown in FIG. 5.
5.2 analysis of neutrophil (CBC) assay results
The neutrophil content was determined 4 days, 18 days after administration of SMT011, SMTB051, SMTB191, SMTB 461. The results are shown in fig. 6 and 7.
5.3 tumor volume Change Condition
The results of the tumor volume change in each group following treatment with MDA-MB-231 tumor-bearing female BALB/c nude mice subject are shown in FIG. 8.
5.4 evaluation index of antitumor drug efficacy
Based on the tumor volume calculation at day 35 after the group administration, the tumor inhibition efficacy evaluation of the test substance on the MDA-MB-231 tumor model is obtained as shown in the following table 10.
Table 10 evaluation of tumor inhibiting efficacy of test substances on MDA-MB-231 tumor modelNote that: a. mean.+ -. SEMb. tumor growth inhibition evaluation index is calculated according to the formulaT/C％＝T treament /T Vehicle X 100 and TGI (%) = [1- (T) i -T 0 )/(V i -V 0 )]X 100 calculation. c. The p-value between each treatment group and the Vechile group was calculated using the one-way ANOVA method.
Results and discussion: SMT011, SMTB051, SMTB191 and SMTB461 show continuous tumor inhibiting effect in the in-vivo efficacy experiment of the MDA-MB-231 model, and the conditions of weight loss and neutrophil reduction are obviously improved. Especially SMT011 showed significantly better tumor inhibiting effect than the positive control SMTB 531. SMT011 high dose group continued to inhibit tumor growth until 70 days after drug withdrawal.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims (15)

  1. An isolated polypeptide or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises modified or unmodified unnatural amino acids, and/or two cysteine residues that are cyclized linked by an intra-chain linker (intro-chain linker), and wherein the polypeptide specifically targets a SORT1 protein;
    Wherein the intrachain linker isOr disulfide bonds.
  2. The polypeptide of claim 1, wherein the polypeptide is a linear peptide or a cyclic peptide.
  3. The polypeptide of claim 1, wherein the polypeptide is a cyclic peptide formed by cyclizing two cysteine residues separated in the polypeptide sequence, wherein the cysteine residues are located in the range of positions 1-6 from the N-terminus of the polypeptide sequence and in the range of positions 1-6 from the C-terminus of the polypeptide sequence, respectively, and the separation between the two cysteine residues is not less than 3 amino acids.
  4. The polypeptide of claim 1, wherein the amino acid sequence structure of the polypeptide is as shown in formula I:
    X 0 -X 1 -X 2 -X 3 -X 4 -X 5 -Ala-X 7 -Val-Arg-X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 (I)
    wherein X is 0 Modified or unmodified Cys or none, or Z for acetylation (Ac) 0 -Cys, wherein Z 0 1-3 amino acid residues;
    X 1 gly, D-Ala, val, asn, arg, gln or none, modified or unmodified for acetylation (Ac);
    X 2 val, 1Nal, 2Nal, D-2-Nal, or Tyr;
    X 3 arg or hArg;
    X 4 is Ala, arg or none;
    X 5 lys, hArg, arg or Cys;
    X 7 gly, arg, D-Ala, leu, phe, or Cys;
    X 10 is Asp or Asn
    X 11 Is Val or Nle;
    X 12 is Phe, 4-Cl-Phe, 4-F-Phe, aib or 1Nal;
    X 13 Lys, hArg, arg or Cys;
    X 14 ser, aib, asn or Cys;
    X 15 glu, arg, lys or Aib;
    X 16 is Ser, arg or Aib;
    X 17 is Tyr or Aib;
    X 18 is Cys or none, or Cys-Z 1 Wherein Z is 1 1-3 amino acid residues;
    and when X 0 X is when it is absent 1 Gly, D-Ala, val, asn, arg or Gln modified for acetylation (Ac); when X is 0 X is not time-free 1 Is unmodified Gly, D-Ala, val, asn, arg or Gln.
  5. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide is set forth in SEQ ID No. 1 or 19.
  6. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide is as set forth in SEQ ID No. 5 or 46.
  7. A drug conjugate, wherein the drug conjugate has a structure according to formula II:
    (D) n -L-P (II)
    wherein D is the payload;
    l is a linker;
    p is a targeting peptide which is a polypeptide according to any one of claims 1 to 6;
    n is a positive integer not less than 1, preferably n=1 to 4, most preferably n=1 or 2.
  8. The drug conjugate of claim 7, wherein the payload is selected from the group consisting of: docetaxel, paclitaxel, or cabazitaxel, or derivatives thereof.
  9. A pharmaceutical composition, comprising: (a) the drug conjugate of claim 7; and (b) a pharmaceutically acceptable carrier.
  10. Use of a polypeptide according to any one of claims 1 to 6, or a drug conjugate according to claim 7, in the manufacture of a medicament for the treatment of a tumor or cancer.
  11. A method of preparing the polypeptide of claim 1, the method comprising the steps of:
    (1) Synthesizing linear polypeptide by adopting a solid-phase synthesis method; and
    (2) Alternatively, the linear polypeptide obtained in step (1) is cyclized with a cyclizing reagent to obtain a cyclic peptide.
  12. A method of preparing the drug conjugate of claim 7, the method comprising the steps of:
    (A) Providing a linker and a payload, reacting the linker with the payload in an organic solvent to obtain an intermediate;
    (B) Activating the intermediate with an activating reagent;
    (C) Subjecting the polypeptide of any one of claims 1-6 to an activated ester reaction with the intermediate of step (a), thereby obtaining the drug conjugate;
    (D) Purifying the drug conjugate of step (C).
  13. A method of detecting a SORT1 protein in a sample, the method comprising the steps of: (i) contacting the sample with the polypeptide of claim 1; (ii) Detecting whether a polypeptide-SORT 1 protein complex is formed, wherein the formation of the complex indicates the presence of SORT1 protein in the sample.
  14. A kit for detecting a SORT1 protein in a sample, the kit comprising the polypeptide of claim 1.
  15. A method of treating a tumor or cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of the drug conjugate of claim 7 or the pharmaceutical composition of claim 9.
CN202380010846.0A 2022-03-01 2023-02-28 Polypeptide compound for SORT1 and drug conjugate thereof Pending CN117098770A (en)

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