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CN119868576A - Intermediate for preparing conjugate of antibody and drug, preparation method and application thereof - Google Patents

Intermediate for preparing conjugate of antibody and drug, preparation method and application thereof Download PDF

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Publication number
CN119868576A
CN119868576A CN202510050328.2A CN202510050328A CN119868576A CN 119868576 A CN119868576 A CN 119868576A CN 202510050328 A CN202510050328 A CN 202510050328A CN 119868576 A CN119868576 A CN 119868576A
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antibody
integer
mmae
group
formula
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王春河
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Shanghai Youmaibo Biotechnology Co ltd
Dashi Pharmaceutical Guangdong Co ltd
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Shanghai Youmaibo Biotechnology Co ltd
Dashi Pharmaceutical Guangdong Co ltd
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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
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K47/68—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
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    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
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Abstract

The application relates to an intermediate for preparing an antibody and drug conjugate, and a preparation method and application thereof. The intermediate for preparing the antibody and drug conjugate has a structure shown in the following formula I. The intermediate designed in the application for preparing the conjugate of the antibody and the drug can effectively couple the monomethyl auristatin and other compounds to the antibody with high DAR value. Compared with the similar antibody and drug conjugate in the prior art, the antibody and drug conjugate prepared by adopting the intermediate provided by the application has stronger killing activity on tumor cells, higher hydrophilicity and thermal stability, better drug substitution performance and lower toxic and side effects, so that the intermediate and the antibody and drug conjugate provided by the application have wide application prospects.

Description

Intermediate for preparing conjugate of antibody and drug, preparation method and application thereof
Technical Field
The invention belongs to the field of medical biology, and particularly relates to an intermediate for preparing an Antibody and Drug Conjugate (ADC), and a preparation method and application thereof.
Background
Typically, antibody Drug Conjugates (ADCs) are one type of tumor targeted therapy, where antibodies targeting tumor antigens are covalently coupled to cytotoxic payloads via chemically synthesized linkers. The choice of drug linker and payload plays a key role in the therapeutic efficacy and safety of the ADC.
Currently, most ADC molecules that enter the clinical development stage utilize cleavable linkers, including acid labile linkers, peptide linkers, and disulfide linkers. Cleavable dipeptide linkers formed by valine-citrulline (Val-Cit, VC) linked to self-cleaving spacer benzyl hydroxyl (PAB) are widely used in the clinical development of ADCs. However, because of the relatively strong hydrophobicity of the VC linker, when combined with the moderately highly hydrophobic payload, it forms a "surfactant-like" structure with the more hydrophilic moiety in the antibody that tends to aggregate, especially when DAR values are higher than 4. In vivo, aggregation of ADC tends to result in rapid plasma clearance, reduced efficacy and increased toxicity, particularly for high DAR ADC molecules.
Monomethyl Auristatin E (MMAE) inhibits tubulin polymerization to exert potent mitotic inhibition, and is widely used as the payload (payload) of ADC drugs, although effective in many refractory solid tumors, due to its poor water solubility, existing linkers exert optimal therapeutic effects when coupled with MMAE at DAR values of-4, while at higher DAR molecules aggregate and lead to rapid clearance. Due to the average DAR value of 4, problems are caused in the aspects of molecular uniformity and batch-to-batch stability, quality control is not facilitated, and the same problems are also caused in other medium-high hydrophobicity compounds.
In view of the foregoing, there is a need in the art to develop new stable linkers and payloads that result in an ADC that is effectively targeted to achieve the highest therapeutic index.
Human trophoblastic cell surface antigen 2 (Trop-2) is highly expressed in various human solid tumors, such as breast cancer, lung cancer, colon cancer, bladder cancer, pancreatic cancer and the like, but has different levels of expression in key normal tissues such as skin, mucous membrane and the like, and is a challenge whether the human trophoblastic cell surface antigen can be prepared as an ADC target. However, the key issue in the success of ADC technology in targeted therapy of Trop-2 positive tumors is to screen the best combination of linker and payload to achieve the highest therapeutic index.
Disclosure of Invention
The invention aims to provide an ADC molecule which contains a medium-high hydrophobicity compound such as monomethyl auristatin and the like as a payload and can be coupled into a high DAR value, and an intermediate (namely a compound in a formula I) for efficiently coupling the compound such as monomethyl auristatin and the like with an antibody.
In a first aspect of the invention, there is provided a compound of formula I as shown below for use in the preparation of an antibody-drug conjugate,
In the formula I, the compound (I),
Z 1 is a linker moiety (moeity) for linking to an antibody;
m 1 is an integer from 1 to 10;
m 2 is an integer from 10 to 50;
X represents a group derived from a small molecule cytotoxic drug.
In another preferred embodiment, Z 1 is a substituted or unsubstituted maleimide group.
In another preferred embodiment, the Z 1 is linked to the antibody through the 1-, 3-or 4-position of the 5-membered ring of maleimide.
In another preferred embodiment, the linkage includes linkage to a sulfhydryl group on an antibody, linkage to an epsilon amino group on an antibody (e.g., epsilon amino group of lysine), linkage to selenium cysteine on an antibody, or a combination thereof.
In another preferred embodiment, the Z 1 is a chemical linkage that is generated by reaction with a naturally occurring reactive group on the antibody or an artificially introduced non-natural reactive group.
In another preferred embodiment, the Z 1, upon attachment to an antibody, forms a chemical attachment structure selected from the group consisting of:
(a) A chemical connection structure formed by coupling reaction of sulfhydryl group on the antibody and maleimide;
(b) A chemical connection structure formed by coupling reaction of sulfhydryl and dibromomaleimide on the antibody;
(c) An epsilon-amino group of lysine on the antibody reacts with an active amine group on a linker to form a chemical connection structure;
(d) Coupling reaction of lysine on the antibody and derivative containing maleimide to form chemical connecting structure;
(e) Coupling reaction of lysine and iodoacetamides on the antibody to form a chemical connecting structure;
(f) Coupling reaction of lysine on the antibody based on iminothiolane to form a chemical connecting structure;
(g) Coupling reaction of lysine on the antibody based on pyridine disulfide to form a chemical connecting structure;
(h) The chemical connection structure is formed by site-directed coupling with artificially introduced selenium cysteine on the antibody;
(i) Any combination of the above;
In another preferred embodiment, in formulas (a) and (b) above, m 3 is a positive integer from 1 to 8 (preferably from 2 to 6).
In another preferred embodiment, in formulas (d) and (e) above, the spacer is selected from- (CH 2) m4-、-(CH2O)m4 -, wherein m4 is an integer from 0 to 10, preferably an integer from 1 to 6.
In another preferred embodiment, the compound of formula I is a compound of formula Ia:
In the formula Ia, the amino acid sequence of the formula,
M 1 is an integer from 1 to 10;
m 2 is an integer from 10 to 50;
X is a hydrophobic compound such as monomethyl auristatin (i.e. a group of the compound such as monomethyl auristatin after losing H atom).
In another preferred embodiment, m 1 is 1-8, more preferably 4,5, most preferably 5.
In another preferred embodiment m 2 is 12-40, more preferably 16-36, most preferably 24.
In another preferred embodiment, X is a monomethyl auristatin E (MMAE) -derived group, i.e
In another preferred embodiment, the compound of formula I has the structure shown in formula III:
The definition of MMAE is as described above.
In a second aspect of the invention there is provided an antibody to drug conjugate (ADC) formed by the conjugation of a compound of formula I according to the first aspect with an antibody.
In another preferred embodiment, the antibody-drug conjugate has a structure represented by formula II:
Ab-(L-X)n II
In the formula II, the compound of the formula I,
Ab represents an antibody which,
L is a divalent linker as shown below:
Wherein Z 1' is the radical remaining after removal of one hydrogen from the Z 1 radical;
n is the average number of couplings of the drug to the antibody;
The definition of m 1 and m 2 is as described above.
In another preferred embodiment, n is an integer or non-integer >0, preferably 1.ltoreq.n.ltoreq.15, e.g. n is an integer or non-integer from 5 to 10, preferably from 6 to 9, more preferably from 7 to 8.
In another preferred embodiment, the antibody is selected from the group consisting of chimeric antibodies, bivalent or bispecific molecules, diabodies, triabodies and tetrabodies.
In another preferred embodiment, the antibody may be an antibody to any target, for example, the target is selected from the group consisting of:
Trop-2、PD-L1、CTLA4、OX40、EpCAM、EGFR、EGFRVIII,HER2、PTPN2、VEGF、CD11a、CD19、CD20、CD22、CD25、CD30、CD33、CD40、CD56、CD64、CD70、CD73、CD74、CD79、CD105、CD138、CD174、CD205、CD227、CD326、CD340、MUC16、GPNMB、PSMA、Cripto、ED-B、TMEFF2、EphB2、Apo2、NMP179、p16INK4A、Nectin-4、B7H3、EMA、CEA、MIB-1、GD2、APRIL Receptors, folate receptors, mesothelin inhibitors, MET, TM4SF1, IL13 receptors, and the like.
In another preferred embodiment, in the antibody-drug conjugate, m 1 is an integer from 1 to 8 in the divalent linker L.
In another preferred embodiment, in the antibody-drug conjugate, m 2 is an integer of 20, 21, 22, 23, 24, 25, 26 in the bivalent linker L.
In another preferred embodiment, in the antibody-drug conjugate, m 1 is 4 and m 2 is 24 in the bivalent linker L.
In another preferred embodiment, in the antibody-drug conjugate, m 1 is 5 and m 2 is 24 in the bivalent linker L.
In another preferred embodiment, the antibody is hRS7.
In another preferred embodiment, the amino acid sequence of the light chain variable region of the antibody hRS7 comprises the sequence shown in SEQ ID No.:1 or is shown in SEQ ID No.:1 and/or the amino acid sequence of the heavy chain variable region of the antibody hRS7 comprises the sequence shown in SEQ ID No.:2 or is shown in SEQ ID No.: 2.
In another preferred embodiment, the antibody is hRS7 and in the L, Z 1' is the residue of maleimide after removal of one hydrogen, m 1 is 5, m 2 is 24, n is an integer between 1 and 15.
In a third aspect the present invention provides a pharmaceutical composition comprising (a) an antibody and drug conjugate according to the second aspect, and (b) a pharmaceutically acceptable carrier.
In a fourth aspect, the invention provides the use of an antibody as described in the second aspect and a drug conjugate or a pharmaceutical composition as described in the third aspect in the manufacture of a medicament for the treatment of a tumour or immune disease.
In another preferred embodiment, the tumor is selected from the group consisting of Trop-2 targeted tumors, HER2 targeted tumors, and EGFR targeted tumors.
In another preferred embodiment, the tumor is a Trop-2 targeted tumor and is selected from the group consisting of breast cancer, lung cancer, colon cancer, bladder cancer, and pancreatic cancer.
In a fifth aspect of the invention, there is provided a method of preparing an antibody-drug conjugate according to the second aspect, the method comprising the steps of:
(1) Providing a reaction system, wherein the reaction system comprises an antibody to be coupled and the compound of the formula I in the first aspect;
(2) In the reaction system, the antibody and the compound of the formula I are subjected to a coupling reaction, so that the antibody and drug conjugate of the second aspect is prepared.
In another preferred embodiment, the pH of the reaction system is from 6.0 to 8.0, preferably from 7.0 to 8.0.
In another preferred embodiment, the molar ratio of the antibody to the compound of formula I (or hydrophobic compound such as monomethyl auristatin) is 1:1-1:20, preferably 1:6-1:15, more preferably 1:8-1:12.
In another preferred embodiment, the reaction time is 1 to 48 hours, preferably 3 to 36 hours.
In another preferred embodiment, the coupling includes site-directed coupling and random coupling.
In another preferred embodiment, the fixed-point coupling comprises two coupling modes of K-Lock and C-Lock. In the K-Lock coupling mode, VK-X is coupled to a lysine (K) residue in the antibody sequence, and in the C-Lock coupling mode, VK-X is coupled to a cysteine (C) residue in the antibody sequence.
In a sixth aspect of the present invention, there is provided a compound represented by formula IV
Wherein W 1 is selected from H, fmoc-
W 2 is selected from H, trt-,Wherein when W 1 isWhen W 2 is notOr when W 2 isWhen W 1 is not
X, m 1、m2、Z1 are defined above.
According to a seventh aspect of the present invention, there is provided a process for the preparation of a compound of formula IV according to the sixth aspect, comprising the steps of:
(S1) reacting the compound 1 with X-H in an inert solvent in the presence of a base to obtain a compound of formula IV;
x, W 1、W2 are defined above.
In another preferred embodiment, in step (S1), the inert solvent is DMF.
In another preferred embodiment, in step (S1), the base is DIEA.
In an eighth aspect of the invention, there is provided the use of a compound of formula I as described in the first aspect for the preparation of an antibody and drug conjugate.
In a ninth aspect of the invention there is provided a method of treating or preventing a tumour, the method comprising the step of administering to a subject in need thereof an antibody as described above and a drug conjugate or a pharmaceutical composition as described above.
In another preferred embodiment, the subject is a mammal, including a human.
In another preferred embodiment, the antibody and drug conjugate are administered at a dose of 0.1 to 60 mg/kg, preferably 0.5 to 15 mg/kg, more preferably 1 to 10 mg/kg.
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.
Advantageous effects
Compared with the prior art, the invention has the main beneficial effects that:
(1) The compounds of formula I of the present invention are effective in coupling monomethyl auristatin and like compounds to antibodies at high DAR values, whereas conventional Val-Cit-PAB linkers are not.
(2) The compound shown in the formula I has better hydrophilicity.
(3) The antibody and drug conjugates resulting from the compounds of formula I of the present invention have optimized DAR values and loadings, resulting in a larger therapeutic window for the ADCs of the present invention.
(4) The antibody and drug conjugate provided by the invention has the activity of obviously killing tumor cells.
(5) The invention develops the ADC containing the X molecule which can be efficiently coupled and stabilized with the antibody (such as hRS7 antibody) for the first time, and the ADC has a synergistic therapeutic effect and can remarkably enlarge the killing activity of X on tumor cells.
(6) The compounds of formula I of the present invention can be conjugated to a variety of different antibodies, and the resulting antibody-drug conjugates have optimized DAR values, thereby providing the ADCs of the present invention with greater hydrophilicity and thermal stability.
(7) The antibody conjugate has higher drug effect.
(8) The antibody and drug conjugate provided by the invention has better drug substitution performance and lower toxic and side effects.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address specific conditions in the examples below, is generally followed by conventional conditions, such as those described in Sam brook et al, molecular cloning, laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or by the manufacturer's recommendations. Percentages and parts are weight percentages and parts unless otherwise indicated.
Drawings
FIG. 1 shows the molecular structures of VK1 (Val-Lys-PAB), VK2 (PEG 4-Val-Lys-PAB), VC (Val-Cit-PAB) linker and hRS7-VK1-MMAE linker, b. Shows the preparation conditions and detection of anti-Trop-2-ADC, c. RP-HPLC analysis of drug to antibody ratio (DAR) in different ADC molecules, d. HIC-HPLC analysis of hydrophobicity of different ADC molecules, e.SEC-HPLC analysis of thermal stability of different ADC molecules, f. HIC-HPLC and SEC-HPLC analysis results are summarized.
FIG. 2 shows changes in body weight of mice treated with hRS7-VK-MMAE and hRS7-VC-MMAE, liver toxicity of hRS7-VK-MMAE and hRS7-VC-MMAE, and efficacy of c and d.hRS7-VK-MMAE and hRS7-VC-MMAE on the model of transplanted tumor.
FIG. 3 affinity of hRS7-VK-MMAE and hRS7-VC-MMAE for human, monkey and murine TROP-2. Antigen expression levels in different tumor cell lines were examined by flow-through techniques, c.killing effects of hRS7-VK-MMAE and hRS7-VC-MMAE on different tumor cell lines, d.endocytic levels of hRS7-VK-MMAE and hRS 7-VC-MMAE.
FIG. 4 is a graph of the minimum dose of hRS7-VK1-MMAE to MDA-MB-468 tumor cell transplantation tumor, tumor growth inhibition and mouse weight, b.hRS7-VK1-MMAE to BxPC-3 tumor cell transplantation tumor, tumor growth inhibition and mouse weight, c.hRS7-VK1-MMAE to NCI-N87 tumor cell transplantation tumor, tumor growth inhibition and mouse weight.
FIG. 5 shows the drug metabolism of hRS7-VK1-MMAE in mice, b.hRS7-VK1-MMAE in cynomolgus monkeys, c.different doses of hRS7-VK1-MMAE in mice, d.different doses of hRS7-VK1-MMAE in total antibody concentration in plasma of mice.
Detailed Description
Through extensive and intensive studies, the present inventors have conducted extensive screening to obtain a highly potent, stable and hydrophobic optimized compound of formula I, which can be used for the preparation of targeted Antibody and Drug Conjugates (ADCs). The incorporation of the compound of formula I in the ADC can solve the problem of coupling X as the ADC payload with high DAR values. Experimental results show that the ADC molecules using the compound of the formula I and the antibody show better drug effect and safety in vitro and in vivo under the condition of DAR 8. The present invention has been completed on the basis of this finding.
Experiments show that the compound of the formula I is a novel stable linker and payload, and is particularly suitable for efficiently coupling drug molecules (such as MMAE, taxol and other compounds) which are difficult to form high DAR value ADC with antibodies, so that stable and effective high DAR value ADC is formed. The ADC prepared based on the intermediate has targeting property, can realize excellent therapeutic index, and can obviously reduce toxic and side effects of the ADC on normal cells or tissues.
Terminology
"DAR value" refers to the ratio of drug to antibody (drug antibody ratio).
In the present invention, when the "hydrophobic compound such as monomethyl auristatin" is used as a part of other compounds, it is a group such as monomethyl auristatin, that is, a group of compounds such as monomethyl auristatin after the compound loses an H atom.
Active ingredient
"Compounds of formula I", "antibody and drug conjugate intermediates", "linker and payload combinations", "linkers and payloads" are used interchangeably.
The present invention provides a compound of formula I having the structure
In the formula,
Z 1、m1、m2, MMAE are as defined above.
In another preferred embodiment, m 1 is an integer from 1 to 10;
X is hydrophobic compound such as monomethyl auristatin.
In another preferred embodiment m 1 is an integer from 2 to 10, such as from 2 to 8, more preferably 4, 5, most preferably 5.
In another preferred embodiment, m 2 is 10 to 50, such as m 2 is 12 to 40, more preferably 15 to 36, most preferably 24.
Preferably, m 1 is an integer from 2 to 10 (i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10);
Preferably, m 2 is an integer from 15 to 30 (i.e., 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30);
preferably, the method comprises the steps of, MMAE is a monomethyl auristatin compound.
Preferably, the compound of formula I is a compound of formula Ia:
wherein m 1、m2 and X are as defined above.
Preferably, the structure of the compound of formula I is as shown in formula III
Wherein MMAE is as defined above.
Antibodies to
As used herein, the term "antibody" or "immunoglobulin" is an iso-tetralin protein of about 150000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H), having identical structural features. Each light chain is linked to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end, the constant region of the light chain being opposite the first constant region of the heavy chain and the variable region of the light chain being opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.
As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH publication No.91-3242, vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.
The "light chain" of a vertebrate antibody (immunoglobulin) can be classified into one of two distinct classes (called kappa and lambda) depending on the amino acid sequence of its constant region. Immunoglobulins can be assigned to different classes based on the amino acid sequence of their heavy chain constant region. There are mainly class 5 immunoglobulins IgA, igD, igE, igG and IgM, some of which can be further divided into subclasses (isotypes) such as IgG1, igG2, igG3, igG4, igA and IgA2. The heavy chains corresponding to different classes of immunoglobulins are constantly distinguished and are designated α, δ, ε, γ, and μ, respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
In general, the antigen binding properties of antibodies can be described by 3 specific regions located in the heavy and light chain variable regions, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of which are relatively conserved and do not directly participate in the binding reaction. These CDRs form a loop structure, the β -sheets formed by the FR therebetween are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody.
The invention includes not only whole antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.
In the present invention, the antibodies of the invention also include conservative variants thereof, meaning that up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature to the amino acid sequence of the antibodies of the invention to form a polypeptide. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.
Table A
Initial residues Representative substitution Preferred substitution
Ala(A) Val;Leu;Ile Val
Arg(R) Lys;Gln;Asn Lys
Asn(N) Gln;His;Lys;Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
G1n(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro;Ala Ala
His(H) Asn;Gln;Lys;Arg Arg
Ile(I) Leu;Val;Met;Ala;Phe Leu
Leu(L) Ile;Val;Met;Ala;Phe Ile
Lys(K) Arg;Gln;Asn Arg
Met(M) Leu;Phe;Ile Leu
Phe(F) Leu;Val;Ile;Ala;Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr;Phe Tyr
Tyr(Y) Trp;Phe;Thr;Ser Phe
Val(V) Ile;Leu;Met;Phe;Ala Leu
The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique such as amplification by PCR or screening of a genomic library. In addition, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.
At present, it is already possible to obtain the DNA sequences encoding the antibodies of the invention (or fragments or derivatives thereof) described, entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.
The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.
The host cell may be a prokaryotic cell, such as a bacterial cell, or a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell.
Typically, the transformed host cell is cultured under conditions suitable for expression of the antibodies of the invention. The antibodies of the invention are then purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, using conventional separation and purification means well known to those skilled in the art.
The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined using immunoprecipitation or in vitro binding assays, such as Radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA). The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis by Munson et al, anal. Biochem.,107:220 (1980).
The antibodies of the invention may be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.
In another preferred embodiment, the antibody is antibody hRS7.
In another preferred embodiment, the light chain variable region (V-Kappa) amino acid sequence of antibody hRS7 is the amino acid sequence shown as SEQ ID NO. 1 (DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGV PDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQ).
In another preferred example, the heavy chain variable region (VH) amino acid sequence of antibody hRS7 is an amino acid sequence as set forth in SEQ ID NO. 2 (QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTY TGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWG QGSLVTVSS).
Antibody and drug conjugate and preparation thereof
As used herein, the terms "antibody of the invention and drug conjugate", "conjugate of the invention" or "ADC of the invention" are used interchangeably to refer to an antibody-drug conjugate having the structure shown in formula II.
In the present invention, the antibody-drug conjugate has a structure represented by formula II:
Ab-(L-X)n II
Wherein,
Ab means an antibody, which may be any of the classes of antibodies described above (e.g., chimeric antibodies (e.g., humanized murine antibodies), bivalent or bispecific molecules (e.g., bispecific antibodies), diabodies, triabodies, and tetrabodies);
X, L, n, m 1、m2 are defined above.
Preferably, in formula II, n is an integer or non-integer from 4 to 12, preferably n is an integer or non-integer from 5 to 10, preferably an integer or non-integer from 6 to 9, more preferably an integer or non-integer from 7 to 8.
Preferably, in formula II above, m 1 is an integer of 2,3, 4, 5, 6, and m 2 is an integer of 20, 21, 22, 23, 24, 25.
Preferably, in formula II above, m 1 is 5;m 2 and 24.
Coupling method
The invention provides a coupling method, which couples a compound shown in the formula I to an antibody, and greatly improves the killing power of the antibody to tumor cells on the basis of not changing the affinity of the antibody.
Typical coupling modes suitable for use in the present invention include C-Lock coupling modes. In the C-Lock coupling mode, the drug molecule is coupled to a cysteine (C) residue in the antibody sequence.
Preferably, the preparation method of the antibody and drug conjugate of the invention is as follows:
An amount (e.g., 0.5 mg) of antibody is pipetted, an amount (e.g., 8-fold material equivalents) of Tris (2-carboxyethyl) phosphine (Tris (2-carboxyethyl) phosphine, TCEP) is added and reacted for a period (e.g., 2.5 hours) at a suitable temperature, e.g., (37 ℃). The compound of formula I (e.g., 15-fold equivalent of substance) is then added directly to perform a coupling reaction (e.g., an overnight reaction at 4 ℃ for, e.g., 17 hours), thereby obtaining the ADC of the invention. Finally, the buffer was replaced by PBS at pH7.0 through an Amicon Ultra 4 10K ultrafiltration tube.
Preferably, the preparation method of the antibody and drug conjugate of the invention is as follows:
An amount (e.g., 0.5 mg) of antibody is pipetted, an amount (e.g., 2-fold material equivalents) of Tris (2-carboxyethyl) phosphine (Tris (2-carboxyethyl) phosphine, TCEP) is added and reacted for a period (e.g., 2.5 hours) at a suitable temperature (e.g., 37 ℃). After the reaction was completed, the reaction mixture was concentrated to 0.5 ml or less by centrifugal ultrafiltration, topped up with a coupling solution (75mM NaAc,pH6.5,1mM DTPA,10%DMSO), and concentrated to 0.5 ml or less by centrifugation, and repeated three times. The compound of formula I (e.g., 8-fold equivalent of substance) is then added directly to perform a coupling reaction (e.g., an overnight reaction at 4 ℃ for, e.g., 17 hours), thereby obtaining the ADC of the invention.
Preferably, the preparation method of the antibody and drug conjugate of the invention is as follows:
An amount (e.g., 0.5 mg) of antibody is pipetted, an amount (e.g., 3 times the equivalent of substance) of Dithiothreitol (DTT) is added, and the reaction is carried out for a period of time (e.g., 2 hours) at a suitable temperature (e.g., 37 ℃). After the reaction was completed, the reaction mixture was concentrated to 0.5 ml or less by centrifugal ultrafiltration, topped up with a coupling solution (75mM NaAc,pH6.5,1mM DTPA,10%DMSO), and concentrated to 0.5 ml or less by centrifugation, and repeated three times. The compound of formula I (e.g., 10-fold equivalent of substance) is then added directly to perform a coupling reaction (e.g., an overnight reaction at 4 ℃, e.g., 17 hours), to obtain the ADC of the invention.
Pharmaceutical compositions and methods of administration
The invention also provides pharmaceutical compositions comprising the ADCs of the invention, and methods of treating mammalian diseases using the ADCs of the invention. Preferably, the disease is a tumor, including a tumor with a specific target, such as Trop-2 targeted tumor, her2 targeted tumor, EGFR targeted tumor.
The invention also provides application of the antibody and drug conjugate in preparing anti-tumor drugs.
In the present invention, the pharmaceutical composition comprises an effective amount of an ADC according to the invention (as active ingredient), together with at least one pharmaceutically acceptable carrier, diluent or excipient. The active ingredient is typically admixed with, or diluted with, an excipient at the time of preparation. When the excipient acts as a diluent, it may employ a solid, semi-solid, or liquid material as the vehicle, carrier, or medium for the active ingredient. Thus, the composition may be a solution, a sterile injectable solution or the like.
Suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water and the like, and the formulation may also include wetting agents, emulsifying agents, preservatives (e.g., methyl and propyl hydroxybenzoates) and the like. The antitumor agent may be formulated in a unit or multiple dosage form, each containing a predetermined amount of the ADC of the present invention calculated to produce the desired therapeutic effect, and a suitable pharmaceutical excipient.
The antitumor agent may be administered by conventional routes including, but not limited to, intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, topical administration, and the like.
When the drug is used, a safe and effective amount of the antibody and drug conjugate is applied to a human, wherein the safe and effective amount is preferably in the range of 0.5-50 mg/kg body weight, more preferably 1-10 mg/kg body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are well within the skill of the skilled practitioner.
In addition, the conjugates of the invention can be used in combination with other therapeutic agents, including, but not limited to, various cytokines such as TNF, IFN, IL-2, etc., various tumor chemotherapeutic agents such as 5-FU, methotrexate, etc., agents that affect nucleic acid biosynthesis, alkylating agents such as nitrogen mustard, cyclophosphamide, etc., agents that interfere with transcription processes to prevent RNA synthesis such as doxorubicin, actinomycin D, etc., agents that affect protein synthesis such as vincristine, camptothecins, and certain hormonal agents, etc.
Abbreviations:
Fmoc 9-fluorenylmethoxycarbonyl
Trt trityl radical
DIEA N-ethyl-N-isopropyl-2-amine
DMF dimethylformamide
TCEP tris (2-carboxyethyl) phosphine hydrochloride
Materials and methods
(A) Cell lines and reagents
MDA-MB-468, bxPC-3, NCI-H2452 and HT-29 were supplied by the China Shanghai cell line Bank.
DMEM was supplemented with 10% new born calf serum (Thermo FISHER SCIENTIFIC, waltherm, ma). Cells were incubated at 37 ℃ in a humid atmosphere of 5% co 2. PE-labeled goat anti-human IgG markers were purchased from Biolegend (san diego, usa). Humanized Trop-2 antibody hRS7 was prepared according to U.S. patent 9931417B 2.
Cell Counting Kit-8 (CCK 8) was purchased from Dojindo Laboratories (Tokyo, japan). Matrigel (# 354234) was purchased from BD Biosciences (san jose, usa). MMAE, m-PEG24 acid and MC-OSu are supplied by Levena Biopharma (Suzhou, china). Anti-LAMP-1 (# 9091), anti-Clathrin (# 4796), anti-GM 130 (# 12480) and 555-Anti-rabbit IgGF (ab') 2 fragments were purchased from CELL SIGNALING Technology (Trask us lahn denfos (LANE DANVERS)), igG H & L from Abcam track (Shanghai) Company ltd, human Trop-2 recombinant protein (6×histag) from Sino Biological (beijing in china), formaldehyde from Polysciences, inc. (catalog No. 18814), normal goat serum #5425 from CELL SIGNALING Technology (us, TRASK LANE DANVERS), proLongTMLive Anti-fade reagent (#p 36974) and TMB substrate from Thermo FISHER SCIENTIFIC inc. TCEP (BondBreakerTM) from Pierce (northford in il in us) and other chemicals are products of (MERCK KGAA).
(B) Preparation of ADC and detection of DAR value
The reduction was performed by adding 6 molar equivalents of TCEP (1 mg/mL) to 8mg/mL of an antibody solution containing 9% sucrose, 10mM acetate, 0.01% Tween-20, pH7.0 at 37℃for 2 hours. After reduction, 20 molar equivalents of linker payload were added per antibody thiol and incubated at 4 ℃ for more than 16 hours. The solution mixture was concentrated by centrifugation (4000 rpm,15 minutes, thermo Fisher ST40R TX 1) with AmiconUltracontainer (50,000MWCO,Millipore Corporation). The concentration of ADC was measured by UV detector (Nanodrop 100,Thermo Fisher Scientific Inc) at 280nm absorbance. DAR values of the final product were confirmed by Reverse Phase (RP) -HPLC and QTOF mass spectrometry.
(C) RP-HPLC analysis of DAR values for ADCs
RP-HPLC analysis was performed on 1260 Infinicity UHPLC (Agilent Technologies) under 1) Agilent 8 μm, 4.6X250 mm, 2) mobile phase A0.01% aqueous trifluoroacetic acid (TFA), 3) mobile phase B0.01% acetonitrile solution, 4) gradient procedure 0-3min (100% A), 3-25min (100% A-100% B), 25-30min (100% B), 5) column temperature 70℃and 6) sample loading 15ul (1 mg/mL). The degree of hydrophobicity of an ADC molecule is proportional to the number of payloads coupled and can be reflected by its retention time. Since the drug linker has ultraviolet absorbability, the average number of conjugated drug molecules per antibody molecule in the ADC can be calculated from the expression that the average number of conjugated drug molecules = (L0 peak area ratio x 0+l1 peak area ratio x 1+h0 peak ratio x 0+h1 peak ratio x 1+h2 peak ratio x 2+h3 peak ratio x 3)/100×2.
(D) SEC-HPLC analysis of ADC aggregates
SEC-HPLC methods were used to monitor the aggregation and degradation of ADCs. After 1 hour incubation at 60 ℃, samples were analyzed on Thermo MAbPac SEC-1 at 5 μm, (7.8X100 mm) P/N088460 using 1260HPLC system (Agilent). Mobile phase Phosphate Buffered Saline (PBS), 50mM sodium phosphate and 300mM sodium chloride, pH6.8. The column temperature was maintained at 26 ℃ during separation. The detector was set at 280nm. The flow rate was 0.7mL/min. Sample introduction amount was 15. Mu.l.
(e)SDS-PAGE
Samples were boiled in 1 Xloading buffer for 10 min, samples containing 5 μg of hRS7 or ADC were separated by SDS-PAGE (10% Bio-Rad Criterion Tris-HCl gel), then stained with Coomassie blue for 5 min, and detected with BioRad ChemiDoc MP.
(F) Detection of tumor cell Trop-2 expression level
Cells were incubated with 5 μg/mL hRS7 in ice-cold staining medium for 30min on ice and washed twice with ice-cold staining medium to remove unbound antibody. Cells were then stained with PE goat anti-human IgG (5 μg/mL) in ice cold staining medium for 30min and washed twice. Cells were examined by flow cytometry.
(G) ELISA detection of antigen binding to antibodies
96-Well immunoplates were coated with 2 μg/mL histidine-tagged antigen protein (Sino Biological inc.) at 4 ℃ overnight. Plates were washed, blocked with 1% casein, and then incubated with 10nM antibody or ADC for 1 hour at room temperature. After washing, anti-human kappa-HRP (Biolegend San Diego, USA) was added and incubated for 1 hour. After washing, TMB substrate was added and detected at 450nm using a SpectraMax M5e (Molecular Devices) microplate reader.
(H) Detection of ADC endocytic rate
To assess the endocytosis rate of the ADCs, MDA-MB-231 or BxPC-3 or HT-29 cells (1X 10 6) were incubated with samples of ADCs (3 μg/mL) in staining medium for 30 minutes on ice and washed twice, then placed in fresh complete medium. The humidification chamber at 37 ℃ initiates internalization. At the end of incubation, cells were washed twice with ice-cold staining medium and then stained with goat anti-human IgG-PE. The cells were analyzed by flow cytometry to detect the level of residual surface ADC.
(I) Cell viability assay
Cell viability was determined using cell counting kit 8 (CCK-8). Cells were seeded into 96-well plates at 3000-10000 cells/well. After overnight incubation, the drug was added at various concentrations. Plates were incubated in a humidity oven at 37 ℃,5% co 2 for 96 hours. CCK8 reagent was added and returned to the incubator until the untreated control cells had an absorbance at 450nm of greater than 1.0. Growth inhibition was measured as percent growth relative to untreated cells. Dose response curves were generated from the average of two or three determinations and IC 50 values were calculated using PRISM GRAPHPAD software.
(J) Detection of efficacy in mice
Female athymic BALB/c nude mice of five weeks of age were purchased from CHARLES RIVER Company (Shanghai, china) and each group was randomly divided into 5-6 mice. Animal handling and procedures were approved and performed as required by the Institutional Animal Care and Use Committee (IACUC) of Shanghai drug research institute, academy of sciences, china. All models were inoculated subcutaneously on the sides of mice. BxPC-3 cells were established by injecting 5X 10 6 cells mixed in Matrigel matrix. MDA-MB-468 cells were established by injecting 1X 10 7 cells suspended in Matrigel matrix. After the tumor volume reached about 200mm 3, tumor-bearing mice were randomly divided into treatment and control groups according to tumor volume. Each drug was administered intraperitoneally. Injected into mice at a dose of 3 or 10mg/kg (10 mL/kg). Tumor volume is defined as length×1/2×width×width. Percent inhibition was calculated by (1-mean tumor volume in treated group/mean tumor volume in control group) ×100%. Toxicity associated with the different treatment groups was assessed by monitoring weight loss.
(K) Dose tolerance assay
Dose tolerance assays were performed with BALB/c mice (5 mice per dose). The drug is administered intravenously in a single administration.
The injection dosages of hRS7-VK-MMAE and hRS7-VC-MMAE were 60 and 40mg/kg, respectively. Toxicity was assessed by observing mouse behavior, weight loss and survival. The weight change rate (%) was calculated as (weight-treatment precursor weight)/(treatment precursor weight) ×100%.
(L) Statistical analysis
PRISM GRAPHPAD version 6 was used for statistical analysis. Results are shown as mean ± SD. Statistical differences between control and experimental groups were calculated by student's t-test, with P <0.05 being considered significant differences.
Example 1
Preparation of antibodies
In this example, hRS7 antibodies were expressed and purified to obtain hRS7 antibodies. The method comprises the following steps:
The amino acid sequences of the hRS7 light and heavy chains were from us patent 9931417B2 and then reverse transcribed into cDNA sequences by Vector NTI software. The DNA sequence was synthesized and subcloned into the pcDNA3.1 vector and amplified in E.coli. Purified plasmids were transfected into HEK293 cells by PEI. Cells were then cultured in FreeStyle 293 expression medium in suspension. After 5 days of culture, cell culture supernatants were collected and antibodies were purified by protein a affinity. The amino acid sequence of the light chain variable region is SEQ ID NO. 1, and the amino acid sequence of the heavy chain variable region is SEQ ID NO. 2.
Example 2
Preparation of Compound VK-MMAE
The structure of the compound VK1-MMAE is shown below:
Reacting monomethyl auristatin E with the VK1 linker to form the compound VK1-MMAE, i.e
Mal-Val-Lys (PEG 24) -PAB-monomethyl Australian statin (VK 1-MMAE) by the following method:
Step 1 Synthesis of Compound 3 (Fmoc-Val-Lys (Trt) -PAB-MMAE)
Fmoc-Val-Lys (Trt) -PAB-PNP (purchased from Levena Biopharma, USA) and MMAE (purchased from Levena Biopharma, USA) were dissolved in anhydrous DMF and DIEA was added to the solution, the mixture was stirred at room temperature 22℃for 2 hours and the desired product Fmoc-Val-Lys (Trt) -PAB-MMAE was purified directly by reverse phase HPLC and freeze-dried to form compound 3 as a white powder.
Step 2 Synthesis of Compound 4 (Fmoc-Val-Lys-PAB-MMAE TFA salt)
Compound 3 from step 1 was dissolved in 10% TFA/DCM and stirred at room temperature for 20min, and after reaction the mixture was concentrated under reduced pressure and purified by reverse phase HPLC to give the TFA salt of compound 4, fmoc-Val-Lys-PAB-MMAE.
Step3 Synthesis of Compound 5
Compound 4, m-PEG24 acid and HATU from step 2 were dissolved in DMF, DIEA was added and stirred at room temperature for 30 min to give product 5a.
Diisopropylamine was added to the reaction mixture, stirred at room temperature for 3 hours, the mixture was concentrated and purified by reverse HPLC after reaction, and waxy compound 5 was obtained after lyophilization.
Step 4 Synthesis of Compound 6 (Mal-val-Lys (m-PEG 24) -PAB-MMAE, VK 1-MMAE)
The compound 5, MC-OSu or m-PEG 4-acid obtained in step 3 and HATU were dissolved in DMF, DIEA was added and stirred for 20 minutes. After purification by reverse HPLC and freeze-drying, the desired compound 6, mal-Val-Lys-PAB-MMAE (VK 1-MMAE) or Mal-PEG4-Val-Lys-PAB-MMAE (VK 2-MMAE) was finally obtained.
Example 3
Preparation of ADC
The VK-MMAE prepared in example 2 was coupled to the antibody prepared in example 1 by a coupling reaction to form the corresponding ADC.
Stable MMAE-loaded ADC molecules with DAR value 8, designated hRS7-VK1-MMAE and hRS7-VK2-MMAE (fig. 1 a), were successfully prepared via VK linker.
For comparison purposes, a Trop-2-ADC with a DAR value of 8 for MMAE payload, hRS7-VC-MMAE (fig. 1 a), was also prepared.
SDS-PAGE of hRS7-VK2-MMAE with DAR values from 0 to 8 under reducing conditions is shown in FIG. 1 b. RP-HPLC detection (C in FIG. 1) and HIC-HPLC hydrophobicity detection (d in FIG. 1) of the Trop-2-ADCs of three DAR8 prepared with the VK or VC linkers demonstrated good homogeneity of the product, and even incubation at 60℃for 1 hour (e in FIG. 1) showed no severe aggregation and degradation, indicating that pegylation of the linkers could significantly increase the hydrophobicity and stability of the ADC molecules.
Comparative example 1
Preparation of control ADC molecules
A Trop-2 targeted hRS7-VC-MMAE molecule was prepared by hydrophobic VC linker (FIG. 1 a). However, this molecule partially precipitates out of solution during the preparation process, and thus its therapeutic ability and safety cannot be evaluated.
Thus, it can be seen that binding the hydrophobic linker to the hydrophobic payload compromises the stability of the ADC molecule, leading to an increase in aggregates and precipitation.
Comparative example 2 study of tolerance of hRS7-VK-MMAE
The safety of relatively high doses of hRS7-VK1-MMAE, hRS7-VK2-MMAE in BALB/c mice was studied by acute toxicity experiments with single intravenous administration in mice. Compared to hRS7-VK2-MMAE and hRS7-VC-MMAE, hRS7-VK1-MMAE Maximum Tolerated Dose (MTD). At a single dose of 60mg/kg, hRS7-VK1-MMAE showed less signs of toxicity or weight loss, hRS7-VK2-MMAE caused some weight loss in mice compared to it, while hRS7-VC-MMAE caused severe adverse reactions and weight loss exceeding 15% (fig. 2 a). Meanwhile, hRS7-VK1-MMAE and hRS7-VK2-MMAE did not show severe liver injury, while hRS7-VC-MMAE caused more severe liver injury (b in FIG. 2).
Thus, hRS7-VK1-MMAE has better safety and tolerability than hRS7-VK2-MMAE and hRS 7-VC-MMAE.
Comparative example 3 inhibition of tumor cells specifically expressing Trop-2 in vivo by hRS7-VK1-MMAE and hRS7-VK2-MMAE
On day 8, mice vaccinated with BxPC-3 cells (n=5 per group) were treated with 1mg/kg ADC or naked antibody, and tumors were measured every two days. On day 25, a two-tailed t-test was used to evaluate statistical significance between the treatment and control groups. * P <0.00001, hRS7-VK1-MMAE/hRS7-VK2-MMAE compared to hRS7 treatment group, no significant differences (ns), hRS7-VK1-MMAE compared to hRS7-VK 2-MMAE. Data = mean ± SD, the results are shown in fig. 2 c.
On day 8, mice vaccinated with BxPC-3 cells (n=5 per group) were treated with 0.5mg/kg with ADC or control (PBS) and tumor volumes were measured every two days. The two-tailed t-test was used to evaluate statistical significance between the treatment and control groups. * P <0.0001, comparing hRS7-VK1-MMAE/hRS7-VK2-MMAE with hRS7, comparing hRS7-VK1-MMAE with hRS7-VK2-MMAE without significant difference (ns), comparing hRS7-VK1-MMAE with hRS7-VK2-MMAE, comparing hRS7-VK1-MMAE/hRS7-VK2-MMAE with hRS 7-VC-MMAE. Data = mean ± SEM, results are shown in fig. 2 d, demonstrating that hRS7-VK1-MMAE is comparable to hRS7-VK2-MMAE in vivo efficacy, but hRS7-VK1-MMAE is better tolerated (fig. 2 a).
Example 4 inhibition of hRS7-VK1-MMAE on tumor cells specifically expressed by Trop-2 in vitro
(A) The hRS7-VK1-MMAE molecule with DAR value 8 has good affinity to human, monkey Trop-2 protein (6 xhis tag), but does not bind to mouse Trop-2, as shown in fig. 3 a.
(B) The three tumor cell lines showed different levels of Trop-2 expression (b in fig. 3).
(C) The antitumor efficacy of hRS7-VK1-MMAE in different tumor cell lines was examined by CCK-8. In tumor cells with different expression levels of Trop-2, hRS7-VK1-MMAE has remarkable killing effect on BxPC-3 and MDA-MB-468 with high expression of Trop-2, but has no killing effect on HT-29 with low expression of Trop-2, as shown in figure 3 c.
(D) Cancer cells (1X 10 6 cells/mL) in PBS were incubated with 3. Mu.g/mL hRS7 at 4℃for 30min in this example, human IgG1 was used as isotype control, and the results are shown in FIG. 3 d. These results indicate that hRS7-VK1-MMAE can trigger faster internalization rates in TROP-2 highly expressing cells, which provides support for higher cell killing efficacy.
Example 5 Minimum Effective Dose (MED) of hRS7-VK1-MMAE in different tumor cell lines
Three tumor cells MDA-MB-468, bxPC-3 and NCI-N87 vaccinated with Trop-2 expressing tumor cells (n=5 per group) were treated with different doses of hRS7-VK1-MMAE at doses of 0.1mg/kg,0.2mg/kg and 0.4mg/kg, respectively, three times per week and tumor volumes were measured every two days. The tumor volume and inhibition rate and the body weight results of the mice are shown in FIG. 4.
In the BxPC-3 and NCI-N87 xenograft tumor model with higher Trop-2 expression, tumor volumes were significantly different from the blank group at the dose of 0.1 mg/kg. The tumor volume is reduced more obviously with the gradual increase of the dose, the drug effect is proportional to the administration dose, and in the MDA-MB-468 xenograft tumor model with high expression in Trop-2, when hRS7-VK1-MMAE is 0.1mg/kg, the difference is slightly different from a blank control group at the beginning, but the difference is gradually reduced or even finally is not different after stopping administration, but the difference is always obviously different from the blank control group in the 0.2mg/kg group. The three models are combined to show that the MED of hRS7-VK1-MMAE is 0.1-0.2mg/kg, which is far lower than the tolerance dose, and has a larger treatment window.
EXAMPLE 6 Pharmacokinetic (PK) study of hRS7-VK1-MMAE
(A) BALB/c mice (n=3) received 3mg/kg of hRS7 and hRS7-VK1-MMAE intravenously, blood was collected from each mouse orbit 1, 4, 24, 72, 180 hours post-dose, and total antibody and ADC concentrations in plasma were detected (fig. 5 a). hRS7-VK1-MMAE exhibited excellent PK profile comparable to unconjugated parental hRS 7.
(B) Male cynomolgus monkeys (n=5) were intravenously infused with 0.2, 0.5, 1.0mg/kg hRS7-VK1-MMAE, blood was collected at 0, 6, 24, 48, 72, 168, 336 and 504 hours post-administration, and total anti-concentration was detected by ELISA, ADC and free MMAE were detected by mass spectrometry (b in fig. 5). hRS7-VK1-MMAE exhibited excellent PK profile comparable to unconjugated parental hRS 7. The metabolic conditions in the female cynomolgus monkey are consistent.
(C) BALB/c mice MBA-MB-468 mice (n=3) received different doses of hRS7 and hRS7-VK1-MMAE were intravenously administered, blood was collected from each mouse orbit at 0, 1, 2, 4, 8, 24, 48, 120, 196, 264, 336, 408, and 504 hours post-administration, and plasma ADC and total antibody concentrations were detected (c, d, respectively, in fig. 5). hRS7-VK1-MMAE exhibited excellent PK profile comparable to unconjugated parental hRS 7.
Therefore, the hRS7-VK1-MMAE has better tolerance than the hRS7-VK2-MMAE, and the inhibition effect on tumor cells specifically expressed by Trop-2 is equivalent to that of the hRS7-VK 2-MMAE. Both safety and tolerability are better than hRS 7-VC-MMAE.
While it is known in the art that the insertion of PEG into a molecule increases the hydrophilicity of the molecule in ADC preparation, the inventors of the present application have unexpectedly found that it is not necessarily advantageous to insert PEG into the backbone of the molecule, as demonstrated by the present application above, that hRS7-VK1-MMAE shows a more excellent effect in terms of aggregation and tolerance than hRS7-VK2-MMAE (see fig. 1 and 2), which is difficult for the skilled person to predict.
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 (10)

1.一种用于制备抗体与药物偶联物的下式I所示的化合物，1. A compound of formula I for preparing an antibody-drug conjugate, 式I中，In Formula I, Z1为用于与抗体进行连接的接头部分； Z1 is a linker moiety used to connect to an antibody; m1为1-10的整数；m 1 is an integer from 1 to 10; m2为10-50的整数； m2 is an integer from 10 to 50; X表示由小分子细胞毒药物衍生的基团。X represents a group derived from a small molecule cytotoxic drug. 2.根据权利要求1所述的化合物，其中，2. The compound according to claim 1, wherein 所述Z1为取代或未取代的马来酰亚胺基团；The Z 1 is a substituted or unsubstituted maleimide group; 例如，所述Z1通过马来酰亚胺的5元环的1位、3位或4位与抗体连接，For example, the Z1 is linked to the antibody via position 1, position 3 or position 4 of the 5-membered ring of maleimide. 例如，所述连接包括：与抗体上的巯基连接、与抗体上的ε-氨基(如赖氨酸的ε-氨基)连接、与抗体上的硒半胱氨酸进行连接、或其组合，For example, the connection includes: connection to a sulfhydryl group on the antibody, connection to an ε-amino group on the antibody (such as the ε-amino group of lysine), connection to a selenocysteine on the antibody, or a combination thereof. 例如，所述Z1与抗体上天然存在的活性基团或人工引入的非天然活性基团发生反应生成化学连接结构，For example, the Z1 reacts with a naturally occurring active group on an antibody or an artificially introduced non-natural active group to form a chemically linked structure. 例如，所述Z1与抗体连接后，形成选自下组的化学连接结构：For example, after Z1 is linked to the antibody, a chemical linking structure selected from the following group is formed: (a)与抗体上巯基与马来酰亚胺通过偶联反应，形成的化学连接结构；(a) a chemical connection structure formed by coupling reaction between the thiol group on the antibody and maleimide; (b)与抗体上巯基与二溴马来酰亚胺通过偶联反应，形成的化学连接结构；(b) a chemical connection structure formed by coupling reaction between the thiol group on the antibody and dibromomaleimide; (c)抗体上赖氨酸的ε-氨基与连接子上的活性胺基团反应，形成的化学连接结构；(c) the ε-amino group of lysine on the antibody reacts with the active amine group on the linker to form a chemical connection structure; (d)抗体上赖氨酸与含马来酰亚胺的衍生物进行的偶联反应，形成的化学连接结构；(d) a coupling reaction between lysine on the antibody and a maleimide-containing derivative to form a chemically linked structure; (e)抗体上赖氨酸与碘乙酰胺类的偶联反应，形成的化学连接结构；(e) a coupling reaction between lysine on the antibody and iodoacetamide to form a chemical connection structure; (f)抗体上赖氨酸基于亚氨基硫烷的偶联反应，形成的化学连接结构；(f) a chemical connection structure formed by a coupling reaction of lysine on the antibody based on iminothiolane; (g)抗体上赖氨酸基于吡啶二硫化物的偶联反应，形成的化学连接结构；(g) a chemical connection structure formed by a pyridine disulfide coupling reaction of lysine on an antibody; (h)与抗体上人工引入的硒半胱氨酸进行的定点偶联，形成的化学连接结构；(h) site-specific coupling with selenocysteine artificially introduced into the antibody to form a chemical connection structure; (i)上述的任意组合；(i) any combination of the above; 其中，上式(a)和(b)中，m3为1-8的正整数，上式(d)和(e)中，spacer选自：-(CH2)m4-、-(CH2O)m4-，其中，m4为0-10的整数，较佳地，1-6的整数。Wherein, in the above formulae (a) and (b), m3 is a positive integer of 1-8, and in the above formulae (d) and (e), spacer is selected from: -(CH2) m4- , -(CH2O) m4- , wherein m4 is an integer of 0-10, preferably, an integer of 1-6. 3.根据权利要求1所述的化合物，其中，所述式I化合物为下式Ia的化合物：3. The compound according to claim 1, wherein the compound of formula I is a compound of the following formula Ia: 式Ia中，In Formula Ia, m1为1-10的整数；m 1 is an integer from 1 to 10; m2为10-50的整数； m2 is an integer from 10 to 50; X为单甲基澳瑞他汀等化合物失去H原子后的基团，X is a group after monomethyl auristatin and other compounds lose H atoms, 优选地，m1为1-8，更佳地为4、5，最佳地为5；m2为12-40，更佳地16-36，最佳地为24；X为如下结构所示的MMAE：Preferably, m1 is 1-8, more preferably 4, 5, and most preferably 5; m2 is 12-40, more preferably 16-36, and most preferably 24; X is MMAE shown in the following structure: 进一步优选地，所述式Ia化合物的结构如下式III所示：Further preferably, the structure of the compound of formula Ia is shown in formula III below: MMAE的定义如上所述。The definition of MMAE is as described above. 4.一种抗体与药物偶联物，所述抗体与药物偶联物是如权利要求1所述的式I化合物与抗体偶联形成的抗体与药物偶联物，4. An antibody-drug conjugate, wherein the antibody-drug conjugate is formed by coupling the compound of formula I according to claim 1 with an antibody, 优选地，所述抗体与药物偶联物具有下式II所示的结构：Preferably, the antibody-drug conjugate has a structure shown in the following formula II: Ab-(L-X)n(II)Ab-(L-X)n(II) 式II中，In formula II, Ab表示抗体，Ab stands for antibody, L为如下所示的二价连接子：L is a divalent linker as shown below: 其中，Z1’为式I化合物中的Z1基团去掉一个氢后剩下的基团；Wherein, Z 1 ' is the group remaining after removing one hydrogen from the Z 1 group in the compound of formula I; n为偶联于所述抗体的所述药物的平均偶联数量；n is the average number of conjugated drugs conjugated to the antibody; X、m1和m2分别如权利要求1中所定义。X, m1 and m2 are as defined in claim 1, respectively. 5.根据权利要求4所述的抗体与药物偶联物，其中，n是>0的整数或非整数，较佳地1≤n≤15，例如，n为5-10的整数或非整数；较佳地，为6-9的整数或非整数；更佳地，为7-8的整数或非整数。5. The antibody-drug conjugate according to claim 4, wherein n is an integer or non-integer>0, preferably 1≤n≤15, for example, n is an integer or non-integer of 5-10; preferably, an integer or non-integer of 6-9; more preferably, an integer or non-integer of 7-8. 6.根据权利要求4所述的抗体与药物偶联物，其中，所述抗体选自下组：嵌合抗体、二价或双特异性分子、双抗体、三抗体和四抗体，6. The antibody-drug conjugate according to claim 4, wherein the antibody is selected from the group consisting of a chimeric antibody, a bivalent or bispecific molecule, a diabody, a triabody and a tetrabody, 所述抗体是任意靶点的抗体，例如所述靶点选自下组：The antibody is an antibody of any target, for example, the target is selected from the following group: Trop-2、PD-L1、CTLA4、OX40、EpCAM、EGFR、EGFRVIII，HER2、PTPN2、VEGF、CD11a、CD19、CD20、CD22、CD25、CD30、CD33、CD40、CD56、CD64、CD70、CD73、CD74、CD79、CD105、CD138、CD174、CD205、CD227、CD326、CD340、MUC16、GPNMB、PSMA、Cripto、ED-B、TMEFF2、EphB2、Apo2、NMP179、p16INK4A、Nectin-4、B7H3、EMA、CEA、MIB-1、GD2、APRIL受体、叶酸受体、间皮素抑制剂、MET、TM4SF1、IL13受体，Trop-2, PD-L1, CTLA4, OX40, EpCAM, EGFR, EGFRVIII, HER2, PTPN2, VEGF, CD11a, CD19, CD20, CD22, CD25, CD30, CD33, CD40, CD56, CD64, CD70, CD73, CD74, CD79, CD105, CD138, CD174, CD205, CD227, CD326, CD340, MUC16, GPNMB, PSMA, Cripto, ED-B, TMEFF2, EphB2, Apo2, NMP179, p16INK4A, Nectin-4, B7H3, EMA, CEA, MIB-1, GD2, APRIL receptor, folate receptor, mesothelin inhibitor, MET, TM4SF1, IL13 receptor, 进一步优选地，所述抗体为hRS7，Further preferably, the antibody is hRS7, 更优选地，所述抗体hRS7的轻链可变区的氨基酸序列包含SEQ ID NO.:1所示的序列或为SEQ ID NO.:1所示的序列；和/或所述抗体hRS7的重链可变区的氨基酸序列包含SEQ IDNO.:2所示的序列或为SEQ ID NO.:2所示的序列。More preferably, the amino acid sequence of the light chain variable region of the antibody hRS7 comprises the sequence shown in SEQ ID NO.: 1 or is the sequence shown in SEQ ID NO.: 1; and/or the amino acid sequence of the heavy chain variable region of the antibody hRS7 comprises the sequence shown in SEQ ID NO.: 2 or is the sequence shown in SEQ ID NO.: 2. 7.根据权利要求4所述的抗体与药物偶联物，其中，所述抗体与药物偶联物中，所述抗体为hRS7，且在所述L中，Z1’为马来酰亚胺去掉一个氢后剩下的基团，m1为5，m2为24，n为7或8，X为MMAE。7. The antibody-drug conjugate according to claim 4, wherein in the antibody-drug conjugate, the antibody is hRS7, and in L, Z 1 ' is the group remaining after removing one hydrogen from maleimide, m 1 is 5, m 2 is 24, n is 7 or 8, and X is MMAE. 8.一种药物组合物，所述药物组合物包含：(a)如权利要求4-7中任一项所述的抗体与药物偶联物，以及(b)药学上可接受的载体。8. A pharmaceutical composition comprising: (a) the antibody-drug conjugate according to any one of claims 4 to 7, and (b) a pharmaceutically acceptable carrier. 9.一种如权利要求4-7中任一项所述的抗体与药物偶联物或如权利要求8所述的药物组合物在制备治疗肿瘤或免疫疾病的药物中的用途，9. Use of the antibody-drug conjugate according to any one of claims 4 to 7 or the pharmaceutical composition according to claim 8 in the preparation of a drug for treating tumors or immune diseases, 优选地，所述肿瘤选自Trop-2靶向的肿瘤、HER2靶向的肿瘤和EGFR靶向的肿瘤，Preferably, the tumor is selected from a Trop-2 targeted tumor, a HER2 targeted tumor and an EGFR targeted tumor, 进一步优选地，所述肿瘤为Trop-2靶向的肿瘤，并且选自乳腺癌、肺癌、结肠癌、膀胱癌和胰腺癌。Further preferably, the tumor is a Trop-2 targeted tumor and is selected from breast cancer, lung cancer, colon cancer, bladder cancer and pancreatic cancer. 10.一种制备如权利要求4-7中任一项所述的抗体与药物偶联物的方法，所述方法包括以下步骤：10. A method for preparing an antibody-drug conjugate according to any one of claims 4 to 7, the method comprising the following steps: (1)提供一反应体系，所述反应体系中包括待偶联的抗体和如权利要求1所述的式I化合物；(1) providing a reaction system, wherein the reaction system comprises an antibody to be coupled and a compound of formula I according to claim 1; (2)在所述反应体系中，将所述抗体和所述的式I化合物进行偶联反应，从而制得所述抗体与药物偶联物。(2) In the reaction system, the antibody and the compound of formula I are subjected to a coupling reaction to obtain the antibody-drug conjugate.
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