CN108066772B

CN108066772B - Antibody and Drug Conjugate (ADC) molecules targeting TACTD 2

Info

Publication number: CN108066772B
Application number: CN201611000747.2A
Authority: CN
Inventors: 王春河
Original assignee: Shanghai Institute of Materia Medica of CAS
Current assignee: Shanghai Institute of Materia Medica of CAS
Priority date: 2016-11-14
Filing date: 2016-11-14
Publication date: 2021-07-13
Anticipated expiration: 2036-11-14
Also published as: WO2018086239A9; WO2018086239A1; CN108066772A

Abstract

The present invention relates to Antibody and Drug Conjugate (ADC) molecules targeting tactd 2. The invention particularly discloses an antibody and drug conjugate of a targeting TACTD 2, and experimental results show that the antibody and drug conjugate have a remarkable anti-tumor effect. The invention also discloses pharmaceutical application of the antibody and drug conjugate of the targeting TACTD 2, and an effect of the antibody and drug conjugate in tumor inhibition or prevention.

Description

Antibody and Drug Conjugate (ADC) molecules targeting TACTD 2

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to an Antibody and Drug Conjugate (ADC) molecule targeting TACTD 2.

Background

Tumor associated calcium signal transducer 2 (TACTD 2), also known as Trop-2, EGP-1, MS-1, Gp-15, T-16 and GA733-1, is a monomeric, transmembrane glycoprotein with a molecular weight of 45 kDa. Its protein sequence is presumed to contain a signal peptide (26 amino acid residues), an extracellular domain (248 amino acid residues), a transmembrane domain (23 amino acid residues) and an intracellular domain (26 amino acid residues) based on the gene sequence.

In the tissues of various solid tumors of epithelial origin (such as breast cancer, cervical cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer and the like), TACTD 2 is overexpressed in high proportion. For example, lesion cells of 76% of breast cancer, 55% of pancreatic cancer and 56% of gastric cancer patients over-express the antigen. Clinical data indicate that expression levels of tactd 2 are positively correlated with tumor invasiveness and negatively correlated with patient prognosis. On cancerous cells, tactd 2 is an oncogene with a signaling function. It may be activated by crosslinking of the antibody or cleavage of the intracellular fragment portion. Upon activation, its intracellular region is phosphorylated by phosphokinase C and mediates the transmission of calcium signaling. Calcium signaling activates or up-regulates a variety of signaling molecules that promote cell division and growth, such as NF κ B, cyclin D-1, and ERKs, to accelerate cell growth, invasion, and metastasis. In addition, a large amount of tactd 2 is expressed in cancer cells, and the expression level varies greatly depending on the cell type, and thus it may be a means for controlling the function of tactd 2. The great difference of expression and function of TACTD 2 between tumor cells and normal cells makes it an ideal target for drug action.

Antibody-Drug conjugates (ADCs) are a class of biomacromolecule drugs that use monoclonal antibodies to direct small molecule compound load (payload) to kill cancerous cells or tumor tissues in a targeted manner.

Those skilled in the art are working to develop new, more effective antibody and drug conjugates targeting tactd 2.

Disclosure of Invention

The invention aims to provide an Antibody and Drug Conjugate (ADC) molecule targeting TACTD 2.

In a first aspect of the invention, there is provided an Antibody and Drug Conjugate (ADC), or a pharmaceutically acceptable salt thereof, having the structure of formula I:

Ab-(L-D)n I

wherein the content of the first and second substances,

ab represents an antibody, and Ab is hRS 7;

d is a small molecule drug with cytotoxicity, and the small molecule drug is selected from the following group: monomethyl auristatin (monomethylauristatin), calicheamicin, maytansinoids, doxorubicin (adriamycin), Pyrrolobenzodiazepines (PBD), duocarmycin (duocarmycin), or combinations thereof;

l is a linker linking the antibody and the drug;

n is the average coupling number of the drugs coupled to the antibody, can be an integer or a positive number of non-integers, and is more than or equal to 0.8 and less than or equal to 8;

"-" is a bond or a linker.

In another preferred embodiment, in formula I, n is the average number of conjugates of the drug to the antibody, and is also the drug-to-antibody ratio (DAR value).

In another preferred embodiment, n is an integer or a non-integer positive number from 1 to 7.

In another preferred embodiment, n is an integer or a non-integer positive number from 1 to 6; preferably, the number is an integer or a positive non-integer number from 2 to 6; more preferably, it is an integer or a positive non-integer number from 2 to 4.

In another preferred embodiment, D is monomethyl auristatin-E (MMAE), monomethyl auristatin-D (MMAD), monomethyl auristatin-F (MMAF), doxorubicin, pyrrolobenzodiazepines, duocarmycin, or combinations thereof.

In another preferred embodiment, L is mc (maleimidocaproyl), vc (valine-citrulline ) -PABC (para-aminobenzyloxycarbonyl).

In another preferred embodiment, L is MCC (maleimidomethyl cyclohexoxane-I-carboxylate, maleimidomethyl cyclohexyl-I-carboxylate) -vc-PABC.

In another preferred embodiment, when L is mc-vc-PABC and D is MMAE, the antibody and drug conjugate is hRS7- [ mc-vc-PABC-MMAE ] n (abbreviated as hRS7- [ vc-MMAE ] n or hRS7-vc-MMAE), wherein n is a positive number of 1 to 8, and the structure thereof is shown in formula II:

in another preferred embodiment, L comprises x oligomeric polyethylene glycol PEGs (which can be represented by PEGx, wherein x is the number of repetitions of polyethylene glycol PEG), and x is an integer from 1 to 28.

In another preferred embodiment, when x oligomeric polyethylene glycol PEGs are included in L, the antibody and drug conjugate can be represented by Ab- (L (pegx) -D) n.

In another preferred embodiment, the PEGx may be selected from one or more of the group consisting of: PEG2, PEG4, PEG6, PEG8, PEG12, PEG16, PEG24, PEG28, and the like, wherein the number following the PEG represents the number of repetitions of the PEG.

In another preferred embodiment, the antibody and drug conjugate is hRS7- [ l (pegx) -MMAE ] n, where x is the number of repetitions of PEG, preferably an integer from 1 to 28; preferably, it is an integer of 4 to 24.

In another preferred embodiment, the antibody and drug conjugate is hRS7- [ mc-PEGx-vc-PABC-MMAE ] n (abbreviated as hRS7-vc (PEGx) -MMAE), where x is the number of repetitions of PEG, preferably an integer from 1 to 64; preferably, it is an integer of 4 to 24.

In another preferred embodiment, PEG may be linked to maleic acid.

In another preferred embodiment, the Antibody and Drug Conjugate (ADC) is according to formula III or IV:

in another preferred embodiment, L may be intercalated with more multimeric polyethylene glycol (PEG) to improve the hydrophilic properties of the molecule, and the Antibody and Drug Conjugate (ADC) is represented by the formulas V-1, V-2, and V-3:

in another preferred embodiment, in formula II, when valine in L is replaced by an amino acid residue having a reactive group such as arginine or aspartic acid, and then (PEG) n is attached to the reactive group of the side chain of the amino acid, the antibody and drug conjugate are represented by structural formula VI, wherein x is an integer from 1 to 28; n is a positive number from 1 to 8;

in another preferred embodiment, the antibody hRS7 has the following characteristics:

(a) the amino acid sequence of the light chain variable region of the antibody hRS7 comprises or is the sequence shown in SEQ ID No. 1; and/or

(b) The amino acid sequence of the heavy chain variable region of the antibody hRS7 comprises or is the sequence shown in SEQ ID No. 2.

In a second aspect, the invention provides a pharmaceutical composition comprising an antibody of the first aspect of the invention and a drug conjugate, and a pharmaceutically acceptable carrier.

In a third aspect, the invention provides the use of an antibody and drug conjugate according to the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention for the manufacture of an anti-tumour agent.

In another preferred example, the tumor includes, but is not limited to, breast cancer (triple negative and non-triple negative), stomach cancer, ovarian cancer, small cell and non-small cell (squamous, adenocarcinoma, adenosquamous, large cell) lung cancer, cervical cancer, uterine cancer, esophageal cancer, head and neck cancer, pancreatic cancer, colorectal cancer, bladder cancer, liver cancer, prostate cancer and other solid tumors that highly express tactd 2.

In a fourth aspect, the present invention provides a method for producing an antibody and drug conjugate according to the first aspect of the present invention, the method comprising the steps of:

(1) providing a reaction system, wherein the reaction system comprises an antibody and a drug molecule, and the drug molecule is connected with a joint;

(2) and (3) performing coupling reaction on the antibody and a drug molecule in the reaction system, thereby preparing the antibody and drug conjugate.

In another preferred embodiment, the pH of the reaction system is 6.0-8.0; preferably 6.0-7.0.

In another preferred embodiment, the molar ratio of the antibody to the drug molecule is 1: 1-1: 20; preferably, 1: 10-1: 20.

in another preferred embodiment, the reaction time is 1-48 h; preferably 3 to 36 hours.

In a fifth aspect, the present invention provides a method of treating or preventing a tumour, the method comprising the steps of: administering to a subject in need thereof an antibody and drug conjugate according to the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention.

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 concentration of 0.3-10 mg/kg; preferably, 1-6 mg/kg; more preferably, 1-4 mg/kg.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows that hRS7-vc-MMAE has significantly higher killing efficacy against pancreatic cancer cells BxPC-3 in vitro than hRS7-CL2A-SN 38. Although MMAE itself has a stronger killing power on tumor cells than SN38, this enhanced killing power is dramatically amplified after compatibility with hRS7, so the antibody hRS7 has a clear synergistic effect with MMAE.

FIG. 2 shows hRS7-vc-MMAE in transplantation of 1x 10⁶、2x 10⁶、5x 10⁶The BALB/c-Nu tumor nude mouse model of BxPC3 shows a strong pharmacodynamic trend. Rx 3 mg/kg, QW, i.v. Tumor volume is length x width²/2。

FIG. 3 shows that hRS7-vc-MMAE shows potent efficacy in BALB/c-Nu tumor nude mouse model transplanted with MBA-MB-468. QW, i.v. Tumor volume is length x width²/2. n is 5. The 1 and 3 mg/kg dose groups showed significant efficacy, while the 0.3 mg/kg dose group showed poor efficacy.

FIG. 4 shows the killing efficiency of hRS7-vc-MMAE on breast cancer cells MBA-MB-468 for different DAR values. Where DAR values of 2, 4 and 6 are similar in efficacy, and when DAR values are below 2, the cell killing effect is poor.

FIG. 5 shows that the ADC molecule hRS7-mc-PEG4-vc-PABC-MMAE (abbreviated as hRS7-vc (PEG4) -MMAE) with PEG4 embedded in the linker (or connecting chain) has more significant drug effect.

Figure 6 shows the results of stability experiments for each ADC molecule.

Figure 7 shows the results of stability experiments for each ADC molecule.

Figure 8 shows the molecular hydrophobicity of each ADC molecule.

Figure 9 shows the molecular hydrophobicity of each ADC molecule.

Figure 10 shows the cell killing activity of each ADC molecule.

Detailed Description

The present inventors have conducted extensive and intensive studies to provide a large number of screens for obtaining a highly effective antibody and drug conjugate targeting tactd 2 with optimized loading capacity, stability and hydrophobicity. The experimental result shows that the antibody and drug conjugate of the invention obtains an abnormally significant anti-tumor effect by reasonably matching the antibody hRS7 with a small molecule drug (such as MMAE); on the other hand, the optimized load capacity is adopted, so that the toxic and side effects of the antibody and drug conjugate on other cells (such as normal cells) are obviously reduced. In addition, polyethylene glycol with proper polymerization number is embedded into the carrier of the conjugate in a specific and optimized mode, so that the physicochemical property of the molecule is further improved, and the drug effect is improved. The present invention has been completed based on this finding.

Definition of

As used herein, the terms "antibody and drug conjugate of the invention", "conjugate of the invention", or "ADC of the invention" are used interchangeably and refer to an antibody-drug conjugate having the structure shown in formula I.

In addition, in the present invention, the same ADC molecule or class of ADC molecules can be represented in various forms.

For example, one class of ADC molecules, hRS7- [ mc-vc-PABC-MMAE]When n is different, it can be called hRS7-vc-PAB-MMAE or hRS7-mc-vc-PABC-MMAE or hRS7-vc-MMAE or hRS 7-mal-vc-PABC-MMAE. hRS7- [ mc-vc-PABC-MMAE]The value of n in n is the DAR value of ADC molecule. For example, in this context, an hRS7-mc-vc-PABC-MMAE or hRS7-vc-MMAE with a DAR of 2, i.e., hRS7- [ mc-vc-PABC-MMAE]₂。

As another example, hRS7-vc (PEG4) -MMAE may be represented as hRS7-mc-PEG4-vc-PABC-MMAE or hRS7-PEG 4-vc-PAB-MMAE.

As another example, Mal-Peg4-Val-Lys (m-dPEG24) -PAB-MMAE may also be denoted as Mal-Peg4-Val-Lys (PEG24-Me) -PABC-MMAE, and particularly the ADC molecule formed with hRS7 may be denoted as hRS7-PEG4-VL (m-dPEG24) -PAB-MMAE;

Mal-Peg4-Lys (Peg24-Me) -Cit-PAB-MMAE can also be expressed as Mal-Peg4-Lys (PEG24-Me) -Cit-PABC-MMAE, and particularly the ADC molecule formed with hRS7 can be expressed as hRS7-PEG4-L (m-dPEG24) -C-PAB-MMAE;

Mal-PEG24-VC-PAB-MMAE can also be expressed as Mal-PEG24-Val-Cit-PABC-MMAE, and especially ADC molecules formed with hRS7 can be expressed as hRS7-PEG 24-VC-PAB-MMAE.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Antibodies

As used herein, the term "antibody" or "immunoglobulin" is an heterotetrameric glycan protein of about 150000 daltons with the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one 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 at one end a variable region (VH) followed by a plurality of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Particular amino acid residues form the 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 segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, which are in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, Vol I, 647-669 (1991)). The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.

The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two distinct classes (termed kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be assigned to different classes based on the amino acid sequence of their heavy chain constant regions. There are mainly 5 classes of 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 IgA 2. The heavy chain constant regions corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The 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 an antibody can be described by 3 specific regions 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 the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, and the β -sheets formed by the FRs between them 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 antibodies of the same type.

The invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.

In the present invention, the antibody of the present invention also includes conservative variants thereof, which means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids having similar or similar properties as compared with the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variants are preferably produced by amino acid substitutions according to Table A.

TABLE A

Initial residue(s)	Representative substitutions	Preferred substitutions
			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
Gln(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, for example, by PCR amplification or genomic library screening. Alternatively, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. 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.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, the DNA sequence encoding the antibody of the invention (or a fragment thereof, or a derivative thereof) has been obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.

Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the invention. The antibody of the invention is 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 by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (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 antibody of the present invention may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These 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 (such as salt precipitation), centrifugation, cell lysis by osmosis, 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 thereof.

In another preferred embodiment, the antibody targeting tactd 2 is antibody hRS 7.

In another preferred example, the light chain variable region (V-Kappa) amino acid sequence of the antibody hRS7 is the amino acid sequence shown in SEQ ID NO. 1 (DIQLTQSPSSLSASVGDRVSITCKASQD VSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQ).

In another preferred example, the heavy chain variable region (VH) amino acid sequence of the antibody hRS7 is the amino acid sequence shown in SEQ ID No. 2 (QVQLQQSGSELKKPGASVKVSCKASG YTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS).

Small molecule drugs

Small molecule drugs suitable for use in the present invention are highly cytotoxic compounds, preferably monomethyl auristatin (monomethylauristatin), calicheamicin, maytansinoids, doxorubicin (adriamycin), Pyrrolobenzodiazepines (PBD), duocarmycin (duocarmycin), or combinations thereof; more preferably selected from: monomethyl auristatin-E (MMAE), monomethyl auristatin-D (MMAD), monomethyl auristatin-F (MMAF), pyrrole benzodiazepines, duocarmycin, or combinations thereof.

Joint

Linkers (L) suitable for use in the present invention are used to link the small molecule drugs and antibodies of the present invention.

In another preferred embodiment, L is mc-vc-PABC (may be referred to simply as vc).

In another preferred embodiment, L is MCC-vc-PABC.

The L of the present invention may contain x oligomeric polyethylene glycols (which may be represented by PEGx, where x is the number of repetitions of polyethylene glycol (PEG)). Wherein x may be an integer from 1 to 28.

In another preferred embodiment, PEG may be linked to maleic acid.

Antibody and drug conjugate and preparation thereof

The present invention provides an Antibody and Drug Conjugate (ADC) comprising (a) an antibody targeting tactd 2 and (b) a small molecule drug that is cytotoxic, linked together by a linker (L).

The invention provides a coupling method, which couples small molecule drugs to an antibody through a specific linker 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 schemes suitable for use in the present invention include both K-Lock and C-Lock coupling schemes. In the K-Lock coupling mode, the drug molecule is coupled to a lysine (K) residue in the antibody sequence, and in the C-Lock coupling mode, the drug molecule is coupled to a cysteine (C) residue in the antibody sequence.

In another preferred embodiment, the invention employs a specific linker (L) moiety to enable conjugation to a specific lysine, thereby obtaining site-directed conjugation and optimized loading of antibody conjugate drugs.

In another preferred embodiment, the antibody and drug conjugate of the present invention is prepared as follows:

a certain amount (e.g., 0.5 mg) of the antibody is aspirated, and Tris (2-carboxyethyl) phosphine (TCEP) is added in an amount of 2 times the mass equivalent, and reacted at 37 ℃ for a certain period of time (e.g., 2.5 hours). After the reaction was completed, the reaction mixture was concentrated to below 0.5 ml by centrifugal ultrafiltration, topped up with a coupling solution (75mM NaAc, pH6.5,1mM DTPA, 10% DMSO), and further concentrated to below 0.5 ml by centrifugation, and repeated three times. We then added 10 times the physical equivalent of antibody to vc-MMAE and reacted overnight (over 17 hours) at 4 ℃. We finally replaced the buffer to PBS solution at pH7.0 by Amicon microliter tra 410K ultrafiltration tubes.

a certain amount (e.g., 0.5 mg) of the antibody is aspirated, and Tris (2-carboxyethyl) phosphine (TCEP) is added in an amount of 3 times the mass equivalent, and reacted at 37 ℃ for a certain period of time (e.g., 2.5 hours). Then, vc-MMAE was added directly in an amount of 20 times the physical equivalent of the antibody, and the reaction was carried out overnight at 4 ℃ (17 hours or more). We finally replaced the buffer to PBS solution at pH7.0 by Amicon Ultra 410K ultrafiltration tubes.

a certain amount (e.g., 0.5 mg) of the antibody is aspirated, and Dithiothreitol (DTT) is added in a 3-fold physical equivalent amount, and reacted at 37 ℃ for a certain period of time (e.g., 2 hours). After the reaction was completed, the reaction mixture was concentrated to below 0.5 ml by centrifugal ultrafiltration, topped up with a coupling solution (75mM NaAc, pH6.5,1mM DTPA, 10% DMSO), and further concentrated to below 0.5 ml by centrifugation, and repeated three times. We then added 10 times the physical equivalent of antibody to vc-MMAE and reacted overnight (over 17 hours) at 4 ℃. We finally replaced the buffer to PBS solution at pH7.0 by Amicon Ultra 410K ultrafiltration tubes.

In another preferred embodiment, the Antibody and Drug Conjugate (ADC) is according to formula II I or IV:

pharmaceutical compositions and methods of administration

The invention also provides pharmaceutical compositions comprising the ADCs of the invention, and methods of using the ADCs of the invention to treat diseases in mammals. Preferably, the disease is a disease targeted by tactd 2, such as a tumor.

The invention also provides application of the antibody and the drug conjugate in preparation of antitumor 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. In preparation, the active ingredient is typically mixed with, or diluted with, excipients or enclosed within a carrier which may be in the form of a capsule or sachet. When the excipient serves as a diluent, it may employ a solid, semi-solid or liquid material as a 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, glucose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, etc.; the formulation may further comprise: wetting agents, emulsifiers, preservatives (such as methyl and propyl hydroxybenzoates), and the like. The antineoplastic drug may be formulated in unit or multi-unit dosage forms, each dosage form containing a predetermined amount of the ADC of the invention calculated to produce the desired therapeutic effect, together with suitable pharmaceutical excipients.

The antineoplastic agent may be administered by conventional routes including, but not limited to: intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, topical administration, and the like.

When the medicament is used, a safe and effective amount of the antibody and the medicament conjugate is administered to a human, wherein the range of the safe and effective amount is preferably 0.5-50 mg/kg of body weight, and more preferably 1-10 mg/kg of body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

In addition, the conjugates of the invention may 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 drugs, such as 5-FU, methotrexate and other drugs affecting nucleic acid biosynthesis; alkylating agents such as mechlorethamine and cyclophosphamide; drugs such as adriamycin and actinomycin D which interfere with the transcription process and prevent RNA synthesis; vincristine, camptothecin and other drugs which affect protein synthesis, and some hormone drugs, etc.

Compared with the prior art, the invention has the main beneficial effects that:

(1) the antibody and drug conjugate provided by the invention has significant activity of killing tumor cells.

(2) The antibodies and drug conjugates provided by the invention have optimized DAR values and better loading, so that the treatment window of the ADC of the invention is larger.

(3) The invention discovers for the first time that the matching of the antibody hRS7 and MMAE has a synergistic effect, and can remarkably expand the killing activity of MMAE on tumor cells.

(4) The antibody and the drug conjugate provided by the invention have optimized DAR value of 2-4, and compared with ADC with high DAR value, the disulfide bond in the antibody is more preserved, so that the ADC provided by the invention has higher stability.

(5) The invention introduces poly-polyethylene glycol into the connecting chain, thereby improving the physicochemical property of the molecule and improving the drug effect.

(6) The antibody and drug conjugate provided by the invention have better pharmacokinetics performance and lower toxic and side effects.

The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specifying the detailed conditions in the following examples, generally followed by conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.

Materials and general methods

isotype refers to an isotype control antibody, here a humanized antibody targeting Toxin B, belonging to the human IgG1 antibody class.

MDA-MB-468 cells are conventional triple negative breast cancer cells, purchased from Shanghai cell Bank of Chinese academy.

BxPC-3 is a conventional pancreatic cancer cell, and is purchased from Shanghai cell bank of Chinese academy of sciences.

HEK293F cells were purchased from seimei fei china ltd.

CHO-K1 cells were purchased from Jianshun Bioresponsibility, Inc., Lanzhou.

mc-vc-PABC-MMAE was purchased from Haoyuan biological medicine technology, Inc. in Shanghai.

hRS7-CL2A-SN38 the preparation method is disclosed in the patent document US 8,758,752B 2.

EXAMPLE 1 preparation of antibody and drug conjugate

I. Preparing an antibody protein solution:

using molecular cloning technology, the light chain (amino acid sequence shown in SEQ ID No.: 1) and the heavy chain (amino acid sequence shown in SEQ ID No.: 2) of the hRS7 antibody were first cloned into the expression vector pcDNA3.1. Then, plasmids carrying the cDNA sequences of the antibody light and heavy chains, respectively, were amplified in E.coli, followed by transfection of HEK293 or CHO cells, which were cultured in suspension in serum-free medium, with the transfection reagent PEI, and 5% CO at 37 ℃%₂Shake culturing for 4-10 days under the condition. The antibodies in the cell culture supernatant were isolated and purified on a GE AKTA Purifier using Protein A and Superdex 200 size exclusion chromatography columns and finally concentrated by ultrafiltration to give an antibody Protein solution with a concentration of more than 1 mg/ml.

II. Preparation of antibody and drug conjugate:

the following experiments can be performed to prepare different DAR values of ADC molecule hRS7-vc-MMAE (i.e. hRS7- [ mc-vc-PABC-MMAE ] n, n is the DAR value representing the molecule) by changing the conditions of reduction and coupling. Hereinafter, vc-MMAE is mc-vc-PABC-MMAE.

(1) The antibody was concentrated to 5-10 mg/ml in PBS (pH7.0) buffer by ultrafiltration. According to the measured concentration, 1mg of the antibody was aspirated, and Tris (2-carboxyethyl) phosphine (TCEP) was added in an amount of 1.5 times the amount of the substance for reaction at 37 ℃ for 2.5 hours. After the reaction was completed, the reaction mixture was concentrated to below 0.5 ml by centrifugal ultrafiltration, topped up with a coupling solution (75mM NaAc, pH6.5,1mM DTPA, 10% DMSO), and further concentrated to below 0.5 ml by centrifugation, and repeated three times. We then added 10 times the physical equivalent of antibody to vc-MMAE and reacted overnight (over 17 hours) at 4 ℃. We finally replaced the buffer to PBS solution at pH7.0 by Amicon Ultra 410K ultrafiltration tubes. The ADC molecule hRS7-vc-MMAE (1) prepared by this method had a DAR value of about 2.

(2) The antibody was concentrated to 5-10 mg/ml in PBS (pH7.0) buffer by ultrafiltration. According to the measured concentration, 1mg of the antibody was aspirated, and Tris (2-carboxyethyl) phosphine (TCEP) was added in an amount of 10 times the amount of the substance to react at 37 ℃ for 2.5 hours. After the reaction was completed, the reaction mixture was concentrated to below 0.5 ml by centrifugal ultrafiltration, topped up with a coupling solution (75mM NaAc, pH6.5,1mM DTPA, 10% DMSO), and further concentrated to below 0.5 ml by centrifugation, and repeated three times. We then added 20 times the physical equivalent of antibody to vc-MMAE and reacted overnight (17 hours more) at 4 ℃. We finally replaced the buffer to PBS solution at pH7.0 by Amicon Ultra 410K ultrafiltration tubes. The ADC molecule hRS7-vc-MMAE (2) prepared by this method had a DAR value of about 4.

(3) The antibody was concentrated to 5-10 mg/ml in PBS (pH7.0) buffer by ultrafiltration. According to the measured concentration, 1mg of the antibody was aspirated, and 25 times the substance equivalent of Tris (2-carboxyethyl) phosphine (TCEP) was added thereto and reacted at 37 ℃ for 2.5 hours. After the reaction was completed, the reaction mixture was concentrated to below 0.5 ml by centrifugal ultrafiltration, topped up with a coupling solution (75mM NaAc, pH6.5,1mM DTPA, 10% DMSO), and further concentrated to below 0.5 ml by centrifugation, and repeated three times. We then added 20 times the physical equivalent of antibody to vc-MMAE and reacted overnight (17 hours more) at 4 ℃. We finally replaced the buffer to PBS solution at pH7.0 by Amicon Ultra 410K ultrafiltration tubes. The ADC molecule hRS7-vc-MMAE (3) prepared by this method had a DAR value of about 6.

(4) The antibody was concentrated to 5-10 mg/ml in PBS (pH7.0) buffer by ultrafiltration. According to the measured concentration, 1mg of the antibody was aspirated, and Tris (2-carboxyethyl) phosphine (TCEP) was added in an amount of 0.5 times the amount of the substance to react at 37 ℃ for 2.5 hours. After the reaction was completed, the reaction mixture was concentrated to below 0.5 ml by centrifugal ultrafiltration, topped up with a coupling solution (75mM NaAc, pH6.5,1mM DTPA, 10% DMSO), and further concentrated to below 0.5 ml by centrifugation, and repeated three times. We then added 5 times the physical equivalent of antibody to vc-MMAE and reacted overnight (17 hours more) at 4 ℃. We finally replaced the buffer to PBS solution at pH7.0 by Amicon Ultra 410K ultrafiltration tubes. The ADC molecule hRS7-vc-MMAE (4) prepared by this method had a DAR value of about 0.5.

(5) ADC molecules hRS7-vc-MMAE (5) were prepared according to the method described above, with a DAR value of about 2.8, except that 6 times the mass equivalent of TCEP reduction was added to the antibody and 10 times the mass equivalent of vc-MMAE was added.

Preparation of control ADC molecules

isotype-vc-MMAE, DAR value about 2.8, was prepared according to experiment (5) of example 1, step II, except that hRS7 isotype control antibody (human toxin B antibody) was used instead of hRS 7.

Example 2 tumor cell inhibition assay

The experimental procedure was as follows:

approximately 1-2 ten thousand cells/100 μ l/well were seeded in 96-well cell culture plates (cell number varied from tumor cell line to tumor cell line, 1 ten thousand cells/well for pancreatic cancer cells BxPC3, and 2 ten thousand cells/well for breast cancer MDA-MB-468).

Drug molecules were diluted with medium at various initial concentrations in a carefully 1:4 equal ratio, and 3 wells per concentration were dosed (total volume 200 microliters).

Administration group: hRS7-vc-MMAE (DAR value about 2.8)

Control group: hRS7-CL2A-SN38(DAR value about 2.8)

MMAE

SN-38

hRS7

isotype-vc-MMAE (DAR value about 2.8)

After incubation at 37 ℃ for 96 hours, 10. mu.l of CCK8 (cell counting kit-8) was added, and the incubation was continued for 1 to 4 hours, after which the absorbance at 450nm was measured. We used the statistical method of two-tailed Student's t-test to compare each dosing group with the control group.

The experimental results are as follows: the killing efficacy of the administration group and each control group on pancreatic cancer cells BxPC3 in vitro was preliminarily compared (results are shown in fig. 1).

From the results, it can be seen that: MMAE is more powerful than SN38, both EC₅₀Values were 0.27 and 6.9nM, respectively, differing by a factor of 20. Surprisingly, the difference in potency between MMAE and SN38 was dramatically amplified when constructed in ADC molecules with hRS7 as the antibody. EC for killing BxPC3 cells by hRS7-vc-MMAE₅₀The value was 0.27nM, whereas hRS7-CL2A-SN38 was greater than 300 nM. Compared with the hRS7-vc-MMAE, the tumor cell killing efficacy of the hRS7-vc-MMAE was increased 1111 times (Table 1).

TABLE 1 comparison of the killing potency of different drugs against BxPC3 cells.

Medicine	EC50(nM)
		MMAE	0.27
SN38	6.9
		hRS7-vc-MMAE	0.27
hRS7-CL2A-SN38	>300

The experimental results show that: in vitro potency, MMAE and antibody hRS7 have significant effect in constructing ADC molecules, but the synergy that is difficult to predict is based on experimental observations and cannot be predicted from the difference in potency between MMAE and SN 38.

Example 3 in vivo antitumor Activity

The experimental procedure was as follows:

pancreatic cancer cell BxPC-3 or breast cancer cell MDA-MB-468 is diluted with physiological saline to required cell concentration, and injected into axillary region of female BALB/c-nu nude mouse of 4-6 weeks of age by subcutaneous injection with 1-5x10⁶One/300. mu.l of tumor cells. Tumor size was measured daily after inoculation with a vernier caliper.

Tumor volume L x omega²/2

Where L is the longest diameter and ω is the shortest diameter.

When the tumor volume approaches 0.25cm or 36 days after inoculation³At that time, the treatment experiment was started. ADC molecules are typically injected once a week (QW), 0.3-3 mg/kg body weight. When the tumor is larger than 1.0cm³At that time, the mouse was considered dead. The therapeutic effect can be divided into partial response (tumor shrinkage of 30% or more), stable (tumor shrinkage of 29% or less, or increase of no more than 20%). TTP (tumor growth time) is the time from the start of treatment to a tumor growth of 20% by volume. Statistical analysis for tumor growth was based on area under the curve (AUC). Statistical method is a two-tailed Student's t-test to compare each dosing group with the control group.

Administration group: ADC (hRS7-vc-MMAE, DAR value approximately 2.8 and 4 in pancreatic and breast cancer, respectively)

Control group: Isotype-ADC (Isotype-vc-MMAE, DAR values of about 2.8 and 4 in pancreatic and breast cancer, respectively)

MMAE

hRS7

hRS7-CL2A-SN38(DAR values of approximately 2.8 and 4 in pancreatic and breast cancer, respectively)

PBS

The experimental results are as follows:

1) hRS7-vc-MMAE showed a very strong tendency to suppress cancer in BxPC3 cell-seeded pancreatic cancer models relative to isotype-vc-MMAE. As shown in fig. 2.

2) The hRS7-vc-MMAE showed very potent cancer-inhibiting effects on the MDA-MB-468 cell-seeded triple negative breast cancer model relative to isotype-vc-MMAE, with the drug effects in the 1 and 3 mg/kg dose groups being significant, while the drug effect at 0.3 mg/kg was not. As shown in fig. 3.

The experimental results show that: hRS7-vc-MMAE has prominent tumor suppression activity in vivo.

Example 4 comparison of the lethality of ADC (hRS7-vc-MMAE) molecules of different DAR values on MBA-MB-468 cells

The ADC molecules prepared in example 1 were each tested for in vivo anti-tumor activity. The experimental method is the same as that of example 4, except that the tumor cells used in the experiment are breast cancer cells MBA-MB-468.

The experimental results are as follows: on MBA-MB-46 cells, there was no significant difference in tumor killing potency for ADC molecules with DAR values of 2, 4 and 6, whereas ADC molecules with DAR values below 2 had poor potency. See fig. 4.

The results show that: the ADC molecule prepared by the invention has excellent tumor killing effect of more than 4.

Example 5 killing of PEG 4-embedded hRS7-vc-MMAE molecules on MBA-MB-468 cells

Preparation of hRS7-vc (PEG4) -MMAE (also known as hRS7-PEG 4-vc-PAB-MMAE):

Mal-PEG4-NHS activated lipid (26 mg,50 umol, purchased from Quanta Biodesign, USA) was dissolved in 1ml of N.N-dimethylformamide. vc-PAB-MMAE (45mg,40 umol, purchased from Levena Biopharma, synthesized as reported in WO 2004/010957) and 9. mu.l DIEA were added with stirring. The reaction was stirred at room temperature (22 ℃ C.) for one hour, purified by reverse phase high pressure liquid phase preparative purification and freeze-dried to give the white solid product Mal-PEG4-vc-PABC-MMAE (47 mg, 78%). MS: M/z 1521.0(M + H)⁺)。

The antibody was conjugated with a small molecule, Mal-PEG4-vc-PABC-MMAE, to hRS7-vc (PEG4) -MMAE of different DAR values, as described in example 1.

The efficacy of hRS7-vc (PEG4) -MMAE and hRS7-vc-MMAE (both DAR values were 4) was compared by MDA-MB-468 tumor cell inhibition assay (see example 4 for experimental methods).

The results show that: the effect of hRS7-vc-MMAE molecules can be remarkably improved by embedding PEG in the linker (or connecting chain). See fig. 5.

Example 6 introduction of 24 polyethylene glycols (PEG24) in the linker chains and preparation of ADC molecules containing these linker chains:

1) synthesis of Mal-Peg4-Val-Lys (m-dPEG24) -PAB-MMAE

Fmoc-Val-Lys (Trt) -PAB-PNP (100mg,0.102mmol, purchased from Levena Biopharma, USA) and MMAE (78mg,0.102mmol, purchased from Levena Biopharma, USA) was dissolved in anhydrous DMF (5mL) and DIEA (7mg) was added to the solution. The mixture was reacted at room temperature (22 ℃ C.) for 2 hours, and the objective product Fmoc-Val-Lys (Trt) -PAB-MMAE (3) was directly purified by reverse phase HPLC. Product (3) formed as a white powder after freeze-drying (123mg, 77%). MS M/z 1559.2(M + H)⁺)。

Compound 3(120mg) was dissolved in 10% TFA/DCM (4mL) solution and stirred at room temperature for 20 min. The mixture was reacted and purified by concentration under reduced pressure and reverse phase HPLC to give compound 4(88 mg). MS M/z 1316.7(M + H)⁺)。

Fmoc-val-Lys-PAB-MMAE TFA salt (i.e., Compound 4) (80mg), M-dPEG24 acid (69mg) and HATU (23mg) were dissolved in DMF (3ml), DIEA (20mg) was added, and stirring was performed at room temperature for 30 minutes to obtain the desired product [ MS: M/z 1208.8(M/2+ H)⁺)]. Diisopropylamine (1mL) was added to the reaction mixture and stirred at room temperature for 3 hours. The mixture was reacted and concentrated and reverse phase HPLC purified to afford compound 5(53mg) as a wax after lyophilization. MS: M/z 1097.3(M/2+ H)⁺)。

Compound 5(50mg), Mal-PEG 4-acid (10mg, purchased from Quanta Biodesign, USA) and HATU were dissolved in DMF (3mL), DIEA (9mg) was added and stirred for 20 min. After purification by reverse phase HPLC and freeze-drying, the desired compound Mal-Peg4-Val-Lys (m-dPEG24) -PAB-MMAE compound 6(21mg) was finally obtained. MS: M/z 1296.1(M/2+ H)⁺)。

Referring to the procedure of example 1, the antibody was coupled with Compound 6 to the ADC molecule hRS7-PEG4-VL (m-dPEG24) -PAB-MMAE.

2) Synthesis of Mal-Peg4-Lys (Peg24-Me) -Cit-PAB-MMAE

Compound 1(80mg, purchased from Levena Biopharma, USA), MMAE (60mg, purchased from Levena Biopharma, USA),and DIEA (8mg) was dissolved in dry DMF (3mL) and stirred at room temperature for 16 h. LC/MS detection of reaction product formation [ MS: M/z 1246.6(M + H)⁺)]. Diisopropylamine (1mL) was added to the reaction solution and stirred at room temperature for 3 hours. Purification by HPLC and lyophilization afforded H-Cit-PAB-MMAE TFA salt (Compound 2') (92mg) as a white powder. MS M/z 1024.8(M + H)⁺)。

H-Cit-PAB-MMAE (i.e.Compound 2') (40mg), Fmoc-Lys (Boc) -OH (18mg, purchased from Chem-Impex Inc., USA) and HATU (15mg) were dissolved in DMF (3mL) and DIEA (15mg) was added and stirred at room temperature for 20 min. The crude product was purified by HPLC to the desired product Fmoc-Lys (Boc) -Cit-PAB-MMAE (41 mg). MS M/z 1474.3(M + H)⁺). The resulting product was dissolved in 10% TFA/DCM (5mL) and stirred at room temperature for 20 min. Concentration and drying afforded the crude product, which was further isolated by HPLC purification to yield Fmoc-Lys-Cit-PAB-MMAE (31mg) (compound 3') as a white powder. MS M/z 1374.2(M + H)⁺)。

Compound 3' (31mg), m-dPEG24(23mg, purchased from Quanta Biodesign, USA) and HATU (8mg) were dissolved in DMF (3ml) and DIEA (10mg) was added and stirred at room temperature for 20 min. More diisopropylamine (1mL) was added and stirring was continued for 3 hours. Excess base was removed under reduced pressure and HPLC purified to give H-Lys (m-dPEG24) -Cit-PAB-MMAE TFA salt (29mg) as a white powder (Compound 4'). MS: M/z 1126.9(M/2+ H)⁺)。

Compound 4' (29mg) and Mal-PEG4-PFP (8mg, from Levena Biopharma, USA) were dissolved in DMF (3mL) and DIEA (10mg) was added and stirred at room temperature for 10 min. Purification was then carried out directly by reverse phase HPLC and after lyophilization the waxy molecule of interest, Mal-PEG4-Lys (m-dPEG24) -Cit-PAB-MMAE (Compound 5') (19mg) was obtained. MS: M/z 1325.6(M/2+ H)⁺)。

Referring to the procedure of example 1, the antibody was 5' conjugated to compound ADC molecule hRS7-PEG4-L (m-dPEG24) -C-PAB-MMAE.

3) Synthesis of Mal-PEG24-VC-PAB-MMAE

Mal-PEG24-NHS ester (Compound i) (70mg, 50. mu. mol, available from Quanta Biodesign, USA), vc-PAB-MMAE (Compound ii) (45mg, 40. mu. mol, available from Levena Biopharma, synthesized according to the published methods of WO 2004/010957) and DIEA (9. mu.L) were dissolved in anhydrous DMF (1 mL). The mixture was stirred at room temperature (22 ℃) for 1 hour and purified directly by reverse phase HPLC to afford the desired product Mal-PEG24-vc-PAB-MMAE (Compound ii i) (70mg, 73%) as a colorless syrup after lyophilization. MS M/z 1202.1(M + 2H)⁺)/2。

Referring to the procedure of example 1, the antibody was conjugated with compound iii to the ADC molecule hRS7-PEG 24-vc-PAB-MMAE.

Example 7 stability testing

Since the DAR properties of the hRS7-CL2A-SN38 molecule are high, resulting in multiple disulfide bonds of the molecule being opened, the stability of the molecule must be affected. The DAR value of hRS7-vc-MMAE prepared by the invention is more reasonable, so we predict that its molecular stability will be better, and use the hypothesis that the antibody stability test verifies.

The assay samples to be stored at 4 ℃ were diluted to a concentration of 1 mg/ml with PBS at room temperature and 50 μ L was taken for testing directly (4 ℃ storage conditions) or after incubation in a 60 ℃ water bath for 1 hour (accelerated stability experiments). We used a Thermo MAbPac SEC-1(7.8 × 300mm) size exclusion chromatography column on a Thermo microliter tiMate 3000UHPLC high pressure liquid chromatograph to quantify the content of normal structural antibodies or ADC molecules (main peak), polymeric molecules, and melted or degraded molecules in the sample.

The mobile phase of UHPLC is 50mM phosphate buffer (pH7.0) +150mM NaCl, column temperature 25 deg.C, injection volume 20 μ L, flow rate 1ml/min, UV detection wavelength 214nm and 280 nm. We finally performed data acquisition and analysis by Chromeleon software.

As shown in fig. 6, we found that the stability of the ADC molecules was reduced by conjugation of small molecules, and more polymerized and melted molecules appeared compared to antibody hRS7(a and B). Among these, DAR ═ 6 molecules show a high number of melting or degradation molecules (I) under storage conditions at 4 ℃, while a high number of aggregates (J) appeared in accelerated stability experiments, indicating that this molecule is not sufficiently stable. The stability of the ADC molecules DAR-4 (E and F) was not significantly reduced compared to the ADC molecules DAR-2 (C and D). In addition, the stability of the ADC molecule (G) with PEG4 was superior to the ADC molecule without PEG (E) at 4 ℃ storage, although there was no significant difference in the performance of the two in the accelerated stability experiments. From the data synthesis of fig. 6, DAR ═ 4, whereas the stability of the molecule with PEG4 added to the linker was optimal.

Next we performed stability analysis of ADC molecules incorporating PEG4 and PEG24 in the linker chain, with hRS7-vc-MMAE and hRS7-PEG4-vc-PAB-MMAE as controls. The molecules compared included:

1.hRS7-PEG24-vc-PAB-MMAE

2.hRS7-PEG4-VL(m-dPEG24)-PAB-MMAE

3.hRS7-PEG4-L(m-dPEG24)-C-PAB-MMAE

4.hRS7-PEG4-vc-PAB-MMAE

5, hRS7-vc-PAB-MMAE (i.e., hRS7-vc-MMAE)

As shown in FIG. 7, 51% of the aggregates appeared in hRS7-vc-MMAE after incubation at 60 ℃ for 1 hour. The insertion of PEG4 (hRS7-PEG4-vc-PAB-MMAE) reduced the fraction of aggregated molecules to 33%. However, after adding PEG24 directly into the connecting chain, the molecule has two polymerization peaks, and the proportion of the polymerization molecules accounts for 45.1 percent of the total amount of protein, and the stability of the molecule is improved lower than that of PEG 4. However, when PEG24 was attached to the ADC molecule through a side chain, the stability of the molecule was significantly improved, wherein the polymerization rate of hRS7-PEG4-VL (m-dPEG24) -PAB-MMAE was reduced to 24.2%, and the polymerization rate of hRS7-PEG4-L (m-dPEG24) -C-PAB-MMAE was reduced to 27.2%. From the data in FIG. 7, the stability of the molecule of hRS7-PEG4-L (m-dPEG24) -C-PAB-MMAE was optimal.

Example 8 hydrophobicity detection

Small molecules such as MMAE have very high hydrophobicity, which results in poor drug-forming properties of the molecule. According to the invention, a plurality of PEGs are added into a connecting chain so as to reduce the hydrophobicity of molecules. PEG can be added to the linking chain in a variety of ways. We used a hydrophobic column, namely, Hydrophic Interaction Chromatography (HIC), and validated this hypothesis on HPLC and optimized the manner of PEG access.

We diluted the sample to be analyzed with chromatographic mobile phase a to a concentration of 1 mg/kg and then performed the sample hydrophobicity analysis with HIC. Detection was performed using Agilent 1260Infinity HPLC, using a TOSOH TSKgel Butyl-NPR 4.6 x 35mm column, and data acquisition analysis was performed by Agilent OpenLAB software.

Chromatographic conditions are as follows: mobile phase A is 2M (NH)₄)₂SO₄in 50mM phosphate buffer (pH7.0), mobile phase B is 50mM phosphate buffer (pH7.0), gradient is 0min 100% buffer A, 5min 100% buffer A, 25min 100% buffer B, 30min 100% buffer B, column temperature 37 deg.C, injection volume is 10 μ L, flow rate is 1mL/min, UV detection wavelength is 214 nm.

The higher the hydrophobicity of the molecule, the later the time of exit in the HIC column and the more to the right on the X-axis. Results As shown in the results of FIG. 8, the hydrophobicity of hRS7-vc-MMAE molecules of different DAR values increased with increasing DAR. Since DAR-4 is similar in potency to DAR-6, and DAR-4 molecules are superior to DAR-4 in hydrophobicity, 4 is the preferred choice for DAR value.

The data in fig. 9 show that pegylation can alter the hydrophobicity of the ADC molecules. Wherein the hydrophobicity of the hRS7-PEG4-vc-PAB-MMAE, the hydrophobicity of the hRS7-PEG4-VL (m-dPEG24) -PAB-MMAE and the hydrophobicity of the hRS7-PEG4-L (m-dPEG24) -C-PAB-MMAE is reduced relative to the hydrophobicity of the hRS 7-vc-PAB-MMAE. However, the improvement effect of hRS7-PEG24-vc-PAB-MMAE on the hydrophilicity of the molecule is not good.

The reduced hydrophobicity may reduce the tendency of ADC molecules to aggregate in solution through hydrophobic small molecule moiety interactions. In addition, the reduction of hydrophobicity can reduce the speed of metabolic discharge of ADC molecules by liver, thereby prolonging the half-life period of the molecules in vivo, enhancing the drug effect and reducing the toxic and side effects on the liver.

Example 9 PEGylation of the linker chain did not affect the tumor killing efficiency of the ADC molecules

We performed in vitro pharmacodynamic comparisons of ADC molecules incorporating PEG4 and PEG24 in the linker chain to confirm that pegylation did not affect target recognition, endocytosis, transport and cargo release of the ADC molecules.

As shown in FIG. 10 and Table 2, the pharmacological effects of hRS7-PEG24-vc-PAB-MMAE on pancreatic cancer cells BxPC3 and breast cancer cells SK-BR-3 are significantly reduced compared with hRS7-vc-MMAE, and may be related to the poor physicochemical properties of the molecule. And compared with the hRS7-vc-MMAE, the cell killing efficiency of the hRS7-PEG4-vc-PAB-MMAE, the hRS7-PEG4-VL (m-dPEG24) -PAB-MMAE and the hRS7-PEG4-L (m-dPEG24) -C-PAB-MMAE is improved or approximate, and the PEG is not influenced by the target recognition, endocytosis, transportation and load release of ADC molecules.

TABLE 2 comparison of cell killing potency (EC50 values, nM) of different PEG-modified ADC molecules against BxPC3 and SK-BR-3.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shanghai pharmaceutical research institute of Chinese academy of sciences

<120> Antibody and Drug Conjugate (ADC) molecule targeting TACTD 2

<130> P2016-0520

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 90

<212> PRT

<213> Intelligent people

<400> 1

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln

85 90

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<211> 121

<212> PRT

<213> Intelligent people

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Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Ser Leu Val Thr Val Ser Ser

115 120

Claims

1. An Antibody and Drug Conjugate (ADC), or a pharmaceutically acceptable salt thereof, having the formula:

Ab-(L(PEGx)-D)n

wherein the content of the first and second substances,

ab represents an antibody, and Ab is hRS 7;

d is small molecule drug monomethyl auristatin with cytotoxicity;

l is a linker linking the antibody and the drug;

the Antibody and Drug Conjugate (ADC) is shown as a formula V-2 or V-3:

PEGx is PEG 28;

n is the average coupling number of the drugs coupled to the antibody, can be an integer or a positive number of non-integers, and is more than or equal to 1 and less than or equal to 8;

"-" is a bond or a linker.

2. The antibody and drug conjugate of claim 1, wherein n is an integer or a positive non-integer number from 1 to 7.

3. The antibody and drug conjugate of claim 1, wherein the antibody hRS7 has the following characteristics:

(a) the amino acid sequence of the light chain variable region of the antibody hRS7 is a sequence shown in SEQ ID NO. 1; and/or

(b) The amino acid sequence of the heavy chain variable region of the antibody hRS7 is the sequence shown in SEQ ID NO. 2.

4. A pharmaceutical composition comprising the antibody of any one of claims 1-3 in combination with a drug conjugate, and a pharmaceutically acceptable carrier.

5. Use of the antibody and drug conjugate according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 4 for the preparation of an anti-tumor medicament.

6. The use of claim 5, wherein the tumor is selected from the group consisting of: breast cancer, gastric cancer, ovarian cancer, small cell and non-small cell lung cancer, cervical cancer, uterine cancer, esophageal cancer, head and neck cancer, pancreatic cancer, colorectal cancer, bladder cancer, liver cancer, prostate cancer and other solid tumors that highly express tactd 2.

7. A method for producing the antibody and drug conjugate of claim 1, comprising the steps of:

(2) in the reaction system, the antibody and a drug molecule are subjected to a coupling reaction, thereby producing an antibody-drug conjugate according to claim 1.

8. The method of claim 7, wherein the reaction system has a pH of 6.0 to 8.0.

9. The method of claim 7, wherein the molar ratio of antibody to drug molecule is 1:1 to 1: 20.

10. the process according to claim 7, wherein the reaction time is from 1 to 48 hours.