Methods for administering anti-AXL antibody-drug conjugates

Abstract

The present disclosure relates to a method of administering an anti-AXL antibody-drug conjugate, wherein the antibody-drug conjugate is administered in combination with a composition comprising a nanoscale cavitation initiator. Application of ultrasound after administration provides effective targeted delivery of the antibody-drug conjugate to disease sites. The therapeutic method is useful for the treatment of solid tumors.

Description

[Technical field]

[0001] The present invention relates to the treatment of solid tumors using a therapy involving an antibody drug conjugate and a nanoscale initiator of inertial cavitation. [Background technology]

[0002] AXL is a member of the human TAM family of receptor tyrosine kinases. The Gas6 / AXL signaling pathway is associated with tumor cell proliferation, metastasis, invasion, epithelial-mesenchymal transition (EMT), angiogenesis, drug resistance, immune regulation, and stem cell maintenance. Increased AXL expression has been associated with a number of cancers, including lung, breast, pancreatic, ovarian, colon, and melanoma, among others. A variety of therapeutic agents targeting AXL have been developed, typically including small molecule inhibitors, monoclonal antibodies (mAbs), nucleotide aptamers, soluble receptors, and several natural compounds. Summary of the Invention [Problem to be solved by the invention]

[0003] The present inventors have previously developed an antibody-drug conjugate comprising an anti-AXL antibody conjugated to a pyrrolobenzodiazepine (PBD) dimer. The present inventors have recently found that co-administration of the conjugate with a nanoscale cavitation initiator as disclosed herein significantly enhances the delivery of the conjugate to malignant cells in solid tumors. The nanoscale cavitation generator delivers stabilized nanoscale gas bubbles. Upon irradiation with focused therapeutic ultrasound, the bubbles expand and collapse, resulting in inertial cavitation. This creates a mechanical streaming effect that significantly enhances the delivery of the co-administered conjugate. [Means for solving the problem]

[0004] Thus, in a first aspect there is provided an antibody drug conjugate for use in a method for treating a solid tumor in a subject, said method comprising administering to the subject: (i) A pharmaceutical composition comprising a plurality of nanoparticles, each of the nanoparticles comprising: a cup having a cavity, the cavity having an opening of at least 50 nm in size; and a gas pocket present within the cavity; wherein the gas pocket is partially enclosed by the cup, wherein the nanoparticle comprises a single nanocup with a single cavity, and wherein the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier or diluent that is a liquid medium; and (ii) an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-AXL antibody conjugated to a pyrrolobenzodiazepine dimer; and exposing the subject to ultrasound.

[0005] In a second aspect, there is provided an antibody drug conjugate for use in a method for treating a solid tumor in a subject, the method comprising administering to the subject: (i) A pharmaceutical composition comprising a plurality of nanoparticles, each of the nanoparticles comprising: a cup having a cavity, the cavity having an opening of at least 50 nm in size; and a gas pocket present within the cavity; wherein the gas pocket is partially enclosed by the cup, wherein the nanoparticle comprises a single nanocup with a single cavity, and wherein the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier or diluent that is a liquid medium; and (ii) an antibody-drug conjugate comprising an anti-AXL antibody conjugated to a pyrrolobenzodiazepine dimer; and exposing the subject to ultrasound.

[0006] In a third aspect, there is provided use of an antibody-drug conjugate in the manufacture of a medicament for use in a method for treating a solid tumor in a subject, comprising administering to the subject: (i) A pharmaceutical composition comprising a plurality of nanoparticles, each of the nanoparticles comprising: a cup having a cavity, the cavity having an opening of at least 50 nm in size; and a gas pocket present within the cavity; wherein the gas pocket is partially enclosed by the cup, wherein the nanoparticle comprises a single nanocup with a single cavity, and wherein the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier or diluent that is a liquid medium; and (ii) an antibody-drug conjugate comprising an anti-AXL antibody conjugated to a pyrrolobenzodiazepine dimer; and exposing the subject to ultrasound. [Brief description of the drawings]

[0007] [Figure 1] FIG. 1 illustrates an exemplary nanoparticle as described herein. [Diagram 2] FIG. 1 shows a schematic diagram of a process for producing nanoparticles as described herein. [Diagram 3] 1 is a graph showing tumor volumes the day before treatment, illustrating the results of the study described in the Examples. [Figure 4] 1 is a graph showing cavitation recorded by passive acoustic mapping in animals treated with nanoparticles and exposed to ultrasound, illustrating the results of the study described in the Examples. [Diagram 5] 1 is a graph showing the mean tumor growth for each treatment group, illustrating the results of the study described in the Examples. [Figure 6] 1 is a graph showing individual tumor growth for different treatments illustrating the results of the study described in the Examples. [Figure 7] 7A-7B are graphs showing the results of the study described in the Examples, where FIG. 7A shows survival curves for all groups and FIG. 7B shows a representative pair of groups. [Figure 8] 1 is a graph showing the average mouse body weights by treatment group, illustrating the results of the study described in the Examples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Antibody-drug conjugates The antibody-drug conjugate for use in the disclosed method comprises an anti-AXL antibody conjugated to a cytotoxin comprising a pyrrolobenzodiazepine (PBD) dimer. The cytotoxin is connected to the antibody via one or more linkers, examples of suitable linkers are described in more detail below. In one embodiment, each linker is connected via the N10 position of one of the PBD moieties conjugated to the antibody as defined below. Thus, various aspects and embodiments of the disclosure herein are suitable for use in providing a PBD compound to a preferred site in a subject, with the conjugate in some embodiments releasing the active PBD compound preferably without retaining any portion of the linker. Prior to transport or delivery to a cell, the antibody-drug conjugate (ADC) is preferably stable and intact, i.e., the antibody remains linked to the drug moiety. The linker in this preferred embodiment is stable outside the target cell and may be cleaved at some effective rate inside the cell. An effective linker in one embodiment (i) maintains the specific binding properties of the antibody, (ii) allows intracellular delivery of the conjugate or drug moiety, (iii) remains stable and intact, i.e., is not cleaved, until the conjugate is delivered or transported to its target site, and (iv) maintains the cytotoxic, cell-killing or cytostatic effect of the PBD drug moiety. The stability of the ADC can be measured by standard analytical techniques, such as mass spectrometry, HPLC, and the separation / analysis technique LC / MS.

[0009] Anti-AXL antibody AXL is a member of the human TAM family of receptor tyrosine kinases. In one embodiment, the AXL polypeptide corresponds to Genbank Accession No. AAH32229, Version No. AAH32229.1 GL21619004, Record Updated: March 6, 2012 01:18 PM (SEQ ID NO: 11). The term "antibody" as used herein is used in the broadest sense and specifically includes monoclonal antibodies, including both complete antibodies and antibody fragments, so long as the monoclonal antibody exhibits the desired biological activity, e.g., the ability to bind AXL. Antibodies may be murine, rat, human, humanized, chimeric, or derived from other species. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to an antigen of a target of interest or portion thereof, including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin may be an immunoglobulin molecule of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or subclass or allotype (e.g., human G1m1, G1m2, G1m3, non-G1m1 [i.e., any allotype other than G1m1], G1m17, G2m23, G3m21, G3m28, G3m11, G3m5, G3m13, G3m14, G3m10, G3m15, G3m16, G3m6, G3m24, G3m26, G3m27, A2m1, A2m2, Km1, Km2 and Km3). Various immunoglobulin variants derived from conventional immunoglobulins, such as bispecific antibodies, scFvs, nanobodies, etc., are known in the art, and are all within the scope of the term "antibody" as long as they retain the CDRs required for AXL binding activity.

[0010] Thus, in one embodiment, the antibody comprises: (i) an immunoglobulin heavy chain variable region having a CDR1 region having an amino acid sequence set forth in SEQ ID NO:3, a CDR2 region having an amino acid sequence set forth in SEQ ID NO:4, and a CDR3 region having an amino acid sequence set forth in SEQ ID NO:5; and (ii) an immunoglobulin light chain variable region having a CDR1 region having an amino acid sequence set forth in SEQ ID NO:6, a CDR2 region having an amino acid sequence set forth in SEQ ID NO:7, and a CDR3 region having an amino acid sequence set forth in SEQ ID NO:8. The CDR sequences disclosed herein are identified and defined using the Kabat numbering scheme (Kabat et al., USDepartment of Health and Human Services, 1991). In one embodiment, the antibody comprises a VH domain having the sequence set forth in SEQ ID NO: 1. In another embodiment, the antibody comprises a VL domain having the sequence set forth in SEQ ID NO: 2. Thus, the antibody may comprise a VH domain and a VL domain, wherein the VH comprises the sequence set forth in SEQ ID NO: 1 and the VL domain comprises the sequence set forth in SEQ ID NO: 2. The VH and VL domain(s) in various embodiments form an antibody antigen-binding site that binds to AXL.

[0011] In one embodiment, the antibody is a complete antibody comprising a VH domain and a VL domain, the VH and VL domains having a sequence as set forth in SEQ ID NO:1 paired with SEQ ID NO:2, such as antibody 1H12 described in WO 2018 / 146189. Other anti-AXL antibodies include antibody 5F11 (and antibodies containing its CDRs) - described in WO 2015 / 193420); and antibodies 107, 148 and 733 (and variants thereof) (and antibodies containing their CDRs) described in WO 2018 / 007592.

[0012] Specific references to the CDRs of antibodies 107, 148 and 733 in WO 2018 / 007592 are reproduced below and incorporated by reference: (a) a VH region comprising the CD1, CDR2 and CDR3 sequences represented by SEQ ID NOs: 36, 37, and 38, respectively; and a VL region comprising the CDR1, CDR2 and CDR3 sequences represented by SEQ ID NOs: 39, GAS, and 40, respectively; (b) a VH region comprising the CDR1, CDR2 and CDR3 sequences represented by SEQ ID NOs: 46, 47 and 48, respectively; and a VL region comprising the CDR1, CDR2 and CDR3 sequences represented by SEQ ID NOs: 49, AAS and 50, respectively; (c) a VH region comprising CDR1, CDR2 and CDR3 sequences represented by SEQ ID NOs: 114, 115 and 116, respectively, and a VL region comprising CDR1, CDR2 and CDR3 sequences represented by SEQ ID NOs: 117, 118 and 119, respectively

[0733] .

[0013] Other anti-AXL antibodies include 20G7-D9 (Leconet et al., 2017, Clin Cancer Res 23(11):2806-2816); YW327.6S2 (Ye et al., 2010 Oncogene 29:5254-5264); and DAXL-88 (Duan et al., 2019, Scand. J. Immunol. 90(2):12777). As used herein, the phrase "binds to AXL" is used to mean that the antibody binds to AXL with higher affinity than a non-specific partner such as bovine serum albumin (BSA, Genbank Accession No. CAA76847, Version No. CAA76847.1 GI:3336842, Archive Last Updated: Jan. 7, 2011 02:30 PM). In certain embodiments, the antibody binds to AXL with a ....1 GI:3336842, Archive Last Updated: Jan. 7, 2011 02:30 PM). In certain embodiments, the antibody binds to AXL with a higher affinity than a non-specific partner such as bovine serum albumin (BSA, Genbank Accession No. CAA76847.1 GI:3336842, Archive Last Updated: Jan. 7, 2011 02:30 PM). In certain embodiments, the antibody binds to AXL with a higher affinity than a non-specific partner such as bovine serum albumin (BSA), as measured under physiological conditions. 4 , 10 5 or 10 6 The antibodies of the present disclosure can bind to AXL with high affinity in some embodiments. For example, in some embodiments, the antibodies can bind to AXL with an association constant (Ka) that is about 10 times higher. -6 M or less, e.g. 1×10 -6 , 10 -7 , 10 -8 , 10 -9 ,10 -10 , 10 -11 , 10 -12 , 10 -13 or 10 -14 It can bind to AXL with a KD of

[0014] An "antibody fragment" comprises a portion of a full-length antibody, generally the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and scFv fragments; diabodies; linear antibodies; fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity determining regions), and epitope-binding fragments of any of the above that immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0015] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical except for naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The term "monoclonal" refers to the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies as used in this disclosure may be produced by hybridoma technology or by recombinant DNA technology. Monoclonal antibodies may also be isolated from phage antibody libraries or from transgenic mice carrying a fully human immunoglobulin system. The monoclonal antibodies of the present specification specifically include "chimeric" antibodies in which portions of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chains are identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies include "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., an Old World monkey or ape) and human constant region sequences.

[0016] A "complete antibody" herein is an antibody comprising VL and VH domains, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. A complete antibody may have one or more "effector functions", which refer to the biological activities attributable to the Fc region (native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down-regulation of cell surface receptors such as B cell receptor and BCR.

[0017] Antibody Modifications The antibodies disclosed herein may be modified, for example, to reduce immunogenicity to a human subject. This may be accomplished using any of a number of techniques well known to those of skill in the art, including humanization to reduce the in vivo immunogenicity of non-human antibodies or antibody fragments. A variety of humanization techniques exist, including "CDR grafting", "guide selection", "deimmunization", "resurfacing" (also known as "veneering"), "composite antibodies", "human string content optimization" and framework shuffling.

[0018] PBD cytotoxin Pyrrolobenzodiazepines (PBDs) suitable for use in the present disclosure, in one embodiment, are capable of recognizing and binding to a specific sequence of DNA, with the preferred sequence being PuGPu. PBDs have the following general structure:

[0019] [ka] There is. They differ in the number, type and position of substituents on both their aromatic A-ring and pyrrolo C-ring, as well as the degree of saturation of the C-ring. The B-ring contains either an imine (N=C), a carbinolamine (NH-CH(OH)), or a carbinolamine methyl ether (NH-CH(OMe)) at the N10-C11 position, which is the electrophilic center responsible for DNA alkylation. All known natural products have an (S)-configuration at the chiral C11a position, which is twisted right-handed when viewed from the C-ring towards the A-ring. This provides a suitable three-dimensional shape for isohelicity with the minor groove of B-form DNA, resulting in a snug fit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Acc., 19, 230-237 (1986)). Their ability to form adducts in the minor groove interferes with DNA processing and can therefore be used as antitumor agents.

[0020] The cytotoxin typically has the following formula (II):

[0021] [ka] (In the formula, R LL is a linker for connecting to an antibody, examples and certain embodiments of which are provided in detail below under "Linkers" There are PBD dimers such as the PBD dimer Linker R LL When released from R LL The bond between N10 and C11, where R was originally attached, usually forms a double bond between the nitrogen and carbon atoms to which they are both attached (an imine (C=N)). Similarly, in the released drug, R 10 and R 11 usually form a double bond between the nitrogen and carbon atoms to which they are both attached. Thus, the released drug has the following formula RelA:

[0022] [ka] It may be.

[0023] R 10 and R 11 In one embodiment, R 10 and R 11 both form a double bond between the nitrogen atom and the carbon atom to which they are attached. In other embodiments, R 10 is a linker R for connecting to an antibody Ab LL A and R 11 is OH, and the definition of RLLA is R LL Similar to RLLA and R LL are the same or different. In other embodiments, R 10 is a capping group RC, and R 11 is OH. Capping groups can be used to reduce or inhibit the cytotoxic activity of the PBD, effectively forming a prodrug, which in this context is not linked to a cell-binding agent. The capping group is then removed, for example, in the target cell or tumor microenvironment, activating the drug. Various capping groups are described in Franzyk and Christiansen, 2021, Molecules 26:1292. (1) Prodrugs that are cleaved in acidic media, such as dithiocarbamates. (2) Prodrugs that are cleaved by reactive oxygen species. (3) Prodrugs that are cleaved by glutathione. (4) Prodrugs that are cleaved by expressed enzymes, such as oxidoreductases, hydrolases and matrix metalloproteinases. (5) Prodrugs that are cleaved by β-glucuronidase, such as R C is a glucuronide, e.g.,

[0024] [ka] (wherein the square brackets indicate that the NO2 group is optional, but in one embodiment, the NO2 group is present). may include: Capping groups can also be used to modify the physicochemical characteristics of an antibody-drug conjugate (e.g., to make the conjugate more stable). For example, capping groups can increase the hydrophilicity of an antibody-drug conjugate.

[0025] m In some embodiments, m is 0. In some embodiments, m is 1.

[0026] R 2 and R 12 In one embodiment, R 2 and R 12 is the same. In one embodiment, there is a double bond between C2 and C3 and between C2′ and C3, and R 2 and R 12 Both are methyl. In one embodiment, there is a single bond between C2 and C3 and between C2′ and C3, and R 2 and R 12 Both are H. In one embodiment, there is a single bond between C2 and C3 and between C2′ and C3, and R 2 and R 12 Both,

[0027] [ka] It is.

[0028] In a preferred embodiment, the drug linker LD has the following formula (III):

[0029] [ka] (In the formula, R LL , R 10 , R 11 and m have the same meaning as above. It is expressed as: In certain embodiments, R10 and R11 together form a double bond between the nitrogen atom and the carbon atom to which they are attached; and m is 0 (ie, SG2000 with a linker at N10).

[0030] In other embodiments, the drug-linker LD has the following formula (IV):

[0031] [ka] (In the formula, R LL , R 10 , R 11 and m is as defined above. It is expressed as: In certain embodiments, R10 and R11 together form a double bond between the nitrogen atom and the carbon atom to which they are attached; and m is 1 (ie, SG3199 with a linker at N10).

[0032] Drug-Linker A variety of linker technologies are available in the art for linking cytotoxins to cell-binding agents. Linkers may incorporate a variety of different moieties to contribute to the stability of antibody-drug conjugates and determine drug release properties. For example, linkers may include cleavable moieties, such as those cleavable by cathepsin B (e.g., valine-alanine or valine-citrulline). Another strategy is to use pH-sensitive linkers, where the lower pH of endosomal and lysosomal compartments leads to hydrolysis of acid-labile groups (e.g., hydrazones) in the linker. Alternative linkers may be non-cleavable, which can avoid or reduce off-target effects and improve plasma stability during circulation. The functionality that can be conjugated to the cell-binding agent is based on the site of conjugation and its chemistry. N-hydroxysuccinimide esters are a common choice for functionalizing amines, especially when coupling to ε-lysine residues. For conjugation to cysteine, thiol-reactive maleimides are the most applicable reactive handles, but disulfide bridges may be created by oxidation with linkers that contain sulfhydryl groups. When aldehyde or keto functional groups, such as oxidized sugar groups or pAcPhe unnatural amino acids, react with hydrazides and alkoxyamines, acid-labile hydrazone or oxime bonds can be generated. Additionally, hydrazines can be coupled to aldehydes via HIPS ligation to generate stable CC bonds.

[0033] A more recent approach is based on N-linked glycosylation sites in antibodies, such as Asn-297 on IgG molecules. GlycoConnect™ (Synaffix) enzymatically trims the N-linked glycans to a GlcNAc core, followed by further enzymatic processing to introduce an azide-containing activation moiety that can be used to incorporate a drug-linker using copper-free click chemistry. In one embodiment, the resulting drug-linker contains a GalNAc residue between the trimmed GlcNAc core and the remainder of the linker (e.g., the linkers shown as B1 or B2 below). Other aspects of linker chemistry include spacers and / or moieties, such as polyethylene glycol (PEG) chains or polar functional groups such as sulfonyl within the linker, that mask the hydrophobicity of the cytotoxic payload, reduce cellular efflux mechanisms, and / or increase overall stability. In one aspect, the antibody drug conjugates of the present disclosure can be described as Ab-LD, where Ab is an anti-AXL antibody, D is a PBD-containing cytotoxin, and L is a linker. The number of drug moieties per Ab (drug loading, p) depends on the number of linkers attached to each Ab and the number of drug moieties per linker. Typically, drug loading p is 1-8, e.g., 1-4, 1-2, or 2-4. In some embodiments, one drug moiety is attached to each linker, while in other embodiments, two or more drug moieties may be attached to each linker (e.g., a branched linker). Because variation can arise from the conjugation process, drug loading is usually considered on an average basis. Methods for determining average drug loading are known in the art.

[0034] In one embodiment, a linker (e.g., R LL (represented as) is represented by the following formula (IIa):

[0035] [ka] (In the formula, Q:

[0036] [ka] (In the formula, Q X wherein Q is an amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue; X(G LL ) is the following:

[0037] [ka] (In the formula, a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, c2=0 or 1, and d=0 to 5, where at least b1 or b2=0 (i.e., only one of b1 and b2 does not have to be 0), and at least c1 or c2=0 (i.e., only one of c1 and c2 does not have to be 0)). Represented by; and G LL is a linker group linked to Ab It is expressed as:

[0038] QX In one embodiment, Q is an amino acid residue. The amino acid may be a natural amino acid or an unnatural amino acid. In one embodiment, Q is selected from Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp, where Cit is citrulline. In one embodiment, Q comprises a dipeptide residue. The amino acids in the dipeptide can be any combination of natural and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. When the linker is a cathepsin-labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide is the recognition site for cathepsin.

[0039] In one embodiment, Q is: NH- Phe-Lys- C=O NH-Val-Ala- C=O NH- Val-Lys- C=O NH- Ala-Lys- C=O NH- Val-Cit- C=O NH- Phe-Cit- C=O NH- Leu-Cit- C=O NH- Ile-Cit- C=O NH- Phe-Arg- C=O NH- Trp-Cit- C=O , and NH- Gly-Val- C=O ; (wherein Cit is citrulline) is selected from.

[0040] Preferably, Q is one of the following: NH- Phe-Lys- C=O ; NH- Val-Ala- C=O ; NH- Val-Lys- C=O ; NH- Ala-Lys- C=O ; and NH- Val-Cit- C=O ; is selected from. Most preferably, Q is NH- Phe-Lys- C=O , NH- Val-Cit- C=O or NH- Val-Ala- C=O is selected from. Other interesting dipeptide combinations include the following: NH- Gly-Gly- C=O NH- Gly-Val- C=O NH- Pro-Pro- C=O , and NH- Val-Glu- C=O Examples include: Other dipeptide combinations may be used, including those described in Dubowchik et al., Bioconjugate Chemistry, 2002, 13, 855-869, incorporated herein by reference.

[0041] In one embodiment, Q is a tripeptide residue. The amino acids in the tripeptide can be any combination of natural and unnatural amino acids. In one embodiment, the tripeptide comprises natural amino acids. When the linker is a cathepsin-labile linker, the tripeptide is the site of action for cathepsin-mediated cleavage. The tripeptide is the recognition site for cathepsin. Particularly interesting tripeptide linkers are the following: NH- Glu-Val-Ala- C=O NH- Glu-Val-Cit- C=O NH- αGlu-Val-Ala- C=O NH- αGlu-Val-Cit- C=O It is.

[0042] In one embodiment, Q is a tetrapeptide residue. The amino acids in the tetrapeptide can be any combination of natural and unnatural amino acids. In one embodiment, the tetrapeptide comprises natural amino acids. When the linker is a cathepsin-labile linker, the tetrapeptide is the site of action for cathepsin-mediated cleavage. The tetrapeptide is the recognition site for cathepsin. Tetrapeptide linkers of particular interest are the following: NH- Gly-Gly-Phe-Gly- C=O (SEQ ID NO: 12); and NH- Gly-Phe-Gly-Gly- C=O (SEQ ID NO:13) It is. In one embodiment, the tetrapeptide has the structure: NH- Gly-Gly-Phe-Gly- C=O It is. In the above representation of peptide residues: NH- represents the N-terminus, -C=O represents the C-terminus of the residue, which is attached to an NH bonded to the benzene ring. Glu is:

[0043] [ka] Represents a glutamic acid residue. αGlu is:

[0044] [ka] Represents the glutamic acid residue when bound via the α chain.

[0045] In one embodiment, the amino acid side chains are chemically protected, if appropriate. The side chain protecting groups may be those discussed above. The protected amino acid sequence may be cleaved by an enzyme. For example, a dipeptide sequence containing a Boc side chain protected Lys residue may be cleaved by cathepsin. Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalog and as discussed above.

[0046] G LL G LL below:

[0047] [Table 1] (Wherein, Ar is C 5-6 represents an arylene group, e.g., phenylene; X is C 1-4 CBA represents an alkyl group linked to an antibody. LL ) can be selected from: In one embodiment, G LL is G LL1-1 and G LL1-2 In some of these embodiments, G LL is G LL1-1 It is. In another embodiment, G LL is G LL 10 and G LL11 In some of these embodiments, G LL is G LL10 It is.

[0048] C 5-6 Arylene: As used herein, the term "C 5-6 "Arylene" refers to a divalent moiety obtained by removing two hydrogen atoms from an aromatic ring atom of an aromatic compound. In this context, prefixes (e.g., C 5-6 ) indicates the number of ring atoms, whether carbon atoms or heteroatoms, or range of number of ring atoms. The ring atoms may be all carbon atoms, such as in a "carbarylene group," in which case the group is phenylene (C6). Alternatively, the ring atoms may contain one or more heteroatoms, such as in a "heteroarylene group." Examples of heteroarylene groups include the following: N1: Pyrrole (azole) (C5), pyridine (azine) (C6); O1: furan(oxoyl)(C5); S1: thiophene (thiol) (C5); N1O1: oxazole (C5), isoxazole (C5), isoxazine (C6); N2O1: oxadiazole (furazan) (C5); N3O1: oxatriazole (C5); N1S1: thiazole (C5), isothiazole (C5); N2: imidazole (1,3-diazole) (C5), pyrazole (1,2-diazole) (C5), pyridazine (1,2-diazine) (C6), pyrimidine (1,3-diazine) (C6) (e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine) (C6); and N3: triazoles (C5), triazines (C6); These include, but are not limited to, those derived from

[0049] C 1-4 Alkyl: As used herein, the term "C 1-4 "Alkyl" refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 4 carbon atoms, which may be aliphatic or alicyclic, and may be saturated or unsaturated (e.g., partially unsaturated, fully unsaturated). 1-n "Alkyl" refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having 1 to n carbon atoms, which may be aliphatic or alicyclic and may be saturated or unsaturated (e.g., partially unsaturated, fully unsaturated). Thus, the term "alkyl" includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., described below.

[0050] X X is:

[0051] [ka] (In the formula, a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, c2=0 or 1, d=0 to 5, at least b1 or b2=0, and at least c1 or c2=0.) It is expressed as: a can be 0, 1, 2, 3, 4, or 5. In some embodiments, a is 0 to 3. In some of these embodiments, a is 0 or 1. In further embodiments, a is 0. In further embodiments, a is 1. b1 may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b1 is 0 to 12. In some of these embodiments, b1 is 0 to 8, and may be 0, 2, 3, 4, 5, or 8. In further embodiments, b1 is 2. b2 may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b2 is 0-12. In some of these embodiments, b2 is 0-8, and may be 0, 2, 3, 4, 5, or 8. In further embodiments, b2 is 8. Only one of b1 and b2 may not be 0. c1 may be 0 or 1. c2 may be 0 or 1. Only one of c1 and c2 may not be 0. d can be 0, 1, 2, 3, 4, or 5. In some embodiments, d is 0 to 3. In some of these embodiments, d is 1 or 2. In further embodiments, d is 2. In further embodiments, d is 5. In some embodiments of X, a is 0, b1 is 0, c1 is 1, c2 is 0, d is 2, and b2 can be 0 to 8. In some of these embodiments, b2 is 0, 2, 3, 4, 5, or 8. In further embodiments, b2 is 8. In some embodiments of X, a is 1, b2 is 0, c1 is 0, c2 is 1, d is 2, and b1 can be 0 to 8. In some of these embodiments, b1 is 0, 2, 3, 4, 5, or 8. In further embodiments, b1 is 2. The linker is typically R in the following example embodiment: LL As shown in position N10, it is connected to the PBD dimer.

[0052] Drug Linker Embodiments In certain embodiments, the Drug-Linker LD is as follows:

[0053] [Table 2] (In the formula, R LL and RLLA are as above) is selected from.

[0054] In one embodiment, the LD comprises the following:

[0055] [Table 3] is selected from.

[0056] Conjugation Sites and Drug Loading Drug-linkers may be conjugated to cell-binding agents such as antibodies at several different sites using a variety of methods known in the art. Conjugation sites include cysteine ​​and lysine residues in the sequence (endogenous or recombinant), as well as sites of N-linked glycosylation after trimming (e.g., GlycoConnect™ or GlyClick™ approaches). Thus, in one embodiment, the drug-linker is conjugated via a trimmed Asn-GlcNAc residue, usually at the endogenous N-linked glycosylation site (Asn-297) in the antibody. With regard to cysteine ​​conjugation, in one embodiment, the cysteine ​​is an endogenous cysteine ​​located in the hinge region or Fc domain. In other embodiments, the cysteine ​​in the recombinant cysteine ​​is introduced into the hinge region or Fc domain. Drug loading is the average number of PBD drugs per cell-binding agent, eg, antibody. The average number of drugs per antibody in a preparation of ADCs derived from a conjugation reaction can be characterized by conventional means such as UV, reversed-phase HPLC, HIC, mass spectrometry, ELISA assay, and electrophoresis. The quantitative distribution of ADCs in terms of p can also be determined. The average value of p in a particular preparation of ADCs can be determined by ELISA (Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al (2005) Clin. Cancer Res. 11:843-852). However, the distribution of p(drug) values ​​is not differentiated by the detection limit of antibody-antigen binding and ELISA. Also, the ELISA assay for detection of antibody-drug conjugates does not determine where the drug moiety is attached to the antibody, e.g., the heavy or light chain fragment, or the specific amino acid residue. In some instances, separation, purification, and characterization of homogeneous ADCs with a particular value of p from ADCs with other drug loadings can be achieved by means such as reversed-phase HPLC or electrophoresis. The technique may also be applied to other types of conjugates. For some antibody-drug conjugates, p may be limited by the number of binding sites on the antibody. For example, an antibody may have only one or a few cysteine ​​thiol groups, or only one or a few sufficiently reactive thiol groups to which a linker may be attached. Higher drug loading, for example p>5, may result in aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates. Usually, less than the theoretical maximum drug moiety is conjugated to the antibody during the conjugation reaction. The antibody may contain many lysine residues that do not react with, for example, the drug-linker intermediate or linker reagent. Only the most reactive lysine groups can react with amine-reactive linker reagents. Also, only the most reactive cysteine ​​thiol groups can react with thiol-reactive linker reagents. In general, the antibody does not contain many, if any, free and reactive cysteine ​​thiol groups that may be linked to the drug moiety. Most cysteine ​​thiol residues in the antibody of the compound exist as disulfide bridges and must be reduced with a reducing agent such as dithiothreitol (DTT) or TCEP under partial or total reducing conditions. The loading (drug / antibody ratio) of the ADC can be controlled in several different ways, including (i) limiting the molar excess of the drug-linker intermediate or linker reagent relative to the antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limited reducing conditions for cysteine ​​thiol modification.

[0057] Some antibodies have reducible interchain disulfides, i.e. cysteine ​​bridges. Antibodies become reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine ​​bridge thus theoretically forms two reactive thiol nucleophilic groups. Additional nucleophilic groups can be introduced into antibodies via the reaction of lysine with 2-iminothiolane (Traut's reagent), resulting in the conversion of an amine to a thiol. Reactive thiol groups may be introduced into an antibody (or a fragment thereof) by engineering one, two, three, four, or more cysteine ​​residues (e.g., by preparing a mutant antibody containing one or more non-natural cysteine ​​amino acid residues). US Pat. No. 7,521,541 teaches recombinant antibodies by the introduction of reactive cysteine ​​amino acids. Cysteine ​​amino acids can be engineered at reactive sites in antibodies so as not to form intrachain or intermolecular disulfide bonds (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al (2009) Blood 114(13):2721-2729; U.S. Patent No. 7,521,541; U.S. Patent No. 7,723,485; WO 2009 / 052249). Engineered cysteine ​​thiols can react with the disclosed linker or drug-linker reagents that have thiol-reactive electrophilic groups, such as maleimides or α-haloamides, to form ADCs with cysteine ​​engineered antibodies and PBD drug moieties. Thus, the location of the drug moiety can be designed, controlled, and known. Engineered cysteine ​​thiol groups usually react with thiol-reactive linker or drug-linker reagents in high yield, allowing for controlled drug loading. Engineering an IgG antibody to introduce a cysteine ​​amino acid by substitution at a single site on the heavy or light chain results in two new cysteines on a symmetric antibody.

[0058] In some embodiments, the antibody may be modified to have reactive groups present on it (e.g., azide). In this case, p is limited by the number of binding sites on the antibody, i.e., the number of azide groups. For example, an antibody may have only one or two azide groups to which a drug linker may be attached.

[0059] When two or more nucleophilic or electrophilic groups on an antibody react with a drug-linker intermediate, or a linker reagent followed by a drug moiety reagent, the resulting product is a mixture of ADC compounds in which the drug moieties attached to the antibody are distributed, e.g., 1, 2, 3, etc. Liquid chromatography methods such as polymeric reversed phase (PLRP) and hydrophobic interaction (HIC) can separate compounds in a mixture by drug loading value. Although preparations of ADCs with a single drug loading value (p) can be isolated, these single loading value ADCs can still be heterogeneous mixtures because the drug moieties can be attached to different sites on the antibody via the linker. Thus, the antibody-drug conjugate compositions of the disclosure include a mixture of antibody-drug conjugate compounds, where the antibody has one or more PBD drug moieties, and the drug moieties may be attached to the antibody at different amino acid residues.

[0060] In one embodiment, the average number of dimeric pyrrolobenzodiazepine groups per antibody is in the range of 1 to 8. In certain embodiments, the range is selected from 1 to 4, 1 to 4, 2 to 4, and 1 to 3. In certain embodiments, there are one or two dimeric pyrrolobenzodiazepine groups per antibody. When there are two linking groups on LD, they are preferably to the same antibody. In some of these embodiments, there is only one LD attached to each antibody, and thus the drug loading is 1.

[0061] Preparation of drug conjugates The antibody drug conjugates of the present disclosure may comprise a drug-linker, such as the following drug linker (as defined herein above in the following formula (II):

[0062] [ka] The antibody can be prepared by conjugating a

[0063] As noted above, (i) the methods described in U.S. Pat. No. 9,889,207 or Flynn et al., 2016. Mol Cancer Ther 15: Several conjugation techniques are known in the art, such as conjugation to endogenous or engineered cysteine ​​residues via maleimides, such as conjugation via a chemoenzymatically trimmed N-linked glycosylation site, e.g., at Asn-297 or equivalent, using GlyClick or GlycoConnect, as described in WO 2018 / 146188, which describes trimming of a glycan isoform to the core GlcNAc using EndoS, followed by enzymatic transfer of an N-acetylgalactose residue to the core GlcNAc, typically with an azide group for conjugation to a drug-linker using a galactose transferase (GalT) or galactose-N-acetyltransferase (GalNAcT) enzyme (resulting in a GalNAc residue between the GlcNAc of the trimmed glycan and the remainder of the linker, such as linkers designated B1 or B2). When using the GalT enzyme, preferably the enzyme incorporates Y289L and / or C342T. Finally, the drug-linker is reacted with the azide group using copper-free click chemistry, such as the method described in van Geel, R. et al., Bioconjugate Chemistry, 2015, 26, 2233-2242. This method may be applied to compounds C1 and C2 below. For drug linkers, see, e.g., Tibergien et al., 2016, ACS Med. Chem. Lett. 7:983-987, WO 2014 / 057074, WO 2018 / 069490, and WO 2018 / 146188. In particular, the following table provides references for each drug-linker of particular interest.

[0064] [Table 4] Cavitation Initiator The nanoscale cavitation initiators described herein are nanoparticles with a size of 1 nm to 1000 nm. The size of the nanoparticles is usually at least 60 nm or at least 100 nm, preferably at least 300 nm and up to 1000 nm, for example up to 600 nm. For example, the size of the nanoparticles may be 60 to 1000 nm or 300 to 600 nm, preferably 400 to 600 nm or 400 to 500 nm. The size of a nanoparticle is usually the average diameter of the nanoparticles (s in Figure 1). The diameter is usually determined by electron microscopy. When multiple nanoparticles are present in a composition, the cumulant average is usually taken as the size of the nanoparticle. This can be determined by dynamic light scattering.

[0065] The nanoparticles contain a single nanocup, each nanocup containing a single gas pocket within its cavity. Using nanoparticles with a single cup or cavity and a single gas pocket stabilized therein provides a more predictable and reliable response to applied ultrasound. By controlling the size of the nanocup and the size of the stabilized gas bubble, the particle can be tailored to respond to specific ultrasound conditions. Shallow or small depressions or defects on the surface of the nanoparticle are not considered cavities in the context of the present invention. Such small depressions or defects are usually unable to stabilize a gas pocket within the cavity. Thus, the opening of the cavity is at least 50 nm in size. Any depression that is usually less than about 5 nm, usually less than about 10 nm or less than about 20 nm deep is not considered a cavity in the context of the present invention. Thus, a nanoparticle with a single cavity may contain one or more such depressions on its surface with an opening size of less than 50 nm and / or a depth of less than about 5 nm, usually less than about 10 nm or less than about 20 nm.

[0066] The nanocups are typically substantially partially spherical. The term "partially spherical" refers to a shape that forms a portion of a sphere. The term "partially spherical" includes shapes such as spherical caps, hemispheres, and a sphere that is truncated on its equator (i.e., the portion of a sphere complementary to a spherical cap). Substantially partially spherical includes shapes such as shapes that form a portion of a spheroid, and shapes that form a portion of a sphere or spheroid with irregularities such as recesses and protrusions.

[0067] The wall thickness of the cup refers to the shortest distance from a point on the surface of the cup facing the cavity, through the cup, to a point on the opposite surface of the cup (w in FIG. 1). The depth of the cup refers to the maximum distance from the height of the rim of the cup to the base of the cup (d in FIG. 1). The wall thickness and depth of the cup can be determined by transmission electron microscopy. The wall thickness (w) of the cup is typically 10-100 nm, for example 30-70 nm or about 50 nm. The wall thickness of the cup can be selected and precisely controlled based on the methods disclosed herein for producing nanoparticles.

[0068] In some embodiments, the wall thickness may vary within a cup or from cup to cup in the composition, and an average can then be determined. In some embodiments, the thickness may be uniform or substantially uniform within a cup or from cup to cup in the composition. The wall thickness of the cup is usually substantially uniform both within a single nanoparticle and from one nanoparticle to another nanoparticle of the composition of the present invention. The wall thickness is usually a constant percentage of the overall cup size. As used herein, the term "substantially uniform thickness" refers to a shape that has a maximum thickness that is no more than 25% greater than its minimum thickness, e.g., no more than 20% greater, no more than 15% greater, no more than 10% greater, or no more than 5% greater.

[0069] The desired size and shape of the cavity containing the gas pocket is determined by the ultrasonic frequency used to minimize the rarefaction pressure amplitude required to initiate inertial cavitation, typically in the range of 0.5 MHz to 5.0 MHz. Ultrasonic pressure amplitudes below 5 MPa are preferred, such as 4 MPa or less, or 3 MPa or less. Ultrasonic pressure amplitudes as low as 0.5 MPa can produce extensive inertial cavitation at frequencies as high as 2 MHz in the presence of nanocups.

[0070] The opening of a nanocup cavity is the largest dimension of the cavity measured at the rim of the cup (p in FIG. 1), typically the portion of the cup that forms the boundary between the enclosed portion of the gas pocket and the unenclosed portion of the gas pocket. In the case of a substantially partially spherical cavity, the opening is substantially circular and the size is measured as the diameter of the opening. The size of the opening of a cavity may be determined by transmission electron microscopy. At least a portion of the cavity, optionally the entire cavity, is typically substantially partially spherical, e.g., substantially hemispherical or semispherical. The cavity typically has an opening size (p) of 50-900 nm, e.g., at least 100 nm and no more than 700 nm, or no more than 600 nm, diameter at the opening for a substantially partially spherical cavity. The cavity may be, for example, 50-700 nm or 100-600 nm. One skilled in the art will be able to select the appropriate cavity shape and size to complement the desired ultrasonic parameters and produce nanoparticles with the desired cavity shape and size based on the methods disclosed herein for producing nanoparticles. The ratio of the size of the opening to the size of the nanoparticle (ratio p:s in FIG. 1) is typically 1:3 to 5:6, for example 1:2 to 2:3. The depth of the cavity is usually greater than 30 nm, preferably greater than 50 nm, for example, the depth of the cavity is usually 60 to 500 nm, for example, 80 to 400 nm.

[0071] The gas pockets are partially encapsulated by the nanocups. A portion of the gas pocket is usually exposed to the liquid medium in which the particles are provided. This can cause the gas pockets to expand and collapse resulting in inertial cavitation. The gas pockets are usually gas bubbles with a diameter of at least 50 nm, preferably at least 100 or 150 nm. The maximum diameter of the gas bubbles may be 300 nm or less. Thus, in one embodiment, the gas bubbles are between 150 nm and 250 nm. The gas present within the gas pockets is typically, but not limited to, air, nitrogen, oxygen, a perfluorocarbon such as perfluoropropane, or a mixture thereof, In one embodiment, the gas pockets are gas bubbles. The gas pocket may be the same size and shape as the cavity. For example, if the cavity is a hemisphere with diameter d at its opening, the gas pocket may be a hemisphere with diameter d. Alternatively, the gas pocket may be larger than the cavity, i.e., protrude from the rim of the cup. It may protrude from the edge of the cup a distance of 10% or more, e.g., 20% or more, 50% or more, 80% or more, or 100% or more of the cup depth. The depth of the cup and the extent to which the gas pocket may protrude may be determined using a transmission electron microscope. In an embodiment, the cavity is partially spherical or substantially partially spherical with a curvature of radius r, and the gas pocket is a sphere with radius r. In this example embodiment, the cavity is a hemisphere with diameter d at its opening, and the gas pocket is a sphere with radius 1 / 2d.

[0072] The cup is typically formed from a polymeric material, but may be formed from any solid material that may be formed into a nanocup, and thus the cup typically comprises a polymer, and preferably consists essentially of a polymer. For example, the cup can be formed of a polymer that can be formed by an emulsion polymerization procedure. Suitable polymers include poly(methyl methacrylate) (PMMA), poly(2-hydroxyethyl methacrylate) (pHEMA), copolymers (e.g., poly(lactic-co-glycolic acid) (PLGA), polystyrene and divinylbenzene), and methyl methacrylate / 2-hydroxyethyl methacrylate copolymers, as well as pH- or temperature-responsive polymers (e.g., poly(N-isopropylacrylamide) (PNIPAM)). Other suitable polymers include, but are not limited to, polymers classified as polyanhydrides. In some embodiments, the cup is formed from a cross-linked polymer. Cross-linking of the polymer can be achieved by introducing a cross-linking agent into the monomer mixture prior to or during polymerization. Suitable cross-linking agents include divinylbenzene (DVB) or vinyl silane. Cross-links can also be present in the polymer in the form of regions of disulfide bonds. Regions of disulfide bonds can be introduced using thiol chemistry. Cross-linked polymers can be used to provide rigidity to the nanoparticles. Rigidity may be desirable, for example, to reduce or prevent shattering of the cup during cavitation. The degree of cross-linking can be determined by various spectroscopic methods, such as Raman spectroscopy or nuclear magnetic resonance spectroscopy, or polymer swelling techniques. The polymeric cup may include a polymer formed from two or more, e.g., two or three, different monomer units. The polymer may be produced by copolymerizing a mixture of monomers and / or by providing one or more prepolymers that are linked by copolymerization or via a crosslinker. The cup is typically biocompatible, i.e., capable of performing its desired function in relation to a medical therapeutic or diagnostic method without inducing therapeutically unacceptable local or systemic effects in the recipient or beneficiary of that treatment or diagnosis.

[0073] Pharmaceutical compositions comprising nanoparticles as described herein include, in addition to the nanoparticles, a pharma- ceutically acceptable carrier or diluent, which is a liquid medium. The nanoparticles are usually provided as a suspension in a liquid medium. This facilitates delivery of the nanoparticles, usually by parenteral administration. The nanoparticles may optionally be stored in a dry form and resuspended before use. Pharmaceutical compositions usually include up to 85% by weight, for example up to 50% by weight, of nanoparticles. Pharmaceutical compositions should usually be non-toxic, sterile, and pyrogen-free.

[0074] Suitable pharma- ceutically acceptable carriers and diluents are suitable for parenteral administration and include aqueous solutions and sterile water. Glucose, saccharose, mannitol, glycerin, sorbitol or combinations thereof may be present in the aqueous solution. Dilute glucose solutions, such as 0.5-10%, preferably 1-8%, for example about 5% glucose solutions, may be used. If desired, the pharmaceutical composition may include one or more additional excipients, carriers, buffers, stabilizers, antioxidants or other materials as required. Pharmaceutically acceptable carriers, diluents and other excipients, buffers, stabilizers, antioxidants or other materials should be non-toxic and should not interfere with the effectiveness of the nanoparticles.

[0075] Pharmaceutical compositions containing a plurality of nanoparticles usually contain substantially uniform nanoparticles, providing greater precision and control of the acoustic properties of the particles. Usually, the particle size distribution is narrow, with a polydispersity index of 0.2 or less. The average particle size of a composition containing a plurality of nanoparticles is defined herein as the modal mean (i.e., the particle size indicated by the largest number of particles). In a preferred embodiment of the invention, at least 50% of the nanoparticles in the composition are within an average of 50 nm in size. More preferably, at least 60%, 70% or 80% of the nanoparticles are within an average of 50 nm in size. Even more preferably, at least 50% of the nanoparticles in the composition are within an average of 20 nm in size. More preferably, at least 60%, 70% or 80% of the nanoparticles are within an average of 20 nm in size. Dynamic light scattering can be used to determine the particle size variation within the composition. The average cavity size (cavity opening size, ρ) of a composition comprising a plurality of nanoparticles is defined herein as the modal mean (i.e., the cavity size exhibited by the largest number of particles). In a preferred embodiment of the present invention, the cavity sizes of at least 50% of the nanoparticles in the composition are within 50 nm on average. More preferably, the cavity sizes of at least 60%, 70% or 80% of the nanoparticles are within 50 nm on average. Even more preferably, the cavity sizes of at least 50% of the nanoparticles in the composition are within 20 nm on average. More preferably, the cavity sizes of at least 60%, 70% or 80% of the nanoparticles are within 20 nm on average. Dynamic light scattering can be used to determine the variation in cavity opening size within a composition. The concentration of nanoparticles in the composition can be determined by one skilled in the art depending on the subject and the ultrasound parameters used. A typical composition contains at least 0.01 mg / mL to 500 mg / mL. Preferably, the concentration of nanoparticles in the composition is in the range of 100-500 mg / mL, for example 100-300 mg / mL.

[0076] Manufacturing method of nanocups The nanoparticles can be produced by forming a cup by a seeded emulsion polymerization technique and drying the cup in the presence of a gas to introduce gas pockets. The size and shape of the cavity may be selected and precisely controlled by choosing the seed particles used. Thus, seeded emulsion polymerization techniques typically use seed particles that have a size and shape complementary to the desired size and shape of the cavity. For example, spherical seed particles may be used to produce cups with partially spherical cavities. Larger seed particles can be used to form larger cavities, and similarly, smaller seed particles can be used to produce smaller cavities. The size of the seed particles used is typically 50 to 900 nm, for example at least 50 or at least 100 nm, and up to 600 nm, up to 400 nm, or up to 300 nm.

[0077] Emulsion polymerization is typically carried out by mixing seed particles, monomers, and optionally a crosslinker in a suitable medium, such as water, mixing to create turbulent flow conditions, and adding a suitable initiator, such as potassium persulfate. The emulsion polymerization reaction is allowed to continue at a suitable temperature for a suitable period of time. Although one skilled in the art can select appropriate temperature and time parameters for the particular reaction mixture used, polymerization is typically carried out at a temperature of 70-90° C. for 4-6 hours, for example, about 80° C. for about 5 hours. The polymerization is usually a free radical polymerization process. The polymer coated on the seed particles can be a solid or liquid polymer that copolymerizes with the seed particles.

[0078] When the cup is formed from two or more monomer units, different monomers may be provided to the emulsion polymerization at the beginning of the polymerization, or alternatively, one or more monomers may be provided during the polymerization process. Alternatively, one or more monomer units may be prepolymerized and the prepolymer provided to the polymerization mixture either at the beginning of the polymerization or during the polymerization. Typically, one or more monomers or prepolymers may have a higher affinity for the seed particles. Thus, the polymerization may provide a cup with one monomer unit (i.e., the monomer with the greatest affinity for the seed particles) formed predominantly on the inner surface of the cup and a different monomer unit formed predominantly on the outer surface of the cup. This may provide a cup with different surface properties on the inner and outer surfaces.

[0079] In the case of polymer nanocups, the seed particles may remain bonded, e.g., chemically bonded, to the inner surface of the nanocups, so that the seed particle material is itself incorporated into the cup. In case of complete reaction of all available seed particles, no seed particles remain that need to be removed. Large aggregates and small by-products are removed by centrifugation and filtration. The final shape, size, and successful formation of the nanocups can be confirmed by transmission electron microscopy (TEM).

[0080] The wall thickness of the cup can be controlled by selecting the amount of monomer mixture relative to the amount of seed particles present in the reaction mixture. Increasing the amount of monomer relative to the number of seed particles will result in a thicker cup wall, while decreasing the amount of monomer relative to the number of seed particles will result in a thinner cup wall. Additionally, the wall thickness of the cup can be adjusted by controlling the duration for which polymerization occurs, i.e., a longer reaction time will result in a thicker cup, provided sufficient reactants are present.

[0081] After emulsion polymerization, the product may be washed, for example by centrifugation, to remove excess polymer. After washing, the product is usually suspended in a suitable medium, such as water, before drying. The product may be dried by any suitable technique, such as freeze-drying or air-drying. In the case of freeze-drying, cryoprotectants are usually not used. Air-drying is preferred and may be carried out, for example, at elevated temperature or in a desiccator. Air-drying may be carried out for 12 to 36 hours, for example 24 hours. In one embodiment, air-drying is carried out at a temperature of 40-50° C. for 6 to 18 hours, for example about 12 hours, followed by 30 hours in a desiccator at 18-24° C. When drying in air, the gas pockets that form in the cavities are gas bubbles. When the drying step is performed in a desired gas, an alternative gas may be used for the gas pockets. Alternatively, drying can be performed in air and the resulting nanoparticles with air gas pockets can be subjected to a vacuum to remove the gas bubbles and contacted with a desired gas to introduce new gas pockets of the desired gas. Drying the nanocups in a gas atmosphere provides the required gas pockets in the cavities of the cups. No gas pockets are present prior to drying.

[0082] The dried nanocups may be resuspended in a liquid medium, such as an aqueous solution or other suitable pharma- ceutically acceptable carrier or diluent, as described herein. The solution is then typically mixed to maximize particle dispersion. The dried nanocups retain their gas pockets upon resuspension. Thus, nanocups dried and resuspended in a gaseous atmosphere differ from those formed directly in solution in that gas pockets are present in the cavities during the drying process.

[0083] After resuspension of the nanocaps, it is preferable to filter the solution to remove any aggregated material. Syringe filtration, for example at 1-5 μm, typically 1.5-3 μm, can be used to remove fine particles. This limits the particles present to those with nanoparticle sizes below the filtration limit. A combination of coarse filtration followed by syringe filtration may be used.

[0084] Figure 2 shows a schematic representation of the process of producing nanocups according to the invention. Figure 2a shows individual template particles coated with a copolymer, inducing swelling and deformation of the spherical particle. The obtained nanocups were dried and suspended to trap gas in the cavity. Figure 2b proposes a schematic representation of the mechanism of inertial cavitation of nanoparticle systems. First, a gas bubble is stabilized in the cavity. With sufficient negative pressure, the gas bubble can spread to the mouth of the cavity and finally detach from the particle. Inertial cavitation occurs when a detached gas bubble grows unsteadily before finally collapsing under the inertia of the surrounding medium.

[0085] Disorders Treated The treatment described herein includes anti-cancer activity applications. In particular, in some embodiments, the treatment includes an antibody conjugated to a PBD drug moiety, i.e., a toxin, i.e., a linker is covalently attached. When the drug is not conjugated to an antibody, the PBD drug has a cytotoxic effect. Thus, the biological activity of the PBD drug moiety is regulated by conjugation to an antibody. The antibody drug conjugate (ADC) of the present disclosure selectively delivers an effective dose of cytotoxic drug to tumor tissue, thereby allowing for higher selectivity, i.e., a smaller effective dose is required.

[0086] Co-administration of ADC with nanoparticles and ultrasound enhances targeted delivery of ADC to tumor sites at low doses. Ultrasound is applied to the affected area after co-administration of ADC and nanoparticles, which induces inertial cavitation in the treatment area. Microstreaming resulting from the inertial cavitation effect enhances transport of nanoparticles and ADC to tumors upon co-administration. Inertial cavitation can deliver ADC further within the tumor mass.

[0087] Accordingly, the present disclosure provides a method of treating a solid tumor comprising administering (i) a composition comprising a plurality of nanoparticles described herein (also referred to as a nanoparticle composition), and (ii) a conjugate described herein, and exposing the subject to ultrasound. Also provided is a method of treating a solid tumor in a subject, comprising administering (i) a nanoparticle composition described herein and (ii) a conjugate described herein, and exposing the subject to ultrasound. A therapeutically effective amount of the conjugate is administered to the subject. The amount of the nanoparticle composition administered to the subject is an amount effective to induce inertial cavitation upon exposure to ultrasound. Also provided herein is the use of a conjugate described herein in the manufacture of a medicament for use in a method of treating a solid tumor in a subject, the method comprising administering (i) a nanoparticle composition described herein, and (ii) a conjugate described herein, and exposing the subject to ultrasound. Also provided are nanoparticle compositions described herein for use in a method of treating a solid tumor in a subject, the method comprising administering (i) a nanoparticle composition described herein, and (ii) a conjugate described herein, and exposing the subject to ultrasound. Also provided herein is the use of a nanoparticle composition described herein for the manufacture of a medicament for use in a method comprising administering (i) a nanoparticle composition described herein, and (ii) a conjugate described herein, and exposing the subject to ultrasound.

[0088] The treatments described herein may be used for the treatment of proliferative disorders. The term "proliferative disorder" relates to unwanted or uncontrolled cell proliferation, either in vitro or in vivo, of unwanted, excessive or abnormal cells, such as neoplastic or hyperplastic growth. The proliferative disorder may be characterized by the presence of a neoplasm that contains both AXL+ve and AXL-ve cells. The target neoplasm or neoplastic cell can be all or part of a solid tumor, for example an advanced solid tumor. Thus, in one embodiment, the neoplasm / cancer is itself essentially AXL+ve. Examples of proliferative conditions that can be treated using the methods and conjugate compounds described herein include, but are not limited to, benign, pre-neoplastic, and malignant cell proliferation, including neoplasms, tumors, and cancers. In one embodiment, the proliferative condition is a solid tumor. Particular proliferative conditions to be treated include lung cancer (such as non-small cell lung cancer), breast cancer (such as triple negative breast cancer), pancreatic cancer, ovarian cancer, prostate cancer, colon cancer, colorectal cancer, renal cell carcinoma, thymic carcinoma, glioblastoma, and melanoma.

[0089] Patient Selection In some embodiments, individuals are selected prior to treatment as being suitable for treatment with that treatment. As used herein, an individual considered suitable for treatment is one who is expected to benefit from or respond to the treatment. The individual may have, be suspected of, or be at risk for cancer. The individual may have been diagnosed with cancer. Typically, the individual is an animal or human subject. In some aspects, the individual is selected based on the amount or pattern of expression of the first target protein, hi some aspects, the selection is based on expression of the first target protein on the cell surface.

[0090] In some cases, the individual is selected based on having or being suspected of having cancer, being at risk of cancer, or having been diagnosed with a proliferative disease characterized by the presence of a neoplasm that includes cells with high levels of surface expression of AXL. The neoplasm may be composed of cells with high levels of surface expression of AXL. In some cases, high levels of surface expression means that the average number of anti-AXL antibodies bound to a single neoplastic cell is 70,000 or more, e.g., 80,000 or more, 90,000 or more, 100,000 or more, 110,000 or more, 120,000 or more, 130,000 or more, 140,000 or more, or 150,000 or more.

[0091] In some cases, the individual is selected based on having or being suspected of having cancer, being at risk of cancer, or having been diagnosed with a proliferative disease characterized by the presence of a neoplasm comprising cells with low levels of surface expression of AXL. The neoplasm may be composed of cells with low levels of surface expression of AXL. In some cases, low levels of surface expression means that the average number of anti-AXL antibodies bound to a single neoplastic cell is less than 20,000, e.g., less than 80,000, less than 70,000, less than 60,000, less than 50,000, less than 40,000, less than 30,000, less than 20,000, less than 10,000, or less than 5,000.

[0092] In some embodiments, the individual is selected based on having a neoplasm that includes both AXL+ve cells and AXL-ve cells. The neoplasm may be composed of AXL-ve neoplastic cells, and optionally, the AXL-ve neoplastic cells are associated with AXL+ve neoplastic cells or non-neoplastic cells. The neoplasm or neoplastic cells may be all or part of a solid tumor. The solid tumor may be partially or entirely AXL-ve. In some cases, expression of AXL is determined in a particular tissue of interest, such as a sample of tumor tissue. In some cases, systemic expression of the target is determined, such as in a sample of a circulating fluid, such as blood, plasma, serum, or lymph.

[0093] In one embodiment, individuals are selected as suitable for treatment based on the presence or absence of AXL expression in the sample, in which case individuals who do not express AXL may be considered unsuitable for treatment. In another embodiment, the level of AXL expression is used to select an individual suitable for treatment. If the expression level of AXL is above a threshold level, the individual is determined to be suitable for treatment. In some embodiments, the presence of AXL in cells in the sample indicates that the individual is suitable for treatment with the ADC disclosed herein. In other embodiments, the amount of AXL expression must exceed a threshold level to indicate that the individual is suitable for treatment. In some embodiments, the observation that AXL localization is altered in the sample compared to the control indicates that the individual is suitable for treatment. In some embodiments, a patient is determined to be suitable for treatment if at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more of the total cells in the sample express the first target protein. In some embodiments disclosed herein, a patient is determined to be suitable for treatment if at least 10% of the cells in the sample express the first target protein. In some embodiments, a patient is determined to be suitable for treatment if at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more of the total cells in the sample express the second target protein. In some embodiments disclosed herein, a patient is determined to be suitable for treatment if at least 10% of the cells in the sample express the second target protein.

[0094] In some embodiments, the sample is taken from a bodily fluid, more preferably a bodily fluid circulating in the body. Thus, the sample may be a blood sample or a lymphatic sample. In some cases, the sample is a urine sample or a saliva sample. In some cases, the sample is a blood sample or a blood-derived sample. A blood-derived sample can be a selected fraction of an individual's blood, such as a selected cell-containing fraction or a plasma or serum fraction. The selected cell-containing fraction may contain a cell type of interest, which may include white blood cells (WBCs), in particular peripheral blood mononuclear cells (PBCs) and / or granulocytes, and / or red blood cells (RBCs). Thus, the methods according to the present disclosure include the detection of a first target polypeptide or nucleic acid in blood, white blood cells, peripheral blood mononuclear cells, granulocytes and / or red blood cells.

[0095] In other embodiments, the sample is a solid tissue biopsy. The sample may be fresh or archived. For example, archived tissue may be from a biopsy at the individual's initial diagnosis or at the time of recurrence. In some embodiments, the sample is a fresh biopsy. The terms "subject," "patient," and "individual" are used interchangeably herein.

[0096] In some embodiments disclosed herein, an individual is identified as having or suspected of having a proliferative disease such as cancer, or as being at risk for cancer. In some embodiments disclosed herein, the individual has already been diagnosed with the disease. A list of relevant diseases is provided above. Glioblastoma (e.g., glioblastoma multiforme), ovarian cancer, breast cancer, colon cancer, pancreatic cancer, thyroid cancer, and melanoma are conditions of particular interest. In some cases, the individual has been diagnosed with a proliferative disease, such as cancer, one of the disorders listed above. Glioblastoma (e.g., glioblastoma multiforme), ovarian cancer, breast cancer, colon cancer, pancreatic cancer, thyroid cancer, and melanoma are conditions of particular interest. In some cases, the individual has been diagnosed with a solid tumor containing AXL+ expressing cells. The individual may be undergoing or may have undergone therapeutic treatment for the cancer. The subject may or may not have previously received an anti-AXL ADC. In some cases, the cancer is glioblastoma (e.g., glioblastoma multiforme), ovarian cancer, breast cancer, colon cancer, pancreatic cancer, thyroid cancer, or melanoma.

[0097] Treatment method The term "treatment / treatment" as used herein in the context of treating a condition generally relates to treatments and therapies, whether in humans or animals (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, such as inhibition of progression of the condition, including slowing the rate of progression, halting the rate of progression, regressing the condition, ameliorating the condition, and curing the condition. Treatment as a prophylactic measure (i.e., prevention, prophylaxis) is also included. The term "therapeutically effective amount" or "effective amount" as used herein relates to an amount of an active compound, or a material, composition or dosage form containing an active compound, that is effective to produce some desired therapeutic effect, commensurate with a reasonable benefit / risk ratio, when administered in accordance with a desired treatment regimen. Similarly, the term "prophylactically effective amount" as used herein relates to an amount of active compound, or material, composition or dosage form containing an active compound, that is effective, when administered in accordance with a desired treatment regimen, for producing some desired prophylactic effect, commensurate with a reasonable benefit / risk ratio.

[0098] Methods of treatment are disclosed herein. Methods of treatment are also provided, comprising administering to a subject in need of treatment a therapeutically effective amount of a composition comprising an ADC and nanoparticles as described herein. The term "therapeutically effective amount" is an amount sufficient to show benefit to the subject. The benefit may be at least an improvement in at least one symptom. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the subject being treated. Prescribing treatment, e.g., determining dosage, is within the responsibility of general practitioners and other medical practitioners. Subjects may be tested to determine eligibility to be treated with the methods disclosed herein. Methods of treatment may include determining whether a subject is eligible for treatment using the methods disclosed herein. The nanoparticle composition is administered in an amount effective to induce inertial cavitation. Inertial cavitation may be monitored and detected using passive acoustic mapping (WO 2010 / 052494). Treatment may involve administration of the ADCs (and nanoparticle compositions) alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition being treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (e.g., administration of active agents including drugs such as chemotherapeutic agents), surgery, and radiation therapy.

[0099] A "chemotherapeutic agent" is a compound useful in the treatment of cancer, regardless of mechanism of action. Classes of chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, spindle poison plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include compounds used in "targeted therapy" and conventional chemotherapy. The term "chemotherapeutic agent" is further defined as: (i) anti-hormonal agents, such as anti-estrogens and selective estrogen receptor modulators (SERMs), which act to regulate or inhibit hormone action on tumors; (ii) aromatase inhibitors, which inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands; (iii) anti-androgens; (iv) protein kinase inhibitors, such as MEK inhibitors; (v) lipid kinase inhibitors; and (vi) anti-angiogenic agents. Also included within the definition of "chemotherapeutic agent" are therapeutic antibodies.

[0100] The composition comprising the conjugate compound is preferably a pharmaceutical composition. Pharmaceutical compositions according to and for use according to the present disclosure may comprise an active ingredient, e.g., a conjugate compound, and a pharma- ceutically acceptable excipient, carrier, buffer, stabilizer, or other material known to those skilled in the art. Such materials should be non-toxic and not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, usually by injection, e.g., cutaneous, subcutaneous, or intravenous. For intravenous, cutaneous or subcutaneous injection, or injection into the affected area, the active ingredient is in the form of a pyrogen-free parenterally acceptable aqueous solution with appropriate pH, isotonicity and stability. Those skilled in the art are well able to prepare suitable solutions using isotonic vehicles such as, for example, Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection, etc. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as necessary. The nanoparticle composition may be administered by injection, for example, intravenously, cutaneously or subcutaneously, typically by intravenous administration. The nanoparticle composition and the complex may typically be administered separately, simultaneously, sequentially, or there may be a period between administration of the two agents. The nanoparticle composition may be administered before the complex. Alternatively, the complex may be administered before the nanoparticle composition.

[0101] After administration of the complex and nanoparticle composition, the subject is exposed to ultrasound at a pressure and frequency suitable for clinical use. Ultrasound is applied to the affected area. Typically, low intensity focused ultrasound is used. The intensity of low intensity ultrasound is typically similar to or lower than that used in routine clinical practice for diagnostics. Ultrasound can be pulsed or continuous. The amplitude of the ultrasonic pressure is preferably less than 5 MPa, for example 4 MPa or less, or 3 MPa or less. Thus, typical peak negative ultrasonic pressures are in the range of 0.5 MPa to 5 MPa, for example 1 to 4 MPa, for example 1.5 to 3 MPa. The ultrasonic frequency (fc) may be up to 5 MHz, for example up to 2 MHz, for example up to 1 MHz, or about 0.5 MHz. Typically, the frequency (fc) used is in the range of 0.1 to 5 MHz, for example 0.1 to 1 MHz. The pulse repetition frequency may be up to 20 kHz, for example up to 1 kHz, or up to 1 Hz. The ultrasonic exposure may last up to 120 minutes, for example up to 60 minutes, or up to 30 minutes. Typically, the ultrasonic exposure is performed when there is effective circulation of the complex and the nanoparticle composition in the bloodstream of the subject. Preferably, the ultrasonic administration is performed during the period of peak circulation of the complex.

[0102] Cavitation may be monitored during exposure, for example, using passive acoustic mapping (WO 2010 / 052494), to maintain cavitation energy within a desired range, for example, below 1 nJ, or within the range of 0.1 pJ to 1 nJ, for example, 0.5 pJ to 0.5 nJ, using passive acoustic mapping. Varying peak negative pressure ultrasound can be used to control cavitation energy. Passive acoustic mapping can also be used to monitor delivery of the complex to the tumor in real time. Cavitation in a subject can be monitored to determine the extent and location of complex delivery.

[0103] Dosage It will be understood by those skilled in the art that the appropriate dosage of the composition comprising the ADC and the active ingredient may vary from subject to subject. Determining the optimal dosage generally involves balancing the degree of therapeutic benefit against any risk or adverse side effects. The selected dosage level will depend on a variety of factors, including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of treatment, other drugs, compounds, and / or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health, and previous medical history of the subject. The amount of compound and the route of administration are ultimately at the discretion of the physician, veterinarian, or clinician, but generally, the dosage will be selected to achieve a local concentration at the site of action that achieves the desired effect without causing substantial adverse or toxic side effects. In some embodiments, the dosage of ADC is determined by the expression of AXL observed in the sample obtained from the subject.Therefore, depending on the level or location of AXL expression in the sample, a larger or smaller dose of ADC may be required.For example, when the expression level of AXL is high, the appropriate dose of ADC may be higher.In some cases, when the expression level of AXL is high, administration of ADC and other agents may be required.For example, co-administration with chemotherapeutic agent ADC may indicate more aggressive treatment when the expression level of AXL is high. In certain embodiments, dosage levels are determined by expression of AXL on neoplastic cells in a sample obtained from a subject, such as when the target neoplasm is composed of or comprises neoplastic cells that express AXL. In some embodiments, the dosage level is determined by the expression of AXL on cells associated with the target neoplasm. For example, the target neoplasm may be a solid tumor that is composed of or contains neoplastic cells that express AXL. For example, the target neoplasm may be a solid tumor that is composed of or contains neoplastic cells that do not express AXL. The cells that express AXL may be neoplastic or non-neoplastic cells associated with the target neoplasm.

[0104] The complex may be administered in a single dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) during treatment. Methods of determining the most effective means and dosage of administration of the complex are well known to those of skill in the art and will vary with the dosage form used for treatment, the purpose of the treatment, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern selected by the treating physician, veterinarian, or clinician. When the complex is administered in multiple doses during treatment, the application of the nanoparticle composition and ultrasound may be provided with one dose of the complex, or two or more doses of the complex may be administered with the nanoparticle composition and ultrasound. For ADCs, the above dosages may apply to the conjugate (including the PBD moiety and the linker to the antibody) or to the effective amount of the PBD compound provided, e.g., the amount of compound releasable after cleavage of the linker. Aspects of the invention 1. An antibody-drug conjugate for use in a method for treating a solid tumor in a subject, said method comprising administering to the subject: (i) A pharmaceutical composition comprising a plurality of nanoparticles, each of the nanoparticles comprising: a cup having a cavity, the cavity having an opening of at least 50 nm in size; and a gas pocket present within the cavity; wherein the gas pocket is partially enclosed by the cup, wherein the nanoparticle comprises a single nanocup with a single cavity, and wherein the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier or diluent that is a liquid medium; and (ii) an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-AXL antibody conjugated to a pyrrolobenzodiazepine dimer; and exposing the subject to ultrasound. Antibody-drug conjugates. 2. The antibody-drug conjugate of [1], wherein the antibody comprises: (i) an immunoglobulin heavy chain variable region having a CDR1 region having the amino acid sequence represented by SEQ ID NO: 3, a CDR2 region having the amino acid sequence represented by SEQ ID NO: 4, and a CDR3 region having the amino acid sequence represented by SEQ ID NO: 5; and (ii) an immunoglobulin light chain variable region having a CDR1 region having the amino acid sequence represented by SEQ ID NO: 6, a CDR2 region having the amino acid sequence represented by SEQ ID NO: 7, and a CDR3 region having the amino acid sequence represented by SEQ ID NO: 8. 3. The antibody-drug conjugate described in [2], wherein the antibody contains an immunoglobulin heavy chain variable region having the amino acid sequence represented by SEQ ID NO:1. 4. The antibody-drug conjugate described in [2] or [3], wherein the antibody contains an immunoglobulin light chain variable region having the amino acid sequence represented by SEQ ID NO:2. 5. The antibody-drug conjugate has the following formula (I): Ab-LD (I) (In the formula, Ab is an antibody that binds to AXL; and LD has the following formula (II):

[0105] [ka] (In the formula, (a)R LL is a linker for linking to an Ab; (b)(i)R 10 and R 11 forms a double bond between the nitrogen atom and the carbon atom to which they are both attached; or (ii) R 10 is RLLA, a linker for connection to Ab, and R 11 is OH; or (iii) R 10 is a capping group RC, and R 11 is OH: (c) m is 0 or 1; and (d) If there is a double bond between C2 and C3, R 2 is methyl; If there is a single bond between C2 and C3, R 2H or

[0106] [ka] and; If there is a double bond between C2' and C3', R 12 is methyl; If there is a single bond between C2' and C3', R 12 H or

[0107] [ka] is) is the drug linker represented by ;) The antibody-drug conjugate according to any one of [1] to [4], wherein the antibody-drug conjugate is represented by the formula: 6. R LL is represented by the following formula (IIa):

[0108] [ka] (In the formula, Q:

[0109] [ka] (In the formula, QX, Q is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue.) X is:

[0110] [ka] (In the formula, a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, c2=0 or 1, d=0 to 5, at least b1 or b2=0, and at least c1 or c2=0.) G LL is a linker group attached to the antibody The antibody-drug conjugate according to [5], 7. The antibody-drug conjugate according to any one of [1] to [6], wherein the linker is a cleavable linker, for example, a linker comprising a cathepsin-cleavable sequence, for example, Val-Ala or Val-Cit. 8. The antibody-drug conjugate according to any one of [1] to [7], wherein a cytotoxin is conjugated to the antibody at an endogenous and / or recombinant N-linked glycosylation site. 9. The antibody-drug conjugate according to any one of [5] to [8], wherein LD is compound B2. 10. The antibody-drug conjugate described in any one of [1] to [9], wherein at least a portion of the cavity of each of the nanoparticles is substantially partially spherical, or the cavity of each of the nanoparticles is substantially partially spherical. 11. The antibody-drug conjugate according to any one of [1] to

[10] , wherein the gas pocket is a bubble having a diameter of at least 50 nm, preferably 150 to 250 nm. 12. The antibody-drug conjugate according to any one of [1] to

[11] , wherein the gas pocket is an air bubble. 13. The antibody-drug conjugate described in any one of [1] to

[12] , wherein the cup of each of the nanoparticles is substantially partially spherical. 14. The antibody-drug conjugate described in any one of [1] to

[13] , wherein the wall thickness of each cup is substantially uniform. 15. The antibody-drug conjugate described in any one of [1] to

[14] , wherein the size of each of the nanoparticles is 300 to 600 nm. 16. The antibody-drug conjugate according to any one of [1] to

[15] , wherein the nanoparticles are polymeric nanoparticles, preferably the nanoparticles comprise a crosslinked polymer. 17. The antibody-drug conjugate according to any one of [1] to

[16] , wherein the ultrasonic pressure amplitude is 1 MPa to 4 MPa. 18. A method for treating a solid tumor in a subject, comprising administering to the subject: (i) A pharmaceutical composition comprising a plurality of nanoparticles, each of the nanoparticles comprising: a cup having a cavity, the cavity having an opening of at least 50 nm in size; and a gas pocket present within the cavity; wherein the gas pocket is partially enclosed by the cup, wherein the nanoparticle comprises a single nanocup with a single cavity, and wherein the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier or diluent that is a liquid medium; and (ii) an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-AXL antibody conjugated to a pyrrolobenzodiazepine dimer; and exposing the subject to ultrasound; A method comprising:

[0111] Sequence Listing SEQ ID NO:1 [1H12 VH, CDR underlined]

[0112] [ka] SEQ ID NO:2 [1H12 VL, CDR underlined]

[0113] [ka] SEQ ID NO:3 [1H12 VH, Kabat CDR1]

[0114] [ka] SEQ ID NO:4 [1H12 VH, Kabat CDR2]

[0115] [ka] SEQ ID NO:5 [1H12 VH, Kabat CDR3]

[0116] [ka] SEQ ID NO:6 [1H12 VL, Kabat CDR1]

[0117] [ka] SEQ ID NO:7 [1H12 VL, Kabat CDR2]

[0118] [ka] SEQ ID NO:8 [1H12 VL, Kabat CDR3]

[0119] [ka] SEQ ID NO:9 [1H12 full length heavy chain]

[0120] [ka] SEQ ID NO: 10 [1H12 full length light chain]

[0121] [ka] Sequence number 11 [AXL]

[0122] [ka] Certain aspects and embodiments of the present disclosure are described in more detail below with reference to the following examples, which are illustrative only and not limiting in any way. EXAMPLES

[0123] Synthesis of ADCs AXL-PBD was constructed as described in WO 2018 / 146189 (using anti-AXL antibody 1H12 and PBD SG3199 - see compound C2). Control antibodies The control antibody was the anti-AXL antibody 1H12. Synthesis of nanoparticles The nanoparticles were prepared using the method of Example 1 of WO 2015 / 075442. The size of the nanoparticles was 480 nm, and 200 nm air bubbles were stabilized in the cavity. After drying, the nanoparticles were provided in a 5% glucose solution at a concentration of 17.5 mg / uL. the purpose In this study, a single low dose of ADC was tested in combination with nanoparticle + sonication in the tumor xenograft model SN12C (human kidney cancer). The treatment regimen was compared with the corresponding non-sonicated antibody (at both low and high doses) to understand whether the combination treatment enhanced the therapeutic effect over the same dose of antibody alone.

[0124] method Cells were thawed, expanded in vitro, and on the day of tumor implantation, harvested, counted, and cultured at 5 × 10 7 The cells were resuspended at a concentration of 1000 cells / mL. The prepared cell suspension was always kept in a Bijou-Davidson tube on ice. Female CB17 SCID mice (7-9 weeks old; n=50) were shaved (dorsal and flank regions) the day before cell implantation. On the day of treatment, the mice were anesthetized and microchips were implanted subcutaneously in the neck / back region. While still anesthetized, SN12C cells (5 × 10 in 100 μL) were instilled into one of their flanks (unilateral tumor) using a 1 mL syringe fitted with a 25 G needle. 6 The animals were monitored 2-3 times per week, including general welfare checks, caliper measurements of the tumors, and weighing the mice. The tumors grew to an average size of approximately 150 mm. 3 When the animals reached the age of 18, they were divided into six cohorts as shown in Table 1. Mice were depilated 4 days before treatment. On the day of treatment, mice were kept in a hot box (40°C) for 10-15 min to induce vasodilation before anesthesia. During anesthesia, all mice were placed in a water bath (37°C) and injected intravenously (via tail vein cannuation) with either nanoparticles (0.875 mg in 50 μl glucose 5%) and PBS (control group), ADC or antigen. All mice were then exposed to ultrasound treatment (US) or mock treatment (no US).

[0125] Table 1: Experimental groups

[0126] [Table 5] At the time of nanoparticle and drug injection, mice undergoing US treatment were exposed to pulsed therapeutic ultrasound (fc = 0.5 MHz, N = 8,000 cycles, repetition 0.5 Hz) for 10 min at variable peak negative ultrasound pressures of 1.5 MPa to 3 MPa. Cavitation was monitored by passive acoustic mapping and maintained at 0.1-0.5 nJ by modifying the ultrasound pressure as necessary. After treatment, all mice were returned to their cages to recover. Mice were monitored twice weekly and tumors were measured with calipers until the mean tumor length and width exceeded 12 mm, after which mice were culled and tumors were harvested and immediately frozen (-20°C).

[0127] result As shown in Figure 3, the mean tumor volume on the day before treatment was approximately 140-150 nm in all groups. 3 In Figure 3, each single point represents an individual tumor, while the bars indicate the group mean ± standard deviation. One-way ANOVA was used for statistical analysis, but no statistical significance was found. Cavitation was recorded by passive acoustic mapping and is shown in Figure 4. Cavitation in the US-treated groups was similar for both groups 3 and 5 (antibody and ADC, respectively) and was within the set target range of 1 x 10-10 to 5 x 10-10 Joules. In Figure 4, each single point represents an individual tumor, while the bars show the group mean ± standard deviation. An unpaired t-test was used for statistical analysis, but no statistical significance was found.

[0128] Tumors were measured twice a week after treatment until the volume exceeded the maximum size given by the formula (length + width) / 2>12 mm. Figure 5A shows the average tumor growth for each group (each curve represents the average tumor growth (± standard error of the mean) for a particular group plotted until the first animal of that group was culled). The analysis shows that groups treated with either a low dose of ADC (0.3 mg / kg) + US or a higher dose of the same antibody (1 mg / kg) had reduced tumor growth compared to all other groups. A two-way ANOVA analysis performed on the data until the first animal was culled confirmed that groups 5 and 6 were statistically different from the other groups at day 12 after treatment (Figure 5B). Individual tumor growth in each group is shown in Figure 6. Each curve represents a single tumor and was plotted until a value of (length + width) / 2 >12 mm and animals had to be culled.

[0129] Although all animals died before the time limit of the experiment, survival analysis (Figure 7) confirmed the observed tumor growth in groups 5 and 6 (0.3 mg / kg + US ADC and 1 mg / kg ADC, respectively) and showed a statistically significantly longer survival than all other groups. No differences were observed between groups 1-4 or between groups 5 and 6. In Figure 7A, survival curves for all groups are shown along with the respective median survival times. In Figure 7B, survival graphs are for representative group pairs. Statistical significance was calculated by log-rank (Mantel-Cox) test. Mice weights and appearance were monitored twice a week after treatment and no welfare effects were observed for antibody and US treatment or their combination. Animal weight values ​​over the course of the study are shown in Figure 8. Figure 8A shows the mean mouse weights for each group. Each curve represents the mean weight (± standard deviation) for a particular group and is plotted until the first animal was culled from that group. Figure 8B is similar to 8A but shows only mouse weights until the first animal dropped out of the study (no statistically significant differences were found by 2-way ANOVA).

[0130] Consideration Analysis of the experimental data obtained in this study shows that the control antibody administered with or without nanoparticle and US combination treatment did not significantly affect tumor growth and animal survival compared to the PBS-treated control group. This was also the case for treatment with a low dose (0.3 mg / kg) of ADC. However, treatment with the same dose of ADC combined with nanoparticles and US treatment significantly reduced tumor growth and ultimately significantly prolonged animal survival. Both of these effects were comparable to those achieved by treatment with a higher dose of ADC (1 mg / kg). No adverse effects were observed in relation to the antibody and nanoparticle or US treatment or their combination, as evidenced by the lack of differences in mouse body weight between all treatment groups and the control group. This study demonstrates that administering ADC with nanoparticles and applying ultrasound US treatment provides significant therapeutic benefit, allowing for the administration of lower doses of ADC while still maintaining efficacy. Several publications are cited above in order to more fully describe and disclose the disclosure and the state of the art to which the invention herein may pertain. Each of the references mentioned in this disclosure is incorporated herein by reference in its entirety.

Claims

1. 1. An antibody-drug conjugate for use in a method for treating a solid tumor in a subject, the method comprising administering to the subject: (i) A pharmaceutical composition comprising a plurality of nanoparticles, each of the nanoparticles comprising: a cup having a cavity, the cavity having an opening of at least 50 nm in size; and a gas pocket present within the cavity; wherein the gas pocket is partially enclosed by the cup, wherein the nanoparticle comprises a single nanocup with a single cavity, and wherein the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier or diluent that is a liquid medium; and (ii) an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-AXL antibody conjugated to a pyrrolobenzodiazepine dimer; and exposing the subject to ultrasound. Antibody-drug conjugates.

2. The antibody-drug conjugate of claim 1, wherein the antibody comprises: (i) an immunoglobulin heavy chain variable region having a CDR1 region having an amino acid sequence represented by SEQ ID NO:3, a CDR2 region having an amino acid sequence represented by SEQ ID NO:4, and a CDR3 region having an amino acid sequence represented by SEQ ID NO:5; and (ii) an immunoglobulin light chain variable region having a CDR1 region having an amino acid sequence represented by SEQ ID NO:6, a CDR2 region having an amino acid sequence represented by SEQ ID NO:7, and a CDR3 region having an amino acid sequence represented by SEQ ID NO:

8.

3. The antibody-drug conjugate of claim 2 , wherein the antibody comprises an immunoglobulin heavy chain variable region having an amino acid sequence represented by SEQ ID NO:

1.

4. The antibody-drug conjugate of claim 2 , wherein the antibody comprises an immunoglobulin light chain variable region having the amino acid sequence represented by SEQ ID NO:

2.

5. The antibody-drug conjugate has the following formula (I): Ab-L-D (I) (In the formula, Ab is an antibody that binds to AXL; and L-D is represented by the following formula (II): 【Chemistry 1】 (In the formula, (a) R ＬＬ is a linker for linking to the Ab; (b)(i)R １０ and R １１ form a double bond between the nitrogen atom and the carbon atom to which they are both attached; or (ii) R １０ is RLLA, a linker for connection to Ab, and R １１ is OH; or (iii) R １０ is a capping group RC, and R １１ is OH: (c) m is 0 or 1; and (d) When a double bond is present between C2 and C3, R ２ is methyl; When a single bond is present between C2 and C3, R ２ is H or 【Chemistry 2】 and When a double bond is present between C2' and C3', R １２ is methyl; When a single bond is present between C2' and C3', R １２ is H or 【Chemistry 3】 (It is) The drug linker is represented by The antibody-drug conjugate of claim 1 ,

6. R ＬＬ is represented by the following formula (IIa): 【Chemistry 4】 (In the formula, Q is the following: 【Chemistry 5】 (In the formula, QX is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue.) X is one of the following: 【Chemistry 6】 (In the formula, a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, c2=0 or 1, d=0 to 5, at least b1 or b2=0, and at least c1 or c2=0.) G ＬＬ is a linker group attached to the antibody The antibody-drug conjugate of claim 5 ,

7. The antibody-drug conjugate of claim 1, wherein the linker is a cleavable linker, e.g., a linker comprising a cathepsin-cleavable sequence, e.g., Val-Ala or Val-Cit.

8. The antibody-drug conjugate of claim 1 , wherein a cytotoxin is conjugated to the antibody at an endogenous and / or recombinant N-linked glycosylation site.

9. The antibody-drug conjugate of claim 5, wherein L-D is compound B2.

10. The antibody-drug conjugate of claim 1 , wherein at least a portion of the cavity of each of the nanoparticles is substantially partially spherical, or wherein the cavity of each of the nanoparticles is substantially partially spherical.

11. The antibody-drug conjugate of claim 1, wherein the gas pocket is a bubble having a diameter of at least 50 nm, preferably 150-250 nm.

12. The antibody-drug conjugate of claim 1, wherein the size of each of the nanoparticles is 300-600 nm.

13. The antibody-drug conjugate of claim 1 , wherein the nanoparticle is a polymeric nanoparticle or comprises a cross-linked polymer.

14. The antibody-drug conjugate of claim 1, wherein the ultrasonic pressure amplitude is 1 MPa to 4 MPa.

15. 1. A method for treating a solid tumor in a subject, comprising administering to the subject: (i) A pharmaceutical composition comprising a plurality of nanoparticles, each of the nanoparticles comprising: a cup having a cavity, the cavity having an opening of at least 50 nm in size; and a gas pocket present within the cavity; wherein the gas pocket is partially enclosed by the cup, wherein the nanoparticle comprises a single nanocup with a single cavity, and wherein the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier or diluent that is a liquid medium; and (ii) an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-AXL antibody conjugated to a pyrrolobenzodiazepine dimer; and exposing the subject to ultrasound; A method comprising:

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