CBM uses
阅读说明：本技术 Cbm用途 (CBM uses ) 是由 格雷姆·罗杰斯 海伦·康纳里斯 于 2020-01-24 设计创作，主要内容包括：本公开提供了调节细胞生长的分子。这些分子包括与碳水化合物结合的那些分子,并且可用于治疗和/或预防细胞增殖和/或分化病症、癌症和/或其迁移和/或扩散。(The present disclosure provides molecules that modulate cell growth. These molecules include those that bind to carbohydrates and are useful in the treatment and/or prevention of cell proliferation and/or differentiation disorders, cancer, and/or migration and/or spread thereof.)
1. A carbohydrate binding module for use in a method of modulating cell growth and/or cell activity, wherein the carbohydrate binding module is selected from the group consisting of:
(i) a carbohydrate binding module of carbohydrate binding module family 32(CBM 32);
(ii) a carbohydrate binding module of carbohydrate binding module family 47(CBM 47);
(iii) a carbohydrate binding module of carbohydrate binding module family 67(CBM 67); and
(iv) carbohydrate binding module of carbohydrate binding module family 70(CBM 70).
2. The carbohydrate binding module of claim 1, for use of claim 1, wherein the carbohydrate binding module of carbohydrate binding module family 32(CBM32) comprises SEQ ID NO 1 or SEQ ID NO 2 or a carbohydrate binding portion thereof.
3. The carbohydrate binding module according to claim 1 or 2, for use according to claim 1 or 2, wherein the carbohydrate binding module is for use in the treatment and/or prevention of cancer.
4. The carbohydrate binding molecule of claim 3, for use of claim 3, wherein the carbohydrate binding module is CBM32.
5. The carbohydrate binding module of claim 4, for use of claim 4, wherein the cancer is selected from the group consisting of:
(a) ovarian cancer;
(b) lung cancer;
(c) colon cancer; and
(d) breast cancer.
6. A molecule comprising a carbohydrate binding module for use in a method of modulating cell growth and/or cell activity, wherein the molecule comprises a single CBM selected from the group consisting of:
(i) a single CBM 32;
(ii) a single CBM 47;
(iii) a single CBM 67;
(iv) a single CBM 70;
(v) CpCBM32 alone;
(vi) single SpCBM 47;
(vii) a single SaCBM 67; and
(viii) single SpCBM 70.
7. A molecule comprising two or more carbohydrate binding modules for use in a method of modulating cell growth and/or cell activity, wherein the molecule comprises two or more CBMs selected from the group consisting of:
(i)CBM32；
(ii)CBM47；
(iii)CBM67；
(iv)CBM70；
(v)CpCBM32；
(vi)SpCBM47；
(vii) SacBM 67; and
(viii)SpCBM70。
8. a molecule for use in a method of modulating cell growth and/or cell activity, wherein the molecule is selected from the group consisting of:
(i) a molecule comprising Cp2CBM32 TD;
(ii) a molecule comprising Sp2CBM47 TD;
(iii) a molecule comprising Sa2CBM67 TD; and
(iv) a molecule comprising Sp2CBM70 TD;
9. the molecule according to any one of claims 6-8 for use according to claims 6-8, wherein the molecule is for the treatment and/or prevention of cancer.
10. The molecule according to claim 9, for use according to claim 9, wherein the molecule is selected from the group consisting of:
(i) a molecule comprising one, two or more CBMs 32;
(ii) a molecule comprising one, two or more CpCBM 32;
(iii) a molecule comprising one, two or more peptides or proteins comprising the sequences of SEQ ID NOs 1,2, or carbohydrate-binding portions thereof; and
(iv) a molecule comprising CpCBM32 TD.
11. The molecule of claim 10, for use of claim 10, wherein the cancer is selected from the group consisting of:
(a) ovarian cancer;
(b) lung cancer;
(c) colon cancer; and
(d) breast cancer.
12. A molecule for use in the treatment and/or prevention of cancer, wherein said molecule is selected from the group consisting of:
(i) a molecule comprising one or more CpCBM 32;
(ii) a molecule comprising one or more SpCBM 47;
(iii) a molecule comprising one or more SaCBM 67;
(iv) a molecule comprising one or more SpCBM 70;
(v) a molecule comprising Cp2CBM32 TD;
(vi) a molecule comprising Sp2CBM47 TD;
(vii) a molecule comprising Sa2CBM67 TD; and
(viii) a molecule comprising Sp2CBM70 TD.
13. A carbohydrate binding module classified as CBM32 and/or CBM40 or a molecule comprising a carbohydrate binding module classified as CBM32 and/or CBM40 for use in the treatment and/or prevention of refractory (or resistant) cancer.
14. The carbohydrate binding module or molecule according to claim 13, for use according to claim 13, selected from the group consisting of:
(i) a molecule comprising one or more CpCBM 32;
(ii) a molecule comprising Cp2CBM32 TD;
(iii) a molecule comprising Vc2CBM40 TD; and
(iv) a molecule comprising Sp2CBM40 TD.
15. The carbohydrate binding module or molecule of claim 13 or 14, for use of claim 13 or 14, wherein the carbohydrate binding module of carbohydrate binding module family 32(CBM32) comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 or a carbohydrate binding portion thereof, and the carbohydrate binding module of carbohydrate binding module family 40(CBM40) comprises a sequence selected from the group consisting of:
(i) 23 or a carbohydrate binding portion thereof,
(ii) 24 or a carbohydrate binding portion thereof,
(iii) 25 or a carbohydrate-binding portion thereof; and
(iv) 26 or a carbohydrate-binding portion thereof.
CpCBM32, a molecule comprising Cp2CBM32 TD; a molecule comprising Vc2CBM40 TD; and/or a molecule comprising Sp2CBM40TD for use in the treatment of refractory (or resistant) ovarian cancer.
Technical Field
The present invention provides uses, methods and compositions for treating cancer.
Background
Some lectins are glycoproteins of non-immunological origin, exhibiting the ability to induce apoptosis in malignant cells and thus exhibit anti-cancer properties. This phenomenon occurs in part through the interaction of these lectins with specific glycan receptors on immune cells (Yau et al, 2015).
One such lectin is Viscum album (Viscumin) from Viscum album, a toxin that binds to a cellular receptor that is glycosylated with α 2,6 sialyllactose (Muthing et al, 2002). Viscosarin is a 57kDa heterodimer comprising two subunits, A and B. The a subunit exerts its toxic effects by disabling the ribosome, thereby interrupting the production of protein, while the B subunit exhibits glycan-binding function. Lectins show picomolar cytotoxicity in vitro and in vivo, and the upper recommended dose for clinical trial subjects is 6 μ g/kg (half-life of 13 minutes) (Zwierzina et al, 2011).
Another lectin that has been shown to be able to prevent cell migration and growth (and therefore has anti-cancer properties) is Maackia amurensis (Maackia amurensis) seed lectin or MASL (Occhoa-Alvarez et al, 2012; Astarita et al, 2012). This lectin also has cytotoxicity exhibiting nanomolar potency (-300 nM) and exerts its effect by binding to podoplanin, an α 2,3 sialylated mucin-type transmembrane glycoprotein, overexpressed in a variety of human cancers (Kato et al, 2005); schacht et al, 2005; shibahara et al, 2006).
WO2018/055373 describes the use of sialic acid binding molecules (including carbohydrate binding modules) in methods of modulating cell growth and in the treatment and/or prevention of cell proliferation and/or differentiation disorders.
Treatment of cell proliferation and differentiation disorders, including for example cancer, requires the provision of additional molecules that are well tolerated in the host and have therapeutic potential.
Summary of The Invention
The present disclosure is based on the following findings: a class of sugar (carbohydrate) binding proteins, known as Carbohydrate Binding Modules (CBMs), can regulate various aspects of cell growth and/or cell activity.
Accordingly, the present disclosure provides a variety of uses, including medical uses of molecules comprising the CBMs described herein.
CBMs are commonly found as part of larger enzymes (e.g., carbohydrate-active enzymes such as glycosidases) where their role is to bind carbohydrate ligands and direct the catalytic domain of the enzyme to its substrate.
The present disclosure specifically relates to those CBMs belonging to the following group:
(i) carbohydrate binding module family 32(CBM 32);
(ii) carbohydrate binding module family 47(CBM47)
(iii) Carbohydrate binding module family 67(CBM67)
(iv) Carbohydrate binding module family 70(CBM70)
In this regard, the present disclosure relates to the discovery that CBMs from all of the CBM families described above (CBM families (i) - (iv)) modulate cell growth and/or cell activity.
As used herein, the terms "CBM 32," "CBM 47," "CBM 67," and "CBM 70" may refer to a compound, composition, or molecule that comprises one or more carbohydrate binding modules and is classified as belonging to carbohydrate binding module families 32, 47, 67, and/or 70. The term "CBM 32" as used herein may include and/or comprise those carbohydrate binding modules designated as belonging to the CBM32 family in a carbohydrate-active enzyme database (freely accessible over the Internet: http:// www.cazy.org /). Furthermore, the terms "CBM 47", "CBM 67" and "CBM 70" as used herein may include and/or comprise those carbohydrate binding modules designated as belonging to the CBM47, CBM67 and CBM70 families in carbohydrate-active enzyme databases (freely accessible over the Internet: http:// www.cazy.org /).
The present disclosure may include the use of molecules such as larger molecules comprising CBMs (e.g., CBM32, CBM47, CBM67, and CBM 70). By way of non-limiting example, the molecules of the present disclosure may not only exhibit the ability to bind sialic acid, but may also have one or more other functions. For example, the molecule can have enzymatic activity.
In some cases, CBMs for various uses described herein may not be provided as part of or included in a molecule (e.g., fusion protein) having enzymatic (e.g., sialidase) activity.
It should be noted that throughout this specification the terms "comprising", "including" and/or "comprising" are used to indicate that aspects and embodiments of the present invention "include" one or more particular features. It should be understood that this/these terms may also encompass aspects and/or embodiments that "consist essentially of" or "consist of the relevant features.
Thus, in the context of the present disclosure, "CBM 32", "CBM 47", "CBM 67" and/or "CBM 70" may comprise, consist essentially of, or consist of one or more molecules classified as belonging to carbohydrate binding module families 32, 47, 67 and/or 70. The terms "CBM 32", "CBM 47", "CBM 67" and/or "CBM 70" may include fragments or portions of native, wild-type or reference CBM32s, CBM47s, CBM67s and/or CBM70s, any such fragments or portions should be functional, that is, they retain the carbohydrate binding capacity of the native, wild-type or reference CBM32, CBM47, CBM67 and/or CBM70 from which they are derived.
Useful CBM32 may be from any suitable source. For example, the CBM32 used may be obtained from microorganisms including bacteria such as the genera cellulovibrio (Cellvibrio), Yersinia (Yersinia), Micromonospora (Micromonospora), Streptococcus (Streptococcus), bifidobacterium (bifidobacterium) and Clostridium (Clostridium). For example, useful CBM32 may be obtained or derived from, for example, vibrio mixus (Cellvibrio mixtus), Yersinia enterocolitica (Yersinia enterocolitica), Clostridium perfringens (Clostridium perfringens), Clostridium thermocellum (Clostridium thermocellum), Streptococcus pneumoniae (Streptococcus pneumoniae), Bifidobacterium longum (Bifidobacterium longum), and pseudomonas aeruginosa. More details on the origin, structure and function of the CBM32 family can be found in carbohydrate active enzyme databases (freely available on the Internet: http:// www.cazy.org/CBM32. html).
An exemplary CBM32 sequence is provided by SEQ ID NO:
SEQ ID NO:1
thus, a CBM for use may comprise a polypeptide having the sequence of SEQ ID NO:1 or a carbohydrate-binding portion thereof, consisting essentially of, or consisting of.
The carbohydrate-binding fragment of SEQ ID NO:1 may comprise any position from between about 5,6,7, 8, 9 or 10 (consecutive or contiguous) amino acids to about 138 (consecutive or contiguous) amino acids of SEQ ID NO: 1. Suitable fragments may comprise about 11, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, or about 135 (consecutive or contiguous) amino acids from SEQ ID No. 1.
CBM32 or a nucleic acid comprising SEQ ID NO: 1. consisting essentially of SEQ ID NO:1 or consists of SEQ ID NO:1 can bind, for example, galactose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and/or lactose. Thus, any fragment for use may also bind galactose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and/or lactose. One skilled in the art will appreciate that the binding affinity of any given CBM32 molecule may depend on the precise CBM32 subtype; as a further example, some CBM32 exhibit affinity for a variety of ligands (examples include blood group II H-trisaccharide (Fuca1-2Galb1-4GlcNAc), N-acetyl-D-lactosamine (LacNAc), galactose, lactose-N-disaccharide, disaccharide GlcNAc- α -1,4-Gal (which can be referred to as N-acetylglucosamine where α 1,4 is linked to galactose) and/or GlcNAc). It should also be noted that multiple CBM32 subtypes may be derived from a single organism; these different CBM subtypes may exhibit the same, similar or different binding specificities. For example, clostridium perfringens contains two sialidases NanJ and NanH; NanJ contains a galactose-specific CBM 32; NanH comprises four putative CBMs 32 with different binding selectivities, e.g., CBM32 encoded by NanH binds GlcNAc. As used herein, the term CBM32 includes all CBM32 variants, derivatives, and subtypes.
SEQ ID NO 1 is derived from the sequence as deposited in the UniProt database under ID number:
a0A2X2YJF 2. This sequence is duplicated as SEQ ID NO:2 below (SEQ ID NO:1 shows residues 42-180, shaded in the sequence below):
SEQ ID NO:2
SEQ ID NOs 1 and 2 are derived from clostridium perfringens.
CBMs for use in various aspects of the present disclosure may include one, two, three, four, or more CBMs 32.
CBM's for use may comprise one, two, three, four or more proteins comprising SEQ ID NO 1 or carbohydrate-binding fragments thereof.
One skilled in the art will appreciate that a useful CBM32 may comprise a sequence exhibiting some degree (e.g., 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, or 60%) of sequence identity or homology with the CBM32 sequence of SEQ ID NOs: 1 and 2. All such variants or divergent sequences are included within the scope of the present disclosure and are encompassed by the term "CBM 32". The same and/or homologous CBM32 sequence may have carbohydrate binding function.
CBM47 may be derived from any suitable source. For example, the CBM47 for use may be obtained from microorganisms including bacteria of the genera Acinetobacter (Acinetobacter), mussel (mussel), Campylobacter (Campylobacter), phytophthora (Planctomycetes), Streptococcus (Streptococcus) and Streptomyces (Streptomyces), for example. For example, a useful CBM47 may be obtained or derived from, for example, a Streptococcus lentus (Streptococcus mitis) or Streptococcus pneumoniae. More detailed information on the potential source and structure and function of the CBM47 family can be found in carbohydrate active enzyme databases (freely available on the Internet: http:// www.cazy.org/CBM47. html).
An exemplary CBM47 sequence is provided by SEQ ID NO: 3:
SEQ ID NO:3
thus, a CBM for use may comprise a polypeptide having the sequence of SEQ ID NO:3 or a carbohydrate-binding portion thereof, consisting essentially of, or consisting of.
The carbohydrate-binding fragment of SEQ ID NO. 3 may comprise any position from between about 5,6,7, 8, 9 or 10 (consecutive or contiguous) amino acids to about 144 (consecutive or contiguous) amino acids of SEQ ID NO. 3. Suitable fragments may comprise about 11, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130 or about 135, about 140 or about 143 (consecutive or consecutive) amino acids from SEQ ID No. 3.
CBM47 or a protein comprising, consisting essentially of, or consisting of SEQ ID NO. 3 may bind L-fucose, fucosyllactose, H-trisaccharide and/or LewisyAn antigen. Thus, any fragment for use may also bind L-fucose, fucosyllactose, H-trisaccharide and/or LewisyAn antigen.
SEQ ID NO 3 is derived from the sequence as deposited in the UniProt database under ID number:
a0A1Q2T 229. This sequence is duplicated as SEQ ID NO:4 below (SEQ ID NO:3 is shown as residue 601-745, shaded in the sequence below):
SEQ ID NO:4
SEQ ID NOs 3 and 4 are derived from streptococcus pneumoniae.
CBMs for use in various aspects of the present disclosure may include one, two, three, four, or more CBMs 47.
CBM's for use may comprise one, two, three, four or more proteins comprising SEQ ID NO 3 or carbohydrate-binding fragments thereof.
One skilled in the art will appreciate that a useful CBM47 may comprise a sequence that exhibits some degree (e.g., 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, or 60%) of sequence identity or homology with the CBM47 sequence of SEQ ID NOs: 3 and 4. All such variants or divergent sequences are included within the scope of the present disclosure and are encompassed by the term "CBM 47". The same and/or homologous CBM47 sequence may have carbohydrate binding function.
CBM67 may be derived from any suitable source. For example, CBM67 for use may be obtained from microorganisms including bacteria of the genera Bacillus (Bacillus), Paenibacillus (Paenibacillus), Mycoplasma (Planctomycetes) and Streptomyces, for example. For example, a useful CBM47 may be obtained or derived from, for example, Streptomyces avermitilis (Streptomyces avermitilis). Further details regarding the potential source and structure and function of the CBM67 family can be found in the carbohydrate active enzyme database (freely available on the Internet: http:// www.cazy.org/CBM67. html).
An exemplary CBM67 sequence is provided by SEQ ID NO:
SEQ ID NO:5
thus, a CBM for use may comprise a polypeptide having the sequence of SEQ ID NO:5 or a carbohydrate-binding portion thereof, consisting essentially of, or consisting of.
The carbohydrate-binding fragment of SEQ ID NO. 5 may comprise any position from between about 5,6,7, 8, 9 or 10 (consecutive or contiguous) amino acids to about 164 (consecutive or contiguous) amino acids of SEQ ID NO. 5. Suitable fragments may comprise about 11, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, or about 163 (consecutive or consecutive) amino acids from SEQ ID No. 5.
CBM67 or a protein comprising, consisting essentially of, or consisting of SEQ ID NO. 5 can bind to L-rhamnose. Thus, any fragment for use may also bind to L-rhamnose.
SEQ ID NO 5 is derived from the sequence as deposited in the UniProt database under ID number:
q82PP 4. This sequence was replicated as SEQ ID NO:6 below (SEQ ID NO:5 is shown as residue 132-296, shaded in the sequence below):
SEQ ID NO:6
SEQ ID NO 5 and 6 are derived from Streptomyces avermitilis (Streptomyces avermitilis).
CBMs for use in various aspects of the present disclosure may include one, two, three, four, or more CBMs 67.
CBM's for use may comprise one, two, three, four or more proteins comprising SEQ ID NO 5 or carbohydrate-binding fragments thereof.
One skilled in the art will appreciate that a useful CBM67 may comprise a sequence exhibiting some degree (e.g., 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, or 60%) of sequence identity or homology with the CBM67 sequence of SEQ ID NOs: 5 and 6. All such variants or divergent sequences are included within the scope of the present disclosure and are encompassed by the term "CBM 67". The same and/or homologous CBM67 sequence may have carbohydrate binding function.
CBM70 may be derived from any suitable source. For example, CBM70 for use may be obtained from microorganisms including, for example, bacteria of the genera Bacillus, Paenibacillus, Planococcus, and Streptococcus. For example, a useful CBM70 may be obtained or derived from, for example, streptococcus pneumoniae. Further details regarding the potential source and structure and function of the CBM70 family can be found in the carbohydrate active enzyme database (freely available on the Internet: http:// www.cazy.org/CBM70. html).
An exemplary CBM70 sequence is provided by SEQ ID NO:
SEQ ID NO:7
thus, a CBM for use may comprise a polypeptide having the sequence of SEQ ID NO:7 or a carbohydrate-binding portion thereof, consisting essentially of, or consisting of.
The carbohydrate-binding fragment of SEQ ID NO. 7 may comprise any position from between about 5,6,7, 8, 9 or 10 (consecutive or contiguous) amino acids to about 158 (consecutive or contiguous) amino acids of SEQ ID NO. 7. Suitable fragments may comprise about 11, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, or about 157 (consecutive or consecutive) amino acids from SEQ ID No. 7.
CBM70 or a protein comprising, consisting essentially of, or consisting of SEQ ID NO. 7 can bind to hyaluronic acid. Thus, any fragment for use may also bind hyaluronic acid.
SEQ ID NO 7 is derived from the sequence as deposited in the UniProt database under ID number:
q54873. This sequence is duplicated as SEQ ID NO:8 below (SEQ ID NO:7 is shown as residues 54-212, shaded in the sequence below):
SEQ ID NO:8
SEQ ID NOs 7 and 8 are derived from streptococcus pneumoniae.
CBMs for use in various aspects of the present disclosure may include one, two, three, four, or more CBMs 70.
CBM's for use may comprise one, two, three, four or more proteins comprising SEQ ID NO 7 or carbohydrate-binding fragments thereof.
One skilled in the art will appreciate that a useful CBM70 may comprise a sequence exhibiting some degree (e.g., 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, or 60%) of sequence identity or homology with the CBM70 sequence of SEQ ID NOs: 7 and 8. All such variants or divergent sequences are included within the scope of the present disclosure and are encompassed by the term "CBM 70". The same and/or homologous CBM70 sequence may have carbohydrate binding function.
In summary, molecules for use in various aspects and embodiments of the present disclosure may comprise one or more CBMs, such as one or more CBMs 32, one or more CBMs 47, one or more CBMs 67, and/or one or more CBMs 70.
The molecule for use may comprise a single CBM selected from the group consisting of:
a single CBM 32;
a single CBM 47;
a single CBM 67; and
a single CBM70.
The molecule for use may comprise a single CBM selected from the group consisting of:
CpCBM32 alone;
single SpCBM 47;
a single SaCBM 67; and
single SpCBM 70.
Wherein "Cp" refers to clostridium perfringens, thus "CpCBM 32" denotes the CBM32 moiety derived from clostridium perfringens; "Sp" represents Streptococcus pneumoniae and thus "SpCBM 47" and "SpCBM 70" represent CBM47 and/or CBM70 moieties derived from Streptococcus pneumoniae; and "SacBM 67" refers to the CBM67 moiety derived from S.avermitilis.
CBMs for use may include a plurality or plurality (i.e., two or more) CBMs. Molecules comprising multiple CBMs may be referred to as "multivalent CBMs. The multivalent CBM may, for example, comprise two or more cpcbms, two or more spcbms, two or more sacbms as described above. The multivalent CBM may comprise a mixture of different CBMs, such as one or more cpcbms and one or more Sp/sacbms.
Multivalent CBM molecules (e.g., molecules comprising two or more CpCBM32 moieties) may be prepared as constructs comprising multiple CBMs linked by an amino acid/peptide linker. Each CBM may be linked to another CBM by, for example, a peptide comprising 5, 10 or 15 amino acids. For example, any one or more of the following peptides may be used to link two or more CBMs to produce a multivalent CBM:
(i) linker of 5 amino acids:
ALNGS(SEQ ID NO:9)
LQALG(SEQ ID NO:10)
GGNSG(SEQ ID NO:11)
GGGSG(SEQ ID NO:12)
GGSLG(SEQ ID NO:13)
GGGSA(SEQ ID NO:14)
(ii) linker of 10 amino acids:
ALNGSGGGSG(SEQ ID NO:15)
LQALGGGGSL(SEQ ID NO:16)
(iii) linker of 15 amino acids:
ALNGSGGGSGGGGSG(SEQ ID NO:17)
thus, various aspects and embodiments (uses, CBMs for use, methods and medicaments) of the present disclosure may utilize molecules comprising (consisting of or consisting essentially of) a CBM selected from the group consisting of:
(i) one or more CpCBM 32;
(ii) one or more SpCBM 47;
(iii) one or more SaCBM 67; and
(iv) one or more SpCBM 70.
The molecules for use may further comprise an oligomerisation domain. Suitable oligomerization domains can exhibit the ability to self-associate to form multimeric structures such as trimers. The oligomerisation domain for use may comprise any molecule or any functional fragment thereof having the oligomerisation properties described above. For example, one or more (e.g., two) CBM molecules may be bound, coupled or fused to an oligomerization domain, and the resulting CBM molecule:: an oligomerization domain "fusion" (with one or more other such "fusions") may be used as a molecule for modulating cell growth and/or activity and/or for treating or preventing any of the diseases and/or conditions disclosed herein.
Suitable oligomerisation domains may be derived, for example, from Pseudomonas aeruginosa (Pseudomonas aeruginosa) pseudoaminase (pseudoaminidase). An exemplary pseudomonas aeruginosa pseudoaminase sequence amino acid sequence has been deposited under accession number PAO579 and is reproduced below as SEQ ID NO:18 (438 amino acids).
The oligomerization domain of SEQ ID NO 18 is from amino acid residues 333 to 438 (highlighted in grey) and the sequence is SEQ ID NO 19.
Thus, the oligomerization domain for use can comprise from about residues 250, 275, 300, 310, 320, 333, 340 to 350 (i.e., about residues 250 to about residues 350, including about any residues therebetween) to about residues 400, 410, 420, 430, or 438 (i.e., about any residues from about residue 400 to residue 438, including about any residues therebetween) of the Pseudomonas aeruginosa pseudoaminase trimerization domain (PaTD) provided by SEQ ID NO: 18. For example, useful molecules may utilize an oligomerization domain comprising residues 333 to 438 of SEQ ID NO 18.
Other molecules useful in various aspects and embodiments of the present disclosure are described below.
One molecule for use is designated "Cp 2CBM32 TD", the construct comprises a Cp2CBM32 unit which itself comprises 2CBM32 molecules derived from clostridium perfringens (highlighted in grey), wherein the 2CBM32 copies are linked in series by a short peptide linker molecule (dashed underline). The construct further comprises a trimerisation domain (TD: underlined) which is itself fused to one of the two CBM32 via another short linker moiety (bold underlined). The sequence of an exemplary Cp2CBM32TD is provided below as SEQ ID NO 19.
SEQ ID NO:19
One molecule for use is designated "Sp 2CBM47 TD", and the construct is an Sp2CBM47 unit which itself comprises 2CBM47 molecules (highlighted in grey) derived from streptococcus pneumoniae, with 2 copies of CBM47 linked in series by a short peptide linker molecule (dashed underline). The construct further comprises a trimerisation domain (TD: underlined) which is itself fused to one of the two CBM47 via another short linker moiety (bold underlined). The sequence of an exemplary Sp2CBM47TD is provided below as SEQ ID NO: 20.
SEQ ID NO:20
One molecule for use is designated "Sa 2CBM67 TD", and the construct comprises a Sa2CBM67 unit which itself comprises 2CBM67 molecules (highlighted in grey) derived from s.avermitilis, wherein the 2CBM67 copies are linked in series by a short peptide linker molecule (dashed underline). The construct further comprises a trimerisation domain (TD: underlined) which is itself fused to one of the two CBM67 via another short linker moiety (bold underlined). The sequence of exemplary Sa2CBM67TD is provided below as SEQ ID NO: 21.
SEQ ID NO:21
One molecule for use is designated "Sp 2CBM70 TD", the construct comprises an Sp2CBM70 unit which itself comprises 2CBM70 molecules (highlighted in grey) derived from streptococcus pneumoniae, wherein the 2 copies of CBM70 are linked in series by a short peptide linker molecule (dashed underline). The construct further comprises a trimerisation domain (TD: underlined) which is itself fused to one of the two CBM70 via another short linker moiety (bold underlined). The sequence of an exemplary Sp2CBM70TD is provided below as SEQ ID NO: 22.
SEQ ID NO:22
In a first aspect, various molecules disclosed herein are useful in therapy. Further, the various molecules disclosed herein may be used as medicaments. In this context, the reader should understand that "the molecules disclosed herein" include at least the following:
(i) a molecule comprising one or more CBMs 32;
(ii) a molecule comprising one or more CBMs 47;
(iii) a molecule comprising one or more CBMs 67;
(iv) a molecule comprising one or more CBMs 70;
(v) a molecule comprising Cp2CBM32 TD;
(vi) a molecule comprising Sp2CBM47 TD;
(vii) a molecule comprising Sa2CBM67 TD; and
(viii) a molecule comprising Sp2CBM70 TD;
in one aspect, molecules comprising CBM32 for use in methods of modulating cell growth and/or cell activity are provided. The molecule comprising CBM32 may comprise Cp2CBM32TD or be Cp2CBM32 TD.
In another aspect, molecules comprising CBM47 for use in methods of modulating cell growth and/or cell activity are provided. Molecules comprising CBM47 may comprise Sp2CBM47TD or be Sp2CBM47 TD.
In a further aspect, there is provided a molecule comprising CBM67 for use in a method of modulating cell growth and/or cell activity. The molecule comprising CBM67 may comprise Sa2CBM67TD or is Sa2CBM67 TD.
Additionally, molecules comprising CBM70 for use in methods of modulating cell growth and/or cell activity are provided. Molecules comprising CBM70 may comprise Sp2CBM70TD or be Sp2CBM70 TD.
In another aspect, a method of modulating growth and/or activity of a cell is provided, the method comprising contacting the cell with a molecule comprising CBM32. The method may be an in vitro method. The molecule comprising CBM32 may comprise Cp2CBM32TD or be Cp2CBM32 TD.
Further aspects provide methods of modulating cell growth and/or activity, comprising contacting a cell with a molecule comprising CBM47. The method may be an in vitro method. Molecules comprising CBM47 may comprise Sp2CBM47TD or be Sp2CBM47 TD.
A further aspect provides a method of modulating growth and/or activity of a cell, the method comprising contacting the cell with a molecule comprising CBM67. The method may be an in vitro method. The molecule comprising CBM67 may comprise Sa2CBM67TD or is Sa2CBM67 TD.
Another aspect provides a method of modulating growth and/or activity of a cell, the method comprising contacting the cell with a molecule comprising CBM70. The method may be an in vitro method. Molecules comprising CBM70 may comprise Sp2CBM70TD or be Sp2CBM70 TD.
As described above, a "molecule comprising CBM 32", "molecule comprising CBM 47", "molecule comprising CBM 67" and/or "molecule comprising CBM 70" may consist essentially of the respective CBM32, CBM47, CBM67 or CBM70, or may consist of the respective CBM32, CBM47, CBM67 or CBM70.
The CBM32, CBM47, CBM67 or CBM70 component of the molecules mentioned in the above aspects may comprise the complete wild-type CBM32, CBM47, CBM67 or CBM70 sequence (as provided above for each of SEQ ID NOs: 1-8). Additionally or alternatively, various aspects of the present disclosure may utilize carbohydrate-binding fragments of any of the CBM32, 47, 67, or 70 sequences disclosed herein (including the sequences of SEQ ID NOs: 1-8).
It should be noted that the terms CBM32, CBM47, CBM67 or CBM70 further include recombinant molecules generated from the reference CBM32, CBM47, CBM67 or CBM70 sequences (including SEQ ID NOs: 1-8). Likewise, these recombinant molecules may comprise full-length CBM32, CBM47, CBM67, or CBM70 sequences, or carbohydrate-binding fragments thereof.
For convenience, the term CBM32/47/67/70 will be used to refer to all of the above CBM molecules (which comprise, consist essentially of, or consist of one or more CBM32, one or more CBM47, one or more CBM67, or one or more CBM70, or carbohydrate-binding fragments of any of these).
The term "modulating" may include any increase or decrease in one or more aspects of cell growth and/or activity. In other words, the molecules described herein (e.g., molecules comprising one or more CBM32/47/67/70) may inhibit certain aspects of cell growth and/or activity, or may induce or stimulate other aspects of cell growth and/or activity.
The terms "growth" and "activity" as applied to a cell may include processes and/or phenomena associated with one or more of cell proliferation, cell viability, cell migration, cell metabolism, cell differentiation, and/or cell morphology/phenotype. The terms "growth" and/or "activity" may further include a response of a cell to certain exogenous and/or endogenous factors or stimuli, including, for example, a response to certain compounds of the immune system, cytokines, chemokines, and one or more environmental factors (light, temperature, pressure, mechanical stress, etc.). Thus, the CBM32/47/67/70 molecules disclosed herein can be used to modulate (inhibit, decrease, or increase) the level of cellular responsiveness.
Given that CBM32/47/67/70 has been shown to modulate cell growth and activity (as described above), it will be appreciated that these molecules may be useful in a number of related medical and veterinary applications and uses.
For example, molecules comprising one or more CBMs 32/47/67/70 may be used to treat and/or prevent diseases or conditions in which abnormal cell growth and/or abnormal cell activity is a factor.
Disclosed herein is a molecule comprising CBM32/47/67/70 for use in the treatment and/or prevention of a disease and/or condition caused, contributed to, and/or characterized by abnormal cell growth and/or activity. The molecule comprising CBM32/47/67/70 may be selected from the group consisting of:
(i) a molecule comprising one or more CpCBM 32;
(ii) a molecule comprising one or more SpCBM 47;
(iii) a molecule comprising one or more SaCBM 67;
(iv) a molecule comprising one or more SpCBM 70;
(v) a molecule comprising Cp2CBM32 TD;
(vi) a molecule comprising Sp2CBM47 TD;
(vii) a molecule comprising Sa2CBM67 TD; and
(viii) a molecule comprising Sp2CBM70 TD.
Further, the use of a molecule comprising CBM32/47/67/70 in the manufacture of a medicament for the treatment and/or prevention of a disease and/or condition caused, contributed to, and/or characterized by abnormal cell growth and/or activity is disclosed.
The molecule comprising CBM32/47/67/70 may be selected from the group consisting of:
(i) a molecule comprising one or more CpCBM 32;
(ii) a molecule comprising one or more SpCBM 47;
(iii) a molecule comprising one or more SaCBM 67;
(iv) a molecule comprising one or more SpCBM 70;
(v) a molecule comprising Cp2CBM32 TD;
(vi) a molecule comprising Sp2CBM47 TD;
(vii) a molecule comprising Sa2CBM67 TD; and
(viii) a molecule comprising Sp2CBM70 TD.
The present disclosure also provides methods of treating and/or preventing a disease and/or condition caused, contributed to, and/or characterized by abnormal cell growth and/or activity, comprising the step of administering to a subject in need thereof a therapeutically effective amount of a molecule comprising CBM 32/47/67/70. The molecule comprising CBM32/47/67/70 may be selected from the group consisting of:
(i) a molecule comprising one or more CpCBM 32;
(ii) a molecule comprising one or more SpCBM 47;
(iii) a molecule comprising one or more SaCBM 67;
(iv) a molecule comprising one or more SpCBM 70;
(v) a molecule comprising Cp2CBM32 TD;
(vi) a molecule comprising Sp2CBM47 TD;
(vii) a molecule comprising Sa2CBM67 TD; and
(viii) a molecule comprising Sp2CBM70 TD.
Diseases caused, contributed to, or characterized by abnormal cell growth and/or activity can include, for example, cell proliferative disorders, including those referred to or classified as benign or malignant conditions. For example, the term "cell proliferative disorder" may include those diseases and/or conditions collectively referred to as "cancer". The term "cancer" may include, but is not limited to, those cancers known as breast cancer, colon cancer, lung cancer, ovarian cancer, glioma and melanoma. In particular, the term cancer (or cell proliferative disorder) may relate to colon, lung and ovarian cancer.
The term "cancer" may also include those diseases and/or conditions collectively referred to as "leukemia" (chronic and acute) as well as any cancer that affects mucosal/mucosa-associated surfaces or tissues.
Thus, the molecules described herein comprising CBM32/47/67/70 may be useful for the treatment and/or prevention of cancer. The molecule comprising CBM32/47/67/70 may be selected from the group consisting of:
(i) a molecule comprising one or more CpCBM 32;
(ii) a molecule comprising one or more SpCBM 47;
(iii) a molecule comprising one or more SaCBM 67;
(iv) a molecule comprising one or more SpCBM 70;
(v) a molecule comprising Cp2CBM32 TD;
(vi) a molecule comprising Sp2CBM47 TD;
(vii) a molecule comprising Sa2CBM67 TD; and
(viii) a molecule comprising Sp2CBM70 TD.
Accordingly, molecules comprising CBM32/47/67/70 for use in the treatment and/or prevention of cancer are provided. The molecule comprising CBM32/47/67/70 may be selected from the group consisting of:
(i) a molecule comprising one or more CpCBM 32;
(ii) a molecule comprising one or more SpCBM 47;
(iii) a molecule comprising one or more SaCBM 67;
(iv) a molecule comprising one or more SpCBM 70;
(v) a molecule comprising Cp2CBM32 TD;
(vi) a molecule comprising Sp2CBM47 TD;
(vii) a molecule comprising Sa2CBM67 TD; and
(viii) a molecule comprising Sp2CBM70 TD.
Further, there is provided the use of a molecule comprising CBM32/47/67/70 in the manufacture of a medicament for the treatment and/or prevention of cancer. The molecule comprising CBM32/47/67/70 may be selected from the group consisting of:
(i) a molecule comprising one or more CpCBM 32;
(ii) a molecule comprising one or more SpCBM 47;
(iii) a molecule comprising one or more SaCBM 67;
(iv) a molecule comprising one or more SpCBM 70;
(v) a molecule comprising Cp2CBM32 TD;
(vi) a molecule comprising Sp2CBM47 TD;
(vii) a molecule comprising Sa2CBM67 TD; and
(viii) a molecule comprising Sp2CBM70 TD.
Also provided are methods of treating and/or preventing cancer, the methods comprising the step of administering to a subject in need thereof a therapeutically effective amount of a molecule comprising CBM 32/47/67/70. The molecule comprising CBM32/47/67/70 may be selected from the group consisting of:
(i) a molecule comprising one or more CpCBM 32;
(ii) a molecule comprising one or more SpCBM 47;
(iii) a molecule comprising one or more SaCBM 67;
(iv) a molecule comprising one or more SpCBM 70;
(v) a molecule comprising Cp2CBM32 TD;
(vi) a molecule comprising Sp2CBM47 TD;
(vii) a molecule comprising Sa2CBM67 TD; and
(viii) a molecule comprising Sp2CBM70 TD.
In addition to the general discovery that molecules comprising CBM32/47/67/70 may be useful in the treatment and/or prevention of cancer, molecules comprising one or more CBM32 may be particularly useful in the treatment and/or prevention of some specific cancers.
Accordingly, the present disclosure further provides a molecule comprising one or more CBMs 32 for use in the treatment and/or prevention of a cancer selected from the group consisting of:
(a) ovarian cancer;
(b) lung cancer;
(c) colon cancer; and
(d) breast cancer.
In view of the above, the present disclosure provides:
(i) a molecule comprising one or more CBMs 32 for use in the treatment and/or prevention of cancer;
(ii) use of a molecule comprising one or more CBMs 32 in the manufacture of a medicament for the treatment and/or prevention of cancer; and
(iii) a method of treating and/or preventing cancer by administering to a subject in need thereof a therapeutically effective amount of a molecule comprising one or more CBM32,
wherein for each of (i), (ii) and (iii), the cancer is one or more selected from the group consisting of:
(a) ovarian cancer;
(b) lung cancer;
(c) colon cancer; and
(d) breast cancer.
In other words, each CBM32 disclosed herein can be used to treat and/or prevent two, three, or all four of cancers (a) through (d), in addition to being useful to treat and/or prevent each of these individual cancers.
Suitable CBM32 for use in the treatment and/or prevention of any one, two, three, or all four of cancers (a) to (d) may include any of those CBM32 disclosed herein. By way of example only, in some cases, molecules for such methods may be selected from the group consisting of:
(i) a molecule comprising one, two or more CBMs 32;
(ii) a molecule comprising one, two or more CpCBM 32;
(iii) a molecule comprising one, two or more peptides comprising the sequences of SEQ ID NOs 1,2, or carbohydrate-binding portions thereof; and
(iv) a molecule comprising CpCBM32 TD.
While the inventors have identified CBM32 as having a general efficacy against a wide range of cancers, in some cases, molecules comprising one or more CBM32 may not exhibit the same level of efficacy against certain specific cancer types. Thus, for molecules comprising one or more CBM32, treatment and/or prevention of cancer may preclude treatment of one or more cancers selected from melanoma, pancreatic cancer, and glioma.
The present disclosure also provides molecules comprising CBM32 and/or CBM40 for use in the treatment and/or prevention of refractory (or resistant) cancer.
In addition to the CBM32 already described herein, an exemplary CBM40 for use may comprise the sialic acid binding domain of Vibrio cholerae (Vibrio cholerae) NanH sialidase (VcCBM: a CBM40) and/or the equivalent (or homologous) domain from Streptococcus pneumoniae NanA sialidase (SpCBM: also CBM 40). Of course, similar or homologous sialic acid binding modules present in other organisms are also encompassed by the terms "CBM" and "CBM 40". An exemplary Vibrio cholerae NanH sialidase amino acid sequence is deposited under accession number A5F7A4 and is reproduced below as SEQ ID NO:23(781 amino acids).
The CBM region of SEQ ID NO. 23 is from amino acid residues 25 to 216, and this sequence may be SEQ ID NO. 24.
An exemplary Streptococcus pneumoniae NanA sialidase amino acid sequence has been deposited under accession number P62575 and is reproduced below as SEQ ID NO:25(1035 amino acids).
The CBM region of SEQ ID NO. 25 is from amino acid residues 121 to 305, and this sequence may be SEQ ID NO. 26.
Thus, a molecule for treating refractory cancer may comprise a polypeptide having SEQ ID NO:23 or 24 or a sialic acid binding fragment thereof. For example, a useful sialic acid binding molecule can comprise a protein portion encoded by the sialic acid binding domain of the nanH gene of Vibrio cholerae (encoding a sialidase) (as provided in SEQ ID NO: 23) or an equivalent or homologous gene present in another organism (e.g., the equivalent/homologous nanA sialidase gene of Streptococcus pneumoniae: see SEQ ID NO: 25). The CBM40 for use in treating refractory cancer may comprise from about residues 1, 5, 10, 15, 25 or 30 (i.e., from 1-30 or any amino acid residue therebetween) to about residues 150, 175, 200, 210, 216, 220-781 (to any residue from 150 to 781, including any residue therebetween) of the Vibrio cholerae sialidase molecule of SEQ ID NO:23 or 24. For example, CBM40 for use in treating refractory cancer may comprise a polypeptide having an amino acid sequence corresponding to SEQ ID NO:23 from residue 25 to about 216 residues.
A further suitable CBM40 for use in treating refractory cancer may comprise a polypeptide having the amino acid sequence of SEQ ID NO:25 or 26 or a sialic acid binding fragment thereof. For example, useful may comprise a protein portion encoded by the sialic acid binding domain of the streptococcus pneumoniae nanA gene (encoding a sialidase). The CBM40 for use in the treatment of cancer may comprise from about residues 80, 90, 100, 110, 120, 121 to 130 (i.e. from any about residues 80 to 130, including any residues therebetween) to about residues 250, 275, 300, 305, 310, 320-1035 (i.e. to any residues from about 250-1035, including to about any residues therebetween) of the Streptococcus pneumoniae sialidase molecules of SEQ ID Nos. 25 and 26. For example, CBM40 for use may comprise a polypeptide having an amino acid sequence corresponding to SEQ ID NO:25 residues 121 to about 305 residues.
One skilled in the art will appreciate that useful CBM40 may comprise a sequence identical to SEQ ID NO: 23. 24, 25 and 26, or a sequence exhibiting some degree (e.g., 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65% or 60%) of sequence identity or homology. All such variants or divergent sequences are included within the scope of the present disclosure and are encompassed by the term "CBM 40". The same and/or homologous CBM40 sequence may have carbohydrate binding function.
A molecule for treating refractory cancer may comprise one or more CBM 40. The molecule may comprise a single or single CBM40, for example a single VcCBM40 or a single SpCBM 40. Alternatively, the co-used molecule may comprise a plurality or plurality (i.e., two or more) of CBM 40. Molecules comprising multiple CBMs may be referred to as "multivalent" molecules or CBMs. A multivalent CBM may, for example, comprise two or more VcCBM40 or two or more SpCBM 40. The multivalent CBM may comprise a mixture of different CBMs, such as one or more vccbms and one or more spcbms.
Molecules comprising CBM32 and/or CBM40 for use in the treatment of refractory (or resistant) cancers may be selected from the group consisting of:
(i) a molecule comprising one or more CpCBM 32;
(ii) a molecule comprising Cp2CBM32 TD;
(iii) a molecule comprising Vc2CBM40 TD; and
(iv) a molecule comprising Sp2CBM40 TD.
Furthermore, the present disclosure provides the use of a molecule comprising CBM32 and/or CBM40 in the manufacture of a medicament for the treatment of refractory (or resistant) cancer. Molecules comprising CBM32 and/or CBM40 may be selected from the group consisting of:
(i) a molecule comprising one or more CpCBM 32;
(ii) a molecule comprising Cp2CBM32 TD;
(iii) a molecule comprising Vc2CBM40 TD; and
(iv) a molecule comprising Sp2CBM40 TD.
Also disclosed are methods of treating refractory (or resistant) cancers, comprising administering to a subject in need thereof a therapeutically effective amount of a molecule comprising CBM32 and/or CBM 40. Molecules comprising CBM32 and/or CBM40 may be selected from the group consisting of:
(i) a molecule comprising one or more CpCBM 32;
(ii) a molecule comprising Cp2CBM32 TD;
(iii) a molecule comprising Vc2CBM40 TD; and
(iv) a molecule comprising Sp2CBM40 TD.
The subject in need or actually to be administered the molecule disclosed herein or a medicament comprising the molecule may be any subject having (or suspected of having) the following diseases: (i) a cell proliferative disorder, (ii) cancer, (iii) any other disease and/or condition described herein; (iv) a disease or condition caused, contributed to, or characterized by abnormal cell growth and/or activity; (v) one or more cancers selected from the group consisting of (a) breast cancer, (b) colon cancer, (c) lung cancer, (d) ovarian cancer, (e) glioma, and (f) melanoma; (vi) one or more cancer cells selected from the group consisting of (a) lung cancer, (b) colon cancer, and (c) ovarian cancer; and/or (v) refractory (or resistant) cancers.
Additionally or alternatively, any subject may be a subject predisposed to or susceptible to the following diseases: (i) a cell proliferative disorder, (ii) cancer, (iii) any other disease and/or condition described herein; (iv) a disease or disorder caused, contributed to, or characterized by abnormal cell growth and/or activity; (v) one or more selected from the group consisting of (a) breast cancer; (b) colon cancer; (c) lung cancer; (d) ovarian cancer; (e) glioma and (f) melanoma: or (vi) one or more cancers selected from the group consisting of (a) lung cancer, (b) colon cancer; and (c) ovarian cancer.
Refractory or resistant cancers may include cancers that are non-responsive to first line or preferred drugs, including standard or preferred chemotherapy or radiotherapy options. Refractory or resistant cancers may include those cancers that are resistant (or non-responsive) to cisplatin. The term refractory or resistant cancer may include refractory or resistant ovarian cancer. The terms refractory (ovarian) cancer and resistant (ovarian) cancer may include ovarian cancer that is resistant to cisplatin.
Accordingly, the present disclosure provides (i) a molecule comprising one or more CpCBM 32; (ii) a molecule comprising Cp2CBM32 TD; (iii) a molecule comprising Vc2CBM40 TD; and/or (iv) a molecule comprising Sp2CBM40TD for use in the treatment of refractory (or resistant) ovarian cancer.
It is to be understood that any treatment described herein may involve the use of one or more molecules of the present disclosure to treat, ameliorate or reduce one or more symptoms of the various diseases and disorders described herein. For example, symptoms of a disease such as cancer may include, for example, the presence of a tumor and/or cell mass. Thus, the molecules described herein can be used to modulate (e.g., stop, delay, inhibit, or reduce) tumor formation and/or metastasis thereof. These molecules can also be used to reduce the overall size of a tumor. Certain tumors, including those that are large and/or invasive, are often easier to remove surgically if they are first reduced in size. Typically, chemical and/or radiation therapy based treatments may be used to reduce the size of the tumor, but such treatments may be replaced and/or supplemented by treatments based on the use of molecules comprising CBM 32/47/67/70. As noted above, molecules comprising CBM32/47/67/70 exhibit the ability to modulate cell growth and/or activity, and thus, without wishing to be bound by theory, it is possible that the mechanism supporting any one of the disclosed molecules' ability to affect tumor size may result from the cell proliferation, differentiation and/or metabolic regulation of that molecule.
In view of the above, successful treatment of tumors is therefore characterized by a reduction in tumor size, observed or detectable/detectable levels of tumor metastasis, angiogenesis within tumorigenic tissues and/or tissue invasion.
The molecules of the present disclosure may be used in methods of modulating (e.g., inhibiting, limiting, or reducing) tumor growth, development, and/or metastasis in a subject in need thereof. The molecules described herein may be formulated as compositions for modulating tumor growth, development and/or metastasis, or for use in the manufacture of a medicament to achieve these objectives. The present disclosure also provides molecules comprising CBM32/47/67/70 for use in treating tumors. Furthermore, the use of a molecule comprising CBM32/47/67/70 in the manufacture of a medicament for the treatment of a tumour is described. In addition, the present disclosure provides methods of treating a tumor comprising administering to a subject (or tumorigenic tissue) in need thereof a molecule comprising CBM 32/47/67/70.
As previously mentioned, the term "subject in need thereof" may include any subject suspected of having a tumor or diagnosed with a tumor and/or identified as having a predisposition to have a tumor and/or being predisposed to having a tumor. The term "tumor" may include refractory or resistant tumors. The term tumor may include refractory or resistant ovarian cancer tumors.
Accordingly, the present disclosure provides various uses of molecules comprising CBM32/47/67/70 and which have been identified as modulators of cell growth and/or activity. Any given molecule (including CBM32/47/67/70) can be identified as a modulator of cell growth and/or activity by the various assays and assays described in the examples section of this patent application. For example, a cellular wound scratch assay is one example of an assay that may be adapted to determine whether any given "test" molecule exhibits the necessary ability to modulate cell growth, cell migration, and/or activity. Additionally or alternatively, BrdU cell proliferation assays can be used to determine any effect of a test agent (e.g., a molecule comprising CBM32/47/67/70) on cell proliferation, growth, and/or activity. Briefly, cells can be cultured in medium supplemented with BrdU, which is incorporated into de novo DNA as a thymidine substitute as the cells grow and proliferate. This will label the progeny cells, and the amount of BrdU incorporation can be used as a measure or indicator of cell proliferation/growth. BrdU detection can be achieved by using antibodies with specificity or affinity for BrdU.
In other cases, based on metabolic determination, e.g.2.0Assay(2.0) can be used to determine any effect of a test agent (e.g., a molecule comprising CBM32/47/67/70) on cell proliferation, growth, and/or activity. In such assays, the amount of ATP (adenosine triphosphate) can be assessed to determine the number of metabolically active cells (and thus can provide a measure of viable cells). The number of viable cells treated with the test agent can be used as a measure or indicator of cell proliferation/growth.
Alternatively or additionally, assays based on DNA staining for exampleDirect cell proliferation assays can be used to determine any effect of a test agent (e.g., a molecule comprising CBM32/47/67/70) on cell proliferation, growth, and/or activity. In such assays, the number of viable cells can be determined using DNA staining (e.g., cell permeable DNA binding dyes). Again, the number of viable cells treated with the test agent can be used as a measure or indicator of cell proliferation/growth.
Accordingly, the present disclosure may relate to those molecules that exhibit the ability to modulate cell growth and/or activity by a cell activity modulation assay (e.g., a cell wound scratch assay). To this end, the present disclosure further provides a method of identifying a molecule (including a molecule comprising CBM32/47/67/70) for use in a method of modulating cell growth and/or activity or for various medical and/or veterinary applications described herein, the method comprising subjecting a test compound to an assay capable of reporting the effect of the test compound on cell growth and/or activity, wherein the test compound is a molecule comprising CBM3247/67/70 and the compound is useful in the treatment and/or prevention of a disease and/or condition of the type described herein if the assay reports that the compound has an effect on any aspect of cell growth and/or activity. Assays capable of reporting the effect of a test compound on cell growth and/or activity may be cell wound scratch assays, BrdU-type, or the like,2.0 type and/orDirect cell proliferation assay, as described herein.
Molecules comprising CBM32/47/67/70 may further be used as molecules that may be conjugated, bound or linked or associated with other entities for the purpose of targeting or delivering the entity to some tissue or cell.
This type of molecule may be otherwise referred to as a "therapeutic warhead" or "conjugate". Without wishing to be bound by theory, the presence of ligands for the various CBMs that may be contained within the molecules of the present disclosure (e.g., CBM32, CBM47, CBM67, and/or CBM70) in certain cell receptors and membrane-binding molecules may allow the various molecules described herein to be used as a means of delivering conjugated heterologous molecules (i.e., molecules other than the molecule containing CBM32/47/67/70) to the cells or tissues containing the cells. Such conjugate molecules are useful for the treatment of cancer, wherein the molecules described herein (which exhibit affinity for carbohydrates expressed on the cell surface) are useful for directing therapeutic and/or cytotoxic moieties thereto.
For example, molecules described herein (including any CBM32/47/67/70 molecule) can be conjugated to one or more (e.g., two, three, four, or more) moieties (e.g., therapeutic and/or cytotoxic). Accordingly, the present disclosure relates to CBM32/47/67/70 molecular conjugates.
The CBM32/47/67/70 molecule conjugates can comprise a CBM32/47/67/70 molecule of the present disclosure conjugated (linked, bound, or otherwise associated) to a heterologous moiety. The heterologous moiety may comprise a therapeutic and/or cytotoxic moiety, which may be conjugated to certain moieties of the CBM32/47/67/70 molecule.
For example, a heterologous moiety may be conjugated to one or both ends of the CBM32/47/67/70 molecule. The heterologous moiety may additionally or alternatively be conjugated (or even fused) to an internal portion of the CBM32/47/67/70 molecule. It will be appreciated that whereas the heterologous moiety is conjugated to the CBM32/47/67/70 molecule, the molecule (or its conjugation) should not (substantially) interfere with or ablate or reduce the carbohydrate binding properties of the CBM32/47/67/70 molecule.
As described above, the heterologous moiety may be a drug for the treatment of a disease affecting a cell or tissue expressing a receptor comprising a ligand of any one of the CBM32, CBM47, CBM67 or CBM70 molecules. For example, the drug may be a chemotherapeutic drug or the like for treating cancer. The heterologous moiety may be a cytotoxic moiety capable of killing cells or inducing apoptosis. The heterologous portion can comprise a molecule capable of recruiting or recruiting a particular cell to a particular tissue. For example, the heterologous moiety can be, e.g., a T Cell Receptor (TCR), which can be used as a means of recruiting T cells to, e.g., a tumor or cancerous tissue.
The present disclosure may provide compositions for various uses, medicaments, and methods described herein. Thus, any of the molecules described herein comprising CBM32/47/67/70 may be formulated for use.
For convenience, and with reference to the following sections describing compositions, formulations, etc., it should be noted that the molecules comprising CBM32/47/67/10 and any conjugates comprising the same (e.g., CBM32/47/67/70:: drug conjugates/fusions) described herein are intended to be encompassed under the generic term "CBM 32/47/67/70-comprising molecules".
The molecules comprising CBM32/47/67/70 may be formulated for use and as therapeutic or pharmaceutical compositions. Various compositions may comprise one or more molecules described herein, and any given treatment may require administration of one or more of these compositions (together, simultaneously or separately). It should be noted that compositions according to the present disclosure may further comprise one or more other therapeutic moieties (e.g., molecules, small molecules, antibodies, oligonucleotides, etc., useful for treating one or more diseases and/or conditions). Additionally or alternatively, the molecule comprising CBM32/47/67/70 may be administered with one or more other (different) therapeutic entities, wherein the one or more other (different) therapeutic entities may be used to treat the same or different diseases. The term "administered together" includes the administration of CBM32/47/67/70 before, after, and/or concurrently with the administration of one or more other therapeutic entities.
The molecules described herein can be formulated for enteral (including oral), parenteral, and/or topical administration, and those skilled in the art will appreciate that the precise formulation can vary depending on the route of administration.
The Pharmaceutical compositions of The invention may be prepared conventionally and comprise materials commonly used in pharmaceuticals and described, for example, in Remington's The Sciences and Practice of Pharmacy, 22 nd edition (Pharmaceutical Press 2012) and/or Handbook of Pharmaceutical Excipients, 7 th edition (compiled by Rowe et al, Pharmaceutical Press 2012), all of which are incorporated herein by reference in their entirety.
The therapeutic or pharmaceutical compositions of the present disclosure (i.e., compositions comprising a molecule comprising CBM32/47/67/70 and used in any of the medicaments or methods described herein, including methods or medicaments for modulating cell growth and/or activity and/or treating cancer) can be formulated with one or more pharmaceutically acceptable excipients, carriers, adjuvants, and buffers. The compositions may be administered, for example, orally (including mucosally), parenterally, intraperitoneally, enterally, intramuscularly, subcutaneously, intravenously, or by any other route useful to achieve the desired effect (in which case the effect includes modulating cell growth/activity, treating or preventing a disease/condition and/or cancer associated with cell growth/activity and/or modulating tumor growth). As noted above, the exact composition of the formulation may vary depending on the route of administration selected.
A therapeutic or pharmaceutical formulation comprising a molecule comprising CBM32/47/67/70 and intended for administration to a subject may be coated, encapsulated or encapsulated in a material that protects the molecule from enzymes, acids and other natural compounds/conditions (including, for example, compounds (including antibodies), cells of the immune system and processes) that may inactivate or denature the compound and/or its carbohydrate binding properties.
Among the various standard and conventional excipients that may be used in compositions comprising the molecules described herein are those pharmaceutically acceptable organic or inorganic carrier materials that are suitable for parenteral, enteral, oral (including mucosal) and other routes of administration without deleterious reaction with the molecule comprising CBM 32/47/67/70.
When the molecule comprising CBM32/47/67/70 is formulated for parenteral administration, the composition may be sterile.
The composition may comprise an oil or aqueous solution, suspension and/or emulsion.
In other embodiments, the composition may take the form of an implant, such as a (dissolvable or biodegradable) film, a pessary, or an implant (including a suppository).
Pharmaceutical formulations comprising the molecules described herein may be mixed with stabilizers, wetting agents, emulsifiers, salts (for influencing osmotic pressure), buffers and/or other substances which do not deleteriously react with the active compound.
One or more of the molecules described herein can be formulated for oral administration and administered orally. As noted above, oral administration includes mucosal administration, which itself includes intranasal administration and/or inhalation administration.
Compositions for use may include solid dosage forms suitable for oral administration. These may include, for example, capsules, tablets, pills, powders, and granules. In any given solid dosage form, the molecule comprising CBM32/47/67/70 (or any conjugate comprising the same) may be mixed with at least one inert pharmaceutically acceptable excipient. Examples of suitable excipients are known to those skilled in the art, but may include, for example, fillers or extenders, wetting agents, binders, disintegrants, solution retarding agents, absorption promoters, adsorbents, lubricants, or mixtures thereof. The tablet, pill or capsule may also contain buffering agents. Solid dosage forms such as tablets, dragees, capsules, pills and/or granules can also be prepared with coatings and shells, e.g. coatings to protect against the gastrointestinal environment and/or stomach acid.
The solid dosage form may contain opacifying agents and may also be formulated to ensure delayed release of the active agent (in this case, the molecule comprising CBM32/47/67/70 or the conjugate comprising it) in or to a specific part of its intestinal tract.
Solid compositions for oral administration may be formulated in unit dosage forms, each dose containing an appropriate dose of a molecule comprising CBM32/47/67/70 (or a conjugate comprising the same). The exact amount of the molecule comprising CBM32/47/67/70 (or a conjugate comprising the same) included in any given solid dosage form will vary depending on the intended use. The solid composition may comprise a "unit dose," i.e., a unit dose comprising an amount of a molecule comprising CBM32/47/67/70 (or a conjugate comprising the same) calculated to produce a desired effect (e.g., modulating cell growth and/or activity) during treatment.
Liquid dosage forms for oral administration may, as stated, include emulsions, solutions, suspensions, syrups and elixirs. In addition to the compound or composition, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers.
Any of the disclosed molecules can be used in any suitable amount. As noted above, the acid molecule may be formulated for oral, mucosal or parenteral administration, and thus, the precise formulation may depend on the intended route of administration. The amount of molecules comprising CBM32/47/67/70 present at any given dose may range from 0.1 μ g to 1000 μ g. E.g., amounts of about 0.1. mu.g, 0.2. mu.g, 0.3. mu.g, 0.4. mu.g, 0.5. mu.g, 1. mu.g, 10. mu.g, 20. mu.g, 25. mu.g, 50. mu.g, 100. mu.g, 200. mu.g, 300. mu.g, 400. mu.g, 500. mu.g, 600. mu.g, 700. mu.g, 800. mu.g or 900. mu.g. A selected amount of CBM32/47/67/70 molecule may be formulated in a specific volume of pharmaceutically acceptable excipient, diluent, and/or buffer. The volume of excipient, diluent or buffer is about 10. mu.L to 5 mL. For example, a desired amount of a CBM32/47/67/70 molecule can be combined (or formulated) with about 15 μ L,20 μ L,25 μ L,30 μ L,35 μ L,40 μ L,45 μ L,50 μ L,55 μ L,60 μ L,65 μ L,70 μ L,75 μ L,80 μ L,85 μ L,90 μ L,95 μ L,100 μ L,200 μ L,250 μ L,300 μ L,400 μ L,500 μ L,600 μ L,700 μ L,800 μ L,900 μ L,1mL,2mL,3mL, or 4 mL. For example, 100 μ g of CBM32/47/67/70 molecules can be mixed with about 250 μ L of excipient to produce a final concentration of 400 μ g/mL. A dose of about 0.1. mu.g/mL to 1mg/mL concentration may be used, including, for example, a dose of 5. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL, 300. mu.g/mL, 500. mu.g/mL, 600. mu.g/mL, 700. mu.g/mL, 800. mu.g/mL, or 900. mu.g/mL.
In use, a dose of CBM32/47/67/70 molecule administered as part of the treatment and/or prevention of a cell proliferation and/or differentiation disorder (e.g., cancer) may be administered multiple times over days, weeks, months, or years. For example, after the initial (or first) administration, one more dose of CBM32/47/67/70 molecules may be administered after about (+/-1 or 2 days) 3,4,5,6,7,17,21,28 and/or 35 days. A particular dose of CBM32/47/67/70 molecule may be administered 1,2, 3 or more times on any given day. Each time, a CBM32/47/67/70 molecule may be administered (by being considered to be the best way to affect appropriate treatment or induction to prevent development of cell proliferation and/or differentiation disorders).
Detailed Description
The invention will now be described in detail with reference to the following drawings, which show:
FIG. 1: proliferation data (BrdU incorporation assay). Different cancer cell lines (melanoma ((a), A2058), breast cancer ((b), MDA. MB.231), pancreas ((c), PANC1) and lung ((d), A549) were treated with Cp2CBM32TD (400. mu.g/mL), PBS (control), sialic acid binding CBM40 reagents (Sp2CBMTD (400 μ g/mL), Vc2CBMTD (400 μ g/mL) or Vc4CBM (400 μ g/mL)) or sialic acid binding null CBM40 mutant (Sp2(R274Q) CBMTD (400 μ g/mL)) were treated and left for 24 hours.
FIG. 2: proliferation data (BrdU incorporation assay). Different cancer cell lines ((a), glioma (T98G), colon ((b), SW620) and ovary ((c), PE014) were treated with Cp2CBM32TD (400 μ g/mL), PBS (control), sialic acid binding CBM40 reagent (Sp2CBMTD (400 μ g/mL), Vc2CBMTD (400 μ g/mL) or Vc4CBM (400 μ g/mL)) or sialic acid binding null CBM40 mutant (Sp2(R274Q) CBMTD (400 μ g/mL)) and left for 24 hours.
FIG. 3: percent inhibition of cell proliferation in human metastatic ovarian carcinoma cell line PE01, PE01 cisplatin resistance (PE01-CR), and PE 014. (a) Cp2CBM32TD (GalNAc/galactose/lactose binding CBM from clostridium perfringens) was used against three ovarian cell lines, including the cisplatin resistant form (PE 01-CR). (b) Vc2CBMTD against three ovarian cell lines (PE01, PE01-CR, PE014) was used. (c) Sp2CBMTD (PE01, PE01-CR, PE014) was used against three ovarian cell lines.
FIG. 4: proliferation data (BrdU incorporation assay). Different cancer cell lines (melanoma ((a), a2058), breast ((b), mda. mb.231), pancreas ((c), PANC1) and lung ((d), a549) were treated with 2CBM47TD (400 μ g/mL), 2CBM67TD (400 μ g/mL), 2CBM70TD (400 μ g/mL), PBS (control) or sialic acid binding CBM40 reagent Sp2CBMTD (400 μ g/mL) and left for 24 hours, then cells were incubated with BrdU for 2-6 hours ELISA assays to measure the amount of BrdU incorporated into the DNA, which are standard assays to measure cell growth/proliferation.
FIG. 5: proliferation data (BrdU incorporation assay). Different cancer cell lines ((a), glioma (T98G), colon ((b), SW620) and ovary ((c), PE014, (d) PE01)) were treated with 2CBM47TD (400 μ g/mL), 2CBM67TD (400 μ g/mL), 2CBM70TD (400 μ g/mL), PBS (control) or sialic acid binding CBM40 reagent Sp2CBMTD (400 μ g/mL) and left for 24 hours. The cells were then subjected to BrdU assay and incubated for 2-6 hours. An ELISA assay was performed to measure the amount of BrdU incorporated into DNA. The assay is a standard assay to measure cell growth/proliferation. Two-way anova was used to show statistical significance compared to control groups (×) for p <0.0001, × for p <0.001, and ×) for p <0.01 and for p < 0.1.
FIG. 6: proliferation data (BrdU incorporation assay). Ovarian cancer cells (PE01 cisplatin-resistant (CR)) were treated with Sp2CBMTD (400. mu.g/mL), 2CBM47TD (400. mu.g/mL), 2CBM67TD (400. mu.g/mL), 2CBM70TD (400. mu.g/mL) or PBS (control) and left for 24 hours. The cells were then subjected to BrdU assay and incubated for 2-6 hours. An ELISA assay was performed to measure the amount of BrdU incorporated into DNA. The assay is a standard assay to measure cell growth/proliferation. Two-way anova was used to show statistical significance compared to control groups (×) for p <0.0001, × for p <0.001, and ×) for p <0.01 and for p < 0.1.
FIG. 7: from (a) CellTiter2.0assay (CTG 2.0); and (b)Proliferation data obtained from direct cell proliferation assays. Ovarian cancer cell lines (SK-OV-3) were treated with Cp2CBM32TD and left for 72 hours, as described below. Dose response curves were generated with nine different concentrations using a 3-fold dilution series from 3mg/mL to 0.457. mu.g/mL. After treatment, use2.0 determination of orDirect cell proliferation assay end-point analysis was performed on the plates.
FIG. 8: from (a) CellTiter2.0assay (CTG 2.0); and (b)Proliferation data obtained from direct cell proliferation assays. Ovarian cancer cell lines (TOV-21G) were treated with Cp2CBM32TD and left for 72 hours, as described below. Dose response curves were generated with nine different concentrations using a 3-fold dilution series from 3mg/mL to 0.457. mu.g/mL. After treatment, use2.0 determination of orDirect cell proliferation assay end-point analysis was performed on the plates.
FIG. 9: from (a) CellTiter2.0assay (CTG 2.0); and (b)Proliferation data obtained from direct cell proliferation assays. Colorectal cancer cell line (Gp2D) was treated with Cp2CBM32TD and left for 72 hours, as described below. Dose response curves were generated with nine different concentrations using a 3-fold dilution series from 3mg/mL to 0.457. mu.g/mL. After treatment, use2.0 determination of orDirect cell proliferation assay end-point analysis was performed on the plates.
FIG. 10: from (a) CellTiter2.0assay (CTG 2.0); and (b)Proliferation data obtained from direct cell proliferation assays. Breast cancer cell lines (MDA-MB-231) were treated with Cp2CBM32TD andthe mixture was left for 72 hours as follows. Dose response curves were generated with nine different concentrations using a 3-fold dilution series from 3mg/mL to 0.457. mu.g/mL. After treatment, use2.0 determination of orDirect cell proliferation assay end-point analysis was performed on the plates.
FIG. 11: from (a) CellTiter2.0assay (CTG 2.0); and (b)Proliferation data obtained from direct cell proliferation assays. The breast cancer cell line (CAMA-1) was treated with Cp2CBM32TD and left for 72 hours as described below. Dose response curves were generated with nine different concentrations using a 3-fold dilution series from 3mg/mL to 0.457. mu.g/mL. After treatment, use2.0 determination of orDirect cell proliferation assay end-point analysis was performed on the plates.
FIG. 12: from (a) CellTiter2.0assay (CTG 2.0); and (b)Proliferation data obtained from direct cell proliferation assays. The lung cancer cell line (NCI-H441) was treated with Cp2CBM32TD and left for 72 hours, as described below. Dose response curves were generated with nine different concentrations using a 3-fold dilution series from 3mg/mL to 0.457. mu.g/mL. After treatment, use2.0 determination of orDirect cell proliferation assay end-point analysis was performed on the plates.
Experiment of
Part A
Many different cancer cell lines were treated with various CBM test agents, each of which comprised one or more molecules containing a CBM selected from the group consisting of CBM32, CBM40, CBM47, CBM67, and CBM70.
These test agents were evaluated in a BrdU cell proliferation assay, as described above for each of fig. 1-6.
The results of these measurements are shown in figures 1 to 6.
Part B
CBM Cp2CBM32TD was selected for further studies using the following assay:
(I)CellTiter2.0assay (CTG 2.0); and
(ii)direct cell proliferation assay.
2.0assay provides a homogeneous method to determine the number of viable cells in culture by quantifying the amount of ATP present, which indicates the presence of metabolically active cells.Direct cell proliferation assays rely on the use of cell-penetrating DNA binding dyes in combination with background inhibitory agents. These procedures are fully described in Promega2.0Assay Technical Manual TM403(2018, 10 months)Direct Cell promotion Assay Molecular Probes manual MP 35011 (7/20/2009).
Cell lines stored in liquid nitrogen were thawed and expanded in growth medium. Once the cells reached the desired doubling time, the screening was started. 25 μ L of cells in growth medium were seeded at a rate of 500-1500 cells per well in black 384-well tissue culture treatment plates. Assay plates were equilibrated by centrifugation and incubated at 37 ℃ for 24 hours prior to compound treatment. At the time of treatment, a set of time points 0 (T) was collected0) Plates were assayed (untreated) and readings of DNA content and viability were measured using CyQUANT Direct (ThermoFisher) and CellTitre-Glo 2.0(Promega), respectively. Add 15 μ L/well of assay reagent to assay plate. Nine different concentrations were evaluated using a 3-fold dilution series from 3mg/mL to 0.457. mu.g/mL.
Time point 0 (T) was read on an Envision microplate reader (Perkin Elmer)0) Fluorescence (CyQUANT) or absorbance (CellTitre-Glo 2.0) of the plate. Assay plates were incubated with compounds for 72 hours and then analyzed using CyQUANT Direct and CellTitre-Glo 2.0.
Growth Inhibition (GI) was used as a measure of cell growth. The GI percentage is calculated by applying the following test and equation:
if it is not
If it is not
Where T is the signal measure for the test article, V is the untreated/vehicle-treated control measure, V0Is an untreated/vehicle control measure at time zero (also colloquially referred to asIs T0A plate). This formula is derived from the growth inhibition calculation used in the National Cancer Institute's NCI-60high throughput screening.
A GI reading of 0% indicates no growth inhibition and will occur if a T reading of 72 hours is comparable to a V reading for the corresponding time period. 100% of GI represents complete growth inhibition (cytostasis), in which case cells treated with compound for 72 hours will have a similar T0The same endpoint reading for control cells. 200% of GI represents complete death (cytotoxicity) of all cells in the culture well, and in this case, the T reading at 72 hours will be lower than the T0And (6) comparison.
Inhibition is also provided as a measure of cell viability. An inhibition level of 0% indicates that the treatment did not inhibit cell growth. Inhibition of 100% indicates that the cell number did not double during the treatment window. Both cytostatic (cytostatic) and cytotoxic treatments can produce percent inhibition of 100%. Percent inhibition was calculated using the following formula:
I＝1-T/U
where T is treated and U is untreated/vehicle control.
In each case, cells were seeded in growth medium in black 384-well tissue culture treatment plates and equilibrated by centrifugation. The treated assay plates were incubated with the test compounds for 72 hours. After treatment, use2.0 determination of orDirect cell proliferation assay end-point analysis was performed on the plates.
The results are shown in fig. 7 to 12.
In these further studies, the inventors determined that Cp2CBM32TD did show antiproliferative activity against the breast cancer cell line mda. mb.231 (see fig. 10a and 10b) and the breast cancer cell line CAMA-1 (see fig. 11a and 11 b). This same level of activity against mda.mb.231 cancer cells was not observed in the BrdU assay.
Without being bound by theory, the inventors hypothesize that this difference may be due to the population doubling time of mda.mb.231 cells (approximately 36 hours). Therefore, it is believed that the shorter treatment time (24 hours duration) in the BrdU assay is not sufficient to correctly demonstrate the antiproliferative activity of Cp2CBM32TD on mda. mb.231 cells.
- 上一篇：一种医用注射器针头装配设备
- 下一篇：表皮生长因子在治疗糖尿病性足溃疡中的用途