Patent Application
20240294590
The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Apr. 17, 2024, is named 56699-723_402.xml and is 233,994 bytes in size.
The present application relates to the field of growth factor variants and methods for controlling the multimerization of such growth factors.
In biological systems, proteins often make up complicated signaling cascades that direct the cell to behave in a particular way. For example, a common way that cells are directed to begin the process of dividing is that a protein (ligand) binds to the extra cellular domain of a transmembrane protein receptor wherein binding of the ligand to the extra cellular domain confers a change in the conformation of the receptor. The ligand-induced conformational change can take place in the extra cellular domain, the intra cellular domain or both and results in a change in which proteins or molecules are able to bind to the receptor. This outside to inside signaling is a common mechanism that is used to signal cells to divide, initiate programmed cell death and many other processes.
One commonly used mechanism that regulates the activity of growth factor receptors is ligand-induced dimerization of the receptor's extra cellular domain which in turn brings the intracellular tails close together which makes a good docking site for modifying proteins such as kinases that initiate a signaling cascade that eventuates in a signal to the cell's nucleus that causes the cell to divide.
Ligand-induced dimerization of the extra cellular domain of growth factor receptors is often accomplished through the binding of ligand dimers; that is two ligands non-covalently bind to each other to form homo- or hetero-dimers which then bind to two receptors that are either the same (homo) or different (hetero).
An important example of ligand-induced receptor dimerization is NM23 dimers binding to and dimerizing the extra cellular domain of MUC1*, which is the truncated form of the MUC1 transmembrane protein that is tumor and stem cell specific. Whether or not the ligand is a monomer, dimer or a higher order multimer is a function of, among other things, its concentration. For many growth factor receptors, only the dimeric form of the ligand activates the growth factor receptor. Additionally, in many biological systems, there are feedback loops wherein the higher order multimers turn off the function that is promoted by the dimer. For example, the NM23 dimer activates pluripotent growth but the NM23 hexamer turns off pluripotent growth and initiates differentiation. Similarly, the CI protein of Phage lambda turns on transcription of one set of genes when it is bound to DNA as a tetramer but turns off transcription of those genes when, as a function of increased concentration, the CI protein becomes an octamer. In many cases, it is desirable to constitutively activate a growth factor receptor, or increase some activity that is mediated by a specific multimerization state of a protein. The problem is that it is very difficult to express and isolate a specific multimer and even more difficult to maintain that multimerization state when it is added to a biological system or expressed within a biological system. Therefore, it would be advantageous to be able to generate ligands that exist exclusively in a specific multimerization state, or prefer that multimerization state, such as dimers or that prefer dimerization.
Although NM23 mutants have been reported that prefer dimer formation, the portion that exists as the active dimer relative to the inactive hexamer varies greatly, particularly when expressed as the recombinant protein, depending on the cell that is expressing it, concentration, and a number protein expression conditions that are difficult or impossible to control. Therefore, it would be beneficial to develop methods, including recombinant methods, which would result in a higher percentage of or more stable populations of dimeric forms of NM23 or NM23 mutants.
The invention overcomes the above-mentioned problems, and provides genetic variants of proteins, chimeras, and single chain constructs that produce proteins that prefer a specific multimerization state.
In one aspect, the present invention is directed to a recombinantly made protein construct that preferentially forms a specific multimer. The multimerization state may be its biologically active state. The multimerization state may be a dimer. Alternatively, the multimerization state may be its inactive state. The multimerization state may be a higher order multimer.
The protein may be a growth factor or a transcription factor. The dimer form may be a homodimer or a heterodimer. The protein may be a mammalian protein, such as human protein or mouse protein.
The protein construct may comprise two monomers or fragments of the monomers, wherein such may be linked together through a linker peptide, thus forming a monomer-linker-monomer construct.
The monomeric protein may be NM23, such as H1, H2 or H7 isoforms. The monomer may be NM23 or a mutant thereof that favors forming dimer and inhibits formation of higher order multimers. The mutant NM23 may be S120G or P96S or NM23 and P96S. The monomer may be NM23 or a mutant thereof that favors forming dimer, wherein one to ten C-terminal amino acids are deleted. One to six C-terminal amino acids may be deleted.
The linker may include GS, GS2, GS3, IgG1 hinge region, or IgG2a hinge region or combination thereof.
In one aspect, the protein construct according may include
NM23 P96SΔC2/S120G IgG2ah noC, or
NM23 P96SΔC6/S120G IgG2ah noC.
In one aspect, the specific multimer may be formed by recombinantly connecting a protein monomer to a second component. The specific multimer may be formed between the second components. The second component may be a linker peptide or a protein or protein fragment.
In one aspect, the multimer may be formed between the second components via a chemical bond. Or, the multimer may be formed between the second components via a covalent bond. The covalent bond may be a disulfide bond. And in particular, the multimer may be a dimer.
In one aspect, the second component may be a IgG1 hinge or IgG2a hinge or a combination thereof. The protein construct may be NM23-S120G-IgG1h, bNM23-S120G-IgG2ah, or NM23 S120G IgG1Fc.
In another aspect, in the protein construct according to above, the multimer may be formed between the second components via a non-covalent bond. The second component may be a protein that has high affinity to bind to another protein. The second component may be all or a portion of Fc region of an antibody, Fos or Jun. The Fc region may be IgG1Fc. In one aspect, the second component may be able to homo-dimerize or hetero-dimerize.
In another aspect, cysteines may be inserted into the protein to promote multimer formation via disulfide bonds. The second component may be a fragment of the Fc domain of an IgM antibody.
In another aspect, in any of the protein constructs discussed above, an amino acid sequence that facilitates entrance into a cell or into the nucleus of the cell may be included.
In yet another aspect, in any of the protein constructs discussed above, an amino acid sequence that facilitates secretion of the protein construct from its expressing host cell may be included
The present invention is also directed to an isolated nucleic acid that includes any of the protein constructs discussed above. The nucleic acid may further comprise a sequence that encodes amino acid sequence that facilitates entrance into a cell or into the nucleus of the cell. The nucleic acid may further include a sequence that encodes amino acid sequence that facilitates secretion of the protein from its expressing host cell. The nucleic acid may include nucleic acid encoding NM23 or a mutant thereof that favors dimer formation.
In another aspect, the present invention is directed to an expression vector that includes any of the nucleic acids discussed above. The vector may be a plasmid or a virus.
In another aspect, the present invention is directed to a host cell that includes the vector as discussed above.
In still another aspect, the present invention is directed to a method for proliferating cells that includes transfecting or transducing the cells with the vectors discussed herein. The cell may be a stem or progenitor cell.
In yet another aspect, the invention is directed to a method for inducing pluripotency in a somatic cell that includes transfecting or transducing the cells with the vector described herein.
In another aspect, the present invention is directed to a method of treating a patient suffering from a condition that would be alleviated by treatment with administration of immature cells, comprising:
The nucleic acid sequence of one or more of the genes in the vector may be native to the cell and may have been modified.
In another aspect, the present invention is directed to a method for altering expression of a targeted gene that includes the steps of:
The above method may include further the step of (iii) designing the nucleic acid to insert at a location near the promoter site of the targeted gene. The targeted cell may be stem or progenitor cell. The progenitor cell may be hematopoietic cell. And the progenitor cell may be B-cell or B-cell precursor.
In another aspect, the invention is directed to the above method, which further comprises engineering multimerization state of the transcription factor so as to promote the active state, if it is desired to have the gene of interest expressed and not in the active state if it is desired to suppress the gene of interest.
In still another aspect, the present invention is directed to a method of healing or alleviating an illness that could benefit from increased production of stem or progenitor cell, that includes administering to a patient suffering from or at risk of developing a disease, genetic defect or unhealthy condition the cell described above. The cell may be a fertilized or unfertilized egg. The cell may be a stem or progenitor cell. The cell may be obtained from the patient to be treated. Or the cell may be an iPS cell. The cell may be an iPS cell from the patient to be treated.
In another aspect, the present invention is directed to a method for identifying growth factor mutant that prefers dimerization and resists formation of higher order multimers, that includes determining affinity of binding of the growth factor to a target receptor, wherein higher order multimers do not bind to the target receptor with the same affinity as the dimer. The growth factor may be NM23 and the target receptor may be MUC1*. The target receptor may be a MUC1* extra cellular domain peptide that may be PSMGFR peptide having the sequence of
In another aspect, the invention is directed to a protein according to above, which is genetically engineered to resist dimer formation. The protein may be genetically engineered to prefer formation of higher order multimers. The protein may be genetically engineered to prefer formation of tetramers, pentamers or hexamers. The higher order multimer comprises protein genetically fused to Fc portions of an IgM antibody. The protein may be a growth factor. The protein may be NM23. The protein may be a transcription factor. The protein may be p53.
The present invention is directed to methods and compositions that increase a specific multimerization state of a protein. The methods can be applied to any protein for which a multimer is the active form, in particular wherein the active form is a dimer.
One method for making ligands multimeric is to make constructs for recombinant proteins that are already connected. For example, single chain proteins wherein two or more monomers are connected either directly or via a linker that may vary in length or sequence to obtain the desired biological activity.
Another method for making ligands that are multimeric is to make recombinant chimeras wherein each monomer is connected to a portion of a protein that multimerizes. For example, the proteins Fos and Jun interact so that these proteins could be genetically connected to ligands that may be the same or different in order to cause the dimerization of the resultant chimeras. Similarly, the Fc regions of antibodies dimerize. When ligand-Fc region chimeras are made, they dimerize and can mimic the activity of a naturally occurring dimeric ligand.
Yet another method for making ligand multimers is via chemical coupling of two or more monomeric ligands. For example, a bifunctional linker can be used to chemically couple two protein ligands to make homo or hetero dimers.
Yet another method for making multimeric ligands is to identify small molecules that bind to the target receptor and then synthesizing multimers of the small molecule.
In a preferred embodiment, the multimerization state that is preferred for enhancing a natural biological interaction, such as activating a growth factor receptor, is a dimer. In a more preferred embodiment, the ligand that is made dimeric or made to prefer dimer formation is NM23. NM23 isoforms H1 (NME1), H2 (NME2) and H7 (NME7) are preferred, with H1 especially preferred. In a yet more preferred embodiment, the NM23 is of human origin.
Because many ligands that bind to and activate receptors and in particular growth factor receptors activate their cognate receptor by dimerizing it, an approach for inhibiting growth is to use one of the above mentioned methods to make multimers of the ligand, wherein more than two ligands are connected together or encouraged to form higher order multimers. It is also common that ligands that bind to specific nucleic acid sequences, only do so when they are in the dimeric state. Once again, the above mentioned methods can be used to make variants of these proteins or small molecules such that they preferentially form dimers, in particular, when binding to nucleic acids. To inhibit nucleic acid binding, variants that prefer formation of higher order multimers can be generated.
In a preferred embodiment, the ligands that are designed to form higher order multimers can interact with the wild type protein to inhibit the ability of the native protein to form dimers. For example, NM23-H1 binds to the MUC1* growth factor receptor and induces dimerization which triggers growth, survival and pluripotency. Native NM23 exists as a monomer, dimer, tetramer or hexamer, depending on its sequence and concentration. Recombinant NM23 can be refolded or purified such that populations of dimers can be isolated. Mutations of NM23-H1 that prefer dimer formation and resist the formation of tetramers and hexamers have been isolated from human cancers. Therefore, an approach for the inhibition of cancerous growth would be to identify NM23 mutants that prefer the formation of higher order multimers, which do not induce growth and pluripotency. Especially preferred would be those mutants that are able to recruit wild type NM23 into their multimers so that they would not form the cancer-associated dimers.
NM23 in dimeric form binds to the MUC1* receptor on stem and progenitor cells. Binding to MUC1* facilitates endocytosis of NM23 dimers after which they are translocated to the nucleus, where NM23 binds to DNA as a dimer to regulate transcription of genes involved in the growth and pluri-as well as multi-potency of stem and progenitor cells. Therefore, it is an important application of the invention to use the methods described to make NM23 variants that prefer dimer formation for use in the growth, maintenance and induction of pluripotency. That is. NM23 variants that prefer dimer formation can be used in vitro, ex vivo and in vivo to promote the growth of stem and progenitor cells and to maintain pluripotency or multipotency.
In addition, the invention also includes administration of these NM23 variants to a patient for the treatment of conditions that would benefit from treatment with immature cells, including stem and progenitor cells. NM23 and NM23 variants can be administered to a patient either systemically or locally.
The invention also includes using methods of the invention, which influence a protein to be in a specific multimerization state to carry out a biological function that is not conferred by the monomer or some other multimer, in vitro, or in vivo in a patient, in an egg fertilized or unfertilized or in stem cells for research or destined for therapeutic use, and for the induction of pluripotency or induction of cells to a less mature state. In such cases, the nucleic acids that encode the NM23 variants are used, for example as a part of an expression vector. In one embodiment, the NM23 variant is designed to be positioned near a target gene. For example, the NM23 variant can be designed to be inserted into or near the gene of interest. In one case, a patient's own cell, which may be an iPS cell or a partial iPS cell, bears a corrected gene or an inserted gene: nucleic acids that code for one or more of the NM23 variants that prefer dimerization are inserted into the same cells, to promote the proliferation of said cells either in vitro, ex vivo or in vivo.
In one embodiment, these methods are used in conjunction with methods that correct a genetic defect or disease condition by selectively propagating a specific cell population. For example, dimeric NM23 can be made to be expressed in cells that express the fetal form of hemoglobin or a corrected gene for the treatment of sickle cell anemia. In another embodiment, these methods are used to stimulate growth of a cell in a gene therapy setting. In another embodiment, such as for the treatment of auto-immune diseases, either the growth promoting multimer or the growth inhibiting multimer is used to increase or decrease production of specific cell types.
NM23 and NM23 variants also promote the growth of stem and progenitor cells of other mammals. For example, mouse stem and progenitor cells proliferate in the presence of NM23 dimers whether wild type protein or a variant of the protein or whether of human or mouse origin.
These and other objects of the invention will be more fully understood from the following description of the invention, the referenced drawings attached hereto and the claims appended hereto.
The present invention will become more fully understood from the detailed description given herein below, and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein:
In the present application, “a” and “an” are used to refer to both single and a plurality of objects.
As used herein, “multimer” refers to a plurality of monomers that are covalently linked together or non-covalently fused to each other.
As used herein, “higher order multimer” refers to a plurality of monomers that are covalently linked together or non-covalently fused to each other, which is greater than a dimer.
As regards the use of nucleotide symbols other than a, g, c, t, they follow the convention set forth in WIPO Standard ST.25, Appendix 2, Table 1, wherein k represents t or g; n represents a, c, t or g; m represents a or c; r represents a or g; s represents c or g; w represents a or t and y represents c or t.
(DNA) (ggtggttctggt)n (n=1 to 3) (other DNA sequences are possible depending on the codon used for each amino acid) (SEQ ID NO: 131)
(corresponding amino acid sequence) (GGSG)n (n=1 to 3) (SEQ ID NO:132)
(DNA) gct(gaagctgctgctaaa)nGCT (other DNA sequences are possible depending on the codon used for each amino acid) (SEQ ID NO:133)
(corresponding amino acid sequence) A(EAAAK)nA (n=2-5) (SEQ ID NO:134)
(DNA) ggtgctggtggtgctggtggtgctggtgctggtggtgctggtgctggtgctggt (other DNA sequences are possible depending on the codon used for each amino acid) (SEQ ID NO:135)
(corresponding amino acid sequence) GAGGAGGAGAGGAGAGAG (SEQ ID NO:136)
(DNA) ggtgctggtggtgctggtggtgctggtgctggtggtgctggtgctggtgctggtgaacttggtgctggtggtgctggtggtgctggtgct ggtggtgctggtgctggtgctggt (other DNA sequences are possible depending on the codon used for each amino acid) (SEQ ID NO:137)
(corresponding amino acid sequence) GAGGAGGAGAGGAGAGAGELGAGGAGGAGAGGAGAGAG (SEQ ID NO:138)
(DNA) ggtggtgctggtgctggtgctggt (other DNA sequences are possible depending on the codon used for each amino acid) (SEQ ID NO: 139)
(corresponding amino acid sequence) GGAGAGAG (SEQ ID NO : 140)
(DNA) ggttctggtggtggtggttctggtggtggtggttctggt (other DNA sequences are possible depending on the codon used for each amino acid) (SEQ ID NO:141)
(corresponding amino acid sequence) GSGGGGSGGGGSG (SEQ ID NO:142)
(DNA) cttgctgctgct (other DNA sequences are possible depending on the codon used for each amino acid) (SEQ ID NO:143)
(corresponding amino acid sequence) LAAA (SEQ ID NO: 144)
(DNA) cttggtggtggtggttctggtggtggtggttctggtggtggtggttctgctgctgct (other DNA sequences are possible depending on the codon used for each amino acid) (SEQ ID NO:145
(corresponding amino acid sequence) LGGGGSGGGGSGGGGSAAA (SEQ ID NO:146)
(DNA)
ctttctggtggtggtggttctggtggtggtggttctggtggtggtggttctggtggtggtggttctgctgctgct (other DNA sequences are possible depending on the codon used for each amino acid) (SEQ ID NO:147)
(corresponding amino acid sequence) LSGGGGSGGGGSGGGGSGGGGSAAA (SEQ ID NO:148)
(DNA) cttgct(gaagctgctgctaaa)ngctgctgct (n=1 to 5) (other DNA sequences are possible depending on the codon used for each amino acid) (SEQ ID NO:149)
(corresponding amino acid sequence) LA(EAAAK)nAAA (n=1 to 5) (SEQ ID NO:150)
(DNA) ctttttaataaagaacaacaaaatgctttttatgaaattcttcatcttcctaatcttaatgaagaacaacgtaatggttttattcaatctcttaaag atgatccttctcaatctgctaat (other DNA sequences are possible depending on the codon used for each amino acid) (SEQ ID NO:151)
(corresponding amino acid sequence) LFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAAA (SEQ ID NO:152)
The present invention discloses methods for making proteins that preferentially form specific multimer(s), wherein the specific multimer has a desired biological function that the monomer or some other multimer of the protein does not have. For example, many growth factors exert their growth promoting function only when they are in the dimeric form. These may be homo or hetero-dimers. In these cases, methods of the invention are used to enhance growth by increasing the amount of dimers of the growth factors. Conversely, if the desired biological function is to inhibit growth, for example, of cancer, then the growth factors would be engineered or mutants selected that resist dimer formation.
Methods of the invention that generate proteins or variants of the native protein that are more likely to exist in the desired multimeric form, that which results in a desired biological function, but which function is not conferred by the monomer or other multimeric form, can be used in vitro, ex vivo or in vivo. In vitro, the variant that prefers a specific multimerization state can be expressed and optionally purified, then used for cell culture, particularly for the culture of stem and progenitor cells, including hematopoietic stem and progenitor cells and other immature cells of the bone marrow or other in vitro uses.
Additionally, the expressed and optionally purified multimer-specific variants can be used to treat a patient by administering topically via a cream, suave, bandage, and the like, or administered as a systemic treatment such as by oral ingestion, injection, and the like. In a preferred embodiment, the parent protein is NM23 and the multimer-specific variant prefers dimer formation, optionally inhibits formation of the tetramer or hexamer and otherwise increases the amount of dimer that is present over a wide range of protein concentrations. In some cases, it may be desirable to recombinantly synthesize the multimer-specific variant with a leader sequence that increases the proteins entrance into the cell.
Alternatively, nucleic acids encoding the multimer-specific variant can be introduced into a cell, using any one of a variety of methods known to those skilled in the art, such as homologous recombination, stable or transient transfection or transduction, viral transduction, such as by using lentiviruses or retroviruses, including self-inactivating vectors, including self-inactivating lentiviral vectors and self-inactivating retroviral vectors. In one embodiment, the cell is a stem cell or an iPS cell, which may be derived from the patient or from a donor. The resultant cells may be transplanted into a patient before or after in vitro expansion. In one embodiment, nucleic acid manipulations are performed to correct a genetic abnormality in the cell that has also been transfected or transduced to express the multimer-specific variant.
In a preferred embodiment, the protein is NM23 and the multimer-specific variant prefers dimerization and inhibits the formation of higher order multimers, with the net effect that the population of the expressed protein has an increased amount of dimer, over a wide range of concentrations, compared to the parent protein. In this way, the NM23 dimers, which are growth factors, will drive the expansion of the cell even after transplantation into the patient. In one embodiment, the genetic disorder to be treated is sickle cell anemia and treatment includes stimulating the growth of cells that express the fetal form of hemoglobin or the corrected gene for the adult form. The growth factor variant can be inserted into the cell so that it is only transiently expressed, for example by using a self-inactivating viral vector. Alternatively, the multimer-specific variant, which in a preferred embodiment is an NM23 variant that prefers dimer formation, can be permanently inserted into the genome. Optionally, the cell can also be transfected or transduced to express MUC1, or a fragment of MUC1, including MUC1 chimeras, or MUC1*, wherein the extra cellular domain has been truncated to included essentially most or all of the PSMGFR sequence.
In some cases, including those in which cells are transfected with nucleic acids encoding a multimer-specific variant, it is desirable to modify the nucleic acid of the variant at the 5′ end to include a leader sequence that causes the cell to secrete the expressed protein. Such sequences are known to those skilled in the art and commonly include sequences derived from antibodies, see Example 13.
The following are exemplary leader sequences that cause an expressed protein to be secreted from the cell. Any of these sequences or others known to those skilled in the art can be added to the nucleic acid sequence of the variant so that the expressed protein is secreted from the cell. Optionally, the sequence is added to the N-terminus or 5′ of the nucleic acid sequence. In this way, the NM23 mutants, deletions, single chain variants and fusion protein chimeras can readily be used in vitro and ex vivo, as well as in vivo.
carotovora) leader sequence (DNA)
Cells transfected or transduced to express the growth factor variant, such as an NM23 variant that prefers dimerization, can be somatic cell, a stem cell, including hematopoietic stem cells and progenitor cells, or iPS cells. In one embodiment, somatic cells such as fibroblasts or dermablasts are transduced with nucleic acids that encode an NM23 variant that prefers dimer formation. The NM23 variant nucleic acids may be transduced along with one or more genes that cause the host cell to revert to a more immature state. These other genes may include but not limited to Oct4, Sox2, Nanog, Klf4, Lin28 and c-Myc. The cells can be transfected or transduced to express the NM23 variant temporarily, such that it will drive the expansion of that cell type for a limited amount of time, or permanently using methods such as stable transfection or homologous recombination and the like.
The methods of the invention can act to provide a growth factor that is constitutively active or more active than the native growth factor to stimulate the proliferation of a specific population of cells, which may carry a genetic mutation or correction. In one embodiment, one recombinant variant that increases the percentage of a specific multimeric state of a growth factor is carried on the same nucleic acid, plasmid, or expression vector that carries the sequence of the corrected gene or gene to be expressed. In another embodiment, if it is desired that a gene be down-regulated, a multimer that does not stimulate growth may be used. The invention also includes the use of these methods, in patients, in cells destined for transplant in patients, in blastocysts and embryos as well as in fertilized or unfertilized eggs, which may be used for in vitro fertilization.
In a preferred embodiment, the growth factor is NM23 wherein the dimer form activates growth and higher order multimers such as tetramers and hexamers turn off the NM23 mediated pathway that stimulates growth and induces or maintains pluripotency. For example, NM23 dimers promote stem and progenitor cell growth and pluripotency and also promote the growth of cancer cells. The hexamer or tetramer form of NM23 does not promote stem or cancer cell growth. Therefore, methods of the invention that result in variants that prefer dimer formation are used to promote stem cell, progenitor cell and/or cancer cell growth. In the case of stem and progenitor cells, the higher order NM23 multimers can be used to induce differentiation.
Conversely, methods of the invention that result in variants that prefer tetramer, hexamer or higher order multimer formation are used to inhibit cancer cell growth. Alternatively, NM23 variants can be further modified to carry a toxin to target the killing of cancer cells. NM23 is a ligand of MUC1* which is present at the surface of many different cancer cells. One could use NM23 to target specifically cancer cells to deliver a drug or toxin to kill the targeted cells. One example is the use of the ribosome-inactivating protein called saporin. By itself, saporin is not able to enter the cell, but when coupled to another protein that binds to the cell surface, the saporin/protein complex can be internalized and is toxic to the cell.
Because NM23 activates the MUC1* growth factor receptor, MUC1 or truncated forms of MUC1, including MUC1* wherein the extra cellular domain includes primarily the sequence of the PSMGFR peptide, can be optionally expressed either in vitro or in vivo along with the NM23 variant. MUC1 or MUC1 truncations, or MUC1 variants can similarly be expressed from the same expression plasmid as NM23 and optionally a gene desired to be expressed.
In another embodiment, the growth factor receptor itself is engineered to be in a specific multimerization state in order to either promote or inhibit growth. In a preferred embodiment, the growth factor receptor is MUC1* and the preferred multimer is a dimer.
One method of generating proteins that prefer a specific multimerization state is to identify mutants of that protein that prefer the formation of that specific multimerization state. For example, Table 1 lists some of the NM23 mutations that encourage the formation of dimers.
Constructs for recombinant proteins are generated such that one or more monomers are already connected. For example single chain proteins wherein two or more monomers are connected either directly or indirectly via for example a linker that may vary in length or sequence to obtain the desired biological activity. Linkers can vary in length and sequence. In a preferred embodiment, the linker is 5-100 amino acids. In a more preferred embodiment the linker is 10-75 amino acids. In a yet more preferred embodiment, the linker is either 10 amino acids or 43 amino acids. Similarly, the sequence of a linker can vary from a flexible (GGGGS)n type linker to any sequence known or suspected to be flexible in a natural protein, including modifications of native sequences, wherein several mutations intended to increase flexibility or solubility are inserted. Table 2 lists some preferred linker sequences.
Table 3 lists some preferred single chain constructs wherein two monomers are recombinantly connected via a linker.
In some cases, the linker sequence itself tends to form homo or hetero dimers. For example, portions of the Fc region of antibodies dimerize with another Fc region. Chimeric proteins consisting of a portion of the parent protein plus a portion of a protein that naturally dimerizes are variants that prefer dimer formation. Alternatively, chimeras that use Fc sequences from IgM proteins would prefer the formation of higher order multimers such as the characteristic pentamer. Modified hinge regions of antibodies fused to NM23 mutant monomers preferentially form dimers and are described in detail herein.
In another approach, protein dimers are achieved by genetically making the protein of interest a fusion chimera wherein it is fused to a protein or protein fragment that has a dimerization domain. Ligands that are multimeric can be generated by making recombinant chimeras wherein each monomer is connected to a portion of a protein that multimerizes. For example, the proteins Fos and Jun interact so that they could be recombinantly connected to ligands that may be the same or different in order to cause the dimerization of the resultant chimeras. Table 3 lists preferred constructs, some of which are chimeras.
Cysteines can be inserted into the protein of interest such that dimer formation is encouraged via formation of disulfide bonds. The invention also includes inserting cysteines into linkers, whether comprised of natural amino acids or unnatural polymers or small molecules, to facilitate dimerization. Table 4 contains a list of preferred variants, whose dimerization is enhanced by the introduction of cysteines.
Yet another method for making ligand multimers is via chemical coupling of two or more monomeric ligands. For example, a bifunctional linker can be used to chemically couple two protein ligands to make homo or hetero dimers. The linker can be a chemical cross linker or similar that facilitates dimerization via covalent coupling of two monomers to form homo or hetero dimers.
The same can be accomplished by chemically coupling the proteins when they are in dimeric state, for example, immobilizing target protein in a defined geometry, for example on a SAM and chemically coupling the proteins either directly or indirectly via a linker while they are confined in a geometry that mimics dimerization state. Alternatively, dimers can be isolated then a coupling agent is added to directly couple two proteins together either directly or via a linker.
Yet another method for making multimeric ligands is to identify small molecules that bind to the target receptor and then synthesize multimers of the small molecule.
In a preferred embodiment, the multimerization state that is preferred for enhancing a natural biological interaction, such as the activity of a growth factor, is a dimer. In a more preferred embodiment, the ligand that is made dimeric or made to prefer dimer formation is NM23. NM23 isoforms H1, H2 and H7 (NME7) are preferred, with H1 especially preferred. In a yet more preferred embodiment, the NM23 is human. Also preferred for use with methods for increasing growth factor activity are mutants that enhance dimerization and optimally resist formation of higher order multimers. For example, the NM23-S120G and NM23-P96S mutants have been reported to prefer dimer formation and to resist the formation of tetramers and hexamers which do not activate the MUC1* growth factor receptor and do not bind to nucleic acids to induce expression of genes involved in pluripotency and cancer. However, for NM23-S120G mutant, we discovered that it is preferred that the protein be denatured and refolded to obtain significant populations of dimer. Similarly, the fraction of the P96S mutant that existed as a dimer was increased by denaturation and refolding. C-terminal deletions disrupt regions that participate in the formation of the higher order multimers and thus increase the portion of the protein that is in dimeric form. Thus C-terminal deletions of NM23 wild type or mutants is preferred for increasing the percentage and stability of dimer populations. C-terminal deletion of mutants such as S120G and/or P96S, that already prefer dimer formation are especially preferred. Deletion of 1-6 amino acids from the C-terminus of NM23 is especially preferred.
Although the S120G and P96S mutations are naturally occurring mutations identified in human cancer and in developmental abnormalities of the fruit fly, respectively, the invention also includes purposely introducing mutations and identifying those that either increase or decrease the propensity to form dimers. For example, to enhance growth factor activity wherein the growth factor is active when in dimeric form, mutations that prefer dimer formation are identified and preferred. For inhibiting growth factor activity, mutations that resist formation of dimers or prefer formation of tetramers or hexamers are preferred. Either site directed or random mutagenesis can be used wherein those that favor dimer formation are identified by a variety of methods including but not limited to structural analysis such as crystal structure, ability to support, induce or maintain pluripotency in stem cells, ability to bind to MUC1* peptide that includes essentially the PSMGFR peptide sequence. In addition, NM23 variants that favor dimer formation can be identified for example by using phage display and standard random mutagenesis wherein the desired mutants are identified by their ability to bind to stem cells or the MUC1* peptide.
Similarly. NM23 variants and similar multimer-specific variants of the invention can be further modified with sequences that increase the variant's ability to penetrate the cell membrane.
As will become evident, the ligand monomers that are genetically engineered to prefer dimer formation may be the wild type protein or a mutant or truncation that prefers dimer formation or resists the formation of higher order multimers. The case of NM23 is meant to be exemplary and the invention also includes the use of these methods, linkers, linker sequences and/or use of portions of other proteins or molecules for increasing activity of other agents that exert their effects when in dimeric form.
Another type of protein for which the multimerization state is important is transcription factors. It is common that transcription factors that bind to specific nucleic acid sequences, only do so when they are in the dimeric state. However, some transcription factors, such as tumor suppressor p53, bind DNA as a tetramer. Still other transcription factors exert a specific transcription function only when they exist as octamers. Methods of the invention can be used to make variants of these transcription factors to increase the desired activity, for example by making transcription factor variants that prefer dimerization.
Because ligands that bind to and activate receptors and especially growth factor receptors, often need to be in the dimeric state to bind to and/or activate its growth factor receptor function, an approach for inhibiting growth is to use one of the above mentioned methods to make multimers of the ligand, wherein more than two ligands are connected together or encouraged to form higher order multimers. To inhibit nucleic acid binding, variants that prefer formation of higher order multimers can be generated.
In a preferred embodiment, the ligands that are designed to form higher order multimers can interact with the wild type protein to inhibit the ability of the native protein to form dimers. For example, NM23 binds to the MUC1* growth factor receptor and induces dimerization which triggers growth, survival and pluripotency. NM23 can exist as a monomer, dimer, tetramer or hexamer, depending on its sequence and concentration. Recombinant NM23 can be refolded or purified such that populations of dimers can be isolated. Mutations of NM23 that prefer dimer formation and resist the formation of tetramers and hexamers have been isolated from human cancers. Therefore, an approach for the inhibition of cancerous growth would be to identify NM23 mutants that prefer the formation of higher order multimers, which do not induce growth and pluripotency. Especially preferred would be those mutants that are able to recruit wild type NM23 into their multimers so that they would not form the cancer-associated dimers.
It is known that MUC1* growth factor receptor is activated by ligand induced dimerization of its extra cellular domain. Bivalent antibodies raised against the extra cellular domain of MUC1* (PSMGFR sequence:
dimerize the MUC1* receptor and stimulate growth in a dose dependent manner. The dose response curve is a classic bell-shaped curve that is characteristic of Class I growth factor receptors that are activated by dimerization. The bell-shaped curve is caused when growth increases as dimerization of MUC1* increases but when the NM23 dimers or the bivalent antibodies are in excess, each receptor is bound by one, rather than two growth factors. This actually blocks dimerization of the growth factor receptor and results in a decrease in growth. Consistent with these findings, the addition of the monovalent Fab of the anti-MUC1*ecd antibody blocks receptor dimerization and consequently inhibits growth, see
An NM23 mutant S120G was previously isolated from a human neuroblastoma. It was reported that the mutant NM23 preferred dimer formation and resisted the formation of higher order multimers, specifically the tetramers and hexamers that wild type NM23 is known to form. Other NM23 mutants that were reported to prefer dimerization were the P96S mutation and deletions at the C-terminus of 1-6 amino acids.
However, when NM23-WT (wild type), S120G or P96S mutants are made as recombinant proteins, their multimerization state depends on sequence, how it is expressed. how it is collected and purified, as well as concentration. For example, despite expressing the S120G mutant, reported to prefer dimer formation, many expression/purification methods resulted in populations comprised exclusively of tetramers and hexamers. Other methods of protein expression produced NM23 populations that were comprised of hexamer, tetramer and a small dimer population. One method for expressing and purifying NM23 mutants that results in significant populations of dimer involved denaturing the expressed protein and then refolding it. In an optional step, the dimer population was further purified by size exclusion chromatography (FPLC) as described in Example 4 herein.
Characterization of NM23-WT or three different preparations of mutant S120G was carried out by FPLC (
Surface Plasmon Resonance (SPR) experiments were carried out to determine if there were differences in binding affinities of the various NM23s multimers (monomers. dimers and hexamers) to the MUC1* peptide. SPR measurements were taken on a Biacore 3000 instrument wherein a histidine-tagged MUC1* peptide (PSMGFR) was immobilized to saturation on an SPR chip that was coated with a self-assembled monolayer coated with 3.8% NTA-Ni in a background of tri-ethylene glycol terminated thiols, see Example 5,
To further test the ability of the various NM23 multimers to bind to its cognate receptor, we performed a nanoparticle experiment, see Example 6 and
All three batches of NM23,-WT, S120G-hexamer and S120G-dimer were tested for their ability to promote undifferentiated stem cell growth. Human embryonic stem cells were cultured in minimal stem cell media that contained 8 nM of one of the NM23 preparations. In one of the wells the free MUC1*ecd peptide (PSMGFR) was added to competitively inhibit binding of NM23-S120G-dimers to the MUC1* receptor which is on all pluripotent stem cells. The results are shown in the photograph of
In another part of this experiment, levels of miR-145, which is the microRNA that signals the stem cell's exit from pluripotency, was measured. This experiment showed that the disruption of the interaction between the NM23 dimer and MUC1* caused a spike in miR-145, which further corroborated the finding that disruption of the interaction between NM23 dimers and MUC1* triggers differentiation and conversely the interaction promotes pluripotency. The proteins used in these experiments to determine their ability to support pluripotent stem cell growth were characterized at the time of the experiment by gel electrophoresis using a non-denaturing native gel. The native gel of
Therefore, NM23 mutants, deletion mutants and engineered variants that prefer dimer formation are ideal for the growth, maintenance and induction of pluripotency or multipotency, for example in somatic cells, as well as for the numerous applications disclosed herein. Nucleic acids encoding these variants can also be transfected into cells to promote the growth of these cells. Exemplary variants of proteins that prefer a specific dimerization state compared to the native protein were made, characterized and tested for their ability to function as the specific multimer. The NM23 variants that have increased populations of stable dimer compared to the wild type protein that were made and tested are listed in Tables 1, 3 and 4.
Tables 1, 3 and 4 list NM23 mutants that prefer dimer formation, engineered constructs wherein two NM23 wild type or mutant monomers are connected via a linker to form a dimer, and NM23 fusion proteins that preferentially form dimers. Table 3 lists and describes NM23 variants that were generated, expressed, characterized and tested for their ability to mimic the behavior of native NM23 dimers, and particularly to test their ability to mimic the behavior of NM23-S120G-dimers that were 80% or greater in dimer form. The methods used to generate the constructs, express the protein, and refold in some cases are described in Examples 2, 3, 9, and 10. Methods used to test the function of the variants produced are described in Examples 5-7 and 11-12.
Pluripotent human stem cells were cultured in NM23 variants and their ability to promote undifferentiated stem cell growth and to inhibit spontaneous differentiation of the cells was determined. Both human BGO1v/hOG and H9 embryonic stem cells were cultured in minimal stem cell media plus 8 nM of either our standard NM23-S120G-RS or an NM23 variant. In all cases, the NM23 variants tested fully supported pluripotent stem cell growth. Cell morphology was typical of undifferentiated stem cells and was devoid of thickened or darkened areas that denote differentiation, see
In addition to assessing stem cell morphology as proof that the NM23 variants functioned as well as the native dimers or the S120G dimers, the growth rate of stem cells cultured in media containing the NM23 variants was compared to the growth rate of identical cells cultured in NM23-S120G “RS” that had been refolded and then purified by FPLC so that the isolated fractions were essentially 100% dimer, see Example 11c. In these experiments, 200,000 stem cells all drawn from the same source (human ES-BGO1v/hOG) were cultured in either NM23-S120G RS or one of the NM23 variants shown in
As another method of assessing the function of the NM23 dimer preferring variants, quantitative PCR was performed to measure expression levels of the pluripotency genes in stem cells cultured in the NM23 variants, see Example 11d.
As a yet further measure of the function of engineered NM23 dimer preferring variants, their migration from cell surface to cell nucleus was tracked and compared to that of NM23-S120G-RS, see Example lle and
These experiments show that connecting two monomers with a linker or fusing the protein of interest with a portion of a strong dimerization domain, results in more stable “dimers” that function as dimers over a wide range of concentrations, whereas the multimerization state of native NM23 is highly dependent on concentration and exists as a dimer only at very low nanomolar concentrations. Mutant NM23 proteins that prefer dimerization are an improvement over the wild type protein for promoting growth factor function and pluripotency, but they vary greatly in the amount of dimer produced and in the stability of those dimers depending on sequence, method of expression and purification. NM23 variants that are either single chain constructs or fusion proteins like those listed in Tables 3 and 4 represent an improvement over the state of the art because they increase the portion of dimer formed, increase dimer stability, and importantly can be expressed as a pseudo dimer in a cell or an organism, where expression and refolding methods required to form dimers of mutants that “prefer dimer formation” could not be done.
Any of the mutants, deletions and/or single chain or fusion chimeras of the invention, including those described in Examples 2, 9 and 10 can be made to be secreted by expressing cells for use in vitro, ex vivo and/or in vivo. Sequences that cause expressed proteins to be secreted are known to those skilled in the art. Particularly, sequences derived from antibodies are added to the N-terminus of the protein or to the 5′end of the gene of interest. In addition to the inclusion of leader sequences, the expression cell type need not be limited to E. coli and also includes mammalian cells, mammalian expression cells, yeast, somatic cells, stem cells, iPS cells or cells undergoing induction of pluripotency or induction to a less mature state than the starting cell.
It is not intended that the use of NM23, mutants or variants thereof, be limited to use with human cells or in humans. MUC1* has great sequence homology among mammals as does NM23.
Table 2 shows the various linkers that can be used with any ligand but with NM23 variants shown in Table 1 in preferred embodiments. Table 2 also lists the portions of proteins that were genetically fused to ligand monomers listed in Table 1 to form constitutively active forms of the protein, which in an especially preferred embodiment is NM23.
In addition to the S120G mutant that prefers dimer formation and resists the formation of the higher order multimers, there are other mutants and variants that also favor dimer formation or stabilization. For example, another NM23 mutant that is easier to express and maintain as stable dimer is the P96S mutation. Another approach to making variants that form or stabilize dimers is to delete regions of the protein that participate in the formation of the higher order multimers such as the tetramers and hexamers. For example, in NM23-H1, the C-terminus promotes formation of tetramers and hexamers. Deletions of 1-9 amino acids at the C-terminus are preferred for generating NM23 variants that have increased activity, including increased growth factor activity, increased binding to MUC1* growth factor receptor and/or increased binding to nucleic acids that regulate expression of other pluripotency and multipotency genes.
In a preferred embodiment, 2 amino acids are deleted (CΔ2) from the C-terminus of human NM23-H1. In an especially preferred embodiment, 6 amino acids are deleted (CΔ6). To maximize solubility, expression of dimers or stabilization of dimers, the protein can be made with one or more mutations plus deletions. For example, one NM23 variant that, when expressed as the recombinant protein, has a soluble fraction that is mostly dimer is NM23-P96S-CΔ6. This NM23 is ideal for stimulation of stem or progenitor cell growth because it does not need to be denatured and refolded, has a major portion of the soluble fraction that is a dimer, and dimer populations that can be further purified by size exclusion chromatography remain stable for long periods of time.
c, d, e, and f show gel and FPLC trace of NM23-P96S-CΔ2 and NM23-P96S-CΔ6, respectively. Construct design and protein expression and purification are described in Example 2c. In addition,
Other NM23 variants listed in Tables 1, 3 and 4 were generated as described in Examples 2 and 9.
Sequence alignment between human NM23-H1 and other mammalian homologues, including but not limited to mouse NM23, will elucidate comparable regions of the homologue protein that should be mutated or deleted.
The NM23 variants described herein can also be used as a vehicle for drug delivery for the treatment of MUC1*-positive cancers. For example, cytotoxic agents can be chemically coupled to the NM23 or NM23 variant. Toxins may be genetically engineered such that they are attached to the NM23. Alternatively, the NM23 variant can be modified, for example with cysteine or with certain enzyme-specific sequences that facilitate specific coupling of therapeutic agents to the NM23 variants.
NM23 in dimeric form binds to the MUC1* receptor on stem and progenitor cells. Binding to MUC1* facilitates endocytosis of NM23 dimers after which they are translocated to the nucleus, where NM23 binds to DNA as a dimer to regulate transcription of genes involved in the growth and maintaining pluripotency as well as multi-potency of stem and progenitor cells. Therefore, in one aspect, the invention is directed to using the methods described herein to make NM23 variants that prefer dimer formation for use in the growth, maintenance and induction of pluripotency. That is, NM23 variants that prefer dimer formation can be used in vitro to promote the growth of stem and progenitor cells and to maintain pluripotency or multipotency. In addition, the invention also includes administration of these NM23 variants to a patient for the treatment of conditions that would benefit from treatment with immature cells, including stem and progenitor cells. NM23 and NM23 variants can be administered to a patient either systemically or locally.
The invention also includes using similar methods to form multimers that are not dimers, such as higher order multimers including but not limited to tetramers or hexamers. In addition, the multimers need not be the active state of the protein. For example, in many cases, it is desirable to treat a patient with a constitutively inactive form of a protein. In a preferred embodiment, a patient is treated with an inactive form of a growth factor. In a yet more preferred embodiment the inactive form is a hexamer. In a still more preferred embodiment, the patient is a cancer patient and is treated with NM23 in hexameric form. NM23 hexamers can be obtained by expressing the natural protein. More preferred are variants in which 4-6 monomers are connected using linkers and chimera strategies described herein. For example, to make an inactive NM23 variant one could make NM23-antibody fragment chimeras wherein the Fc portion or the hinge region is taken from an IgM antibodies such that the NM23 would favor formation of pentamers.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. The following examples are offered by way of illustration of the present invention, and not by way of limitation.
Class I growth factor receptors are activated by ligand-induced dimerization of their extra cellular domain. To demonstrate that MUC1* is activated by ligand-induced dimerization of its extra cellular domain, we treated MUC1* positive cells, ZR75-30 breast cancer cells with either the bivalent anti-MUC1* antibody or the monovalent Fab of the same antibody. The graph of
NM23wt was amplified by polymerase chain reaction (PCR) using the following primers:
The fragment was then purified, digested (NdeI, XhoI) and cloned between NdeI and XhoI restriction sites of the expression vector pET21b.
NM23-H1 mutant S120G (serine #120 mutated to a glycine) was made using the GeneTailor™ Site-directed mutagenesis system (Life Technologies) following the manufacturer instructions using the following primers: 5′-gcaggaacattatacatggcggtgattctg-3′ (SEQ ID NO:5) and 5′-gccatgtataatgttcctgccaacttgtat-3′ (SEQ ID NO:6).
We generated the NM23-H1 mutant P96S (proline #96 mutated to a serine) using the QuickChange site-directed mutagenesis kit (Agilent) following the manufacturer instructions using the following primers: 5′-teggggagaccaactctgcagactccaag-3′ (SEQ ID NO:7) and 5′-cttggagtctgcagagttggtctccccga-3″ (SEQ ID NO:8). The template used for the PCR reaction was NM23 wild type cloned between NdeI and XhoI restriction sites. After sequence confirmation, the deletion constructs were generated by PCR. NM23 P96S ΔC1 was amplified using the following primers: 5′-atcgatcatatggccaactgtgagcgtaccttc-3′ (SEQ ID NO:9) and 5′-gtggtgaccggtatagatccagttctgagcaca-3′ (SEQ ID NO:10). NM23 P96S ΔC2 was amplified using the following primers: 5′-atcgatcatatggccaactgtgagcgtaccttc-3″ (SEQ ID NO:11) and 5′-gtggtgaccggtgatccagttctgagcacagct-3′ (SEQ ID NO:12). NM23 P96S ΔC6 was amplified using the following primers: 5′-atcgatcatatggccaactgtgagcgtaccttc-3′ (SEQ ID NO:13) and 5′-gtggtgaccggtagcacagctcgtgtaatctacca-3′ (SEQ ID NO:14). The resulting fragments were purified, digested (NdeI, AgeI) and cloned between NdeI and AgeI restriction sites of the expression vector pET21b. The pET21b was previously modified by replacing the XhoI restriction by AgeI using an overlap PCR method. Optimal dimer formation was observed when NM23-P96S was cloned between NdeI and XhoI. Optimal dimer formation for all deletion mutants was observed when cloned between NdeI and AgeI.
LB broth (Luria-Bertani broth) was inoculated with 1/10 of an overnight culture and cultured at 37° C. until OD600 reached ˜0.5. At this point, recombinant protein expression was induced with 0.4 mM Isopropyl-β-D-thio-galactoside (IPTG, Gold Biotechnology) and culture was stopped after 5 h. After harvesting the cells by centrifugation (6000 rpm for 10 min at 4° C.), cell pellet was resuspended with running buffer: PBS pH7.4, 360 mM NaCl and 80 mM imidazole. Then lysozyme (1 mg/mL, Sigma), MgCl2 (0.5 mM) and DNAse (0.5 ug/mL, Sigma) was added. Cell suspension was incubated on a rotating platform (275 rpm) for 30 min at 37° C. and sonicated on ice for 5 min. Insoluble cell debris was removed by centrifugation (20000 rpm for 30 min at 4° C.). The cleared lysate was then applied to a Ni-NTA column (Qiagen) equilibrated with the running buffer. The column was washed (8 CV) before eluting the protein off the column with the running buffer (6 CV) supplemented with 420 mM imidazole.
For protein denaturation, the elution fractions were pooled and denatured by adding Ivol of 100 mM Tris pH 8.0+8M urea, the solution was concentrated by half and another vol of 100 mM Tris pH 8.0+8M urea was added. This cycle was repeated until final urea concentration was ˜7M. The protein was then refolded by dialysis.
Denatured protein was dialysed overnight against 100 mM Tris pH 8.0, 4M urea, 0.2 mM imidazole, 0.4M L-arginine, 1 mM EDTA and 5% glycerol; then dialysed for 24 h against 100 mM Tris pH 8.0, 2M urea, 0.2 mM imidazole, 0.4M L-arginine, 1 mM EDTA and 5% glycerol; next, the protein was dialysed for 24 h against 100 mM Tris pH 8.0, 1M urea, 0.2 mM imidazole, 0.4M L-arginine, 1 mM EDTA and 5% glycerol; then dialysed for 8 h against 100 mM Tris pH 8.0, 0.2 mM imidazole, 0.4M L-arginine, 1 mM EDTA and 5% glycerol; then, the protein was dialysed overnight against 25 mM Tris pH 8.0, 0.2 mM imidazole, 0.1M L-arginine, 1 mM EDTA and 5% glycerol: dialysed 3×3 h against PBS pH 7.4, 0.2 mM imidazole, 1 mM EDTA and 5% glycerol; dialysed overnight against PBS pH 7.4, 0.2 mM imidazole, 1 mM EDTA and 5% glycerol; finally the refolded protein was centrifuged (18.500 rpm) 30 min at 4° C. and supernatant was collected.
Specific multimers can be further purified from a mixed pool using size exclusion chromatography, also called FPLC. The dimer was further purified by size exclusion chromatography (Superdex 200) “FPLC”. FPLC fractions that were essentially 100% dimer were collected and pooled and aliquoted and stored at −80° C. and are referred to herein as NM23-S120G-RS or S120G-RS. The fractions containing the dimer were pooled.
Typically, 500 uL of each sample to be characterized were loaded onto a Superdex 200 10/300 GL column. The molecular weight of each species peak is determined by comparison to an FPLC trace made by a molecular weight standard that is injected before characterizing the sample proteins.
Three different preparations of recombinant NM23-S120G plus NM23-WT (wild type) were tested for their ability to bind to a MUC1* extra cellular domain peptide (PSMGFR sequence) using techniques of surface plasmon resonance. The different preparations were first analyzed by FPLC to characterize them according to which multimers they formed. FPLC analysis of the NM23 species that were tested is shown in
Surface Plasmon Resonance (SPR) experiments were carried out using a Biacore 3000 instrument. Bare gold Biacore chips were coated with self-assembled monolayers (SAMs) according to methods of Bamdad. C. The use of variable density self-assembled monolayers to probe the structure of a target molecule. Biophys J. 1998 Oct;75(4): 1989-96. NTA-Ni-tri-ethylene glycol SAMs were formed to present 3.8% NTA-Ni, which binds to and captures histidine-tagged proteins. Histidine tagged PSMGFR peptide (MUC1*ecd peptide) was flowed over the chip surface and immobilized to saturation. Next, each of four different preparations of NM23-S120G were injected over a stable peptide surface.
The overlay of Surface Plasmon Resonance (SPR) measurements of
A similar SPR experiment was performed and results are shown in
NTA-Ni SAMs were formed on gold nanoparticles according to the methods of Thompson et al, dx.doi.org/10.1021/am200459a | ACS Appl. Mater. Interfaces 2011, 3, 2979-2987. In this method, gold nanoparticles are coated with self-assembled monolayers (SAMs) that have NTA-Ni-thiols incorporated into the SAM. The NTA-Ni moiety captures histidine-tagged proteins. If proteins immobilized on the nanoparticles recognize each other and draw the attached nanoparticles close together, an intrinsic property of the nano gold causes the solution to turn from the characteristic pink to blue. The same thing happens if a dimeric protein added in solution binds to particle-immobilized proteins. NM23-S120G-dimer. NM23-WT, or NM23-S120G-hexamer were separately added to nanoparticles bearing MUC1*ecd peptides (PSMGFR).
The photograph of
Three batches of NM23,-WT, S120G-hexamer and S120G-dimer were tested for their ability to promote undifferentiated stem cell growth. Human embryonic stem cells (H9) were cultured in minimal stem cell media (this media described in Example 8 below) that contained 8 nM of one of the NM23 preparations. In one of the wells the free MUC1*ecd peptide (PSMGFR) was added to competitively inhibit binding of NM23-S120G-dimers to the MUC1* receptor which is on all pluripotent stem cells. The cells were plated at 200,000 cells per well of a 6-well plate that had been coated with an anti-MUC1* monoclonal antibody (MN-C3) to make the stem cells adhere. Media was changed every 48 hours as is typical. Photographs were taken on Day 4. Olympus IX 71 inverted microscope The results are shown in the photograph of
In another part of this experiment, levels of miR-145, which is the microRNA that signals the stem cell's exit from pluripotency, was measured. That experiment showed that the disruption of the interaction between the NM23 dimer and MUC1* caused a spike in miR-145, which further corroborated the finding that disruption of the interaction between NM23 dimers and MUC1* triggers differentiation and conversely the interaction promotes pluripotency. The proteins used in these experiments to determine their ability to support pluripotent stem cell growth were characterized at the time of the experiment by gel electrophoresis using a non-denaturing native gel. The native gel of
400 ml DME/F12/GlutaMAX I (Invitrogen #10565-018)
100 ml Knockout Serum Replacement (Invitrogen #10828-028)
5 ml 100× MEM Non-essential Amino Acid Solution (Invitrogen #11140-050)
0.9 ml (0.1 mM) beta-mercaptoethanol (55 mM stock, Invitrogen #21985-023)
2.5 ml PSA (penicillin, streptomycin, amphotericin) MP Biochem ( #1674049)
NM23 with or without the mutations such as S120G, P96S, or C-terminal deletions can be engineered to prefer dimer formation by making a construct that links two protein monomers. NM23-S120G or other mutation that makes the protein resist formation of tetramers and hexamers is preferred. Table 2 gives the DNA sequence followed by the encoded amino acid sequence.
A (GGGGS)x2 linker was introduced in frame of NM23 S120G (3′) by PCR using the following primers:
The resulting fragment was purified, digested (NdeI, NheI) and cloned between NdeI and NheI restriction sites of the expression vector pET21b.
Another NM23 S120G fragment was amplified by polymerase chain reaction (PCR) using the following primers:
The fragment was then purified, digested (NheI/XhoI) and cloned in frame, between the NheI and XhoI restrictions sites of the previously cloned NM23 S120G containing the (GGGGS)x2 linker. The expressed protein can be purified and/or refolded, for in vitro applications, using the optional refolding protocol of Example 3b, with optional addition of 1-5 mM DTT.
A (GGGGS)x3 linker was introduced in frame of NM23 S120G (3′) by PCR using the following primers:
The resulting fragment was purified, digested (NdeI, NheI) and cloned between NdeI and NheI restriction sites of the expression vector pET21b.
Another NM23 S120G fragment was amplified by polymerase chain reaction (PCR) using the following primers:
The fragment was then purified, digested (NheI/XhoI) and cloned in frame, between the NheI and XhoI restrictions sites of the previously cloned NM23 S120G containing the (GGGGS)x3 linker. The expressed protein can be purified and/or refolded, for in vitro applications, using the optional refolding protocol of Example 3b, with optional addition of 1-5 mM DTT.
A modified hinge region of IgG1 without cysteine was introduced in frame of NM23 S120G (3′) by 2 successive PCR reactions. First the following couple of primers were used:
The PCR fragments was purified and used as template in a second PCR reaction using the following primers:
The resulting fragment was purified, digested (NdeI, NheI) and cloned between NdeI and NheI restriction sites of the expression vector pET21b.
Another NM23 S120G fragment was amplified by polymerase chain reaction (PCR) using the following primers:
The fragment was then purified, digested (NheI/XhoI) and cloned in frame, between the NheI and XhoI restrictions sites of the previously cloned NM23 S120G containing the modified hinge region linker. The expressed protein can be purified and/or refolded, for in vitro applications, using the optional refolding protocol of Example 3b, with optional addition of 1-5 mM DTT.
A modified hinge region of IgG2a without cysteine was introduced in frame of NM23 S120G (3′) by 2 successive PCR reactions. First the following couple of primers were used:
The PCR fragments was purified and used as template in a second PCR reaction using the following primers:
The resulting fragment was purified, digested (NdeI, NheI) and cloned between NdeI and NheI restriction sites of the expression vector pET21b.
Another NM23 S120G fragment was amplified by polymerase chain reaction (PCR) using the following primers:
The fragment was then purified, digested (NheI/XhoI) and cloned in frame, between the NheI and XhoI restrictions sites of the previously cloned NM23 S120G containing the modified hinge region linker. The expressed protein can be purified and/or refolded, for in vitro applications, using the optional refolding protocol of Example 3b, with optional addition of 1-5 mM DTT.
A modified hinge region of IgG1 and IgG2a without cysteine was introduced in frame of NM23 S120G (3′) by 4 successive PCR reactions. First the following couple of primers were used:
The PCR fragment was purified and used as template in a second PCR reaction using the following primers:
The PCR fragment was purified and used as template in a third PCR reaction using the following primers:
The PCR fragment was purified and used as template in a fourth PCR reaction using the following primers:
The resulting fragment was purified, digested (NdeI, NheI) and cloned between NdeI and NheI restriction sites of the expression vector pET21b.
Another NM23 S120G fragment was amplified by polymerase chain reaction (PCR) using the following primers:
The fragment was then purified, digested (NheI/XhoI) and cloned in frame, between the NheI and XhoI restrictions sites of the previously cloned NM23 S120G containing both IgG1 and IgG2a modified hinge region linker. The expressed protein can be purified and/or refolded, for in vitro applications, using the optional refolding protocol of Example 3b, with optional addition of 1-5 mM DTT.
A fusion protein in which NM23-wt or preferably S120G mutant is genetically fused to a protein that naturally dimerizes is another method of producing an NM23 variant that prefers dimer formation and if the S120G mutation is included the resultant dimers will resist forming the higher order multimers such as tetramers and hexamers. One way to do this is to fuse NM23, or a portion of NM23 that binds to the MUC1* peptide, to the Fc portion of an antibody. The Fc portion of antibodies homo-dimerizes via disulfide bonds between cysteines. This construct consists of the NM23-S120G protein connected to the Fc region of an IgG antibody. Inclusion of a histidine tag enables purification over an NTA-Ni affinity column. Alternatively, the fusion protein can be purified over a protein A or Protein G column. Cleavage with Pepsin cleaves the Fc region and releases the portion just below the Cysteines.
NM23 S120G was amplified by polymerase chain reaction (PCR) using the following primers:
The fragment was then purified, digested (NdeI, XhoI) and cloned between NdeI and XhoI restriction sites of the expression vector pET21b.
The Fc region of IgG1 was amplified by PCR using the following primers:
The fragment was then purified, digested (XhoI) and cloned in frame (at the XhoI restriction site) of the previously cloned NM23 S120G. The expressed protein can be purified and/or refolded, for in vitro applications. If protein is in inclusion bodies: after harvesting the cells by centrifugation (6000 rpm for 10 min at 4° C.), cell pellet was resuspended with running buffer: 100 mM NaH2PO4, 10 mM Tris pH 8.0, 10 mM imidazole and 8M urea. The solution was incubated on a rotating platform (275 rpm) for 30 min at 37° C. and sonicated on ice for 5 min. Insoluble cell debris was removed by centrifugation (20000 rpm for 30 min at 4° C.). The cleared lysate was then applied to a Ni-NTA column (Qiagen) equilibrated with the running buffer. The column was washed (8 CV) before eluting the protein off the column with the running buffer (6 CV) supplemented with 420 mM imidazole. Before refolding, NTA-Ni elution fractions were pooled and 5 mM reduced glutathione (GSSH) and 0.5 mM oxidized glutathione (GSSG) was added and incubated over night at 4° C. with stirring. Then the protein was refolded using the optional refolding protocol of Example 3b. To avoid protein being sequestered in inclusion bodies, necessitating denaturation and refolding, the following is performed: protein expression can be directed to the periplasm of the expressing cell which has been shown to favor disulfide bond formation and allowing the protein to be folded correctly and soluble.
The IgG1 hinge region was fused 3′ to NM23 S120G by polymerase chain reaction (PCR) using the following primers:
The fragment was then purified, digested (NdeI, XhoI) and cloned between NdeI and XhoI restriction sites of the expression vector pET21b. The expressed protein can be purified and/or refolded, for in vitro applications as described in Example 10a.
The IgG2a hinge region was fused 3′ to NM23 S120G by polymerase chain reaction (PCR) using the following primers:
The fragment was then purified, digested (NdeI, XhoI) and cloned between NdeI and XhoI restriction sites of the expression vector pET21b. The expressed protein can be purified and/or refolded, for in vitro applications as described in Example 10a.
NM23 variants that were generated were first tested for their ability to form dimers. The single chain constructs should migrate through a reducing SDS-PAGE gel with the molecular weight of the monomer-linker-monomer. On a non-reducing gel, the single chain variants migrate with the apparent molecular weight of the dimer, while the higher order multimers generally run with the apparent molecular weight of the monomer. The characteristic tetramers and hexamers do not depend on disulfide bonds to multimerize, while dimers of native NM23, NM23-S120G and NM23-P96S and other variants do depend on disulfide bonds. Native gels and FPLC were also used to determine the multimerization state of the variants. Reducing and non-reducing SDS-PAGE gels showing formation of the dimer are shown in
Either H9 or BGO1v/hOG embryonic stem cells were plated onto either Matrigel coated or anti-MUC1* (MN-C3) antibody coated 6-well cell culture plates at a density of 200,000 cells per well. The cells were cultured in minimal stem cell media (see Example 8) to which was added 8 nM NM23-S120G-RS or an NM23 variant of the invention. Media was changed every 24 hours for the BGO1v/hOG cells and every 48 hours for the H9 cells. A Rho kinase inhibitor was added at each media change for the BGO1v/hOG cells but only for the first 48 hours for the H9 cells. Cells were cultured for four days and then photographed under magnification.
In addition to assessing stem cell morphology as proof that the NM23 variants functioned as the native dimers or the S120G dimers, the growth rate of stem cells cultured in media containing the NM23 variants was compared to the growth rate of identical cells cultured in NM23-S120G “RS” that had been refolded and then purified by FPLC so that the isolated fractions were essentially 100% dimer. In these experiments, 200,000 stem cells all drawn from the same source (human ES—BGO1v/hOG) were cultured in either NM23-S120G RS or one of the NM23 variants shown in
As another method of assessing the function of the NM23 dimer preferring variants, quantitative PCR was performed to measure expression levels of the pluripotency genes in stem cells cultured in the NM23 variants.
Experimental details: Stem cells grown in different NM23 variants were collected. The cells were pelleted and frozen at −70° C. Total RNA was extracted from the samples using TRIzol® Reagent. Quantification of NANOG, OCT4, MUC1, NM23 and GAPDH in the RNA samples was performed using TaqMan® One Step RT-PCR Master Mix Reagents. The real-time PCR data were analyzed using the comparative Ct method. The relative amount of each transcript in each sample was obtained by computing the difference between the target Ct and the corresponding GAPDH (ΔCt). A second normalization was performed by subtracting the RS sample ΔCt from all the others in the data set (ΔΔCt).
As a yet further measure of the function of engineered NM23 dimer preferring variants, their migration from cell surface to cell nucleus was tracked and compared to that of NM23-S120G RS. It is known that NM23 dimers mediate the growth of MUC1*-positive cancer cells and human pluripotent stem cells, which are all MUC1*-positive. When MUC1*-positive cancer cells are incubated in media that contains NM23 in dimer form, the NM23 dimers bind to the MUC1* receptor, become internalized and within 30-60 minutes are translocated to the nucleus where they likely function as transcription factors.
Experimental details: T47D breast cancer cells were initially plated onto collagen-coated 8-well chambers containing 10% FBS RPMI media for 24 hrs followed by serum-starvation (1% FBS RPMI) for 24 hrs, at 37° C., 5% CO2. Subsequently T47D cells were incubated with 16 nM or 128 nM NM23S120GRS or NM23IgG1/IgG2a in 10% FBS RPMI for 30 minutes. T47D cells were then fixed in 4% paraformaldehyde. Cells were blocked for one hour in PBS+1% BSA+5% normal goat serum+0.01% Triton-x (“blocking buffer”) and then incubated in blocking buffer containing primary antibody for one hour at room temperature. Cells were then washed with PBS followed by incubation with the appropriate secondary antibody (Alexa-Fluor, Invitrogen) for one hour at room temperature (kept in the dark). Following washing with PBS, cells were mounted with Prolong Gold+DAPI (Invitrogen) and coverslip. T47D cells were visualized on a Zeiss LSM 510 laser scanning confocal microscope.
NM23 supports proliferation of mouse ES cells with pluripotent colony morphology.
Mouse ES cells (129/S6, EMD Millipore, Billerica, MA) were cultured on inactivated MEF feeder cell layers for two days in mouse ES cell minimal medium (mESC-MM) supplemented with either 1,000 U/mL recombinant mLIF (a, c) (EMD Millipore) or 16 nM NM23-S120G-RS (b, d), and photographed at low magnification under phase-contrast illumination. Size bars indicate 500 microns. In both cases, single cells and colonies consisting of just a few cells on day 1 give rise to larger multicellular oval colonies with bright, defined edges typical of pluripotent mouse ES cells. mESC-MM consists of KnockOut D-MEM basal medium, 15% KnockOut Serum Replacement, 1× GlutaMax I, 1× OptiMEM non-essential amino acids, 0.1 mM B-ME (Life Technologies, Carlsbad, CA), and 1× Penicillin/Streptomycin (Lonza, Allendale, NJ).
Results are shown in
Any of the mutants, deletions and/or single chain or fusion chimeras of the invention, including those described in Examples 2, 9 and 10 can be made to be secreted by the expressing cells for use in vitro, ex vivo and/or in vivo. Sequences that cause expressed proteins to be secreted are known to those skilled in the art. Particularly, sequences derived from antibodies are added to the N-terminus of the protein or to the 5′end of the gene of interest. In addition to the inclusion of leader sequences, the expression cell type need not be limited to E. coli and also includes mammalian cells, mammalian expression cells, yeast, somatic cells, stem cells, iPS cells or cells undergoing induction of pluripotency or induction to a less mature state than the starting cell.
One of the benefits of NM23 variants of the invention is that they are designed to spontaneously form stable dimers without the necessity of denaturation and refolding which cannot be done in an in vivo setting. To demonstrate the advantage of single chain and fusion chimeras that naturally dimerize, NM23-WT, NM23-S120G and variants NM23-S120G-GS2, IgG1h-noC, IgG1h/IgG2ah-noC were expressed and without denaturation or refolding were characterized by non-reducing SDS-PAGE. The gel of
Mutations that promote cancer or stem cell growth can be identified by sequencing NM23 from several cancers. The S120G mutant was isolated from a neuroblastoma. Alternatively, one can randomly mutate NM23 encoding DNA then test the resultant proteins to screen for the mutants that promote cancer or stem cell growth. The limiting factor is the amount of time to screen the many possible mutations in a cell-based assay. A convenient method for testing mutants for their ability to form dimers and also resist formation of higher order multimers is to test the mutants for their ability to bind to the MUC1* peptide. As we have shown, NM23 tetramers and hexamers do not bind to MUC1* peptide. An assay that simultaneously identifies mutants that form dimers but not tetramers and hexamers is a nanoparticle assay in which the MUC1* peptide is loaded onto gold nanoparticles. In multi-well plates each mutant is added to the peptide-bearing nanoparticles. If the mutant readily forms dimers, the dimeric NM23 binds to the particle-immobilized MUC1* peptides and draws the particles close together which causes the particle solution to change from pink to blue. If the mutants are added at high concentration, the mutants that are still able to form tetramers and hexamers will do so, which will not bind to the peptides and the solution will remain pink. Therefore, mutants that prefer dimer formation and do not form the inactive hexamers are readily identified because they will turn the nanoparticle solution blue even when added at high concentrations of for example more than 500 nM.
Example 16. Human BGO1v/hOG embryonic stem cells were plated on to Vita plates coated with 12.5 ug per well of a monoclonal anti-MUC1* antibody (MN-C3) at a cell density of 100,000 cells per well of a 6-well cell culture plate. The cells were cultured for 2 days in NM23 variants that had been refolded according to the optional refolding protocol of Example 3. The NM23 variants used at 8 nM in minimal stem cell media (see Example 8) were NM23-S120G-RS (a,e), NM23-S120G-GS2 (“R” in figure denotes refolded) (b,f), NM23-S120G-IgG1h noC (c,g), and NM23-S120G-IgG1h/IgG2ah noC (d,h). As can be seen from the cell morphology, all the stem cells grew as pluripotent stem cells, devoid of differentiating, fibroblast like cells and also devoid of thickening and darkening which are also indicative of differentiation.
All of the references cited herein are incorporated by reference in their entirety.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically described herein. Such equivalents are intended to be encompassed in the scope of the claims.
This application is a division of U.S, patent application Ser. No. 16/447,812, filed Jun. 20, 2019, which is a division of U.S, patent application Ser. No. 14/077,061, filed Nov. 11, 2013, now abandoned, which is a continuation of International Patent Application No. PCT/US2012/036975, filed May 8, 2012, which claims the benefit of U.S. Provisional Application No. 61/484,052, filed May 9, 2011, each of which are incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61484052 | May 2011 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16447812 | Jun 2019 | US |
| Child | 18640690 | US | |
| Parent | 14077061 | Nov 2013 | US |
| Child | 16447812 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2012/036975 | May 2012 | WO |
| Child | 14077061 | US |
