Patent Application
20230416400
This invention generally relates to immunohistochemistry (IHC) and cancer diagnosis. In alternative embodiments, provided are chimeric or recombinant anti-human BRAF V600E antibodies, including products of manufacture and kits comprising them, and methods for making and using them, including for example their use in the detection or diagnosis of a cancer or other conditions. In alternative embodiments, anti-BRAF V600E antibodies as provided herein are used together with an agent for determining whether MLH1 expression or activity is reduced or absent. In further alternative embodiments, anti-BRAF V600E antibodies as provided herein are used together with antibodies which specifically bind at least one protein selected from the group consisting of MLH1, PMS2, MSH2 and MSH6 to differentiate between Sporadic Colorectal Cancer (CRC) and probable Lynch syndrome.
BRAF (also referred to as serine/threonine-protein kinase B-Raf, proto-oncogene B-Raf, v-Raf murine sarcoma viral oncogene homolog B) is a human gene that encodes a protein called B-Raf. The B-Raf protein is involved in directing cell growth by sending intracellular signals.
Mutation in the human BRAF gene has been associated with certain types of cancer. The most frequent mutation is where valine (V) is substituted by glutamic acid (E) at amino acid position 600 (called V600E). Oncogenic mutations in the kinase domain of the BRAF gene (such as BRAF V600E), results in constitutive activation of the B-raf protein which renders B-raf active in the absence of activating stimuli and can lead to excessive cell proliferation, apoptosis resistance and cancer. BRAF V600E is the result of a single point mutation (T1799A) in the kinase domain of the BRAF gene, which results in substitution of valine (V) to glutamic acid (E) at position 600 in the amino acid sequence of the B-raf protein.
The V600E mutation can be a driver mutation for melanoma, hairy cell leukemia, papillary thyroid carcinoma, colorectal cancer, non-small-cell lung cancer, Langerhans cell histiocytosis and ameloblastoma, and detection of the BRAF V600E mutation, for example, by immunohistochemistry (IHC), can aid in the diagnosis of these cancers.
However, antibodies targeting the human BRAF V600E mutation are very difficult to make due to only a single amino acid mutation needing to be differentiated by the antibody; this also makes their application in IHC problematic.
In alternative embodiments, provided are chimeric or recombinant anti-human BRAF V600E antibodies, including products of manufacture and kits comprising them, and methods for making and using them, including for example their use in the detection or diagnosis of a cancer.
In alternative embodiments, provided are chimeric or recombinant antibodies (Ab) or antigen binding proteins (ABP) capable of specifically binding a human BRAF V600E protein, selected from the group consisting of:
In alternative embodiments of the chimeric or recombinant antibodies (Ab) or antigen binding proteins (ABP) as provided herein:
In alternative embodiments, provided are chimeric or recombinant nucleic acids encoding a chimeric or recombinant antibody as provided herein. In alternative embodiments the chimeric or recombinant nucleic acids further comprise and are operatively linked to a transcriptional regulatory element; and optionally the transcriptional regulatory element comprises a promoter; and optionally the promoter is a inducible promoter or a constitutive promoter.
In alternative embodiments, provided are expression cassettes, vectors, recombinant viruses, artificial chromosomes, cosmids, phagemids or plasmids comprising a chimeric or recombinant nucleic acid as provided herein.
In alternative embodiments, provided are cells comprising a chimeric or recombinant antibody as provided herein, a chimeric or recombinant nucleic acid as provided herein, or an expression cassette, vector, recombinant virus, artificial chromosome, cosmid, phagemid or plasmid as provided herein. In alternative embodiments, the cell is a bacterial, fungal, mammalian, yeast, insect or plant cell.
In alternative embodiments, provided are methods for detecting the presence of a human BRAF V600E protein in a cell or a tissue or organ or a portion of any of the foregoing comprising contacting the cell, tissue or organ or a portion of any of the foregoing with a chimeric or recombinant antibody as provided herein. In alternative embodiments of the methods:
In alternative embodiments, provided are methods for determining whether a colorectal cancer (CRC) in a subject is the result of a sporadic mutation or probable Lynch syndrome comprising:
In alternative embodiments, provided are methods for differentiating between Sporadic Colorectal Cancer (CRC) and Lynch syndrome in a CRC patient, or to identify probable Lynch syndrome in a CRC patient having loss of DNA mismatch repair gene MLH1, comprising conducting an immunohistochemistry assay using a chimeric or recombinant antibody as provided herein to detect the expression or presence of a human BRAF V600E protein in a cell or tissue sample from the CRC patient. In alternative embodiments of the methods:
In alternative embodiments, provided are methods for detecting or diagnosing a cancer, wherein the method comprises:
In alternative embodiments of these methods:
In alternative embodiments, provided are methods for treating, ameliorating or preventing a cancer comprising first detecting or diagnosing the cancer using a method as provided herein, followed by treatment of the individual in need thereof for the treatment, amelioration or prevention of the cancer.
In alternative embodiments, provided are methods for detecting or diagnosing an inflammatory disease, wherein the method comprises: detection of expression or presence of a human BRAF V600E protein in a cell, tissue or organ sample, using of chimeric or recombinant antibody as provided herein, to detect the expression or presence of the human BRAF V600E protein in the cell, tissue or organ sample; and detecting the expression or presence of the human BRAF V600E protein in the tissue or organ sample detects or diagnoses the inflammatory disease. In alternative embodiments, the inflammatory disease is Erdheim-Chester disease or polyostotic sclerosing histiocytosis. In alternative embodiments, the method comprises or further comprises detection of expression or presence of a human BRAF V600E protein in a cell, tissue or organ sample from an individual in need thereof. In alternative embodiments, the detection comprises conducting an immunohistochemistry (IHC) assay.
In alternative embodiments, provided are methods for treating, ameliorating or preventing an inflammatory disease comprising first detecting or diagnosing the inflammatory disease using a method as provided herein, followed by treatment of the individual in need thereof for the treatment, amelioration or prevention of the inflammatory disease.
In alternative embodiments, provided are uses of a chimeric or recombinant antibody as provided herein for detecting or diagnosing a cancer or an inflammatory disease. In alternative embodiments, the uses comprising use of an immunohistochemistry (IHC) assay.
In alternative embodiments, provided are kits comprising a chimeric or recombinant antibody as provided herein, and optionally the kit comprises components needed for an immunohistochemistry (IHC) assay, or comprises instructions for practicing a method as provided herein.
In alternative embodiments, provided are chimeric or recombinant antigen binding protein (ABP) capable of specifically binding a human BRAF V600E protein comprising: a rabbit antibody (Ab) heavy chain Fc region or portion thereof which is capable of specifically binding to a detectable secondary antibody or portion thereof; and, an antigen (Ag) binding region or an Ag-binding portion thereof comprising an Ab variable region from a species other than rabbit, wherein the variable region specifically binds to the human BRAF V600E protein. In alternative embodiments, of these chimeric or recombinant antigen binding proteins:
In alternative embodiments, provided are chimeric or recombinant antigen binding proteins comprising two Fab regions joined by a linker, wherein each Fab region comprises a heavy chain region comprising a rabbit H C1 region fused to a mouse VH region and wherein said heavy chain region is associated with a light chain region comprising a rabbit L C1 region fused to a mouse VL region.
The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
All publications, patents, patent applications cited herein are hereby expressly incorporated by reference in their entireties for all purposes.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The drawings set forth herein are illustrative of exemplary embodiments provided herein and are not meant to limit the scope of the invention as encompassed by the claims.
Like reference symbols in the various drawings indicate like elements.
In alternative embodiments, provided are chimeric or recombinant anti-human BRAF V600E antibodies, including products of manufacture and kits comprising them, and methods for making and using them, including for example their use in the detection or diagnosis of a cancer such as: colorectal cancer (see ref 2, 3) melanoma (see ref. 2, 4, 5) (including malignant melanoma), non-Hodgkin lymphoma (see ref. 6), hairy cell leukemia (see ref. 7), papillary thyroid carcinoma (see ref. 8), adenocarcinoma of the lung (see ref. 9), non-small-cell lung cancer (NSCLC) (see ref. 4), Langerhans cell histiocytosis (see ref 10), ameloblastoma (see ref. 11), brain tumors including glioblastoma (see ref. 2, 12) and pleomorphic xanthoastrocytoma (see ref. 13), or hereditary nonpolyposis colorectal cancer (HNPCC) (see ref 6).
The BRAF mutation is most frequent in melanoma and thyroid cancers. Almost 60% of melanomas are reported to be BRAF mutant. In papillary thyroid carcinoma, 60% of cases show activating somatic alterations of genes encoding effectors in the MAPK signaling pathway, including BRAF. In colorectal cancer and NSCLC BRAF mutation has also been shown as a recurrent alteration.
Mutations of BRAF that lead to a constitutive activation of BRAF that is independent of upstream stimuli for growth and proliferation in cancer, such as BRAF V600E, has been under investigation for treatment with BRAF and/or MEK inhibitors.
Vemurafenib, one of the first BRAF inhibitors, is specific for BRAF V600E (see references 14 and 15). It was initially approved for advanced-stage melanoma treatment. In melanoma MEK inhibitors as single agents has been shown to be relatively modest, whereas the combination of BRAF and MEK inhibitors in BRAF-mutant melanomas has shown great success (see references 16 and 17).
In BRAF-mutant colorectal and thyroid cancers, RAF inhibitors have not show much clinical effect as single agents (see references 14 and 15).
In NSCLC clinical trial of dabrafenib and trametinib combination treatment achieved 64% objective response rate (see reference 18). The most common BRAF mutation in lung cancer is the BRAF V600E mutation, accounting for roughly 50% of BRAF-mutant NSCLC (see reference 19).
In metastatic colorectal cancer combination of BRAF and MEK inhibitors have achieved partial or complete response in a subset of patients (see reference 19). In a follow-up study, they also showed that combinatorial inactivation of BRAF, MEK, and EGFR achieves a higher response rate in BRAFV600E-positive patients (see reference 21). In Non-Hodgkin Lymphoma, Langerhans cell histiocytosis (see reference 22), glioblastoma (see references 23 and 24), ameloblastoma (see reference 25), and Hairy cell leukemia (see reference 26), pleomorphic xanthoastrocytoma (see reference 27) BRAF inhibitor alone or in combination with MEK inhibitors and or immunotherapy are in clinical trials or cases have been reported of treatment.
In alternative embodiments, chimeric or recombinant anti-human BRAF V600E antibodies as provided herein, including products of manufacture and kits comprising them, are used in the detection or diagnosis of an inflammatory disease such as erdheim-chester disease (see reference 28) (also called polyostotic sclerosing histiocytosis).
In alternative embodiments, chimeric or recombinant anti-human BRAF V600E antibodies as provided herein, including products of manufacture and kits comprising them, are used in the detection or diagnosis of Lynch syndrome, which is an autosomal dominant genetic condition that is associated with a high risk of colon cancer, endometrial cancer, or cancers of the ovary, stomach, small intestine, hepatobiliary tract, upper urinary tract, brain.
In alternative embodiments, provided are methods for treating, ameliorating or preventing a cancer such as: colorectal cancer, melanoma (including malignant melanoma), non-Hodgkin lymphoma, hairy cell leukemia, papillary thyroid carcinoma, adenocarcinoma of the lung, non-small-cell lung cancer, Langerhans cell histiocytosis, ameloblastoma, brain tumors including glioblastoma and pleomorphic xanthoastrocytoma, or hereditary nonpolyposis colorectal cancer (HNPCC), comprising use of chimeric or recombinant anti-human BRAF V600E antibodies as provided herein, including products of manufacture and kits comprising them, for the detection and/or diagnosis of the cancer, followed by the appropriate treatment or therapy, wherein optionally the appropriate treatment or therapy comprises administration of BRAF and/or MEK inhibitors.
In alternative embodiments, provided are methods for treating, ameliorating or preventing an inflammatory disease such as Erdheim-Chester disease (also called polyostotic sclerosing histiocytosis), comprising use of chimeric or recombinant anti-human BRAF V600E antibodies as provided herein, including products of manufacture and kits comprising them, for the detection and/or diagnosis of the inflammatory disease (for example, Erdheim-Chester disease), followed by the appropriate treatment or therapy, wherein optionally the appropriate treatment or therapy comprises administration of BRAF and/or MEK inhibitors.
In alternative embodiments, provided are methods for treating, ameliorating or preventing Lynch syndrome, including the possibly associated colon cancer, endometrial cancer, or cancers of the ovary, stomach, small intestine, hepatobiliary tract, upper urinary tract, brain, comprising use of chimeric or recombinant anti-human BRAF V600E antibodies as provided herein, including products of manufacture and kits comprising them, for the detection and/or diagnosis of the Lynch syndrome or associated cancer, followed by the appropriate treatment or therapy, wherein optionally the appropriate treatment or therapy comprises administration of BRAF and/or MEK inhibitors.
In alternative embodiments, exemplary anti-human BRAF V600E antibodies as provided herein are used to differentiate between sporadic colorectal cancer (CRC) and probable Lynch syndrome.
Lynch syndrome is an autosomal dominant genetic condition the causes a high risk for CRC as well as cancers in other sites. Lynch syndrome accounts for 2% to 5% of all CRCs. Lynch syndrome pertains to those individuals in whom a germline mutation in one of the DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6 or PMS2) has been identified.
In Lynch syndrome immunohistochemistry (IHC) can be used to show abnormal MMR (MLH1, MSH2, MSH6 and/or PMS2) protein expression (a MMR (MLH1, PMS2, MSH2 and MSH6) IHC panel can be used). Lynch syndrome sufferers inherit one germline mutant allele and one normal, wild type allele of MLH1, MSH2, MSH6 or PMS2. During tumor formation, the normal allele is inactivated by mutation or loss, leading to no expression of functional alleles. MMR (MLH1, MSH2, MSH6 and PMS2) abnormal expression in Lynch tumors may be observed in IHC in two staining patterns: either complete loss of expression of MMR protein or a patchy/weak expression depending on the effect on protein expression of a given mutation.
However, loss of MLH1 protein can also be due to hypermethylation of the promotor in the MLH1 gene and indicates a sporadic occurrence of CRC compared to Lynch syndrome. In addition, the presence of BRAF V600E is close correlated with hypermethylation in the promoter of the MLH1 gene in sporadic CRC (see references 1, 2, 3).
Thus, a MMR (MLH1, MSH2, MSH6 and/or PMS2) IHC panel can be used to identify probable Lynch syndrome in CRC patients and, in cases with loss of MLH1, a negative BRAF V600E IHC test result indicates probable Lynch syndrome. A positive BRAF V600E IHC test result indicates sporadic CRC (non-Lynch syndrome). As illustrated in
While the invention is not limited by any particular mechanism of action, the presence of the V600E mutation in melanoma is directly linked to a treatment. BRAF is a serine/threonine protein kinase activating the MAP kinase/ERK-signaling pathway, as illustrated in
In alternative embodiments, use of chimeric or recombinant anti-human BRAF V600E antibodies as provided herein in immunohistochemistry (IHC) protocols results in improved results. In IHC, a primary antibody (IgG) is used to specifically bind a protein of interest (for example, BRAF V600E) in a tissue sample, for example, a Formalin-Fixed Paraffin-Embedded (FFPE) tissue, or equivalent. Visualization of this primary antibody (Ab) in the tissue sample, e.g., the FFPE tissue, can be made using a secondary antibody (Ab) or other polypeptide capable of specifically binding the primary Ab (for example, BRAF V600E). Frequently, the IHC primary Ab is an IgG immunoglobulin, for example, a mouse IgG immunoglobulin, where the IgG can be one of the IgG or IgM isotypes, for example, a mouse IgG1, IgG2a, IgG2b, IgG3 and IgM. In alternative embodiments, for increased effectiveness in detecting a mix of mouse IgG isotypes, the secondary Ab is or comprises a polyclonal anti-mouse IgG and/or IgM antibody, and the secondary Ab can be a mixture of Abs specific towards the constant region of each of the primary antibody isotypes such that one secondary Ab reagent (for example, a polyclonal serum) can be used for detecting all the primary Abs. The secondary Ab can be any mixture of IgG isotypes, and can also include IgM isotype Abs.
In alternative embodiments, provided is an improved primary Ab in the form of a chimeric or recombinant anti-human BRAF V600E Ab where the constant regions of the Ab are exchanged with that of another species, for example, in one embodiment the mouse constant regions are replaced with rabbit constant regions. This allows for usage of a secondary Ab raised against (or specific for) the constant region of a different species than that of the primary Ab, for example, rabbit in place of mouse. Rabbits have only one isotype of IgG in contrast to the several IgG isotypes in mouse. Thus, in some embodiments, the chimeric or recombinant antibody contains a constant region from a species that has only one isotype of IgG. This constant region shift can be an advantage for multiplexing (where more than one primary antibody is used, for example, from different species) such as double staining with different colors to specifically look at co-localization of multiple markers, for example, different primary antibodies.
The chimeric or recombinant anti-human BRAF V600E Ab as provided herein has resulted in a great reduction of the antibody titer which provides optimal performance in IHC. For example, in alternative embodiments, if the mouse antibody which provides optimal performance is titrated to 500 ng/mL, the chimeric or recombinant antibody as provided herein can be titrated to between about 30 ng/mL to 62.5 ng/mL depending on method of antibody quantification; thus, use of an exemplary chimeric or recombinant anti-human BRAF V600E Ab in IHC results in an about 8 to 16-fold reduction of antibody needed in the assay. The diagnostic usage of the exemplary chimeric or recombinant anti-human BRAF V600E Ab as provided herein is at least as good, if not better, as the mouse version of the antibody.
An exemplary sequence of an exemplary chimeric or recombinant anti-human BRAF V600E Ab as provided herein is (the complementarity determining regions, or CDRs, are bolded):
METGLRWLLLVAVLKGVQCQAYLQQSGAELVRPGASVKMSCKASGYTFT
SYN
MHWIKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDRSSNTA
CGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLY
SLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLG
GPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVR
TARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEK
TISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEK
NGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEAL
HNHYTQKSISRSPGK
DGISPWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYL
where METGLRWLLLVAVLKGVQC (SEQ ID NO:3) is the rabbit IgG heavy chain signal peptide; and
the rabbit IgG heavy chain constant domain is
Summary heavy chain: Amino acids (aa) 1 to 19=signal peptide; aa 20 to 133=mouse heavy chain variable domain; aa 134 to 456=Rabbit heavy chain IgG constant domains. CDR regions according to IMGT numbering: CDR1 aa 45 to 52 (GYTFTSYN) (SEQ ID NO:9), CDR2 aa 70 to 77 (IYPGNGDT) (SEQ ID NO:10), and CDR3 aa 116 to 122 (ARDGISP) (SEQ ID NO:11).
MDTRAPTQLLGLLLLWLPGATFDIKMTQSPSSMYASLGERVTITCKASQ
DINRYLSWFQQKPGKSPKTLIYRANRVLDGVPSRFSGSGSGQDYSLTIS
RANRVLDGVPSRFSGSGSGQDYSLTISSLESEDMGIYYCLHYDEYPYTY
where MDTRAPTQLLGLLLLWLPGATF (SEQ ID NO:7) is the rabbit IgG heavy chain signal peptide; and
the rabbit kappa1 light chain constant domain is:
Summary light chain: Amino acids (aa) 1 to 22=Signal peptide, aa 23 to 129=mouse light chain variable domain; aa 130 to 233=rabbit Kappa1 constant domain.
CDR regions according to IMGT numbering: CDR1 aa 49 to 54 (QDINRY) (SEQ ID NO:12), CDR2 aa 72 to 74 (RAN) (SEQ ID NO:13), and CDR3 aa 111 to 119 (LHYDEYPYT) (SEQ ID NO:14).
In alternative embodiments, chimeric or recombinant Abs as provided herein, including the exemplary chimeric or recombinant anti-human BRAF V600E Ab comprising heavy chain SEQ ID NO:1 and light chain SEQ ID NO:5, with signal peptide, or without the signal peptide (SEQ ID NO:2 and SEQ ID NO:6)) can be expressed as a recombinant Ab using a plasmid (or any expression vehicle) encoding the respective heavy and light chains, or the heavy chain and the light chain can be encoded in separate expression vehicles. In some embodiments, the heavy and light chains can be (cis- or trans-) expressed from a pTT5™ vector(s) (National Research Council Canada, NRC-CNRC, Canada) in HEK293-6E cells. In alternative embodiment, the vector or vectors expressing the heavy and/or light chains are episomal or are chromosomally integrated, for example, in a stable cell line capable of synthesizing, optionally inducibly synthesizing, the heavy and/or light chains.
In alternative embodiments, provided are nucleic acids encoding chimeric or recombinant Abs as provided herein. Nucleic acids as provided herein can be made, isolated and/or manipulated by, for example, cloning and expression of cDNA libraries, amplification of message or genomic DNA by PCR, and the like. Nucleic acids used to practice embodiments as provided herein, whether RNA, cDNA, genomic DNA, vectors, viruses or hybrids thereof, may be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/generated recombinantly. Recombinant polypeptides generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including bacterial, fungal, mammalian, yeast, insect or plant cell expression systems.
Alternatively, these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, for example, Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066.
Techniques for the manipulation of nucleic acids, such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, for example, Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).
Another useful means of obtaining and manipulating nucleic acids used to practice embodiments as provided herein comprises screening and re-cloning inserts isolated or amplified from, e.g., genomic clones or cDNA clones. Sources of nucleic acids include recombinant nucleic acid sequences, genomic or cDNA libraries contained and/or expressed in, for example, mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos. 5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld (1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC); bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see, e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see, e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinant viruses, phages, phagemids or plasmids.
In alternative embodiments, nucleic acids as provided herein are operably linked to transcriptional regulatory elements, including promoters, with can be constitutive or inducible transcriptional regulatory elements.
In alternative aspects, provided are “expression cassettes” comprising a nucleotide sequence as provided herein, for example encoding a chimeric or recombinant antibody as provided herein. Expression cassettes can include at least a transcriptional regulatory element, for example, a promoter, operably linked with an antibody coding sequence, and optionally can also include transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used, for example, enhancers.
In alternative aspects, expression cassettes used to practice embodiments as provided herein include plasmids, expression vectors, recombinant viruses, any form of recombinant “naked DNA” vector, and the like. In alternative aspects, a “vector” used to practice embodiments as provided herein can comprise a nucleic acid that can infect, transfect, transiently or permanently transduce a cell. In alternative aspects, a vector used to practice embodiments as provided herein can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid. In alternative aspects, vectors used to practice embodiments as provided herein can comprise viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.). In alternative aspects, vectors used to practice embodiments as provided herein can include, but are not limited to replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated. Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No. 5,217,879), and can include both the expression and non-expression plasmids. In alternative aspects, the vector used to practice embodiments as provided herein can be stably replicated by the cells during mitosis as an autonomous structure, or can be incorporated within the host's genome.
In alternative aspects, “promoters” used to practice embodiments as provided herein include all sequences capable of driving transcription of a coding sequence in a cell, for example, a bacterial, yeast, fungal, plant, insect (e.g., baculovirus) or mammalian cell. Thus, promoters used in the constructs include cis-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. For example, a promoter used to practice embodiments as provided herein can be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5′ and 3′ untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences can interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) transcription.
“Constitutive” promoters used to practice embodiments as provided herein can be those that drive expression continuously under most environmental conditions and states of development or cell differentiation. “Inducible” or “regulatable” promoters used to practice embodiments as provided herein can direct expression of a nucleic acid as provided herein under the influence of environmental conditions or developmental conditions. Examples of environmental conditions that may affect transcription by inducible promoters used to practice embodiments as provided herein include the presence of an inducing factor administered to a cell.
In alternative embodiments, peptides and polypeptides used to practice embodiments as provided herein can comprise any “mimetic” and/or “peptidomimetic” form. In alternative embodiments, peptides and polypeptides used to practice embodiments as provided herein can comprise synthetic chemical compounds which have substantially the same structural and/or functional characteristics of the natural polypeptide, for example, a chimeric or recombinant antibody as provided herein. The mimetic used to practice embodiments as provided herein can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic's structure and/or activity. Routine experimentation will determine whether a mimetic is effective for practicing the invention, for example, if a mimetic composition is effective in specifically binding BRAF V600E protein. Methodologies detailed herein and others known to persons skilled in the art may be used to select or guide one to choose effective mimetic for practicing the compositions and/or methods of this invention.
Polypeptide mimetic compositions for practicing embodiments as provided herein can comprise any combination of non-natural structural components. In alternative aspects, mimetic compositions for practicing embodiments as provided herein can comprise one or all of the following three structural groups: a) residue linkage groups other than the natural amide bond (“peptide bond”) linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like. For example, a polypeptide can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds.
Provided are products of manufacture and kits for practicing methods as provided herein comprising chimeric or recombinant anti-human BRAF V600E Abs as provided herein; and optionally the products of manufacture and kits can further comprise some or all reagents needed to perform an IHC, and optionally can comprise instructions for practicing methods as provided herein.
The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples.
Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols, for example, as described in Sambrook et al. (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for polymerase chain reactions (PCRs) can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR—Basics: From Background to Bench, First Edition, Springer Verlag, Germany.
This example demonstrates that anti-BRAF V600E antibodies as provided herein, and methods and compositions incorporating these exemplary Abs, are effective and can be used to detect BRAF V600E. For example, the anti-BRAF V600E antibodies provided herein may be used to detect BRAF V600E in IHC on colorectal carcinomas (CRC) to differentiate between sporadic CRC and Lynch syndrome. Anti-BRAF V600E antibodies as provided herein also can be used to identify BRAF V600E positive melanomas, papillary thyroid carcinomas and hairy cell leukemia, and other cancers.
Tissues used for assessing the ability of the anti-BRAF V600E antibodies to detect BRAF V600E include various clinical tissues, including colorectal cancers, expressing different protein levels of BRAF V600E and cases that are negative for BRAF V600E. Clinical tissues (CT) also includes CRC cases with known BRAF V600E molecular status.
Level of background staining obtained with the exemplary BRAF V600E (the antibody comprising of heavy chain SEQ ID NO:2 and the light chain SEQ ID NO:6) Dako OMNIS™ IHC staining protocol was tested/evaluated on a tissue array including various tissues, including tonsil, liver, breast cancer, carcinoid, colon cancer, colon, melanomas, prostate, cerebellum, pancreas and kidney.
In addition, performance of the BRAF V600E antibody (chimeric or the antibody comprising heavy chain SEQ ID NO:2 and SEQ ID NO:6) was tested on colorectal carcinomas, melanomas, papillary thyroid carcinomas and hairy cell leukemia to support the efficacy of the antibody.
Clone Selection
Recombinant antibody specifically targeting the BRAF V600E mutation, represented by the peptide sequence of the amino acids 596 to 606 (GLATEKSRWSG) (SEQ ID NO:17) was obtained as follows: Vectors were synthesized by GENEART™ (Invitrogen) to contain the DNA sequence encoding the antibody heavy and light chains respectively. Cloning was made into pTT5™ vectors for transient expression in HEK293-6E cells.
Plasmids were generated encoding mouse IgG2a heavy chain version and mouse kappa1 light chain version for production of mouse IgGs. Additionally, plasmids were generated encoding chimeric antibody containing the constant regions from rabbit heavy and light chains linked to the mouse variable region, respectively. More specifically, the mouse heavy chain the constant domains 1, 2, and 3, and the hinge region were replaced with rabbit IgG heavy chain constant domains. For the light chain the mouse kappa constant domain was replaced with a rabbit light chain kappa1 constant domain. The remaining part of the respective heavy and light chains (variable domains) remained unaltered, having the same variable region as for the mouse antibody versions.
The plasmids were used to express recombinant mouse and chimeric mouse/rabbit antibodies, respectively.
The antibodies were quantified and used, non-purified, for testing in TIC in FLEX protocol (ENVISION™ FLEX System™, Dako). Specific staining of BRAF V600E was observed for both the recombinant mouse and recombinant chimeric antibodies.
Both antibodies were Protein A purified and tested again in IHC, both in FLEX+ and FLEX++ protocols on Dako OMNIS™.
Purification had given a slight improvement of signal to noise ratio, and therefore it was decided to proceed only with the Protein A purified antibodies. Furthermore, from this test the chimeric antibody version had an optimal performance concentration of 62.5 ng/mL and the mouse antibody of 0.5 μg/ml.
For all the clone selection part, protocol optimization, and DoE (Design o Experiment), the concentration of the antibody used has been determined by ELISA.
Protocols
Initial testing of the chimeric antibody was a 5-step 2-fold titration to narrow down an approximate optimal concentration (1×) for further testing. 1× was assumed to be around 60 ng/ml.
A titration of the performance benchmark antibody was performed on a target specific multiblock, and slides were evaluated by consensus of a group of trained observers.
Preliminary testing showed that the signal to noise ratio was improved by pretreatment in high pH and by a FLEX++ protocol. Further testing of primary antibody concentration, diluent and incubation time was performed. The best tradeoff between signal and noise was found to be a primary antibody concentration at 60 ng/ml diluted in S0809 and incubated for 20 min.
The double of the 1× concentration, 120 ng/ml, was tested on the full tissue package containing 33 positive specimens and 18 negative specimens. Slides were evaluated and results clearly showed that the primary antibody titer of 120 ng/mL was too high with excessive background staining and increased chances of false positive specimens. Slides was evaluated side by side with performance benchmark slides.
A small tissue package with 12 selected specimens was now stained with both 30 ng/ml and 60 ng/ml as primary antibody concentration, and from these tests it was concluded that the optimal BRAF V600E performance was somewhere in between the two tested concentrations. A compromise was made, and a full tissue package was stained with 45 ng/ml as primary antibody concentration.
This protocol (BRAF V600E, chimeric antibody diluted in S0809 to 45 ng/ml) seemed to have “good” signal to noise ratio with a low chance of detecting false positive specimens, as well as a strong concordance to Ventana's benchmark assay. All slides were evaluated side by side with performance benchmark slides.
Table 1, below, shows the performance of chimeric BRAF V600E antibody in colorectal cancer (CRC) specimens with optimal protocol on Dako OMNIS™. The BRAF V600E staining is localized to the cytoplasm. The staining intensity in CRC tumor cells varies from 0 to 2.75.
In rare cases weak nuclear staining was observed, which should be considered as non-specific. Furthermore, in some CRC specimens a weak grey/yellow cytoplasmic background staining was observed in tumor cells, normal epithelium, smooth muscle cells and other stromal cells. This weak grey/yellow staining was also observed with negative control reagent.
CRC Stained with Chimeric BRAF V600E Antibody
As used in this specification and the claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
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Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”
The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Incorporation by reference of these documents, standing alone, should not be construed as an assertion or admission that any portion of the contents of any document is considered to be essential material for satisfying any national or regional statutory disclosure requirement for patent applications. Notwithstanding, the right is reserved for relying upon any of such documents, where appropriate, for providing material deemed essential to the claimed subject matter by an examining authority or court.
Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and yet these modifications and improvements are within the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms. Thus, the terms and expressions which have been employed are used as terms of description and not of limitation, equivalents of the features shown and described, or portions thereof, are not excluded, and it is recognized that various modifications are possible within the scope of the invention. Embodiments of the invention are set forth in the following claims.
A number of embodiments of the invention have been described. Nevertheless, it can be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
This Patent Convention Treaty (PCT) International Patent Application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application Ser. No. 63/114,123, Nov. 16, 2020. The aforementioned application is expressly incorporated herein by reference in their entirety and for all purposes
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/059368 | 11/15/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63114123 | Nov 2020 | US |
