Therapeutic Antibodies – Christine Pietsch, Jianying Dong, Rosa Cardoso, Hong Zhou, Janssen Pharmaceutica NV
Abstract for “CD47 Antibodies, Methods, and Uses”
“The present disclosure pertains generally to monoclonal antibody that specifically binds to CD47, and more specifically to CD47 antibodies that don’t have significant platelet activity or hemagglutination activity. These monoclonal antibody therapeutics can also be generated using methods that are described.
Background for “CD47 Antibodies, Methods, and Uses”
“The protein cluster for differentiation 47 (CD47), also known by integrin associated protein IAP, ovarian cancer antibody OA3, and Rh related antigen, is a universally expressed member of the cell surface pentaspan transmembrane Ig superfamily. CD47 interacts on macrophages with SIRP Alpha (signal-regulatory proteins alpha), and thus dampens phagocytosis. This pathway is co-opted by cancer cells, which evade phagocytosis, and promotes survival of these cells (Jaiswal S. et al, Trends In Immunol 31: 212-219 2010, 2010). This mechanism is new and can be used therapeutically to target CD47 in many cancers.
CD47 expression is associated with poorer clinical outcomes in a variety of malignancies, including Non-Hodgkin lymphoma and acute lymphocytic lymphoma (NHL), acute myelogenous (AML), ovarian carcinoma, glioma and glioblastoma. CD47 has also been shown to be a cancer stem-cell marker in leukemias as well as solid tumors (Jaiswal et.al., 2009 Cell; 138(2): 271?85; Chan et.al., 2009 Proc Natl Acad Sci USA; 106(33),: 14016-21; Chan and colleagues, 2010 Curr Opin Urrol, 20(5); 393-7; Majeti R et.al., 2011 Oncogene; 30(9): 1009-19; 9: 1009-19; 1009-19
However, CD47 antibodies have been shown to cause platelet aggregation as well as hemagglutination in red blood cells. Homotypic interactions are when two CD47-expressing cells become homotypic and cause them to clump or aggregate when they are treated with a bivalent CD47 binding agent. MABL, a CD47 antibody (full IgG, or F(ab)), can cause hemagglutination of CD47-expressing cells. The hemagglutination in erythrocytes caused by a CD47 antibody, MABL (a full IgG or F(ab)), was only mitigated when MABL was made into an scFv/bivalent scFv. (See e.g., Uno S, Kinoshita Y, Azuma Y, et al., 2007 Oncol Rep, 17(5):1189-94). Similar to the CD47 antibody B6H12, it has been shown that direct aggregation of platelets of subjects with certain polymormisphms of Fc receptor, Fc.RII (Dorahy, Thorne, Fecondo, J V, and Burns, G F, 1997 Journal of Biol. Chem. 272:1323-1330). This is why therapeutically targeting CD47 with CD47 antibodies can be limited by platelet aggregation or hemagglutination.
“Here are antibodies that specifically bind CD47 and inhibit CD47’s interaction with SIRP alpha. They also have no significant platelet aggregation activitiy.”
“In certain embodiments, the antibody binds specifically to CD47 by interfacing with CD47 (SEQID NO: 21 excluding signal sequence) amino acids residues: Q1, N27, E29 and E97. Some embodiments interact with CD47 (SEQID NO: 21 excluding signal sequence). The antibody’s amino acids residues are Y37, K39 and R45. Some embodiments interact with CD47 (SEQID NO: 21 excluding signal sequence). The antibody reacts with the amino acid residues E35 and K39, Y37. The antibody may interact with CD47 (SEQID NO: 21 excluding signal sequence) amino acids residues E29 and Q31, T34, D35, V36.
“Another aspect of the described embodiments features an antibody that specifically binds human or cyno CD47 and comprises a variable heavy chain from SEQ ID Nos: 4-6. Optionally, the antibody may contain a variable light (VL), chain region from SEQ ID Nos. 7 and 8. The antibody may contain a VH region from SEQID NOS: 4 to 6 and a VL region from SEQID NOS: 7 to 8. Other aspects of the antibody include a VH region consisting of SEQID NO: 4 or 5 and a VL region consisting of SEQID NO: 7. Another aspect of the antibody is that it has a VH region consisting of SEQID NO: 6 and a VL region consisting of SEQID NO: 8.
“In certain embodiments, the CD47 antibodies comprises a sequence of VH complementarity-determining region 1 (CDR1) set forth by SEQID NO. 9 or 10, a sequence of VH CDR2 set forth by SEQID NO. 11 or 12, a sequence VH CDR2 set forth SEQID NO. 13 or 14, a sequence VH H CDR1 set forth SEQID NO. 15 or 16, a sequence VL L CDR2 set forth SEQID NO. 17 or 18 and a sequence and a sequence in either 19 or 20. The CD47 antibody, for example, includes a VH CDR1 (SEQ ID No: 9), a SEQ CDR2 (SEQ ID NOT: 11), a SEQ CDR3 (SEQ ID ID NO. 13), a SEQ CDR3 (SEQ ID ID NO. 14), a SEQ CDR1 (1 or 2) sequence in SEQID NO. 15, a SEQ I NO. 13, and a SEQID NO. 15 VL CDR2 (2 or 17) and a CDR3 (3L CDR3) sequence in the 19 or 20). Another example is the CD47 antibody, which includes a sequence of VH CDR1 in SEQID NO: 10, a sequence of VH CDR2 in SEQID NO: 12, and a sequence VH CDR3 in SEQID NO: 13, a sequence VH CDR2 in SEQID NO: 14, and a sequence VL CDR3 in SEQID NO: 17, a sequence VL CDR3 in SEQID NO: 19, and a sequence VL CDR3 set forthin SEQID NO: 20, a sequence in SEQUAKE EQ ID PARTIAL
“Some embodiments of the antibodies bind CD47 in a location where a VH and VL chains have more extensive contacts with CD47 than they do with CD47. In this position, the VH chain is near the membrane of CD47-expressing cells, and the VL chain of antibody blocks a SIRPalpha binding site on CD47.
“Some embodiments of the antibodies lack significant hemagglutination activities. Some embodiments have a platelet-aggregation activity that is less than 10% of what would be observed in the absence or presence of the antibody. Some embodiments of the antibody are chimeric, humanized or fully human. The antibody can bind to human CD47 and cynomolgus CD47 in some embodiments. The antibody may promote macrophage-mediated destruction of CD47-expressing cells. Some embodiments contain an IgG type selected from IgG1 and IgG2 IgG.
“Described herein is also a pharmaceutical composition that includes an antibody described herein, and a pharmaceutical-acceptable carrier. Kits can include the described antibodies. These include polynucleotides that encode the antibodies, as well as vectors for the cells and cells suitable to express the polynucleotides.
“Another embodiment consists of methods for relieving a symptom associated with cancer or another neoplastic condition. The subject is administered one or more of the antibodies described herein. However, the antibody has no significant platelet aggregation activity. The amount of antibody administered is sufficient to relieve the subject’s symptoms. The subject may be a human in some embodiments. Some embodiments of the antibody are chimeric, humanized or fully human. The antibody may inhibit CD47’s interaction with SIRP alpha in some embodiments. The antibody may contain an IgG type selected from the IgG1 and IgG2 groups. Some embodiments include chemotherapy.
“Some characteristics of antibodies are described herein:
“Specific binding to human CD47 or cyno CD47”
“Blocking CD47 interaction by SIRP alpha”
“Do not have significant human platelet accumulation activity.”
“Do not have significant Hemagglutination Activity.”
“Are capable of encouraging phagocytosis by tumor cells via macrophages.”
“Display potent anti-tumor activities in a mouse model for human cancers.”
“The antibodies discussed herein are of great value in the treatment and prevention of many types of cancer.”
“Many CD47 antibodies exist that block SIRP alpha. But, before the subject matter was discussed herein, full IgG CD47 antibody that blocked SIRP Alpha caused side effects of platelet accumulation and/or hemagglutination. This is an undesirable characteristic. While 2D3 and other antibodies do not cause hemagglutination they do not block SIRP Alpha. There were no CD47 antibodies that could block SIRP alpha in full IgG format without causing platelet or red blood cell clumping.
“In some embodiments, the IgG CD47 antibody described herein does not show significant platelet accumulation and hemagglutination. This increases the effectiveness of therapeutically targeting CD47 and preserves the ability to block the interaction between CD47 and SIRP alpha which promotes phagocytosis for CD47-expressing cells. The full IgG CD47 antibody described in the disclosure, e.g. C47B157-C47B161 or C47B222, do not exhibit significant cell agglutination activities. The CD47 antibodies discussed herein, for example, do not exhibit significant platelet aggregation or hemagglutination activities. The combination of the CD47 antibodies (e.g. C47B157 and C47B161 as well as derivatives thereof) is unique in that they can block SIRPalpha without significant platelet accumulation and hemagglutination activity.
“General”
“Those who are skilled in the art will recognize that the disclosures herein can be modified and varied beyond the ones specifically described. The present disclosure includes all variations and modifications. All of the steps, features and compositions, as well as all combinations, are included in the present disclosure.
The specific embodiments herein are not intended to limit the scope of the present disclosure. They are only meant to serve as an example. Functionally-equivalent products, compositions, and methods are clearly within the scope of the present disclosure.”
“The compositions and methods described in this document are made or performed without undue experimental use, except where indicated, of conventional molecular biology, microbiology technology, virology and peptide synthesis in solution, solid phase and pharmaceutical chemistry, medicinal and immunology. For the preparation, formulation, delivery, and treatment patients, standard techniques are used.
“Definitions”
“Since they are not defined otherwise, the scientific and technical terms that are used in connection to the present disclosure will have the meanings that are common to those with ordinary skill in art.”
“The following terms shall have the following meanings, except where otherwise noted, when they are used in accordance to the present disclosure:
“The term ‘and/or? (e.g.?X/or Y) is defined as: “X and Y” shall be understood as meaning either?X or Y? either?X and Y? and shall be considered to support both the literal or implied meanings.
“Unless otherwise stated or required by context, any reference to a single, composition of matter, group or number of steps, or group of compositions, shall be taken as encompassing one or more (i.e. One or more of these steps, compositions, of matter, groups or groups of steps, or both, of those compositions, of matter, shall be considered. As used herein, the singular forms of?a? and?an? The singular forms?a?,?an?? und?the? are used herein. Unless the context explicitly dictates otherwise, plural aspects are included. Referring to?a is an example. Refer to?an? for a reference to both a single and multiple. Includes a single and two or more; refer to?the? Includes a single as much as two or more; reference to?the?
“As used herein the terms?”CD47?”,?”integrin-associated protein IAP?,??ovarian cancer antigen OA3?, and?Rh-related antibody? are interchangeable. They can be interchangeably used.
“The terms’red blood cell(s),? and?erythrocyte(s)? “The terms?red blood cell(s)?” and?erythrocyte (s)? are interchangeable. They are interchangeable and can be used interchangeably in this article.
“Platelet aggregation” is the term used herein. The adhesion between activated plateslets results in the formation or clumps (or aggregates) of activated plateslets. As shown in the Examples, platelet aggregation can be measured using an aggregometer. This measures the increase of transmittance of light when platelet aggregation takes place. Significant platelet aggregate activity? This occurs when there is a significant increase in light transmittance by at least 25% within 6 minutes following the addition of the antibody described herein. It is different from light transmittance before antibody addition.
“The term “agglutination” refers to cellular clumping. Cellular clumping is referred to as ‘agglutination’, while cellular hemoglobination is referred to as?hemagglutination. Refers to the clumping of a particular subset of cells (i.e. red blood cells). Hemagglutination can also be called a form of agglutination.
“In the hemagglutination test described herein, patterns are formed by erythrocytes that can be either buttons or? ?halos,? ?halos,? The term “significant hemagglutination activ? Refers to any halo pattern found in a well after the addition of the antibody described herein.
“Antibody” is used in this document. The term “antibody” refers to immunoglobulin molecules as well as immunologically active immunoglobulin (Ig), i.e. molecules that have an antigen binding site that specifically binds to (immunoreacts) with an antigen. By “specifically bind?” or?immunoreacts? or?directed against? This means that the antibody reacts with antigenic determinants of the desired antigen and doesn’t react with or bind at a lower affinity (Kd>106) to other polypeptides. Monoclonal, single-chain, Fab, F(ab), chimeric, and dAb (domain antibodies) are all examples of antibodies. )2 fragments and Fv, scFvs and an Fab expression collection.
The tetramer is the basic structural unit of an antibody. Each tetramer consists of two identical pairs polypeptide chains. Each pair has one?light? (approximately 25 kDa) & one?heavy?” Chain (between 50 and 70 kDa). Each chain’s amino-terminal section contains a variable area of around 100 to 110 amino acids that is primarily responsible in antigen recognition. Each chain’s carboxy-terminal section contains a region that is primarily responsible to effector function. The majority of antibody molecules that are obtained from people relate to the IgG, IgM and IgA classes. They differ by the nature of each heavy chain in the molecule. There are subclasses for certain classes, like IgG1, IgG2, or others. The light chain can also be called a kappa or a lambda in humans.
“Monoclonal antibody” is a term that refers to monoclonal antibodies. “Monoclonal antibody” (mAb) is a term that refers to an antibody population consisting of one molecular species. It is composed of a unique light-chain gene product and a unique heavier chain gene product. The monoclonal antibody’s complementarity determining areas (CDRs), are identical in all molecules. MAbs have an antigen binding area that can immunoreact with a specific epitope of antigen.
“Abdominal molecules obtained from human beings are generally of any of the following classes: IgG, IgM and IgA. They differ by the nature of the heavy chains in the molecule. There are subclasses for certain classes, like IgG1, IgG2, or others. The light chain can also be called a kappa or a lambda in humans.
“The term “antigen binding site”? or ?binding portion? This refers to the immunoglobulin molecule which participates in antigen-binding. The N-terminal variable (??) amino acid residues form the antigen binding site. Regions of the heavy (??H?) Regions of the heavy (?H?) and light chains (?L?) chains. Three highly divergent stretches are found within the V regions for the heavy and lighter chains. These are known as the?hypervariable areas? They are located between more stable flanking stretches, known as the?framework regions? or?FRs?. The term ‘FR?????? FR? refers to amino acids sequences that are found naturally between and adjacent to hypervariable regions of immunoglobulins. The three hypervariable areas of a light chain and three of the hypervariable regions in a heavy chain form an antigen-binding space. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as ?complementarity-determining regions,? CDRs. Each domain has an assigned amino acid according to the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health Bethesda Md.). (1987 and 1991), Chothia & Lesk (J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989).
“Epitope” is the term used herein. Any protein determinant that can be used to specificbind to an immunoglobulin, fragment thereof, or T-cell receptor. The term “epitope” is used. Any protein determinant that can be used to specifically bind to an immunoglobulin receptor or T-cell receptor is called an “epitope”. The epitopic determinants are usually composed of chemically active surface groups of molecules, such as sugar side chains or amino acids. They also have specific three-dimensional structural characteristics and specific charge characteristics. When the dissociation constant of an antibody is?1?M, it is considered to be able to bind antigens.
“Specifically Binds” is the term used herein. The non-covalent interactions between immunoglobulin molecules and antigens that are specific to them refer to these types of interactions. A dissociation constant (Kd), which is the value of an interaction between immunological binding molecules and antigens, can be used to express the strength or affinity of these interactions. A smaller Kd indicates greater affinity.
The immune binding properties of certain polypeptides can easily be quantified by using well-known methods. One method is to measure the rates of antigen-binding sites/antigen complex formation and dissociation. These rates are dependent on the concentrations and affinity of the interactions and the geometric parameters that equally affect the rate in both directions. The?on rate constant and the?off rate constant are therefore equal. The?on rate constant (kon) as well as the?offrate constant (koff) can be calculated. Calculating the concentrations and actual rates of dissociation and association can determine the value of koff. Nature 361:186-87 (1993). The ratio of Koff/kon allows the cancellation of all parameters that are not associated with affinity and is equal to Kd, the dissociation constant. (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). The present disclosure identifies an antibody that binds to CD47 when the equilibrium binding constant, (Kd), is?1?M as determined by various assays like radioligand binding assays or surface plasmon resonance, (SPR), flow cell cytometry binding assays or other similar assays.
“An ?isolated? An antibody is one that has been isolated from its environment and is able to be recovered. It is a component of its environment that contains contaminants. These components can include enzymes, hormones and other non-proteinaceous solutes. Preferable embodiments will purify the antibody (1) to greater 95% by weight of the antibody, most preferably more that 99%, (2) to a degree sufficient for at least 15 residues N-terminal and internal amino acids sequences by using a spinning cup sequenator or (3) to homogeneity via SDS-PAGE under reduced or nonreducing conditions using Coomassie or, preferably silver stain. Since at least one component will be absent from the antibody’s natural environment, isolated antibody refers to the antibody found in situ within recombinant cell. Normally, however, isolated antibodies will only be obtained by at least one purification step.
“Polypeptide” is a generic term that refers to native protein, fragments, or analogs of a polypeptide sequence. “Polypeptide” is used herein to describe native protein fragments or analogs of a particular polypeptide sequence. Thus, analogs and native protein fragments are species within the polypeptide geneus.
“The term ?naturally-occurring? “Naturally-occurring” is a term that refers to objects found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory or otherwise is naturally-occurring.”
“The term ‘oligonucleotide’ is used. The term?oligonucleotide? refers to both naturally occurring and modified nucleotides that are linked by non-naturally occurring and naturally occurring oligonucleotide links. The oligonucleotide is a subset of polynucleotides that generally has a length of 200 or less. Preferably, oligonucleotides have a length of 10-60 bases. Most preferably, they are between 12, 13, 14, 15, 16, 17, 18, 19, 20 or 40 bases. Most oligonucleotides have a single strand, which is useful for probing, but oligonucleotides can be double-stranded to aid in the construction of gene mutants. The oligonucleotides discussed herein can be either antisense or sense.
“Sequence identity” is a term that refers to the identification of a sequence. means that two polynucleotide or amino acid sequences are identical (i.e., on a nucleotide-by-nucleotide or residue-by-residue basis) over the comparison window. What is the percentage of sequence identity? It is calculated by comparing two sequences that are optimally aligned over a window. To determine the number matched positions, divide the number matched positions by all positions in the window (i.e. the window size) and multiply the result by 100. The term “substantial identity” is used herein. The term “substantial identity” is used herein to denote a characteristic of a polynucleotide sequence or amino acid sequence. This means that the sequence has at minimum 85 percent sequence identification, preferably between 90 and 95 percent sequence ID, more often at 99 percent sequence identitate, when compared to a reference sequencing over a comparison window consisting of at most 18 (6 amino acid) positions. A comparison window may also include deletions or additions totalling 20 percent or less over the comparison window. A subset of a larger sequence could be used as the reference sequence.
“The twenty standard amino acids and their abbreviations are used as such in this document. See Immunology?A Synthesis (2nd Ed., E. S. Golub & D. R. Gren), Sinauer Associates Sunderland? Mass. (1991)). (1991). Examples of unconventional amino acids include: 4 hydroxyproline, ?-carboxyglutamate, ?-N,N,N-trimethyllysine, ?-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ?-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). According to convention and standard usage, the left-hand direction of the polypeptide notation is the amino terminal direction. The right-hand direction corresponds with the carboxy-terminal directions.
“Similarly, except where specified otherwise, the left hand end of single-stranded mononucleotide sequencings is the 5. End The left-hand direction for double-stranded mononucleotide sequencings is called the 5? direction. Direction of 5? To 3? The transcription direction sequence regions are the sequences that add nascent transcripts to the DNA strand. They have the same sequence as the original RNA sequence and are 5? To the 5? The sequence regions at the end of the transcript of RNA are called?upstream sequences?. They are sequence regions on DNA strands with the same sequence as RNA. The 3? The?downstream sequences’ are at the end of the transcript of RNA. The term “substantial identity” is used to describe polypeptides. Two peptide sequences must share at least 80 percent of their sequence identity when they are optimally aligned using programs GAP and BESTFIT with default gap weights. Preferably, at least 90 percent sequence identification, more preferably at minimum 95 percent sequence identitate, and most preferably, at least 99 percent sequence ID.
“Preferably, the residue positions that are not identical differ using conservative amino acid substitutions.”
“?Conservative? “?Conservative?” refers to the interchangeability between residues with similar side chains. Glycine, alanine-valine, leucine and isoleucine are examples of amino acid side chains that have aliphatic side chain; serine, threonine, and threonine are examples of side chains with aliphatic sidechains; glycine and alanine are side chains that have aliphatic sidechains; serine, threonine and threonine are side chains that have amide-containing sidechainsine, arginine-containing sidechainsine-containing sidechainsine; and methionine and cysteine-containing sidechainsine-containing sidechainsine-isoleucine-valine, glutamic-alanine-alanine-alanine-alanine-alanine-isoleucine-isoleucine-valine-isoleucine-alanine-valine-isoleucine-arginine-leucine-alanine-e-tyleucine-alanine-leucine-sine-sine-rich-alanine-rich-sine-glutysine-sine-sine-sine-gluty-gluten-gluten-gluco-glutine-gluco-glutar-sine-valine-glutine-sine-glutine-it-le-ty-glutine-ty-g-g-isomic-gluten-ar-gluten-g-gluco-g-hecine-g-ty-g-le-ty-g-ty-g-ty-g-ty-g-ty-g-ty-g-ty-glucocine-g-glucocine-g-g-g-alanine-g-a-g-alanine-g-gluco-g-g-alanine-g-g-g-g-sine-ty-sine-alanine-alanine-glysine-ty-leucine-leucine-le-alanine-leucine-ty-sine-ale-ty-leucine-ty-leucine Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.”
“As discussed herein minor variations in amino acid sequences for antibodies or immunoglobulin molecular are considered to be included in the present disclosure. Provided that these variations in the sequence of amino acids maintain at least 75% and more preferably at minimum 80%, 90%, 95%, 95%, and most importantly at least 99 percent, the modifications in the amino acids sequences remain at least 75%. Particularly, conservative replacements of amino acids are possible. Conservative replacements are those which occur within a family that is related in their side chain. The genetically encoded amino acid families are: (1) aspartate, glutamate;(2) basic amino acids, lysine and arginine; (3) non-polar amino compounds are alanine (valine), leucine (isoleucine), proline, phenylalanine), methionine or tryptophan; (4) uncharged, polar amino acids include glycine. Hydrophilic amino acids are arginine and asparagine as well as glutamine, glutamines, glutamate. Histidine, lysine. serine, and the threonine. Cysteine, cysteine (isoleucine), leucine and methionine; proline, tryptophan; tyrosine, tyrosine, and valine are the hydrophobic amino acid families. The aliphatic-hydroxy families include serine, threonine, cysteine, isoleucine or leucine; (iii), asparagine (amide containing family); (iv) phenylalanine (tryptophan), and tyrosine (aromatic family). It is reasonable to assume that an isolated substitution of a leucine for an isoleucine, valine, aspartate with glutamate, or a threonine and serine with a threonine, or similar replacement of an amino acids with a structurally related one, will not have any major effect on binding or properties of the resulting molecules, particularly if it does not involve an amino-acid within a framework. Assaying the specific activity can easily determine whether an amino acid change leads to a functional protein. These assays are described in great detail. Those with ordinary skill can easily prepare fragments or analogs of antibodies and immunoglobulin molecules. The preferred amino- and carboxytermini of fragments and analogs are located near the boundaries of functional domains. Using public and proprietary sequence databases, the nucleotide sequence data and/or amino acids sequence data can help identify structural and functional domains. Computerized comparison methods are preferred to identify sequence motifs and predicted conformation domains in proteins with known structure and/or functions. Bowie et. al. have identified methods to identify protein sequences that are folded into a three-dimensional structure. Science 253(164) (1991). The examples above demonstrate that skilled art-workers can recognize sequence motifs as well as structural conformations. These may be used to create structural and functional domains according to the present disclosure.
“Preferred amino acids are those that (1) reduce susceptibility for proteolysis, (2) decrease susceptibility at oxidation and (3) alter binding affinity to form protein complexes. (4) alter bindingaffinities. (5) confer or modify other physicochemical, functional properties of analogs. Analogs may include muteins from a different sequence than the naturally occurring peptide sequence. One or more amino acid substitutions (preferably conservative ones) can be made in the naturally occurring sequence. Preferably, the substitution should occur in the part of the polypeptide that does not form intermolecular contacts. The structural characteristics of the parent sequence should not be significantly altered by a conservative amino acid substitution. For example, the replacement amino acid should not break the helix in the parent sequence or alter other secondary structures that make up the parent sequence. Art-recognized polypeptide secondary and tertiary structure examples are described in Proteins, Structures and Molecular Principles, Creighton, Ed., W. H. Freeman and Company, New York (1984); Introduction to Protein Structure, C. Branden and J. Tooze, Garland Publishing, New York, N.Y (1991); and Thornton et.al. Nature 354:105 (1991).
“The term agent? “Agent” can be used to refer to a chemical compound, mixture of chemical compounds, or extract made from biological material.
“As used herein the terms ‘label? “Label” or “labeled” are terms that can be used herein. Refers to incorporation or “labeled” The label or marker may also be therapeutic in certain cases. There are many methods for labeling glycoproteins and polypeptides. Some examples of labels for polypeptides are radioisotopes and radionuclides, including but not limited to: radioisotopes (e.g. 3H, 14C. 15N, 35S), 90Y, 99Tc. 111In. 125I. 131I), fluorescent labels (e.g. FITC. rhodamine. luciferase. alkalinephosphatase. luciferase. luciferase. To reduce steric hindrance, some labels may be attached to spacer arms of different lengths. What is the term “pharmaceutical drug”? The term “pharmaceutical agent or drug” is used herein to refer to any chemical composition or composition that can induce a desired therapeutic effect when administered to a patient.
“Antineoplastic agent” is a term that refers to agents that inhibit the development or progression of a neoplasm in a human. This term refers to agents that inhibit the development or progression (or both) of a neoplasm in a person, especially a malignant (cancerous), lesion such as a cancer, sarcoma or lymphoma. Antineoplastic agents often have the ability to inhibit metastasis.
“Other chemical terms are used herein according to the convention usage in art, as illustrated by The McGraw-Hill Dictionary of Chemical Terms. (Parker, S. Ed., McGraw-Hill San Francisco (1985 )).”).
“CD47 Antibodies”
“Monoclonal antibody described herein can bind CD47 and inhibit SIRP alpha binding to CD47. They also decrease CD47-SIRP beta-mediated signaling and promote phagocytosis. They also inhibit tumor growth and/or migrating. For example, the Examples cellular assay can be used to determine inhibition.
“Exemplary antibodies” include an antibody with a variable heavy chain (VH), selected from SEQID NOs: 4-6, and an antibody with a variable light chain (VL), selected from SEQID NOs 7 and 8. Exemplary antibodies are specifically those listed in Table 1.
“TABLE 1\nVariable heavy Variable light\nAntibody (VH) chain (VL) chain\nC47B157 SEQ ID NO: 4 SEQ ID NO: 7\nC47B161 SEQ ID NO: 5 SEQ ID NO: 7\nC47B222 SEQ ID NO: 6 SEQ ID NO: 8”
Below are highlighted the complementarity determining areas (CDRs), of the VH chain for CD47 antibodies. Some embodiments have the VH CDR1 amino acid sequence as DYNMH (SEQID NO: 9) and DYWIG (10 SEQ ID NO. 10). Some embodiments have the VH CDR2 amino acid sequence as DIYPYNGGTGYNQKFKG or IIYPGDSDTRYSPSFQG. (SEQID NO: 11). “In some embodiments, VH CDR3’s amino acid sequence is either GGWHAMDS (SEQID NO: 13), or VGRFASHQLDY(SEQ ID No: 14).
“In some embodiments the amino acid sequence for VL CDR1 may be RSRQSIVHTNRYTYLA, (SEQID NO: 15), or RASQSVNNRLA (16). Some embodiments have the VL CDR2 amino acid sequence as KVSNRFS (SEQID NO: 17), or WASTRAI(SEQ ID No: 18). “In some embodiments, VL CDR3’s amino acid sequence is either FQGSHVPYT or QQGASWPFT (SEQID NO: 20).
“This disclosure also includes antibodies that bind the same epitope to CD47 antibodies as those described herein. The antibodies described in this patent bind to the epitope that contains one or more amino acids of an exemplary human CD47. Q08722.1 (GI.1171879) is incorporated by reference. Underlined is the signal sequence (amino acid 1-18).
“SEQ?ID?NO:?21\nMWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNME\nAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKG\nDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNE\nNILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITV\nIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLT\nSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQ\nLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE\n\nIn some embodiments the antibodies interact with Q1, L3, N27, E29, E97, L101, T102, R103, and E104 of SEQ ID NO: 21. Some embodiments of the antibodies interact with K39, R45 and D46. Some embodiments of the antibodies interact with E35 and K39, 49, 46, 49, E51, A53. L54. K56. T58, V59. T99. L101. T102. Some embodiments of the antibodies interact with E29 and Q31, T34, E35. V36.
“Those who are skilled in the art will know that it is possible to determine without undue experimental whether an antibody has the same particularity as one described herein (e.g. C47B157 or C47B161 or an antibody with a variable heavy chains selected from SEQID NOs: 4-8 and a variable light chains selected from SEQ ID NOs 7 and 8 respectively) by determining if the former blocks CD47 binding. The antibody that is being tested may compete with the one described in this disclosure. This can be shown by a decrease of binding by the antibody.
An alternative method to test whether an antibody has the same specificity as the one described herein is to incubate it with soluble CD47 proteins (with which it is normally reactive) and then add the antibody to be tested to see if it is inhibited in its ability bind CD47. If the antibody is unable to bind CD47, then it will likely have the same or equivalent epitope specificity.
“Antibodies of The Present Disclosure”
“The ability of the antibodies described herein to modulate or block, inhibit, reduce and antagonize CD47- or CD47/SIRP-alpha-mediated signaling can be evaluated. These assessments could include the measurement of CD47- or CD47/SIRPalpha-mediated signaling when one or more of these antibodies are present. These assays may include competitive binding assays, or they can measure a biologic readout such as the ability to promote the phagocytosis by a CD47-expressing cell by a macrophage (as described in Example 7).
“Various methods are known in the art for the production monoclonal antibodies against CD47 or derivatives, fragments analogs homologs and orthologs thereof. (See, for instance, Antibodies A Laboratory Manual, Harlow D, and Lane D 1988, Cold Spring Harbor Laboratory Press Cold Spring Harbor, N.Y.; incorporated herein as reference). Fully human antibodies are those antibody molecules that contain the complete sequence of the light and heavy chains, as well as the CDRs. These antibodies are called ‘human antibodies’. These antibodies are also known as?human antibodies? herein. For example, human monoclonal antibodies can be prepared using the methods described in the Examples below. You can also prepare human monoclonal antibody using the trioma method; the human-B-cell hybridoma procedure (see Kozbor, and al., 1983 Immunol Now 4: 72); or the EBV hybridoma to create human monoclonal antibody (see Cole, and al., 1985 in: MONOCLONAL CANCER THERAPY, Alan R. Liss, Inc., pages. 77-96). Human monoclonal antibodies can be used and produced using human hybridomas (see Cote et al. 1983). Proc Natl Acad Sci USA 1980: 2026-2030). Or by in vitro transforming human B cells with Epstein Barr virus (see Cole, and al., 1985 In MONOCLONAL ANOBODYS AND CANCER THEAPY, Alan R. Liss, Inc., pages. 77-96).”
“Antibodies can be purified using well-known methods, such as affinitychromatography using protein A and protein G. These provide primarily the IgG portion of immune serum. The immunoaffinity chromatography method uses immunoaffinity columns to immobilize the immune specific antibody. D. Wilkinson discusses the purification of immunoglobulins in The Scientist, Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).”
“The monoclonal CD47 antibodies in this disclosure are monoclonal antibodies. Monoclonal antibodies are monoclonal antibodies that block, inhibit, reduce or neutralize CD47- or CD47/SIRP-mediated cell signaling. They can be generated by immunizing animals with membrane bound or soluble CD47 such as human CD47 or an immunogenic fraction, derivative, or variant thereof. The animal can also be immunized by cells transfected to express CD47. The antibodies can also be obtained by screening a library containing antigen binding domain or antibody sequences that bind to CD47. This library can be prepared in bacteriophage, as protein or peptide-fusions to a Bacteria coat protein. It is then expressed on the surface phage particles assembled with the encoding DNA sequences (i.e., the?phage displayed librarian?). The hybridomas resulting from myeloma/B-cell fusions are then tested for CD47 reactivity.
“Monoclonal antibody preparations can be made, for instance, by hybridoma methods such as those described in Kohler and Milstein Nature, 256,495 (1975). A hybridoma is when a mouse, hamster or other suitable host animal is immunized using an immunizing drug to produce antibodies that are specific to the immunizing agents. The lymphocytes may also be immunized in vitro.
“The immunizing agent will usually include the protein antigen, a portion thereof or a combination thereof. If cells of human origin are required, peripheral blood lymphocytes will be used. However, spleen cells and lymph node cells can also be used. To form a hybridoma, the lymphocytes are fused with an immortalized line of cells using a suitable glue agent such as polyethylene glycol (Goding Monoclonal Antibodies Principles and Practice Academic Press, 1986, pp. 59-103). Immortalized cells are often transformed mammalian cells, especially myeloma cell lines of rodent, bovine, and human origin. Most commonly, myeloma cells from mice or rats are used. Hybridoma cells can then be grown in suitable culture media that contains at least one substance that inhibits the growth or survival unfused immortalized cells. Hypoxanthine, aminopterin and thymidine are some examples of culture media that can be used to grow hybridomas. These substances are necessary to prevent HGPRT-deficient cells from growing.
“Preferred immortalized cells lines are those that fuse well, support stable high-level expression of antibody by selected antibody-producing cell types, and are sensitive in a medium like HAT medium. For the production of monoclonal antibody-producing cells, murine myeloma line is preferred. They can be purchased from the Salk Institute Cell Distribution Center in San Diego, Calif. or the American Type Culture Collection in Manassas. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63)).”
The culture medium in which hybridoma cells were cultured can be tested for monoclonal antigen-specific antibodies. The binding specificity of monoclonal antibody produced by hybridoma cells can be determined either by immunoprecipitation (RIA) or an in vitro binding test (ELISA). These techniques and assays have been developed. Scatchard analysis of Munson, Pollard, and Anal can be used to determine the binding affinity of monoclonal antibodies. Biochem., 107:220 (1980). It is also important to find monoclonal antibodies that are specific and have a high affinity for the target antigen in therapeutic applications.
After the desired hybridoma cells have been identified, the clones may be subcloned using limiting dilution protocols and grown according to standard methods. (See Goding. Monoclonal Antibodies Principles and Practice. Academic Press, 1986, pp. 59-103). Suitable media include Dulbecco’s Modified Eagle’s Medium or RPMI-1640 medium. Alternately, hybridoma cells may be grown in vivo in mammal as ascites.
“The monoclonal antibody subclones secreted can be isolated from the culture medium or ascites liquid by using conventional immunoglobulin purification processes such as protein A-Sepharose or hydroxylapatite Chromatography, gel electrophoresis or dialysis.
“Human Antibodies and Humanization of Antibodies.”
“The antibodies described in this document include either fully human antibodies or humanized antibody. These antibodies can be administered to people without creating an immune reaction by the person against the administered immunoglobulin.
“A CD47 antibody can be generated by phage-display techniques using only human sequences. These approaches are well-known in art, e.g. in U.S. Pat. No. No. 6,521,404 are herein incorporated by reference. This method uses a combinatorial library that contains random pairs of light- and heavy-chained phages. Another method is to produce a CD47 antibody by immunizing a non-human transgenic animal with human CD47 proteins. This approach renders some of the non-human endogenous heavy and/or light chain loci, as well as the kappa and kappa light chains, inoperable and prevents them from generating the genes that encode immunoglobulins to respond to an antigen. The animal has been transfected with at least one of the human heavy chain loci and at least one of the human light chain loci. In response to an administered antibody, the human loci are rearranged to encode genes for human variable regions. The xenomouse then produces fully human immunoglobulin-secreting B-cells upon immunization.
“Xenogenic non-human animals can be produced using a variety of techniques. See U.S. Pat. Nos. Nos. 6,075,181 & 6,150,584, which are hereby incorporated in their entirety by reference. This strategy was used in the creation of the first XenoMouse? These strains were published in 1994. See Green et al. Nature Genetics 7 (13-21) (1994), is herein incorporated by reference. U.S. Pat. Nos. 6,162,963, 6,150,584 and 6,114,598, 6,075,181 and 6,075,181 respectively, and 5,939.598 and Japanese Patent Numbers. 3 068 180B2, 3 068 5006 B2, and a 3 068 507 B2 European Patent No. EP 0 463 51 151 B1 International Patent Applications No. WO94/02602, 3 068 506 B2, and 3 068 507 B2 and European Patent No., EP 0 463 151 B1 and International Patent Applications No.
Humanization, chimerization, and display of appropriate libraries are all methods that can be used to produce antibodies with lower immunogenicity. You will see that murine antibodies and antibodies from other species can also be humanized or primatized using well-known techniques. Winter and Harris Immunol Today, 14:43 46 (1993), and Wright et. al. Crit. Review in Immunol. 12125-168 (1992). Recombinant DNA techniques can be used to engineer the antibody of interest. This allows for the substitution of the CH1, CH2, and CH3 hinge domains and/or the framework with the human sequence. (See WO 922102190 and U.S. Pat. Nos. 5,530,101; 5,585,089, 5,693,761 and 5,693,792 respectively; 5,714,350, and 5,777,085). Liu et. al. also know that Ig cDNA can be used to construct chimeric immunoglobulin gene (Chimeric Immunglobulin Gene Construction). P.N.A.S. 84:3439 (1987), and J. Immunol. 139:3521 (1987)). cDNA is made from the mRNA obtained from a hybridoma cell or another cell that produces the antibody. The polymerase chain reaction with specific primers may be used to amplify the cDNA of interest (U.S. Patent. Nos. Nos. A library can also be made and screened in order to identify the sequence of interest. The DNA sequence that encodes the variable region of an antibody is then fused with human constant region sequences. Kabat et al. may provide the sequences of human constant region genes. (1991) Sequences for Proteins of Immunological Interest, N.I.H. publication no. 91-3242. There are many clones that contain human C region genes. The desired effecter functions (e.g. complement fixation or activity in antibody-dependent cell cytotoxicity) will guide the choice of an isotype. IgG1 or IgG2 are the preferred isotypes. One of the constant human light chains, kappa, or lambda regions, can be used. Conventional methods are used to express the chimeric, humanized anti-body.
Antibody fragments such as Fv and F(ab) may be prepared. )2 or Fab can be prepared by cleaving the intact protein, e.g. by protease, chemical cleavage, or by other methods. A truncated gene can also be created. A chimeric gene that encodes a portion F(ab) could be an example. The ‘2 fragment would contain DNA sequences that encode the CH1 domain, hinge region and H chain. A translational stop codon will then be used to produce the truncated molecules.
“Consensus sequences from H and L J may be used for designing oligonucleotides to use as primers for inserting useful restriction sites in the J region for linkage of human C region segments to V region segments. Site directed mutation can be used to modify C region cDNA and place a restriction site in the same position in the human sequence.
“Expression vectors can be plasmids or retroviruses as well as YACs and EBV derived episomes. Convenient vectors encode a functionally complete human CH/CL immunoglobulin sequence. They also have appropriate restriction sites that allow for easy inserting and expression of any VH/VL sequence. Splicing occurs in such vectors between the splice donor sites in the J region and the site that precedes the human C region. It also takes place at the splice areas within the human CH exons. The native chromosomal sites that are downstream of the coding areas are where polyadenylation occurs and transcription termination takes place. The chimeric antibody can be linked to any strong promoter (e.g. retroviral LTRs or SV-40 early promoters) (Okayama et.al. Mol. Cell. Bio. 3:280 (1983)), Rous sarcoma virus LTR (Gorman et al. P.N.A.S. P.N.A.S. Cell 41:885 (85). Native Ig promoters, and other similar substances, may also be used, it will be appreciated.”
These techniques allow for the generation of antibodies to CD47-expressing cells, soluble CD47 forms, epitopes and peptides, as well as expression libraries (see e.g. U.S. Pat. No. No.
“The CD47 antibodies can be expressed using a vector that contains a DNA segment encoded for the single chain antibody. These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene gun, catheters, etc. Chemical conjugates, such as those described in WO 93/4701, are considered vectors. They have a targeting moiety (e.g. a ligand for a cell surface receptor), and an nucleic acid binding moiety. polylysine), viral vector (e.g. A DNA or RNA viral vector, fusion proteins such that described in PCT/US95/02140 and WO 95/22618. This is a fusion-protein containing a target moiety. An antibody that is specific for a target cells and a nucleic acids binding moiety (e.g. a protamine), plasmids, phage, etc. You can have chromosomal vectors, non-chromosomal vectors, or synthetic ones.
“Preferred vectors are viral vectors and fusion proteins. Retroviral vectors are moloney murine virus. Prefer DNA viral vectors. These vectors include orthopox and avipox vectors as well as herpesvirus vectors like the herpes simplex I virus vector (HSV). (see Geller A. I. et.al., J. Neurochem 64:487 (1995); Lim F., et.al. in DNA Cloning. Mammalian Systems. D. Glover Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I. et al., Proc Natl. Acad. Sci. : U.S.A. 90:7603 (1993); Geller, A. I., et al., Proc Natl. Acad. Sci. USA 87:1149 (1990), Adenovirus Vectors (see LeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat. Genet. 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995); Yang, et. al., J. Virol. Genet. 8:148 (1994).”
“Pox virus vectors introduce the gene to the cells’ cytoplasm. The vectors of Avipox virus cause a very short-term expression of the nucleic acids. For introducing the nucleic acids into neural cells, vectors such as adenovirus vectors or adenoassociated virus vectors (HSV), are preferable. Adenovirus vectors have a shorter expression time (approximately 2 months) than the adenovirus vectors (approximately 4 months), which is in turn shorter than HSV vectors. The condition being treated and the target cell will determine the vector that is used. Standard techniques can be used to introduce the virus, e.g. Transfection, transduction, or transformation can be used to introduce the gene. There are many ways to transfer genes, including naked DNA, CaPO4 precipitation and DEAE dextran.
The vector can be used to target any cell. Stereotaxic injection, for example, can be used to guide the vectors (e.g. adenovirus, HSV) to a desired location. Additionally, the particles can be delivered by intracerebroventricular (icy) infusion using a minipump infusion system, such as a SynchroMed Infusion System. Convection, a method that relies on bulk flow, is also effective in delivering large molecules to larger areas of the brain. It may also be helpful in delivering the vector to the targeted cell. (See Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al., Am. J. Physiol. 266:292-305 (1994)). You can also use catheters, intravenous parenteral, intraperitoneal, subcutaneous injections and oral routes of administration.
These vectors can be used for large amounts of antibodies that can then be used in many different ways. You can detect CD47 in samples, for example. You can also use the antibody to try and bind to or disrupt CD47-, CD47/SIRPalpha-mediated signaling, and CD47/SIRPalpha-mediated interaction.
“Techniques are possible to be modified for the production single-chain antibodies specific for an antigenic protein (see, e.g. U.S. Pat. No. 4,946,778). Methods can also be adapted to construct Fab expression libraries (see e.g. Huse, et. al. 1989 Science 246; 1275-1281). This allows for rapid identification of monoclonal Fab Fab fragments that have the desired specificity for a particular protein, derivative, fragment, analog, or homolog. Techniques for producing antibody fragments that are compatible with a protein antigen can be used in the art. These include: (i) an F? Pepsin digestion produces an F(ab)2fragment; (ii), an F(ab)2fragment that is reduced in disulfide bridges from an F? )2 fragment; iii) an Fab fraction generated by treating the antibody molecule using papain and a reducing agents and (iv). Fv fragments. There are many variations possible to the above-described embodiments, including Fv, Fab, Fab and Fab? F(ab) F(ab)
“Fc Modifications”
It is possible to modify the antibody described in this article with regard to effector function to improve, e.g. the effectiveness of the antibody when treating disorders and diseases associated with aberrant CD47 signalsing. Because CD47 expression is widespread, mutations can be introduced to the Fc region of an antibody to suppress effector function and decrease the chance of normal cell-killing.
“In some embodiments, this antibody is an IgG type. Some embodiments have the constant region of antibody as human IgG1 isotype. This sequence contains the amino acids SEQ ID NO. 1.
Summary for “CD47 Antibodies, Methods, and Uses”
“The protein cluster for differentiation 47 (CD47), also known by integrin associated protein IAP, ovarian cancer antibody OA3, and Rh related antigen, is a universally expressed member of the cell surface pentaspan transmembrane Ig superfamily. CD47 interacts on macrophages with SIRP Alpha (signal-regulatory proteins alpha), and thus dampens phagocytosis. This pathway is co-opted by cancer cells, which evade phagocytosis, and promotes survival of these cells (Jaiswal S. et al, Trends In Immunol 31: 212-219 2010, 2010). This mechanism is new and can be used therapeutically to target CD47 in many cancers.
CD47 expression is associated with poorer clinical outcomes in a variety of malignancies, including Non-Hodgkin lymphoma and acute lymphocytic lymphoma (NHL), acute myelogenous (AML), ovarian carcinoma, glioma and glioblastoma. CD47 has also been shown to be a cancer stem-cell marker in leukemias as well as solid tumors (Jaiswal et.al., 2009 Cell; 138(2): 271?85; Chan et.al., 2009 Proc Natl Acad Sci USA; 106(33),: 14016-21; Chan and colleagues, 2010 Curr Opin Urrol, 20(5); 393-7; Majeti R et.al., 2011 Oncogene; 30(9): 1009-19; 9: 1009-19; 1009-19
However, CD47 antibodies have been shown to cause platelet aggregation as well as hemagglutination in red blood cells. Homotypic interactions are when two CD47-expressing cells become homotypic and cause them to clump or aggregate when they are treated with a bivalent CD47 binding agent. MABL, a CD47 antibody (full IgG, or F(ab)), can cause hemagglutination of CD47-expressing cells. The hemagglutination in erythrocytes caused by a CD47 antibody, MABL (a full IgG or F(ab)), was only mitigated when MABL was made into an scFv/bivalent scFv. (See e.g., Uno S, Kinoshita Y, Azuma Y, et al., 2007 Oncol Rep, 17(5):1189-94). Similar to the CD47 antibody B6H12, it has been shown that direct aggregation of platelets of subjects with certain polymormisphms of Fc receptor, Fc.RII (Dorahy, Thorne, Fecondo, J V, and Burns, G F, 1997 Journal of Biol. Chem. 272:1323-1330). This is why therapeutically targeting CD47 with CD47 antibodies can be limited by platelet aggregation or hemagglutination.
“Here are antibodies that specifically bind CD47 and inhibit CD47’s interaction with SIRP alpha. They also have no significant platelet aggregation activitiy.”
“In certain embodiments, the antibody binds specifically to CD47 by interfacing with CD47 (SEQID NO: 21 excluding signal sequence) amino acids residues: Q1, N27, E29 and E97. Some embodiments interact with CD47 (SEQID NO: 21 excluding signal sequence). The antibody’s amino acids residues are Y37, K39 and R45. Some embodiments interact with CD47 (SEQID NO: 21 excluding signal sequence). The antibody reacts with the amino acid residues E35 and K39, Y37. The antibody may interact with CD47 (SEQID NO: 21 excluding signal sequence) amino acids residues E29 and Q31, T34, D35, V36.
“Another aspect of the described embodiments features an antibody that specifically binds human or cyno CD47 and comprises a variable heavy chain from SEQ ID Nos: 4-6. Optionally, the antibody may contain a variable light (VL), chain region from SEQ ID Nos. 7 and 8. The antibody may contain a VH region from SEQID NOS: 4 to 6 and a VL region from SEQID NOS: 7 to 8. Other aspects of the antibody include a VH region consisting of SEQID NO: 4 or 5 and a VL region consisting of SEQID NO: 7. Another aspect of the antibody is that it has a VH region consisting of SEQID NO: 6 and a VL region consisting of SEQID NO: 8.
“In certain embodiments, the CD47 antibodies comprises a sequence of VH complementarity-determining region 1 (CDR1) set forth by SEQID NO. 9 or 10, a sequence of VH CDR2 set forth by SEQID NO. 11 or 12, a sequence VH CDR2 set forth SEQID NO. 13 or 14, a sequence VH H CDR1 set forth SEQID NO. 15 or 16, a sequence VL L CDR2 set forth SEQID NO. 17 or 18 and a sequence and a sequence in either 19 or 20. The CD47 antibody, for example, includes a VH CDR1 (SEQ ID No: 9), a SEQ CDR2 (SEQ ID NOT: 11), a SEQ CDR3 (SEQ ID ID NO. 13), a SEQ CDR3 (SEQ ID ID NO. 14), a SEQ CDR1 (1 or 2) sequence in SEQID NO. 15, a SEQ I NO. 13, and a SEQID NO. 15 VL CDR2 (2 or 17) and a CDR3 (3L CDR3) sequence in the 19 or 20). Another example is the CD47 antibody, which includes a sequence of VH CDR1 in SEQID NO: 10, a sequence of VH CDR2 in SEQID NO: 12, and a sequence VH CDR3 in SEQID NO: 13, a sequence VH CDR2 in SEQID NO: 14, and a sequence VL CDR3 in SEQID NO: 17, a sequence VL CDR3 in SEQID NO: 19, and a sequence VL CDR3 set forthin SEQID NO: 20, a sequence in SEQUAKE EQ ID PARTIAL
“Some embodiments of the antibodies bind CD47 in a location where a VH and VL chains have more extensive contacts with CD47 than they do with CD47. In this position, the VH chain is near the membrane of CD47-expressing cells, and the VL chain of antibody blocks a SIRPalpha binding site on CD47.
“Some embodiments of the antibodies lack significant hemagglutination activities. Some embodiments have a platelet-aggregation activity that is less than 10% of what would be observed in the absence or presence of the antibody. Some embodiments of the antibody are chimeric, humanized or fully human. The antibody can bind to human CD47 and cynomolgus CD47 in some embodiments. The antibody may promote macrophage-mediated destruction of CD47-expressing cells. Some embodiments contain an IgG type selected from IgG1 and IgG2 IgG.
“Described herein is also a pharmaceutical composition that includes an antibody described herein, and a pharmaceutical-acceptable carrier. Kits can include the described antibodies. These include polynucleotides that encode the antibodies, as well as vectors for the cells and cells suitable to express the polynucleotides.
“Another embodiment consists of methods for relieving a symptom associated with cancer or another neoplastic condition. The subject is administered one or more of the antibodies described herein. However, the antibody has no significant platelet aggregation activity. The amount of antibody administered is sufficient to relieve the subject’s symptoms. The subject may be a human in some embodiments. Some embodiments of the antibody are chimeric, humanized or fully human. The antibody may inhibit CD47’s interaction with SIRP alpha in some embodiments. The antibody may contain an IgG type selected from the IgG1 and IgG2 groups. Some embodiments include chemotherapy.
“Some characteristics of antibodies are described herein:
“Specific binding to human CD47 or cyno CD47”
“Blocking CD47 interaction by SIRP alpha”
“Do not have significant human platelet accumulation activity.”
“Do not have significant Hemagglutination Activity.”
“Are capable of encouraging phagocytosis by tumor cells via macrophages.”
“Display potent anti-tumor activities in a mouse model for human cancers.”
“The antibodies discussed herein are of great value in the treatment and prevention of many types of cancer.”
“Many CD47 antibodies exist that block SIRP alpha. But, before the subject matter was discussed herein, full IgG CD47 antibody that blocked SIRP Alpha caused side effects of platelet accumulation and/or hemagglutination. This is an undesirable characteristic. While 2D3 and other antibodies do not cause hemagglutination they do not block SIRP Alpha. There were no CD47 antibodies that could block SIRP alpha in full IgG format without causing platelet or red blood cell clumping.
“In some embodiments, the IgG CD47 antibody described herein does not show significant platelet accumulation and hemagglutination. This increases the effectiveness of therapeutically targeting CD47 and preserves the ability to block the interaction between CD47 and SIRP alpha which promotes phagocytosis for CD47-expressing cells. The full IgG CD47 antibody described in the disclosure, e.g. C47B157-C47B161 or C47B222, do not exhibit significant cell agglutination activities. The CD47 antibodies discussed herein, for example, do not exhibit significant platelet aggregation or hemagglutination activities. The combination of the CD47 antibodies (e.g. C47B157 and C47B161 as well as derivatives thereof) is unique in that they can block SIRPalpha without significant platelet accumulation and hemagglutination activity.
“General”
“Those who are skilled in the art will recognize that the disclosures herein can be modified and varied beyond the ones specifically described. The present disclosure includes all variations and modifications. All of the steps, features and compositions, as well as all combinations, are included in the present disclosure.
The specific embodiments herein are not intended to limit the scope of the present disclosure. They are only meant to serve as an example. Functionally-equivalent products, compositions, and methods are clearly within the scope of the present disclosure.”
“The compositions and methods described in this document are made or performed without undue experimental use, except where indicated, of conventional molecular biology, microbiology technology, virology and peptide synthesis in solution, solid phase and pharmaceutical chemistry, medicinal and immunology. For the preparation, formulation, delivery, and treatment patients, standard techniques are used.
“Definitions”
“Since they are not defined otherwise, the scientific and technical terms that are used in connection to the present disclosure will have the meanings that are common to those with ordinary skill in art.”
“The following terms shall have the following meanings, except where otherwise noted, when they are used in accordance to the present disclosure:
“The term ‘and/or? (e.g.?X/or Y) is defined as: “X and Y” shall be understood as meaning either?X or Y? either?X and Y? and shall be considered to support both the literal or implied meanings.
“Unless otherwise stated or required by context, any reference to a single, composition of matter, group or number of steps, or group of compositions, shall be taken as encompassing one or more (i.e. One or more of these steps, compositions, of matter, groups or groups of steps, or both, of those compositions, of matter, shall be considered. As used herein, the singular forms of?a? and?an? The singular forms?a?,?an?? und?the? are used herein. Unless the context explicitly dictates otherwise, plural aspects are included. Referring to?a is an example. Refer to?an? for a reference to both a single and multiple. Includes a single and two or more; refer to?the? Includes a single as much as two or more; reference to?the?
“As used herein the terms?”CD47?”,?”integrin-associated protein IAP?,??ovarian cancer antigen OA3?, and?Rh-related antibody? are interchangeable. They can be interchangeably used.
“The terms’red blood cell(s),? and?erythrocyte(s)? “The terms?red blood cell(s)?” and?erythrocyte (s)? are interchangeable. They are interchangeable and can be used interchangeably in this article.
“Platelet aggregation” is the term used herein. The adhesion between activated plateslets results in the formation or clumps (or aggregates) of activated plateslets. As shown in the Examples, platelet aggregation can be measured using an aggregometer. This measures the increase of transmittance of light when platelet aggregation takes place. Significant platelet aggregate activity? This occurs when there is a significant increase in light transmittance by at least 25% within 6 minutes following the addition of the antibody described herein. It is different from light transmittance before antibody addition.
“The term “agglutination” refers to cellular clumping. Cellular clumping is referred to as ‘agglutination’, while cellular hemoglobination is referred to as?hemagglutination. Refers to the clumping of a particular subset of cells (i.e. red blood cells). Hemagglutination can also be called a form of agglutination.
“In the hemagglutination test described herein, patterns are formed by erythrocytes that can be either buttons or? ?halos,? ?halos,? The term “significant hemagglutination activ? Refers to any halo pattern found in a well after the addition of the antibody described herein.
“Antibody” is used in this document. The term “antibody” refers to immunoglobulin molecules as well as immunologically active immunoglobulin (Ig), i.e. molecules that have an antigen binding site that specifically binds to (immunoreacts) with an antigen. By “specifically bind?” or?immunoreacts? or?directed against? This means that the antibody reacts with antigenic determinants of the desired antigen and doesn’t react with or bind at a lower affinity (Kd>106) to other polypeptides. Monoclonal, single-chain, Fab, F(ab), chimeric, and dAb (domain antibodies) are all examples of antibodies. )2 fragments and Fv, scFvs and an Fab expression collection.
The tetramer is the basic structural unit of an antibody. Each tetramer consists of two identical pairs polypeptide chains. Each pair has one?light? (approximately 25 kDa) & one?heavy?” Chain (between 50 and 70 kDa). Each chain’s amino-terminal section contains a variable area of around 100 to 110 amino acids that is primarily responsible in antigen recognition. Each chain’s carboxy-terminal section contains a region that is primarily responsible to effector function. The majority of antibody molecules that are obtained from people relate to the IgG, IgM and IgA classes. They differ by the nature of each heavy chain in the molecule. There are subclasses for certain classes, like IgG1, IgG2, or others. The light chain can also be called a kappa or a lambda in humans.
“Monoclonal antibody” is a term that refers to monoclonal antibodies. “Monoclonal antibody” (mAb) is a term that refers to an antibody population consisting of one molecular species. It is composed of a unique light-chain gene product and a unique heavier chain gene product. The monoclonal antibody’s complementarity determining areas (CDRs), are identical in all molecules. MAbs have an antigen binding area that can immunoreact with a specific epitope of antigen.
“Abdominal molecules obtained from human beings are generally of any of the following classes: IgG, IgM and IgA. They differ by the nature of the heavy chains in the molecule. There are subclasses for certain classes, like IgG1, IgG2, or others. The light chain can also be called a kappa or a lambda in humans.
“The term “antigen binding site”? or ?binding portion? This refers to the immunoglobulin molecule which participates in antigen-binding. The N-terminal variable (??) amino acid residues form the antigen binding site. Regions of the heavy (??H?) Regions of the heavy (?H?) and light chains (?L?) chains. Three highly divergent stretches are found within the V regions for the heavy and lighter chains. These are known as the?hypervariable areas? They are located between more stable flanking stretches, known as the?framework regions? or?FRs?. The term ‘FR?????? FR? refers to amino acids sequences that are found naturally between and adjacent to hypervariable regions of immunoglobulins. The three hypervariable areas of a light chain and three of the hypervariable regions in a heavy chain form an antigen-binding space. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as ?complementarity-determining regions,? CDRs. Each domain has an assigned amino acid according to the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health Bethesda Md.). (1987 and 1991), Chothia & Lesk (J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989).
“Epitope” is the term used herein. Any protein determinant that can be used to specificbind to an immunoglobulin, fragment thereof, or T-cell receptor. The term “epitope” is used. Any protein determinant that can be used to specifically bind to an immunoglobulin receptor or T-cell receptor is called an “epitope”. The epitopic determinants are usually composed of chemically active surface groups of molecules, such as sugar side chains or amino acids. They also have specific three-dimensional structural characteristics and specific charge characteristics. When the dissociation constant of an antibody is?1?M, it is considered to be able to bind antigens.
“Specifically Binds” is the term used herein. The non-covalent interactions between immunoglobulin molecules and antigens that are specific to them refer to these types of interactions. A dissociation constant (Kd), which is the value of an interaction between immunological binding molecules and antigens, can be used to express the strength or affinity of these interactions. A smaller Kd indicates greater affinity.
The immune binding properties of certain polypeptides can easily be quantified by using well-known methods. One method is to measure the rates of antigen-binding sites/antigen complex formation and dissociation. These rates are dependent on the concentrations and affinity of the interactions and the geometric parameters that equally affect the rate in both directions. The?on rate constant and the?off rate constant are therefore equal. The?on rate constant (kon) as well as the?offrate constant (koff) can be calculated. Calculating the concentrations and actual rates of dissociation and association can determine the value of koff. Nature 361:186-87 (1993). The ratio of Koff/kon allows the cancellation of all parameters that are not associated with affinity and is equal to Kd, the dissociation constant. (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). The present disclosure identifies an antibody that binds to CD47 when the equilibrium binding constant, (Kd), is?1?M as determined by various assays like radioligand binding assays or surface plasmon resonance, (SPR), flow cell cytometry binding assays or other similar assays.
“An ?isolated? An antibody is one that has been isolated from its environment and is able to be recovered. It is a component of its environment that contains contaminants. These components can include enzymes, hormones and other non-proteinaceous solutes. Preferable embodiments will purify the antibody (1) to greater 95% by weight of the antibody, most preferably more that 99%, (2) to a degree sufficient for at least 15 residues N-terminal and internal amino acids sequences by using a spinning cup sequenator or (3) to homogeneity via SDS-PAGE under reduced or nonreducing conditions using Coomassie or, preferably silver stain. Since at least one component will be absent from the antibody’s natural environment, isolated antibody refers to the antibody found in situ within recombinant cell. Normally, however, isolated antibodies will only be obtained by at least one purification step.
“Polypeptide” is a generic term that refers to native protein, fragments, or analogs of a polypeptide sequence. “Polypeptide” is used herein to describe native protein fragments or analogs of a particular polypeptide sequence. Thus, analogs and native protein fragments are species within the polypeptide geneus.
“The term ?naturally-occurring? “Naturally-occurring” is a term that refers to objects found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory or otherwise is naturally-occurring.”
“The term ‘oligonucleotide’ is used. The term?oligonucleotide? refers to both naturally occurring and modified nucleotides that are linked by non-naturally occurring and naturally occurring oligonucleotide links. The oligonucleotide is a subset of polynucleotides that generally has a length of 200 or less. Preferably, oligonucleotides have a length of 10-60 bases. Most preferably, they are between 12, 13, 14, 15, 16, 17, 18, 19, 20 or 40 bases. Most oligonucleotides have a single strand, which is useful for probing, but oligonucleotides can be double-stranded to aid in the construction of gene mutants. The oligonucleotides discussed herein can be either antisense or sense.
“Sequence identity” is a term that refers to the identification of a sequence. means that two polynucleotide or amino acid sequences are identical (i.e., on a nucleotide-by-nucleotide or residue-by-residue basis) over the comparison window. What is the percentage of sequence identity? It is calculated by comparing two sequences that are optimally aligned over a window. To determine the number matched positions, divide the number matched positions by all positions in the window (i.e. the window size) and multiply the result by 100. The term “substantial identity” is used herein. The term “substantial identity” is used herein to denote a characteristic of a polynucleotide sequence or amino acid sequence. This means that the sequence has at minimum 85 percent sequence identification, preferably between 90 and 95 percent sequence ID, more often at 99 percent sequence identitate, when compared to a reference sequencing over a comparison window consisting of at most 18 (6 amino acid) positions. A comparison window may also include deletions or additions totalling 20 percent or less over the comparison window. A subset of a larger sequence could be used as the reference sequence.
“The twenty standard amino acids and their abbreviations are used as such in this document. See Immunology?A Synthesis (2nd Ed., E. S. Golub & D. R. Gren), Sinauer Associates Sunderland? Mass. (1991)). (1991). Examples of unconventional amino acids include: 4 hydroxyproline, ?-carboxyglutamate, ?-N,N,N-trimethyllysine, ?-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ?-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). According to convention and standard usage, the left-hand direction of the polypeptide notation is the amino terminal direction. The right-hand direction corresponds with the carboxy-terminal directions.
“Similarly, except where specified otherwise, the left hand end of single-stranded mononucleotide sequencings is the 5. End The left-hand direction for double-stranded mononucleotide sequencings is called the 5? direction. Direction of 5? To 3? The transcription direction sequence regions are the sequences that add nascent transcripts to the DNA strand. They have the same sequence as the original RNA sequence and are 5? To the 5? The sequence regions at the end of the transcript of RNA are called?upstream sequences?. They are sequence regions on DNA strands with the same sequence as RNA. The 3? The?downstream sequences’ are at the end of the transcript of RNA. The term “substantial identity” is used to describe polypeptides. Two peptide sequences must share at least 80 percent of their sequence identity when they are optimally aligned using programs GAP and BESTFIT with default gap weights. Preferably, at least 90 percent sequence identification, more preferably at minimum 95 percent sequence identitate, and most preferably, at least 99 percent sequence ID.
“Preferably, the residue positions that are not identical differ using conservative amino acid substitutions.”
“?Conservative? “?Conservative?” refers to the interchangeability between residues with similar side chains. Glycine, alanine-valine, leucine and isoleucine are examples of amino acid side chains that have aliphatic side chain; serine, threonine, and threonine are examples of side chains with aliphatic sidechains; glycine and alanine are side chains that have aliphatic sidechains; serine, threonine and threonine are side chains that have amide-containing sidechainsine, arginine-containing sidechainsine-containing sidechainsine; and methionine and cysteine-containing sidechainsine-containing sidechainsine-isoleucine-valine, glutamic-alanine-alanine-alanine-alanine-alanine-isoleucine-isoleucine-valine-isoleucine-alanine-valine-isoleucine-arginine-leucine-alanine-e-tyleucine-alanine-leucine-sine-sine-rich-alanine-rich-sine-glutysine-sine-sine-sine-gluty-gluten-gluten-gluco-glutine-gluco-glutar-sine-valine-glutine-sine-glutine-it-le-ty-glutine-ty-g-g-isomic-gluten-ar-gluten-g-gluco-g-hecine-g-ty-g-le-ty-g-ty-g-ty-g-ty-g-ty-g-ty-g-ty-glucocine-g-glucocine-g-g-g-alanine-g-a-g-alanine-g-gluco-g-g-alanine-g-g-g-g-sine-ty-sine-alanine-alanine-glysine-ty-leucine-leucine-le-alanine-leucine-ty-sine-ale-ty-leucine-ty-leucine Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.”
“As discussed herein minor variations in amino acid sequences for antibodies or immunoglobulin molecular are considered to be included in the present disclosure. Provided that these variations in the sequence of amino acids maintain at least 75% and more preferably at minimum 80%, 90%, 95%, 95%, and most importantly at least 99 percent, the modifications in the amino acids sequences remain at least 75%. Particularly, conservative replacements of amino acids are possible. Conservative replacements are those which occur within a family that is related in their side chain. The genetically encoded amino acid families are: (1) aspartate, glutamate;(2) basic amino acids, lysine and arginine; (3) non-polar amino compounds are alanine (valine), leucine (isoleucine), proline, phenylalanine), methionine or tryptophan; (4) uncharged, polar amino acids include glycine. Hydrophilic amino acids are arginine and asparagine as well as glutamine, glutamines, glutamate. Histidine, lysine. serine, and the threonine. Cysteine, cysteine (isoleucine), leucine and methionine; proline, tryptophan; tyrosine, tyrosine, and valine are the hydrophobic amino acid families. The aliphatic-hydroxy families include serine, threonine, cysteine, isoleucine or leucine; (iii), asparagine (amide containing family); (iv) phenylalanine (tryptophan), and tyrosine (aromatic family). It is reasonable to assume that an isolated substitution of a leucine for an isoleucine, valine, aspartate with glutamate, or a threonine and serine with a threonine, or similar replacement of an amino acids with a structurally related one, will not have any major effect on binding or properties of the resulting molecules, particularly if it does not involve an amino-acid within a framework. Assaying the specific activity can easily determine whether an amino acid change leads to a functional protein. These assays are described in great detail. Those with ordinary skill can easily prepare fragments or analogs of antibodies and immunoglobulin molecules. The preferred amino- and carboxytermini of fragments and analogs are located near the boundaries of functional domains. Using public and proprietary sequence databases, the nucleotide sequence data and/or amino acids sequence data can help identify structural and functional domains. Computerized comparison methods are preferred to identify sequence motifs and predicted conformation domains in proteins with known structure and/or functions. Bowie et. al. have identified methods to identify protein sequences that are folded into a three-dimensional structure. Science 253(164) (1991). The examples above demonstrate that skilled art-workers can recognize sequence motifs as well as structural conformations. These may be used to create structural and functional domains according to the present disclosure.
“Preferred amino acids are those that (1) reduce susceptibility for proteolysis, (2) decrease susceptibility at oxidation and (3) alter binding affinity to form protein complexes. (4) alter bindingaffinities. (5) confer or modify other physicochemical, functional properties of analogs. Analogs may include muteins from a different sequence than the naturally occurring peptide sequence. One or more amino acid substitutions (preferably conservative ones) can be made in the naturally occurring sequence. Preferably, the substitution should occur in the part of the polypeptide that does not form intermolecular contacts. The structural characteristics of the parent sequence should not be significantly altered by a conservative amino acid substitution. For example, the replacement amino acid should not break the helix in the parent sequence or alter other secondary structures that make up the parent sequence. Art-recognized polypeptide secondary and tertiary structure examples are described in Proteins, Structures and Molecular Principles, Creighton, Ed., W. H. Freeman and Company, New York (1984); Introduction to Protein Structure, C. Branden and J. Tooze, Garland Publishing, New York, N.Y (1991); and Thornton et.al. Nature 354:105 (1991).
“The term agent? “Agent” can be used to refer to a chemical compound, mixture of chemical compounds, or extract made from biological material.
“As used herein the terms ‘label? “Label” or “labeled” are terms that can be used herein. Refers to incorporation or “labeled” The label or marker may also be therapeutic in certain cases. There are many methods for labeling glycoproteins and polypeptides. Some examples of labels for polypeptides are radioisotopes and radionuclides, including but not limited to: radioisotopes (e.g. 3H, 14C. 15N, 35S), 90Y, 99Tc. 111In. 125I. 131I), fluorescent labels (e.g. FITC. rhodamine. luciferase. alkalinephosphatase. luciferase. luciferase. To reduce steric hindrance, some labels may be attached to spacer arms of different lengths. What is the term “pharmaceutical drug”? The term “pharmaceutical agent or drug” is used herein to refer to any chemical composition or composition that can induce a desired therapeutic effect when administered to a patient.
“Antineoplastic agent” is a term that refers to agents that inhibit the development or progression of a neoplasm in a human. This term refers to agents that inhibit the development or progression (or both) of a neoplasm in a person, especially a malignant (cancerous), lesion such as a cancer, sarcoma or lymphoma. Antineoplastic agents often have the ability to inhibit metastasis.
“Other chemical terms are used herein according to the convention usage in art, as illustrated by The McGraw-Hill Dictionary of Chemical Terms. (Parker, S. Ed., McGraw-Hill San Francisco (1985 )).”).
“CD47 Antibodies”
“Monoclonal antibody described herein can bind CD47 and inhibit SIRP alpha binding to CD47. They also decrease CD47-SIRP beta-mediated signaling and promote phagocytosis. They also inhibit tumor growth and/or migrating. For example, the Examples cellular assay can be used to determine inhibition.
“Exemplary antibodies” include an antibody with a variable heavy chain (VH), selected from SEQID NOs: 4-6, and an antibody with a variable light chain (VL), selected from SEQID NOs 7 and 8. Exemplary antibodies are specifically those listed in Table 1.
“TABLE 1\nVariable heavy Variable light\nAntibody (VH) chain (VL) chain\nC47B157 SEQ ID NO: 4 SEQ ID NO: 7\nC47B161 SEQ ID NO: 5 SEQ ID NO: 7\nC47B222 SEQ ID NO: 6 SEQ ID NO: 8”
Below are highlighted the complementarity determining areas (CDRs), of the VH chain for CD47 antibodies. Some embodiments have the VH CDR1 amino acid sequence as DYNMH (SEQID NO: 9) and DYWIG (10 SEQ ID NO. 10). Some embodiments have the VH CDR2 amino acid sequence as DIYPYNGGTGYNQKFKG or IIYPGDSDTRYSPSFQG. (SEQID NO: 11). “In some embodiments, VH CDR3’s amino acid sequence is either GGWHAMDS (SEQID NO: 13), or VGRFASHQLDY(SEQ ID No: 14).
“In some embodiments the amino acid sequence for VL CDR1 may be RSRQSIVHTNRYTYLA, (SEQID NO: 15), or RASQSVNNRLA (16). Some embodiments have the VL CDR2 amino acid sequence as KVSNRFS (SEQID NO: 17), or WASTRAI(SEQ ID No: 18). “In some embodiments, VL CDR3’s amino acid sequence is either FQGSHVPYT or QQGASWPFT (SEQID NO: 20).
“This disclosure also includes antibodies that bind the same epitope to CD47 antibodies as those described herein. The antibodies described in this patent bind to the epitope that contains one or more amino acids of an exemplary human CD47. Q08722.1 (GI.1171879) is incorporated by reference. Underlined is the signal sequence (amino acid 1-18).
“SEQ?ID?NO:?21\nMWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNME\nAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKG\nDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNE\nNILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITV\nIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLT\nSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQ\nLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE\n\nIn some embodiments the antibodies interact with Q1, L3, N27, E29, E97, L101, T102, R103, and E104 of SEQ ID NO: 21. Some embodiments of the antibodies interact with K39, R45 and D46. Some embodiments of the antibodies interact with E35 and K39, 49, 46, 49, E51, A53. L54. K56. T58, V59. T99. L101. T102. Some embodiments of the antibodies interact with E29 and Q31, T34, E35. V36.
“Those who are skilled in the art will know that it is possible to determine without undue experimental whether an antibody has the same particularity as one described herein (e.g. C47B157 or C47B161 or an antibody with a variable heavy chains selected from SEQID NOs: 4-8 and a variable light chains selected from SEQ ID NOs 7 and 8 respectively) by determining if the former blocks CD47 binding. The antibody that is being tested may compete with the one described in this disclosure. This can be shown by a decrease of binding by the antibody.
An alternative method to test whether an antibody has the same specificity as the one described herein is to incubate it with soluble CD47 proteins (with which it is normally reactive) and then add the antibody to be tested to see if it is inhibited in its ability bind CD47. If the antibody is unable to bind CD47, then it will likely have the same or equivalent epitope specificity.
“Antibodies of The Present Disclosure”
“The ability of the antibodies described herein to modulate or block, inhibit, reduce and antagonize CD47- or CD47/SIRP-alpha-mediated signaling can be evaluated. These assessments could include the measurement of CD47- or CD47/SIRPalpha-mediated signaling when one or more of these antibodies are present. These assays may include competitive binding assays, or they can measure a biologic readout such as the ability to promote the phagocytosis by a CD47-expressing cell by a macrophage (as described in Example 7).
“Various methods are known in the art for the production monoclonal antibodies against CD47 or derivatives, fragments analogs homologs and orthologs thereof. (See, for instance, Antibodies A Laboratory Manual, Harlow D, and Lane D 1988, Cold Spring Harbor Laboratory Press Cold Spring Harbor, N.Y.; incorporated herein as reference). Fully human antibodies are those antibody molecules that contain the complete sequence of the light and heavy chains, as well as the CDRs. These antibodies are called ‘human antibodies’. These antibodies are also known as?human antibodies? herein. For example, human monoclonal antibodies can be prepared using the methods described in the Examples below. You can also prepare human monoclonal antibody using the trioma method; the human-B-cell hybridoma procedure (see Kozbor, and al., 1983 Immunol Now 4: 72); or the EBV hybridoma to create human monoclonal antibody (see Cole, and al., 1985 in: MONOCLONAL CANCER THERAPY, Alan R. Liss, Inc., pages. 77-96). Human monoclonal antibodies can be used and produced using human hybridomas (see Cote et al. 1983). Proc Natl Acad Sci USA 1980: 2026-2030). Or by in vitro transforming human B cells with Epstein Barr virus (see Cole, and al., 1985 In MONOCLONAL ANOBODYS AND CANCER THEAPY, Alan R. Liss, Inc., pages. 77-96).”
“Antibodies can be purified using well-known methods, such as affinitychromatography using protein A and protein G. These provide primarily the IgG portion of immune serum. The immunoaffinity chromatography method uses immunoaffinity columns to immobilize the immune specific antibody. D. Wilkinson discusses the purification of immunoglobulins in The Scientist, Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).”
“The monoclonal CD47 antibodies in this disclosure are monoclonal antibodies. Monoclonal antibodies are monoclonal antibodies that block, inhibit, reduce or neutralize CD47- or CD47/SIRP-mediated cell signaling. They can be generated by immunizing animals with membrane bound or soluble CD47 such as human CD47 or an immunogenic fraction, derivative, or variant thereof. The animal can also be immunized by cells transfected to express CD47. The antibodies can also be obtained by screening a library containing antigen binding domain or antibody sequences that bind to CD47. This library can be prepared in bacteriophage, as protein or peptide-fusions to a Bacteria coat protein. It is then expressed on the surface phage particles assembled with the encoding DNA sequences (i.e., the?phage displayed librarian?). The hybridomas resulting from myeloma/B-cell fusions are then tested for CD47 reactivity.
“Monoclonal antibody preparations can be made, for instance, by hybridoma methods such as those described in Kohler and Milstein Nature, 256,495 (1975). A hybridoma is when a mouse, hamster or other suitable host animal is immunized using an immunizing drug to produce antibodies that are specific to the immunizing agents. The lymphocytes may also be immunized in vitro.
“The immunizing agent will usually include the protein antigen, a portion thereof or a combination thereof. If cells of human origin are required, peripheral blood lymphocytes will be used. However, spleen cells and lymph node cells can also be used. To form a hybridoma, the lymphocytes are fused with an immortalized line of cells using a suitable glue agent such as polyethylene glycol (Goding Monoclonal Antibodies Principles and Practice Academic Press, 1986, pp. 59-103). Immortalized cells are often transformed mammalian cells, especially myeloma cell lines of rodent, bovine, and human origin. Most commonly, myeloma cells from mice or rats are used. Hybridoma cells can then be grown in suitable culture media that contains at least one substance that inhibits the growth or survival unfused immortalized cells. Hypoxanthine, aminopterin and thymidine are some examples of culture media that can be used to grow hybridomas. These substances are necessary to prevent HGPRT-deficient cells from growing.
“Preferred immortalized cells lines are those that fuse well, support stable high-level expression of antibody by selected antibody-producing cell types, and are sensitive in a medium like HAT medium. For the production of monoclonal antibody-producing cells, murine myeloma line is preferred. They can be purchased from the Salk Institute Cell Distribution Center in San Diego, Calif. or the American Type Culture Collection in Manassas. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63)).”
The culture medium in which hybridoma cells were cultured can be tested for monoclonal antigen-specific antibodies. The binding specificity of monoclonal antibody produced by hybridoma cells can be determined either by immunoprecipitation (RIA) or an in vitro binding test (ELISA). These techniques and assays have been developed. Scatchard analysis of Munson, Pollard, and Anal can be used to determine the binding affinity of monoclonal antibodies. Biochem., 107:220 (1980). It is also important to find monoclonal antibodies that are specific and have a high affinity for the target antigen in therapeutic applications.
After the desired hybridoma cells have been identified, the clones may be subcloned using limiting dilution protocols and grown according to standard methods. (See Goding. Monoclonal Antibodies Principles and Practice. Academic Press, 1986, pp. 59-103). Suitable media include Dulbecco’s Modified Eagle’s Medium or RPMI-1640 medium. Alternately, hybridoma cells may be grown in vivo in mammal as ascites.
“The monoclonal antibody subclones secreted can be isolated from the culture medium or ascites liquid by using conventional immunoglobulin purification processes such as protein A-Sepharose or hydroxylapatite Chromatography, gel electrophoresis or dialysis.
“Human Antibodies and Humanization of Antibodies.”
“The antibodies described in this document include either fully human antibodies or humanized antibody. These antibodies can be administered to people without creating an immune reaction by the person against the administered immunoglobulin.
“A CD47 antibody can be generated by phage-display techniques using only human sequences. These approaches are well-known in art, e.g. in U.S. Pat. No. No. 6,521,404 are herein incorporated by reference. This method uses a combinatorial library that contains random pairs of light- and heavy-chained phages. Another method is to produce a CD47 antibody by immunizing a non-human transgenic animal with human CD47 proteins. This approach renders some of the non-human endogenous heavy and/or light chain loci, as well as the kappa and kappa light chains, inoperable and prevents them from generating the genes that encode immunoglobulins to respond to an antigen. The animal has been transfected with at least one of the human heavy chain loci and at least one of the human light chain loci. In response to an administered antibody, the human loci are rearranged to encode genes for human variable regions. The xenomouse then produces fully human immunoglobulin-secreting B-cells upon immunization.
“Xenogenic non-human animals can be produced using a variety of techniques. See U.S. Pat. Nos. Nos. 6,075,181 & 6,150,584, which are hereby incorporated in their entirety by reference. This strategy was used in the creation of the first XenoMouse? These strains were published in 1994. See Green et al. Nature Genetics 7 (13-21) (1994), is herein incorporated by reference. U.S. Pat. Nos. 6,162,963, 6,150,584 and 6,114,598, 6,075,181 and 6,075,181 respectively, and 5,939.598 and Japanese Patent Numbers. 3 068 180B2, 3 068 5006 B2, and a 3 068 507 B2 European Patent No. EP 0 463 51 151 B1 International Patent Applications No. WO94/02602, 3 068 506 B2, and 3 068 507 B2 and European Patent No., EP 0 463 151 B1 and International Patent Applications No.
Humanization, chimerization, and display of appropriate libraries are all methods that can be used to produce antibodies with lower immunogenicity. You will see that murine antibodies and antibodies from other species can also be humanized or primatized using well-known techniques. Winter and Harris Immunol Today, 14:43 46 (1993), and Wright et. al. Crit. Review in Immunol. 12125-168 (1992). Recombinant DNA techniques can be used to engineer the antibody of interest. This allows for the substitution of the CH1, CH2, and CH3 hinge domains and/or the framework with the human sequence. (See WO 922102190 and U.S. Pat. Nos. 5,530,101; 5,585,089, 5,693,761 and 5,693,792 respectively; 5,714,350, and 5,777,085). Liu et. al. also know that Ig cDNA can be used to construct chimeric immunoglobulin gene (Chimeric Immunglobulin Gene Construction). P.N.A.S. 84:3439 (1987), and J. Immunol. 139:3521 (1987)). cDNA is made from the mRNA obtained from a hybridoma cell or another cell that produces the antibody. The polymerase chain reaction with specific primers may be used to amplify the cDNA of interest (U.S. Patent. Nos. Nos. A library can also be made and screened in order to identify the sequence of interest. The DNA sequence that encodes the variable region of an antibody is then fused with human constant region sequences. Kabat et al. may provide the sequences of human constant region genes. (1991) Sequences for Proteins of Immunological Interest, N.I.H. publication no. 91-3242. There are many clones that contain human C region genes. The desired effecter functions (e.g. complement fixation or activity in antibody-dependent cell cytotoxicity) will guide the choice of an isotype. IgG1 or IgG2 are the preferred isotypes. One of the constant human light chains, kappa, or lambda regions, can be used. Conventional methods are used to express the chimeric, humanized anti-body.
Antibody fragments such as Fv and F(ab) may be prepared. )2 or Fab can be prepared by cleaving the intact protein, e.g. by protease, chemical cleavage, or by other methods. A truncated gene can also be created. A chimeric gene that encodes a portion F(ab) could be an example. The ‘2 fragment would contain DNA sequences that encode the CH1 domain, hinge region and H chain. A translational stop codon will then be used to produce the truncated molecules.
“Consensus sequences from H and L J may be used for designing oligonucleotides to use as primers for inserting useful restriction sites in the J region for linkage of human C region segments to V region segments. Site directed mutation can be used to modify C region cDNA and place a restriction site in the same position in the human sequence.
“Expression vectors can be plasmids or retroviruses as well as YACs and EBV derived episomes. Convenient vectors encode a functionally complete human CH/CL immunoglobulin sequence. They also have appropriate restriction sites that allow for easy inserting and expression of any VH/VL sequence. Splicing occurs in such vectors between the splice donor sites in the J region and the site that precedes the human C region. It also takes place at the splice areas within the human CH exons. The native chromosomal sites that are downstream of the coding areas are where polyadenylation occurs and transcription termination takes place. The chimeric antibody can be linked to any strong promoter (e.g. retroviral LTRs or SV-40 early promoters) (Okayama et.al. Mol. Cell. Bio. 3:280 (1983)), Rous sarcoma virus LTR (Gorman et al. P.N.A.S. P.N.A.S. Cell 41:885 (85). Native Ig promoters, and other similar substances, may also be used, it will be appreciated.”
These techniques allow for the generation of antibodies to CD47-expressing cells, soluble CD47 forms, epitopes and peptides, as well as expression libraries (see e.g. U.S. Pat. No. No.
“The CD47 antibodies can be expressed using a vector that contains a DNA segment encoded for the single chain antibody. These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene gun, catheters, etc. Chemical conjugates, such as those described in WO 93/4701, are considered vectors. They have a targeting moiety (e.g. a ligand for a cell surface receptor), and an nucleic acid binding moiety. polylysine), viral vector (e.g. A DNA or RNA viral vector, fusion proteins such that described in PCT/US95/02140 and WO 95/22618. This is a fusion-protein containing a target moiety. An antibody that is specific for a target cells and a nucleic acids binding moiety (e.g. a protamine), plasmids, phage, etc. You can have chromosomal vectors, non-chromosomal vectors, or synthetic ones.
“Preferred vectors are viral vectors and fusion proteins. Retroviral vectors are moloney murine virus. Prefer DNA viral vectors. These vectors include orthopox and avipox vectors as well as herpesvirus vectors like the herpes simplex I virus vector (HSV). (see Geller A. I. et.al., J. Neurochem 64:487 (1995); Lim F., et.al. in DNA Cloning. Mammalian Systems. D. Glover Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I. et al., Proc Natl. Acad. Sci. : U.S.A. 90:7603 (1993); Geller, A. I., et al., Proc Natl. Acad. Sci. USA 87:1149 (1990), Adenovirus Vectors (see LeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat. Genet. 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995); Yang, et. al., J. Virol. Genet. 8:148 (1994).”
“Pox virus vectors introduce the gene to the cells’ cytoplasm. The vectors of Avipox virus cause a very short-term expression of the nucleic acids. For introducing the nucleic acids into neural cells, vectors such as adenovirus vectors or adenoassociated virus vectors (HSV), are preferable. Adenovirus vectors have a shorter expression time (approximately 2 months) than the adenovirus vectors (approximately 4 months), which is in turn shorter than HSV vectors. The condition being treated and the target cell will determine the vector that is used. Standard techniques can be used to introduce the virus, e.g. Transfection, transduction, or transformation can be used to introduce the gene. There are many ways to transfer genes, including naked DNA, CaPO4 precipitation and DEAE dextran.
The vector can be used to target any cell. Stereotaxic injection, for example, can be used to guide the vectors (e.g. adenovirus, HSV) to a desired location. Additionally, the particles can be delivered by intracerebroventricular (icy) infusion using a minipump infusion system, such as a SynchroMed Infusion System. Convection, a method that relies on bulk flow, is also effective in delivering large molecules to larger areas of the brain. It may also be helpful in delivering the vector to the targeted cell. (See Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al., Am. J. Physiol. 266:292-305 (1994)). You can also use catheters, intravenous parenteral, intraperitoneal, subcutaneous injections and oral routes of administration.
These vectors can be used for large amounts of antibodies that can then be used in many different ways. You can detect CD47 in samples, for example. You can also use the antibody to try and bind to or disrupt CD47-, CD47/SIRPalpha-mediated signaling, and CD47/SIRPalpha-mediated interaction.
“Techniques are possible to be modified for the production single-chain antibodies specific for an antigenic protein (see, e.g. U.S. Pat. No. 4,946,778). Methods can also be adapted to construct Fab expression libraries (see e.g. Huse, et. al. 1989 Science 246; 1275-1281). This allows for rapid identification of monoclonal Fab Fab fragments that have the desired specificity for a particular protein, derivative, fragment, analog, or homolog. Techniques for producing antibody fragments that are compatible with a protein antigen can be used in the art. These include: (i) an F? Pepsin digestion produces an F(ab)2fragment; (ii), an F(ab)2fragment that is reduced in disulfide bridges from an F? )2 fragment; iii) an Fab fraction generated by treating the antibody molecule using papain and a reducing agents and (iv). Fv fragments. There are many variations possible to the above-described embodiments, including Fv, Fab, Fab and Fab? F(ab) F(ab)
“Fc Modifications”
It is possible to modify the antibody described in this article with regard to effector function to improve, e.g. the effectiveness of the antibody when treating disorders and diseases associated with aberrant CD47 signalsing. Because CD47 expression is widespread, mutations can be introduced to the Fc region of an antibody to suppress effector function and decrease the chance of normal cell-killing.
“In some embodiments, this antibody is an IgG type. Some embodiments have the constant region of antibody as human IgG1 isotype. This sequence contains the amino acids SEQ ID NO. 1.
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