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Delivery of pharmaceutical agents via the human insulin receptorRelated Patent Categories: Chemistry: Natural Resins Or Derivatives; Peptides Or Proteins; Lignins Or Reaction Products Thereof, Proteins, I.e., More Than 100 Amino Acid Residues, Blood Proteins Or Globulins, E.g., Proteoglycans, Platelet Factor 4, Thyroglobulin, Thyroxine, Etc., Globulins, Immunoglobulin, Antibody, Or Fragment Thereof, Other Than Immunoglobulin Antibody, Or Fragment Thereof That Is Conjugated Or Absorbed, Chimeric, Mutated, Or Recombined Hybrid (e.g., Bifunctional, Bispecific, Rodent-human Chimeric, Single Chain, Rfv, Immunoglobulin Fusion Protein, Etc.)Delivery of pharmaceutical agents via the human insulin receptor description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20080051564, Delivery of pharmaceutical agents via the human insulin receptor. Brief Patent Description - Full Patent Description - Patent Application Claims
BACKGROUND OF THE INVENTION [0001] 1. Field of the Invention [0002] The present invention relates generally to the delivery of pharmaceutical agents from the blood stream to the human brain and other organs or tissues that express the human insulin receptor. More particularly, the present invention involves the development of "humanized" monoclonal antibodies (MAb) that may be attached to pharmaceutical agents to form compounds that are able to readily bind to the human insulin receptor (HIR). The compounds are able to cross the human blood brain barrier (BBB) by way of insulin receptors located on the brain capillary endothelium. Once across the BBB, the humanized monoclonal antibody/pharmaceutical agent compounds are also capable of undergoing receptor mediated endocytosis into brain cells via insulin receptors located on the brain cells. [0003] 2. Description of Related Art [0004] The publications and other reference materials referred to herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference. For convenience, the reference materials are identified by author and date and grouped in the appended bibliography. [0005] The BBB is a system-wide membrane barrier that prevents the brain uptake of circulating drugs, protein therapeutics, antisense drugs, and gene medicines. Drugs or genes can be delivered to the human brain for the treatment of serious brain disease either (a) by injecting the drug or gene directly into the brain, thus bypassing the BBB, or (b) by injecting the drug or gene into the bloodstream so that the drug or gene enters the brain via the transvascular route across the BBB. With intra-cerebral administration of the drug, it is necessary to drill a hole in the head and perform a procedure called craniotomy. In addition to being expensive and highly invasive, this craniotomy based drug delivery to the brain approach is ineffective, because the drug or gene is only delivered to a tiny volume of the brain at the tip of the injection needle. The only way the drug or gene can be distributed widely in the brain is the transvascular route following injection into the bloodstream. However this latter approach requires the ability to undergo transport across the BBB The BBB has proven to be a very difficult and stubborn barrier to traverse safely. [0006] Prior work has shown that drugs or gene medicines can be ferried across the BBB using molecular Trojan horses that bind to BBB receptor/transport systems. These Trojan horses may be modified proteins, endogenous peptides, or peptidomimetic monoclonal antibodies (MAb's). For example, HIR MAb 83-14 is a murine MAb that binds to the human insulin receptor (HIR). This binding triggers transport across the BBB of MAb 83-14 (Pardridge et al., 1995), and any drug or gene payload attached to the MAb (Wu et al., 1997). [0007] The use of molecular Trojan horses to ferry drugs or genes across the BBB is described in U.S. Pat. Nos. 4,801,575 and 6,372,250. The linking of drugs to MAb transport vectors is facilitated with use of avidin-biotin technology. In this approach, the drug or protein therapeutic is monobiotinylated and bound to a conjugate of the antibody vector and avidin or streptavidin. The use of avidin-biotin technology to facilitate linking of drugs to antibody-based transport vectors is described in U.S. Pat. No. 6,287,792. Fusion proteins have also been used where a drug is genetically fused to the MAb transport vector. [0008] HIRMAb 83-14 has been shown to rapidly undergo transport across the BBB of a living Rhesus monkey, and to bind avidly to isolated human brain capillaries, which are the anatomical substrate of the human BBB (see Pardridge et al., 1995). In either case, the activity of the HIRMAb 83-14 with respect to binding and transport at the primate or human BBB is more than 10-fold greater than the binding or transport of other peptidomimetic MAb's that may target other BBB receptors such as the transferrin receptor (Pardridge, 1997). To date, HIRMAb 83-14 is the most active BBB transport vector known (Pardridge, 1997). On this basis, the HIRMAb 83-14 has proven to be a very useful agent for the delivery of drugs to the primate brain in vivo, and would also be highly active for brain drug or gene delivery to the brain in humans. [0009] HIRMAb 83-14 cannot be used in humans because this mouse protein will be immunogenic. Genetically engineered forms of HIRMAb 83-14 might be used in humans in either the form of a chimeric antibody or a genetically engineered "humanized" HIRMAb. However, in order to perform the genetic engineering and production of either a chimeric or a humanized antibody, it is necessary to first clone the variable region of the antibody heavy chain (VH) and the variable region of the antibody light chain (VL). Following cloning of the VH and VL genes, the genes must be sequenced and the amino acid sequence deduced from the nucleotide sequence. With this amino acid sequence, using technologies known to those skilled in the art (Foote et al., 1992), it may be possible to perform humanization of the murine HIRMAb 83-14. However, HIRMAb 83-14 may lose biological activity following the humanization (Pichla et al. 1997). Therefore, it is uncertain as to whether the murine HIRMAb can be humanized with retention of biological activity. [0010] A chimeric form of the HIRMAb 83-14 has been genetically engineered, and the chimeric antibody binds to the HIR and is transported into the primate brain (Coloma et al., 2000). However, a chimeric antibody retains the entire mouse FR for both the VH and the VL, and because of this, chimeric antibodies are still immunogenic in humans (Bruggemann et al., 1989). In contrast to the chimeric antibody, a humanized antibody would use the human FR amino acid sequences for both the VH and the VL and retain only the murine amino acids for the 3 complementarity determining regions (CDRs) of the VH and 3 CDRs of the VL. Not all murine MAb's can be humanized, because there is a loss of biological activity when the murine FR's are replaced by human FR sequences (Pichla et al., 1997). The biological activity of the antibody can be restored by substituting back certain mouse FR amino acids (see U.S. Pat. No. 5,585,089). Nevertheless, even with FR amino acid back-substitution, certain antibodies cannot be humanized with retention of biological activity (Pichla et al., 1997). Therefore, there is no certainty that the murine HIRMAb 83-14 can be humanized even once the key murine CDR and FR amino acid sequences are known. SUMMARY OF THE INVENTION [0011] In accordance with the present invention, it was discovered that the murine HIRMAb 83-14 antibody can be humanized to provide a biologically active humanized insulin receptor (HIR) antibody that may be used in combination with drugs and diagnostic agents to treat human beings in vivo. The HIR antibody may be conjugated to the drug or diagnostic agent using avidin-biotin conjugation or the HIR antibody/drug combination may be prepared as a fusion protein using genetic engineering techniques. The HIR antibody is especially well suited for delivering neuropharmaceutical agents to the human brain across the BBB. The humanized character of the HIR antibody significantly reduces immunogenic reactions in humans. [0012] The humanized murine antibody of the present invention is capable of binding to the HIR and includes a heavy chain (HC) of amino acids and a light chain (LC) of amino acids which both include variable and constant regions. The variable regions of the HC and LC include complementarity determining regions (CDRs) that are interspersed between framework regions (FRs). [0013] The HC includes a first CDR located at the amino end of the variable region, a third CDR located at the carboxyl end of the HC variable region and a second CDR located between said first and third CDRs. The amino acid sequences for the first CDR, the second CDR, and the third CDR are SEQ. ID. NOS. 31, 33 and 35, respectively, and combined equivalents thereof. The HC framework regions include a first FR located adjacent to the amino end of the first CDR, a second FR located between said first and second CDRs, a third FR located between said second and third CDRs and a fourth FR located adjacent to the carboxyl end of said third CDR. In accordance with the present invention, the four FRs of the HC are humanized such that the overall antibody retains biological activity with respect to the HIR and is not immunogenic in humans. [0014] The LC also includes a first CDR located at the amino end of the variable region, a third CDR located at the carboxyl end of the variable region and a second CDR located between said first and third CDRs. The amino acid sequences for the first CDR, the second CDR, and the third CDR are SEQ. ID. NOS. 38, 40, and 42, respectively, and combined equivalents thereof. The LC framework regions include a first FR located adjacent to the amino end of said first CDR, a second FR located between said first and second CDRs, a third FR located between said second and third CDRs and a fourth FR located adjacent to the carboxyl end of said third CDR. Pursuant to the present invention, the four FRs of the LC are humanized such that the overall antibody retains biological activity with respect to the HIR and has minimal immunogenicity in humans. [0015] The constant regions of the murine antibody are also modified to minimize immunogenicity in humans. The murine HC constant region is replaced with the HC constant region from a human immunoglobulin such as IgG1. The murine LC constant region is replaced with a constant region from the LC of a human immunoglobulin such as a kappa (.kappa.) LC constant region. Replacement of the murine HC and LC constant regions with human constant regions was found to not adversely affect the biological activity of the humanized antibody with respect to HIR binding. [0016] The present invention not only covers the humanized murine antibodies themselves, but also covers pharmaceutical compositions that are composed of the humanized antibody linked to a drug or diagnostic agent. The humanized antibody is effective in delivering the drug or diagnostic agent to the HIR in vivo to provide transport across the BBB and/or endocytosis into cells via the HIR. The compositions are especially well suited for intra venous (iv) injection into humans for delivery of neuropharmaceutical agents to the brain. [0017] The above discussed and many other features and attendant advantages of the present invention will become better understood by reference to the detailed description when taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS [0018] FIG. 1 shows the nucleotide sequence for the murine VH (SEQ. ID. NO. 1) and murine VL (SEQ. ID. NO. 2) and deduced amino acid sequence of the murine VH (SEQ. ID. NO. 3) and the murine VL (SEQ. ID. NO. 4), which shows the 3 framework (FR) regions and the 4 complementarity determining regions (CDRs) of both the heavy chain (HC) and the light chain (LC) of the 83-14 murine HIRMAb. The amino acids denoted by an asterisk (*) were confirmed by amino acid sequencing of either the intact murine LC or tryptic peptides of the intact murine HC; for amino acid sequencing, the intact murine HC or LC were purified from gels following purification of the intact murine IgG from the hybridoma conditioned medium. [0019] FIGS. 2A and 2B graphically show the results of a radio-receptor assay on isolated human brain capillaries that were obtained with a mechanical homogenization procedure from human autopsy brain. These capillaries were incubated with [.sup.25I]-labeled chimeric HIRMAb (Coloma et al., 2000) (FIG. 2A) or [.sup.125I]-version 5 humanized HIRMAb (FIG. 2B). The data show that both antibodies bind equally well to human brain capillaries, which form the anatomical basis of the BBB in humans. [0020] FIG. 3 shows the brain scan of a Rhesus monkey treated with a humanized monoclonal antibody in accordance with the present invention. The [.sup.125I]-labeled version 5 HIRMAb was injected intravenously in an anesthetized rhesus monkey, and the animal was euthanized 120 minutes later. The brain was rapidly removed and cut into coronal hemispheric slabs, which were immediately frozen. Cryostat sections (20 .mu.m) were cut and exposed to x-ray film. The film was scanned to yield the image shown in FIG. 3. This image shows the clear demarcations between the gray matter and white matter of the primate brain. Owing to the higher vascular density in gray matter, there is a greater uptake of the humanized HIRMAb, relative to white matter. [0021] FIG. 4 shows a comparison of the amino acid sequence for the 3 FRs and 3 CDRs of both the heavy chain and the light chain for the following: (a) the version 5 humanized HIRMAb, (v) the original murine 83-14 HIRMAb, and (c) the VH of the B43 human IgG or the VL of the REI human IgG. Continue reading about Delivery of pharmaceutical agents via the human insulin receptor... Full patent description for Delivery of pharmaceutical agents via the human insulin receptor Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Delivery of pharmaceutical agents via the human insulin receptor patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. 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