ANTICARBOXYMETHYL LYSINE ANTIBODIES AND ULTRASOUND FOR REMOVING AGE-MODIFIED CELLS
20210253739 · 2021-08-19
Inventors
Cpc classification
C07K2317/24
CHEMISTRY; METALLURGY
A61K47/6925
HUMAN NECESSITIES
A61K47/6843
HUMAN NECESSITIES
C07K2317/92
CHEMISTRY; METALLURGY
C07K16/44
CHEMISTRY; METALLURGY
International classification
C07K16/44
CHEMISTRY; METALLURGY
A61K47/68
HUMAN NECESSITIES
A61K47/69
HUMAN NECESSITIES
Abstract
A method of killing AGE-modified cells comprises applying ultrasound to a subject and administering to the subject a composition comprising an anti-AGE antibody. The applying of ultrasound may occur before or after the administering of the anti-AGE antibody. The AGE-modified cells may be in a restricted site.
Claims
1. A method of killing AGE-modified cells, comprising: applying ultrasound to a subject; and administering to the subject a composition comprising an anti-AGE antibody.
2. (canceled)
3. A method of treating an AGE disorder, comprising: applying ultrasound to a subject having an AGE disorder; and administering to the subject a composition comprising an anti-AGE antibody.
4. A method of treating a subject with a glioma, comprising: disrupting the blood-brain barrier of the subject; followed by administering to the subject a composition comprising an anti-AGE antibody.
5. A method of treating a subject with osteoarthritis, comprising: applying ultrasound to the affected joint; followed by administering to the subject a composition comprising an anti-AGE antibody, wherein the composition is administered intravenously.
6. The method of claim 3, wherein: the anti-AGE antibody, is conjugated to a microbubble.
7-10. (canceled)
11. The method of claim 1, wherein the administering of the anti-AGE antibody occurs before the applying of ultrasound.
12. The method of claim 1, wherein the AGE-modified cells are in a restricted site.
13. The method of claim 1, wherein the restricted site is selected from the group consisting of the brain, a joint, the eyes, the testes, and the prostate.
14-18. (canceled)
19. The method of claim 1, wherein the subject is selected from the group consisting of humans, goats, sheep, pigs, cows, horses, camels, alpacas, dogs and cats.
20. The method of claim 1, wherein the subject is a human.
21. The method of claim 1, wherein the anti-AGE antibody is non-immunogenic to a species selected from the group consisting of humans, cats, dogs, horses, camels, alpaca, cattle, sheep, pigs, and goats.
22-23. (canceled)
24. The method of claim 1, wherein the anti-AGE antibody binds an AGE-modified cell comprising at least one protein or peptide that exhibits AGE modifications selected from the group consisting of FFI, pyrraline, AFGP, ALI, carboxymethyllysine, carboxyethyllysine and pentosidine.
25. The method of claim 1, wherein the anti-AGE antibody binds a carboxymethyllysine-modified protein or peptide.
26. (canceled)
27. The method of claim 1, wherein the AGE-modified cell is a senescent cell.
28-34. (canceled)
35. The method of claim 1, wherein the antibody is conjugated to an agent that causes the destruction of AGE-modified cells.
36-37. (canceled)
38. The method of claim 1, wherein the antibody is monoclonal.
39. The method of claim 1, wherein the antibody is substantially non-immunogenic to humans.
40. The method of claim 1, wherein the antibody has a rate of dissociation (k.sub.d) of at most 9×10.sup.−3 sec.sup.−1.
41-43. (canceled)
44. The method of claim 3, wherein the AGE disorder comprises at least one AGE disorder selected from the group consisting of brain cancer, brain tumors, gliomas, meningiomas, pituitary adenomas and nerve sheath tumors, meningitis, brain/cerebral abscess, late-stage neurological trypanosomiasis (sleeping sickness), cerebral edema, prion diseases, encephalitis, rabies, arthritis/osteoarthritis, degenerative joint disease, synovitis, bursitis, prostate cancer, eye cancer, and eye disorders.
45. The method of claim 1, wherein the ultrasound is applied at a frequency of 0.5-10 MHz, an intensity of 0.05-5 W/cm.sup.2, and a pulse repetition frequency (PRF) of 0.05-10 kHz.
46-49. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWING
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[0044]
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DETAILED DESCRIPTION
[0050] It has been discovered that ultrasound can allow temporary access to restricted sites. Applying diagnostic ultrasound in combination with administration of microbubbles disrupts the blood-brain barrier (Zhao, B. et al., “Blood-brain barrier disruption induced by diagnostic ultrasound combined with microbubbles in mice”, Oncotarget, Vol. 9, No. 4, pp. 4897-4914 (2018)). Focused ultrasound applied in the presence of microbubbles disrupts the blood-brain barrier to allow delivery of the chemotherapy drugs 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU or carmustine) and doxorubicin to glioblastomas in rats (Liu, H-L. et al., “Blood-brain barrier disruption with focused ultrasound enhances delivery of chemotherapeutic drugs for glioblastoma treatment”, Radiology, Vol. 255, No. 2, pp. 415-425 (2010); Sun, T. et al., “Closed-loop control of targeted ultrasound drug delivery across the blood-brain/tumor barriers in a rat glioma model”, Proceedings of the National Academy of Sciences, pp. 1-10 (2017)). Ultrasound applied in the presence of microbubbles is able to disrupt the blood-brain barrier to allow single chain antibodies and the anti-EGFR monoclonal antibody trastuzumab (HERCEPTIN®) to access the brains of mice (Nisbet, R. M. et al., “Combined effects of scanning ultrasound and a tau-specific single chain antibody in a tau transgenic mouse model”, Brain, Vol. 140, pp. 1220-1230 (2017); Kinoshita, M. et al., “Noninvasive localized delivery of Herceptin to the mouse brain by MRI-guided focused ultrasound-induced blood-brain barrier disruption”, Proceedings of the National Academy of Sciences, Vol. 103, No. 31, pp. 11719-11723 (2006)). These studies suggest that a combination therapy including application of ultrasound and administration of antibodies is an effective treatment regimen for targeting cells and tissues in restricted sites.
[0051] A number of AGE disorders affect restricted sites. For example, recent studies have identified an association between cellular senescence and gliomas. Glioma cells become spontaneously senescent in vitro (Stoczynska-Fidelus, E., et al., “Spontaneous in vitro senescence of glioma cells confirmed by an antibody against IDH1.sup.R132H”, Anticancer Research, Vol. 34, pp. 2859-2868 (2014)). Similarly, advanced glycation end-products cause a dose-dependent increase of nitrite accumulation in glioma cells in vitro, which results in increased nitric oxide release and a corresponding increase in oxidative damage (Lin, C-H. et al., “Advanced glycosylation end products induce nitric oxide synthase expression in C6 glioma cells involvement of a p38 MAP kinase-dependent mechanism”, Life Sciences, Vol. 69, pp. 2503-2515 (2001)). In addition, glioma invasiveness is accompanied by reduced cellular proliferative activity, a sign of senescence (Berens, M. E. et al., “‘. . . those left behind.’ Biology and oncology of invasive glioma cells”, Neoplasia, Vol. 1, No. 3, pp. 208-219 (1999)). By identification of a common link between cellular senescence and gliomas, the Present Application shows that antibodies that bind to advanced glycation end-products are an effective treatment for gliomas. Application of ultrasound may be used to disrupt the blood-brain barrier and permit anti-AGE antibodies to target and kill glioma cells.
[0052] The present invention uses a combination of ultrasound and anti-AGE antibodies to specifically target and kill cells expressing AGE-modified proteins or peptides (AGE-modified cells) in a restricted site. The ultrasound provides access to the restricted site, which then allows the anti-AGE antibodies to access AGE-modified cells within the restricted site. The ultrasound provides access to the restricted site with or without contrast media such as microbubbles.
[0053] The combination of ultrasound and anti-AGE antibodies may also be used to enhance the destruction of AGE-modified cells. Ultrasound increases antibody binding to a target antigen, which will improve the binding of anti-AGE antibodies to AGE-modified cells. Once bound, ultrasound also enhances cell killing through the antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism. In addition, AGE-modified cells that have bound to anti-AGE antibodies are more susceptible to destruction by ultrasound. The ability of ultrasound to interfere with inflammatory processes will also reduce inflammation, a key driver of cellular senescence. The combination of ultrasound and anti-AGE antibodies is synergistic and produces a greater than additive effect at killing AGE-modified cells, such as senescent cells. These enhanced cell-killing effects may be realized in any location of the body or in in vitro settings.
[0054] A method of killing AGE-modified cells includes applying ultrasound and administering an antibody that binds to a glycation end-product (anti-AGE antibody). The ultrasound may be applied before the antibodies are administered to facilitate access to a restricted site. Alternatively, the ultrasound may be applied after the antibodies are administered and/or during administration.
[0055] The AGE-modified cells may be found in any part of the body. Preferably, the AGE-modified cells are in a restricted site. Examples of restricted sites include the brain, joints, eyes, testes and prostate. Alternatively, the AGE-modified cells may be in an in vitro setting, such as a tissue culture or cell culture.
[0056] The ultrasound may be applied at a frequency of 20,000 Hz-15 MHz. Preferably, the ultrasound is applied at a frequency of 0.5-10 MHz, including 0.6 MHz, 0.7 MHz, 0.8 MHz, 0.9 MHz, 1.0 MHz, 1.5 MHz, 2.0 MHz, 2.5 MHz, 3.0 MHz, 3.5 MHz, 4.0 MHz, 4.5 MHz, 5.0 MHz, 5.1 MHz, 5.2 MHz, 5.3 MHz, 5.4 MHz, 5.5 MHz, 5.6 MHz, 5.7 MHz, 5.8 MHz, 5.9 MHz, 6.0 MHz, 6.1 MHz, 6.2 MHz, 6.3 MHz, 6.4 MHz, 6.5 MHz, 6.6 MHz, 6.7 MHz, 6.8 MHz, 6.9 MHz, 7.0 MHz, 7.5 MHz, 8.0 MHz, 8.5 MHz, 9.0 MHz and 9.5 MHz.
[0057] The ultrasound may be applied at an intensity of 0.01-5 W/cm.sup.2. Preferably, the ultrasound is applied at an intensity of 0.05-5 W/cm.sup.2, including 0.06 W/cm.sup.2, 0.07 W/cm.sup.2, 0.08 W/cm.sup.2, 0.09 W/cm.sup.2, 0.1 W/cm.sup.2, 0.2 W/cm.sup.2, 0.3 W/cm.sup.2, 0.4 W/cm.sup.2, 0.5 W/cm.sup.2, 0.6 W/cm.sup.2, 0.7 W/cm.sup.2, 0.8 W/cm.sup.2, 0.9 W/cm.sup.2, 1.0 W/cm.sup.2, 1.1 W/cm.sup.2, 1.2 W/cm.sup.2, 1.3 W/cm.sup.2, 1.4 W/cm.sup.2, 1.5 W/cm.sup.2, 1.6 W/cm.sup.2, 1.7 W/cm.sup.2, 1.8 W/cm.sup.2, 1.9 W/cm.sup.2, 2.0 W/cm.sup.2, 2.5 W/cm.sup.2, 3.0 W/cm.sup.2, 3.5 W/cm.sup.2, 4.0 W/cm.sup.2 and 4.5 W/cm.sup.2.
[0058] The ultrasound may be continuous or may be pulsed. Preferably, the ultrasound is pulsed ultrasound. Pulsed ultrasound may have a pulse repetition frequency (PRF) of 0.01-20 kHz. Preferably, the pulsed ultrasound has a PRF of 0.05-10 kHz, including 0.06 kHz, 0.07 kHz, 0.08 kHz, 0.09 kHz, 0.1 kHz, 0.2 kHz, 0.3 kHz, 0.4 kHz, 0.5 kHz, 1.0 kHz, 1.5 kHz, 2.0 kHz, 3.0 kHz, 3.5 kHz, 4.0 kHz, 4.5 kHz, 5.0 kHz, 5.5 kHz, 6.0 kHz, 6.5 kHz, 7.0 kHz, 7.5 kHz, 8.0 kHz, 8.5 kHz, 9.0 kHz and 9.5 kHz.
[0059] The ultrasound may be applied once or multiple times per day. For example, the ultrasound may be applied 1-100 times per day. The ultrasound may be applied at regular intervals, such as once per hour (24 times per day), or may be applied multiple times within a fixed time period, such as 60 times per hour. Less frequent administration is also possible, including once per week or once per month.
[0060] The ultrasound may be focused or unfocused. Focused ultrasound typically delivers higher intensities than unfocused ultrasound.
[0061] Other ultrasound parameters, such as wavelength, velocity, wave shape, duration of application, pulse duration, spatial pulse length and duty factor, may be varied to provide a specific therapeutic effect. Preferably, the ultrasound parameters are selected so as to target AGE-modified cells while sparing other cells.
[0062] Appropriate ultrasound parameters may be determined by simple experimentation. Cells may be isolated, such as in a sample from a patient or in a cell culture. Physical differences between cells, such as stiffening or changes in elasticity, allows target cells to be identified (see, for example, U.S. Pat. Nos. 6,067,859 and 7,751,057). Ultrasound may be applied to the cells until a desired outcome is achieved. The cells are observed before and after ultrasound application to determine the effect of the ultrasound. One or more ultrasound parameters may be varied and the ultrasound reapplied. The cells are then observed to determine the effect of the modified ultrasound parameters. This process may be repeated until a desired therapeutic effect is achieved. For example, AGE-modified cells may be isolated and ultrasound may be applied to determine the resonant harmonic frequency that will result in cell destruction. The frequency of the ultrasound may be gradually increased until the resonant harmonic frequency is determined. Determining appropriate ultrasound parameters is a simple process for a skilled artisan since ultrasound is a well-studied mechanical technique that produces predictable results.
[0063] Contrast media may optionally be administered prior to or simultaneously with application of the ultrasound. Preferred contrast media include gas-filled microbubbles. Microbubbles may have a shell composed of albumin (ALBUNEX®, BISPHERE®, ECHOGEN® and OPTISON®), phospholipids (DEFINITY®, IMAGENT®, MICROMARKER®, SONAZOID®, SONOVUE® and TARGESTAR®), galactolipids (LEVOVIST®) or PLGA/phospholipids (IMAGIFY®). Microbubbles that have a nanometer-scale diameter are also referred to as nanobubbles. Contrast media may be administered near the location where the ultrasound will be applied.
[0064] Microbubbles may also be used for ultrasonic drug delivery. Therapeutic agents may be conjugated to the exterior of the microbubbles or may be contained in the gas-filled interior of the microbubbles. Microbubbles may be conjugated to targeting ligands. Preferred targeting ligands include monoclonal antibodies, which may also act as therapeutic agents. The monoclonal antibodies are preferably humanized (or similarly modified as appropriate for the species being treated) to reduce their immunogenicity. A particularly preferred targeting ligand is a humanized monoclonal anti-AGE antibody. Other drug carriers for ultrasonic drug delivery include liposomes and micelles. Liposomes and micelles may also be conjugated to monoclonal antibodies, such as anti-AGE antibodies.
[0065] The ultrasound may be generated and applied using any suitable ultrasound transducer. A preferred ultrasound transducer is an ultrasound probe. A water-based gel may be applied to the area of treatment to improve transmission of ultrasound into the body. Alternatively, ultrasound generators may be implanted in the body to further facilitate access to a restricted site (see, for example, U.S. Pat. No. 8,977,361).
[0066] The anti-AGE antibody may bind to one or more AGE-modified proteins or peptides having an AGE modification such as FFI, pyrraline, AFGP, ALI, carboxymethyllysine (CML), carboxyethyllysine (CEL) and pentosidine, and mixtures of such antibodies. Preferably, the antibody is non-immunogenic to the animal in which it will be used, such as non-immunogenic to humans; companion animals including cats, dogs and horses; and commercially important animals, such camels (or alpaca), cattle (bovine), sheep, pig, and goats. More preferably, the antibody has the same species constant region as antibodies of the animal to reduce the immune response against the antibody, such as being humanized (for humans), felinized (for cats), caninized (for dogs), equuinized (for horses), camelized (for camels or alpaca), bovinized (for cattle), ovinized (for sheep), porcinized (for pigs), or caperized (for goats). Most preferably, the antibody is identical to that of the animal in which it will be used (except for the variable region), such as a human antibody, a cat antibody, a dog antibody, a horse antibody, a camel antibody, a bovine antibody, a sheep antibody, a pig antibody, or a goat antibody. Details of the constant regions and other parts of antibodies for these animals are described below. The antibody may be monoclonal or polyclonal. Preferably, the antibody is a monoclonal antibody.
[0067] Preferred anti-AGE antibodies include those which bind to proteins or peptides that exhibit a carboxymethyllysine or carboxyethyllysine AGE modification. Carboxymethyllysine (also known as N(epsilon)-(carboxymethyl)lysine, N(6)-carboxymethyllysine, or 2-Amino-6-(carboxymethylamino)hexanoic acid) and carboxyethyllysine (also known as N-epsilon-(carboxyethyl)lysine) are found on proteins or peptides and lipids as a result of oxidative stress and chemical glycation. CML- and CEL-modified proteins or peptides are recognized by the receptor RAGE which is expressed on a variety of cells. CML and CEL have been well-studied and CML- and CEL-related products are commercially available. For example, Cell Biolabs, Inc. sells CML-BSA antigens, CML polyclonal antibodies, CML immunoblot kits, and CML competitive ELISA kits (www.cellbiolabs.com/cml-assays) as well as CEL-BSA antigens and CEL competitive ELISA kits (www.cellbiolabs.com/cel-n-epsilon-carboxyethyl-lysine-assays-and-reagents). A preferred antibody includes the variable region of the commercially available mouse anti-glycation end-product antibody raised against carboxymethyl lysine conjugated with keyhole limpet hemocyanin, the carboxymethyl lysine MAb (Clone 318003) available from R&D Systems, Inc. (Minneapolis, Minn.; catalog no. MAB3247), modified to have a human constant region (or the constant region of the animal into which it will be administered). Commercially-available antibodies, such as the carboxymethyl lysine antibody corresponding to catalog no. MAB3247 from R&D Systems, Inc., may be intended for diagnostic purposes and may contain material that is not suited for use in animals or humans. Preferably, commercially-available antibodies are purified and/or isolated prior to use in animals or humans to remove toxins or other potentially-harmful material.
[0068] The anti-AGE antibody preferably has a low rate of dissociation from the antibody-antigen complex, or k.sub.d (also referred to as k.sub.back or off-rate), preferably at most 9×10.sup.−6, 8×10.sup.−3, 7×10.sup.−3 or 6×10 (sec.sup.−1). The anti-AGE antibody preferably has a high affinity for the AGE-modified protein of a cell, which may be expressed as a low dissociation constant K.sub.D of at most 9×10.sup.−6, 8×10.sup.−6, 7×10.sup.−6, 6×10.sup.−6, 5×10.sup.−6, 4×10.sup.−6 or 3×10.sup.−6 (M). Preferably, the binding properties of the anti-AGE antibody are similar to, the same as, or superior to the carboxymethyl lysine MAb (Clone 318003) available from R&D Systems, Inc. (Minneapolis, Minn.; catalog no. MAB3247), illustrated in
[0069] The anti-AGE antibody may destroy AGE-modified cells through antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC is a mechanism of cell-mediated immune defense in which an effector cell of the immune system actively lyses a target cell whose membrane-surface antigens have been bound by specific antibodies. ADCC may be mediated by natural killer (NK) cells, macrophages, neutrophils or eosinophils. The effector cells bind to the Fc portion of the bound antibody. The anti-AGE antibody may also destroy AGE-modified cells through complement-dependent cytotoxicity (CDC). In CDC, the complement cascade of the immune system is triggered by an antibody binding to a target antigen.
[0070] The anti-AGE antibody may be conjugated to an agent that causes the destruction of AGE-modified cells. Such agents may be a toxin, a cytotoxic agent, magnetic nanoparticles, and magnetic spin-vortex discs.
[0071] A toxin, such as pore-forming toxins (PFT) (Aroian R. et al., “Pore-Forming Toxins and Cellular Non-Immune Defenses (CNIDs),” Current Opinion in Microbiology, 10:57-61 (2007)), conjugated to an anti-AGE antibody may be injected into a patient to selectively target and remove AGE-modified cells. The anti-AGE antibody recognizes and binds to AGE-modified cells. Then, the toxin causes pore formation at the cell surface and subsequent cell removal through osmotic lysis.
[0072] Magnetic nanoparticles conjugated to the anti-AGE antibody may be injected into a patient to target and remove AGE-modified cells. The magnetic nanoparticles can be heated by applying a magnetic field in order to selectively remove the AGE-modified cells.
[0073] As an alternative, magnetic spin-vortex discs, which are magnetized only when a magnetic field is applied to avoid self-aggregation that can block blood vessels, begin to spin when a magnetic field is applied, causing membrane disruption of target cells. Magnetic spin-vortex discs, conjugated to anti-AGE antibodies specifically target AGE-modified cell types, without removing other cells.
[0074] A humanized anti-AGE antibody according to the present invention may have the human constant region sequence of amino acids shown in SEQ ID NO: 22. The heavy chain complementarity determining regions of the humanized anti-AGE antibody may have one or more of the protein sequences shown in SEQ ID NO: 23 (CDR1H), SEQ ID NO: 24 (CDR2H) and SEQ ID NO: 25 (CDR3H). The light chain complementarity determining regions of the humanized anti-AGE antibody may have one or more of the protein sequences shown in SEQ ID NO: 26 (CDR1L), SEQ ID NO: 27 (CDR2L) and SEQ ID NO: 28 (CDR3L).
[0075] The heavy chain of a humanized anti-AGE antibody may have or may include the protein sequence of SEQ ID NO: 1. The variable domain of the heavy chain may have or may include the protein sequence of SEQ ID NO: 2. The complementarity determining regions of the variable domain of the heavy chain (SEQ ID NO: 2) are shown in SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43. The kappa light chain of a humanized anti-AGE antibody may have or may include the protein sequence of SEQ ID NO: 3. The variable domain of the kappa light chain may have or may include the protein sequence of SEQ ID NO: 4. Optionally, the arginine (Arg or R) residue at position 128 of SEQ ID NO: 4 may be omitted. The complementarity determining regions of the variable domain of the light chain (SEQ ID NO: 4) are shown in SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46. The variable regions may be codon-optimized, synthesized and cloned into expression vectors containing human immunoglobulin G1 constant regions. In addition, the variable regions may be used in the preparation of non-human anti-AGE antibodies.
[0076] The antibody heavy chain may be encoded by the DNA sequence of SEQ ID NO: 12, a murine anti-AGE immunoglobulin G2b heavy chain. The protein sequence of the murine anti-AGE immunoglobulin G2b heavy chain encoded by SEQ ID NO: 12 is shown in SEQ ID NO: 16. The variable region of the murine antibody is shown in SEQ ID NO: 20, which corresponds to positions 25-142 of SEQ ID NO: 16. The antibody heavy chain may alternatively be encoded by the DNA sequence of SEQ ID NO: 13, a chimeric anti-AGE human immunoglobulin G1 heavy chain. The protein sequence of the chimeric anti-AGE human immunoglobulin G1 heavy chain encoded by SEQ ID NO: 13 is shown in SEQ ID NO: 17. The chimeric anti-AGE human immunoglobulin includes the murine variable region of SEQ ID NO: 20 in positions 25-142. The antibody light chain may be encoded by the DNA sequence of SEQ ID NO: 14, a murine anti-AGE kappa light chain. The protein sequence of the murine anti-AGE kappa light chain encoded by SEQ ID NO: 14 is shown in SEQ ID NO: 18. The variable region of the murine antibody is shown in SEQ ID NO: 21, which corresponds to positions 21-132 of SEQ ID NO: 18. The antibody light chain may alternatively be encoded by the DNA sequence of SEQ ID NO: 15, a chimeric anti-AGE human kappa light chain. The protein sequence of the chimeric anti-AGE human kappa light chain encoded by SEQ ID NO: 15 is shown in SEQ ID NO: 19. The chimeric anti-AGE human immunoglobulin includes the murine variable region of SEQ ID NO: 21 in positions 21-132.
[0077] A humanized anti-AGE antibody according to the present invention may have or may include one or more humanized heavy chains or humanized light chains. A humanized heavy chain may be encoded by the DNA sequence of SEQ ID NO: 30, 32 or 34. The protein sequences of the humanized heavy chains encoded by SEQ ID NOs: 30, 32 and 34 are shown in SEQ ID NOs: 29, 31 and 33, respectively. A humanized light chain may be encoded by the DNA sequence of SEQ ID NO: 36, 38 or 40. The protein sequences of the humanized light chains encoded by SEQ ID NOs: 36, 38 and 40 are shown in SEQ ID NOs: 35, 37 and 39, respectively. Preferably, the humanized anti-AGE antibody maximizes the amount of human sequence while retaining the original antibody specificity. A complete humanized antibody may be constructed that contains a heavy chain having a protein sequence chosen from SEQ ID NOs: 29, 31 and 33 and a light chain having a protein sequence chosen from SEQ ID NOs: 35, 37 and 39.
[0078] Particularly preferred anti-AGE antibodies may be obtained by humanizing murine monoclonal anti-AGE antibodies. Murine monoclonal anti-AGE antibodies have the heavy chain protein sequence shown in SEQ ID NO: 47 (the protein sequence of the variable domain is shown in SEQ ID NO: 52) and the light chain protein sequence shown in SEQ ID NO: 57 (the protein sequence of the variable domain is shown in SEQ ID NO: 62). A preferred humanized heavy chain may have the protein sequence shown in SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51 (the protein sequences of the variable domains of the humanized heavy chains are shown in SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56, respectively). A preferred humanized light chain may have the protein sequence shown in SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 or SEQ ID NO: 61 (the protein sequences of the variable domains of the humanized light chains are shown in SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66, respectively). Preferably, a humanized anti-AGE monoclonal antibody is composed a heavy chain having a protein sequence selected from the group consisting of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51 and a light chain having a protein sequence selected from the group consisting of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61. Humanized monoclonal anti-AGE antibodies composed of these protein sequences may have better binding and/or improved activation of the immune system, resulting in greater efficacy.
[0079] The protein sequence of an antibody from a non-human species may be modified to include the variable domain of the heavy chain having the sequence shown in SEQ ID NO: 2 or the kappa light chain having the sequence shown in SEQ ID NO: 4. The non-human species may be a companion animal, such as the domestic cat or domestic dog, or livestock, such as cattle, the horse or the camel. Preferably, the non-human species is not the mouse. The heavy chain of the horse (Equus caballus) antibody immunoglobulin gamma 4 may have or may include the protein sequence of SEQ ID NO: 5 (EMBUGenBank accession number AY445518). The heavy chain of the horse (Equus caballus) antibody immunoglobulin delta may have or may include the protein sequence of SEQ ID NO: 6 (EMBUGenBank accession number AY631942). The heavy chain of the dog (Canis familiaris) antibody immunoglobulin A may have or may include the protein sequence of SEQ ID NO: 7 (GenBank accession number L36871). The heavy chain of the dog (Canis familiaris) antibody immunoglobulin E may have or may include the protein sequence of SEQ ID NO: 8 (GenBank accession number L36872). The heavy chain of the cat (Felis catus) antibody immunoglobulin G2 may have or may include the protein sequence of SEQ ID NO: 9 (DDBJ/EMBL/GenBank accession number KF811175).
[0080] Animals of the camelid family, such as camels (Camelus dromedarius and Camelus bactrianus), llamas (Lama glama, Lama pacos and Lama vicugna), alpacas (Vicugna pacos) and guanacos (Lama guanicoe), have a unique antibody that is not found in other mammals. In addition to conventional immunoglobulin G antibodies composed of heavy and light chain tetramers, camelids also have heavy chain immunoglobulin G antibodies that do not contain light chains and exist as heavy chain dimers. These antibodies are known as heavy chain antibodies, HCAbs, single-domain antibodies or sdAbs, and the variable domain of a camelid heavy chain antibody is known as the VHH. The camelid heavy chain antibodies lack the heavy chain CH1 domain and have a hinge region that is not found in other species. The variable region of the Arabian camel (Camelus dromedarius) single-domain antibody may have or may include the protein sequence of SEQ ID NO: 10 (GenBank accession number AJ245148). The variable region of the heavy chain of the Arabian camel (Camelus dromedarius) tetrameric immunoglobulin may have or may include the protein sequence of SEQ ID NO: 11 (GenBank accession number AJ245184).
[0081] In addition to camelids, heavy chain antibodies are also found in cartilaginous fishes, such as sharks, skates and rays. This type of antibody is known as an immunoglobulin new antigen receptor or IgNAR, and the variable domain of an IgNAR is known as the VNAR. The IgNAR exists as two identical heavy chain dimers composed of one variable domain and five constant domains each. Like camelids, there is no light chain.
[0082] The protein sequences of additional non-human species may be readily found in online databases, such as the International ImMunoGeneTics Information System (www.imgt.org), the European Bioinformatics Institute (www.ebi.ac.uk), the DNA Databank of Japan (ddbj.nig.ac.jp/arsa) or the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov).
[0083] An anti-AGE antibody or a variant thereof may include a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51, including post-translational modifications thereof. A heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity may contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-AGE antibody including that sequence retains the ability to bind to AGE.
[0084] An anti-AGE antibody or a variant thereof may include a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56, including post-translational modifications thereof. A variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity may contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-AGE antibody including that sequence retains the ability to bind to AGE. The substitutions, insertions, or deletions may occur in regions outside the variable region.
[0085] An anti-AGE antibody or a variant thereof may include a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 or SEQ ID NO: 61, including post-translational modifications thereof. A light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity may contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-AGE antibody including that sequence retains the ability to bind to AGE. The substitutions, insertions, or deletions may occur in regions outside the variable region.
[0086] An anti-AGE antibody or a variant thereof may include a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 or SEQ ID NO: 66, including post-translational modifications thereof. A variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity may contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-AGE antibody including that sequence retains the ability to bind to AGE. The substitutions, insertions, or deletions may occur in regions outside the variable region.
[0087] Alternatively, the antibody may have the complementarity determining regions of commercially available mouse anti-glycation end-product antibody raised against carboxymethyl lysine conjugated with keyhole limpet hemocyanin (CML-KLH), the carboxymethyl lysine MAb (Clone 318003) available from R&D Systems, Inc. (Minneapolis, Minn.; catalog no. MAB3247).
[0088] The antibody may have or may include constant regions which permit destruction of targeted cells by a subject's immune system.
[0089] Mixtures of antibodies that bind to more than one type AGE of AGE-modified proteins may also be used.
[0090] Bi-specific antibodies, which are anti-AGE antibodies directed to two different epitopes, may also be used. Such antibodies will have a variable region (or complementary determining region) from those of one anti-AGE antibody, and a variable region (or complementary determining region) from a different antibody.
[0091] Antibody fragments may be used in place of whole antibodies. For example, immunoglobulin G may be broken down into smaller fragments by digestion with enzymes. Papain digestion cleaves the N-terminal side of inter-heavy chain disulfide bridges to produce Fab fragments. Fab fragments include the light chain and one of the two N-terminal domains of the heavy chain (also known as the Fd fragment). Pepsin digestion cleaves the C-terminal side of the inter-heavy chain disulfide bridges to produce F(ab′).sub.2 fragments. F(ab′).sub.2 fragments include both light chains and the two N-terminal domains linked by disulfide bridges. Pepsin digestion may also form the Fv (fragment variable) and Fc (fragment crystallizable) fragments. The Fv fragment contains the two N-terminal variable domains. The Fc fragment contains the domains which interact with immunoglobulin receptors on cells and with the initial elements of the complement cascade. Pepsin may also cleave immunoglobulin G before the third constant domain of the heavy chain (C.sub.H3) to produce a large fragment F(abc) and a small fragment pFc′. Antibody fragments may alternatively be produced recombinantly. Preferably, such antibody fragments are conjugated to an agent that causes the destruction of AGE-modified cells.
[0092] If additional antibodies are desired, they can be produced using well-known methods. For example, polyclonal antibodies (pAbs) can be raised in a mammalian host by one or more injections of an immunogen, and if desired, an adjuvant. Typically, the immunogen (and adjuvant) is injected in a mammal by a subcutaneous or intraperitoneal injection. The immunogen may be an AGE-modified protein of a cell, such as AGE-antithrombin III, AGE-calmodulin, AGE-insulin, AGE-ceruloplasmin, AGE-collagen, AGE-cathepsin B, AGE-albumin such as AGE-bovine serum albumin (AGE-BSA), AGE-human serum albumin and ovalbumin, AGE-crystallin, AGE-plasminogen activator, AGE-endothelial plasma membrane protein, AGE-aldehyde reductase, AGE-transferrin, AGE-fibrin, AGE-copper/zinc SOD, AGE-apo B, AGE-fibronectin, AGE-pancreatic ribose, AGE-apo A-I and II, AGE-hemoglobin, AGE-Na.sup.+/K.sup.+-ATPase, AGE-plasminogen, AGE-myelin, AGE-lysozyme, AGE-immunoglobulin, AGE-red cell Glu transport protein, AGE-β-N-acetyl hexominase, AGE-apo E, AGE-red cell membrane protein, AGE-aldose reductase, AGE-ferritin, AGE-red cell spectrin, AGE-alcohol dehydrogenase, AGE-haptoglobin, AGE-tubulin, AGE-thyroid hormone, AGE-fibrinogen, AGE-β.sub.2-microglobulin, AGE-sorbitol dehydrogenase, AGE-α.sub.1-antitrypsin, AGE-carbonate dehydratase, AGE-RNAse, AGE-low density lipoprotein, AGE-hexokinase, AGE-apo C-I, AGE-RNAse, AGE-hemoglobin such as AGE-human hemoglobin, AGE-low density lipoprotein (AGE-LDL) and AGE-collagen IV. AGE-modified cells, such as AGE-modified erythrocytes, whole, lysed, or partially digested, may also be used as AGE antigens. Examples of adjuvants include Freund's complete, monophosphoryl Lipid A synthetic-trehalose dicorynomycolate, aluminum hydroxide (alum), heat shock proteins HSP 70 or HSP96, squalene emulsion containing monophosphoryl lipid A, α2-macroglobulin and surface active substances, including oil emulsions, pleuronic polyols, polyanions and dinitrophenol. To improve the immune response, an immunogen may be conjugated to a polypeptide that is immunogenic in the host, such as keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, cholera toxin, labile enterotoxin, silica particles or soybean trypsin inhibitor. A preferred immunogen conjugate is AGE-KLH. Alternatively, pAbs may be made in chickens, producing IgY molecules.
[0093] Monoclonal antibodies (mAbs) may also be made by immunizing a host or lymphocytes from a host, harvesting the mAb-secreting (or potentially secreting) lymphocytes, fusing those lymphocytes to immortalized cells (for example, myeloma cells), and selecting those cells that secrete the desired mAb. Other techniques may be used, such as the EBV-hybridoma technique. Non-human antibodies may be made less immunogenic to humans by engineering the antibodies to contain a combination of non-human and human antibody components. A chimeric antibody may be produced by combining the variable region of a non-human antibody with a human constant region. A humanized antibody may be produced by replacing the complementarity determining regions (CDRs) of a human antibody with those of a non-human antibody. Similarly, antibodies may be made less immunogenic to other species by being substantially “ized” to a given animal, such as cat, dog, horse, camel or alpaca, cattle, sheep, pig, or goat, at the amino acid level. If desired, the mAbs may be purified from the culture medium or ascites fluid by conventional procedures, such as protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ammonium sulfate precipitation or affinity chromatography. Additionally, human monoclonal antibodies can be generated by immunization of transgenic mice containing a third copy IgG human trans-loci and silenced endogenous mouse Ig loci or using human-transgenic mice. Production of humanized monoclonal antibodies and fragments thereof can also be generated through phage display technologies.
[0094] A “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Preferred examples of such carriers or diluents include water, saline, Ringer's solutions and dextrose solution. Supplementary active compounds can also be incorporated into the compositions. Solutions and suspensions used for parenteral administration can include a sterile diluent, such as water for injection, saline solution, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
[0095] The antibodies may be administered by injection, such as by intravenous injection or locally, such as by intra-articular injection into a joint. Pharmaceutical compositions suitable for injection include sterile aqueous solutions or dispersions for the extemporaneous preparation of sterile injectable solutions or dispersion. Various excipients may be included in pharmaceutical compositions of antibodies suitable for injection. Suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL® (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid so as to be administered using a syringe. Such compositions should be stable during manufacture and storage and must be preserved against contamination from microorganisms such as bacteria and fungi. Various antibacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal, can contain microorganism contamination. Isotonic agents such as sugars, polyalcohols, such as mannitol, sorbitol, and sodium chloride can be included in the composition. Compositions that can delay absorption include agents such as aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating antibodies, and optionally other therapeutic components, in the required amount in an appropriate solvent with one or a combination of ingredients as required, followed by sterilization. Methods of preparation of sterile solids for the preparation of sterile injectable solutions include vacuum drying and freeze-drying to yield a solid.
[0096] For administration by inhalation, the antibodies may be delivered as an aerosol spray from a nebulizer or a pressurized container that contains a suitable propellant, for example, a gas such as carbon dioxide. Antibodies may also be delivered via inhalation as a dry powder, for example using the iSPERSE™ inhaled drug delivery platform (PULMATRIX, Lexington, Mass.). The use of anti-AGE antibodies which are chicken antibodies (IgY) may be non-immunogenic in a variety of animals, including humans, when administered by inhalation.
[0097] An appropriate dosage level of each type of antibody will generally be about 0.01 to 500 mg per kg patient body weight. Preferably, the dosage level will be about 0.1 to about 250 mg/kg; more preferably about 0.5 to about 100 mg/kg. A suitable dosage level may be about 0.01 to 250 mg/kg, about 0.05 to 100 mg/kg, or about 0.1 to 50 mg/kg. Within this range the dosage may be 0.05 to 0.5, 0.5 to 5 or 5 to 50 mg/kg. Although each type of antibody may be administered on a regimen of 1 to 4 times per day, such as once or twice per day, antibodies typically have a long half-life in vivo. Accordingly, each type of antibody may be administered once a day, once a week, once every two or three weeks, once a month, or once every 60 to 90 days.
[0098] Unit dosage forms can be created to facilitate administration and dosage uniformity. Unit dosage form refers to physically discrete units suited as single dosages for the subject to be treated, containing a therapeutically effective quantity of one or more types of antibodies in association with the required pharmaceutical carrier. Preferably, the unit dosage form is in a sealed container and is sterile.
[0099] A subject that receives application of ultrasound and administration of an anti-AGE antibody may be tested to determine if the treatment has been effective to kill AGE-modified cells. A subject may be considered to have received an effective treatment if he or she demonstrates a reduction in one or more symptoms of an AGE disorder between subsequent measurements or over time. For example, a subject with glioblastoma may be considered to have received an effective treatment based on a reduction in tumor size. Alternatively, the concentration and/or number of senescent cells may be measured over time. Treatment and subsequent testing may be repeated until the desired therapeutic result is achieved.
[0100] Any mammal may be treated by the methods herein described. Humans are a preferred mammal for treatment. Other mammals that may be treated include mice, rats, goats, sheep, pigs, cows, horses and companion animals, such as dogs or cats. Alternatively, any of the mammals or subjects identified above may be excluded from the patient population in need of treatment for pain associated with inflammation. The methods may also be applied in vitro, such as to a cell culture or tissue culture.
[0101] A subject may be identified as in need of treatment based on a diagnosis of having an AGE disorder. Examples of AGE disorders include Alzheimer's disease, amyotrophic lateral sclerosis (ALS or Lou Gehrig's Disease), chronic obstructive pulmonary disease (COPD), Huntington's chorea, idiopathic pulmonary fibrosis, muscular dystrophy (including Becker's, Duchenne, Limb-Girdle and Yamamoto's muscular dystrophy), macular degeneration, cataracts, diabetic retinopathy, Parkinson's disease, progeria (including Werner Syndrome and Hutchinson Gilford progeria), vitiligo, cystic fibrosis, atopic dermatitis, eczema, arthritis (including osteoarthritis, rheumatoid arthritis and juvenile rheumatoid arthritis), atherosclerosis, cancer and metastatic cancer (including, for example, breast cancer, triple negative breast cancer, lung cancer, melanoma, colon cancer, renal cell carcinoma, prostate cancer, cancer of the cervix, bladder cancer, rectal cancer, esophageal cancer, liver cancer, mouth and throat cancer, multiple myeloma, ovarian cancer, stomach cancer, pancreatic cancer and retinal blastoma cancers), cancer therapy-related disability or cancer therapy side effects, hypertension, glaucoma, osteoporosis, sarcopenia, cachexia, stroke, myocardial infarction, atrial fibrillation, transplantation rejection, diabetes mellitus—Type I, diabetes mellitus—Type II, radiation exposure, HIV treatment side effects, chemical weapons exposure, poisoning, inflammation, nephropathy, Lewy body dementia, prion disease (including bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, scrapie, chronic wasting disease, kuru and fatal familial insomnia), lordokyphosis, auto-immune disorders, loss of adipose tissue, psoriasis, Crohn's disease, asthma, the physiological effects of aging (including “cosmetic” effects, such as wrinkling, age spots, hair loss, reduction in subcutaneous adipose tissue and thinning of the skin), idiopathic myopathy (including, for example, idiopathic inflammatory myopathy, idiopathic inflammatory myositis, polymyositis, dermatomyositis, sporadic inclusion body myositis and juvenile myositis), multiple sclerosis, neuromyelitis optica (NMO, Devic's disease or Devic's syndrome), epilepsy and adrenoleukodystrophy (ALD, X-linked adrenoleukodystrophy, X-ALD, cerebral ALD or cALD).
[0102] A particularly preferred treatment group includes subjects who have been diagnosed with an AGE disorder affecting a restricted site of the body. Examples of AGE disorders that affect a restricted site of the body include brain tumors (including gliomas such as anaplastic astrocytoma, glioblastoma multiforme, oligodendroglioma and diffuse intrinsic pontine glioma, meningiomas, pituitary adenomas and nerve sheath tumors), meningitis, brain/cerebral abscess, late-stage neurological trypanosomiasis (sleeping sickness), cerebral edema, prion diseases, encephalitis, rabies, arthritis/osteoarthritis, degenerative joint disease, synovitis, bursitis, prostate cancer, eye cancer and eye disorders.
[0103] The Present Application includes 66 nucleotide and amino acid sequences in the Sequence Listing filed herewith. Variants of the nucleotide and amino acid sequences are possible. Known variants include substitutions, deletions and additions to the sequences shown in SEQ ID NO: 4, 16 and 20. In SEQ ID NO: 4, the arginine (Arg or R) residue at position 128 may optionally be omitted. In SEQ ID NO: 16, the alanine residue at position 123 may optionally be replaced with a serine residue, and/or the tyrosine residue at position 124 may optionally be replaced with a phenylalanine residue. SEQ ID NO: 20 may optionally include the same substitutions as SEQ ID NO: 16 at positions 123 and 124. In addition, SEQ ID NO: 20 may optionally contain one additional lysine residue after the terminal valine residue.
EXAMPLES
Example 1: In Vivo Study of the Administration of Anti-Glycation End-Product Antibody
[0104] To examine the effects of an anti-glycation end-product antibody, the antibody was administered to the aged CD1(1CR) mouse (Charles River Laboratories), twice daily by intravenous injection, once a week, for three weeks (Days 1, 8 and 15), followed by a 10 week treatment-free period. The test antibody was a commercially available mouse anti-glycation end-product antibody raised against carboxymethyl lysine conjugated with keyhole limpet hemocyanin, the carboxymethyl lysine MAb (Clone 318003) available from R&D Systems, Inc. (Minneapolis, Minn.; catalog no. MAB3247). A control reference of physiological saline was used in the control animals.
[0105] Mice referred to as “young” were 8 weeks old, while mice referred to as “old” were 88 weeks (±2 days) old. No adverse events were noted from the administration of the antibody. The different groups of animals used in the study are shown in Table 1.
TABLE-US-00001 TABLE 1 The different groups of animals used in the study Number of Animals Treatment- Group Test Dose Level Main Study Free No. Material Mice (pg/gm/BID/week) Females Females 1 Saline young 0 20 — 2 Saline old 0 20 20 3 Antibody old 2.5 20 20 4 None old 0 20 pre 5 Antibody old 5.0 20 20 — = Not Applicable, Pre = Subset of animals euthanized prior to treatment start for collection of adipose tissue.
[0106] P16.sup.INK4a mRNA, a marker for senescent cells, was quantified in adipose tissue of the groups by Real Time-qPCR. The results are shown in Table 2. In the table ΔΔCt=ΔCt mean control Group (2)—ΔCt mean experimental Group (1 or 3 or 5); Fold Expression=2.sup.−ΔΔCt.
TABLE-US-00002 TABLE 2 P16.sup.INK4a mRNA quantified in adipose tissue Calculation Group 2 vs Group 2 vs Group 2 vs (unadjusted to Group 1 Group 3 Group 5 Group 4: 5.59) Group 2 Group 1 Group 2 Group 3 Group 2 Group 5 Mean ΔCt 5.79 7.14 5.79 6.09 5.79 7.39 ΔΔCt −1.35 −0.30 −1.60 Fold 2.65 1.23 3.03 Expression
[0107] The table above indicates that untreated old mice (Control Group 2) express 2.55-fold more p16.sup.Ink4a mRNA than the untreated young mice (Control Group 1), as expected. This was observed when comparing Group 2 untreated old mice euthanized at end of recovery Day 85 to Group 1 untreated young mice euthanized at end of treatment Day 22. When results from Group 2 untreated old mice were compared to results from Group 3 treated old mice euthanized Day 85, it was observed that p16.sup.Ink4a mRNA was 1.23-fold higher in Group 2 than in Group 3. Therefore, the level of p16.sup.Ink4a mRNA expression was lower when the old mice were treated with 2.5 μg/gram/BID/week of antibody.
[0108] When results from Group 2 (Control) untreated old mice were compared to results from Group 5 (5 μg/gram) treated old mice euthanized Day 22, it was observed that p16.sup.Ink4a mRNA was 3.03-fold higher in Group 2 (controls) than in Group 5 (5 μg/gram). This comparison indicated that the Group 5 animals had lower levels of p16.sup.Ink4a mRNA expression when they were treated with 5.0 μg/gram/BID/week, providing p16.sup.Ink4a mRNA expression levels comparable to that of the young untreated mice (i.e. Group 1). Unlike Group 3 (2.5 μg/gram) mice that were euthanized at end of recovery Day 85, Group 5 mice were euthanized at end of treatment Day 22.
[0109] These results indicate the antibody administration resulted in the killing of senescent cells.
[0110] The mass of the gastrocnemius muscle was also measured, to determine the effect of antibody administration on sarcopenia. The results are provided in Table 3. The results indicate that administration of the antibody increased muscle mass as compared to controls, but only at the higher dosage of 5.0 μg/gm/BID/week.
TABLE-US-00003 TABLE 3 Effect of antibody administration on mass of the gastrocnemius muscle Weight relative to Summary Absolute weight of body mass of Group Information Gastrocnemius Muscle Gastrocnemius Muscle 1 Mean 0.3291 1.1037 SD 0.0412 0.1473 N 20 20 2 Mean 0.3304 0.7671 SD 0.0371 0.1246 N 20 20 3 Mean 0.3410 0.7706 SD 0.0439 0.0971 N 19 19 5 Mean 0.4074 0.9480 SD 0.0508 0.2049 N 9 9
[0111] These results demonstrate that administration of antibodies that bind to AGEs of a cell resulted in a reduction of cells expressing p16.sup.Ink4a, a biomarker of senescence. The data show that reducing senescent cells leads directly to an increase in muscle mass in aged mice. These results indicate that the loss of muscle mass, a classic sign of sarcopenia, can be treated by administration of antibodies that bind to AGEs of a cell.
Example 2: Affinity and Kinetics of Test Antibody
[0112] The affinity and kinetics of the test antibody used in Example 1 were analyzed using Nα,Nα-bis(carboxymethyl)-L-lysine trifluoroacetate salt (Sigma-Aldrich, St. Louis, Mo.) as a model substrate for an AGE-modified protein of a cell. Label-free interaction analysis was carried out on a BIACORE™ T200 (GE Healthcare, Pittsburgh, Pa.), using a Series S sensor chip CM5 (GE Healthcare, Pittsburgh, Pa.), with Fc1 set as blank, and Fc2 immobilized with the test antibody (molecular weigh of 150,000 Da). The running buffer was a HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% P-20, pH of 7.4), at a temperature of 25° C. Software was BIACORE™ T200 evaluation software, version 2.0. A double reference (Fc2-1 and only buffer injection), was used in the analysis, and the data was fitted to a Langmuir 1:1 binding model.
TABLE-US-00004 TABLE 4 Experimental set-up of affinity and kinetics analysis Association and dissociation Flow path Fc1 and Fc2 Flow rate (μl/min.) 30 Association time (s) 300 Dissociation time (s) 300 Sample concentration (μM) 20 − 5 − 1.25 (x2) − 0.3125 − 0.078 − 0
[0113] A graph of the response versus time is illustrated in
Example 3: Construction and Production of Murine Anti-AGE IgG2b Antibody and Chimeric Anti-AGE IgG1 Antibody
[0114] Murine and chimeric human anti-AGE antibodies were prepared. The DNA sequence of murine anti-AGE antibody IgG2b heavy chain is shown in SEQ ID NO: 12. The DNA sequence of chimeric human anti-AGE antibody IgG1 heavy chain is shown in SEQ ID NO: 13. The DNA sequence of murine anti-AGE antibody kappa light chain is shown in SEQ ID NO: 14. The DNA sequence of chimeric human anti-AGE antibody kappa light chain is shown in SEQ ID NO: 15. The gene sequences were synthesized and cloned into high expression mammalian vectors. The sequences were codon optimized. Completed constructs were sequence confirmed before proceeding to transfection.
[0115] HEK293 cells were seeded in a shake flask one day before transfection, and were grown using serum-free chemically defined media. The DNA expression constructs were transiently transfected into 0.03 liters of suspension HEK293 cells. After 20 hours, cells were sampled to obtain the viabilities and viable cell counts, and titers were measured (OCTET® QKe, ForteBio). Additional readings were taken throughout the transient transfection production runs. The cultures were harvested on day 5, and an additional sample for each was measured for cell density, viability and titer.
[0116] The conditioned media for murine and chimeric anti-AGE antibodies were harvested and clarified from the transient transfection production runs by centrifugation and filtration. The supernatants were run over a Protein A column and eluted with a low pH buffer. Filtration using a 0.2 μm membrane filter was performed before aliquoting. After purification and filtration, the protein concentrations were calculated from the OD280 and the extinction coefficient. A summary of yields and aliquots is shown in Table 5:
TABLE-US-00005 TABLE 5 Yields and aliquots Concentration Volume No. of Total Yield Protein (mg/mL) (mL) vials (mg) Murine anti-AGE 0.08 1.00 3 0.24 Chimeric anti-AGE 0.23 1.00 3 0.69
[0117] Antibody purity was evaluated by capillary electrophoresis sodium-dodecyl sulfate (CE-SDS) analysis using LabChip® GXII, (PerkinElmer).
Example 4: Binding of Murine (Parental) and Chimeric Anti-AGE Antibodies
[0118] The binding of the murine (parental) and chimeric anti-AGE antibodies described in Example 3 was investigated by a direct binding ELISA. An anti-carboxymethyl lysine (CML) antibody (R&D Systems, MAB3247) was used as a control. CML was conjugated to KLH (CML-KLH) and both CML and CML-KLH were coated overnight onto an ELISA plate. HRP-goat anti-mouse Fc was used to detect the control and murine (parental) anti-AGE antibodies. HRP-goat anti-human Fc was used to detect the chimeric anti-AGE antibody.
[0119] The antigens were diluted to 1 μg/mL in 1× phosphate buffer at pH 6.5. A 96-well microtiter ELISA plate was coated with 100 μL/well of the diluted antigen and let sit at 4° C. overnight. The plate was blocked with 1×PBS, 2.5% BSA and allowed to sit for 1-2 hours the next morning at room temperature. The antibody samples were prepared in serial dilutions with 1×PBS, 1% BSA with the starting concentration of 50 μg/mL. Secondary antibodies were diluted 1:5,000. 100 μL of the antibody dilutions was applied to each well. The plate was incubated at room temperature for 0.5-1 hour on a microplate shaker. The plate was washed 3 times with 1×PBS. 100 μL/well diluted HRP-conjugated goat anti-human Fc secondary antibody was applied to the wells. The plate was incubated for 1 hour on a microplate shaker. The plate was then washed 3 times with 1×PBS. 100 μL HRP substrate TMB was added to each well to develop the plate. After 3-5 minutes elapsed, the reaction was terminated by adding 100 μL of 1N HCl. A second direct binding ELISA was performed with only CML coating. The absorbance at OD450 was read using a microplate reader.
[0120] The OD450 absorbance raw data for the CML and CML-KLH ELISA is shown in the plate map below. 48 of the 96 wells in the well plate were used. Blank wells in the plate map indicate unused wells.
TABLE-US-00006 Plate map of CML and CML-KLH ELISA: Conc. (μg/mL) 1 2 3 4 5 6 7 50 0.462 0.092 0.42 1.199 0.142 1.852 16.67 0.312 0.067 0.185 0.31 0.13 0.383 5.56 0.165 0.063 0.123 0.19 0.115 0.425 1.85 0.092 0.063 0.088 0.146 0.099 0.414 0.62 0.083 0.072 0.066 0.108 0.085 0.248 0.21 0.075 0.066 0.09 0.096 0.096 0.12 0.07 0.086 0.086 0.082 0.098 0.096 0.098 0 0.09 0.085 0.12 0.111 0.083 0.582 R&D Parental Chimeric R&D Parental Chimeric Positive Anti- Anti- Positive Anti- Anti- Control AGE AGE Control AGE AGE CML-KLH Coat CML Coat
[0121] The OD450 absorbance raw data for the CML-only ELISA is shown in the plate map below. 24 of the 96 wells in the well plate were used. Blank wells in the plate map indicate unused wells.
TABLE-US-00007 Plate map of CML-only ELISA: Conc. (μg/mL) 1 2 3 4 5 6 7 50 1.913 0.165 0.992 16.66667 1.113 0.226 0.541 5.555556 0.549 0.166 0.356 1.851852 0.199 0.078 0.248 0.617284 0.128 0.103 0.159 0.205761 0.116 0.056 0.097 0.068587 0.073 0.055 0.071 0 0.053 0.057 0.06 R&D Parental Chimeric Positive Anti- Anti- Control AGE AGE
[0122] The control and chimeric anti-AGE antibodies showed binding to both CML and CML-KLH. The murine (parental) anti-AGE antibody showed very weak to no binding to either CML or CML-KLH. Data from repeated ELISA confirms binding of the control and chimeric anti-AGE to CML. All buffer control showed negative signal.
Example 5: Humanized Antibodies
[0123] Humanized antibodies were designed by creating multiple hybrid sequences that fuse select parts of the parental (mouse) antibody sequence with the human framework sequences. Acceptor frameworks were identified based on the overall sequence identity across the framework, matching interface position, similarly classed CDR canonical positions, and presence of N-glycosylation sites that would have to be removed. Three humanized light chains and three humanized heavy chains were designed based on two different heavy and light chain human acceptor frameworks. The amino acid sequences of the heavy chains are shown in SEQ ID NO: 29, 31 and 33, which are encoded by the DNA sequences shown in SEQ ID NO: 30, 32 and 34, respectively. The amino acid sequences of the light chains are shown in SEQ ID NO: 35, 37 and 39, which are encoded by the DNA sequences shown in SEQ ID NO: 36, 38 and 40, respectively. The humanized sequences were methodically analyzed by eye and computer modeling to isolate the sequences that would most likely retain antigen binding. The goal was to maximize the amount of human sequence in the final humanized antibodies while retaining the original antibody specificity. The light and heavy humanized chains could be combined to create nine variant fully humanized antibodies.
[0124] The three heavy chains and three light chains were analyzed to determine their humanness. Antibody humanness scores were calculated according to the method described in Gao, S. H., et al., “Monoclonal antibody humanness score and its applications”, BMC Biotechnology, 13:55 (Jul. 5, 2013). The humanness score represents how human-like an antibody variable region sequence looks. For heavy chains a score of 79 or above is indicative of looking human-like; for light chains a score of 86 or above is indicative of looking human-like. The humanness of the three heavy chains, three light chains, a parental (mouse) heavy chain and a parental (mouse) light chain are shown below in Table 6:
TABLE-US-00008 TABLE 6 Antibody humanness Antibody Humanness (Framework + CDR) Parental (mouse) heavy chain 63.60 Heavy chain 1 (SEQ ID NO: 29) 82.20 Heavy chain 2 (SEQ ID NO: 31) 80.76 Heavy chain 3 (SEQ ID NO: 33) 81.10 Parental (mouse) light chain 77.87 Light chain 1 (SEQ ID NO: 35) 86.74 Light chain 2 (SEQ ID NO: 37) 86.04 Light chain 3 (SEQ IN NO: 39) 83.57
[0125] Full-length antibody genes were constructed by first synthesizing the variable region sequences. The sequences were optimized for expression in mammalian cells. These variable region sequences were then cloned into expression vectors that already contain human Fc domains; for the heavy chain, the IgG1 was used.
[0126] Small scale production of humanized antibodies was carried out by transfecting plasmids for the heavy and light chains into suspension HEK293 cells using chemically defined media in the absence of serum. Whole antibodies in the conditioned media were purified using MabSelect SuRe Protein A medium (GE Healthcare).
[0127] Nine humanized antibodies were produced from each combination of the three heavy chains having the amino acid sequences shown in SEQ ID NO: 29, 31 and 33 and three light chains having the amino acid sequences shown in SEQ ID NO: 35, 37 and 39. A comparative chimeric parental antibody was also prepared. The antibodies and their respective titers are shown below in Table 7:
TABLE-US-00009 TABLE 7 Antibody titers Antibody Titer (mg/L) Chimeric parental 23.00 SEQ ID NO: 29 + SEQ ID NO: 35 24.67 SEQ ID NO: 29 + SEQ ID NO: 37 41.67 SEQ ID NO: 29 + SEQ ID NO: 39 29.67 SEQ ID NO: 31 + SEQ ID NO: 35 26.00 SEQ ID NO: 31 + SEQ ID NO: 37 27.33 SEQ ID NO: 31 + SEQ ID NO: 39 35.33 SEQ ID NO: 33 + SEQ ID NO: 35 44.00 SEQ ID NO: 33 + SEQ ID NO: 37 30.33 SEQ ID NO: 33 + SEQ ID NO: 39 37.33
[0128] The binding of the humanized antibodies may be evaluated, for example, by dose-dependent binding ELISA or cell-based binding assay.
Example 6: In Vivo Study of the Administration of a Carboxymethyl Lysine Monoclonal Antibody
[0129] The effect of a carboxymethyl lysine antibody on tumor growth, metastatic potential and cachexia was investigated. In vivo studies were carried out in mice using a murine breast cancer tumor model. Female BALB/c mice (BALB/cAnNCrl, Charles River) were eleven weeks old on Day 1 of the study.
[0130] 4T1 murine breast tumor cells (ATCC CRL-2539) were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 2 mM glutamine, 25 μg/mL gentamicin, 100 units/mL penicillin G Na and 100 μg/mL streptomycin sulfate. Tumor cells were maintained in tissue culture flasks in a humidified incubator at 37° C. in an atmosphere of 5% CO.sub.2 and 95% air.
[0131] The cultured breast cancer cells were then implanted in the mice. 4T1 cells were harvested during log phase growth and re-suspended in phosphate buffered saline (PBS) at a concentration of 1×10.sup.6 cells/mL on the day of implant. Tumors were initiated by subcutaneously implanting 1×10.sup.5 4 T1 cells (0.1 mL suspension) into the right flank of each test animal. Tumors were monitored as their volumes approached a target range of 80-120 mm.sup.3. Tumor volume was determined using the formula: tumor volume=(tumor width).sup.2(tumor length)/2. Tumor weight was approximated using the assumption that 1 mm.sup.3 of tumor volume has a weight of 1 mg. Thirteen days after implantation, designated as Day 1 of the study, mice were sorted into four groups (n=15/group) with individual tumor volumes ranging from 108 to 126 mm.sup.3 and a group mean tumor volume of 112 mm.sup.3. The four treatment groups are shown in Table 8 below:
TABLE-US-00010 TABLE 8 Treatment groups Group Description Agent Dosing (μg/g) 1 Control phosphate buffered saline N/A (PBS) 2 Low-dose carboxymethyl lysine 5 monoclonal antibody 3 High-dose carboxymethyl lysine 10 monoclonal antibody 4 Observation only None N/A
[0132] An anti-carboxymethyl lysine monoclonal antibody was used as a therapeutic agent. 250 mg of carboxymethyl lysine monoclonal antibody was obtained from R&D Systems (Minneapolis, Minn.). Dosing solutions of the carboxymethyl lysine monoclonal antibody were prepared at 1 and 0.5 mg/mL in a vehicle (PBS) to provide the active dosages of 10 and 5 μg/g, respectively, in a dosing volume of 10 mL/kg. Dosing solutions were stored at 4° C. protected from light.
[0133] All treatments were administered intravenously (i.v.) twice daily for 21 days, except on Day 1 of the study where the mice were administered one dose. On Day 19 of the study, i.v. dosing was changed to intraperitoneal (i.p.) dosing for those animals that could not be dosed i.v. due to tail vein degradation. The dosing volume was 0.200 mL per 20 grams of body weight (10 mL/kg), and was scaled to the body weight of each individual animal.
[0134] The study continued for 23 days. Tumors were measured using calipers twice per week. Animals were weighed daily on Days 1-5, then twice per week until the completion of the study. Mice were also observed for any side effects. Acceptable toxicity was defined as a group mean body weight loss of less than 20% during the study and not more than 10% treatment-related deaths. Treatment efficacy was determined using data from the final day of the study (Day 23).
[0135] The ability of the anti-carboxymethyl lysine antibody to inhibit tumor growth was determined by comparing the median tumor volume (MTV) for Groups 1-3. Tumor volume was measured as described above. Percent tumor growth inhibition (% TGI) was defined as the difference between the MW of the control group (Group 1) and the MTV of the drug-treated group, expressed as a percentage of the MW of the control group. % TGI may be calculated according to the formula: % TGI=(1−MTV.sub.treated/MTV.sub.control)×100.
[0136] The ability of the anti-carboxymethyl lysine antibody to inhibit cancer metastasis was determined by comparing lung cancer foci for Groups 1-3. Percent inhibition (% Inhibition) was defined as the difference between the mean count of metastatic foci of the control group and the mean count of metastatic foci of a drug-treated group, expressed as a percentage of the mean count of metastatic foci of the control group. % Inhibition may be calculated according to the following formula: % Inhibition=(1−Mean Count of Foci.sub.treated/Mean Count of Foci.sub.control)×100.
[0137] The ability of the anti-carboxymethyl lysine antibody to inhibit cachexia was determined by comparing the weights of the lungs and gastrocnemius muscles for Groups 1-3. Tissue weights were also normalized to 100 g body weight.
[0138] Treatment efficacy was also evaluated by the incidence and magnitude of regression responses observed during the study. Treatment may cause partial regression (PR) or complete regression (CR) of the tumor in an animal. In a PR response, the tumor volume was 50% or less of its Day 1 volume for three consecutive measurements during the course of the study, and equal to or greater than 13.5 mm.sup.3 for one or more of these three measurements. In a CR response, the tumor volume was less than 13.5 mm.sup.3 for three consecutive measurements during the course of the study.
[0139] Statistical analysis was carried out using Prism (GraphPad) for Windows 6.07. Statistical analyses of the differences between Day 23 mean tumor volumes (MTVs) of two groups were accomplished using the Mann-Whitney U test. Comparisons of metastatic foci were assessed by ANOVA-Dunnett. Normalized tissue weights were compared by ANOVA. Two-tailed statistical analyses were conducted at significance level P=0.05. Results were classified as statistically significant or not statistically significant.
[0140] The results of the study are shown below in Table 9:
TABLE-US-00011 TABLE 9 Results Gastroc. Lung % weight/ weight/ MTV % Lung Inhibi- normalized normalized Group (mm.sup.3) TGI foci tion PR CR (mg) (mg) 1 1800 N/A 70.4 N/A 0 0 353.4/19.68 2799.4/ 292.98 2 1568 13% 60.3 14% 0 0 330.4/21.62 2388.9/ 179.75 3 1688 6% 49.0 30% 0 0 398.6/24.91 2191.6/ 214.90
[0141] All treatment regimens were acceptably tolerated with no treatment-related deaths. The only animal deaths were non-treatment-related deaths due to metastasis. The % TGI trended towards significance (P>0.05, Mann-Whitney) for the 5 μg/g (Group 2) and 10 μg/g treatment group (Group 3). The % Inhibition trended towards significance (P>0.05, ANOVA-Dunnett) for the 5 μg/g treatment group. The % Inhibition was statistically significant (P≤0.01, ANOVA-Dunnett) for the 10 μg/g treatment group. The ability of the carboxymethyl lysine antibody to treat cachexia trended towards significance (P>0.05, ANOVA) based on a comparison of the organ weights of the lung and gastrocnemius between treatment groups and the control group. The results indicate that administration of an anti-carboxymethyl lysine monoclonal antibody is able to reduce cancer metastases. This data provides additional evidence that in vivo administration of anti-AGE antibodies can provide therapeutic benefits safely and effectively.
Example 7: Treatment of Glioma in a Canine Model
[0142] Dogs with spontaneous gliomas will be selected for treatment. Gliomas will be verified by magnetic resonance imaging (MRI) and evaluation of physical symptoms. The dogs will be divided into five treatment groups as shown in Table 10 below:
TABLE-US-00012 TABLE 10 Treatment groups Group Description Agent 1 Anti-AGE antibody Anti-CML antibody 2 Anti-AGE antibody Anti-CML antibody and low-frequency and ultrasound low intensity pulsed ultrasound 3 Control antibody Canine IgG1 antibody and low-frequency and ultrasound low intensity pulsed ultrasound 4 Ultrasound Low-frequency low intensity pulsed ultrasound 5 Control None
[0143] The anti-AGE antibody is a carboxymethyllysine antibody. The anti-AGE antibody is a chimeric antibody, caninized antibody or canine antibody. The control antibody is a canine IgG1 antibody (Novus Biologicals). The antibodies are administered at a dose of 10 mg/kg. The antibodies are administered intravenously at day 0 and once weekly for three weeks.
[0144] The ultrasound is administered simultaneously with the antibodies. The ultrasound is administered at a frequency of 5.7 MHz, an intensity of 90 mW/cm.sup.2 and a pulse repetition frequency (PRF) of 6.0 kHz. The ultrasound is administered by a probe positioned perpendicular to the temporal window of each dog's cranium.
[0145] At the end of the three-week administration regimen all groups are evaluated by MRI for the presence and amount of glioma tumor and for physical symptoms. The animals in group 2 (anti-AGE antibody and ultrasound) will have the least amount of glioma and the fewest physical symptoms, followed by the animals in group 1 (anti-AGE antibody), the animals in group 4 (ultrasound) and the animals in group 3 (control antibody and ultrasound). The animals in group 5 (control) will have no improvement in symptoms. The combination of ultrasound and anti-AGE antibody is able to access the restricted site of the brain and cross the blood-brain barrier to provide the most effective therapy.
Example 8: Treatment of Degenerative Joint Disease (Osteoarthritis) in a Canine Model
[0146] Dogs with spontaneous degenerative joint disease (osteoarthritis) in the stifle joint (the canine equivalent to the human knee) will be selected for treatment. Degenerative joint disease will be verified by magnetic resonance imaging (MRI) and evaluation of physical symptoms. The dogs will be divided into five treatment groups as shown in Table 11 below:
TABLE-US-00013 TABLE 11 Treatment groups Group Description Agent 1 Anti-AGE antibody Anti-CML antibody 2 Anti-AGE antibody Anti-CML antibody and low-frequency and ultrasound low intensity pulsed ultrasound 3 Control antibody Canine IgG1 antibody and low-frequency and ultrasound low intensity pulsed ultrasound 4 Ultrasound Low-frequency low intensity pulsed ultrasound 5 Control None
[0147] The anti-AGE antibody is a carboxymethyllysine antibody. The anti-AGE antibody is a chimeric antibody, caninized antibody or canine antibody. The control antibody is a canine IgG1 antibody (Novus Biologicals). The antibodies are administered at a dose of 10 mg/kg. The antibodies are administered intravenously at day 0 and once weekly for three weeks.
[0148] The ultrasound is administered simultaneously with the antibodies. The ultrasound is administered at a frequency of 5.7 MHz, an intensity of 90 mW/cm.sup.2 and a pulse repetition frequency (PRF) of 6.0 kHz. The ultrasound is administered by a probe positioned on the affected stifle joint.
[0149] At the end of the three-week administration regimen all groups are evaluated by MRI for the presence and amount of degenerative joint disease and for physical symptoms. The animals in group 2 (anti-AGE antibody and ultrasound) will have the least amount of degenerative joint disease and the fewest physical symptoms, followed by the animals in group 1 (anti-AGE antibody), the animals in group 4 (ultrasound) and the animals in group 3 (control antibody and ultrasound). The animals in group 5 (control) will have no improvement in symptoms. The combination of ultrasound and anti-AGE antibody is able to access the restricted site of the joint to provide the most effective therapy.
Example 9: In Vitro Administration of Antibodies and Ultrasound to Glioblastoma and Glioblastoma/Astrocytoma Cells
[0150] Cell lines of glioblastoma cells (CRL-1620) and glioblastoma/astrocytoma cells (HTB-15) were exposed to antibodies (AB912), followed by administration of ultrasound. AB912 binds to proliferating cell nuclear antigen (PCNA), which is a protein involved in the DNA replication process. Expression of PCNA is associated with proliferation or neoplastic transformation.
[0151] The antibody was applied to the cells at a final concentration of 0.2 mg/ml, 30 minutes prior to the ultrasound exposure. The cells were exposed to ultrasound for 1 hour, at a frequency of 1 Mhz at 0.4 w/cm.sup.2 and a 50% pulse. The base of the dish was approximately 3 mm from the ultrasound head. After exposure, the control group and the 1 hour without AB912 group were seeded at 2×10.sup.5 cells/cm.sup.2 and the 1 hour with AB912 group was seeded at 1.1×10.sup.5.
[0152] Approximately 90% of CRL-1620 cells bound with the antibody showed significant detachment from the culture dish. Only approximately 10% of the cells not bound with antibody detached from the culture plate. The samples were incubated at 37° C. and supplemented with 5% CO.sub.2 and allowed to culture for approximately 64 hours. After the incubation period, the cell cultures were observed under a microscope and no reattachment was observed. Several large cell clusters were observed floating in the media. These cells were manually disrupted and no viable cells were found. The results are shown in Table 12 below:
TABLE-US-00014 TABLE 12 Treatment of CRL-1620 cells Group Percentage of detached cells Control 10% 1 hr ultrasound 10% 1 hr ultrasound and antibody 90%
[0153] The same antibody administration and ultrasound administration procedures described above were repeated with HTB-15 cells. Approximately 85% of cells bound with the antibody showed significant detachment from the culture dish. Only approximately 10% of the cells not bound with the antibody detached from the culture plate. After the 64-hour incubation period, the cell cultures were observed under a microscope and no reattachment was observed. The results are shown in Table 13 below:
TABLE-US-00015 TABLE 13 Treatment of HTB-15 cells Group Percentage of detached cells Control 10% 1 hr ultrasound 10% 1 hr ultrasound and antibody 85%
[0154] After 24 hours, the cultures of both cell lines that were bound with the antibody, but did not have ultrasound administered to them showed no visible signs of toxicity. The cultures exposed to ultrasound but were not bound with the antibody also showed no toxicity. The cells that were exposed to the antibody and ultrasound did exhibit toxicity.
[0155] These results are a proof-of-principle that the administration of antibodies and ultrasound together may be used to kill cells. The combination therapy selectively targeted and killed glioblastoma and glioblastoma/astrocytoma cells, two types of cancer found in the restricted site of the brain when present in vivo. The results demonstrate that the combination of antibody and ultrasound administration results in enhanced cell killing as compared to administration of ultrasound alone.
Example 10—Immunofluorescent Staining of Glioma Cell Panel
[0156] Glioma cells from the ATCC glioma cell line panel were trypsinized in 0.25% trypsin EDTA for approximately five minutes, then washed and resuspended to approximately 10.sup.5 cells/mL. 100 μL aliquots of the suspension were transferred to a round bottom 96-well plate and centrifuged to remove the medium. Each cell line was resuspended to 200 μL of 0.1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) in phosphate-buffered saline (PBS) for 10 minutes, and then washed three times. The cells were then resuspended in 10 μg/mL of AB912-T2-17G6 antibody (a proliferating cell nuclear antigen (PCNA) antibody) in PBS and incubated at 2-8° C. for 30 minutes. The cells were rinsed three times in PBS then suspended in 10 μg/mL of anti-human IgG (H&L) conjugated with Texas Red (a secondary antibody). For each wash, cells were centrifuged at 200×g for five minutes and supernatant was aspirated from each well. Immunofluorescent detection was used to verify binding of the antibody to the glioma cells.
Example 11—In Vitro Anti-AGE Antibody Binding Studies
[0157] In vitro experiments investigated the binding of the humanized monoclonal anti-advanced glycation end-product (anti-AGE) antibody referred to as SIWA 318H (produced by SIWA Therapeutics, Inc.) to glioma and pancreatic cancer cells. SIWA 318H is known to bind to carboxymethyllysine-modified proteins. Binding was verified by immunofluorescent staining as described in Example 10.
[0158] Pancreatic cancer cells from the human pancreatic cancer PANC-1 cell line were exposed to SIWA 318H.
[0159] Glioma cells from the ATCC glioma cell line panel were exposed to SIWA 318H. SIWA 318H bound to all cell lines included in the panel (CRL-1620 human glioblastoma cells, HTB-12 human astrocytoma cells, HTB-148 human neuroglioma cells, HTB-15 human glioblastoma cells and HTB-14 human glioblastoma cells). Binding of SIWA 318H antibody to the glioma cells was confirmed by flow cytometry.
[0160] These results demonstrate that SIWA 318H, an anti-AGE antibody, binds to pancreatic cancer cells and glioma cells.
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