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AU2016336959A1 - Anti-age antibodies and methods of use thereof - Google Patents

Anti-age antibodies and methods of use thereof Download PDF

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AU2016336959A1
AU2016336959A1 AU2016336959A AU2016336959A AU2016336959A1 AU 2016336959 A1 AU2016336959 A1 AU 2016336959A1 AU 2016336959 A AU2016336959 A AU 2016336959A AU 2016336959 A AU2016336959 A AU 2016336959A AU 2016336959 A1 AU2016336959 A1 AU 2016336959A1
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Lewis S. Gruber
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Siwa Corp
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Abstract

An anti-AGE antibody comprises a protein or peptide that comprises at least one amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with specific amino acid sequences. The anti-AGE antibody binds to a protein or peptide that exhibits a carboxymethyllysine modification. The anti-AGE antibody may be used for killing senescent cells, killing partially-functional or non-functional cells, treating sarcopenia, promoting tissue or organ regeneration, promoting regenerative processes or overcoming aging effects, treating atherosclerosis, preventing or delaying the onset of cataracts, preventing or delaying the onset of loss of adipose tissue, increasing health span, preventing or delaying the onset of lordokyphosis, treating inflammation or autoimmune disorders, treating neurodegenerative disorders or treating cancer.

Description

invention is an anti-AGE antibody, comprising a protein or peptide comprising at least one amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 39. The antibody binds to a protein or peptide that exhibits a carboxymethyllysine modification.
[12] In a second aspect, the present invention is an anti-AGE antibody, comprising a protein or peptide comprising at least one amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with an amino acid sequence selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID
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NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28. The antibody binds to a protein or peptide that exhibits a carboxymethyllysine modification.
[13] In a third aspect, the present invention is an anti-AGE antibody comprising a heavy chain and a light chain. The heavy chain comprises an amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 29, SEQ ID NO:
31, and SEQ ID NO: 33, or the light chain comprises an amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 19, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 39. The antibody binds to a protein or peptide that exhibits a carboxymethyllysine modification.
[14] In a fourth aspect, the present invention is an anti-AGE antibody comprising a heavy chain and a light chain. The heavy chain comprises an amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 29, SEQ ID NO:
31, and SEQ ID NO: 33, and the light chain comprises an amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 19, SEQ ID NO: 35, SEQ ID NO:
37, and SEQ ID NO: 39. The antibody binds to a protein or peptide that exhibits a carboxymethyllysine modification.
[15] In a fifth aspect, the present invention is an anti-AGE antibody, comprising complementary determining region comprising at least one amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with an amino acid sequence selected from
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PCT/US2016/034880 the group consisting of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO:
26, SEQ ID NO: 27, and SEQ ID NO: 28. The antibody binds to a protein or peptide that exhibits a carboxymethyllysine modification. The antibody is substantially nonimmunogenic to a species selected from the group consisting of mice, rats, goats, sheep, cows, horses, dogs and cats.
[16] In a sixth aspect, the present invention is an antibody conjugate, comprising an anti-AGE antibody fragment comprising a protein or peptide comprising at least one amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 39, and an agent that causes the destruction of AGE-modified cells. The agent that causes the destruction of AGE-modified cells is conjugated to the anti-AGE antibody fragment. The antibody binds to a protein or peptide that exhibits a carboxymethyllysine modification.
[17] Definitions [18] The term “peptide” means a molecule composed of 2-50 amino acids.
[19] The term “protein” means a molecule composed of more than 50 amino acids.
[20] The term “sarcopenia” means the syndrome characterized by the presence of (1) low muscle mass and (2) low muscle function (low muscle strength or reduced physical performance). Muscle mass may be measured by body imaging techniques, such as computed tomography scanning (CT scan), magnetic resonance imaging (MRI) or dual energy X-ray absorptiometry (DXA or DEXA); bioimpedance analysis (BIA); body potassium measurement, such as total body potassium (TBK) or partial body potassium (PBK); or anthropometric measurements, such as mid-upper arm circumference, skin fold thickness or calf circumference. Preferably, muscle mass is measured by CT scan,
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MRI or DXA. Muscle strength may be measured by handgrip strength, knee flexion/extension or peak expiratory flow. Preferably, muscle strength is measured by handgrip strength. Physical performance may be measured by the Short Physical Performance Battery, gait speed measurement, timed get-up-and-go (TGUG) or the stair climb power test. Preferably, physical performance is measured by gait speed measurement. A subject may be identified as having sarcopenia or in need of treatment if (1) the subject is at least 25 years old and (2) his or her measured muscle mass and measured muscle function are two standard deviations or more below the mean value for healthy 25 year olds of the same gender and no alternative pathology has been identified to account for the reduced muscle mass and reduced muscle function. Preferably, a subject being treated for sarcopenia is at least 40 years old. More preferably, a subject being treated for sarcopenia is at least 50 years old. Most preferably, a subject being treated for sarcopenia is at least 60 years old. Alternatively, a subject may be identified as having sarcopenia or in need of treatment if (1) his or her gait speed is less than 1.0 m/s across a 4 m course and (2) he or she has an objectively measured low muscle mass, such as, for example, an appendicular mass relative to the square of height less than or equal to 7.23 kg/m2 for male subjects or less than or equal to 5.67 kg/m2 for female subjects (Fielding, R. A., et al., “Sarcopenia: an undiagnosed condition in older adults. Current consensus definition: prevalence, etiology, and consequences”, Journal of the American Medical Directors Association, Vol. 12(4), pp. 249-256 (May 2011).
The term “neurodegenerative disorder means disorders which result in neurons loosing function and/or dying, in the central nervous system including the brain. Such disorders included central nervous system neurodegenerative disorders such as AD,
PD, Lewy body dementia, MS, prion diseases (also known as transmissible spongiform encephalopathies (TSEs), including Creutzfeldt-Jakob disease, variant CreutzfeldtJakob disease, bovine spongiform encephalopathy (“mad cow” disease), scrapie (in sheep and goats), chronic wasting disease (in deer and elk), kuru and fatal familial insomnia), and ALS.
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PCT/US2016/034880 [22] “Neurodegenerative proteins” are proteins which accumulate in a patient having a neurodegenerative disorders and which are associated with the neurodegenerative disorder. Examples include, beta-amyloid protein plaques (associated with AD), tau protein tangles (associated with AD), mutated superoxide dismutase-1 (associated with ALS), prion protein aggregates (associated with TSEs) and alpha-synuclein protein fibrils (associated with PD and Lewy Body dementia). A “neurodegenerative protein” is the form of the protein which accumulates during the neurodegenerative disorder, typically a mutant or mis-folded form.
[23] The terms “advanced glycation end-product,” “AGE,” “AGE-modified protein or peptide,” “glycation end-product” and “AGE antigen” refer to modified proteins or peptides that are formed as the result of the reaction of sugars with protein side chains that further rearrange and form irreversible cross-links. This process begins with a reversible reaction between a reducing sugar and an amino group to form a Schiff base, which proceeds to form a covalently-bonded Amadori rearrangement product. Once formed, the Amadori product undergoes further rearrangement to produce AGEs. AGEmodified proteins and antibodies to AGE-modified proteins are described in U.S. 5,702,704 to Bucala (“Bucala”) and U.S. 6,380,165 to Al-Abed et al. (“Al-Abed”). Glycated proteins or peptides that have not undergone the necessary rearrangement to form AGEs, such as N-deoxyfructosyllysine found on glycated albumin, are not AGEs. AGEs may be identified by the presence of AGE modifications (also referred to as AGE epitopes or AGE moieties) such as 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI); 5hydroxymethyl-1-alkylpyrrole-2-carbaldehyde (Pyrraline); 1-alkyl-2-formyl-3,4diglycosyl pyrrole (AFGP), a non-fluorescent model AGE; carboxymethyllysine; and pentosidine. ALI, another AGE, is described in Al-Abed.
[24] “An antibody that binds to an AGE-modified protein on a cell”, “anti-AGE antibody” or “AGE antibody” means an antibody, antibody fragment or other protein or peptide that binds to an AGE-modified protein or peptide which preferably includes a constant region of an antibody, where the protein or peptide which has been AGE-10WO 2017/065837
PCT/US2016/034880 modified is a protein or peptide normally found bound on the surface of a cell, preferably a mammalian cell, more preferably a human, cat, dog, horse, camelid (for example, camel or alpaca), cattle, sheep, or goat cell. “An antibody that binds to an AGEmodified protein on a cell”, “anti-AGE antibody” or “AGE antibody” does not include an antibody or other protein which binds with the same specificity and selectivity to both the AGE-modified protein or peptide, and the same non-AGE-modified protein or peptide (that is, the presence of the AGE modification does not increase binding). AGEmodified albumin is not an AGE-modified protein on a cell, because albumin is not a protein normally found bound on the surface of cells. “An antibody that binds to an AGE-modified protein on a cell”, “anti-AGE antibody” or “AGE antibody” only includes those antibodies which lead to removal, destruction, or death of the cell. Also included are antibodies which are conjugated, for example to a toxin, drug, or other chemical or particle. Preferably, the antibodies are monoclonal antibodies, but polyclonal antibodies are also possible.
[25] The term “senescent cell” means a cell which is in a state of irreversible proliferative arrest and expresses one or more biomarkers of senescence, such as activation of p16lnk4a or expression of senescence-associated β-galactosidase. Also included are cells which express one or more biomarkers of senescence, do not proliferate in vivo, but may proliferate in vitro under certain conditions, such as some satellite cells found in the muscles of ALS patients.
[26] The term “increasing health span” means reducing age-related phenotypes. Age-related phenotypes include, for example, sarcopenia, cataracts, loss of adipose tissue and lordokyphosis.
[27] The term “variant” means a nucleotide, protein or amino acid sequence different from the specifically identified sequences, wherein one or more nucleotides, proteins or amino acid residues is deleted, substituted or added. Variants may be naturallyoccurring allelic variants, or non-naturally-occurring variants. Variants of the identified
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PCT/US2016/034880 sequences may retain some or all of the functional characteristics of the identified sequences.
[28] The term percent (%) sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Preferably, % sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is publicly available from Genentech, Inc. (South San Francisco, CA), or may be compiled from the source code, which has been filed with user documentation in the U.S. Copyright Office and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
[29] In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. Where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.
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Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program.
Brief Description Of The Drawing [30] FIG. 1 is a graph of the response versus time in an antibody binding experiment.
Detailed Description [31] The identification of a link between cellular senescence and sarcopenia allows for new treatment possibilities. For example, if anti-AGE antibodies are administered to a subject, the antibodies will specifically and selectively target senescent cells, and kill or induce apoptosis in such cells expressing an AGE-modified protein or peptide.
[32] The present invention makes use of the discovery that enhanced clearance of cells expressing AGE-modified proteins or peptides (AGE-modified cells) is beneficial in treating or ameliorating sarcopenia. This may be accomplished by administering antiAGE antibodies to a subject.
[33] Administering anti-AGE antibodies to a subject may also be used for increasing health span. Health span may be increased by reducing age-related phenotypes. Administering anti-AGE antibodies may be used, for example, to prevent or delay the onset of cataracts, lordokyphosis or loss of adipose tissue.
[34] Other diseases or disorders that are associated with cellular senescence may also be treated or ameliorated with anti-AGE antibodies. For example, anti-AGE antibodies may be used therapeutically to treat neurodegenerative disorders or cancer.
[35] An antibody that binds to an AGE-modified protein on a cell (“anti-AGE antibody” or “AGE antibody”) is known in the art. Examples include those described in U.S. 5,702,704 (Bucala) and U.S. 6,380,165 (Al-Abed et al.). Examples include an antibody that binds to one or more AGE-modified proteins having an AGE modification such as
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PCT/US2016/034880 [36]
FFI, pyrraline, AFGP, ALI, carboxymethyllysine, carboxyethyllysine and pentosidine, and mixtures of such antibodies. Preferably, the antibody binds carboxymethyllysinemodified proteins. 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, 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), 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 or a goat antibody. Details of the constant regions and other parts of antibodies for these animals are described below. Preferably, the antibody is a monoclonal antibody.
A particularly preferred anti-AGE antibody is an antibody which binds to a protein or peptide that exhibits a carboxymethyllysine modification. Carboxymethyllysine (also known as CML, N(epsilon)-(carboxymethyl)lysine, N(6)-carboxymethyllysine, or 2Amino-6-(carboxymethylamino)hexanoic acid) is found on proteins or peptides and lipids as a result of oxidative stress and chemical glycation, and has been correlated with aging. CML-modified proteins or peptides are recognized by the receptor RAGE which is expressed on a variety of cells. CML has been well-studied and CML-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). A particularly 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, MN; catalog no. MAB3247), modified to have a human constant region (or
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PCT/US2016/034880 the constant region of the animal into which it will be administered). Commerciallyavailable 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.
[37] The anti-AGE antibody has low rate of dissociation from the antibody-antigen complex, or kd (also referred to as kback or off-rate), preferably at most 9 x 10'3, 8 x 10'3, 7 x 10'3 or 6 x 10'3 (sec'1). The anti-AGE antibody has a high affinity for the AGEmodified protein of a cell, which may be expressed as a low dissociation constant KD of at most 9 x 10'6, 8 x 10'6, 7 x 10'6, 6 x 10'6, 5 x 10'6, 4 x 10'6 or 3 x 10'6 (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, MN; catalog no. MAB3247), illustrated in FIG. 1.
[38] The anti-AGE antibody may destroy AGE-modified cells through antibodydependent 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.
[39] 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.
[40] A toxin, such as pore-forming toxins (PFT) (Aroian R. et al., “Pore-Forming Toxins and Cellular Non-lmmune 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
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PCT/US2016/034880 and binds to AGE-modified cells. Then, the toxin causes pore formation at the cell surface and subsequent cell removal through osmotic lysis.
[41] 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.
[42] 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 AGEmodified cell types, without removing other cells.
[43] Antibodies typically comprise two heavy chains and two light chains of polypeptides joined to form a Ύ shaped molecule. The constant region determines the mechanism used to target the antigen. The amino acid sequence in the tips of the Y (the variable region) varies among different antibodies. This variation gives the antibody its specificity for binding antigen. The variable region, which includes the ends of the light and heavy chains, is further subdivided into hypervariable (HV - also sometimes referred to as complementarity determining regions, or CDRs) and framework (FR) regions. When antibodies are prepared recombinantly, it is also possible to have a single antibody with variable regions (or complementary determining regions) that bind to two different antigens, with each tip of the Ύ” being specific to each antigen; these are referred to as bi-specific antibodies.
[44] 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
-16WO 2017/065837
PCT/US2016/034880 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).
[45] The heavy chain of human (Homo sapiens) antibody immunoglobulin G1 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 human (Homo sapiens) antibody immunoglobulin G1 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 humanization of non-human antibodies.
[46] 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
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PCT/US2016/034880 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.
[47] 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.
[48] 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
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PCT/US2016/034880
NO: 5 (EMBL/GenBank 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 (EMBL/GenBank 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).
[49] 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).
[50] 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
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PCT/US2016/034880 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.
[51] 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).
[52] 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 or SEQ ID NO:
20, 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.
[53] 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 or SEQ ID NO:
21, 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.
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PCT/US2016/034880 [54] 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, MN; catalog no. MAB3247).
[55] The antibody may have or may include constant regions which permit destruction of targeted cells by a subject’s immune system.
[56] Mixtures of antibodies that bind to more than one type AGE of AGE-modified proteins may also be used.
[57] 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.
[58] 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’)2 fragments. F(ab’)2 fragments include both light chains and the two Nterminal 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 (CH3) to produce a large fragment F(abc) and a small fragment pFc’. Antibody fragments may alternatively be produced recombinantly.
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PCT/US2016/034880 [59]
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, AGEcollagen, AGE-cathepsin B, AGE-albumin, 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-l and II, AGE-hemoglobin, AGE-Na+/K+-ATPase, AGE-plasminogen, AGE-myelin, AGE-lysozyme, AGE-immunoglobulin, AGE-red cell Glu transport protein, AGE-P-N-acetyl hexominase, AGE-apo E, AGE-red cell membrane protein, AGE-aldose reductase, AGE-ferritin, AGE-red cell spectrin, AGE-alcohol dehydrogenase, AGEhaptoglobin, AGE-tubulin, AGE-thyroid hormone, AGE-fibrinogen, AGE-p2microglobulin, AGE-sorbitol dehydrogenase, AGE-ai-antitrypsin, AGE-carbonate dehydratase, AGE-RNAse, AGE-low density lipoprotein, AGE-hexokinase, AGE-apo ΟΙ, AGE-RNAse, AGE-hemoglobin such as AGE-human hemoglobin, AGE-albumin such as AGE-bovine serum albumin (AGE-BSA) and AGE-human serum albumin, AGE-low density lipoprotein (AGE-LDL) and AGE-collagen IV. AGE-modified cells, such as AGEmodified 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, a2macroglobulin 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. Alternatively, pAbs may be made in chickens, producing IgY molecules.
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PCT/US2016/034880 [60] 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. Techniques for the generation of chimeric antibodies by splicing genes encoding the variable domains of antibodies to genes of the constant domains of human (or other animal) immunoglobulin result in chimeric antibodies that are substantially human (humanized) or substantially “ized” to another animal (such as cat, dog, horse, camel or alpaca, cattle, sheep, 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, hydroxylapatite 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 lg loci or using human-transgenic mice. Production of humanized monoclonal antibodies and fragments thereof can also be generated through phage display technologies.
[61] 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 bisulfite; 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
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PCT/US2016/034880 be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
[62] 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. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL® (BASF; Parsippany, NJ) 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 manitol, 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.
[63] 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.
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PCT/US2016/034880 [64] 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.
[65] A subject that receives administration of an anti-AGE antibody may be tested to determine if it has been effective to treat the sarcopenia, by measuring changes in muscle mass over time. For example, a baseline muscle mass in a subject may be measured followed by administration of the anti-AGE antibody. The effectiveness of the treatment may be determined by periodically measuring muscle mass in the subject and comparing the subsequent measurements to the baseline measurement. A subject may be considered to have effective treatment of sarcopenia if he or she does not demonstrate loss of muscle mass between subsequent measurements or over time. Alternatively, the concentration and/or number of senescent cells in fat or muscle tissue may also be monitored. Administration of antibody and subsequent testing may be repeated until the desired therapeutic result is achieved.
[66] 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.
[67] Any mammal that could develop sarcopenia or other diseases or disorders associated with cellular senescence may be treated by the methods herein described. Humans are a preferred mammal for treatment. Other mammals that may be treated
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PCT/US2016/034880 include mice, rats, goats, sheep, cows, horses and companion animals, such as dogs or cats. A subject in need of treatment may be identified by the diagnosis of a disease or disorder that is known to cause elevated levels of AGEs such as, for example, diabetes (both Type 1 and Type 2), or the presence of a pathological condition associated with AGEs such as, for example, atherosclerosis, inflammation, retinopathy, nephropathy, stroke, endothelial cell dysfunction, neurodegenerative disorders or cancer. In addition, subjects may be identified for treatment based on their age. For example, a human over 75 years of age may be treated for sarcopenia, while a human under 30 years of age might not be identified as in need of treatment for sarcopenia. Alternatively, any of the mammals or subjects identified above may be excluded from the patient population in need of treatment for sarcopenia.
[68] A subject may be identified as having sarcopenia or in need of treatment if (1) the subject is at least 25 years old and (2) his or her measured muscle mass and measured muscle function are two standard deviations or more below the mean value for healthy 25 year olds of the same gender and no alternative pathology has been identified to account for the reduced muscle mass and reduced muscle function. Preferably, a subject being treated for sarcopenia is at least 40 years old. More preferably, a subject being treated for sarcopenia is at least 50 years old. Most preferably, a subject being treated for sarcopenia is at least 60 years old. Alternatively, a subject may be identified as having sarcopenia or in need of treatment if (1) his or her gait speed is less than 1.0 m/s across a 4 m course and (2) he or she has an objectively measured low muscle mass, such as, for example, an appendicular mass relative to the square of height less than or equal to 7.23 kg/m2 for male subjects or less than or equal to 5.67 kg/m2 for female subjects.
[69] In the case of central nervous system neurodegenerative disorders, it may be preferable to administer a composition containing the anti-AGE antibody directly into the central nervous system. Examples of such administration include intrathecal administration; administration into the ventricular system of the brain (intraventricular
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PCT/US2016/034880 [70] administration), for example, through a catheter or a permanent shunt, or other administration device which may be placed during a ventriculostomy (see, for example, Takami, A. et al. “Treatment of primary central nervous system lymphoma with induction of complement-dependent cytotoxicity by intraventricular administration of autologousserum-supplemented rituximab”, Cancer Sci. Vol. 97, pp. 80-83 (January 2006)); and administered by convection enhanced delivery (CED) (see, for example, Chen, K.S., et al. “MONOCLONAL ANTIBODY THERAPY FOR MALIGNANT GLIOMA” chapter 10 of Glioma: Immunotherapeutic Approaches, pp. 132-141 (ed. R. Yamanaka; Landes Bioscience and Springer Science+Business Media, 2012)). All such central nervous system administration may optionally also include administration of a serum supplement (such as autologous serum), to enhance the cell killing properties of the AGE antibody; administration of serum supplement may be prior to, simultaneous with, or subsequent to, the administration of the AGE antibody. Optionally, any of the composition containing AGE antibodies described herein may further contain a serum supplement (such as an autologous serum supplement). In place of a serum supplement, or in addition to a serum supplement, purified immune system cells may also be used, either autologous immune system cells, or immune system cells from a donor; examples of such cells include natural killer cells. In addition to, or instead of, the patient’s or a donor’s natural killer cells, artificial natural killer cells such as those of NANTKWEST®, engineered to bind directly to antibodies, or engineered to bind directly to an AGE antigen (such as carboxymethyllysine) (seewww.nantkwest.com).
In the case of cancer, a mammal that could develop metastatic cancer 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, cows, horses and companion animals, such as dogs or cats. A subject in need of treatment may be identified by the diagnosis of a cancer. Cancers which are particularly subject to metastasis include 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, and stomach
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PCT/US2016/034880 [71] cancer. Treatment may be of patients experiencing metastatic cancer. Treatment may also be administered to patients who have cancer, but prior to any identified metastasis, in order to prevent metastasis. A subject that receives administration of an anti-AGE antibody may be tested to determine if it has been effective to treat the cancer by examining the patient for the spread of the cancer to different parts of the body, particularly in lymph nodes. Administration of antibody and subsequent testing may be repeated until the desired therapeutic result is achieved.
The anti-AGE antibodies may be used in cellular purification processes, such as immunopanning and immunoadsorption. Purification processes are useful in isolating desirable or unwanted cells from tissue cultures, cell cultures or blood. Cellular purification may be used in transplantations, such as a bone marrow transplant, or transfusions, such as a blood transfusion. Cellular purification is especially useful in autologous stem cell transplantation during chemotherapy to remove malignant cells and concentrate beneficial stem cells, such as hematopoietic cells expressing the CD34 protein (CD34+ cells). Immunopanning or immunoadsorption using an anti-AGE antibody may isolate partially-functional or non-functional cells, such as senescent cells, from a tissue culture, cell culture or blood sample. For example, an immunopanning process may involve immobilizing the anti-AGE antibody on a surface, such as a cell culture plate. A tissue culture or cell culture may then be applied to the surface. Any senescent cells present in the tissue culture or cell culture will bind to the anti-AGE antibody, leaving a purified tissue culture or cell culture that is free from senescent cells. Similarly, an immunoadsorption process may involve binding the anti-AGE antibody to senescent cells in a cell culture. The cells may then be passed through a column packed with beads that are coated with a protein that binds to the anti-AGE labeled senescent cells. The cells which pass through the column without binding will be cells that do not express AGE, such as fully-functional cells. An immunoadsorption process may be carried out with a CEPRATE SC Stem Cell Concentration System (CellPro, Inc., Bothell, WA) or similar apparatus.
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PCT/US2016/034880 [72] The one-letter amino acid sequence that corresponds to SEQ ID NO: 1 is shown below:
20 30 40 50
MNLLLILTFV AAAVAQVQLL QPGAELVKPG ASVKLACKAS GYLFTTYWMH
70 80 90
WLKQRPGQGL EWIGEISPTN GRAYYNARFK SEATLTVDKS
100 110 120 130
SNTAYMQLSS LTSEASAVYY CARAYGNYEF AYWGQGTLVT
140 150 160 170
VSVASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV
180 190 200 210 220
TVSWNSGALT SGVHTFPAVL QSSGLYSLSS WTVPSSSLG TQTYICNVNH
230 240 250 260
KPSNTKVDKK VEPKSCDKTH TCPPCPAPEL LGGPSVFLFP 270 280 290 300
PKPKDTLMIS RTPEVTCVW DVSHEDPEVK FNWYVDGVEV
310 320 330 340
HNAKTKPREE QYNSTYRWS VLTVLHQDWL NGKEYKCKVS
350 360 370 380 390
NKALPAPIEK TISKAKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP
400 410 420 430
SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK
440 450 460
SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK [73] Positions 16-133 of the above amino acid sequence correspond to SEQ ID NO: 2. Positions 46-50 of the above amino acid sequence correspond to SEQ ID NO: 41. Positions 65-81 of the above amino acid sequence correspond to SEQ ID NO: 42. Positions 114-122 of the above amino acid sequence correspond to SEQ ID NO: 43.
[74] The one-letter amino acid sequence that corresponds to SEQ ID NO: 3 is shown below:
SUBSTITUTE SHEET (RULE 26)
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PCT/US2016/034880
20 30 40 50
MNLLLILTFV AAAVADWMT QTPLSLPVSL GDQASISCRS RQSLVNSNGN
70 80 90 100
TFLQWYLQKP GQSPKLLIYK VSLRFSGVPD RFSGSGSGTD FTLKISRVEA
110 120 130 140 150
EDLGLYFCSQ STHVPPTFGG GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA
160 170 180 190
SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD
200 210 220 230
STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC [75] Positions 16-128 of the above amino acid sequence correspond to SEQ ID NO: 4. Optionally, the arginine (Arg or R) residue at position 128 of SEQ ID NO: 4 may be omitted. Positions 39-54 of the above amino acid sequence correspond to SEQ ID NO: 44. Positions 70-76 of the above amino acid sequence correspond to SEQ ID NO: 45. Positions 109-117 of the above amino acid sequence correspond to SEQ ID NO: 46.
[76] The DNA sequence that corresponds to SEQ ID NO: 12 is shown below:
ATGGACCCCAAGGGCAGCCTGAGCTGGAGAATCCTGCTGTTCCTGAGC
CTGGCCTTCGAGCTGAGCTACGGCCAGGTGCAGCTGCTGCAGCCAGGT
GCCGAGCTCGTGAAACCTGGCGCCTCTGTGAAGCTGGCCTGCAAGGCT
TCCGGCTACCTGTTCACCACCTACTGGATGCACTGGCTGAAGCAGAGGC
CAGGCCAGGGCCTGGAATGGATCGGCGAGATCTCCCCCACCAACGGCA
GAGCCTACTACAACGCCCGGTTCAAGTCCGAGGCCACCCTGACCGTGG
ACAAGTCCTCCAACACCGOCTACATGCAGCTGTCCTCCCTGACCTCTGA
GGCCTCCGCCGTGTACTACTGCGCCAGAGCTTACGGCAACTACGAGTTC
GCCTACTGGGGCCAGGGCACCCTCGTGACAGTGTCTGTGGCTAAGACC
ACCCCTCCCTCCGTGTACCCTCTGGCTCCTGGCTGTGGCGACACCACCG
GATCCTCTGTGACCCTGGGCTGCCTCGTGAAGGGCTACTTCCCTGAGTC
CGTGACCGTGACCTGGAACTCCGGCTCCCTGTCCTCCTCCGTGCACACC
SUBSTITUTE SHEET (RULE 26)
WO 2017/065837
PCT/US2016/034880
TTTCCAGCCCTGCTGCAGTCCGGCCTGTACACCATGTCCTCCAGCGTGA
CAGTGCCCTCCTCCACCTGGCCTTCCCAGACCGTGACATGCTCTGTGGC
CCACCCTGCCTCTTCCACCACCGTGGACAAGAAGCTGGAACCCTCCGGC
CCCATCTCCACCATCAACCCTTGCCCTCCCTGCAAAGAATGCCACAAGT
GCCCTGCCCCCAACCTGGAAGGCGGCCCTTCCGTGTTCATCTTCCCACC
CAACATCAAGGACGTGCTGATGATCTCCCTGACCCCCAAAGTGACCTGC
GTGGTGGTGGACGTGTCCGAGGACGACCCTGACGTGCAGATCAGTTGG
TTCGTGAACAACGTGGAAGTGCACACCGCCCAGACCCAGACACACAGAG
AGGACTACAACAGCACCATCAGAGTGGTGTCTACCCTGCCCATCCAGCA
CCAGGACTGGATGTCCGGCAAAGAATTCAAGTGCAAAGTGAACAACAAG
GACCTGCCCAGCCCCATCGAGCGGACCATCTCCAAGATCAAGGGCCTC
GTGCGGGCTCCCCAGGTGTACATTCTGCCTCCACCAGCCGAGCAGCTG
TCCCGGAAGGATGTGTCTCTGACATGTCTGGTCGTGGGCTTCAACCCCG
GCGACATCTCCGTGGAATGGACCTCCAACGGCCACACCGAGGAAAACTA
CAAGGACACCGCCCCTGTGCTGGACTCCGACGGCTCCTACTTCATCTAC
TCCAAGCTGAACATGAAGACCTCCAAGTGGGAAAAGACCGACTCCTTCT
CCTGCAACGTGCGGCACGAGGGCCTGAAGAACTACTACCTGAAGAAAAC
CATCTCCCGGTCCCCCGGCTAG [77] The DNA sequence that corresponds to SEQ ID NO: 13 is shown below:
ATGGACCCCAAGGGCAGCCTGAGCTGGAGAATCCTGCTGTTCCTGAGC
CTGGCCTTCGAGCTGAGCTACGGCCAGGTGCAGCTGCTGCAGCCAGGT
GCCGAGCTCGTGAAACCTGGCGCCTCTGTGAAGCTGGCCTGCAAGGCT
TCCGGCTACCTGTTCACCACCTACTGGATGCACTGGCTGAAGCAGAGGC
CAGGCCAGGGCCTGGAATGGATCGGCGAGATCTCCCCCACCAACGGCA
GAGCCTACTACAACGCCCGGTTCAAGTCCGAGGCCACCCTGACCGTGG
ACAAGTCCTCCAACACCGCCTACATGCAGCTGTCCTCCCTGACCTCTGA
GGCCTCCGCCGTGTACTACTGCGCCAGAGCTTACGGCAACTACGAGTTC
GCCTACTGGGGCCAGGGCACCCTCGTGACAGTGTCTGTGGCTAGCACC
AAGGGCCCCAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACCAGC
GGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAG
CCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTGCAC
SUBSTITUTE SHEET (RULE 26)
WO 2017/065837
PCT/US2016/034880
ACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGC
GTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGC
AACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAG
CCTAAGAGCTGCGACAAGACCCACACCTGCCCTCCCTGCCCCGCCCCC
GAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCTCCCAAGCCCAAG
GACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTG
GACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGAC
GGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTAC
AACTCCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGAC
TGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTG
CCCGCTCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGG
GAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCTGACCAAG
AACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACA
TCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGA
CCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAA
GCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTG
CAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCT
GAGCCTGAGCCCCGGATAG [78] The DNA sequence that corresponds to SEQ ID NO: 14 is shown below:
ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCOC
GGCTCCACCGGAGACGTCGTGATGACCCAGACCCCTCTGTCCCTGCCT
GTGTCTCTGGGCGACCAGGCCTCCATCTCCTGCCGGTCTAGACAGTCCC
TCGTGAACTCCAACGGCAACACCTTCCTGCAGTGGTATCTGCAGAAGCC
CGGCCAGTCCCCCAAGCTGCTGATCTACAAGGTGTCCCTGCGGTTCTCC
GGCGTGCCCGACAGATTTTCCGGCTCTGGCTCTGGCACCGACTTCACCC
TGAAGATCTCCCGGGTGGAAGCCGAGGACCTGGGCCTGTACTTCTGCA
GCCAGTCCACCCACGTGCCCCCTACATTTGGCGGAGGCACCAAGCTGG
AAATCAAACGGGCAGATGCTGCACCAACTGTATCCATCTTCCCACCATCC
AGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACA
ACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAA
CGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACA
SUBSTITUTE SHEET (RULE 26)
WO 2017/065837
PCT/US2016/034880
GCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGA
ACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCAC
CCATTGTCAAGAGCTTCAACAGGAATGAGTGTTGA [79] The DNA sequence that corresponds to SEQ ID NO: 15 is shown below:
ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCC
GGCTCCACCGGAGACGTCGTGATGACCCAGACCCCTCTGTCCCTGCCT
GTGTCTCTGGGCGACCAGGCCTCCATCTCCTGCCGGTCTAGACAGTCCC
TCGTGAACTCCAACGGCAACACCTTCCTGCAGTGGTATCTGCAGAAGCC
CGGCCAGTCCCCCAAGCTGCTGATCTACAAGGTGTCCCTGCGGTTCTCC
GGCGTGCCCGACAGATTTTCCGGCTCTGGCTCTGGCACCGACTTCACCC
TGAAGATCTCCCGGGTGGAAGCCGAGGACCTGGGCCTGTACTTCTGCA
GCCAGTCCACCCACGTGCCCCCTACATTTGGCGGAGGCACCAAGCTGG
AAATCAAGCGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAG
CGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAA
CAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGC
CCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAA
GGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGA
CTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACT
GTCTAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCTAA [80] The one-letter amino acid sequence that corresponds to SEQ ID NO:
is shown below:
MDPKGSLSWRILLFLSLAFELSYGQVQLLQPGAELVKPGASVKLACKASGYL
FTTYWMHWLKQRPGQGLEWIGEISPTNGRAYYNARFKSEATLTVDKSSNTA
YMQLSSLTSEASAVYYCARAYGNYEFAYWGQGTLVTVSVAKTTPPSVYPLA
PGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYT
MSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKEC
HKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVWDVSEDDPDVQISWFV
NNVEVHTAQTQTHREDYNSTIRWSTLPIQHQDWMSGKEFKCKVNNKDLPS
PIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLWGFNPGDISVEWTSN
32-1
SUBSTITUTE SHEET (RULE 26)
WO 2017/065837
PCT/US2016/034880
GHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNY
YLKKTISRSPG* [81] The shaded region of the above amino acid sequence corresponds to SEQ ID NO: 20.
[82] The one-letter amino acid sequence that corresponds to SEQ ID NO: 17 is shown below:
MDPKGSLSWRILLFLSLAFELSYGQVQLLQPGAELVKPGASVKLACKASGYL
FTTYWMHWLKQRPGQGLEWIGEISPTNGRAYYNARFKSEATLTVDKSSNTA
YMQLSSLTSEASAVYYCARAYGNYEFAYWGQGTLVTVSVASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY
SLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVE
VHN AKTKPREEQYN STYRWSVLTVLH Q DWLNG KEYKC KVS N KALPAPIEK
TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY
TQKSLSLSPG* [83] The one-letter amino acid sequence that corresponds to SEQ ID NO:
is shown below:
METDTLLLVWLLLWVPGSTGDWMTQTPLSLPVSLGDQASISCRSRQSLVN
SNGNTFLQWYLQKPGQSPKLLIYKVSLRFSGVPDRFSGSGSGTDFTLKISRV
EAEDLGLYFCSQSTHVPPTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGA
SWCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTL
TKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC* [84] The shaded region of the above amino acid sequence corresponds to SEQ ID NO: 21.
[85] The one-letter amino acid sequence that corresponds to SEQ ID NO:
is shown below:
32-2
SUBSTITUTE SHEET (RULE 26)
WO 2017/065837
PCT/US2016/034880
METDTLLLVWLLLVWPGSTGDWMTQTPLSLPVSLGDQASISCRSRQSLVN
SNGNTFLQWYLQKPGQSPKLLIYKVSLRFSGVPDRFSGSGSGTDFTLKISRV
EAEDLGLYFCSQSTHVPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA
SWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* [86] The one-letter amino acid sequence that corresponds to SEQ ID NO:
is shown below:
20 30 40 50
ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV
70 80 90 100
HTFPAVLQSS GLYSLSSWT VPSSNFGTQT YTCNVDHKPS NTKVDKTVER
110 120 130 140 150
KCCVECPPCP APPVAGPSVF LFPPKPKDTL MISRTPEVTC VWDVSHEDP
160 170 180 190
EVQFNWYVDG VEVHNAKTKP REEQFNSTFR WSVLTWHQ
200 210 220 230 240
DWLNGKEYKC KVSNKGLPAP IEKTISKTKG QPREPQVYTL PPSREEMTKN 250 260 270 280 290
QVSLTCLVKG FYPSDISVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT
300 310 320
VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK
32-3
SUBSTITUTE SHEET (RULE 26)
WO 2017/065837
PCT/US2016/034880 [87] The one-letter amino acid sequence that corresponds to SEQ ID NO: 23 is SYTMGVS.
[88] The one-letter amino acid sequence that corresponds to SEQ ID NO: 24 is TISSGGGSTYYPDSVKG.
[89] The one-letter amino acid sequence that corresponds to SEQ ID NO: 25 is QGGWLPPFAX, where X may be any naturally occurring amino acid.
[90] The one-letter amino acid sequence that corresponds to SEQ ID NO: 26 is RASKSVSTSSRGYSYMH.
[91] The one-letter amino acid sequence that corresponds to SEQ ID NO: 27 is LVSNLES.
[92] The one-letter amino acid sequence that corresponds to SEQ ID NO: 28 is QHIRELTRS.
[93] The one-letter amino acid sequence that corresponds to SEQ ID NO: 29 is MDPKGSLSWRILLFLSLAFELSYGQVQLVQSGAEVKKPGASVKVSCKASGYLFTTYW MHWVRQAPGQGLEWMGEISPTNGRAYYNQKFQGRVTMTVDKSTNTVYMELSSLRS EDTAVYYCARAYGNYFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG.
[94] The DNA sequence that corresponds to SEQ ID NO: 30 is ATGGACCCCAAGGGCAGCCTGAGCTGGAGAATCCTGCTGTTCCTGAGCCTGGCCT TCGAGCTGAGCTACGGCCAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGA
-33WO 2017/065837
PCT/US2016/034880 [95]
AACCTGGCGCCTCCGTGAGGTGTCCTGCAAGGCTTCCGGCTACCTGTTCACCACC
TACTGGATGCACTGGGTGCGACAGGCCCCTGGACAGGGCCTGGAATGGATGGGC
GAGATCTCCCCTACCAACGGCAGAGCCTACTACAACAGAAATTCCAGGGCAGAGT
GACCATGACCGTGGACAAGTCCACCAACACCGTGTACATGGAACTGTCCTCCCTG
CGGAGCGAGGACACCGCCGTGTACTACTGCGCTAGAGCCTACGGCAACTACGATT
CGCCTACTGGGGCCAGGGCACCCTCGTGACAGTGTCCTCTGCTAGCACCAAGGG
CCCCAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGAACCGC
CGCCCTGGGCTGCCTGGGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGG
AACAGCGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCCGTGCTGCAGAGC
AGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCAGCAGCAGCCTGGGCA
CCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAA
GAAGGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGCCCTCCCTGCCCCGC
CCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGAC
ACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGC
CACGAGGACCCCGAGGTGAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACA
ACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTACCGCGTGGTGA
GCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAGGAGTACAAGTGCAA
GGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAGGCCAA
GGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCT
GACAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACA
TCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCC
CTCCCGTGCTGGACAGCGACGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGAC
AAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCC
CTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCGGATAGTAA.
The one-letter amino acid sequence that corresponds to SEQ ID NO: 31 is MDPKGSLSWRILLFLSLAFELSYGQVQLVQSGAEVKKPGASVKVSCKASGYLFTTYW MHWVRQAPGQGLEWMGEISPTNGRAYYNAKFQGRVTMTVDKSTNTAYMELSSLRSE DTAVYYCARAYGNYFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNH
-34WO 2017/065837
PCT/US2016/034880 [96]
KPSNTKVDKKVEPKSCDKTHTCPPCPPELLGGPSVFLFPPKPKDTLMISRTPEVTCW
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH
YTQKSLSLSPG.
The DNA sequence that corresponds to SEQ ID NO: 32 is ATGGACCCCAAGGGCAGCCTGAGCTGGAGAATCCTGCTGTTCCTGAGCCTGGCCT TCGAGCTGAGCTACGGCCAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGA AACCTGGCGCCTCCGTGAGGTGTCCTGCAAGGCTTCCGGCTACCTGTTCACCACC TACTGGATGCACTGGGTGCGACAGGCCCCTGGACAGGGCCTGGAATGGATGGGC GAGATCTCCCCTACCAACGGCAGAGCCTACTACAACCAAAATTCCAGGGCAGAGT GACCATGACCGTGGACAAGTCCACCAACACCGCTTACATGGAACTGTCCTCCCTG CGGAGCGAGGACACCGCCGTGTACTACTGCGCTAGAGCCTACGGCAACTACGATT CGCCTACTGGGGCCAGGGCACCCTCGTGACAGTGTCCTCTGCTAGCACCAAGGG CCCCAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGAACCGC CGCCCTGGGCTGCCTGGGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGG AACAGCGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCCGTGCTGCAGAGC AGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCAGCAGCAGCCTGGGCA CCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAA GAAGGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGCCCTCCCTGCCCCGC CCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGAC ACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGC CACGAGGACCCCGAGGTGAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACA ACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTACCGCGTGGTGA GCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAGGAGTACAAGTGCAA GGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAGGCCAA GGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCT GACAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACA TCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCC
-35WO 2017/065837
PCT/US2016/034880
CTCCCGTGCTGGACAGCGACGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGAC
AAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCC
CTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCGGATAGTAA.
[97] The one-letter amino acid sequence that corresponds to SEQ ID NO: 33 is MDPKGSLSWRILLFLSLAFELSYGQVQLVQSGAEVKKPGASVKVSCKASGYLFTTYW MHWVRQAPGQGLEWMGEISPTNGRAYYNAKFQGRVTMTVDKSINTAYMELSRLRSD DTAVYYCARAYGNYFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPPELLGGPSVFLFPPKPKDTLMISRTPEVTCW VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG.
[98] The DNA sequence that corresponds to SEQ ID NO: 34 is ATGGACCCCAAGGGCAGCCTGAGCTGGAGAATCCTGCTGTTCCTGAGCCTGGCCT TCGAGCTGAGCTACGGCCAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGA AACCTGGCGCCTCCGTGAGGTGTCCTGCAAGGCTTCCGGCTACCTGTTCACCACC TACTGGATGCACTGGGTGCGACAGGCCCCTGGACAGGGCCTGGAATGGATGGGC GAGATCTCCCCTACCAACGGCAGAGCCTACTACAACCAAAATTCCAGGGCAGAGT GACCATGACCGTGGACAAGTCCATCAACACCGCTTACATGGAACTGTCCAGACTG CGGAGCGATGACACCGCCGTGTACTACTGCGCTAGAGCCTACGGCAACTACGATT CGCCTACTGGGGCCAGGGCACCCTCGTGACAGTGTCCTCTGCTAGCACCAAGGG CCCCAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGAACCGC CGCCCTGGGCTGCCTGGGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGG AACAGCGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCCGTGCTGCAGAGC AGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCAGCAGCAGCCTGGGCA CCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAA GAAGGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGCCCTCCCTGCCCCGC
-36WO 2017/065837
PCT/US2016/034880
CCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGAC
ACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGC
CACGAGGACCCCGAGGTGAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACA
ACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTACCGCGTGGTGA
GCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAGGAGTACAAGTGCAA
GGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAGGCCAA
GGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCT
GACAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACA
TCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCC
CTCCCGTGCTGGACAGCGACGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGAC
AAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCC
CTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCGGATAGTAA.
[99] The one-letter amino acid sequence that corresponds to SEQ ID NO: 35 is METDTLLLWVLLLWVPGSTGDWMTQSPLSLPVTLGQPASISCRSSQSLVNSNGNTFL QWYQQRPGQSPRLLIYKVSLRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQ STHVPPTFGGGTVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC.
[100] The DNA sequence that corresponds to SEQ ID NO: 36 is ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCC ACCGGAGACGTCGTGATGACCCAGTCCCCTCTGTCCCTGCCTGTGACCCTGGGAC AGCCTGCCTCCATCTCCTCAGATCCTCCCAGTCCCTCGTGAACTCCAACGGCAACA CCTTCCTGCAGTGGTATCAGCAGCGGCCTGGCCAGAGCCCCAGACTGCTGATCTA CAAGGTGTCCCTGCGGTTCTCCGGCGTGCCCGACGATTTTCCGGCTCTGGCTCTG GCACCGACTTCACCCTGAAGATCTCCCGGGTGGAAGCCGAGGACGTGGGCGTGT ACTACTGCTCCCAGAGCACCCACGTGCCCCCTACATTTGGCGGAGGCACCAAGTG GAAATCAAGCGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACG AGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCC
-37WO 2017/065837
PCT/US2016/034880
CCGCGAGGCCAAGGGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAG
CCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAG
CACCCTGACCCTGAGCAAGGCCGACTACGAGAAGACAAGGTGTACGCCTGCGAG
GTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAG
TGCTAA.
[101] The one-letter amino acid sequence that corresponds to SEQ ID NO: 37 is METDTLLLWVLLLWVPGSTGDWMTQSPLSLPVTLGQPASISCRSRQSLVNSNGNTFL QWYQQRPGQSPRLLIYKVSLRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQ STHVPPTFGGGTVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC.
[102] The DNA sequence that corresponds to SEQ ID NO: 38 is ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCC ACCGGAGACGTCGTGATGACCCAGTCCCCTCTGTCCCTGCCTGTGACCCTGGGAC AGCCTGCCTCCATCTCCTCAGATCCAGGCAGTCCCTCGTGAACTCCAACGGCAAC ACCTTCCTGCAGTGGTATCAGCAGCGGCCTGGCCAGAGCCCCAGACTGCTGATCT ACAAGGTGTCCCTGCGGTTCTCCGGCGTGCCCGACGATTTTCCGGCTCTGGCTCT GGCACCGACTTCACCCTGAAGATCTCCCGGGTGGAAGCCGAGGACGTGGGCGTG TACTACTGCTCCCAGAGCACCCACGTGCCCCCTACATTTGGCGGAGGCACCAAGT GGAAATCAAGCGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGAC GAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACC CCCGCGAGGCCAAGGGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACA GCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCA GCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGACAAGGTGTACGCCTGCGA GGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAACCGGGGCGA GTGCTAA.
[103] The one-letter amino acid sequence that corresponds to SEQ ID NO: 39 is METDTLLLVWLLLWVPGSTGDWMTQSPLSSPVTLGQPASISCRSSQSLVNSNGNTFL
-38WO 2017/065837
PCT/US2016/034880
QWYHQRPGQPPRLLIYKVSLRFSGVPDRFSGSGAGKDFTLKISRVEAEDVGVYYCSQ
STHVPPTFGQGTLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
FNRGEC.
[104] The DNA sequence that corresponds to SEQ ID NO: 40 is ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCC ACCGGAGACGTCGTGATGACCCAGTCCCCTCTGTCCAGTCCTGTGACCCTGGGAC AGCCTGCCTCCATCTCCTCAGATCCTCCCAGTCCCTCGTGAACTCCAACGGCAACA CCTTCCTGCAGTGGTATCACCAGCGGCCTGGCCAGCCTCCCAGACTGCTGATCTA CAAGGTGTCCCTGCGGTTCTCCGGCGTGCCCGACGATTTTCCGGCTCTGGCGCTG GCAAGGACTTCACCCTGAAGATCTCCCGGGTGGAAGCCGAGGACGTGGGCGTGT ACTACTGCTCCCAGAGCACCCACGTGCCCCCTACATTTGGCCAGGGCACCAACTG GAAATCAAGCGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACG AGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCC CCGCGAGGCCAAGGGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAG CCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAG CACCCTGACCCTGAGCAAGGCCGACTACGAGAAGACAAGGTGTACGCCTGCGAG GTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAG TGCTAA.
[105] Examples [106] Example 1: In vivo study of the administration of anti-glycation end-product antibody [107] To examine the effects of an anti-glycation end-product antibody, the antibody was administered to the aged CD1(ICR) 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
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PCT/US2016/034880 lysine conjugated with keyhole limpet hemocyanin, the carboxymethyl lysine MAb (Clone 318003) available from R&D Systems, Inc. (Minneapolis, MN; catalog no.
MAB3247). A control reference of physiological saline was used in the control animals.
[108] 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.
[109] Table 1: The different groups of animals used in the study
Group No. Test Material Mice Dose Level (pg/gm/BID/ week) Number of Animals
Main Study Treatment- Free
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.
[110] p-lgiNK4a 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 AACt = ACt mean control Group (2) - ACt mean experimental Group (1 or 3 or 5); Fold Expression= 2 “AACt.
[111] Table 2: pi6INK4a mRNA quantified in adipose tissue
Calculation (unadjusted to Group 4: 5.59) Group 2 vs Group 1 Group 2 vs Group 3 Group 2 vs Group 5
Group 2 Group 1 Group 2 Group 3 Group 2 Group 5
Mean ACt 5.79 7.14 5.79 6.09 5.79 7.39
AACt -1.35 -0.30 .60
Fold Expression 2.55 1.23 3.03
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PCT/US2016/034880 [112] The table above indicates that untreated old mice (Control Group 2) express 2.55-fold more p16lnk4a 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 p16lnk4a mRNA was 1.23-fold higher in Group 2 than in Group 3. Therefore, the level of p16lnk4a mRNA expression was lower when the old mice were treated with 2.5 pg/gram/BID/week of antibody.
[113] When results from Group 2 (Control) untreated old mice were compared to results from Group 5 (5 pg/gram) treated old mice euthanized Day 22, it was observed that p16lnk4a mRNA was 3.03-fold higher in Group 2 (controls) than in Group 5 (5 pg/gram). This comparison indicated that the Group 5 animals had lower levels of p16lnk4a mRNA expression when they were treated with 5.0 pg/gram/BID/week, providing p16lnk4a mRNA expression levels comparable to that of the young untreated mice (i.e. Group 1). Unlike Group 3 (2.5 pg/gram) mice that were euthanized at end of recovery Day 85, Group 5 mice were euthanized at end of treatment Day 22.
[114] These results indicate the antibody administration resulted in the killing of senescent cells.
[115] 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 pg/gm/BID/ week.
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PCT/US2016/034880 [116] Table 3: Effect of antibody administration on mass of the gastrocnemius muscle
Group Summary Information Absolute weight of Gastrocnemius Muscle Weight relative to body mass of 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
[117] Example 2: Affinity and kinetics of test antibody [118] The affinity and kinetics of the test antibody used in Example 1 were analyzed using Na,Na-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 immodilized with the test antibody (molecular weigh of 150,000 Da). The running buffer was a HBS-EP buffer (10 mM HEPES, 150 mM NaCI, 3 mM EDTA and 0.05% P20, 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.
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PCT/US2016/034880 [119] Table 4: Experimental set-up of affinity and kinetics analysis
Association and dissociation
Flow path Fc1 and Fc2
Flow rate (pl/min.) 30
Association time (s) 300
Dissociation time (s) 300
Sample concentration (μΜ) 20 - 5 - 1.25 (x2) - 0.3125 - 0.078 - 0
[120] A graph of the response versus time is illustrated in FIG. 1. The following values were determined from the analysis: ka (1/Ms) = 1.857 χ 103; kd (1/s) = 6.781 χ 10'3; KD (M) = 3.651 χ 10'6; Rmax (RU) = 19.52; and Chi2 = 0.114. Because the Chi2 value of the fitting is less than 10% of Rmax, the fit is reliable.
[121] Example 3: Construction and production of murine anti-AGE lgG2b antibody and chimeric anti-AGE lgG1 antibody [122] Murine and chimeric human anti-AGE antibodies were prepared. The DNA sequence of murine anti-AGE antibody lgG2b heavy chain is shown in SEQ ID NO: 12. The DNA sequence of chimeric human anti-AGE antibody lgG1 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.
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PCT/US2016/034880 [123] 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.
[124] 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 pm 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:
[125] Table 5: Yields and aliquots
Protein Concentration (mg/ml_) Volume (mL) No. of vials Total Yield (mg)
Murine anti-AGE 0.08 1.00 3 0.24
Chimeric anti-AGE 0.23 1.00 3 0.69
[126] Antibody purity was evaluated by capillary electrophoresis sodium-dodecyl sulfate (CE-SDS) analysis using LabChip® GXII, (PerkinElmer).
[127] Example 4: Binding of murine (parental) and chimeric anti-AGE antibodies
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PCT/US2016/034880 [128] 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.
[129] The antigens were diluted to 1 pg/mL in 1x phosphate buffer at pH 6.5. A 96-well microtiter ELISA plate was coated with 100 pL/well of the diluted antigen and let sit at 4°C overnight. The plate was blocked with 1x 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 1x PBS, 1% BSA with the starting concentration of 50 pg/mL. Secondary antibodies were diluted 1:5,000. 100 pL 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 1x PBS. 100 pL/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 1x PBS. 100 pL HRP substrate TMB was added to each well to develop the plate. After 3-5 minutes elapsed, the reaction was terminated by adding 100 pL of 1N HCI. A second direct binding ELISA was performed with only CML coating. The absorbance at OD450 was read using a microplate reader.
[130] 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.
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PCT/US2016/034880 [131] Plate map of CML and CML-KLH ELISA:
Cone.
(ug/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
[132] 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.
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Plate map of CML-only ELISA:
Cone.
(ug/mL) 1 2
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- AntiControl AGE AGE [134] 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.
[135] Example 5: Humanized antibodies [136] 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:
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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.
[137] 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 (July 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:
[138] 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
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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
[139] 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 lgG1 was used.
[140] 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).
[141] 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:
[142] 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
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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
[143] The binding of the humanized antibodies may be evaluated, for example, by dose-dependent binding ELISA or cell-based binding assay.
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PCT/US2016/034880 [144] [145] [146] [147] [148] [149] [150] [151] [152] [153] [154] [155]
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Claims (32)

  1. What is claimed is:
    1. An anti-AGE antibody, comprising a protein or peptide comprising at least one amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3,
    SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33,
    SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 39, wherein the antibody binds to a protein or peptide that exhibits a carboxymethyllysine modification.
  2. 2. An anti-AGE antibody, comprising a protein or peptide comprising at least one amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with an amino acid sequence selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, wherein the antibody binds to a protein or peptide that exhibits a carboxymethyllysine modification.
  3. 3. An anti-AGE antibody, comprising a heavy chain, and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 29, SEQ ID NO: 31, and SEQ ID NO: 33, or the light chain comprises an amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98%
    -57WO 2017/065837
    PCT/US2016/034880 sequence identity, with an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 19, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 39; and the antibody binds to a protein or peptide that exhibits a carboxymethyllysine modification.
  4. 4. An anti-AGE antibody, comprising a heavy chain, and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 29, SEQ ID NO: 31, and SEQ ID NO: 33, and the light chain comprises an amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 19, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 39; and the antibody binds to a protein or peptide that exhibits a carboxymethyllysine modification.
  5. 5. An anti-AGE antibody, comprising complementary determining region comprising at least one amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with an amino acid sequence selected from the group consisting of SEQ ID NO 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, wherein the antibody binds to a protein or peptide that exhibits a carboxymethyllysine modification, and
    -58WO 2017/065837
    PCT/US2016/034880 the antibody is substantially non-immunogenic to a species selected from the group consisting of mice, rats, goats, sheep, cows, horses, dogs and cats.
  6. 6. An antibody conjugate, comprising:
    an anti-AGE antibody fragment comprising a protein or peptide comprising at least one amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO 3, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 39, and an agent that causes the destruction of AGE-modified cells, conjugated to the anti-AGE antibody fragment, wherein the anti-AGE antibody fragment binds to a protein or peptide that exhibits a carboxymethyllysine modification.
  7. 7. An anti-AGE antibody of any of the preceding claims for use in killing senescent cells.
  8. 8. An anti-AGE antibody of any of the preceding claims for use in killing partially-functional or non-functional cells.
  9. 9. An anti-AGE antibody of any of the preceding claims for use in treating sarcopenia in a subject by killing partially-functional or non-functional cells.
  10. 10. An anti-AGE antibody of any of the preceding claims for use in promoting tissue or organ regeneration in a subject by killing partially-functional or non-functional cells.
    -59WO 2017/065837
    PCT/US2016/034880
  11. 11. An anti-AGE antibody of any of the preceding claims for use in promoting regenerative processes or overcoming aging effects in a subject by killing partiallyfunctional or non-functional cells.
  12. 12. An anti-AGE antibody of any of the preceding claims for use in treating atherosclerosis in a subject by killing partially-functional or non-functional cells.
  13. 13. An anti-AGE antibody of any of the preceding claims for use in preventing or delaying the onset of cataracts in a subject.
  14. 14. An anti-AGE antibody of any of the preceding claims for use preventing or delaying the onset of loss of adipose tissue in a subject.
  15. 15. An anti-AGE antibody of any of the preceding claims for use in preventing or delaying the onset of lordokyphosis in a subject.
  16. 16. An anti-AGE antibody of any of the preceding claims for use in treating inflammation or auto-immune disorders in a subject.
  17. 17. An anti-AGE antibody of any of the preceding claims for use in treating neurodegenerative disorders in a subject.
  18. 18. An anti-AGE antibody of any of the preceding claims for use in treating cancer in a subject.
  19. 19. Use of an anti-AGE antibody of any of the preceding claims for the manufacture of a medicament for killing senescent cells.
    -60WO 2017/065837
    PCT/US2016/034880
  20. 20. Use of an anti-AGE antibody of any of the preceding claims for the manufacture of a medicament for treating sarcopenia.
  21. 21. The anti-AGE antibody of any of the preceding claims, wherein the subject has a neurodegenerative disorder selected from the group consisting of AD, PD, Lewy body dementia, MS, prion diseases and ALS.
  22. 22. The anti-AGE antibody of any of the preceding claims, wherein the treating comprises administering the anti-AGE antibody to the central nervous system of the subject.
  23. 23. The anti-AGE antibody of any of the preceding claims, wherein the subject has ALS or MD, and the treating comprises administering the anti-AGE antibody to muscles of the subject.
  24. 24. The anti-AGE antibody of any of the preceding claims, wherein the antibody is substantially non-immunogenic to a species selected from the group consisting of humans, mice, rats, goats, sheep, cows, horses, dogs and cats.
  25. 25. The anti-AGE antibody of any of the preceding claims, wherein the antibody is a humanized antibody.
  26. 26. The anti-AGE antibody of any of the preceding claims, wherein the antibody is monoclonal.
  27. 27. The anti-AGE antibody of any of the preceding claims, wherein the antibody is substantially non-immunogenic to humans.
    -61 WO 2017/065837
    PCT/US2016/034880
  28. 28. The anti-AGE antibody of any of the preceding claims, wherein the antibody has a rate of dissociation (kd) of at most 9 x 103 sec'1.
  29. 29. The anti-AGE antibody of any of the preceding claims, wherein the antibody is conjugated to an agent that causes the destruction of AGE-modified cells.
  30. 30. The anti-AGE antibody of any of the preceding claims, wherein the agent is selected from the group consisting of a toxin, a cytotoxic agent, magnetic nanoparticles, and magnetic spin-vortex discs.
  31. 31. The anti-AGE antibody of any of the preceding claims, wherein the antibody includes constant regions which permit destruction of targeted cells by a subject’s immune system.
  32. 32. The anti-AGE antibody of any of the preceding claims, wherein the antibody is a bi-specific antibody.
    -62WO 2017/065837
    PCT/US2016/034880
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    SUBSTITUTE SHEET (RULE 26)
    SIW01-007-CIP-WO_Sequence_Listing.txt
    Sequence Listing <110> Lewis S. Gruber <120> Anti-AGE antibodies and methods of use thereof <130> SIW01-007-CIP-WO <160> 46 <210> 1 <211> 463 <212> PRT <213> Artificial sequence <220>
    <223> Modified Homo sapiens immunoglobulin G1 heavy chain <400> 1
    Met Asn 1 Leu Leu Leu 5 Ile Leu Thr Phe Val 10 Ala Ala Ala Val Ala 15 Gln Val Gln Leu Leu Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala Ser 20 25 30 Val Lys Leu Ala Cys Lys Ala Ser Gly Tyr Leu Phe Thr Thr Tyr Trp 35 40 45 Met His Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 50 55 60 Glu Ile Ser Pro Thr Asn Gly Arg Ala Tyr Tyr Asn Ala Arg Phe Lys 65 70 75 80 Ser Glu Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr Met 85 90 95 Gln Leu Ser Ser Leu Thr Ser Glu Ala Ser Ala Val Tyr Tyr Cys Ala 100 105 110 Arg Ala Tyr Gly Asn Tyr Glu Phe Ala Tyr Trp Gly Gln Gly Thr Leu 115 120 125 Val Thr Val Ser Val Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 130 135 140 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 145 150 155 160 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 165 170 175
    Page 1
    SIW01-007-CIP-WO_Sequence_Listing.txt
    Gly Ala Leu Thr Ser Gly Val His Thr 185 Phe Pro Ala Val Leu 190 Gln Ser 180 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 195 200 205 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 210 215 220 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 225 230 235 240 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 245 250 255 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 260 265 270 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 275 280 285 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 290 295 300 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 305 310 315 320 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 325 330 335 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 340 345 350 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 355 360 365 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 370 375 380 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 385 390 395 400 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 405 410 415 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 420 425 430
    Page 2
    SIW01-007-CIP-WO_Sequence_Listing.txt
    Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 435 440 445 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460 <210> 2 <211> 118 <212> PRT <213> Mus musculus <400> 2 Gln Val Gln Leu Leu Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ala Cys Lys Ala Ser Gly Tyr Leu Phe Thr Thr Tyr 20 25 30 Trp Met His Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Ser Pro Thr Asn Gly Arg Ala Tyr Tyr Asn Ala Arg Phe 50 55 60 Lys Ser Glu Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Ala Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Tyr Gly Asn Tyr Glu Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Val
    115 <210> 3 <211> 234 <212> PRT <213> Artificial sequence <220>
    <223> Modified Homo sapiens immunoglobulin G1 kappa light chain <400> 3
    Met Asn Leu Leu Leu Ile Leu Thr Phe Val Ala Ala Ala Val Ala Asp 1 5 10 15
    Page 3
    SIW01-007-CIP-WO_Sequence_Listing.txt
    Val Val Met Thr Gln 20 Thr Pro Leu Ser 25 Leu Pro Val Ser Leu 30 Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Arg Gln Ser Leu Val Asn Ser Asn 35 40 45 Gly Asn Thr Phe Leu Gln Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro 50 55 60 Lys Leu Leu Ile Tyr Lys Val Ser Leu Arg Phe Ser Gly Val Pro Asp 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser 85 90 95 Arg Val Glu Ala Glu Asp Leu Gly Leu Tyr Phe Cys Ser Gln Ser Thr 100 105 110 His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230
    <210> 4 <211> 113 <212> PRT <213> Mus musculus
    Page 4
    SIW01-007-CIP-WO_Sequence_Listing.txt <400> 4
    Asp Val 1 Val Met Thr Gln 5 Thr Pro Leu Ser 10 Leu Pro Val Ser Leu 15 Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Arg Gln Ser Leu Val Asn Ser 20 25 30 Asn Gly Asn Thr Phe Leu Gln Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Leu Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Leu Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
    100 105 110
    Arg <210> 5 <211> 327 <212> PRT
    <213> Equus caballus <400> 5 Ala Ser Thr Thr Ala Pro Lys Val Phe Pro Leu Ala Ser His Ser Ala 1 5 10 15 Ala Thr Ser Gly Ser Thr Val Ala Leu Gly Cys Leu Val Ser Ser Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ser Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Met Val Thr Val Pro Ala Ser Ser Leu Lys Ser Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys P age 5
    SIW01-007-CIP-WO_Sequence_Listing.txt 85 90 95
    Lys Ile Val Ile 100 Lys Glu Cys Asn Gly Gly Cys 105 Pro Ala Glu 110 Cys Leu Gln Val Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val 115 120 125 Leu Met Ile Ser Arg Thr Pro Thr Val Thr Cys Val Val Val Asp Val 130 135 140 Gly His Asp Phe Pro Asp Val Gln Phe Asn Trp Tyr Val Asp Gly Val 145 150 155 160 Glu Thr His Thr Ala Thr Thr Glu Pro Lys Gln Glu Gln Phe Asn Ser 165 170 175 Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gln His Lys Asp Trp Leu 180 185 190 Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Ala Leu Pro Ala 195 200 205 Pro Val Glu Arg Thr Ile Ser Lys Pro Thr Gly Gln Pro Arg Glu Pro 210 215 220 Gln Val Tyr Val Leu Ala Pro His Arg Asp Glu Leu Ser Lys Asn Lys 225 230 235 240 Val Ser Val Thr Cys Leu Val Lys Asp Phe Tyr Pro Thr Asp Ile Asp 245 250 255 Ile Glu Trp Lys Ser Asn Gly Gln Pro Glu Pro Glu Thr Lys Tyr Ser 260 265 270 Thr Thr Pro Ala Gln Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Ser 275 280 285 Lys Leu Thr Val Glu Thr Asn Arg Trp Gln Gln Gly Thr Thr Phe Thr 290 295 300 Cys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Glu Lys Ser 305 310 315 320 Val Ser Lys Ser Pro Gly Lys
    325
    Page 6
    SIW01-007-CIP-WO_Sequence_Listing.txt <210> 6 <211> 415 <212> PRT <213> Equus caballus <400> 6
    Ser 1 Leu Glu Asp Thr Ala 5 Val Ile Pro Leu 10 Phe Ser Glu Cys Lys 15 Ala Pro Lys Glu Asp Asp Val Val Ser Leu Ala Cys Leu Val Lys Gly Tyr 20 25 30 Phe Pro Glu Pro Val Gln Val Thr Trp Glu Pro Glu Met Gln Asn Gln 35 40 45 Lys Pro Trp Thr Phe Pro Ala Met Lys Lys Gly Gln Glu Tyr Ile His 50 55 60 Val Phe Ser Leu Thr Thr Trp Trp Lys Pro Gly Ser His Ser Cys Thr 65 70 75 80 Val His His Lys Ala Ser Ser Phe Arg Lys Lys Met Thr Phe Gln Glu 85 90 95 Pro Ala Ser Trp Ala Pro Gln Arg Thr Ser Ala Leu Pro Val Thr Ser 100 105 110 Lys Glu Pro Thr Pro Ala Pro Thr Thr Leu Arg Lys Ser Glu Pro Ser 115 120 125 Thr Arg His Thr Gln Pro Glu Thr Gln Lys Pro Arg Ile Pro Val Asp 130 135 140 Thr Pro Leu Lys Glu Cys Gln Ser His Thr His Pro Pro Ser Ile Tyr 145 150 155 160 Leu Leu His Pro Pro Leu Gln Gly Leu Trp Leu Lys Gly Glu Ala Thr 165 170 175 Phe Thr Cys Leu Val Val Gly Asp Asp Leu Lys Asp Ala His Leu Ser 180 185 190 Trp Glu Leu Ser Glu Arg Ser Asn Gly Met Phe Val Glu Ser Gly Pro 195 200 205 Leu Glu Lys His Thr Asn Gly Ser Gln Ser Arg Ser Ser Arg Leu Ala
    210 215 220
    Page 7
    SIW01-007-CIP-WO_Sequence_Listing.txt
    Leu 225 Pro Arg Ser Ser Trp Ala Met Gly Thr Ser Val Thr Cys Lys Leu 240 230 235 Ser Tyr Pro Asn Leu Leu Ser Ser Met Glu Val Val Gly Leu Lys Glu 245 250 255 His Ala Ala Ser Ala Pro Arg Ser Leu Thr Val His Ala Leu Thr Thr 260 265 270 Pro Gly Leu Asn Ala Ser Pro Gly Ala Thr Ser Trp Leu Gln Cys Lys 275 280 285 Val Ser Gly Phe Ser Pro Pro Glu Ile Val Leu Thr Trp Leu Glu Gly 290 295 300 Gln Arg Glu Val Asp Pro Ser Trp Phe Ala Thr Ala Arg Pro Thr Ala 305 310 315 320 Gln Pro Gly Asn Thr Thr Phe Gln Thr Trp Ser Ile Leu Leu Val Pro 325 330 335 Thr Ile Pro Gly Pro Pro Thr Ala Thr Tyr Thr Cys Val Val Gly His 340 345 350 Glu Ala Ser Arg Gln Leu Leu Asn Thr Ser Trp Ser Leu Asp Thr Gly 355 360 365 Gly Leu Ala Met Thr Pro Glu Ser Lys Asp Glu Asn Ser Asp Asp Tyr 370 375 380 Ala Asp Leu Asp Asp Ala Gly Ser Leu Trp Leu Thr Phe Met Ala Leu 385 390 395 400 Phe Leu Ile Thr Leu Leu Tyr Ser Gly Phe Val Thr Phe Ile Lys 405 410 415
    <210> 7 <211> 334 <212> PRT <213> Canis familiaris <400> 7
    Ser 1 Lys Thr Ser Pro 5 Ser Val Phe Pro Leu 10 Ser Leu Cys His Gln 15 Glu Ser Glu Gly Tyr Val Val Ile Gly Cys Leu Val Gln Gly Phe Phe Pro 20 25 30
    Page 8
    SIW01-007-CIP-WO_Sequence_Listing.txt
    Pro Glu Pro Val 35 Asn Val Thr Trp Asn Ala Gly Lys Asp Ser Thr Ser 40 45 Val Lys Asn Phe Pro Pro Met Lys Ala Ala Thr Gly Ser Leu Tyr Thr 50 55 60 Met Ser Ser Gln Leu Thr Leu Pro Ala Ala Gln Cys Pro Asp Asp Ser 65 70 75 80 Ser Val Lys Cys Gln Val Gln His Ala Ser Ser Pro Ser Lys Ala Val 85 90 95 Ser Val Pro Cys Lys Asp Asn Ser His Pro Cys His Pro Cys Pro Ser 100 105 110 Cys Asn Glu Pro Arg Leu Ser Leu Gln Lys Pro Ala Leu Glu Asp Leu 115 120 125 Leu Leu Gly Ser Asn Ala Ser Leu Thr Cys Thr Leu Ser Gly Leu Lys 130 135 140 Asp Pro Lys Gly Ala Thr Phe Thr Trp Asn Pro Ser Lys Gly Lys Glu 145 150 155 160 Pro Ile Gln Lys Asn Pro Glu Arg Asp Ser Cys Gly Cys Tyr Ser Val 165 170 175 Ser Ser Val Leu Pro Gly Cys Ala Asp Pro Trp Asn His Gly Asp Thr 180 185 190 Phe Ser Cys Thr Ala Thr His Pro Glu Ser Lys Ser Pro Ile Thr Val 195 200 205 Ser Ile Thr Lys Thr Thr Glu His Ile Pro Pro Gln Val His Leu Leu 210 215 220 Pro Pro Pro Ser Glu Glu Leu Ala Leu Asn Glu Leu Val Thr Leu Thr 225 230 235 240 Cys Leu Val Arg Gly Phe Lys Pro Lys Asp Val Leu Val Arg Trp Leu 245 250 255 Gln Gly Thr Gln Glu Leu Pro Gln Glu Lys Tyr Leu Thr Trp Glu Pro 260 265 270 Leu Lys Glu Pro Asp Gln Thr Asn Met Phe Ala Val Thr Ser Met Leu Page 9
    SIW01-007-CIP-WO_Sequence_Listing.txt
    275 280 285 Arg Val Thr Ala Glu Asp Trp Lys Gln Gly Glu Lys Phe Ser Cys Met 290 295 300 Val Gly His Glu Ala Leu Pro Met Ser Phe Thr Gln Lys Thr Ile Asp 305 310 315 320 Arg Leu Ala Gly Lys Pro Thr His Val Asn Val Ser Val Val
    325
    330 <210> 8 <211> 426 <212> PRT
    <213> Canis fam liaris <400> 8 Thr Ser Gln Asp Leu Ser Val Phe Pro Leu Ala Ser Cys Cys Lys Asp 1 5 10 15 Asn Ile Ala Ser Thr Ser Val Thr Leu Gly Cys Leu Val Thr Gly Tyr 20 25 30 Leu Pro Met Ser Thr Thr Val Thr Trp Asp Thr Gly Ser Leu Asn Lys 35 40 45 Asn Val Thr Thr Phe Pro Thr Thr Phe His Glu Thr Tyr Gly Leu His 50 55 60 Ser Ile Val Ser Gln Val Thr Ala Ser Gly Lys Trp Ala Lys Gln Arg 65 70 75 80 Phe Thr Cys Ser Val Ala His Ala Glu Ser Thr Ala Ile Asn Lys Thr 85 90 95 Phe Ser Ala Cys Ala Leu Asn Phe Ile Pro Pro Thr Val Lys Leu Phe 100 105 110 His Ser Ser Cys Asn Pro Val Gly Asp Thr His Thr Thr Ile Gln Leu 115 120 125 Leu Cys Leu Ile Ser Gly Tyr Val Pro Gly Asp Met Glu Val Ile Trp 130 135 140 Leu Val Asp Gly Gln Lys Ala Thr Asn Ile Phe Pro Tyr Thr Ala Pro 145 150 155 160
    Page 10
    SIW01-007-CIP-WO_Sequence_Listing.txt
    Gly Thr Lys Glu Gly Asn Val 165 Thr Ser Thr 170 His Ser Glu Leu Asn 175 Ile Thr Gln Gly Glu Trp Val Ser Gln Lys Thr Tyr Thr Cys Gln Val Thr 180 185 190 Tyr Gln Gly Phe Thr Phe Lys Asp Glu Ala Arg Lys Cys Ser Glu Ser 195 200 205 Asp Pro Arg Gly Val Thr Ser Tyr Leu Ser Pro Pro Ser Pro Leu Asp 210 215 220 Leu Tyr Val His Lys Ala Pro Lys Ile Thr Cys Leu Val Val Asp Leu 225 230 235 240 Ala Thr Met Glu Gly Met Asn Leu Thr Trp Tyr Arg Glu Ser Lys Glu 245 250 255 Pro Val Asn Pro Gly Pro Leu Asn Lys Lys Asp His Phe Asn Gly Thr 260 265 270 Ile Thr Val Thr Ser Thr Leu Pro Val Asn Thr Asn Asp Trp Ile Glu 275 280 285 Gly Glu Thr Tyr Tyr Cys Arg Val Thr His Pro His Leu Pro Lys Asp 290 295 300 Ile Val Arg Ser Ile Ala Lys Ala Pro Gly Lys Arg Ala Pro Pro Asp 305 310 315 320 Val Tyr Leu Phe Leu Pro Pro Glu Glu Glu Gln Gly Thr Lys Asp Arg 325 330 335 Val Thr Leu Thr Cys Leu Ile Gln Asn Phe Phe Pro Ala Asp Ile Ser 340 345 350 Val Gln Trp Leu Arg Asn Asp Ser Pro Ile Gln Thr Asp Gln Tyr Thr 355 360 365 Thr Thr Gly Pro His Lys Val Ser Gly Ser Arg Pro Ala Phe Phe Ile 370 375 380 Phe Ser Arg Leu Glu Val Ser Arg Val Asp Trp Glu Gln Lys Asn Lys 385 390 395 400 Phe Thr Cys Gln Val Val His Glu Ala Leu Ser Gly Ser Arg Ile Leu 405 410 415
    Page 11
    SIW01-007-CIP-WO_Sequence_Listing.txt
    Gln Lys Trp Val Ser Lys Thr Pro Gly Lys 420 425 <210> 9 <211> 335 <212> PRT <213> Felis catus <400> 9
    Ala Ser 1 Thr Thr Ala 5 Ser Ser Val Phe Pro 10 Leu Ala Pro Ser Cys 15 Gly Thr Thr Ser Gly Ala Thr Val Ala Leu Ala Cys Leu Val Leu Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ser Val Leu Gln Ala Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Met Val Thr Val Pro Ser Ser Arg Trp Leu Ser Asp Thr 65 70 75 80 Phe Thr Cys Asn Val Ala His Arg Pro Ser Ser Thr Lys Val Asp Lys 85 90 95 Thr Val Pro Lys Thr Ala Ser Thr Ile Glu Ser Lys Thr Gly Glu Gly 100 105 110 Pro Lys Cys Pro Val Pro Glu Ile Pro Gly Ala Pro Ser Val Phe Ile 115 120 125 Phe Pro Pro Lys Pro Lys Asp Thr Leu Ser Ile Ser Arg Thr Pro Glu 130 135 140 Val Thr Cys Leu Val Val Asp Leu Gly Pro Asp Asp Ser Asn Val Gln 145 150 155 160 Ile Thr Trp Phe Val Asp Asn Thr Glu Met His Thr Ala Lys Thr Arg 165 170 175 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 180 185 190 Pro Ile Leu His Gln Asp Trp Leu Lys Gly Lys Glu Phe Lys Cys Lys 195 200 205
    Page 12
    SIW01-007-CIP-WO_Sequence_Listing.txt
    Val Asn 210 Ser Lys Ser Leu Pro Ser Ala Met Glu Arg Thr Ile Ser Lys 215 220 Ala Lys Gly Gln Pro His Glu Pro Gln Val Tyr Val Leu Pro Pro Thr 225 230 235 240 Gln Glu Glu Leu Ser Glu Asn Lys Val Ser Val Thr Cys Leu Ile Lys 245 250 255 Gly Phe His Pro Pro Asp Ile Ala Val Glu Trp Glu Ile Thr Gly Gln 260 265 270 Pro Glu Pro Glu Asn Asn Tyr Gln Thr Thr Pro Pro Gln Leu Asp Ser 275 280 285 Asp Gly Thr Tyr Phe Leu Tyr Ser Arg Leu Ser Val Asp Arg Ser His 290 295 300 Trp Gln Arg Gly Asn Thr Tyr Thr Cys Ser Val Ser His Glu Ala Leu 305 310 315 320 His Ser His His Thr Gln Lys Ser Leu Thr Gln Ser Pro Gly Lys
    325 330 335 <210> 10 <211> 96 <212> PRT <213> Camelus dromedarius <400> 10
    Glu 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly Leu Val 10 Gln Pro Gly 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Arg Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
    Page 13
    SIW01-007-CIP-WO_Sequence_Listing.txt 85 90 95 <210> 11 <211> 96 <212> PRT <213> Camelus dromedarius <400> 11
    Glu 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly Leu Val 10 Gln Pro Gly 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Met Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95
    <210> 12 <211> 1434 <212> DNA <213> Artificial sequence <220>
    <223> Murine anti-AGE IgG2b heavy chain <400> 12
    atggacccca agggcagcct gagctggaga atcctgctgt tcctgagcct ggccttcgag 60 ctgagctacg gccaggtgca gctgctgcag ccaggtgccg agctcgtgaa acctggcgcc 120 tctgtgaagc tggcctgcaa ggcttccggc tacctgttca ccacctactg gatgcactgg 180 ctgaagcaga ggccaggcca gggcctggaa tggatcggcg agatctcccc caccaacggc 240 agagcctact acaacgcccg gttcaagtcc gaggccaccc tgaccgtgga caagtcctcc 300 aacaccgcct acatgcagct gtcctccctg acctctgagg cctccgccgt gtactactgc Page 14 360
    SIW01-007-CIP-WO_Sequence_Listing.txt
    gccagagctt acggcaacta cgagttcgcc tactggggcc agggcaccct cgtgacagtg 420 tctgtggcta agaccacccc tccctccgtg taccctctgg ctcctggctg tggcgacacc 480 accggatcct ctgtgaccct gggctgcctc gtgaagggct acttccctga gtccgtgacc 540 gtgacctgga actccggctc cctgtcctcc tccgtgcaca cctttccagc cctgctgcag 600 tccggcctgt acaccatgtc ctccagcgtg acagtgccct cctccacctg gccttcccag 660 accgtgacat gctctgtggc ccaccctgcc tcttccacca ccgtggacaa gaagctggaa 720 ccctccggcc ccatctccac catcaaccct tgccctccct gcaaagaatg ccacaagtgc 780 cctgccccca acctggaagg cggcccttcc gtgttcatct tcccacccaa catcaaggac 840 gtgctgatga tctccctgac ccccaaagtg acctgcgtgg tggtggacgt gtccgaggac 900 gaccctgacg tgcagatcag ttggttcgtg aacaacgtgg aagtgcacac cgcccagacc 960 cagacacaca gagaggacta caacagcacc atcagagtgg tgtctaccct gcccatccag 1020 caccaggact ggatgtccgg caaagaattc aagtgcaaag tgaacaacaa ggacctgccc 1080 agccccatcg agcggaccat ctccaagatc aagggcctcg tgcgggctcc ccaggtgtac 1140 attctgcctc caccagccga gcagctgtcc cggaaggatg tgtctctgac atgtctggtc 1200 gtgggcttca accccggcga catctccgtg gaatggacct ccaacggcca caccgaggaa 1260 aactacaagg acaccgcccc tgtgctggac tccgacggct cctacttcat ctactccaag 1320 ctgaacatga agacctccaa gtgggaaaag accgactcct tctcctgcaa cgtgcggcac 1380 gagggcctga agaactacta cctgaagaaa accatctccc ggtcccccgg ctag 1434
    <210> 13 <211> 1416 <212> DNA <213> Artificial sequence <220>
    Page 15
    SIW01-007-CIP-WO_Sequence_Listing.txt <223> Chimeric anti-AGE human IgG1 antibody heavy chain <400> 13
    atggacccca agggcagcct gagctggaga atcctgctgt tcctgagcct ggccttcgag 60 ctgagctacg gccaggtgca gctgctgcag ccaggtgccg agctcgtgaa acctggcgcc 120 tctgtgaagc tggcctgcaa ggcttccggc tacctgttca ccacctactg gatgcactgg 180 ctgaagcaga ggccaggcca gggcctggaa tggatcggcg agatctcccc caccaacggc 240 agagcctact acaacgcccg gttcaagtcc gaggccaccc tgaccgtgga caagtcctcc 300 aacaccgcct acatgcagct gtcctccctg acctctgagg cctccgccgt gtactactgc 360 gccagagctt acggcaacta cgagttcgcc tactggggcc agggcaccct cgtgacagtg 420 tctgtggcta gcaccaaggg ccccagcgtg ttccctctgg cccccagcag caagagcacc 480 agcggcggaa ccgccgccct gggctgcctg gtgaaggact acttccccga gcccgtgacc 540 gtgtcctgga acagcggcgc tctgaccagc ggagtgcaca ccttccctgc cgtgctgcag 600 agcagcggcc tgtactccct gagcagcgtg gtgaccgtgc ccagcagcag cctgggcacc 660 cagacctaca tctgcaacgt gaaccacaag ccctccaaca ccaaggtgga caagaaggtg 720 gagcctaaga gctgcgacaa gacccacacc tgccctccct gccccgcccc cgagctgctg 780 ggcggaccca gcgtgttcct gttccctccc aagcccaagg acaccctgat gatcagccgc 840 acccccgagg tgacctgcgt ggtggtggac gtgagccacg aggaccccga ggtgaagttc 900 aactggtacg tggacggcgt ggaggtgcac aacgccaaga ccaagcctcg ggaggagcag 960 tacaactcca cctaccgcgt ggtgagcgtg ctgaccgtgc tgcaccagga ctggctgaac 1020 ggcaaggagt acaagtgcaa ggtgagcaac aaggccctgc ccgctcccat cgagaagacc 1080 atcagcaagg ccaagggcca gccccgggag cctcaggtgt acaccctgcc ccccagccgc 1140 gacgagctga ccaagaacca ggtgagcctg acctgcctgg tgaagggctt ctacccctcc 1200
    Page 16
    SIW01-007-CIP-WO_Sequence_Listing.txt gacatcgccg tggagtggga gagcaacggc cagcctgaga acaactacaa gaccacccct 1260 cccgtgctgg acagcgacgg cagcttcttc ctgtacagca agctgaccgt ggacaagtcc 1320 cggtggcagc agggcaacgt gttcagctgc agcgtgatgc acgaggccct gcacaaccac 1380 tacacccaga agagcctgag cctgagcccc ggatag 1416 <210> 14 <211> 720 <212> DNA <213> Artificial sequence
    <220> <223> Murine anti-AGE Kappa light chain <400> 14 atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60 gacgtcgtga tgacccagac ccctctgtcc ctgcctgtgt ctctgggcga ccaggcctcc 120 atctcctgcc ggtctagaca gtccctcgtg aactccaacg gcaacacctt cctgcagtgg 180 tatctgcaga agcccggcca gtcccccaag ctgctgatct acaaggtgtc cctgcggttc 240 tccggcgtgc ccgacagatt ttccggctct ggctctggca ccgacttcac cctgaagatc 300 tcccgggtgg aagccgagga cctgggcctg tacttctgca gccagtccac ccacgtgccc 360 cctacatttg gcggaggcac caagctggaa atcaaacggg cagatgctgc accaactgta 420 tccatcttcc caccatccag tgagcagtta acatctggag gtgcctcagt cgtgtgcttc 480 ttgaacaact tctaccccaa agacatcaat gtcaagtgga agattgatgg cagtgaacga 540 caaaatggcg tcctgaacag ttggactgat caggacagca aagacagcac ctacagcatg 600 agcagcaccc tcacgttgac caaggacgag tatgaacgac ataacagcta tacctgtgag 660 gccactcaca agacatcaac ttcacccatt gtcaagagct tcaacaggaa tgagtgttga 720
    <210> 15 <211> 720
    Page 17
    SIW01-007-CIP-WO_Sequence_Listing.txt <212> DNA <213> Artificial sequence <220>
    <223> Chimeric anti-AGE human kappa light chain <400> 15
    atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60 gacgtcgtga tgacccagac ccctctgtcc ctgcctgtgt ctctgggcga ccaggcctcc 120 atctcctgcc ggtctagaca gtccctcgtg aactccaacg gcaacacctt cctgcagtgg 180 tatctgcaga agcccggcca gtcccccaag ctgctgatct acaaggtgtc cctgcggttc 240 tccggcgtgc ccgacagatt ttccggctct ggctctggca ccgacttcac cctgaagatc 300 tcccgggtgg aagccgagga cctgggcctg tacttctgca gccagtccac ccacgtgccc 360 cctacatttg gcggaggcac caagctggaa atcaagcgga ccgtggccgc ccccagcgtg 420 ttcatcttcc ctcccagcga cgagcagctg aagtctggca ccgccagcgt ggtgtgcctg 480 ctgaacaact tctacccccg cgaggccaag gtgcagtgga aggtggacaa cgccctgcag 540 agcggcaaca gccaggagag cgtgaccgag caggactcca aggacagcac ctacagcctg 600 agcagcaccc tgaccctgag caaggccgac tacgagaagc acaaggtgta cgcctgcgag 660 gtgacccacc agggactgtc tagccccgtg accaagagct tcaaccgggg cgagtgctaa 720
    <210> 16 <211> 477 <212> PRT <213> Artificial sequence <220>
    <223> Murine anti-AGE IgG2b heavy chain <400> 16
    Met 1 Asp Pro Lys Gly 5 Ser Leu Ser Trp Arg 10 Ile Leu Leu Phe Leu 15 Ser Leu Ala Phe Glu Leu Ser Tyr Gly Gln Val Gln Leu Leu Gln Pro Gly 20 25 30
    Page 18
    Ala Glu Leu 35 Val Lys SIW01-007-CIP-WO_Sequence_Listing.txt Pro Gly Ala Ser Val 40 Lys Leu Ala Cys 45 Lys Ala Ser Gly 50 Tyr Leu Phe Thr Thr Tyr Trp Met 55 His Trp Leu Lys 60 Gln Arg Pro 65 Gly Gln Gly Leu Glu Trp Ile Gly Glu 70 Ile 75 Ser Pro Thr Asn Gly 80 Arg Ala Tyr Tyr Asn 85 Ala Arg Phe Lys Ser 90 Glu Ala Thr Leu Thr Val 95 Asp Lys Ser Ser 100 Asn Thr Ala Tyr Met Gln 105 Leu Ser Ser Leu 110 Thr Ser Glu Ala Ser 115 Ala Val Tyr Tyr Cys Ala Arg 120 Ala Tyr Gly Asn 125 Tyr Glu Phe Ala 130 Tyr Trp Gly Gln Gly Thr Leu Val 135 Thr Val Ser Val 140 Ala Lys Thr 145 Thr Pro Pro Ser Val Tyr Pro Leu Ala 150 Pro 155 Gly Cys Gly Asp Thr 160 Thr Gly Ser Ser Val 165 Thr Leu Gly Cys Leu 170 Val Lys Gly Tyr Phe Pro 175 Glu Ser Val Thr 180 Val Thr Trp Asn Ser Gly 185 Ser Leu Ser Ser 190 Ser Val His Thr Phe 195 Pro Ala Leu Leu Gln Ser Gly 200 Leu Tyr Thr Met 205 Ser Ser Ser Val 210 Thr Val Pro Ser Ser Thr Trp Pro 215 Ser Gln Thr Val 220 Thr Cys Ser 225 Val Ala His Pro Ala Ser Ser Thr Thr 230 Val 235 Asp Lys Lys Leu Glu 240 Pro Ser Gly Pro Ile 245 Ser Thr Ile Asn Pro 250 Cys Pro Pro Cys Lys Glu 255 Cys His Lys Cys 260 Pro Ala Pro Asn Leu Glu 265 Gly Gly Pro Ser 270 Val Phe Ile Phe Pro 275 Pro Asn Ile Lys Asp Val Leu 280 Met Ile Ser Leu 285 Thr Pro
    Page 19
    SIW01-007-CIP-WO_Sequence_Listing.txt
    Lys Val 290 Thr Cys Val Val Val 295 Asp Val Ser Glu Asp 300 Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr 305 310 315 320 Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Ile Arg Val Val Ser Thr 325 330 335 Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys 340 345 350 Lys Val Asn Asn Lys Asp Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser 355 360 365 Lys Ile Lys Gly Leu Val Arg Ala Pro Gln Val Tyr Ile Leu Pro Pro 370 375 380 Pro Ala Glu Gln Leu Ser Arg Lys Asp Val Ser Leu Thr Cys Leu Val 385 390 395 400 Val Gly Phe Asn Pro Gly Asp Ile Ser Val Glu Trp Thr Ser Asn Gly 405 410 415 His Thr Glu Glu Asn Tyr Lys Asp Thr Ala Pro Val Leu Asp Ser Asp 420 425 430 Gly Ser Tyr Phe Ile Tyr Ser Lys Leu Asn Met Lys Thr Ser Lys Trp 435 440 445 Glu Lys Thr Asp Ser Phe Ser Cys Asn Val Arg His Glu Gly Leu Lys 450 455 460 Asn Tyr Tyr Leu Lys Lys Thr Ile Ser Arg Ser Pro Gly 465 470 475
    <210> 17 <211> 471 <212> PRT <213> Artificial sequence <220>
    <223> Chimeric anti-AGE human IgG1 heavy chain <400> 17
    Met Asp Pro Lys Gly Ser Leu Ser Trp Arg Ile Leu Leu Phe Leu Ser 1 5 10 15
    Page 20
    SIW01-007-CIP-WO_Sequence_Listing.txt
    Leu Ala Phe Glu 20 Leu Ser Tyr Gly Gln Val 25 Gln Leu Leu Gln 30 Pro Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ala Cys Lys Ala 35 40 45 Ser Gly Tyr Leu Phe Thr Thr Tyr Trp Met His Trp Leu Lys Gln Arg 50 55 60 Pro Gly Gln Gly Leu Glu Trp Ile Gly Glu Ile Ser Pro Thr Asn Gly 65 70 75 80 Arg Ala Tyr Tyr Asn Ala Arg Phe Lys Ser Glu Ala Thr Leu Thr Val 85 90 95 Asp Lys Ser Ser Asn Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser 100 105 110 Glu Ala Ser Ala Val Tyr Tyr Cys Ala Arg Ala Tyr Gly Asn Tyr Glu 115 120 125 Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Val Ala Ser 130 135 140 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 145 150 155 160 Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 165 170 175 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 180 185 190 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 195 200 205 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 210 215 220 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 225 230 235 240 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 245 250 255 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 260 265 270
    Page 21
    SIW01-007-CIP-WO_Sequence_Listing.txt
    Lys Asp Thr 275 Leu Met Ile Ser Arg Thr 280 Pro Glu Val Thr 285 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 290 295 300 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 305 310 315 320 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 325 330 335 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 340 345 350 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 355 360 365 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 370 375 380 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 385 390 395 400 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 405 410 415 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 420 425 430 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 435 440 445 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 450 455 460 Ser Leu Ser Leu Ser Pro Gly
    465 470 <210> 18 <211> 239 <212> PRT <213> Artificial sequence <220>
    <223> Murine anti-AGE kappa light chain
    Page 22
    SIW01-007-CIP-WO_Sequence_Listing.txt <400> 18
    Met 1 Glu Thr Asp Thr 5 Leu Leu Leu Trp Val 10 Leu Leu Leu Trp Val 15 Pro Gly Ser Thr Gly Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro 20 25 30 Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Arg Gln Ser 35 40 45 Leu Val Asn Ser Asn Gly Asn Thr Phe Leu Gln Trp Tyr Leu Gln Lys 50 55 60 Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Leu Arg Phe 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Leu Tyr Phe 100 105 110 Cys Ser Gln Ser Thr His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys 115 120 125 Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro 130 135 140 Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe 145 150 155 160 Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp 165 170 175 Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys 195 200 205 Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys 210 215 220 Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 225 230 235
    Page 23
    SIW01-007-CIP-WO_Sequence_Listing.txt <210> 19 <211> 239 <212> PRT <213> Artificial sequence <220>
    <223> Chimeric anti-AGE human kappa light chain <400> 19
    Met Glu Thr 1 Asp Thr 5 Leu Leu Leu Trp Val 10 Leu Leu Leu Trp Val 15 Pro Gly Ser Thr Gly Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro 20 25 30 Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Arg Gln Ser 35 40 45 Leu Val Asn Ser Asn Gly Asn Thr Phe Leu Gln Trp Tyr Leu Gln Lys 50 55 60 Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Leu Arg Phe 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Leu Tyr Phe 100 105 110 Cys Ser Gln Ser Thr His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys 115 120 125 Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
    Page 24
    210
    SIW01-007-CIP-WO_Sequence_Listing.txt 215 220
    Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 20 <211> 118 <212> PRT <213> Artificial sequence <220>
    <223> Murine anti-AGE IgG2b heavy chain (variable region) <400> 20
    Gln 1 Val Gln Leu Leu Gln 5 Pro Gly Ala Glu 10 Leu Val Lys Pro Gly 15 Ala Ser Val Lys Leu Ala Cys Lys Ala Ser Gly Tyr Leu Phe Thr Thr Tyr 20 25 30 Trp Met His Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Ser Pro Thr Asn Gly Arg Ala Tyr Tyr Asn Ala Arg Phe 50 55 60 Lys Ser Glu Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Ala Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Tyr Gly Asn Tyr Glu Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Val
    115 <210> 21 <211> 112 <212> PRT <213> Artificial sequence <220>
    <223> Murine anti-AGE kappa light chain (variable region) <400> 21
    Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Page 25
    SIW01-007-CIP-WO_Sequence_Listing.txt
    1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Arg Gln Ser Leu Val Asn Ser 20 25 30 Asn Gly Asn Thr Phe Leu Gln Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Leu Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Leu Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
    100 105 110 <210> 22 <211> 326 <212> PRT <213> Artificial sequence
    <220> <223> Human constant region <400> 22 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Page 26
    SIW01-007-CIP-WO_Sequence_Listing.txt
    100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys
    <210> 23 <211> 7 <212> PRT <213> Artificial sequence
    325
    Page 27
    SIW01-007-CIP-WO_Sequence_Listing.txt <220>
    <223> CDR1H (heavy chain) <400> 23
    Ser Tyr Thr Met Gly Val Ser 1 5 <210> 24 <211> 17 <212> PRT <213> Artificial sequence <220>
    <223> CDR2H (heavy chain) <400> 24
    Thr Ile Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val Lys 1 5 10 15
    Gly <210> 25 <211> 10 <212> PRT <213> Artificial sequence <220>
    <223> CDR3H (heavy chain) <220>
    <221> misc_feature <222> (10)..(10) <223> Xaa can be any naturally occurring amino acid <400> 25
    Gln Gly Gly Trp Leu Pro Pro Phe Ala Xaa
    1 5 10 <210> 26 <211> 17 <212> PRT <213> Artificial sequence <220>
    <223> CDR1L (light chain) <400> 26
    Arg Ala Ser Lys Ser Val Ser Thr Ser Ser Arg Gly Tyr Ser Tyr Met 1 5 10 15
    His
    Page 28
    SIW01-007-CIP-WO_Sequence_Listing.txt <210> 27 <211> 7 <212> PRT <213> Artificial sequence <220>
    <223> CDR2L (light chain) <400> 27
    Leu Val Ser Asn Leu Glu Ser 1 5 <210> 28 <211> 9 <212> PRT <213> Artificial sequence <220>
    <223> CDR3L (light chain) <400> 28
    Gln His Ile Arg Glu Leu Thr Arg Ser 1 5 <210> 29 <211> 468 <212> PRT <213> Artificial Sequence <220>
    <223> Humanized heavy chain <400> 29
    Met 1 Asp Pro Lys Gly 5 Ser Leu Ser Trp Arg 10 Ile Leu Leu Phe Leu 15 Ser Leu Ala Phe Glu Leu Ser Tyr Gly Gln Val Gln Leu Val Gln Ser Gly 20 25 30 Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala 35 40 45 Ser Gly Tyr Leu Phe Thr Thr Tyr Trp Met His Trp Val Arg Gln Ala 50 55 60 Pro Gly Gln Gly Leu Glu Trp Met Gly Glu Ile Ser Pro Thr Asn Gly 65 70 75 80 Arg Ala Tyr Tyr Asn Gln Lys Phe Gln Gly Arg Val Thr Met Thr Val 85 90 95
    Page 29
    SIW01-007-CIP-WO_Sequence_Listing.txt
    Asp Lys Ser Thr Asn 100 Thr Val Tyr Met Glu 105 Leu Ser Ser Leu 110 Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Tyr Gly Asn Tyr Phe 115 120 125 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145 150 155 160 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 225 230 235 240 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Page 30
    SIW01-007-CIP-WO_Sequence_Listing.txt 340 345 350
    Ala Pro Ile 355 Glu Lys Thr Ile Ser 360 Lys Ala Lys Gly Gln 365 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Lys Asn Gln 370 375 380 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395 400 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
    450 455 460
    Leu Ser Pro Gly 465 <210> 30 <211> 1408 <212> DNA <213> Artificial Sequence <220>
    <223> Humanized heavy chain <400> 30
    atggacccca agggcagcct gagctggaga atcctgctgt tcctgagcct ggccttcgag 60 ctgagctacg gccaggtgca gctggtgcag tctggcgccg aagtgaagaa acctggcgcc 120 tccgtgaggt gtcctgcaag gcttccggct acctgttcac cacctactgg atgcactggg 180 tgcgacaggc ccctggacag ggcctggaat ggatgggcga gatctcccct accaacggca 240 gagcctacta caacagaaat tccagggcag agtgaccatg accgtggaca agtccaccaa 300 caccgtgtac atggaactgt cctccctgcg gagcgaggac accgccgtgt actactgcgc 360 tagagcctac ggcaactacg attcgcctac tggggccagg gcaccctcgt gacagtgtcc 420 tctgctagca ccaagggccc cagcgtgttc cctctggccc ccagcagcaa gagcaccagc 480 ggcggaaccg ccgccctggg ctgcctggga aggactactt ccccgagccc gtgaccgtgt 540
    Page 31
    cctggaacag cggcgctctg SIW01-007- accagcggag -CIP-WO_Sequence_Listing.txt tgcacacctt ccctgccgtg ctgcagagca 600 gcggcctgta ctccctgagc agcgtggtga ccgtgccagc agcagcctgg gcacccagac 660 ctacatctgc aacgtgaacc acaagccctc caacaccaag gtggacaaga aggtggagcc 720 taagagctgc gacaagaccc acacctgccc tccctgcccc gccccgagct gctgggcgga 780 cccagcgtgt tcctgttccc tcccaagccc aaggacaccc tgatgatcag ccgcaccccc 840 gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc ccgaggtgag ttcaactggt 900 acgtggacgg cgtggaggtg cacaacgcca agaccaagcc tcgggaggag cagtacaact 960 ccacctaccg cgtggtgagc gtgctgaccg tgctgcacca ggactggctg aacggcagga 1020 gtacaagtgc aaggtgagca acaaggccct gcccgctccc atcgagaaga ccatcagcaa 1080 ggccaagggc cagccccggg agcctcaggt gtacaccctg ccccccagcc gcgacgagct 1140 gacaagaacc aggtgagcct gacctgcctg gtgaagggct tctacccctc cgacatcgcc 1200 gtggagtggg agagcaacgg ccagcctgag aacaactaca agaccacccc tcccgtgctg 1260 gacagcgacg cagcttcttc ctgtacagca agctgaccgt ggacaagtcc cggtggcagc 1320 agggcaacgt gttcagctgc agcgtgatgc acgaggccct gcacaaccac tacacccaga 1380 agagcctgag cctgagcccg gatagtaa 1408
    <210> 31 <211> 468 <212> PRT <213> Artificial Sequence
    <220> <223> Humanized heavy chain <400> 31 Met Asp Pro Lys Gly Ser Leu Ser Trp Arg Ile Leu Leu Phe Leu Ser 1 5 10 15 Leu Ala Phe Glu Leu Ser Tyr Gly Gln Val Gln Leu Val Gln Ser Gly 20 25 30 Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala 35 40 45 Ser Gly Tyr Leu Phe Thr Thr Tyr Trp Met His Trp Val Arg Gln Ala 50 55 60 Pro Gly Gln Gly Leu Glu Trp Met Gly Glu Ile Ser Pro Thr Asn Gly 65 70 75 80 Arg Ala Tyr Tyr Asn Ala Lys Phe Gln Gly Arg Val Thr Met Thr Val
    85 90 95
    Page 32
    SIW01-007-CIP-WO_Sequence_Listing.txt
    Asp Lys Ser Thr Asn 100 Thr Ala Tyr Met Glu 105 Leu Ser Ser Leu 110 Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Tyr Gly Asn Tyr Phe 115 120 125 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145 150 155 160 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 225 230 235 240 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Page 33
    SIW01-007-CIP-WO_Sequence_Listing.txt 340 345 350
    Ala Pro Ile 355 Glu Lys Thr Ile Ser 360 Lys Ala Lys Gly Gln 365 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Lys Asn Gln 370 375 380 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395 400 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
    450 455 460
    Leu Ser Pro Gly 465 <210> 32 <211> 1408 <212> DNA <213> Artificial Sequence <220>
    <223> Humanized heavy chain <400> 32
    atggacccca agggcagcct gagctggaga atcctgctgt tcctgagcct ggccttcgag 60 ctgagctacg gccaggtgca gctggtgcag tctggcgccg aagtgaagaa acctggcgcc 120 tccgtgaggt gtcctgcaag gcttccggct acctgttcac cacctactgg atgcactggg 180 tgcgacaggc ccctggacag ggcctggaat ggatgggcga gatctcccct accaacggca 240 gagcctacta caaccaaaat tccagggcag agtgaccatg accgtggaca agtccaccaa 300 caccgcttac atggaactgt cctccctgcg gagcgaggac accgccgtgt actactgcgc 360 tagagcctac ggcaactacg attcgcctac tggggccagg gcaccctcgt gacagtgtcc 420 tctgctagca ccaagggccc cagcgtgttc cctctggccc ccagcagcaa gagcaccagc 480 ggcggaaccg ccgccctggg ctgcctggga aggactactt ccccgagccc gtgaccgtgt 540
    Page 34
    cctggaacag cggcgctctg SIW01-007- accagcggag -CIP-WO_Sequence_Listing.txt tgcacacctt ccctgccgtg ctgcagagca 600 gcggcctgta ctccctgagc agcgtggtga ccgtgccagc agcagcctgg gcacccagac 660 ctacatctgc aacgtgaacc acaagccctc caacaccaag gtggacaaga aggtggagcc 720 taagagctgc gacaagaccc acacctgccc tccctgcccc gccccgagct gctgggcgga 780 cccagcgtgt tcctgttccc tcccaagccc aaggacaccc tgatgatcag ccgcaccccc 840 gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc ccgaggtgag ttcaactggt 900 acgtggacgg cgtggaggtg cacaacgcca agaccaagcc tcgggaggag cagtacaact 960 ccacctaccg cgtggtgagc gtgctgaccg tgctgcacca ggactggctg aacggcagga 1020 gtacaagtgc aaggtgagca acaaggccct gcccgctccc atcgagaaga ccatcagcaa 1080 ggccaagggc cagccccggg agcctcaggt gtacaccctg ccccccagcc gcgacgagct 1140 gacaagaacc aggtgagcct gacctgcctg gtgaagggct tctacccctc cgacatcgcc 1200 gtggagtggg agagcaacgg ccagcctgag aacaactaca agaccacccc tcccgtgctg 1260 gacagcgacg cagcttcttc ctgtacagca agctgaccgt ggacaagtcc cggtggcagc 1320 agggcaacgt gttcagctgc agcgtgatgc acgaggccct gcacaaccac tacacccaga 1380 agagcctgag cctgagcccg gatagtaa 1408
    <210> 33 <211> 468 <212> PRT <213> Artificial Sequence
    <220> <223> Humanized heavy chain <400> 33 Met Asp Pro Lys Gly Ser Leu Ser Trp Arg Ile Leu Leu Phe Leu Ser 1 5 10 15 Leu Ala Phe Glu Leu Ser Tyr Gly Gln Val Gln Leu Val Gln Ser Gly 20 25 30 Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala 35 40 45 Ser Gly Tyr Leu Phe Thr Thr Tyr Trp Met His Trp Val Arg Gln Ala 50 55 60 Pro Gly Gln Gly Leu Glu Trp Met Gly Glu Ile Ser Pro Thr Asn Gly 65 70 75 80 Arg Ala Tyr Tyr Asn Ala Lys Phe Gln Gly Arg Val Thr Met Thr Val
    85 90 95
    Page 35
    SIW01-007-CIP-WO_Sequence_Listing.txt
    Asp Lys Ser Ile 100 Asn Thr Ala Tyr Met Glu 105 Leu Ser Arg Leu 110 Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Tyr Gly Asn Tyr Phe 115 120 125 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145 150 155 160 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 225 230 235 240 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Page 36
    SIW01-007-CIP-WO_Sequence_Listing.txt 340 345 350
    Ala Pro Ile 355 Glu Lys Thr Ile Ser 360 Lys Ala Lys Gly Gln 365 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Lys Asn Gln 370 375 380 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395 400 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
    450 455 460
    Leu Ser Pro Gly 465 <210> 34 <211> 1408 <212> DNA <213> Artificial Sequence <220>
    <223> Humanized heavy chain <400> 34
    atggacccca agggcagcct gagctggaga atcctgctgt tcctgagcct ggccttcgag 60 ctgagctacg gccaggtgca gctggtgcag tctggcgccg aagtgaagaa acctggcgcc 120 tccgtgaggt gtcctgcaag gcttccggct acctgttcac cacctactgg atgcactggg 180 tgcgacaggc ccctggacag ggcctggaat ggatgggcga gatctcccct accaacggca 240 gagcctacta caaccaaaat tccagggcag agtgaccatg accgtggaca agtccatcaa 300 caccgcttac atggaactgt ccagactgcg gagcgatgac accgccgtgt actactgcgc 360 tagagcctac ggcaactacg attcgcctac tggggccagg gcaccctcgt gacagtgtcc 420 tctgctagca ccaagggccc cagcgtgttc cctctggccc ccagcagcaa gagcaccagc 480 ggcggaaccg ccgccctggg ctgcctggga aggactactt ccccgagccc gtgaccgtgt 540
    Page 37
    cctggaacag cggcgctctg SIW01-007 accagcggag CIP-WO_Sequence_Listing.txt tgcacacctt ccctgccgtg ctgcagagca 600 gcggcctgta ctccctgagc agcgtggtga ccgtgccagc agcagcctgg gcacccagac 660 ctacatctgc aacgtgaacc acaagccctc caacaccaag gtggacaaga aggtggagcc 720 taagagctgc gacaagaccc acacctgccc tccctgcccc gccccgagct gctgggcgga 780 cccagcgtgt tcctgttccc tcccaagccc aaggacaccc tgatgatcag ccgcaccccc 840 gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc ccgaggtgag ttcaactggt 900 acgtggacgg cgtggaggtg cacaacgcca agaccaagcc tcgggaggag cagtacaact 960 ccacctaccg cgtggtgagc gtgctgaccg tgctgcacca ggactggctg aacggcagga 1020 gtacaagtgc aaggtgagca acaaggccct gcccgctccc atcgagaaga ccatcagcaa 1080 ggccaagggc cagccccggg agcctcaggt gtacaccctg ccccccagcc gcgacgagct 1140 gacaagaacc aggtgagcct gacctgcctg gtgaagggct tctacccctc cgacatcgcc 1200 gtggagtggg agagcaacgg ccagcctgag aacaactaca agaccacccc tcccgtgctg 1260 gacagcgacg cagcttcttc ctgtacagca agctgaccgt ggacaagtcc cggtggcagc 1320 agggcaacgt gttcagctgc agcgtgatgc acgaggccct gcacaaccac tacacccaga 1380 agagcctgag cctgagcccg gatagtaa 1408
    <210> 35 <211> 238 <212> PRT <213> Artificial Sequence
    <220> <223> Humanized light chain <400> 35 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro 20 25 30 Val Thr Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 Leu Val Asn Ser Asn Gly Asn Thr Phe Leu Gln Trp Tyr Gln Gln Arg 50 55 60 Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Lys Val Ser Leu Arg Phe 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
    85 90 95
    Page 38
    SIW01-007-CIP-WO_Sequence_Listing.txt
    Thr Leu Lys Ile 100 Ser Arg Val Glu Ala Glu 105 Asp Val Gly Val 110 Tyr Tyr Cys Ser Gln Ser Thr His Val Pro Pro Thr Phe Gly Gly Gly Thr Val 115 120 125 Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 130 135 140 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170 175 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180 185 190 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 195 200 205 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 210 215 220 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235
    <210> 36 <211> 715 <212> DNA <213> Artificial Sequence <220>
    <223> Humanized light chain <400> 36
    atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60 gacgtcgtga tgacccagtc ccctctgtcc ctgcctgtga ccctgggaca gcctgcctcc 120 atctcctcag atcctcccag tccctcgtga actccaacgg caacaccttc ctgcagtggt 180 atcagcagcg gcctggccag agccccagac tgctgatcta caaggtgtcc ctgcggttct 240 ccggcgtgcc cgacgatttt ccggctctgg ctctggcacc gacttcaccc tgaagatctc 300 ccgggtggaa gccgaggacg tgggcgtgta ctactgctcc cagagcaccc acgtgccccc 360 tacatttggc ggaggcacca agtggaaatc aagcggaccg tggccgcccc cagcgtgttc 420 atcttccctc ccagcgacga gcagctgaag tctggcaccg Page ccagcgtggt 39 gtgcctgctg 480
    SIW01-007-CIP-WO_Sequence_Listing.txt aacaacttct acccccgcga ggccaagggc agtggaaggt ggacaacgcc ctgcagagcg gcaacagcca ggagagcgtg accgagcagg actccaagga cagcacctac agcctgagca gcaccctgac cctgagcaag gccgactacg agaagacaag gtgtacgcct gcgaggtgac ccaccaggga ctgtctagcc ccgtgaccaa gagcttcaac cggggcgagt gctaa
    540
    600
    660
    715 <210> 37 <211> 238 <212> PRT <213> Artificial Sequence <220>
    <223> Humanized light chain <400> 37
    Met 1 Glu Thr Asp Thr 5 Leu Leu Leu Trp Val 10 Leu Leu Leu Trp Val 15 Pro Gly Ser Thr Gly Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro 20 25 30 Val Thr Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Arg Gln Ser 35 40 45 Leu Val Asn Ser Asn Gly Asn Thr Phe Leu Gln Trp Tyr Gln Gln Arg 50 55 60 Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Lys Val Ser Leu Arg Phe 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 Cys Ser Gln Ser Thr His Val Pro Pro Thr Phe Gly Gly Gly Thr Val 115 120 125 Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 130 135 140 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170 175
    Page 40
    SIW01-007-CIP-WO_Sequence_Listing.txt
    Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180 185 190 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 195 200 205 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 210 215 220 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
    225 230 235 <210> 38 <211> 715 <212> DNA <213> Artificial Sequence <220>
    <223> Humanized light chain <400> 38
    atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60 gacgtcgtga tgacccagtc ccctctgtcc ctgcctgtga ccctgggaca gcctgcctcc 120 atctcctcag atccaggcag tccctcgtga actccaacgg caacaccttc ctgcagtggt 180 atcagcagcg gcctggccag agccccagac tgctgatcta caaggtgtcc ctgcggttct 240 ccggcgtgcc cgacgatttt ccggctctgg ctctggcacc gacttcaccc tgaagatctc 300 ccgggtggaa gccgaggacg tgggcgtgta ctactgctcc cagagcaccc acgtgccccc 360 tacatttggc ggaggcacca agtggaaatc aagcggaccg tggccgcccc cagcgtgttc 420 atcttccctc ccagcgacga gcagctgaag tctggcaccg ccagcgtggt gtgcctgctg 480 aacaacttct acccccgcga ggccaagggc agtggaaggt ggacaacgcc ctgcagagcg 540 gcaacagcca ggagagcgtg accgagcagg actccaagga cagcacctac agcctgagca 600 gcaccctgac cctgagcaag gccgactacg agaagacaag gtgtacgcct gcgaggtgac 660 ccaccaggga ctgtctagcc ccgtgaccaa gagcttcaac cggggcgagt gctaa 715
    <210> 39 <211> 238 <212> PRT <213> Artificial Sequence <220>
    <223> Humanized light chain <400> 39
    Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro Page 41
    SIW01-007-CIP-WO_Sequence_Listing.txt
    1 5 10 15 Gly Ser Thr Gly Asp Val Val Met Thr Gln Ser Pro Leu Ser Ser Pro 20 25 30 Val Thr Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 Leu Val Asn Ser Asn Gly Asn Thr Phe Leu Gln Trp Tyr His Gln Arg 50 55 60 Pro Gly Gln Pro Pro Arg Leu Leu Ile Tyr Lys Val Ser Leu Arg Phe 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ala Gly Lys Asp Phe 85 90 95 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 Cys Ser Gln Ser Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Leu 115 120 125 Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 130 135 140 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170 175 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180 185 190 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 195 200 205 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 210 215 220 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
    225 230 235 <210> 40
    <211> 715 <212> DNA <213> Artificial Sequence
    Page 42
    SIW01-007-CIP-WO_Sequence_Listing.txt <220>
    <223> Humanized light chain <400> 40
    atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60 gacgtcgtga tgacccagtc ccctctgtcc agtcctgtga ccctgggaca gcctgcctcc 120 atctcctcag atcctcccag tccctcgtga actccaacgg caacaccttc ctgcagtggt 180 atcaccagcg gcctggccag cctcccagac tgctgatcta caaggtgtcc ctgcggttct 240 ccggcgtgcc cgacgatttt ccggctctgg cgctggcaag gacttcaccc tgaagatctc 300 ccgggtggaa gccgaggacg tgggcgtgta ctactgctcc cagagcaccc acgtgccccc 360 tacatttggc cagggcacca actggaaatc aagcggaccg tggccgcccc cagcgtgttc 420 atcttccctc ccagcgacga gcagctgaag tctggcaccg ccagcgtggt gtgcctgctg 480 aacaacttct acccccgcga ggccaagggc agtggaaggt ggacaacgcc ctgcagagcg 540 gcaacagcca ggagagcgtg accgagcagg actccaagga cagcacctac agcctgagca 600 gcaccctgac cctgagcaag gccgactacg agaagacaag gtgtacgcct gcgaggtgac 660 ccaccaggga ctgtctagcc ccgtgaccaa gagcttcaac cggggcgagt gctaa 715
    <210> 41 <211> 5 <212> PRT <213> Mus musculus <400> 41
    Thr Tyr Trp Met His 1 5 <210> 42 <211> 17 <212> PRT <213> Mus musculus <400> 42
    Glu Ile Ser Pro Thr Asn Gly Arg Ala Tyr Tyr Asn Ala Arg Phe Lys
    1 5 10 15 Ser
    <210> 43 <211> 9 <212> PRT <213> Mus musculus <400> 43
    Page 43
    Ala Tyr Gly Asn Tyr Glu SIW01-007-CIP Phe Ala Tyr :>-WO_Sequence_Listing .txt 1 5 <210> 44 <211> 16 <212> PRT <213> Mus musculus <400> 44 Arg Ser Arg Gln Ser Leu Val Asn Ser Asn Gly Asn Thr Phe Leu Gln 1 5 10 15 <210> 45 <211> 7 <212> PRT <213> Mus musculus <400> 45 Lys Val Ser Leu Arg Phe Ser 1 5 <210> 46 <211> 9 <212> PRT <213> Mus musculus <400> 46 Ser Gln Ser Thr His Val Pro Pro Thr 1 5
    Page 44
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