Patent application title: GLUCAGON RECEPTOR ANTAGONISTS
Inventors:
Andrew Ihor Korytko (Oceanside, CA, US)
Rohn Lee Millican, Jr. (Indianapolis, IN, US)
Assignees:
ELI LILLY AND COMPANY
IPC8 Class: AA61K39395FI
USPC Class:
4241331
Class name: Drug, bio-affecting and body treating compositions immunoglobulin, antiserum, antibody, or antibody fragment, except conjugate or complex of the same with nonimmunoglobulin material structurally-modified antibody, immunoglobulin, or fragment thereof (e.g., chimeric, humanized, cdr-grafted, mutated, etc.)
Publication date: 2011-09-01
Patent application number: 20110212092
Abstract:
The present invention relates to glucagon receptor polypeptide
antagonists which inhibit the binding of the hormone glucagon to its
receptor. More particularly, the present invention relates to high
affinity glucagon receptor antibodies or Fab fragments thereof that
inhibit binding of glucagon to its receptor and their use in the
treatment or prevention of type 2 diabetes (NIDDM) and related disorders
in mammalian species.Claims:
1-15. (canceled)
16. A humanized monoclonal antibody that inhibits binding of glucagon to a glucagon receptor, wherein the antibody comprises a light chain and a heavy chain, wherein (i) the light chain comprises TABLE-US-00009 a CDRL1: S X S S S V S Y X1 H, SEQ ID NO: 53 a CDRL2: T T S X2 L A H, SEQ ID NO: 54 a CDRL3: X3 X4 R S T X5 P P T; SEQ ID NO: 55 and
(ii) the heavy chain comprises TABLE-US-00010 SEQ ID NO: 56 a CDRH1: G D D I T S G Y X6 X7, SEQ ID NO: 57 a CDRH2: Y I S Y S G S T X8 Y X9 P S L K S, SEQ ID NO: 58 a CDRH3: P P X10 Y Y G F G P Y A X11 D Y,
wherein: X is Y or A; X1 is M or I; X2 is N or Y; X3 is Q or L; X4 is Q or W; X5 is L or I; X6 is W or H; X7 is N, D or E; X8 is Y, Q, S or V; X9 is N, S or I; X10 is G or A; X11 is M or L.
17. The humanized monoclonal antibody according to claim 16, wherein X is Y or A; X1 is I; X2 is Y; X3 is Q or L; X4 is Q or W; X5 is L or I; X6 is W or H; X7 is D or E; X8 is Y, Q, S or V; X9 is S; X10 is G or A; X11 is L.
18. The humanized monoclonal antibody according to claim 17, wherein X is A; X1 is I; X2 is Y; X3 is Q; X4 is Q; X5 is L; X6 is H; X7 is D or E; X8 is Y, Q or S; X9 is S; X10 is G or A; X11 is L.
19. A pharmaceutical composition comprising an effective amount of the humanized monoclonal antibody according to claim 18 and a pharmaceutically acceptable excipient.
20. A method of treating type 1 diabetes in a human, wherein the method comprises administrating an effective amount of the humanized monoclonal antibody according to claim 18 to a human patient in need thereof.
21. A method of treating type 2 diabetes in a human, wherein the method comprises administrating an effective amount of the humanized monoclonal antibody according to claim 18 to a human patient in need thereof.
22. A method for achieving weight loss in a human, wherein the method comprises administrating an effective amount of the humanized monoclonal antibody according to claim 18 to a human patient in need thereof.
Description:
FIELD OF THE INVENTION
[0001] The present invention relates to glucagon receptor polypeptide antagonists which inhibit the binding of the hormone glucagon to its receptor. More particularly, the present invention relates to high affinity glucagon receptor antibodies or Fab fragments thereof that inhibit binding of glucagon to its receptor and their use in the treatment or prevention of type 2 diabetes (NIDDM) and related disorders in mammalian species.
BACKGROUND TO THE INVENTION
[0002] Glucagon is 29 amino acid peptide hormone produced by pancreatic α-cells in response to low blood glucose levels. Glucagon binds to a membrane-associated glucagon receptor on the surface of hepatocytes, which triggers a G-protein signal transduction cascade, activating intracellular cyclic AMP and leading to release of glucose through denovo synthesis (gluconeogenesis) and glycogen breakdown (glycogenolysis).
[0003] Unson et al., disclose polyclonal antibodies raised against synthetic peptides corresponding to two extracellular portions of the rat receptor. In the assay disclosed, polyclonal antibodies raised against amino acid residues 126-137 and 206-219 were found to block binding of glucagon to the receptor in rat liver membranes (Unson et al., PNAS Vol. 93, pp. 310-315, January 1996).
[0004] Buggy et al., discloses the preparation of a monoclonal antibody that is said to compete with glucagon for the hormone binding site of the receptor in an in vitro assay (Buggy et al., Horm. Metab. Res. 28 (1996) 215-219). In the assay disclosed the antibody, given the designation CIV395.7A, recognizes the human and rat glucagon receptors, but not mouse. In order to develop antibodies for human therapeutic treatments it is commonly necessary to perform pre-clinical efficacy and safety studies in validated rat and/or murine animal models. It would therefore greatly facilitate drug development of a therapeutic antibody and thus be highly desirable to provide a pre-clinical therapeutic antibody candidates that are able to bind rat, murine and human forms of the glucagon receptor.
[0005] Wright et al., disclose a monoclonal antibody designated hGR-2 F6 and the amino acid sequence of a Fab fragment thereof. This antibody has been raised in a mouse against the human glucagon receptor and described in the disclosed assay as a competitive antagonist at this receptor (Wright et al., Acta Cryst. (2000) D56, 573-580). The applicant has found that hGR-2 F6 binds to the rat and murine forms of the glucagon receptor with only low affinity, and no therapeutic efficacy has been found for hGR-2F6 in a diabetic rat in vivo model at high doses. In particular, this antibody was unable to reduce blood serum glucose in the rat model with any statistical significance (unpublished).
[0006] The applicant has identified a need to provide therapeutic monoclonal antibodies that will bind with high affinity to the glucagon receptor and thereby inhibit the binding of glucagon thereto, to provide effective treatments for diabetes, preferably type 2 diabetes and related disorders. Furthermore, in order to allow pre-clinical drug development of an antibody it is clearly desirable to provide monoclonal antibodies that can bind to the human, rat and murine forms of the glucagon receptor to allow obligatory pre-clinical safety and efficacy studies to be undertaken.
SUMMARY OF INVENTION
[0007] The present invention relates to novel monoclonal antibodies or Fab fragments thereof that are able to specifically bind with a high affinity to native glucagon receptors of human, rat and murine origin. Furthermore, the present inventors provide monoclonal antibodies or Fab fragments thereof that not only bind glucagon receptors of multiple origins, but for the first time provide glucagon receptor binding monoclonal antibodies that show in vivo efficacy in their ability to reduce blood serum glucose.
[0008] In a first aspect the present invention relates to a Fab fragment or humanized monoclonal antibody comprising said Fab fragment, wherein said Fab fragment capable of binding to human, rat and murine glucagon receptors and inhibits glucagon binding to each receptor with a Ki of less than 50 nM.
[0009] In a further unexpected finding the inventors for the first time provide antibodies or Fab fragments thereof capable of specifically binding to the glucagon receptor that are able to significantly increase in vivo serum concentrations of GLP-1. Increasing serum levels of GLP-1 is known in the art to enhance β-cell function, reduce glucagon secretion and delay gastric emptying and is recognized as being highly advantageous in the treatment of diabetes type 2 and associated conditions.
[0010] In a second aspect, the invention relates to a pharmaceutical composition comprising an effective amount of a Fab fragment or humanized monoclonal antibody according to the present invention and a pharmaceutically acceptable excipient.
[0011] In a third aspect, the invention provides a method of treating type 1 or type 2 diabetes and in the achievement of weight loss in a human, wherein said method comprises administrating an effective amount of a Fab fragment or humanized monoclonal antibody according to the present invention, to a patient in need thereof.
[0012] A fourth aspect of the invention comprises a Fab fragment or humanized monoclonal antibody according to the present invention for use as a medicament.
[0013] A fifth aspect of the invention of relates to the use of a Fab fragment or humanized monoclonal antibody according to the present invention in the manufacture of a medicament for the treatment or prevention of type 1 or type 2 diabetes or in the achievement of weight loss in a human.
DETAILED DESCRIPTION
[0014] The present invention relates to a Fab fragment or humanized monoclonal antibody comprising said Fab fragment, wherein said Fab fragment is capable of binding to human, rat and murine glucagon receptors and inhibits glucagon binding to each receptor with a Ki of less than 50 nM.
[0015] The invention also provides monoclonal antibodies, which in addition to the glucagon receptors of human, rat and murine origin, are also able to bind with a high affinity to the glucagon receptor of a cynomologous monkey. Preferably, the Fab fragment or humanized monoclonal antibody comprising said Fab fragment therefore also inhibits glucagon binding to a cynomologous monkey glucagon receptor with a Ki of less than 50 nM. Preferably the Fab fragment or humanized monoclonal antibody comprising said Fab fragment has a Ki at each of the named glucagon receptors of less than 30 nM, more preferably less than 20 nM, further preferred less than 10 nM. Further preferred the Fab fragment has an in vitro Ki at the rat, murine and cyno receptors of less than 20 nM and in vitro Ki at the human receptor of less than 5 nM. More preferably the Ki of the Fab fragment, and in particular at the human glucagon receptor, is from 0.1 nM to 15 nM, most preferably from 1 to 10 nM.
[0016] A Fab fragment or humanized monoclonal antibody comprising said Fab fragment according to the present invention preferably has a functional binding affinity (Kb) at the human and rat glucagon receptors of at least 100 nM. Further preferred the Fab fragment or humanized monoclonal antibody comprising said Fab fragment has a functional binding affinity at these receptors of at least 50 nM, more preferably at least 10 nM. In a most preferred embodiment the Fab fragment or humanized monoclonal antibody comprising said Fab fragment has a functional binding affinity at the human and rat glucagon receptor of 1 to 10 nM.
[0017] In a particularly unexpected finding, the applicant has noted a very rapid rate at which serum GLP-1 is increased and serum blood glucose decreased on in vivo exposure to the Fab fragment or humanized monoclonal antibody comprising said Fab fragment having the binding properties identified in accordance with this invention. In order to maximize this favourable effect it is preferable that the Fab fragment or humanized monoclonal antibody comprising said Fab fragment does not appreciably bind to the GLP receptor i.e. Ki greater than 5000 nM.
DESCRIPTION OF THE SEQUENCES
[0018] SEQ ID NOS 1 to 22 refer to the light and heavy chain CDRs of Table 1: SEQ ID NOS 23 to 30 refer to preferred human framework regions described herein: SEQ ID NOS 31 and 32 are amino acid and cDNA sequences of the human GluR: SEQ ID NOS 33 and 34 are amino acid and cDNA sequences of the rat GluR: SEQ ID NOS 35 and 36 are amino acid and cDNA sequences of the murine GluR: SEQ ID NOS 37 and 38 are amino acid and cDNA sequences of the cyno GluR: SEQ ID NOS 39 and 40 are the variable region amino acid sequences of example 1 (Ab1) SEQ ID NOS 41 and 42 are the variable region amino acid sequences of example 2 (Ab2) SEQ ID NOS 43 and 44 are the variable region amino acid sequences of example 3 (Ab3) SEQ ID NOS 45 and 46 are the variable region amino acid sequences of example 4 (Ab4) SEQ ID NOS 45 and 47 are the variable region amino acid sequences of example 5 (Ab5) SEQ ID NOS 45 and 48 are the variable region amino acid sequences of example 6 (Ab6) SEQ ID NOS 45 and 49 are the variable region amino acid sequences of example 7 (Ab7) SEQ ID NO: 50 refers to a preferred kappa light chain IgG4 constant region. SEQ ID NO: 51 refers to a preferred heavy chain CH1 constant domain. SEQ ID NO: 52 refers to a preferred modified human IgG4 Fc region. SEQ ID NOS 53 to 55 refer to preferred light chain CDRs. SEQ ID NOS 56 to 68 refer to preferred heavy chain CDRs. SEQ ID NOS 59 and 60 are light and heavy chain protein sequences of example 1 (Ab1). SEQ ID NOS 61 and 62 are light and heavy chain protein sequences of example 2 (Ab2). SEQ ID NOS 63 and 64 are light and heavy chain protein sequences of example 3 (Ab3). SEQ ID NOS 65 and 66 are light and heavy chain protein sequences of example 4 (Ab4). SEQ ID NOS 65 and 67 are light and heavy chain protein sequences of example 5 (Ab5). SEQ ID NOS 65 and 68 are light and heavy chain protein sequences of example 6 (Ab6). SEQ ID NOS 65 and 69 are light and heavy chain protein sequences of example 7 (Ab7). SEQ ID NOS 70 and 71 are light and heavy chain DNA sequences of example 1 (Ab1). SEQ ID NOS 72 and 73 are light and heavy chain DNA sequences of example 2 (Ab2). SEQ ID NOS 74 and 75 are light and heavy chain DNA sequences of example 3 (Ab3). SEQ ID NOS 76 and 77 are light and heavy chain DNA sequences of example 4 (Ab4). SEQ ID NOS 76 and 78 are light and heavy chain DNA sequences of example 5 (Ab5). SEQ ID NOS 76 and 79 are light and heavy chain DNA sequences of example 6 (Ab6). SEQ ID NOS 76 and 80 are light and heavy chain DNA sequences of example 7 (Ab7).
DEFINITIONS
[0019] The "glucagon receptor" also referred to herein as "GluR" belongs to the G protein-coupled receptor class 2 family consisting of a long amino terminal extracellular domain, seven transmembrane segments, and an intracellular C-terminal domain. Glucagon receptors are notably expressed on the surface of hepatocytes where they bind to glucagon and transduce the signal provided thereby into the cell. DNA sequences encoding glucagon receptors of rat and human origin have been isolated and disclosed in the art (EP0658200B1). The murine and cynomologous monkey homologues have also been isolated and sequenced (Burcelin, et al., Gene 164 (1995) 305-310); McNally et al., Peptides 25 (2004) 1171-1178).
[0020] The term "inhibits" as used herein with respect to an activity of an antibody or Fab fragment thereof of the invention means the ability to substantially antagonize the biological activity of the glucagon receptor. This ability is reflected in the Ki values calculated from the [125I] glucagon binding assay described herein.
[0021] The term "humanized" as used in reference to a monoclonal antibody of the invention refers to an antibody with at least human frameworks and constant regions (CL, CH domains (e.g., CH1, CH2, CH3), and hinge), and CDRs derived from glucagon receptor binding antibodies. Human frameworks comprise frameworks that correspond to human germline sequences as well as sequences with somatic mutations. Human frameworks and constant regions may be fully human or may vary from the native sequences by one or more amino acid substitutions, terminal and intermediate additions and deletions, and the like. CDRs may be derived from one or more CDRs that bind to the glucagon specific receptors in this application in the context of any antibody framework. For example, the CDRs of the humanized antibody of the present invention may be derived from CDRs that bind glucagon receptors in the context of a mouse antibody framework and then are engineered to bind glucagon receptors in the context of a human framework.
[0022] The term "monoclonal antibody" refers to an antibody that is derived from a single copy or clone, including e.g., any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Preferably a monoclonal antibody of the invention exists in a homogeneous or substantially homogeneous population.
[0023] The term "in vivo efficacy" as used herein with respect to an antibody of the invention means the ability of the antibody to impart a positive biological effect in a human or animal model. Preferably in vivo efficacy refers to a glucose normalization effect on an animal showing elevated blood glucose in response to an antibody of the present invention as compared to a control response. A diabetic Zucker diabetic fatty rat (ZDF) model (Horm Metab Res. 2005 February; 37(2):79-83) may be appropriately used to assess in vivo efficacy, wherein in vivo efficacy preferably denotes 100% blood glucose normalization on exposure of the animal to ≦30 mg/kg dosage of humanized antibody according to the present invention. More preferably in vivo efficacy denotes 100% blood glucose normalization on exposure of the animal to a dosage of ≦15 mg/kg of antibody, further preferred at a dosage of ≦10 mg/kg, more preferably 0.1 to 5 mg/kg. In a most preferred embodiment in vivo efficacy is used to denote 100% blood glucose normalization in a diabetic ZDF rat model on exposure of the animal to 1 to 3 mg/kg dosage of humanized antibody according to the present invention.
[0024] The term "glucose normalization" refers to mean plasma glucose values in a ZDF rat model of less than 120 mg/dL, preferably in the range of 110 to 120 mg/dL. Plasma glucose may be determined in accordance with Etgen et al., (Metabolism 2000; 49(5): 684-688) or calculated from a conversion of whole blood glucose concentration in accordance with D'Orazio et al., (Clin. Chem. Lab. Med. 2006; 44(12): 1486-1490).
[0025] As used herein, "Fab fragment" refers to that portion of an antibody molecule, within the variable region, which contains the amino acid residues of the light and heavy chain CDR and framework sequences in addition to CL and CH1 domain.
[0026] The 3 CDRs of the heavy chain are herein referred to as "CDRH1, CDRH2, and CDRH3" and the 3 CDRs of the light chain are referred to as "CDRL1, CDRL2 and CDRL3". Assignment of amino acids to each domain is in accordance with a well-known convention (Kabat, et al., Ann. NY Acad. Sci. 190:382-93 (1971); Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991)). The antigen-binding domain, or the CDRs of the antigen-binding domain, can be derived from other non-human species including, but not limited to, rabbit, mouse, rat or hamster.
[0027] The present inventors have identified heavy and light chain CDR sequences which may be used in combination to prepare antibody Fab fragments which demonstrate particularly high affinity for glucagon receptors of murine, rat, cynomologous monkey and human origin. Fab fragments preferably comprise;
TABLE-US-00001 (i) a light chain S X S S S V S Y SEQ ID NO: 53 CDRL1: X1 H (ii) a light chain T T S X2 L A H SEQ ID NO: 54 CDRL2: (iii) a light chain X3 X4 R S T X5 P SEQ ID NO: 55 CDRL3: P T (iv) a heavy chain G D D I T S G Y SEQ ID NO: 56 CDRH1: X6 X7 (v) a heavy chain Y I S Y S G S T X8 SEQ ID NO: 57 CDRH2: Y X9 P S L K S (vi) a heavy chain P P X10 Y Y G F G SEQ ID NO: 58 CDRH3: P Y A X11 D Y
wherein:
TABLE-US-00002 X = Y or A X1 = M or I X2 = N or Y X3 = Q or L X4 = Q or W X5 = L or I X6 = W or H X7 = N, D or E X8 = Y, Q, S or V X9 = N, S or I X10 = G or A X11 = M or L
More preferably, X is Y or A; X1 is I; X2 is Y; X3 is Q or L; X4 is Q or W; X5 is L or I; X6 is W or H; X7 is D or E; X8 is Y, Q, S or V; X9 is S; X10 is G or A; X11 is L. Further preferred, X is A; X1 is I; X2 is Y; X3 is Q; X4 is Q; X5 is L; X6 is H; X7 is D or E; X8 is Y, Q or S; X9 is S; X10 is G or A; X11 is L.
[0028] Preferably a Fab fragment or humanized monoclonal antibody comprising said Fab fragment of the present invention comprises the CDR sequences:
TABLE-US-00003 CDRL1 1 2 3 4 5 6 7 8 9 10 S A S S S V S Y I H CDRL2 1 2 3 4 5 6 7 T T S Y L A H CDRL3 1 2 3 4 5 6 7 8 9 Q Q R S T L P P T CDRH1 1 2 3 4 5 6 7 8 9 10 G D D I T S G Y H D CDRH2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Y I S Y S G S T Y Y S P S L K S CDRH3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 P P G Y Y G F G P Y A L D Y
wherein said Fab fragment has one, two or three amino acid substitutions selected from the group consisting of:
CDRL1: A2Y, 19M;
CDRL2: Y4N;
CDRL3: Q1L, Q2W, L6I;
CDRH1: H9W, D10E, D10N;
CDRH2: Y9Q, Y9S, Y9V, S11N, S11I;
CDRH3: G3A, L12M.
[0029] More preferably said Fab fragment comprises one, two or three amino acid substitutions selected from: CDRH1: D10E; CDRH2: Y9Q, Y9S; CDRH3: G3A.
[0030] A Fab fragment or humanized monoclonal antibody comprising said Fab fragment according to the present invention preferably comprises CDR sequences selected from:
[0031] (i) a light chain with a CDRL1 of SEQ ID NO 2; CDRL2 of SEQ ID NO: 4; CDRL3 of SEQ ID NO: 7; and a heavy chain with a CDRH1 of SEQ ID NO: 11; CDRH2 of SEQ ID NO: 15; CDRH3 of SEQ ID NO: 21;
[0032] (ii) a light chain with a CDRL1 of SEQ ID NO 1; CDRL2 of SEQ ID NO: 4; CDRL3 of SEQ ID NO: 6; and a heavy chain with a CDRH1 of SEQ ID NO: 10; CDRH2 of SEQ ID NO: 16; CDRH3 of SEQ ID NO: 20;
[0033] (iii) a light chain with a CDRL1 of SEQ ID NO: 3; CDRL2 of SEQ ID NO: 5; CDRL3 of SEQ ID NO: 8; and a heavy chain with a CDRH1 of SEQ ID NO: 11; CDRH2 of SEQ ID NO: 15; CDRH3 of SEQ ID NO: 21;
[0034] (iv) a light chain with a CDRL1 of SEQ ID NO 3; CDRL2 of SEQ ID NO: 5; CDRL3 of SEQ ID NO: 6; and a heavy chain with a CDRH1 of SEQ ID NO: 12; CDRH2 of SEQ ID NO: 15; CDRH3 of SEQ ID NO: 21;
[0035] (v) a light chain with a CDRL1 of SEQ ID NO 3; CDRL2 of SEQ ID NO: 5; CDRL3 of SEQ ID NO: 6; and a heavy chain with a CDRH1 of SEQ ID NO: 12; CDRH2 of SEQ ID NO: 17; CDRH3 of SEQ ID NO: 21;
[0036] (vi) a light chain with a CDRL1 of SEQ ID NO 3; CDRL2 of SEQ ID NO: 5; CDRL3 of SEQ ID NO: 6; and a heavy chain with a CDRH1 of SEQ ID NO: 12; CDRH2 of SEQ ID NO: 15; CDRH3 of SEQ ID NO: 22; and
[0037] (vii) a light chain with a CDRL1 of SEQ ID NO 3; CDRL2 of SEQ ID NO: 5; CDRL3 of SEQ ID NO: 6; and a heavy chain with a CDRH1 of SEQ ID NO: 13; CDRH2 of SEQ ID NO: 18; CDRH3 of SEQ ID NO: 22.
[0038] It is particularly desirable that an antibody in accordance with the present invention shows in vivo efficacy at a low plasma concentration. In vivo efficacy should be observed at a dosage of 30 mg/kg in a ZDF rat model, preferably at a dosage of less than 15 mg/kg, more preferably less than 5 mg/kg, further preferred with the range 0.1 to 5 mg/kg. Most preferably an antibody in accordance with the present invention achieves 100% glucose normalization in a ZDF rat model at a dosage of about 1 to 3 mg/kg. It has been found that particularly preferred antibodies in accordance with the present invention are able to show 100% glucose normalization in an in vivo ZDF rat model at the low dosage of 3 mg/kg. The present invention therefore preferably comprises a Fab fragment or humanized monoclonal antibody comprising said Fab fragment, wherein said Fab fragment comprises:
[0039] (i) a light chain with a CDRL1 of SEQ ID NO 3; CDRL2 of SEQ ID NO: 5; CDRL3 of SEQ ID NO: 6; and a heavy chain with a CDRH1 of SEQ ID NO: 12; CDRH2 of SEQ ID NO: 15; CDRH3 of SEQ ID NO: 21;
[0040] (ii) a light chain with a CDRL1 of SEQ ID NO 3; CDRL2 of SEQ ID NO: 5; CDRL3 of SEQ ID NO: 6; and a heavy chain with a CDRH1 of SEQ ID NO: 12; CDRH2 of SEQ ID NO: 17; CDRH3 of SEQ ID NO: 21;
[0041] (iii) a light chain with a CDRL1 of SEQ ID NO 3; CDRL2 of SEQ ID NO: 5; CDRL3 of SEQ ID NO: 6; and a heavy chain with a CDRH1 of SEQ ID NO: 12; CDRH2 of SEQ ID NO: 15; CDRH3 of SEQ ID NO: 22; or
[0042] (iv) a light chain with a CDRL1 of SEQ ID NO 3; CDRL2 of SEQ ID NO: 5; CDRL3 of SEQ ID NO: 6; and a heavy chain with a CDRH1 of SEQ ID NO: 13; CDRH2 of SEQ ID NO: 18; CDRH3 of SEQ ID NO: 22.
[0043] In a further preferred embodiment the present invention relates to a Fab fragment or humanized monoclonal antibody comprising said Fab fragment, wherein said Fab fragment comprises a light chain with a CDRL1 of SEQ ID NO 3; CDRL2 of SEQ ID NO: 5; CDRL3 of SEQ ID NO: 6; and a heavy chain with a CDRH1 of SEQ ID NO: 12; CDRH2 of SEQ ID NO: 17; CDRH3 of SEQ ID NO: 21. It has been found that an antibody comprising Fab fragments in accordance with this embodiment has a particularly advantageous property of maintaining in vivo efficacy over an extended period as compared to other similar antibodies within the genus being described.
[0044] A Fab fragment or humanized monoclonal antibody comprising said Fab fragment of the present invention, preferably comprises light and heavy variable chain framework regions of human origin. Moreover, a variety of different human framework sequences may be used singly or in combination as a basis for the humanized immunoglobulins of the present invention. Preferably, the framework regions of the Fab fragment or humanized monoclonal antibody of the invention are of human origin or substantially of human origin (at least 95%, 97% or 99% of human origin). The sequences of framework regions of human origin may be obtained from ImMunoGenetics (IMGT) via their website http://imgt.cines.fr/textes/IMGTindex/FR.html or from The immunoglobulin Factsbook, by Marie-Paule Lefranc, Gerard Lefranc, Academic Press 2001, ISBN 012441351. For example, germline light chain frameworks may be selected from the group consisting of: A11, A17, A18, A19, A20, A27, A30, L1, L1I, L12, L2, L5, L15 L6, L8, O12, O2, and O8 and germline heavy chain framework regions may be selected from the group consisting of: VH2-5, VH2-26, VH2-70, VH3-20, VH3-72, VHI-46, VH3-9, VH3-66, VH3-74, VH4-31, VH I-18, VH I-69, VI-13-7, VH3-11, VH3-15, VH3-21, VH3-23, VH3-30, VH3-48, VH4-39, VH4-59, and VH5-5I.
[0045] The specific antibodies disclosed herein can be used as a template or parent antibody to make additional antibodies of the invention. In one approach the parent antibody CDRs are grafted into a human framework that has a high sequence identity with the parent antibody framework. The sequence identity of the new framework will generally be at least 80%, at least 85%, or at least 90% with the corresponding framework in the parent antibody. This grafting may result in a reduction in binding affinity compared to the parent antibody. If this is the case, the framework can be back-mutated to the parent framework at certain positions based on specific criteria published by Queen et al., [Queen, et al., Proc. Natl. Acad. Sci. USA 88, 2869 (1991)]. Further methods that may be used include, for example, Jones et al., Nature, 321:522 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534 (1988).
[0046] Most preferably the Fab fragment or humanized monoclonal antibody comprising said Fab fragment according to the present invention comprises the following light chain framework (FR) sequences: FR1 SEQ ID NO: 23; FR2 SEQ ID NO: 24; FR3 SEQ ID NO: 25; FR4 SEQ ID NO: 26; and heavy variable chain frameworks sequences: FR5 SEQ ID NO: 27; FR6 SEQ ID NO: 28; FR7 SEQ ID NO: 29; FR8 SEQ ID NO: 30; wherein these are arranged as light chain variable sequence FR1-CDRL1-FR2-CDRL2-FR3-CDRL3-FR4 and heavy chain variable sequence FR5-CDRH1-FR6-CDRH2-FR7-CDRH3-FR8.
[0047] The applicant has surprisingly determined that when predicting the in vivo efficacy of a glucagon receptor antagonism antibody through the use of an in vitro competitive glucagon binding assay, that it is the affinity of the Fab fragment that is positively correlated with in vivo efficacy. Conversely, the binding affinity (Ki) of a full antibody to the glucagon receptor is not necessarily a valid predictor of in vivo efficacy. A process of preparing monoclonal antibodies having the favourable properties sought herein, therefore preferably comprises selecting a Fab fragment that binds to each glucagon receptor with a Ki of less than 50 nM in an in vitro competitive glucagon binding assay using heterologously expressed glucagon receptor gene. More preferably said process comprises selecting a Fab fragment that has an in vitro Ki at each of the glucagon receptors of less than 30 nM, more preferably less than 20 nM, further preferred less than 10 nM. More preferably, the Fab fragment is selected by an in vitro Ki at the rat, murine and cyno receptors of less than 20 nM and in vitro Ki at the human receptor of less than 5 nM. More preferably the Ki of the selected Fab fragment, and in particular at the human glucagon receptor, is from 0.1 nM to 15 nM, most preferably from 1 to 10 nM. Fab fragments identified by this process may then be suitably expressed as full antibodies for therapeutic use by techniques commonly known in the art.
[0048] It will be appreciated that applying the teaching of the present invention the person skilled in the art may use common techniques e.g. site directed mutagenesis, to substitute amino acids within the specific CDR and framework sequences herein disclosed and in so doing generate further variable region amino acid sequences derived from the sequences herein provided. Up to all 20 alternative naturally occurring amino acids may be introduced at a specific substitution site. The in vitro selection process defined here above may then be suitably used to screen these additional variable region amino acid sequences for Fab fragments having the claimed cross reactivity and in vitro Ki that has been found by the present applicants to be indicative of in vivo efficacy. In this way further Fab fragments are identified that are suitable for preparing a humanized antibody in accordance with the present invention. Preferably the amino acid substitution within the frameworks is restricted to one, two or three positions within one or each of the framework sequences disclosed herein. Preferably amino acid substitution within the CDRs is restricted to one to three positions within one or each CDR, more preferably substitution at one or two amino acid positions within one or each CDR is performed. Further preferred, amino acid substitution is performed at one or two amino acid positions in the CDRs of the heavy chain variable region. Most preferably amino acid substitution is performed at one or two amino acid positions within CDRH2.
[0049] A suitable methodology for combining CDR and framework substitutions to prepare alternative antibodies according to the present invention, using an antibody described herein as a parent antibody, is provided in Wu et al., J. Mol. Biol., 294:151-162.
[0050] As used herein, the Fc portion of an immunoglobulin refers to the constant region of an antibody from both heavy chains, which associate through non-covalent interactions and disulfide bonds. The Fc portion can include the hinge regions and extend through the CH2 and CH3 domains to the C-terminus of the antibody. The Fc portion can further include one or more glycosylation sites. Monoclonal antibodies of the present invention may have a heavy chain constant region selected from any of the immunoglobulin classes IgA, IgD, IgG, IgM and IgE. Preferably antibodies of the invention contain an Fc portion which is derived from human IgG4 Fc region because of its reduced ability to bind FcγR and complement factors as compared to other IgG sub-types. More preferably, the IgG4 Fc region of an antibody of the present invention contains substitutions that further reduce effector function [Issacs et al., (1996) Clin. Exp. Immunol. 106:427-433]. These may be selected from one or more of the group comprising proline for glutamate at residue 233, alanine or valine for phenylalanine at residue 234 and alanine or glutamate for leucine at residue 235 (EU numbering, Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. U.S. Dept. of Health and Human Services, Bethesda, Md., NIH Publication no. 91-3242). These residues corresponds to positions 15, 16 and 17 in SEQ ID NO: 52 and positions 235, 236 and 237 of SEQ ID NO: 67. Further, removing the N-linked glycosylation site in the IgG4 Fc region by substituting Ala for Asn at residue 297 (EU numbering) which corresponds to position 79 of SEQ ID NO:52 is another way to ensure that residual effector activity is eliminated in the context of a humanized antibody.
[0051] In addition, the IgG4 Fc portion for use with a humanized monoclonal antibody of present invention preferably contains a substitution that stabilizes heavy chain dimer formation and prevents the formation of half-IgG4 Fc chains. This construct consists of serine at position at 228 (EU numbering) being substituted by proline (amino acid residue 10 in SEQ ID NO:52). The C-terminal lysine residue present in the native molecule may also be deleted in the IgG4 derivative Fc portion of the antibodies discussed herein (position 229 of SEQ ID NO:52; deleted lysine referred to as des-K). A most preferred IgG4 Fc portion is provided by amino acids 221 to 448 of SEQ ID NO: 67.
[0052] The invention is further directed to an isolated nucleic acid sequence encoding an antibody of the invention; a vector (or vectors) comprising that nucleic acid, optionally operably linked to control sequences recognized by a host cell transformed with the vector; a host cell comprising that vector; a process for producing an antibody or Fab fragment thereof according to the invention comprising culturing the host cell so that the nucleic acid is expressed and, optionally, recovering the antibody from the host cell culture medium.
[0053] In another embodiment, the invention provides a pharmaceutical composition comprising the Fab fragment or humanized monoclonal antibody of the invention. The pharmaceutical composition of the invention may further comprise a pharmaceutically acceptable carrier. In said pharmaceutical composition, the Fab fragment or humanized monoclonal antibody of the invention is the active ingredient. Preferably the pharmaceutical composition comprises a homogeneous or substantially homogeneous population of the Fab fragment or humanized monoclonal antibody of the invention. The composition for therapeutic use is sterile and may be lyophilized, optionally supplied with an appropriate diluent.
[0054] A further embodiment of the present invention comprises a host cell or cell culture that is a recipient of any isolated polynucleotide of the invention or any recombinant vector(s) comprising a sequence encoding a HCVR, LCVR, monoclonal antibody or Fab fragment of the invention. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transformed, transduced or infected in vivo or in vitro with one or more a recombinant vectors or a polynucleotide expressing a monoclonal antibody of the invention or a light chain or heavy chain thereof. A host cell which comprises a recombinant vector of the invention (either stably incorporated into the host chromosome or not) may also be referred to as a "recombinant host cell". Preferred host cells for use in the invention are CHO cells, NS0 cells, HeLa, SP2/0 cells or COS cells. Additional host cells for use in the invention include plant cells, yeast cells, other mammalian cells and prokaryotic cells.
[0055] The invention embodies an article of manufacture comprising a packaging material and a Fab fragment or humanized monoclonal antibody of the invention contained within said packaging material and wherein the packaging material comprises a package insert which indicates that the Fab fragment or humanized monoclonal antibody neutralizes a GluR or decreases the level of GluR activity in the patient.
Biological Assays:
Glucagon Receptor (GlucR) Membrane Preparations
[0056] Membrane preparations for binding studies are prepared from 293HEK cells expressing cloned human, mouse, cynomologous monkey or rat glucagon receptor. Each clonal cell line is first grown as a suspension culture and the frozen cell pellet is resuspended in membrane prep buffer consisting of 25 mM Tris, pH 7.5, 1 mM MgCl2, Complete® EDTA-free protease inhibitor tablets (Roche Applied Science), and 20 U/ml DNase I (Sigma Chemical Company) at 4° C. The cells are homogenized with a motor-driven Teflon-glass Potter-Elvehjem homogenizer using 25 strokes, followed by centrifugation at 1800×g for 15 minutes at 4° C. The supernatant is collected and the pellet resuspended in membrane prep buffer, rehomogenized and centrifuged. The second supernatant is combined with the first supernatant and recentrifuged at 1800×g for 15 mins to clarify. The clarified supernatant is transferred to high speed tubes and centrifuged at 25000×g for 30 minutes at 4° C. The membrane pellet (P2) is resuspended in the membrane prep buffer (without DNAase), aliquoted, quick frozen on dry ice and stored at -80° C. until needed.
[125I]Glucagon Binding by Scintillation Proximity Assay (SPA).
[0057] A competitive receptor/ligand binding experiment is adapted to a scintillation proximity assay (SPA) format. Incubations are performed in clear bottom, opaque 96-well microplates. Compound is serially diluted 3-fold in binding buffer consisting of 25 mM Hepes, pH 7.4, 2.5 mM CaCl2, 1 mM MgCl2, 0.1% fatty acid free BSA, 0.003% tween-20 and Complete® EDTA-free protease inhibitor tablets. P2 membranes (prepared above) are diluted in binding buffer from each receptor preparation, then added to the diluted compound followed by addition of 0.15 mgs of wheat germ agglutinin (WGA) SPA beads (GE Healthcare) previously blocked with 1% fatty acid free BSA, and 0.15 nM [125I]-Glucagon (Perkin-Elmer). Plates are sealed with adhesive sealing tape, mixed end over end, and incubated at room temperature for 12 hours. The radioactivity bound to the receptor (in close proximity to the WGA SPA bead) is quantified on a PE Life and Analytical Sciences Trilux Microbeta plate scintillation counter and expressed as counts per minute (CPM). Total binding is determined in the absence of added compound and nonspecific binding is determined by adding 1 uM glucagon (Lilly Research Labs). The final concentration of 1 uM of unlabeled glucagon is capable of completely inhibiting [125]-glucagon binding to background levels.
[125I]Glucagon Binding Data Analysis.
[0058] Raw CPM data for concentration curves of compound are converted to percent inhibition by subtracting nonspecific binding from the individual CPM values and dividing by the total binding signal, also corrected for nonspecific binding. Data is analyzed using four-parameter (curve maximum, curve minimum, IC50, Hill slope) nonlinear regression routines (XLFit version 3.0: Activity Base, IDBS). The equilibrium dissociation constant determined by competitor inhibited radioligand binding, Ki, is calculated from the absolute IC50 value based upon the equation [Ki=IC50/(1+D/Kd)] where D equals the concentration of radioligand used in the experiment and Kd equals the equilibrium binding affinity constant of [125I]glucagon, in the assay for each individual receptor species.
Glucagon-Stimulated cAMP Functional Antagonist Assay.
[0059] The functional antagonist activity is determined from the dose-dependent inhibition of increases in intracellular cAMP with a sub-maximal dose of glucagon using the same clonal rat, mouse, cynomologous, and human glucagon receptor-293HEK cell lines. Quantitation of the intracellular cAMP level is done with an Amplified Luminescent Proximity Homogeneous Assay, (Alpha Screen) from Perkin Elmer (6760625R). Briefly, cAMP generated within the cell competes for binding of a biotinylated cAMP-streptavidin coated Donor bead and a coated anti-cAMP antibody Acceptor bead. As the cAMP level within the cell increases, a disruption of the Acceptor bead-biotinylated cAMP-Donor bead complex occurs. The functional assay is performed in 10 mM Hepes, pH 7.4, with 0.25 mM IBMX in HBSS containing Mg+2 and Ca+2. The clonal glucagon receptor-293HEK cells are suspended at 2500 cells per well and 1 unit/well of biotinylated cAMP from the kit in a total volume of 20 uls. The cells are pre-incubated for 30 minutes at room temperature with 20 uls of either 3-fold serially diluted compounds or of 3-fold serially diluted cAMP for use as a standard curve. The reaction is started by the addition of 20 uls of 300 pM glucagon (3×), a dose sufficient to produce 90% of the maximal intracellular cAMP. After 60 minutes at room temperature in the dark, the reaction is stopped by the addition of 30 uls of lysis buffer made of 1% IGEPAL CA630 (Sigma) and 0.1% fatty-acid free BSA (Gibco) in 10 mM Hepes, pH 7.4 containing 1 unit each of the kit Donor and Acceptor beads per well. The plates are wrapped in foil to protect the Donor and Acceptor beads from light and mixed on Titertek shaker medium speed for 30 secs. After incubation overnight at room temperature, the plates are read on a Packard Fusion®-α Instrument.
Data Analysis for Functional cAMP Activity.
[0060] The alpha screen units are converted to pmoles cAMP generated per well based upon the cAMP standard curve. The pmoles cAMP produced in the presence of compound are converted to % of a maximal response with the submaximal dose of glucagon alone. Within each experiment, the concentration of glucagon needed to produce a 50% response in pmoles cAMP is determined. This EC50 concentration is used to normalize results between runs to a Kb where Kb=(EC50 compound)/[1+(pM glucagon used/EC50 in pM for glucagon dose response)]. The data is analyzed using four-parameter (curve maximum, curve minimum, IC50, Hill slope) nonlinear regression routines (XLFit version 3.0: Activity Base, IDBS).
EXAMPLES
[0061] Antibody examples Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6 and Ab-7 are made and purified as known in the art. An appropriate host cell, such as HEK 293 EBNA or CHO, is either transiently or stably transfected with an expression system for secreting antibodies using an optimal predetermined light chain to heavy chain vector ratio or a single vector system encoding both a light chain (set out in SEQ ID NOS: 70, 72, 74, 76) and a heavy chain (set out in SEQ ID NOS: 71, 73, 75, 77, 78, 79, 80). Clarified medium into which the antibody has been secreted is purified using any of many commonly-used techniques. For example, the medium may be conveniently applied to a Protein A or G Sepharose FF column that has been equilibrated with a compatible buffer, such as phosphate buffered saline (pH 7.4). The column is washed to remove nonspecific binding components. The bound antibody is eluted, for example, by pH gradient (such as 0.1 M sodium phosphate buffer pH 6.8 to 0.1 M sodium citrate buffer pH 2.5). Antibody fractions are detected, such as by SDS-PAGE, and then are pooled. Further purification is optional, depending on the intended use. The antibody may be concentrated and/or sterile filtered using common techniques. Soluble aggregate and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The purity of the antibody after these chromatography steps is greater than 99%. The product may be immediately frozen at -70° C. or may be lyophilized.
[0062] Fab expression is achieved in E. coli wherein the Fab molecules are secreted into the periplasmic space. The cell wall is disrupted by osmotic shock, and the Fab containing a His tag is purified on an IMAC column.
[0063] Table 1 sets out the CDR combinations used in the antibody examples according to the present invention. The full antibody light chain combines the light chain framework sequences interspaced by three light chain CDRs; Framework 1 (SEQ ID NO: 23)-CDRL1-Framework 2 (SEQ ID NO: 24)-CDRL2-Framework 3 (SEQ ID NO: 25)-CDRL3-Framework 4 (SEQ ID NO: 26) and the light chain constant region (SEQ ID NO: 53). The heavy chain framework sequences are interspaced by three heavy chain CDRs Framework 5 (SEQ ID NO: 27)-CDRH1-Framework 6 (SEQ ID NO: 28)-CDRH2-Framework 7 (SEQ ID NO: 29)-CDRH3-Framework 8 (SEQ ID NO: 30) and then the heavy chain CH1 constant region (SEQ ID NO: 51) followed by Fc domain for the Ab, absent in Fab fragment (SEQ ID NO: 52, wherein X10=P; X15=E; X16=A; X17=A; X79=N; X229=is absent).
TABLE-US-00004 TABLE 1 CDRL 1 CDRL 2 CDRL 3 CDRH 1 CDRH 2 CDRH 3 Example SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID Ab-1 2 4 7 11 15 21 Ab-2 1 4 6 10 16 20 Ab-3 3 5 8 11 15 21 Ab-4 3 5 6 12 15 21 Ab-5 3 5 6 12 17 21 Ab-6 3 5 6 12 15 22 Ab-7 3 5 6 13 18 22
[0064] Antibody examples 1 through 7 and Fab fragments thereof all inhibit glucagon binding to human, mouse, cynomologous monkey and rat glucagon receptors in the above glucagon receptor binding assay with a Ki of less than 50 nM.
In Vitro Activity of Antibody of Example 4 (Ab4)
TABLE-US-00005 [0065] TABLE 2 Functional cAMP Antagonism rat GluR human GluR Compound Kb (nM) n Kb (nM) Stdev n Ab4 6.85 1 6.49 0.34 2
[0066] This assay demonstrates that at nanomolar concentrations an Ab4 binding to the GluR can block down stream activities of the rat or human glucagon receptor cell line, and reduce cAMP production by the cells.
TABLE-US-00006 TABLE 3 In vitro Ki (nM), for full antibody of Example 4 and respective Fab human mouse rat cyno human Compound GlucR GlucR GlucR GlucR GLP-1 R Glucagon 3.13 2.72 12.77 5.78 Ab4 1.34 0.83 1.24 6.00 >5000 Fab4 2.90 2.43 3.30 9.42 ND
[0067] This assay demonstrates that in an in vitro glucagon competition binding assay Ab4 binds with high affinity (Ki) to glucagon receptor of human, mouse, rat, cynomologous monkey origin and low affinity to the human GLP-1 receptor.
In Vivo Activity of Antibody of Example 4 (Ab4)
[0068] ZDF rats approximately 8 weeks of age and approximately 400 g in weight are dosed with a single subcutaneous injection of antibody according to example 4 (i.e. Ab4) or a human IgG (hIgG4) control. Each treatment group consists of 6 animals. Blood samples are taken for glucose measurements pre-dose and daily for 13 days following a single subcutaneous 3 or 15 mg/kg dose of the Ab4 or 15 mg/kg of a hIgG control. Blood samples for GLP-1 analysis are taken pre-dose and at 2, 4, 6 and 8 days following the 3 or 15 mg/kg subcutaneous dose of the Ab4 or 15 mg/kg of the hIgG control.
TABLE-US-00007 TABLE 4 Blood glucose levels following a single 3 or 15 mg/kg subcutaneous dose of Ab4 or 15 mg/kg negative control to ZDF rats. Ab4, Ab4, Control 3 mg/kg 15 mg/kg (15 mg/kg hIgG) Mean Mean Mean glucose glucose glucose Time conc. conc. conc. (days) (mg/dL) S.D. (mg/dL) S.D. (mg/dL) S.D. 0 345.3 37.8 359 29.2 366.5 22.9 1 225.7 53.5 143.8 27.2 374.3 22.4 2 97.5 4.3 85.7 5.7 389.3 22.3 3 92.0 7.8 89.8 9.6 361 51.5 4 103.7 20.6 90.0 14.2 381.7 69.0 5 105.3 8.9 97.3 5.2 400.8 62.2 6 108.3 13.8 99.3 10.0 401.2 61.3 7 113.7 7.8 97.5 12.2 451.7 45.5 8 135.8 15.5 104.3 11.4 452.8* 33.6 9 151.0 21.1 109.2 7.0 394.3 40.8 10 149.0 9.2 103.0 15.3 418.3 33.3 11 159.3 28.9 106.3 10.3 410.0 34.4 12 151.7 29.9 108.2 9.5 417.7 58.8 13 218.5 88.1 114.8 11.5 443.0 48.9 There were 6 rats per group except for one value (*) where one animal measured AQL therefore n = 5.
TABLE-US-00008 TABLE 5 Plasma GLP-1 levels following a single 3 or 15 mg/kg subcutaneous dose of Ab4 or 15 mg/kg negative control to ZDF rats. Control Ab4, 3 mg/kg Ab4, 15 mg/kg (15 mg/kg hIgG) Mean Mean Mean Time GLP-1 GLP-1 GLP-1 (days) (pM) SD n (pM) SD n (pM) SD n 0 <6 N.D. 0 <6 N.D. 0 <6 N.D. 0 2 <6 N.D. 0 10 3 2 <6 N.D. 0 4 15 N.D. 1 13 3 5 <6 N.D. 0 6 21 2 3 29 2 5 <6 N.D. 0 8 36 5 3 112 54 6 <6 N.D. 0
[0069] The average and sd are determined only from those rats that had quantifiable GLP-1 levels and the n denotes the number of animals per group that had quantifiable GLP-1 levels. If no animals had quantifiable GLP-1 levels the result is listed as <6 pM. N.D. signifies the value was not determined.
Sequence CWU
1
80110PRTArtificial SequenceSynthetic sequence Humanized CDRL 1 1Ser Tyr
Ser Ser Ser Val Ser Tyr Met His1 5
10210PRTArtificial SequenceSynthetic sequence Humanized CDRL 1 2Ser Ala
Ser Ser Ser Val Ser Tyr Met His1 5
10310PRTArtificial SequenceSynthetic sequence Humanized CDRL 1 3Ser Ala
Ser Ser Ser Val Ser Tyr Ile His1 5
1047PRTArtificial SequenceSynthetic sequence Humanized CDRL 2 4Thr Thr
Ser Asn Leu Ala His1 557PRTArtificial SequenceSynthetic
sequence Humanized CDRL 2 5Thr Thr Ser Tyr Leu Ala His1
569PRTArtificial SequenceSynthetic sequence Humanized CDRL 3 6Gln Gln Arg
Ser Thr Leu Pro Pro Thr1 579PRTArtificial SequenceSynthetic
sequence Humanized CDRL 3 7Gln Trp Arg Ser Thr Leu Pro Pro Thr1
589PRTArtificial SequenceSynthetic sequence Humanized CDRL 3 8Leu Gln
Arg Ser Thr Leu Pro Pro Thr1 599PRTArtificial
SequenceSynthetic sequence Humanized CDRL 3 9Gln Gln Arg Ser Thr Ile Pro
Pro Thr1 51010PRTArtificial SequenceSynthetic sequence
Humanized CDRH 1 10Gly Asp Asp Ile Thr Ser Gly Tyr Trp Asn1
5 101110PRTArtificial SequenceSynthetic sequence
Humanized CDRH 1 11Gly Asp Asp Ile Thr Ser Gly Tyr Trp Asp1
5 101210PRTArtificial SequenceSynthetic sequence
Humanized CDRH 1 12Gly Asp Asp Ile Thr Ser Gly Tyr His Asp1
5 101310PRTArtificial SequenceSynthetic sequence
Humanized CDRH 1 13Gly Asp Asp Ile Thr Ser Gly Tyr His Glu1
5 101416PRTArtificial SequenceSynthetic sequence
Humanized CDRH 2 14Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser
Leu Lys Ser1 5 10
151516PRTArtificial SequenceSynthetic sequence Humanized CDRH 2 15Tyr Ile
Ser Tyr Ser Gly Ser Thr Tyr Tyr Ser Pro Ser Leu Lys Ser1 5
10 151616PRTArtificial
SequenceSynthetic sequence Humanized CDRH 2 16Tyr Ile Ser Tyr Ser Gly Ser
Thr Tyr Tyr Ile Pro Ser Leu Lys Ser1 5 10
151716PRTArtificial SequenceSynthetic sequence Humanized
CDRH 2 17Tyr Ile Ser Tyr Ser Gly Ser Thr Gln Tyr Ser Pro Ser Leu Lys Ser1
5 10 151816PRTArtificial
SequenceSynthetic sequence Humanized CDRH 2 18Tyr Ile Ser Tyr Ser Gly Ser
Thr Ser Tyr Ser Pro Ser Leu Lys Ser1 5 10
151916PRTArtificial SequenceSynthetic sequence Humanized
CDRH 2 19Tyr Ile Ser Tyr Ser Gly Ser Thr Val Tyr Ser Pro Ser Leu Lys Ser1
5 10 152014PRTArtificial
SequenceSynthetic sequence Humanized CDRH 3 20Pro Pro Gly Tyr Tyr Gly Phe
Gly Pro Tyr Ala Met Asp Tyr1 5
102114PRTArtificial SequenceSynthetic sequence Humanized CDRH 3 21Pro Pro
Gly Tyr Tyr Gly Phe Gly Pro Tyr Ala Leu Asp Tyr1 5
102214PRTArtificial SequenceSynthetic sequence Humanized CDRH 3
22Pro Pro Ala Tyr Tyr Gly Phe Gly Pro Tyr Ala Leu Asp Tyr1
5 102323PRTHomo sapiens 23Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys 202415PRTHomo
sapiens 24Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr1
5 10 152532PRTHomo sapiens
25Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr1
5 10 15Leu Thr Ile Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25
302610PRTHomo sapiens 26Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys1 5 102725PRTHomo sapiens 27Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1
5 10 15Thr Leu Ser Leu Thr Cys Thr
Val Ser 20 252814PRTHomo sapiens 28Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly1 5
102932PRTHomo sapiens 29Arg Val Thr Ile Ser Val Asp Thr Ser Lys
Asn Gln Phe Ser Leu Lys1 5 10
15Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg
20 25 303011PRTHomo sapiens
30Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser1 5
1031451PRTHomo sapiens 31Gln Val Met Asp Phe Leu Phe Glu Lys Trp
Lys Leu Tyr Gly Asp Gln1 5 10
15Cys His His Asn Leu Ser Leu Leu Pro Pro Pro Thr Glu Leu Val Cys
20 25 30Asn Arg Thr Phe Asp Lys
Tyr Ser Cys Trp Pro Asp Thr Pro Ala Asn 35 40
45Thr Thr Ala Asn Ile Ser Cys Pro Trp Tyr Leu Pro Trp His
His Lys 50 55 60Val Gln His Arg Phe
Val Phe Lys Arg Cys Gly Pro Asp Gly Gln Trp65 70
75 80Val Arg Gly Pro Arg Gly Gln Pro Trp Arg
Asp Ala Ser Gln Cys Gln 85 90
95Met Asp Gly Glu Glu Ile Glu Val Gln Lys Glu Val Ala Lys Met Tyr
100 105 110Ser Ser Phe Gln Val
Met Tyr Thr Val Gly Tyr Ser Leu Ser Leu Gly 115
120 125Ala Leu Leu Leu Ala Leu Ala Ile Leu Gly Gly Leu
Ser Lys Leu His 130 135 140Cys Thr Arg
Asn Ala Ile His Ala Asn Leu Phe Ala Ser Phe Val Leu145
150 155 160Lys Ala Ser Ser Val Leu Val
Ile Asp Gly Leu Leu Arg Thr Arg Tyr 165
170 175Ser Gln Lys Ile Gly Asp Asp Leu Ser Val Ser Thr
Trp Leu Ser Asp 180 185 190Gly
Ala Val Ala Gly Cys Arg Val Ala Ala Val Phe Met Gln Tyr Gly 195
200 205Ile Val Ala Asn Tyr Cys Trp Leu Leu
Val Glu Gly Leu Tyr Leu His 210 215
220Asn Leu Leu Gly Leu Ala Thr Leu Pro Glu Arg Ser Phe Phe Ser Leu225
230 235 240Tyr Leu Gly Ile
Gly Trp Gly Ala Pro Met Leu Phe Val Val Pro Trp 245
250 255Ala Val Val Lys Cys Leu Phe Glu Asn Val
Gln Cys Trp Thr Ser Asn 260 265
270Asp Asn Met Gly Phe Trp Trp Ile Leu Arg Phe Pro Val Phe Leu Ala
275 280 285Ile Leu Ile Asn Phe Phe Ile
Phe Val Arg Ile Val Gln Leu Leu Val 290 295
300Ala Lys Leu Arg Ala Arg Gln Met His His Thr Asp Tyr Lys Phe
Arg305 310 315 320Leu Ala
Lys Ser Thr Leu Thr Leu Ile Pro Leu Leu Gly Val His Glu
325 330 335Val Val Phe Ala Phe Val Thr
Asp Glu His Ala Gln Gly Thr Leu Arg 340 345
350Ser Ala Lys Leu Phe Phe Asp Leu Phe Leu Ser Ser Phe Gln
Gly Leu 355 360 365Leu Val Ala Val
Leu Tyr Cys Phe Leu Asn Lys Glu Val Gln Ser Glu 370
375 380Leu Arg Arg Arg Trp His Arg Trp Arg Leu Gly Lys
Val Leu Trp Glu385 390 395
400Glu Arg Asn Thr Ser Asn His Arg Ala Ser Ser Ser Pro Gly His Gly
405 410 415Pro Pro Ser Lys Glu
Leu Gln Phe Gly Arg Gly Gly Gly Ser Gln Asp 420
425 430Ser Ser Ala Glu Thr Pro Leu Ala Gly Gly Leu Pro
Arg Leu Ala Glu 435 440 445Ser Pro
Phe 450322034DNAHomo sapiens 32ggatctggca gcgccgcgaa gacgagcggt
caccggcgcc cgacccgagc gcgcccagag 60gacggcgggg agccaagccg acccccgagc
agcgccgcgc gggccctgag gctcaaaggg 120gcagcttcag gggaggacac cccactggcc
aggacgcccc aggctctgct gctctgccac 180tcagctgccc tcggaggagc gtacacacac
accaggactg cattgcccca gtgtgcagcc 240cctgccagat gtgggaggca gctagctgcc
cagaggcatg cccccctgcc agccacagcg 300acccctgctg ctgttgctgc tgctgctggc
ctgccagcca caggtcccct ccgctcaggt 360gatggacttc ctgtttgaga agtggaagct
ctacggtgac cagtgtcacc acaacctgag 420cctgctgccc cctcccacgg agctggtgtg
caacagaacc ttcgacaagt attcctgctg 480gccggacacc cccgccaata ccacggccaa
catctcctgc ccctggtacc tgccttggca 540ccacaaagtg caacaccgct tcgtgttcaa
gagatgcggg cccgacggtc agtgggtgcg 600tggaccccgg gggcagcctt ggcgtgatgc
ctcccagtgc cagatggatg gcgaggagat 660tgaggtccag aaggaggtgg ccaagatgta
cagcagcttc caggtgatgt acacagtggg 720ctacagcctg tccctggggg ccctgctcct
cgccttggcc atcctggggg gcctcagcaa 780gctgcactgc acccgcaatg ccatccacgc
gaatctgttt gcgtccttcg tgctgaaagc 840cagctccgtg ctggtcattg atgggctgct
caggacccgc tacagccaga aaattggcga 900cgacctcagt gtcagcacct ggctcagtga
tggagcggtg gctggctgcc gtgtggccgc 960ggtgttcatg caatatggca tcgtggccaa
ctactgctgg ctgctggtgg agggcctgta 1020cctgcacaac ctgctgggcc tggccaccct
ccccgagagg agcttcttca gcctctacct 1080gggcatcggc tggggtgccc ccatgctgtt
cgtcgtcccc tgggcagtgg tcaagtgtct 1140gttcgagaac gtccagtgct ggaccagcaa
tgacaacatg ggcttctggt ggatcctgcg 1200gttccccgtc ttcctggcca tcctgatcaa
cttcttcatc ttcgtccgca tcgttcagct 1260gctcgtggcc aagctgcggg cacggcagat
gcaccacaca gactacaagt tccggctggc 1320caagtccacg ctgaccctca tccctctgct
gggcgtccac gaagtggtct ttgccttcgt 1380gacggacgag cacgcccagg gcaccctgcg
ctccgccaag ctcttcttcg acctcttcct 1440cagctccttc cagggcctgc tggtggctgt
cctctactgc ttcctcaaca aggaggtgca 1500gtcggagctg cggcggcgtt ggcaccgctg
gcgcctgggc aaagtgctat gggaggagcg 1560gaacaccagc aaccacaggg cctcatcttc
gcccggccac ggccctccca gcaaggagct 1620gcagtttggg aggggtggtg gcagccagga
ttcatctgcg gagaccccct tggctggtgg 1680cctccctaga ttggctgaga gccccttctg
aaccctgctg ggaccccagc tagggctgga 1740ctctggcacc cagaggcgtc gctggacaac
ccagaactgg acgcccagct gaggctgggg 1800gcgggggagc caacagcagc ccccacctac
cccccacccc cagtgtggct gtctgcgaga 1860ttgggcctcc tctccctgca cctgccttgt
ccctggtgca gaggtgagca gaggagtcca 1920gggcgggagt gggggctgtg ccgtgaactg
cgtgccagtg tccccacgta tgtcggcacg 1980tcccatgtgc atggaaatgt cctccaacaa
taaagagctc aagtggtcac cgtg 203433458PRTRattus rattus 33Gln Val
Met Asp Phe Leu Phe Glu Lys Trp Lys Leu Tyr Ser Asp Gln1 5
10 15Cys His His Asn Leu Ser Leu Leu
Pro Pro Pro Thr Glu Leu Val Cys 20 25
30Asn Arg Thr Phe Asp Lys Tyr Ser Cys Trp Pro Asp Thr Pro Pro
Asn 35 40 45Thr Thr Ala Asn Ile
Ser Cys Pro Trp Tyr Leu Pro Trp Tyr His Lys 50 55
60Val Gln His Arg Leu Val Phe Lys Arg Cys Gly Pro Asp Gly
Gln Trp65 70 75 80Val
Arg Gly Pro Arg Gly Gln Ser Trp Arg Asp Ala Ser Gln Cys Gln
85 90 95Met Asp Asp Asp Glu Ile Glu
Val Gln Lys Gly Val Ala Lys Met Tyr 100 105
110Ser Ser Tyr Gln Val Met Tyr Thr Val Gly Tyr Ser Leu Ser
Leu Gly 115 120 125Ala Leu Leu Leu
Ala Leu Val Ile Leu Leu Gly Leu Arg Lys Leu His 130
135 140Cys Thr Arg Asn Tyr Ile His Gly Asn Leu Phe Ala
Ser Phe Val Leu145 150 155
160Lys Ala Gly Ser Val Leu Val Ile Asp Trp Leu Leu Lys Thr Arg Tyr
165 170 175Ser Gln Lys Ile Gly
Asp Asp Leu Ser Val Ser Val Trp Leu Ser Asp 180
185 190Gly Ala Val Ala Gly Cys Arg Val Ala Thr Val Ile
Met Gln Tyr Gly 195 200 205Ile Ile
Ala Asn Tyr Cys Trp Leu Leu Val Glu Gly Val Tyr Leu Tyr 210
215 220Ser Leu Leu Ser Ile Thr Thr Phe Ser Glu Lys
Ser Phe Phe Ser Leu225 230 235
240Tyr Leu Cys Ile Gly Trp Gly Ser Pro Leu Leu Phe Val Ile Pro Trp
245 250 255Val Val Val Lys
Cys Leu Phe Glu Asn Val Gln Cys Trp Thr Ser Asn 260
265 270Asp Asn Met Gly Phe Trp Trp Ile Leu Arg Ile
Pro Val Leu Leu Ala 275 280 285Ile
Leu Ile Asn Phe Phe Ile Phe Val Arg Ile Ile His Leu Leu Val 290
295 300Ala Lys Leu Arg Ala His Gln Met His Tyr
Ala Asp Tyr Lys Phe Arg305 310 315
320Leu Ala Arg Ser Thr Leu Thr Leu Ile Pro Leu Leu Gly Val His
Glu 325 330 335Val Val Phe
Ala Phe Val Thr Asp Glu His Ala Gln Gly Thr Leu Arg 340
345 350Ser Thr Lys Leu Phe Phe Asp Leu Phe Phe
Ser Ser Phe Gln Gly Leu 355 360
365Leu Val Ala Val Leu Tyr Cys Phe Leu Asn Lys Glu Val Gln Ala Glu 370
375 380Leu Leu Arg Arg Trp Arg Arg Trp
Gln Glu Gly Lys Ala Leu Gln Glu385 390
395 400Glu Arg Met Ala Ser Ser His Gly Ser His Met Ala
Pro Ala Gly Thr 405 410
415Cys His Gly Asp Pro Cys Glu Lys Leu Gln Leu Met Ser Ala Gly Ser
420 425 430Ser Ser Gly Thr Gly Cys
Glu Pro Ser Ala Lys Thr Ser Leu Ala Ser 435 440
445Ser Leu Pro Arg Leu Ala Asp Ser Pro Thr 450
455341875DNARattus rattus 34gaattcgcgg ccgccgccgg gccccagatc
ccagtgcgcg aggagcccag tcctagaccc 60agcaacctga ggagaggtgc acacaccccc
aaggacccag gcacccaacc tctgccagat 120gtgggggggt ggctacccag aggcatgctc
ctcacccagc tccactgtcc ctacctgctg 180ctgctgctgg tggtgctgtc atgtctgcca
aaggcaccct ctgcccaggt aatggacttt 240ttgtttgaga agtggaagct ctatagtgac
cagtgccacc acaacctaag cctgctgccc 300ccacctactg agctggtctg caacagaact
ttcgacaagt actcctgctg gcctgacacc 360cctcccaaca ccactgccaa catttcctgc
ccctggtacc taccttggta ccacaaagtg 420cagcaccgcc tagtgttcaa gaggtgtggg
cctgatgggc agtgggttcg agggccacgg 480gggcagtcat ggcgcgacgc ctcccaatgt
cagatggatg atgacgagat cgaggtccag 540aagggggtag ccaagatgta tagcagctac
caggtgatgt acactgtggg ctacagtctg 600tccctggggg ccttgctcct ggcgctggtc
atcctgctgg gcctcaggaa gctgcactgc 660acccggaact acatccacgg gaacctgttc
gcgtccttcg tgctcaaggc tggctctgtg 720ctggtcattg attggctgct caagacacgc
tatagccaga agattggaga tgacctcagt 780gtgagcgtct ggctcagtga tggggcggtg
gctggctgca gagtggccac agtgatcatg 840cagtacggca tcatagccaa ctactgctgg
ttgctggtgg agggtgtgta cctgtacagc 900ctgctgagca tcaccacctt ctcggagaag
agcttcttct ccctctatct gtgcatcggc 960tggggatctc ccctgctgtt tgtcatcccc
tgggtggtgg tcaagtgtct gtttgagaat 1020gtccagtgct ggaccagcaa tgacaatatg
ggattctggt ggatcctgcg tatccctgta 1080ctcctggcca tactgatcaa ttttttcatc
tttgtccgca tcattcatct tcttgtggcc 1140aagctgcgtg cccatcagat gcactatgct
gattacaagt tccggctagc caggtccacg 1200ctgaccctca ttcctctgct gggagtccac
gaagtggtct ttgcctttgt gactgatgag 1260catgcccagg gcaccctgcg ctccaccaag
ctcttttttg acctgttctt cagctccttt 1320cagggtctgc tggtggctgt tctctactgt
ttcctcaaca aggaggtgca ggcagagcta 1380ctgcggcgtt ggaggcgatg gcaagaaggc
aaagctcttc aggaggaaag gatggccagc 1440agccatggca gccacatggc cccagcaggg
acttgtcatg gtgatccctg tgagaaactt 1500cagcttatga gtgcaggcag cagcagtggg
actggctgtg agccctctgc gaagacctca 1560ttggccagta gtctcccaag gctggctgac
agccccacct gaatctccac tggactccag 1620ccaagttgga ttcagaaagg gcctcacaag
acaacccaga aacagatgcc tggccaaggc 1680tgaagaggca aagcagcaag acagcagctt
gtactatcca cactccccta acctgtcctg 1740gccgggtaca ggccacattg atggagtagg
ggctggatat gatggagtag ccatgctatg 1800aactatgggt gttcccatga gtgttgccat
gttccatgca cacagatatg accttcagta 1860aagagctccc gtagg
187535458PRTMus musculus 35Gln Val Met
Asp Phe Leu Phe Glu Lys Trp Lys Leu Tyr Ser Asp Gln1 5
10 15Cys His His Asn Leu Ser Leu Leu Pro
Pro Pro Thr Glu Leu Val Cys 20 25
30Asn Arg Thr Phe Asp Asn Tyr Ser Cys Trp Pro Asp Thr Pro Pro Asn
35 40 45Thr Thr Ala Asn Ile Ser Cys
Pro Trp Tyr Leu Pro Trp Cys His Lys 50 55
60Val Gln His Arg Leu Val Phe Lys Arg Cys Gly Pro Asp Gly Gln Trp65
70 75 80Val Arg Gly Pro
Arg Gly Gln Pro Trp Arg Asn Ala Ser Gln Cys Gln 85
90 95Leu Asp Asp Glu Glu Ile Glu Val Gln Lys
Gly Val Ala Lys Met Tyr 100 105
110Ser Ser Gln Gln Val Met Tyr Thr Val Gly Tyr Ser Leu Ser Leu Gly
115 120 125Ala Leu Leu Leu Ala Leu Val
Ile Leu Leu Gly Leu Arg Lys Leu His 130 135
140Cys Thr Arg Asn Tyr Ile His Gly Asn Leu Phe Ala Ser Phe Val
Leu145 150 155 160Lys Ala
Gly Ser Val Leu Val Ile Asp Trp Leu Leu Lys Thr Arg Tyr
165 170 175Ser Gln Lys Ile Gly Asp Asp
Leu Ser Val Ser Val Trp Leu Ser Asp 180 185
190Gly Ala Met Ala Gly Cys Arg Val Ala Thr Val Ile Met Gln
Tyr Gly 195 200 205Ile Ile Pro Asn
Tyr Cys Trp Leu Leu Val Glu Gly Val Tyr Leu Tyr 210
215 220Ser Leu Leu Ser Leu Ala Thr Phe Ser Glu Arg Ser
Phe Phe Ser Leu225 230 235
240Tyr Leu Gly Ile Gly Trp Gly Ala Pro Leu Leu Phe Val Ile Pro Trp
245 250 255Val Val Val Lys Cys
Leu Phe Glu Asn Val Gln Cys Trp Thr Ser Asn 260
265 270Asp Asn Met Gly Phe Trp Trp Ile Leu Arg Ile Pro
Val Phe Leu Ala 275 280 285Leu Leu
Ile Asn Phe Phe Ile Phe Val His Ile Ile Gln Leu Leu Val 290
295 300Ala Lys Leu Arg Ala His Gln Met His Tyr Ala
Asp Tyr Lys Phe Arg305 310 315
320Leu Ala Arg Ser Thr Leu Thr Leu Ile Pro Leu Leu Gly Val His Glu
325 330 335Val Val Phe Ala
Phe Val Thr Asp Glu His Ala Gln Gly Thr Leu Arg 340
345 350Ser Thr Lys Leu Phe Phe Asp Leu Phe Leu Ser
Ser Phe Gln Gly Leu 355 360 365Leu
Val Ala Val Leu Tyr Cys Phe Leu Asn Lys Glu Val Gln Ala Glu 370
375 380Leu Met Arg Arg Trp Arg Gln Trp Gln Glu
Gly Lys Ala Leu Gln Glu385 390 395
400Glu Arg Leu Ala Ser Ser His Gly Ser His Met Ala Pro Ala Gly
Pro 405 410 415Cys His Gly
Asp Pro Cys Glu Lys Leu Gln Leu Met Ser Ala Gly Ser 420
425 430Ser Ser Gly Thr Gly Cys Val Pro Ser Met
Glu Thr Ser Leu Ala Ser 435 440
445Ser Leu Pro Arg Leu Ala Asp Ser Pro Thr 450
455361944DNAMus musculus 36cagggtctcc cttgcaacct gaggagaggt gcacacactc
tgaggaccta ggtgtgcaac 60ctctgccaga tgtggggcgt ggctacccag aggcatgccc
ctcacccagc tccactgtcc 120ccacctgctg ctgctgctgt tggtgctgtc atgtctgcca
gaggcaccct ctgcccaggt 180aatggacttt ttgtttgaga agtggaagct ctatagtgac
caatgtcacc acaacctaag 240cctgctgccc ccacctactg agctggtctg taacagaacc
ttcgacaact actcctgctg 300gcctgacacc cctcccaaca ccactgccaa catttcctgc
ccctggtacc taccttggtg 360ccacaaagtg cagcaccgcc tagtgttcaa gaggtgtggg
cccgatgggc agtgggttcg 420agggccacgg gggcagccgt ggcgcaacgc ctcccaatgt
cagttggatg atgaagagat 480cgaggtccag aagggggtgg ccaagatgta tagcagccag
caggtgatgt acaccgtggg 540ctacagtctg tccctggggg ccttgctcct tgcgctggtc
atcctgctgg gcctcaggaa 600gctgcactgc acccgaaact acatccatgg gaacctgttt
gcgtcctttg tgctcaaggc 660tggctctgtg ttggtcatcg attggctgct gaagacacgg
tacagccaga agattggcga 720tgacctcagt gtgagcgtct ggctcagtga cggggcgatg
gccggctgca gagtggccac 780agtgatcatg cagtacggca tcatacccaa ctattgctgg
ttgctggtag agggcgtgta 840cctgtacagc ctgctgagcc ttgccacctt ctctgagagg
agcttctttt ccctctacct 900gggcattggc tggggtgcgc ccctgctgtt tgtcatcccc
tgggtggtgg tcaagtgtct 960gtttgagaat gttcagtgct ggaccagcaa tgacaacatg
ggattctggt ggatcctgcg 1020tattcctgtc ttcctggcct tactgatcaa ttttttcatc
tttgtccaca tcattcaact 1080tcttgtggcc aagctgcgtg cccatcagat gcactatgct
gattacaagt tccggctggc 1140caggtccacg ctgaccctca tccctctgct gggggtccac
gaggtggtct ttgcctttgt 1200gactgacgag catgcccaag gcaccctgcg ctccaccaag
ctcttttttg acctgttcct 1260cagctccttc cagggtctgc tggtggctgt tctctactgt
ttcctcaaca aggaggtgca 1320ggcagagctg atgcggcgtt ggaggcaatg gcaagaaggc
aaagctcttc aggaggaaag 1380gttggccagc agccatggca gccacatggc cccagcaggg
ccttgtcatg gtgatccctg 1440tgagaaactt cagcttatga gtgcaggcag cagcagtggg
actggctgtg tgccctctat 1500ggagacctcg ctggccagta gtctcccaag gttggctgac
agccccacct gaatctccac 1560ttggagccta ggcaggttgt gttcaagaaa gggcctcaga
ggacaaccca gagccagatg 1620cccggccaag gttgaagagc caaagcagca agacagcagc
ttgtactgtg cacactcccc 1680taacctgtcc tagcctggca caggccacag tgacagagta
ggggttggat atgatggaga 1740agccatgtta tctatgaact ctgagtgttc ccatgtgtgt
tgacatggtc cctgtaccca 1800gatatgtcct tcagtaaaaa gctcgagtgg agctgctgca
cagctcgtgg acagcaggct 1860tgaagccccc agggacgggg tttgggaggc cggggatgag
cagcacactc agcaggtgga 1920gcgctagtgc aacccaggaa agaa
194437451PRTMacaca fascicularis 37Gln Val Met Asp
Phe Leu Phe Glu Lys Trp Lys Leu Tyr Gly Asp Gln1 5
10 15Cys His His Asn Leu Ser Leu Leu Pro Pro
Pro Thr Glu Leu Val Cys 20 25
30Asn Arg Thr Phe Asp Lys Tyr Ser Cys Trp Pro Asp Thr Pro Ala Asn
35 40 45Thr Thr Ala Asn Ile Ser Cys Pro
Trp Tyr Leu Pro Trp His His Lys 50 55
60 Val Gln His Arg Phe Val Phe Lys Arg Cys Gly Pro Asp Gly Gln Trp65
70 75 80Val Arg Gly Pro
Arg Gly Gln Pro Trp Arg Asp Ala Ser Gln Cys Gln 85
90 95Met Asp Gly Glu Glu Leu Glu Val Gln Lys
Glu Val Ala Lys Met Tyr 100 105
110Ser Ser Phe Gln Val Met Tyr Thr Val Gly Tyr Ser Leu Ser Leu Gly
115 120 125Ala Leu Leu Leu Ala Leu Ala
Val Leu Gly Gly Ile Ser Lys Leu His 130 135
140Cys Thr Arg Asn Ala Ile His Ala Asn Leu Phe Val Ser Phe Val
Leu145 150 155 160Lys Ala
Ser Ser Val Leu Val Ile Asp Gly Leu Leu Arg Thr Arg Tyr
165 170 175Ser Gln Lys Ile Gly Asp Asp
Leu Ser Val Ser Ile Trp Leu Ser Asp 180 185
190Gly Ala Val Ala Gly Cys Arg Val Ala Ala Val Phe Met Gln
Tyr Gly 195 200 205Val Val Ala Asn
Tyr Cys Trp Leu Leu Val Glu Gly Leu Tyr Leu His 210
215 220Asn Leu Leu Gly Leu Ala Thr Leu Pro Glu Arg Ser
Phe Phe Ser Leu225 230 235
240Tyr Leu Gly Ile Gly Trp Gly Ala Pro Met Leu Phe Ile Ile Pro Trp
245 250 255Val Val Val Arg Cys
Leu Phe Glu Asn Ile Gln Cys Trp Thr Ser Asn 260
265 270Asp Asn Met Gly Phe Trp Trp Ile Leu Arg Phe Pro
Val Phe Leu Ala 275 280 285Ile Leu
Ile Asn Phe Phe Ile Phe Ile Arg Ile Val His Leu Leu Val 290
295 300Ala Lys Leu Arg Ala Arg Glu Met His His Thr
Asp Tyr Lys Phe Arg305 310 315
320Leu Ala Lys Ser Thr Leu Thr Leu Ile Pro Leu Leu Gly Val His Glu
325 330 335Val Val Phe Ala
Phe Val Thr Asp Glu His Ala Gln Gly Thr Leu Arg 340
345 350Phe Ala Lys Leu Phe Phe Asp Leu Phe Leu Ser
Ser Phe Gln Gly Leu 355 360 365Leu
Val Ala Val Leu Tyr Cys Phe Leu Asn Lys Glu Val Gln Ser Glu 370
375 380Leu Arg Arg His Trp His Arg Trp Arg Leu
Gly Lys Val Leu Gln Glu385 390 395
400Glu Arg Gly Thr Ser Asn His Lys Ala Pro Ser Ala Pro Gly Gln
Gly 405 410 415Leu Pro Gly
Lys Lys Leu Gln Ser Gly Arg Asp Gly Gly Ser Gln Asp 420
425 430Ser Ser Ala Glu Ile Pro Leu Ala Gly Gly
Leu Pro Arg Leu Ala Glu 435 440
445Ser Pro Phe 450381434DNAMacaca fascicularis 38atgccgccgt gccagccgcg
ccgcccgctg ctgctgctgc tgctgctgct ggcgtgccag 60ccgcaggcgc cgagcgcgca
ggtgatggat tttctgtttg aaaaatggaa actgtatggc 120gatcagtgcc atcataacct
gagcctgctg ccgccgccga ccgaactggt gtgcaaccgc 180acctttgata aatatagctg
ctggccggat accccggcga acaccaccgc gaacattagc 240tgcccgtggt atctgccgtg
gcatcataaa gtgcagcatc gctttgtgtt taaacgctgc 300ggcccggatg gccagtgggt
gcgcggcccg cgcggccagc cgtggcgcga tgcgagccag 360tgccagatgg atggcgaaga
actggaagtg cagaaagaag tggcgaaaat gtatagcagc 420tttcaggtga tgtataccgt
gggctatagc ctgagcctgg gcgcgctgct gctggcgctg 480gcggtgctgg gcggcattag
caaactgcat tgcacccgca acgcgattca tgcgaacctg 540tttgtgagct ttgtgctgaa
agcgagcagc gtgctggtga ttgatggcct gctgcgcacc 600cgctatagcc agaaaattgg
cgatgatctg agcgtgagca tttggctgag cgatggcgcg 660gtggcgggct gccgcgtggc
ggcggtgttt atgcagtatg gcgtggtggc gaactattgc 720tggctgctgg tggaaggcct
gtatctgcat aacctgctgg gcctggcgac cctgccggaa 780cgcagctttt ttagcctgta
tctgggcatt ggctggggcg cgccgatgct gtttattatt 840ccgtgggtgg tggtgcgctg
cctgtttgaa aacattcagt gctggaccag caacgataac 900atgggctttt ggtggattct
gcgctttccg gtgtttctgg cgattctgat taactttttt 960atttttattc gcattgtgca
tctgctggtg gcgaaactgc gcgcgcgcga aatgcatcat 1020accgattata aatttcgcct
ggcgaaaagc accctgaccc tgattccgct gctgggcgtg 1080catgaagtgg tgtttgcgtt
tgtgaccgat gaacatgcgc agggcaccct gcgctttgcg 1140aaactgtttt ttgatctgtt
tctgagcagc tttcagggcc tgctggtggc ggtgctgtat 1200tgctttctga acaaagaagt
gcagagcgaa ctgcgccgcc attggcatcg ctggcgcctg 1260ggcaaagtgc tgcaggaaga
acgcggcacc agcaaccata aagcgccgag cgcgccgggc 1320cagggcctgc cgggcaaaaa
actgcagagc ggccgcgatg gcggcagcca ggatagcagc 1380gcggaaattc cgctggcggg
cggcctgccg cgcctggcgg aaagcccgtt ttaa 143439106PRTArtificial
SequenceSynthetic sequence Humanized variable light chain 39Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Ser
Ala Ser Ser Ser Val Ser Tyr Met 20 25
30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
Tyr 35 40 45Thr Thr Ser Asn Leu
Ala His Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55
60Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro Glu65 70 75 80Asp
Phe Ala Thr Tyr Tyr Cys Gln Trp Arg Ser Thr Leu Pro Pro Thr
85 90 95Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys 100 10540122PRTArtificial
SequenceSynthetic sequence Humanized variable heavy chain 40Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5
10 15Thr Leu Ser Leu Thr Cys Thr Val
Ser Gly Asp Asp Ile Thr Ser Gly 20 25
30Tyr Trp Asp Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
Ile 35 40 45Gly Tyr Ile Ser Tyr
Ser Gly Ser Thr Tyr Tyr Ser Pro Ser Leu Lys 50 55
60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
Ser Leu65 70 75 80Lys
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Pro Pro Gly Tyr Tyr Gly
Phe Gly Pro Tyr Ala Leu Asp Tyr Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
12041106PRTArtificial SequenceSynthetic sequence Humanized
variable light chain 41Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Ser Tyr Ser Ser Ser Val Ser Tyr Met
20 25 30His Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40
45Thr Thr Ser Asn Leu Ala His Gly Val Pro Ser Arg Phe Ser Gly
Ser 50 55 60Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu65 70
75 80Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser
Thr Leu Pro Pro Thr 85 90
95Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
10542122PRTArtificial SequenceSynthetic sequence Humanized variable heavy
chain 42Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser
Glu1 5 10 15Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Asp Asp Ile Thr Ser Gly 20
25 30Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly
Lys Gly Leu Glu Trp Ile 35 40
45Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Ile Pro Ser Leu Lys 50
55 60Ser Arg Val Thr Ile Ser Val Asp Thr
Ser Lys Asn Gln Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95Arg Pro
Pro Gly Tyr Tyr Gly Phe Gly Pro Tyr Ala Met Asp Tyr Trp 100
105 110Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 12043106PRTArtificial SequenceSynthetic
sequence Humanized variable light chain 43Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser
Val Ser Tyr Ile 20 25 30His
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35
40 45Thr Thr Ser Tyr Leu Ala His Gly Val
Pro Ser Arg Phe Ser Gly Ser 50 55
60Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu65
70 75 80Asp Phe Ala Thr Tyr
Tyr Cys Leu Gln Arg Ser Thr Leu Pro Pro Thr 85
90 95Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 10544122PRTArtificial SequenceSynthetic sequence
Humanized variable heavy chain 44Gln Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser Glu1 5 10
15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Asp Ile Thr Ser
Gly 20 25 30Tyr Trp Asp Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35
40 45Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Ser
Pro Ser Leu Lys 50 55 60Ser Arg Val
Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70
75 80Lys Leu Ser Ser Val Thr Ala Ala
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90
95Arg Pro Pro Gly Tyr Tyr Gly Phe Gly Pro Tyr Ala Leu Asp
Tyr Trp 100 105 110Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115
12045106PRTArtificial SequenceSynthetic sequence Humanized variable light
chain 45Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Ile 20
25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile Tyr 35 40
45Thr Thr Ser Tyr Leu Ala His Gly Val Pro Ser Arg Phe Ser Gly Ser 50
55 60Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro Glu65 70 75
80Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Thr Leu Pro
Pro Thr 85 90 95Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys 100
10546122PRTArtificial SequenceSynthetic sequence Humanized variable heavy
chain 46Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser
Glu1 5 10 15Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Asp Asp Ile Thr Ser Gly 20
25 30Tyr His Asp Trp Ile Arg Gln Pro Pro Gly
Lys Gly Leu Glu Trp Ile 35 40
45Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Ser Pro Ser Leu Lys 50
55 60Ser Arg Val Thr Ile Ser Val Asp Thr
Ser Lys Asn Gln Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95Arg Pro
Pro Gly Tyr Tyr Gly Phe Gly Pro Tyr Ala Leu Asp Tyr Trp 100
105 110Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 12047122PRTArtificial SequenceSynthetic
sequence Humanized variable heavy chain 47Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5
10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Asp
Ile Thr Ser Gly 20 25 30Tyr
His Asp Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35
40 45Gly Tyr Ile Ser Tyr Ser Gly Ser Thr
Gln Tyr Ser Pro Ser Leu Lys 50 55
60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65
70 75 80Lys Leu Ser Ser Val
Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85
90 95Arg Pro Pro Gly Tyr Tyr Gly Phe Gly Pro Tyr
Ala Leu Asp Tyr Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
12048122PRTArtificial SequenceSynthetic sequence Humanized variable heavy
chain 48Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser
Glu1 5 10 15Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Asp Asp Ile Thr Ser Gly 20
25 30Tyr His Asp Trp Ile Arg Gln Pro Pro Gly
Lys Gly Leu Glu Trp Ile 35 40
45Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Ser Pro Ser Leu Lys 50
55 60Ser Arg Val Thr Ile Ser Val Asp Thr
Ser Lys Asn Gln Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95Arg Pro
Pro Ala Tyr Tyr Gly Phe Gly Pro Tyr Ala Leu Asp Tyr Trp 100
105 110Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 12049122PRTArtificial SequenceSynthetic
sequence Humanized variable heavy chain 49Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5
10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Asp
Ile Thr Ser Gly 20 25 30Tyr
His Glu Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35
40 45Gly Tyr Ile Ser Tyr Ser Gly Ser Thr
Ser Tyr Ser Pro Ser Leu Lys 50 55
60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65
70 75 80Lys Leu Ser Ser Val
Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85
90 95Arg Pro Pro Ala Tyr Tyr Gly Phe Gly Pro Tyr
Ala Leu Asp Tyr Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
12050107PRTHomo sapiens 50Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu1 5 10
15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40
45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser 50 55 60Thr Tyr Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70
75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser 85 90
95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100
1055198PRTHomo sapiens 51Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg1 5 10
15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70
75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90
95Arg Val52229PRTArtificial SequenceSynthetic sequence Modified human
IgG4 Fc sequence 52Glu Ser Lys Tyr Gly Pro Pro Cys Pro Xaa Cys Pro
Ala Pro Xaa Xaa1 5 10
15Xaa Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
20 25 30Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val 35 40
45Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val 50 55 60Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Xaa Ser65 70
75 80Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu 85 90
95Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
100 105 110Ser Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120
125Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln 130 135 140Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala145 150
155 160Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr 165 170
175Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
180 185 190Thr Val Asp Lys Ser
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195
200 205Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser 210 215 220Leu Ser Leu
Gly Xaa2255310PRTArtificial SequenceSynthetic sequence Light chain CDR1
53Ser Xaa Ser Ser Ser Val Ser Tyr Xaa His1 5
10547PRTArtificial SequenceSynthetic sequence Light chain CDR2 54Thr
Thr Ser Xaa Leu Ala His1 5559PRTArtificial
SequenceSynthetic sequence Light chain CDR3 55Xaa Xaa Arg Ser Thr Xaa Pro
Pro Thr1 55610PRTArtificial SequenceSynthetic sequence
Heavy chain CDR1 56Gly Asp Asp Ile Thr Ser Gly Tyr Xaa Xaa1
5 105716PRTArtificial SequenceSynthetic sequence Heavy
chain CDR2 57Tyr Ile Ser Tyr Ser Gly Ser Thr Xaa Tyr Xaa Pro Ser Leu Lys
Ser1 5 10
155814PRTArtificial SequenceSynthetic sequence Heavy chain CDR3 58Pro Pro
Xaa Tyr Tyr Gly Phe Gly Pro Tyr Ala Xaa Asp Tyr1 5
1059213PRTArtificial SequenceSynthetic sequence Humanized
monoclonal antibody light chain 59Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser
Tyr Met 20 25 30His Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35
40 45Thr Thr Ser Asn Leu Ala His Gly Val Pro Ser
Arg Phe Ser Gly Ser 50 55 60Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu65
70 75 80Asp Phe Ala Thr Tyr Tyr Cys
Gln Trp Arg Ser Thr Leu Pro Pro Thr 85 90
95Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val
Ala Ala Pro 100 105 110Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115
120 125Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala Lys 130 135 140Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu145
150 155 160Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165
170 175Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr Ala 180 185 190Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195
200 205Asn Arg Gly Glu Cys
21060448PRTArtificial SequenceSynthetic sequence Humanized monoclonal
antibody heavy chain 60Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Glu1 5 10
15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Asp Ile Thr Ser Gly
20 25 30Tyr Trp Asp Trp Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Ser Pro Ser Leu
Lys 50 55 60Ser Arg Val Thr Ile Ser
Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70
75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90
95Arg Pro Pro Gly Tyr Tyr Gly Phe Gly Pro Tyr Ala Leu Asp Tyr Trp
100 105 110Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr 130 135 140Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150
155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro 165 170
175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190Val Pro Ser Ser Ser
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195
200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Ser Lys Tyr 210 215 220Gly Pro Pro
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro225
230 235 240Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser 245
250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser Gln Glu Asp 260 265 270Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275
280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Tyr Arg Val 290 295
300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305
310 315 320Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325
330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr 340 345
350Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375
380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu385 390 395 400Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
405 410 415Ser Arg Trp Gln Glu Gly Asn
Val Phe Ser Cys Ser Val Met His Glu 420 425
430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Leu Gly 435 440
44561213PRTArtificial SequenceSynthetic sequence Humanized monoclonal
antibody light chain 61Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Ser Tyr Ser Ser Ser Val Ser Tyr Met
20 25 30His Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40
45Thr Thr Ser Asn Leu Ala His Gly Val Pro Ser Arg Phe Ser Gly
Ser 50 55 60Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu65 70
75 80Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser
Thr Leu Pro Pro Thr 85 90
95Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro
100 105 110Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120
125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
Ala Lys 130 135 140Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu145 150
155 160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser Ser 165 170
175Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195
200 205Asn Arg Gly Glu Cys 21062448PRTArtificial
SequenceSynthetic sequence Humanized monoclonal antibody heavy chain
62Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1
5 10 15Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Asp Asp Ile Thr Ser Gly 20 25
30Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu
Glu Trp Ile 35 40 45Gly Tyr Ile
Ser Tyr Ser Gly Ser Thr Tyr Tyr Ile Pro Ser Leu Lys 50
55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn
Gln Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Pro Pro Gly Tyr Tyr
Gly Phe Gly Pro Tyr Ala Met Asp Tyr Trp 100
105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro 115 120 125Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130
135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr145 150 155
160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180
185 190Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr
Thr Cys Asn Val Asp 195 200 205His
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210
215 220Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
Glu Ala Ala Gly Gly Pro225 230 235
240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser 245 250 255Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260
265 270Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn 275 280
285Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290
295 300Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu305 310
315 320Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
Ser Ile Glu Lys 325 330
335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350Leu Pro Pro Ser Gln Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360
365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu 370 375 380Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390
395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Arg Leu Thr Val Asp Lys 405 410
415Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435
440 44563213PRTArtificial SequenceSynthetic sequence
Humanized monoclonal antibody light chain 63Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser
Val Ser Tyr Ile 20 25 30His
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35
40 45Thr Thr Ser Tyr Leu Ala His Gly Val
Pro Ser Arg Phe Ser Gly Ser 50 55
60Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu65
70 75 80Asp Phe Ala Thr Tyr
Tyr Cys Leu Gln Arg Ser Thr Leu Pro Pro Thr 85
90 95Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
Thr Val Ala Ala Pro 100 105
110Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135
140Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
Glu145 150 155 160Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185
190Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
Ser Phe 195 200 205Asn Arg Gly Glu
Cys 21064448PRTArtificial SequenceSynthetic sequence Humanized
monoclonal antibody heavy chain 64Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Glu1 5 10
15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Asp Ile Thr
Ser Gly 20 25 30Tyr Trp Asp
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35
40 45Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr
Ser Pro Ser Leu Lys 50 55 60Ser Arg
Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65
70 75 80Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90
95Arg Pro Pro Gly Tyr Tyr Gly Phe Gly Pro Tyr Ala Leu
Asp Tyr Trp 100 105 110Gly Gln
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115
120 125Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser Thr 130 135 140Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145
150 155 160Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165
170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr 180 185 190Val
Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195
200 205His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg Val Glu Ser Lys Tyr 210 215
220Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro225
230 235 240Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245
250 255Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp 260 265
270Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285Ala Lys Thr Lys Pro Arg Glu
Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295
300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu305 310 315 320Tyr Lys
Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
325 330 335Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345
350Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr 355 360 365Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370
375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu385 390 395
400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
405 410 415Ser Arg Trp Gln Glu
Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420
425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Leu Gly 435 440
44565213PRTArtificial SequenceSynthetic sequence Humanized monoclonal
antibody light chain 65Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Ile
20 25 30His Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40
45Thr Thr Ser Tyr Leu Ala His Gly Val Pro Ser Arg Phe Ser Gly
Ser 50 55 60Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu65 70
75 80Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser
Thr Leu Pro Pro Thr 85 90
95Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro
100 105 110Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120
125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
Ala Lys 130 135 140Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu145 150
155 160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser Ser 165 170
175Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195
200 205Asn Arg Gly Glu Cys 21066448PRTArtificial
SequenceSynthetic sequence Humanized monoclonal antibody heavy chain
66Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1
5 10 15Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Asp Asp Ile Thr Ser Gly 20 25
30Tyr His Asp Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu
Glu Trp Ile 35 40 45Gly Tyr Ile
Ser Tyr Ser Gly Ser Thr Tyr Tyr Ser Pro Ser Leu Lys 50
55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn
Gln Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Pro Pro Gly Tyr Tyr
Gly Phe Gly Pro Tyr Ala Leu Asp Tyr Trp 100
105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro 115 120 125Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130
135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr145 150 155
160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180
185 190Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr
Thr Cys Asn Val Asp 195 200 205His
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210
215 220Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
Glu Ala Ala Gly Gly Pro225 230 235
240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser 245 250 255Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260
265 270Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn 275 280
285Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290
295 300Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu305 310
315 320Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
Ser Ile Glu Lys 325 330
335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350Leu Pro Pro Ser Gln Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360
365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu 370 375 380Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390
395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Arg Leu Thr Val Asp Lys 405 410
415Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435
440 44567448PRTArtificial SequenceSynthetic sequence
Humanized monoclonal antibody heavy chain 67Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5
10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Asp
Ile Thr Ser Gly 20 25 30Tyr
His Asp Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35
40 45Gly Tyr Ile Ser Tyr Ser Gly Ser Thr
Gln Tyr Ser Pro Ser Leu Lys 50 55
60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65
70 75 80Lys Leu Ser Ser Val
Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85
90 95Arg Pro Pro Gly Tyr Tyr Gly Phe Gly Pro Tyr
Ala Leu Asp Tyr Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125Ser Val Phe Pro Leu Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185
190Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
Val Asp 195 200 205His Lys Pro Ser
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210
215 220Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala
Ala Gly Gly Pro225 230 235
240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260
265 270Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290
295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu305 310 315
320Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
325 330 335Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340
345 350Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr 355 360 365Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370
375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu385 390 395
400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
Lys 405 410 415Ser Arg Trp
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420
425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Leu Gly 435 440
44568448PRTArtificial SequenceSynthetic sequence Humanized monoclonal
antibody heavy chain 68Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Glu1 5 10
15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Asp Ile Thr Ser Gly
20 25 30Tyr His Asp Trp Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Ser Pro Ser Leu
Lys 50 55 60Ser Arg Val Thr Ile Ser
Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70
75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90
95Arg Pro Pro Ala Tyr Tyr Gly Phe Gly Pro Tyr Ala Leu Asp Tyr Trp
100 105 110Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr 130 135 140Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150
155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro 165 170
175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190Val Pro Ser Ser Ser
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195
200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Ser Lys Tyr 210 215 220Gly Pro Pro
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro225
230 235 240Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser 245
250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser Gln Glu Asp 260 265 270Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275
280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Tyr Arg Val 290 295
300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305
310 315 320Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325
330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr 340 345
350Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375
380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu385 390 395 400Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
405 410 415Ser Arg Trp Gln Glu Gly Asn
Val Phe Ser Cys Ser Val Met His Glu 420 425
430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Leu Gly 435 440
44569448PRTArtificial SequenceSynthetic sequence Humanized monoclonal
antibody heavy chain 69Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Glu1 5 10
15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Asp Ile Thr Ser Gly
20 25 30Tyr His Glu Trp Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Ser Pro Ser Leu
Lys 50 55 60Ser Arg Val Thr Ile Ser
Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70
75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90
95Arg Pro Pro Ala Tyr Tyr Gly Phe Gly Pro Tyr Ala Leu Asp Tyr Trp
100 105 110Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr 130 135 140Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150
155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro 165 170
175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190Val Pro Ser Ser Ser
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195
200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Ser Lys Tyr 210 215 220Gly Pro Pro
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro225
230 235 240Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser 245
250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser Gln Glu Asp 260 265 270Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275
280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Tyr Arg Val 290 295
300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305
310 315 320Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325
330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr 340 345
350Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375
380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu385 390 395 400Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
405 410 415Ser Arg Trp Gln Glu Gly Asn
Val Phe Ser Cys Ser Val Met His Glu 420 425
430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Leu Gly 435 440
44570639DNAArtificial SequenceSynthetic sequence DNA sequence encoding
the humanized monoclonal antibody light chain of SEQ ID NO 59
70gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc
60attacctgca gcgcgagcag cagcgtgagc tatatgcatt ggtatcagca gaaaccgggc
120aaagcgccga aactgctgat ttataccacc agcaacctgg cgcatggcgt gccgagccgc
180tttagcggca gcggcagcgg caccgatttt accctgacca ttagcagcct gcagccggaa
240gattttgcga cctattattg ccagtggcgc agcaccctgc cgccgacctt tggcggcggc
300accaaagtgg aaattaaacg caccgtggcg gcgccgagcg tgtttatttt tccgccgagc
360gatgaacagc tgaaaagcgg caccgcgagc gtggtgtgcc tgctgaacaa cttttatccg
420cgcgaagcga aagtgcagtg gaaagtggat aacgcgctgc agagcggcaa cagccaggaa
480agcgtgaccg aacaggatag caaagatagc acctatagcc tgagcagcac cctgaccctg
540agcaaagcgg attatgaaaa acataaagtg tatgcgtgcg aagtgaccca tcagggcctg
600agcagcccgg tgaccaaaag ctttaaccgc ggcgaatgc
639711344DNAArtificial SequenceSynthetic sequence DNA sequence encoding
the humanized monoclonal antibody heavy chain of SEQ ID NO 60
71caggtgcagc tgcaggaaag cggcccgggc ctggtgaaac cgagcgaaac cctgagcctg
60acctgcaccg tgagcggcga tgatattacc agcggctatt gggattggat tcgccagccg
120ccgggcaaag gcctggaatg gattggctat attagctata gcggcagcac ctattatagc
180ccgagcctga aaagccgcgt gaccattagc gtggatacca gcaaaaacca gtttagcctg
240aaactgagca gcgtgaccgc ggcggatacc gcggtgtatt attgcgcgcg cccgccgggc
300tattatggct ttggcccgta tgcgctggat tattggggcc agggcaccct ggtgaccgtg
360agcagcgcga gcaccaaagg cccgagcgtg tttccgctgg cgccgtgcag ccgcagcacc
420agcgaaagca ccgcggcgct gggctgcctg gtgaaagatt attttccgga accggtgacc
480gtgagctgga acagcggcgc gctgaccagc ggcgtgcata cctttccggc ggtgctgcag
540agcagcggcc tgtatagcct gagcagcgtg gtgaccgtgc cgagcagcag cctgggcacc
600aaaacctata cctgcaacgt ggatcataaa ccgagcaaca ccaaagtgga taaacgcgtg
660gaaagcaaat atggcccgcc gtgcccgccg tgcccggcgc cggaagcggc gggcggcccg
720agcgtgtttc tgtttccgcc gaaaccgaaa gataccctga tgattagccg caccccggaa
780gtgacctgcg tggtggtgga tgtgagccag gaagatccgg aagtgcagtt taactggtat
840gtggatggcg tggaagtgca taacgcgaaa accaaaccgc gcgaagaaca gtttaacagc
900acctatcgcg tggtgagcgt gctgaccgtg ctgcatcagg attggctgaa cggcaaagaa
960tataaatgca aagtgagcaa caaaggcctg ccgagcagca ttgaaaaaac cattagcaaa
1020gcgaaaggcc agccgcgcga accgcaggtg tataccctgc cgccgagcca ggaagaaatg
1080accaaaaacc aggtgagcct gacctgcctg gtgaaaggct tttatccgag cgatattgcg
1140gtggaatggg aaagcaacgg ccagccggaa aacaactata aaaccacccc gccggtgctg
1200gatagcgatg gcagcttttt tctgtatagc cgcctgaccg tggataaaag ccgctggcag
1260gaaggcaacg tgtttagctg cagcgtgatg catgaagcgc tgcataacca ttatacccag
1320aaaagcctga gcctgagcct gggc
134472639DNAArtificial SequenceSynthetic sequence DNA sequence encoding
the humanized monoclonal antibody light chain of SEQ ID NO 61
72gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc
60attacctgca gctatagcag cagcgtgagc tatatgcatt ggtatcagca gaaaccgggc
120aaagcgccga aactgctgat ttataccacc agcaacctgg cgcatggcgt gccgagccgc
180tttagcggca gcggcagcgg caccgatttt accctgacca ttagcagcct gcagccggaa
240gattttgcga cctattattg ccagcagcgc agcaccctgc cgccgacctt tggcggcggc
300accaaagtgg aaattaaacg caccgtggcg gcgccgagcg tgtttatttt tccgccgagc
360gatgaacagc tgaaaagcgg caccgcgagc gtggtgtgcc tgctgaacaa cttttatccg
420cgcgaagcga aagtgcagtg gaaagtggat aacgcgctgc agagcggcaa cagccaggaa
480agcgtgaccg aacaggatag caaagatagc acctatagcc tgagcagcac cctgaccctg
540agcaaagcgg attatgaaaa acataaagtg tatgcgtgcg aagtgaccca tcagggcctg
600agcagcccgg tgaccaaaag ctttaaccgc ggcgaatgc
639731344DNAArtificial SequenceSynthetic sequence DNA sequence encoding
the humanized monoclonal antibody heavy chain of SEQ ID NO 62
73caggtgcagc tgcaggaaag cggcccgggc ctggtgaaac cgagcgaaac cctgagcctg
60acctgcaccg tgagcggcga tgatattacc agcggctatt ggaactggat tcgccagccg
120ccgggcaaag gcctggaatg gattggctat attagctata gcggcagcac ctattatatt
180ccgagcctga aaagccgcgt gaccattagc gtggatacca gcaaaaacca gtttagcctg
240aaactgagca gcgtgaccgc ggcggatacc gcggtgtatt attgcgcgcg cccgccgggc
300tattatggct ttggcccgta tgcgatggat tattggggcc agggcaccct ggtgaccgtg
360agcagcgcga gcaccaaagg cccgagcgtg tttccgctgg cgccgtgcag ccgcagcacc
420agcgaaagca ccgcggcgct gggctgcctg gtgaaagatt attttccgga accggtgacc
480gtgagctgga acagcggcgc gctgaccagc ggcgtgcata cctttccggc ggtgctgcag
540agcagcggcc tgtatagcct gagcagcgtg gtgaccgtgc cgagcagcag cctgggcacc
600aaaacctata cctgcaacgt ggatcataaa ccgagcaaca ccaaagtgga taaacgcgtg
660gaaagcaaat atggcccgcc gtgcccgccg tgcccggcgc cggaagcggc gggcggcccg
720agcgtgtttc tgtttccgcc gaaaccgaaa gataccctga tgattagccg caccccggaa
780gtgacctgcg tggtggtgga tgtgagccag gaagatccgg aagtgcagtt taactggtat
840gtggatggcg tggaagtgca taacgcgaaa accaaaccgc gcgaagaaca gtttaacagc
900acctatcgcg tggtgagcgt gctgaccgtg ctgcatcagg attggctgaa cggcaaagaa
960tataaatgca aagtgagcaa caaaggcctg ccgagcagca ttgaaaaaac cattagcaaa
1020gcgaaaggcc agccgcgcga accgcaggtg tataccctgc cgccgagcca ggaagaaatg
1080accaaaaacc aggtgagcct gacctgcctg gtgaaaggct tttatccgag cgatattgcg
1140gtggaatggg aaagcaacgg ccagccggaa aacaactata aaaccacccc gccggtgctg
1200gatagcgatg gcagcttttt tctgtatagc cgcctgaccg tggataaaag ccgctggcag
1260gaaggcaacg tgtttagctg cagcgtgatg catgaagcgc tgcataacca ttatacccag
1320aaaagcctga gcctgagcct gggc
134474639DNAArtificial SequenceSynthetic sequence DNA sequence encoding
the humanized monoclonal antibody light chain of SEQ ID NO 63
74gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc
60attacctgca gcgcgagcag cagcgtgagc tatattcatt ggtatcagca gaaaccgggc
120aaagcgccga aactgctgat ttataccacc agctatctgg cgcatggcgt gccgagccgc
180tttagcggca gcggcagcgg caccgatttt accctgacca ttagcagcct gcagccggaa
240gattttgcga cctattattg cctgcagcgc agcaccctgc cgccgacctt tggcggcggc
300accaaagtgg aaattaaacg caccgtggcg gcgccgagcg tgtttatttt tccgccgagc
360gatgaacagc tgaaaagcgg caccgcgagc gtggtgtgcc tgctgaacaa cttttatccg
420cgcgaagcga aagtgcagtg gaaagtggat aacgcgctgc agagcggcaa cagccaggaa
480agcgtgaccg aacaggatag caaagatagc acctatagcc tgagcagcac cctgaccctg
540agcaaagcgg attatgaaaa acataaagtg tatgcgtgcg aagtgaccca tcagggcctg
600agcagcccgg tgaccaaaag ctttaaccgc ggcgaatgc
639751344DNAArtificial SequenceSynthetic sequence DNA sequence encoding
the humanized monoclonal antibody heavy chain of SEQ ID NO 64
75caggtgcagc tgcaggaaag cggcccgggc ctggtgaaac cgagcgaaac cctgagcctg
60acctgcaccg tgagcggcga tgatattacc agcggctatt gggattggat tcgccagccg
120ccgggcaaag gcctggaatg gattggctat attagctata gcggcagcac ctattatagc
180ccgagcctga aaagccgcgt gaccattagc gtggatacca gcaaaaacca gtttagcctg
240aaactgagca gcgtgaccgc ggcggatacc gcggtgtatt attgcgcgcg cccgccgggc
300tattatggct ttggcccgta tgcgctggat tattggggcc agggcaccct ggtgaccgtg
360agcagcgcga gcaccaaagg cccgagcgtg tttccgctgg cgccgtgcag ccgcagcacc
420agcgaaagca ccgcggcgct gggctgcctg gtgaaagatt attttccgga accggtgacc
480gtgagctgga acagcggcgc gctgaccagc ggcgtgcata cctttccggc ggtgctgcag
540agcagcggcc tgtatagcct gagcagcgtg gtgaccgtgc cgagcagcag cctgggcacc
600aaaacctata cctgcaacgt ggatcataaa ccgagcaaca ccaaagtgga taaacgcgtg
660gaaagcaaat atggcccgcc gtgcccgccg tgcccggcgc cggaagcggc gggcggcccg
720agcgtgtttc tgtttccgcc gaaaccgaaa gataccctga tgattagccg caccccggaa
780gtgacctgcg tggtggtgga tgtgagccag gaagatccgg aagtgcagtt taactggtat
840gtggatggcg tggaagtgca taacgcgaaa accaaaccgc gcgaagaaca gtttaacagc
900acctatcgcg tggtgagcgt gctgaccgtg ctgcatcagg attggctgaa cggcaaagaa
960tataaatgca aagtgagcaa caaaggcctg ccgagcagca ttgaaaaaac cattagcaaa
1020gcgaaaggcc agccgcgcga accgcaggtg tataccctgc cgccgagcca ggaagaaatg
1080accaaaaacc aggtgagcct gacctgcctg gtgaaaggct tttatccgag cgatattgcg
1140gtggaatggg aaagcaacgg ccagccggaa aacaactata aaaccacccc gccggtgctg
1200gatagcgatg gcagcttttt tctgtatagc cgcctgaccg tggataaaag ccgctggcag
1260gaaggcaacg tgtttagctg cagcgtgatg catgaagcgc tgcataacca ttatacccag
1320aaaagcctga gcctgagcct gggc
134476639DNAArtificial SequenceSynthetic sequence DNA sequence encoding
the humanized monoclonal antibody light chain of SEQ ID NO 65
76gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc
60attacctgca gcgcgagcag cagcgtgagc tatattcatt ggtatcagca gaaaccgggc
120aaagcgccga aactgctgat ttataccacc agctatctgg cgcatggcgt gccgagccgc
180tttagcggca gcggcagcgg caccgatttt accctgacca ttagcagcct gcagccggaa
240gattttgcga cctattattg ccagcagcgc agcaccctgc cgccgacctt tggcggcggc
300accaaagtgg aaattaaacg caccgtggcg gcgccgagcg tgtttatttt tccgccgagc
360gatgaacagc tgaaaagcgg caccgcgagc gtggtgtgcc tgctgaacaa cttttatccg
420cgcgaagcga aagtgcagtg gaaagtggat aacgcgctgc agagcggcaa cagccaggaa
480agcgtgaccg aacaggatag caaagatagc acctatagcc tgagcagcac cctgaccctg
540agcaaagcgg attatgaaaa acataaagtg tatgcgtgcg aagtgaccca tcagggcctg
600agcagcccgg tgaccaaaag ctttaaccgc ggcgaatgc
639771344DNAArtificial SequenceSynthetic sequence DNA sequence encoding
the humanized monoclonal antibody heavy chain of SEQ ID NO 66
77caggtgcagc tgcaggaaag cggcccgggc ctggtgaaac cgagcgaaac cctgagcctg
60acctgcaccg tgagcggcga tgatattacc agcggctatc atgattggat tcgccagccg
120ccgggcaaag gcctggaatg gattggctat attagctata gcggcagcac ctattatagc
180ccgagcctga aaagccgcgt gaccattagc gtggatacca gcaaaaacca gtttagcctg
240aaactgagca gcgtgaccgc ggcggatacc gcggtgtatt attgcgcgcg cccgccgggc
300tattatggct ttggcccgta tgcgctggat tattggggcc agggcaccct ggtgaccgtg
360agcagcgcga gcaccaaagg cccgagcgtg tttccgctgg cgccgtgcag ccgcagcacc
420agcgaaagca ccgcggcgct gggctgcctg gtgaaagatt attttccgga accggtgacc
480gtgagctgga acagcggcgc gctgaccagc ggcgtgcata cctttccggc ggtgctgcag
540agcagcggcc tgtatagcct gagcagcgtg gtgaccgtgc cgagcagcag cctgggcacc
600aaaacctata cctgcaacgt ggatcataaa ccgagcaaca ccaaagtgga taaacgcgtg
660gaaagcaaat atggcccgcc gtgcccgccg tgcccggcgc cggaagcggc gggcggcccg
720agcgtgtttc tgtttccgcc gaaaccgaaa gataccctga tgattagccg caccccggaa
780gtgacctgcg tggtggtgga tgtgagccag gaagatccgg aagtgcagtt taactggtat
840gtggatggcg tggaagtgca taacgcgaaa accaaaccgc gcgaagaaca gtttaacagc
900acctatcgcg tggtgagcgt gctgaccgtg ctgcatcagg attggctgaa cggcaaagaa
960tataaatgca aagtgagcaa caaaggcctg ccgagcagca ttgaaaaaac cattagcaaa
1020gcgaaaggcc agccgcgcga accgcaggtg tataccctgc cgccgagcca ggaagaaatg
1080accaaaaacc aggtgagcct gacctgcctg gtgaaaggct tttatccgag cgatattgcg
1140gtggaatggg aaagcaacgg ccagccggaa aacaactata aaaccacccc gccggtgctg
1200gatagcgatg gcagcttttt tctgtatagc cgcctgaccg tggataaaag ccgctggcag
1260gaaggcaacg tgtttagctg cagcgtgatg catgaagcgc tgcataacca ttatacccag
1320aaaagcctga gcctgagcct gggc
1344781344DNAArtificial SequenceSynthetic sequence DNA sequence encoding
the humanized monoclonal antibody heavy chain of SEQ ID NO 67
78caggtgcagc tgcaggaaag cggcccgggc ctggtgaaac cgagcgaaac cctgagcctg
60acctgcaccg tgagcggcga tgatattacc agcggctatc atgattggat tcgccagccg
120ccgggcaaag gcctggaatg gattggctat attagctata gcggcagcac ccagtatagc
180ccgagcctga aaagccgcgt gaccattagc gtggatacca gcaaaaacca gtttagcctg
240aaactgagca gcgtgaccgc ggcggatacc gcggtgtatt attgcgcgcg cccgccgggc
300tattatggct ttggcccgta tgcgctggat tattggggcc agggcaccct ggtgaccgtg
360agcagcgcga gcaccaaagg cccgagcgtg tttccgctgg cgccgtgcag ccgcagcacc
420agcgaaagca ccgcggcgct gggctgcctg gtgaaagatt attttccgga accggtgacc
480gtgagctgga acagcggcgc gctgaccagc ggcgtgcata cctttccggc ggtgctgcag
540agcagcggcc tgtatagcct gagcagcgtg gtgaccgtgc cgagcagcag cctgggcacc
600aaaacctata cctgcaacgt ggatcataaa ccgagcaaca ccaaagtgga taaacgcgtg
660gaaagcaaat atggcccgcc gtgcccgccg tgcccggcgc cggaagcggc gggcggcccg
720agcgtgtttc tgtttccgcc gaaaccgaaa gataccctga tgattagccg caccccggaa
780gtgacctgcg tggtggtgga tgtgagccag gaagatccgg aagtgcagtt taactggtat
840gtggatggcg tggaagtgca taacgcgaaa accaaaccgc gcgaagaaca gtttaacagc
900acctatcgcg tggtgagcgt gctgaccgtg ctgcatcagg attggctgaa cggcaaagaa
960tataaatgca aagtgagcaa caaaggcctg ccgagcagca ttgaaaaaac cattagcaaa
1020gcgaaaggcc agccgcgcga accgcaggtg tataccctgc cgccgagcca ggaagaaatg
1080accaaaaacc aggtgagcct gacctgcctg gtgaaaggct tttatccgag cgatattgcg
1140gtggaatggg aaagcaacgg ccagccggaa aacaactata aaaccacccc gccggtgctg
1200gatagcgatg gcagcttttt tctgtatagc cgcctgaccg tggataaaag ccgctggcag
1260gaaggcaacg tgtttagctg cagcgtgatg catgaagcgc tgcataacca ttatacccag
1320aaaagcctga gcctgagcct gggc
1344791344DNAArtificial SequenceSynthetic sequence DNA sequence encoding
the humanized monoclonal antibody heavy chain of SEQ ID NO 68
79caggtgcagc tgcaggaaag cggcccgggc ctggtgaaac cgagcgaaac cctgagcctg
60acctgcaccg tgagcggcga tgatattacc agcggctatc atgattggat tcgccagccg
120ccgggcaaag gcctggaatg gattggctat attagctata gcggcagcac ctattatagc
180ccgagcctga aaagccgcgt gaccattagc gtggatacca gcaaaaacca gtttagcctg
240aaactgagca gcgtgaccgc ggcggatacc gcggtgtatt attgcgcgcg cccgccggcg
300tattatggct ttggcccgta tgcgctggat tattggggcc agggcaccct ggtgaccgtg
360agcagcgcga gcaccaaagg cccgagcgtg tttccgctgg cgccgtgcag ccgcagcacc
420agcgaaagca ccgcggcgct gggctgcctg gtgaaagatt attttccgga accggtgacc
480gtgagctgga acagcggcgc gctgaccagc ggcgtgcata cctttccggc ggtgctgcag
540agcagcggcc tgtatagcct gagcagcgtg gtgaccgtgc cgagcagcag cctgggcacc
600aaaacctata cctgcaacgt ggatcataaa ccgagcaaca ccaaagtgga taaacgcgtg
660gaaagcaaat atggcccgcc gtgcccgccg tgcccggcgc cggaagcggc gggcggcccg
720agcgtgtttc tgtttccgcc gaaaccgaaa gataccctga tgattagccg caccccggaa
780gtgacctgcg tggtggtgga tgtgagccag gaagatccgg aagtgcagtt taactggtat
840gtggatggcg tggaagtgca taacgcgaaa accaaaccgc gcgaagaaca gtttaacagc
900acctatcgcg tggtgagcgt gctgaccgtg ctgcatcagg attggctgaa cggcaaagaa
960tataaatgca aagtgagcaa caaaggcctg ccgagcagca ttgaaaaaac cattagcaaa
1020gcgaaaggcc agccgcgcga accgcaggtg tataccctgc cgccgagcca ggaagaaatg
1080accaaaaacc aggtgagcct gacctgcctg gtgaaaggct tttatccgag cgatattgcg
1140gtggaatggg aaagcaacgg ccagccggaa aacaactata aaaccacccc gccggtgctg
1200gatagcgatg gcagcttttt tctgtatagc cgcctgaccg tggataaaag ccgctggcag
1260gaaggcaacg tgtttagctg cagcgtgatg catgaagcgc tgcataacca ttatacccag
1320aaaagcctga gcctgagcct gggc
1344801344DNAArtificial SequenceSynthetic sequence DNA sequence encoding
the humanized monoclonal antibody heavy chain of SEQ ID NO 69
80caggtgcagc tgcaggaaag cggcccgggc ctggtgaaac cgagcgaaac cctgagcctg
60acctgcaccg tgagcggcga tgatattacc agcggctatc atgaatggat tcgccagccg
120ccgggcaaag gcctggaatg gattggctat attagctata gcggcagcac cagctatagc
180ccgagcctga aaagccgcgt gaccattagc gtggatacca gcaaaaacca gtttagcctg
240aaactgagca gcgtgaccgc ggcggatacc gcggtgtatt attgcgcgcg cccgccggcg
300tattatggct ttggcccgta tgcgctggat tattggggcc agggcaccct ggtgaccgtg
360agcagcgcga gcaccaaagg cccgagcgtg tttccgctgg cgccgtgcag ccgcagcacc
420agcgaaagca ccgcggcgct gggctgcctg gtgaaagatt attttccgga accggtgacc
480gtgagctgga acagcggcgc gctgaccagc ggcgtgcata cctttccggc ggtgctgcag
540agcagcggcc tgtatagcct gagcagcgtg gtgaccgtgc cgagcagcag cctgggcacc
600aaaacctata cctgcaacgt ggatcataaa ccgagcaaca ccaaagtgga taaacgcgtg
660gaaagcaaat atggcccgcc gtgcccgccg tgcccggcgc cggaagcggc gggcggcccg
720agcgtgtttc tgtttccgcc gaaaccgaaa gataccctga tgattagccg caccccggaa
780gtgacctgcg tggtggtgga tgtgagccag gaagatccgg aagtgcagtt taactggtat
840gtggatggcg tggaagtgca taacgcgaaa accaaaccgc gcgaagaaca gtttaacagc
900acctatcgcg tggtgagcgt gctgaccgtg ctgcatcagg attggctgaa cggcaaagaa
960tataaatgca aagtgagcaa caaaggcctg ccgagcagca ttgaaaaaac cattagcaaa
1020gcgaaaggcc agccgcgcga accgcaggtg tataccctgc cgccgagcca ggaagaaatg
1080accaaaaacc aggtgagcct gacctgcctg gtgaaaggct tttatccgag cgatattgcg
1140gtggaatggg aaagcaacgg ccagccggaa aacaactata aaaccacccc gccggtgctg
1200gatagcgatg gcagcttttt tctgtatagc cgcctgaccg tggataaaag ccgctggcag
1260gaaggcaacg tgtttagctg cagcgtgatg catgaagcgc tgcataacca ttatacccag
1320aaaagcctga gcctgagcct gggc
1344
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