CGRP ANTIBODIES

Abstract

The present invention provides human engineered calcitonin gene related peptide (CGRP) antibodies or antigen-binding fragment thereof. In addition, the present invention provides the use of the human engineered calcitonin gene related peptide (CGRP) antibodies or antigen-binding fragment thereof for the treatment of osteoarthritis pain.

Claims

1-26. (canceled)

27. A method of treating migraines comprising administering to a human patient in need thereof a human engineered calcitonin gene related peptide (CGRP) antibody comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein said LCVR comprises a LCDR1 amino acid sequence RASKDISKYLN (SEQ ID NO: 6), LCDR2 amino acid sequence YTSGYHS (SEQ ID NO: 7), and LCDR3 amino acid sequence QQGDALPPT (SEQ ID NO:5), and wherein the HCVR comprises a HCDR1 amino acid sequence GYTFGNYWMQ (SEQ ID NO: 12), HCDR2 amino acid sequence AIYEGTGKTVYIQKFAD (SEQ ID NO: 16), and HCDR3 amino acid sequence LSDYVSGFGY (SEQ ID NO: 39).

28. The method according to claim 1, wherein the human engineered CGRP antibody is in a pharmaceutical composition.

Description

EXAMPLE 1

Production of Antibodies

[0055] Antibodies I-V can be made and purified as follows. 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 HC:LC vector ratio or a single vector system encoding both LC, such as SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31, and HC, such as SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 or SEQ ID NO: 36. Clarified media, 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 3.0). 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 The amino acid sequences for these exemplified antibodies are provided below.

TABLE-US-00002 TABLE 1 Antibody SEQ ID NOs Heavy Light Antibody Chain Chain LCVR HCVR I 32 27 17 22 II 33 28 18 23 III 34 29 19 24 IV 35 30 20 25 V 36 31 21 26 Antibody HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 I 12 13 14 3 4 5 II 12 15 14 3 4 5 III 12 16 39 6 7 5 IV 12 15 39 8 4 5 V 12 15 39 9 7 5

EXAMPLE 2

Affinity (Kd) Measurements for Human Engineered CGRP antibodies

[0056] Binding affinity of the exemplified antibodies to CGRP is determined using a surface plasmon resonance assay on a Biacore T100 instrument primed with HBS-EP+(GE Healthcare, 10 mM Hepes pH7.4 +150 mM NaCl+3 mM EDTA+0.05% surfactant P20) running buffer and analysis temperature set at 37 C. A CM5 chip containing immobilized protein A (generated using standard NHS-EDC amine coupling) on all four flow cells (Fc) is used to employ a capture methodology. Antibody samples are prepared at 2 g/mL by dilution into running buffer. Human CGRP samples are prepared at final concentrations of 5.0, 2.5, 1.3, 0.63, 0.31, and 0 (blank) nM by dilution into running buffer. A fresh, single-use aliquot of CGRP is used for each replicate experiment to avoid multiple freeze-thaw cycles. Each analysis cycle consists of (1) capturing antibody samples on separate flow cells (Fc2, Fc3, and Fc4), (2) injection of 350 L (210-sec) of CGRP over all Fc at 100 L/min, (3) return to buffer flow for 10 min to monitor dissociation phase, (4) regeneration of chip surfaces with a 5 L (15-sec) injection of glycine, pH1.5, (5) equilibration of chip surfaces with a 5 L (15-sec) injection of HBS-EP+. Each CGRP concentration is injected in duplicate. Data are processed using standard double-referencing and fit to a 1:1 binding model using Biacore T100 Evaluation software, version 2.0.1, to determine the association rate (k.sub.on, M.sup.1s.sup.1 units), dissociation rate (k.sub.off, s.sup.1 units), and Rmax (RU units). The equilibrium dissociation constant (K.sub.D) is calculated as from the relationship K.sub.D=k.sub.offk.sub.on, and is in molar units. Values provided in Table 2 are means of n number of experiments. Table 2 demonstrates that the exemplified antibodies of the present invention bind to CGRP with affinities<200 pM.

TABLE-US-00003 TABLE 2 Antibody Binding Affinities. K.sub.D (pM) Antibody Mean SD n I 190 1 II 26 6 3 III 31 19 6 IV 24 10 4 V 24 6 2

EXAMPLE 3

Reduction of Pain in an MIA Model

[0057] The injection of monoiodoacetic acid (MIA) into the knee joint of rats produces an acute inflammatory insult which then develops into chronic degeneration of the joint tissues in the injected joint. The pain resulting from the joint injury can be measured via differential weight bearing of the hind legs using an incapacitance tester. The MIA model has been well-described in the literature and has been used to demonstrate efficacy vs. pain for a variety of mechanisms and compounds. Efficacy is routinely measured by the ability of a compound to partially normalize weight distribution.

[0058] To determine the ability of the exemplified antibodies of the present invention to reduce pain, male Lewis rats (Harlan, Indianapolis, Ind.) of approximately eight weeks of age at the time of MIA injection are used. The rats are housed in groups of two or three per cage and maintained in a constant temperature and on a 12 hour light/12 hour dark cycle. Animals have free access to food and water at all times except during data collection. All experiments are carried out according to protocols approved by the Eli Lilly Institutional Animal Care and Use Committee.

[0059] The right knees of each rat are injected with 0.3 mg MIA in 50 L of saline and the left knees with 50 L of saline on day 0. On a set day post MIA, human IgG4 control or test CGRP antibody is subcutaneously administered in PBS (n=6 per group). Three days post dosing with test CGRP antibody, pain is measured using incapacitance testing which measures the difference in hind paw weight bearing between the MIA treated and saline injected knees. Each measurement is the average of three separate measurements each measured over 1 second.

[0060] Data are presented as percent inhibition of pain calculated by dividing the mean of the CGRP antibody treated group by the mean of the control antibody group, subtracting this value from 1 and multiplying this resulting value by 100. CGRP treated groups are also compared to vehicle groups by one way analysis of means and Dunnett's test using JMP (versions 5.1 and 6) statistical analysis program (SAS Institute Inc., Cary, N.C.). Differences are considered to be significant if the P value is less than 0.05. As shown in Table 3, the data demonstrate that the exemplified CGRP antibodies of the present invention significantly reduce pain.

TABLE-US-00004 TABLE 3 Percent Inhibition of MIA Induced Pain Significant by MIA Dunnett's test Antibody Dose and route % inhibition (p < 0.05) I 20 mg/kg sc 20% Yes II 4 mg/kg sc 30% Yes III 4 mg/kg sc 37% Yes IV 4 mg/kg sc 37% Yes V 4 mg/kg sc 19% yes

EXAMPLE 4

Inhibition of CGRP Induced cAMP Formation in SK-N-MC Cells

[0061] To compare the ability of an antibody of the present invention to Antibody G1 (LCVR-SEQ ID NO: 40 and HCVR-SEQ ID NO: 41), inhibition of CGRP induced cAMP formation in SK-N-MC is determined. Binding of CGRP to its receptor results in the stimulation of cAMP production. The CGRP receptor is a hetero-trimeric complex consisting of the Calcitonin receptor-Like Receptor (CLR, a G-protein coupled receptor) and Receptor Activity Modifying Protein (RAMP)-1, coupled cytoplasmically to Receptor Component Protein (RCP). The human neuroepithelioma cell line SK-N-MC expresses these 3 molecules naturally and can therefore be used to assess the effect of CGRP on signal transduction events. Production of cAMP is a standard measure for G-protein coupled receptor activation.

[0062] SK-N-MC cells are cultured in MEM, containing 10% FBS, lx MEM non-essential amino acids, 1100 mM MEM Sodium Pyruvate, 1Pen/Strep, and 2 mM L-glutamine. After harvesting, cells are washed once and resuspended in assay buffer (stimulation buffer (HBSS with Mg and Ca, 5 mM HEPES, 0.1% BSA, 100 uM Ascorbic acid) diluted 1:2 with Dulbecco's PBS containing a final concentration of 0.5 mM IBMX) and plated in 96-well plates at 15,000 cells per well. Test antibody or a control human IgG4 antibody are added (serial 4-fold dilutions in assay buffer, 10 concentrations) to the cells, followed by a fixed amount of human -CGRP (2 nM; Bachem H1470). Plates are incubated for 1 hr at room temperature. Levels of cAMP are measured by a homogeneous time resolved fluorescence (HTRF) assay system (Cisbio).

[0063] The amount of cAMP induced by 2 nM human CGRP in the presence of varying concentrations of antibody is calculated as a percentage inhibition compared to CGRP alone. The antibody concentration producing 50% inhibition of cAMP production (IC50) is then calculated from a 4-parameter curve fit model. Following the procedures as described herein, Antibody III (IC.sub.50 of 0.41 nM) inhibited the amount of CGRP induced cAMP to a much greater extent than Antibody G1 (IC.sub.50 of 5.36 nM) possibly due to Antibody III's faster K.sub.on rate (K.sub.on rate as measured by Biacore)

EXAMPLE 5

Rat Dural Plasma Protein Extravasation (PPE) Model

[0064] The rat PPE model is a well established pre-clinical model that may be used to evaluate the efficacy of anti-CGRP antibodies for the treatment of migraine.

[0065] A 2 mg/mL solution of an anti-CGRP antibody (Ab) is prepared in saline solution. All subsequent dilutions are made with saline. A 2 mg/mL solution of monoclonal isotype control antibody (IgG) is also prepared in saline.

[0066] Male Sprague-Dawley rats from Harlan Laboratories (250 to 350 g) are anesthetized with Nembutal (60 mg/kg, ip.) and placed in a stereotaxic frame (David Kopf Instruments) with the incisor bar set at 2.5 mm Following a mid-line sagittal scalp incision, two pairs of bilateral holes are drilled through the skull (3.2 mm posteriorly, 1.8 and 3.8 mm laterally, all coordinates referenced to bregma). Pairs of stainless steel stimulating electrodes (Rhodes Medical Systems Inc), insulated except at the tips, are lowered through the holes in both hemispheres to a depth of 9.2 mm below the dura. Test antibodies or saline vehicle are administered intravenously via the femoral vein (1 mL/kg). Eight minutes later a solution of fluoroscein isothiocyanate (FITC) dye-labeled bovine serum albumin (BSA) (FITC-BSA, Sigma A9771) (20 mg/kg, iv.) is injected into the femoral vein to function as a marker for protein extravasation. Ten minutes following dosing with test antibodies or vehicle, the left trigeminal ganglion is stimulated for 5 minutes at a current intensity of 1.0 mA (5 Hz, 5 ms duration) with a Model S48 Grass Instrument Stimulator. Five minutes following stimulation, the rats are killed by exsanguination with 40 mL of saline. The exsanguination also rinses residual FITC/BSA out of the blood vessels. The top of the skull is removed to collect the dural membranes. The membrane samples are removed from both hemispheres, rinsed with water, and spread flat on microscope slides. The slides are dried for 15 minutes on a slide warmer and cover-slipped with a 70% glycerol/water solution.

[0067] A fluorescence microscope (Zeiss) equipped with a grating monochromator and a spectrophotometer are used to quantify the amount of FITC-BSA dye in each dural sample. The microscope is equipped with a motorized stage interfaced with a personal computer. This facilitates the computer-controlled movement of the stage, with fluorescence measurements at 25 points (500 m steps) on each dural sample. The extravasation induced by electrical stimulation of the trigeminal ganglion is an ipsilateral effect (i.e. occurs only on the side of the dura in which the trigeminal ganglion is stimulated). This allows the other (unstimulated) half of the dura to be used as a control. The extravasation ratio (i.e. the ratio of the amount of extravasation in the dura from the stimulated side compared to the unstimulated side) is calculated.

TABLE-US-00005 SEQUENCES (SEQIDNO:1) ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF (SEQIDNO:2) ACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF Antibody LCDR1 LCDR2 LCDR3 I RASQDIDNYLN YTSEYHS QQGDALPPT (SEQIDNO:3) (SEQIDNO:4) (SEQIDNO:5) II RASQDIDNYLN YTSEYHS QQGDALPPT (SEQIDNO:3) (SEQIDNO:4) (SEQIDNO:5) III RASKDISKYLN YTSGYHS QQGDALPPT (SEQIDNO:6) (SEQIDNO:7) (SEQIDNO:5) IV RASRPIDKYLN YTSEYHS QQGDALPPT (SEQIDNO:8) (SEQIDNO:4) (SEQIDNO:5) V RASQDIDKYLN YTSGYHS QQGDALPPT (SEQIDNO:9) (SEQIDNO:7) (SEQIDNO:5) con- RASX.sub.1X.sub.2IX.sub.3X.sub.4YLN YTSX.sub.5YHS QQGDALPPT sensus (SEQIDNO:10) (SEQIDNO:11) (SEQIDNO:5) X.sub.1isQ,RorK; X.sub.2isDorP; X.sub.3isDorS; X.sub.4isNorK; and X.sub.5isGorE. Antibody HCDR1 HCDR2 HCDR3 I GYTFGNYWMQ AIYEGTGDTRYIQKFAG LSDYVSGFSY (SEQIDNO:12) (SEQIDNO:13) (SEQIDNO:14) II GYTFGNYWMQ AIYEGTGKTVYIQKFAG LSDYVSGFSY (SEQIDNO:12) (SEQIDNO:15) (SEQIDNO:14) III GYTFGNYWMQ AIYEGTGKTVYIQKFAD LSDYVSGFGY (SEQIDNO:12) (SEQIDNO:16) (SEQIDNO:39) IV GYTFGNYWMQ AIYEGTGKTVYIQKFAG LSDYVSGFGY (SEQIDNO:12) (SEQIDNO:15) (SEQIDNO:39) V GYTFGNYWMQ AIYEGTGKTVYIQKFAG LSDYVSGFGY (SEQIDNO:12) (SEQIDNO:15) (SEQIDNO:39) con- GYTFGNYWMQ AIYEGTGX.sub.6TX.sub.7YIQKFAX.sub.8 LSDYVSGFX.sub.9Y sensus (SEQIDNO:12) (SEQIDNO:37) (SEQIDNO:38) X.sub.6isKorD; X.sub.7isVorR; X.sub.8isDorG; and X.sub.9isGorS. (SEQIDNO:17) DIQMTQSPSSLSASVGDRVTITCRASQDIDNYLNWYQQKPGKAPKLLIYYTSEYH SGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGDALPPTFGQGTKLEIK (SEQIDNO:18) DIQMTQSPSSLSASVGDRVTITCRASQDIDNYLNWYQQKPGKAPKLLIYYTSEYH SGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGDALPPTFGQGTKLEIK (SEQIDNO:19) DIQMTQSPSSLSASVGDRVTITCRASKDISKYLNWYQQKPGKAPKLLIYYTSGYH SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDALPPTFGGGTKVEIK (SEQIDNO:20) DIQMTQSPSSLSASVGDRVTITCRASRPIDKYLNWYQQKPGKAPKLLIYYTSEYH SGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGDALPPTFGQGTKLEIK (SEQIDNO:21) DIQMTQSPSSLSASVGDRVTITCRASQDIDKYLNWYQQKPGKAPKLLIYYTSGYH SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDALPPTFGGGTKVEIK (SEQIDNO:22) QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGT GDTRYIQKFAGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLSDYVSGFSYWG QGTLVTVSS (SEQIDNO:23) QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGT GKTVYIQKFAGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLSDYVSGFSYWG QGTLVTVSS (SEQIDNO:24) QVQLVQSGAEVKKPGSSVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGT GKTVYIQKFADRVTITADKSTSTAYMELSSLRSEDTAVYYCARLSDYVSGFGYWG QGTTVTVSS (SEQIDNO:25) QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGT GKTVYIQKFAGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLSDYVSGFGYWG QGTLVTVSS (SEQIDNO:26) QVQLVQSGAEVKKPGSSVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGT GKTVYIQKFAGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLSDYVSGFGYWG QGTTVTVSS (SEQIDNO:27) DIQMTQSPSSLSASVGDRVTITCRASQDIDNYLNWYQQKPGKAPKLLIYYTSEYH SGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGDALPPTFGQGTKLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQIDNO:28) DIQMTQSPSSLSASVGDRVTITCRASQDIDNYLNWYQQKPGKAPKLLIYYTSEYH SGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGDALPPTFGQGTKLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQIDNO:29) DIQMTQSPSSLSASVGDRVTITCRASKDISKYLNWYQQKPGKAPKLLIYYTSGYH SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDALPPTFGGGTKVEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQIDNO:30) DIQMTQSPSSLSASVGDRVTITCRASRPIDKYLNWYQQKPGKAPKLLIYYTSEYH SGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGDALPPTFGQGTKLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQIDNO:31) DIQMTQSPSSLSASVGDRVTITCRASQDIDKYLNWYQQKPGKAPKLLIYYTSGYH SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDALPPTFGGGTKVEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQIDNO:32) QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGT GDTRYIQKFAGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLSDYVSGFSYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK YGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLG (SEQIDNO:33) QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGT GKTVYIQKFAGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLSDYVSGFSYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK YGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLG (SEQIDNO:34) QVQLVQSGAEVKKPGSSVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGT GKTVYIQKFADRVTITADKSTSTAYMELSSLRSEDTAVYYCARLSDYVSGFGYWG QGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK YGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLG (SEQIDNO:35) QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGT GKTVYIQKFAGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLSDYVSGFGYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK YGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLG (SEQIDNO:36) QVQLVQSGAEVKKPGSSVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGT GKTVYIQKFAGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLSDYVSGFGYWG QGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK YGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLG (SEQIDNO:40) EIVLTQSPATLSLSPGERATLSCKASKRVTTYVSWYQQKPGQAPRLLIYGASNRY LGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCSQSYNYPYTFGQGTKLEIK (SEQIDNO:41) EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWISWVRQAPGKGLEWVAEIRSES DASATHYAEAVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCLAYFDYGLAIQN YWGQGTLVTVSS (SEQIDNO:42) DIQMTQSPSSLSASVGDRVTITCRASX.sub.1X.sub.2IX.sub.3X.sub.4YLNWYQQKPGKAPKLLIYYT SX.sub.5YHSGYPSRFSGSGSGTDFTX.sub.6TISSLQPEDX.sub.7ATYYCQQGDALPPTFGX.sub.8GT KX.sub.9EIK X.sub.1= Q,K,orR; X.sub.2= DorP; X.sub.3= DorS; X.sub.4= KorN; X.sub.5= EorG; X.sub.6= ForL; X.sub.7= IorF; X.sub.8= QorG; and X.sub.9= LorV. (SEQIDNO:43) QVQLVQSGAEVKKPGX.sub.1SVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEG TGX.sub.2TX.sub.3YIQKFAX.sub.4RVTX.sub.5TX.sub.6DX.sub.7STSTX.sub.8YMELSSLRSEDTAVYYCARLSDY VSGFX.sub.9YWGQGTX.sub.10VTVSS X.sub.1= AorS; X.sub.2= KorD; X.sub.3= VorR; X.sub.4= GorD; X.sub.5= MorI; X.sub.6= RorA; X.sub.7= TorK; X.sub.8= VorA; X.sub.9= GorS; and X.sub.10= LorT.