CN100422214C - Emitter-binding peptides that produce a change in the spectral emission properties of the emitter - Google Patents

Emitter-binding peptides that produce a change in the spectral emission properties of the emitter Download PDF

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CN100422214C
CN100422214C CNB2004800196219A CN200480019621A CN100422214C CN 100422214 C CN100422214 C CN 100422214C CN B2004800196219 A CNB2004800196219 A CN B2004800196219A CN 200480019621 A CN200480019621 A CN 200480019621A CN 100422214 C CN100422214 C CN 100422214C
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antibody
radiator
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CN1820027A (en
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米夏尔多·席尔纳
凯·利哈
安德烈亚斯·门拉德
斯特凡尼·乌尔林格
科尼莉亚·哈内尔
博多·布罗克斯
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Morphosys AG
Bayer Pharma AG
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Morphosys AG
Schering AG
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL

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Abstract

The invention relates to emitter-binding peptides causing a change in the spectral emission properties of the emitter during interaction between the antigen-binding pocket and the emitter. The inventive emitter-binding peptides particularly represent components of antibodies and antibody fragments.

Description

Make the mutagenic radiator binding peptide of spectral emissions characteristic of radiator
The present invention relates to radiator binding peptide (Emitter-bindende Peptide), it makes the spectral emissions characteristic of radiator produce change when its antigen binding pocket and radiator interaction.Radiator binding peptide of the present invention is the component of antibody or antibody fragment particularly.
Background of invention
Be some material of diagnostic detection and definite its concentration, what now use under many circumstances is the in-vitro diagnosis measuring method, and it is based on having the biomolecules of high-affinity for the material that will measure, as for example peptide, protein, antibody or oligonucleotide.For this reason, preferably use protein and peptide, especially preferably use antibody and antibody fragment.
Some in-vitro diagnosis method is based on combination at the different antibodies that will measure material as for example electrochemiluminescence, and wherein a kind of antibody is used for separating the material that will measure from the sample of being studied, and another kind of antibody carries the detection signal molecule of diagnosis usefulness.In the diagnostic method of electrochemiluminescence, the antibody of mark is by optical detection [Grayeski, M.L., Anal.Chem.1987,59,1243].
Except electrochemiluminescence, the optical characteristics that photoinduced phosphorescence and fluorescence also can be used as molecule is used for diagnosing and measuring method.Compare with phosphorescence with electrochemiluminescence, particularly fluorescence is had the high detection susceptibility and the high linear advantage of measurement signal as the optical characteristics of molecule in big dynamicrange.
For detecting the fluorescence of fluorophore, developed the multiple measuring method of in fluorescent method, using different principle.Existing measuring method application examples such as polarized light reduction (fluorescence polarization=FP), measurement of photon work-ing life (fluorescence measures work-ing life-FLM), bleaching characteristic (the fluorescence photobleaching recovers-FRR) and the energy between the different fluorophore shift the ([Williams of fluorescence-resonance-energy transfer-FRET), A.T., et al, Methods Immunol.Anal.1993,1,446; Youn, H.J.et al, Anal.Biochem.1995 Oswald, B.et al, Anal.Biochem.2000,280,272; Szollosi, J.et al, Cytometry 1998,34, and 159].
Other detection method is based on the change of polarization plane or detection phosphorescence.
In the aforesaid method, the anti-material antibody of use has been realized different purposes.On the one hand, they are used for separating the material that will measure from described sample, and on the other hand, and they also can realize purpose that the unlike signal mediator that is applied on the material to be detected is positioned.Be the antibody in test example such as the sample, main optics and radiometry method are established, and known acoustics (is seen for example Cooper, M.A.et al, Direct and Sensitive Detection of aHuman Virus By Rupture Event Scanning.Nat Biotechnol.2001 Sep; 19 (9): 833-7) with the magnetics measuring method.Measuring method has obtained to use the most widely [Nakamura, R.M., Dito, W.R., Tucker, E.S. (Eds.) .Immunoassays:Clinical Laboratory Techniques for the 1980s.A.R.Liss, NewYork.Edwards, R. (ed.) .Immunoassays:Essential Data, 1996, WileyEurope].
In the existing measuring method of majority, anti-material antibody is with a kind of fluorophore mark.This mark is by special finishing with non-specific chemical coupling.The antibody excess of mark adds in the sample that will study.For the material molecule in conjunction with all mensuration, this is essential.In addition, this method is used to separate the material that will measure based on a kind of anti-material antibody usually and detects the another kind of anti-material antibody signaling molecule mark that will study material at another binding site.Like this, can avoid because unconjugated, but the distortion that produces the measuring result that the antibody of signal causes.Yet owing to there is separating step, this method needs complicated methodology and technical requirements and higher cost.Require to have stoped this method to be used for diagnosis at a high speed than complicated technology, be proved to be disadvantageous especially.
Antibody and peptide at low-molecular-weight molecule are known.These also comprise at the antibody of dye molecule and peptide.Simeonov, A.et al., Science 2000,290, and 307-313 has described the antibody (" blue-fluorescence antibody ") at Stilbene.The photoisomeric change process that these antibody catalysis are special also causes the interior red shift absorption of UV-VIS spectral range and fluorescence maximum value (absorb and move maximum 12nm, fluorescence moves 22nm).Yet, the red shift during for the fluorescence quantum yield that in the 600-1200nm wavelength region, keeps cyanine dyes, Simeonov, A.et al. do not provide any relevant prompting.
Watt, (Immunochemistry 1977,14,533-541) described the spectral response curve of known anti-fluorescein antibody construct for R.M.et al..Behind the combined with fluorescent element, antibody has taken place that absorption and fluorescence are peaked to be moved in visible spectrum range, but has only 12nm or 5nm.In addition, fluorescence quantum yield sharply reduces (about 90%).
Rozinov, M.N.et al. (Chem.Biol.1998,5,713-728) selection 12 aggressiveness (12-mer) peptide from phage library, its combination dye texas Red (TexasRed), rhodamine red (Rhodamine Red), Oregon Green 514 and fluorescein have been described.For texas Red, observed the red shift of absorption and fluorescence, but had only 2.8nm or 1.4nm.
In addition, antibody at different dyes can commercially obtain, for example at antibody (the MolecularProbes Company of fluorescein, tetramethyl-rhodamine, texas Red, Alexa fluorine 488, BODIPYFL, lucifer yellow (Lucifer Yellow) and Cascade Blue, Oregon Green, Inc., USA).Yet these are the polyclone IgG antibody that is used for the bioanalysis purpose, and it has the cross reactivity of certain uncontrollable degree and it is not to produce from the chosen process of strictness.
Also need to be applicable to the radiator binding peptide of the improvement of above-mentioned measuring method, particularly specific antibody.At this moment, particularly those are in the 600-1200nm wavelength region, and the radiator binding peptide that produces red shift when keeping the fluorescence quantum yield of cyanine dyes is favourable.
The present invention of this target is by following realization, a kind of radiator binding peptide, it is characterized in that described peptide changes the spectral emissions characteristic of radiator when its antigen binding pocket and radiator interaction, a kind of method of producing radiator binding peptide of the present invention, comprise with the suitable organism of a kind of radiator immunity, comprise a kind of polymethine dyestuff that is selected from, as dicarbocyanines, three carbonyl cyanines, indigo three carbonyl cyanines (indotricarbocyanine), merocyanine, styryl, the dyestuff of squarilium and oxonol dye and rhodamine phenoxazine or thiodiphenylamine dyestuff and respective application radiator binding peptide of the present invention, nucleic acid, host cell or antibody or conjugate are used for in-vitro diagnosis as diagnostic reagent.Quoted suitable embodiment in the dependent claims.
Therefore, a first aspect of the present invention relates to a kind of radiator binding peptide, it is characterized in that described peptide changes the spectral emissions characteristic of radiator when its antigen binding pocket and radiator interaction.
Radiator binding peptide preferably of the present invention, wherein radiator comprises and a kind ofly has at least one absorb maximum value and/or fluorescence maximum value, preferably have at least one in 750 to 900nm spectral range and absorb maximum value and the peaked dyestuff of fluorescence in 700 to 1000nm spectral range.
Radiator binding peptide more preferably of the present invention, wherein the change of the emission characteristic of emitter portion is selected from polarization plane change, fluorescence intensity change, phosphorescence intensity change, fluorescence change in work-ing life and absorbs maximum value and/or the peaked red shift of fluorescence.The invention is not restricted to these spectrum phenomenons, yet term " occurrence features change " comprises all physical phenomenons or effect within the scope of the invention, wherein the high-energy radiation that takes place of radiator on its characteristic, changes and the quantitative property of this change at this moment depend on the combining of material-radiator conjugate or material-detection reagent-radiator-conjugate and its radiator binding partners and material/non-binding.In one embodiment, described material for example is peptide, protein, oligonucleotide and particularly antibody or antibody fragment.Within the scope of the invention, described antibody fragment is that those comprise the fragment that contains what is called " complementarity-determining region " antigen binding domain territory (CDR) at least.At this moment, the antigen binding domain territory most preferably comprises complete variable chains VL and VH.
In aspect of radiator binding peptide of the present invention is particularly preferred, antibody or antibody fragment are selected from polyclone or monoclonal antibody, humanized antibody, Fab fragment, particularly monomer Fab fragment, scFv fragment, synthetic and recombinant antibodies, scTCR chain and composition thereof.
Particularly preferably be when synthetic and recombinant antibodies or antibody fragment that (WO 97/08320 from those of HuCAL library; Knappik, (2000), J.Mol.Biol.296,57-86; Krebs et al.J Immunol Methods.2001 August 1; 254 (1-2): 67-84).These can be the complete immunoglobulin (Ig) or the antibody (IgA of one of natural form, IgD, IgE, IgG, IgM) or antibody fragment, wherein antibody fragment comprises 5 to 109 of VL amino acid position 4 to 103 and VH at least, 4 to 111 and the preferred complete especially variable chains VL of the amino acid position 3 to 107 of preferred VL and VH and VH (amino acid position 1 to 109 of VL and VH 1 to 113) (numbering is according to WO 97/08320).
In a preferred embodiment, antibody or antibody fragment comprise at least one CDR zone, it is included in (VL:CDR1 position 24-34 among the sequence SEQ-ID No.:1,2,5,6,9,10,13,14,17,19,21,23,25,27,29,31,33,35,37 and 39, CDR2 position 50-56, CDR3 position 89-96; VH:CDR1 position 26-35, CDR2 position 50-65, CDR3 position 95-102), particularly VL CDR3 or VH CDR3.Particularly preferably be the antibody that comprises a variable chains VL this moment, and described variable chains is included among the sequence SEQ-ID No.:2,6,10,14,17,19,21,23,25,27,29,31,33,35,37 and 39 or is included in (or fragment of this antibody) among the sequence SEQ-ID No.:1, one of 5,9 and 13.Most preferably comprise the antibody that VH/VL is right that is included in following sequence centering, described sequence is to being: SEQ-ID No.:1+2; SEQ-ID No.:5+6; SEQ-ID No.:9+10; SEQ-ID No.:13+14; SEQ-ID No.:5+17; SEQ-ID No.:5+19; SEQ-ID No.:5+37; SEQ-ID No.:9+21; SEQ-ID No.:9+23; SEQ-ID No.:9+25; SEQ-ID No.:9+27; SEQ-ID No.:9+29; SEQ-ID No.:9+31; SEQ-ID No.:9+33; SEQ-ID No.:9+39; SEQ-ID No.:13+35 (or fragment of this antibody).Particularly preferably be Fab fragment MOR02628, MOR02965, MOR02977, MOR02969, MOR03263, MOR03325, MOR03285, MOR03201, MOR03267, MOR03268, MOR03292, MOR03294, MOR03295, MOR03309, MOR03293 and MOR03291 this moment.From antibody of the present invention, the multiple possibility of antibody that obtains to illustrate the new modification of characteristic of the present invention is conspicuous for those skilled in the art.
Avidity and selectivity and specificity are responsible in the CDR zone of known particularly heavy chain of those skilled in the art and light chain.At this moment, particularly work in the CDR3 zone of the CDR3 of VH zone and VL, is the CDR2 of VH and the CDR1 of VL then, and in most cases the CDR2 of the CDR1 of VH and VL plays a secondary role.Therefore, be avidity and the selectivity and the specificity of optimizing antibody, special suggestion CDR zone (also referring to for example Schieret al., J.Mol.Biol. (1996) 263,551).At this moment, for example one or more CDR zone can for example be exchanged for the CDR zone of other other antibody that characteristic of the present invention has been shown or specifically by the library of corresponding C DR sequence exchange (seeing the optimization of embodiment 1) for this reason, and described library has produced the variation of completely random or contained more or less strong preference (trend (Tendenz)) to special amino acid or its combination.And those skilled in the art also can be exchanged for complete variable chains the corresponding chain of other clear and definite antibody or the diverse libraries of this chain in the same manner.In addition, be well known by persons skilled in the art by one or more the amino acid based method among the mutagenesis specificity exchange CDR.The evaluation of this change amino-acid residue for example is based on sequence of contrast different antibodies and identifies conservative or carry out at corresponding position height homologous residue at least at this.When changing CDR, those skilled in the art known so-called " norm structure (kanonischen Strukturen) " this moment (Al-Lazikani et al., J.Mol.Biol. (2000) 295,979); Knappik et al.J.Mol.Biol. (2000) 296,57), it influences the three-dimensional arrangement in CDR zone, therefore can consider the optimal strategy that designs.
And the skeleton district that changes antibody or antibody fragment also is that well known to those skilled in the art (WO 92/01787 to obtain technology more stable or more effable molecule; Nieba et al. (1997) Protein Eng.10,435; Ewert et al. (2003) Biochemistry 42,1517).
Except being used for the strategy of having described of modified antibodies or antibody fragment, the measuring method that the knowledge of antibody and the application describe according to the present invention, those skilled in the art also can carry out extra change to the composition of aminoacid sequence or described antibody and by using described analysis and measuring method to determine whether to produce the modified antibodies of the characteristic of its characteristic conforms antibody of the present invention.
Another aspect of the present invention relates to the nucleic acid molecule of one of coding antibody of the present invention or antibody fragment.In a preferred embodiment, this moment, these were nucleic acid molecule of one of coding variable chains VL or variable chains VH, one of described variable chains VL is included among the sequence SEQ-IDNo.:2,6,10,14,17,19,21,23,25,27,29,31,33,35,37 and 39, and described variable chains VH is included among the sequence SEQ-ID No.:1,5,9 and 13.Particularly preferably be SEQ-ID No.:3,4,7,8,11,12,15,16,18,20,22,24,26,28,30,32,34,36,38 and 40 sequence this moment.
The most desirable displaying of radiator binding peptide of the present invention is less than 50nm and preferred binding affinity less than 10nm.
Another aspect is a radiator binding peptide of the present invention, and wherein said radiator comprises and a kind ofly has at least one absorb maximum value and/or fluorescence maximum value, preferably have at least one in the spectral range of 750-900nm and absorb maximum value and the peaked dyestuff of fluorescence in the spectral range of 700-1000nm.Select the red shift of dyestuff, so that shifting to higher wavelength with the reagent interaction described absorption maximum value in back and/or the fluorescence maximum value that detect radiator, movement value is preferably greater than 25nm and reaches most preferably approximately 30nm greater than 15nm.At this moment, moving is the characteristic of dyestuff, there is no need to take in moving by this way.Usually, move the change that is measured as dyestuff emission value, promptly at certain single wavelength.For this reason, provide the suitable optical agents that is used to measure well known by persons skilled in the art.This also be applied to measure change, the phosphorescence intensity of change, the fluorescence intensity of polarization plane change, fluorescence work-ing life change and absorb maximum value and/or the peaked red shift of fluorescence.
For radiator binding peptide of the present invention, the preferred radiator that uses comprises and is selected from the polymethine dyestuff, as a kind of dyestuff of dicarbocyanines, three carbonyl cyanines, indigo three carbonyl cyanines, merocyanine, styryl, squarilium and oxonol dye and rhodamine, phenoxazine or thiodiphenylamine dyestuff.Usually, the radiator of material-radiator of the present invention-conjugate can comprise a kind of cyanine dyes of general formula (I)
Figure C20048001962100141
Wherein D represents group (II) or (III)
Figure C20048001962100151
The position that wherein indicates asterisk is represented the site that is connected with group B and can be represented group (IV), (V), (VI), (VII) or (VIII)
Figure C20048001962100152
R wherein 1And R 2, represent C respectively independently 1-C 4-sulfoalkyl chain, saturated or undersaturated, side chain or straight chain C 1-C 50-alkyl chain, it is optional by 0 to 15 Sauerstoffatom and/or by 0 to 3 carbonyl interval and/or by 0 to 5 hydroxyl replacement; R 3And R 4, represent group-COOE respectively independently 1,-CONE 1E 2,-NHCOE 1,-NHCONHE 1,-NE 1E 2,-OE 1,-OSO 3E 1,-SO 3E 1,-SO 2NHE 1Or-E 1, E wherein 1And E 2Represent hydrogen atom, C respectively independently 1-C 4-sulfoalkyl chain, saturated or undersaturated, side chain or straight chain C 1-C 50-alkyl chain, it is optional by 0 to 15 Sauerstoffatom and/or by 0 to 3 carbonyl interval and/or by 0 to 5 hydroxyl replacement, R 5Represent hydrogen atom, methyl, ethyl or propyl group or fluorine, chlorine, bromine or iodine atom, b represents numeral 2 or 3, and X and Y represent independently O, S ,=C (CH 3) 2Or-(CH=CH)-, and the salt of these compounds and solvate.
May be surprisingly found out that, antibody with near infrared spectral range (＞750nm) have and absorb and after the cyanine dyes high affinity of fluorescence combined, absorption maximum value and fluorescence maximum value had moved about 30nm (red shift) to upper wavelength.Therefore use this principle, may for example in a big concentration range, directly detect and from whole blood sample the spectral separation signal, wherein the signal about the concentration of material that will be determined is linear.
Radiator can form conjugate with material.Within the scope of the invention, what have general formula S-E is material-radiator conjugate, and wherein the S representative wants detected material and E to represent radiator, and it comprises and occurs the part that emission characteristic changes when the reagent with the detection radiator interacts.As the component of conjugate of the present invention, i.a., it is suitable having at least one absorption maximum value and the peaked dyestuff of fluorescence in the spectral range of 600-1200nm.At this moment, preferably in the spectral range of 700-1000nm, have at least one and absorb maximum value and the peaked dyestuff of fluorescence.The dyestuff that meets these conditions for example is following those: the polymethine dyestuff, as dicarbocyanines, three carbonyl cyanines, merocyanine and oxonol dye, rhodamine, phenoxazine or thiodiphenylamine dyestuff, tetrapyrrole dyestuff (tetrapyrrolfarbstoffe), especially benzene porphyrin, chlorine, bacterium chlorine (bacteriochlorine), pheophorbide, bacterium pheophorbide (bacteriopheophorbides), purpurin (purpurines) and phthalocyanine.
Preferred dyestuff be 750 and 900nm between have the peaked cyanine dyes of absorption, and indigo three carbonyl cyanines have special advantage.The structural constituent of conjugate of the present invention also is to determine the material of concentration by method of the present invention.
These are selected from for example antigen, and as protein, peptide, nucleic acid, oligonucleotide, blood constitutent, serum component, lipid, pharmaceutical preparation and low-molecular-weight compound, particularly sugar, dyestuff or other molecular weight are lower than 500 daltonian compounds.
Preferred dyestuff be 750 and 900nm between have the peaked cyanine dyes of absorption, and indigo three carbonyl cyanines have special advantage.
Dyestuff contains structural element, carries out covalent coupling by itself and the structure of matter.These are the joints that for example have carboxyl, amino and hydroxyl.
When opticmeasurement, this can carry out by different way, and depends on the type of the characteristic change of radiator (for example fluorophore) spectral response curve.Usually preferably detect moving or under major part detects wavelength with the part of the fluorophore of antibodies, measure and absorbing and/or fluorescence intensity of absorbing wavelength and emission wavelength.According to the change of the spectral response curve of antibodies fluorophore, other characteristic also can be used for opticmeasurement as photon for example work-ing life, polarization and bleaching characteristic.
The special benefit of fluorophore in the spectral range of near infrared light is the few overlapping (geringen by the blood constitutent generation
Figure C20048001962100171
).Thus, can carry out deep penetration (Tiefeneindringung
Figure C20048001962100172
), and can not make too intensive change of signal to be detected.
And, the known antibody at fluorophore that after combined with fluorescent group, can change spectral response curve in its UV scope of those skilled in the art.In the antigen binding pocket that fluorophore is combined in antibody, can mainly change fluorescence intensity, absorb maximum value, emission maximum and photon work-ing life [Simeonov, A., et al., Science (2000) 307-313].These known antibody are at radiator (fluorophore), yet it has in the visible light of light and UV scope and absorbs and fluorescent emission.
Another aspect of the present invention relates to the application that radiator binding peptide of the present invention is used for in-vitro diagnosis.
For this reason, radiator binding peptide of the present invention also may reside in the diagnostic kit, and optional and other adjuvant together.In addition, all these test kits of the present invention can contain special guidance and file (for example typical curve, quantitative guidance etc.).
The present invention is described in more detail based on embodiment and appended accompanying drawing and sequence now, but the present invention does not limit therewith.Here
Fig. 1: CysDisplay-Screening carrier pMORPH23 (carrier figure and sequence),
Fig. 2: expression vector pMORPHX9 MS (carrier figure and sequence), and
The dyestuff of Fig. 3: embodiment 2 is absorption spectrum (left side) and the fluorescence spectrum when existing and not having antibody MOR02965 in PBS.
Embodiment:
Embodiment 1: select; produce and qualitative radiator binding antibody: select at cyanine dyes Fuji 6-4 (ZK203468) [trisodium-3; 3-dimethyl-2-{4-methyl-7-[3; 3-dimethyl-5-alkylsulfonyl-1-(2-sulphonyl ethyl)-3H-indoles-2-yl] seven-2; 4; 6-triolefin-1-base subunit }-1-(2-sulphonyl ethyl)-2; 3-dihydro-1H-indoles-5-sulfonate; inner salt] ([Trisodium-3; 3-dimethyl-2-{4-methyl-7-[3; 3-dimethyl-5-sulfonato-1-(2-sulfonatoethyl)-3H-indolium-2-yl] hepta-2; 4; 6-trien-1-ylidene}-1-(2-sulfonatoethyl)-2; 3-dihydro-1H-indole-5-sulfonate, Inner Salt]) HuCAL GOLD Fab antibody fragment
HuCAL GOLD antibody library:
Antibody library HuCAL GOLD:HuCAL GOLD is modularization people's antibody library of complete synthetic, Fab antibody fragment form.(WO 97/08320 for HuCAL-consensus sequence-antibody gene that HuCAL GOLD describes based on the HuCAL-scFv1 library; Knappik, (2000), J.Mol.Biol.296,57-86; Krebs et al.J Immunol Methods.2001 Aug 1; 254 (1-2): 67-84). in HuCAL GOLD, all 6 CDR zones forming corresponding to these zones in people's antibody by use so-called trinucleotide mutagenesis (trinukleotidmutagenese) by variation ( Et al. (1994) NucleicAcids Res.1994 Dec 25; 22 (25): 5600-7), and in HuCAL library early (HuCAL-scFv1 and HuCAL-Fab1), have only among VH and the VL regional by variation (seeing Knappik et al., 2000) corresponding to the CDR3-of natural composition.And the screening method (WO 01/05950) that is called a CysDisplay modification also is found among the HuCALGOLD.The carrier pMORPH23 that is used for screening method sees Fig. 1.
2 of 1 and of V λ. primary HuCAL key-gene makes up with its believable (authentischen) N-is terminal: VL λ 1:QS (CAGAGC), VL λ 2:QS (CAGAGC) and VL λ 3:SY (AGCTAT). and these sequences see WO 97/08320.When producing the HuCAL-scFv1-library, these two amino-acid residues are changed to " DI " and clone (EcoRI site) with promotion.These residues have been retained when producing HuCAL-Fab1 and HuCAL GOLD.Therefore, the VL λ gene that has EcoRV cleavage site GATATC (DI) at 5 '-end is all contained in all HuCAL libraries.All HuCAL kappa genes (all genes in key-gene and the library) under any circumstance all contain DI at 5 '-end, because these have represented believable N-end (WO 97/08320).
1 of VH. primary HuCAL-key-gene is produced with its believable N-is terminal: VH1A, VH1B, VH2, VH4 and VH6 have Q (=CAG) amino acid based as first, VH3 and VH5 have E (=GAA).Corresponding sequence sees WO 97/08320.When clone HuCAL-Fab1 and HuCAL GOLD library, amino acid Q (CAG) is impregnated among this 1 of all VH genes.
The production of phagemid
A large amount of phagemids infects from HuCAL GOLD antibody library or from intestinal bacteria (E.coli) TOP10F ' cells produce in ripe library with concentrate by helper phage.At last, HuCAL GOLD or ripe library (in TOP10F ' cell) are cultured to OD at 37 ℃ in 2 * YT substratum of tsiklomitsin/1% glucose of the paraxin with 34 μ g/ml/10 μ g/ml 600Be 0.5.Then, infect at 37 ℃ with the VCSM13 helper phage.The cell that precipitation infects also is resuspended among 2 * YT/34 μ g/ml paraxin/10 μ g/ml tsiklomitsins/50 μ g/ml kantlex/0.25mmol IPTG and 22 ℃ of overnight incubation.With PEG from supernatant, precipitate phage 2x and by centrifugal collection (Ausubel (1998) Current Protocols inMolecular Biology.John Wiley Sons, Inc., New York, USA).Phage is resuspended in PBS/20% glycerine and is stored in-80 ℃.
Each selects following the carrying out of amplification of phagemid in the circulation: logarithmic phase e. coli tg1 cell with the phage-infect of selection and be plated on have 1% glucose/the LB-agar plate of the paraxin of 34 μ g/ml on.After being incubated overnight, scrape bacterial colony, cultivate again and infect with the VCSM13 helper phage.
Initial option is at the antibody of dyestuff Fuji 6-4 (ZK203468)
The spissated phagemid of HuCAL GOLD antibody library purifying is used for the Standard Selection method.As antigen, BSA-or transferrin-link coupled ZK203468 is used alternatingly.Antigen added among the PBS and add Maxisorp with the concentration of 50 μ g/ml TMAmong the microtiter plate F96 (Nunc).The Maxisorp flat board is incubated overnight at 4 ℃ (" bag by ").Maxisorp dull and stereotyped with the PBS sealing that contains 5% milk powder after, about 2E+13 HuCAL GOLD phage joined in (antigen-beladenen), the blind hole of antigen load and and is incubated overnight or 2 hours in room temperature.After several washing steps (washing step becomes stricter along with progressive selection circulation), the bonded phage is used 20mmol DTT or the unconjugated ZK203468 wash-out of 100 μ mol.In a word, carry out three successive and select circulation, wherein selecting to carry out the phage amplification between the circulation, as above-mentioned.
The Fab fragment that subclone is selected is used for expressing
Comprise after 3 round-robin antibody select, isolating HuCAL clone's Fab-coding inset subclone to expression vector pMORPHX9_MS (see figure 2) to promote the expression subsequently of Fab fragment.At last, plasmid-DNA of the HuCAL Fab of selection clone's purifying digests with Restriction Enzyme XbaI and EcoRI.Purifying Fab-coding inset also is connected among the carrier pMORPHX9_MS of corresponding digestion.This clone's step has produced Fab-expression vector pMORPHX9_Fab_MS.The Fab fragment of this vector expression is carried 2 C-end marks (Myc mark and Strep mark II) and is used for purifying and detection.
Screening and qualitative ZK203468-are in conjunction with the Fab fragment
The antigen ZK203468-BSA that has separated several thousand clones and subclone after the selection and in 384 orifice plates, used in for elutriation by ELISA and-specific detection of transferrin tests.Studied it among inhibition-ELISA (inhibitions-ELISA) to not puting together effective combination of dyestuff being cloned in of this evaluation.
This has produced parenteral Fab fragment MOR02628 (protein sequence SEQ-ID NO:1 (VH-CH) and SEQ-ID NO:2 (VL-CL); Dna sequence dna SEQ-ID NO:3 (VH-CH) and SEQ-ID NO:4 (VL-CL)), MOR02965 (protein sequence SEQ-ID NO:5 (VH-CH) and SEQ-ID NO:6 (VL-CL); Dna sequence dna SEQ-ID NO:7 (VH-CH) and SEQ-ID NO:8 (VL-CL)) and MOR02977 (protein sequence SEQ-IDNO:9 (VH-CH) and SEQ-ID NO:10 (VL-CL); Dna sequence dna SEQ-ID NO:11 (VH-CH) and SEQ-ID NO:12 (VL-CL)) and MOR02969 (protein sequence SEQ-ID NO:13 (VH-CH) and SEQ-ID NO:14 (VL-CL); Dna sequence dna SEQ-ID NO:15 (VH-CH) and SEQ-ID NO:16 (VL-CL)), it is effectively in conjunction with unconjugated dyestuff ZK203468.
Embodiment 2: by exchange LCDR3 zone optimizing parenteral antibody fragment
Clone LCDR3 library
Plasmid-DNA of 4 parenterals (parenteralen) clone MOR02628, MOR02965, MOR02969 and MOR02977 digests with Restriction Enzyme EcoRI and XbaI, and among complete Fab-inset subclone display carrier (display vektor) pMORPH23 that extremely corresponding enzyme is cut from expression vector pMORPHX9_MS that will produce.It is essential that this step is used for presenting the segmental gene III of Fab (GenIII) at phage surface for preparation.In another step, 4 parenteral clones (among the existing pMORPH23) digest with BpiI and SphI.At this, LCDR3 district and Clambda constant region are removed from carrier framework.The corresponding carrier-DNA fragment of separation and purification.Simultaneously, separate complementary BpiI/SphI fragment from HuCAL-Fab 2 libraries, it contains diversified LCDR3 zone (mutability with about 3E+8) and Clambda constant region (=inset-DNA).The carrier-DNA of a few μ g and compatible inset-DNA are connected with the T4-DNA-ligase enzyme with 1: 2 mol ratio and transform in electroreception attitude (elektrokompetente) TOP10F ' cell behind purifying.At this moment, each parenteral antibody has obtained the library of 5E+8 to about 1E+9 clone's size.
Combination clone MOR02628, MOR02969 and MOR02977 based on library (" Pool ").Individual treated MOR02965 library (" Lead ").As described, infect the corresponding phagemid of production by these TOP10F '-ripe library with the VCSM13 helper phage.
Maxisorp TMThe antibody of microtiter plate is selected
The purifying in " lead " and " pool " library and spissated phagemid are used for the ripe system of selection (long washing time, be purified, parenteral Fab albumen displacement) under stringent condition.As antigen, BSA-or transferrin-link coupled ZK203468 is used alternatingly.These antigens add among the PBS and add Maxisorp with the lower concentration of 100-250ng/ml TMAmong the microtiter plate F96 (Nunc).The Maxisorp flat board is incubated overnight at 4 ℃ (" bag by ").Maxisorp dull and stereotyped with the PBS sealing that contains 5% milk powder after, with about 2E+13 HuCAL GOLD phage join the antigen load, be incubated overnight or 2 hours in the blind hole and in room temperature.For improving severity, in the incubation process, add purifying Fab fragment extra 100nm or 500nm, the parenteral clone.After abundant several times (ausgedehnten) washing step, the bonded phage is used the DTT wash-out of 20mmol.In a word, carry out two successive and select circulation, wherein selecting to carry out the phage amplification between the circulation, as above-mentioned.
Antibody on Neutavidin Strip is selected
The purifying in " lead " and " pool " library be used for second kind of ripe system of selection (long washing time, Fab albumen that be purified, parenteral or free dye displacement) under the stringent condition in addition with spissated phagemid.As antigen, use the ZK203468 (perhaps having alkyl or ether joint) of biotin-conjugated.These antigens add among the PBS and with 60 or lower concentration and approximately 2E+11 phage mixing of 12ng/ml.Antigen-phage solution is incubated overnight or 2 hours in room temperature.For improving severity, in the incubation process, add extra 0.5 μ g/ml parenteral clone's purifying Fab fragment or the ZK203468 of 40nm/ml.The neutravidin strip that will contain antigen-add sealing in conjunction with the solution of phage then go up and incubation 30 minutes so that by antigenic biotinyl in conjunction with possible solid phase.Behind the washing step, the bonded phage is used the DTT wash-out of 20mmol several times.In a word, carry out two successive and select circulation, wherein selecting to carry out the phage amplification between the circulation, as above-mentioned.
The Fab fragment that subclone is selected is used for expressing
Select (" maturation ") afterwards, isolating HuCAL clone's Fab-coding inset subclone advances expression vector pMORPHX9_MS to promote expression subsequently.At last, the HuCAL Fab of selection clone's purifying treatment-DNA digests with Restriction Enzyme XbaI and EcoRI.Purifying Fab-coding inset also connects into by among the carrier pMORPHX9_MS of corresponding digestion.This clone's step has produced Fab-expression vector pMORPHX9_Fab_MS.The Fab fragment of this vector expression is carried 2 C-end marks (Myc mark and Strep mark II) and is used for purifying and detection.
Identify optimized antibody fragment
For identifying the antibody that dyestuff Fuji 6-4 is had the avidity of improvement, separating clone and in the 384-orifice plate, screening from select by ELISA.At last, ZK203468-BSA is added in the ELISA-microtiter plate.Want detected Fab fragment to add as unpurified bacterial lysate.For research except with the combining of antigen conjugate with the combining of free dye, the identical screening flat board that will have bacterial lysate and extra free dye mixes with two kinds of different concns.Because the Fab that unconjugated dyestuff causes shows not specific detection dyestuff conjugate of antibody in conjunction with the inhibition of solid phase, but free dye.At this, suppress accurate qualitative isolating clone in test and ELISA form and the Luminex device at solution, and measured its avidity Fuji 6-4.
Following clone shows the avidity of improvement than parenteral antibody:
Figure C20048001962100241
In a word, the avidity of parenteral MOR02977 may improve 140 times than MOR03267.The clone of all other evaluations improves 2-70 doubly than dividing other parenteral Fab to show.
Embodiment 3: qualitative dyestuff-antibody complex of photophysics and mensuration spectrum move/fluorescence quantum yield
Detected based on indigo three carbonyl cyanine dyes trisodiums-3; 3-dimethyl-2-{4-methyl-7-[3; 3-dimethyl-5-alkylsulfonyl-1-(2-sulphonyl ethyl)-3H-indoles-2-yl] seven-2; 4; 6-triolefin-1-base subunit }-1-(2-sulphonyl ethyl)-2; 3-dihydro-1H-indoles-5-sulfonate, the dyestuff-antibody complex of inner salt bonded antibody (seeing embodiment 1 and 2).Be prepared in concentration among the PBS and be the above-mentioned dyestuff of 1 μ mol/l and 2.4 μ mol/l antibody separately solution and room temperature incubation 2 hours.(Perkin-Elmer Lambda2) measures the absorption maximum value with spectrial photometer.With SPEX fluorolog (by lamp and detector the susceptibility that relies on wavelength is calibrated,
Figure C20048001962100242
Empfindlichkeit von Lampe und Detektor kalibriert) measures with respect to Fox Green (Q=0.13 among the DMSO, J Chem Eng Data 1977,22,379, Bioconjugate Chem2001,12,44) fluorescence maximum value and fluorescence quantum yield.According to absorbing and the fluorescence maximum value, calculating spectrum with respect to above-mentioned dye solution when the antibody (1 μ mol/l) that does not have in PBS moves (absorbing maximum value 754nm, fluorescence maximum value 783nm, fluorescence quantum yield 10%).
Figure C20048001962100251
The result is summarised in the following table:
Antibody Parenteral antibody Absorb maximum value (nm) Fluorescence maximum value (nm) Absorb and move (nm) Fluorescence moves (nm) Fluorescence quantum yield (%)
Free dye - 754 783 - - 10.0
MOR02965 - 799 815 45 32 13.0
MOR03263 MOR02965 798 810 44 27 10.0
MOR03325 MOR02965 803 819 49 36 18.0
MOR02977 - 773 788 19 5 24.5
MOR03201 MOR02977 786 804 32 21 21.0
MOR03267 MOR02977 783 802 29 19 38.5
MOR03268 MOR02977 786 805 32 22 30.0
MOR03292 MOR02977 781 800 27 17 25.0
MOR03294 MOR02977 783 803 29 20 22.0
MOR03295 MOR02977 784 803 30 20 23.0
MOR03309 MOR02977 784 803 30 20 21.5
Embodiment 4: make up the expression vector that is used to express the HuCAL immunoglobulin (Ig)
Clone's heavy chain: " multiple clone site " of removing carrier pCDNA3.1+ (Invitrogen) (NheI/ApaI), and the platzhalter compatible with the restricted cleavage site of HuCAL design is used to be connected leader sequence (NheI/EcoRI), the segmental VH structural domain of Fab (MunI/) and constant region for immunoglobulin (BlpI/ApaI).Leader sequence (EMBL 83133) has a Kozak sequence (Kozak, 1987).The constant region of human IgG (PIR J00228), IgG4 (EMBL K01316) and serum-IgA1 (EMBL J00220) is divided into and has length and be the overlapping oligonucleotide of 70 bases for example." silent mutation " importing is designed inconsistent restricted cleavage site to remove with HuCAL.Oligonucleotide connects by " overlapping extension-PCR ".
In the process of IgG molecule, the segmental heavy chain of Fab cuts and is connected in the carrier of using the EcoRI/BlpI opening by MfeI/BlpI in subclone Fab fragment.EcoRI (g/aattc) and MfeI (c/aattg) have two compatible sticky ends (aatt), and the sequence of the original MfeI cleavage site in the Fab fragment in being connected to the IgG expression vector time from: c/aattg changes into g/aattg, thus, MfeI cleavage site and EcoRI cleavage site are all destroyed on the one hand, and on the other hand, amino acid has taken place from Q (codon: caa) to E (codon: change gaa).
Clone's light chain. replace pCDNA3.1/Zeo+ (Invitrogen) " multiple clone site " by two different platzhalter.K-platzhalter contains the restricted cleavage site that is useful on the constant region (BsiWI/ApaI) of mixing k-leader sequence (NheI/EcoRV), HuCAL Fab Vk structural domain (EcoRV/BsiWI) and k-chain.Corresponding cleavage site is NheI/EcoRV (1-leader sequence), EcoRV/HpaI (V1-structural domain) and HpaI/ApaI (constant region 1-chain) among the l-platzhalter.K-leader sequence (EMBL Z00022) and 1-leader sequence (EMBL J00241) are all provided by the Kozak sequence.The constant region of people k-(EMBL L00241) and 1-chain (EMBL M18645) is all passed through " overlapping extension (overlap extension)-PCR " assembling, as above-mentioned.
Producing IgG-expresses Chinese hamster ovary celI .CHO-K1 cell and waits the molar mixture cotransfection with heavy and light IgG chain expression vector.Select dual resistance transfectant with the G418 of 600mg/ml and the Zeocin of 300mg/ml (Invitrogen).The IgG that detects in each clone's supernatant by " catching ELISA (capture-ELISA) " expresses.Positive colony is cultivated in the RPMI-1640 substratum with 10% " ultra-low IgG-FCS " (Life Technologies).The pH of supernatant be after 8.0 and sterile filtration after, solution is carried out A albumen-column chromatography (Standard-Protein A-of standard ) (Poros 20 A, PEBiosystems).
Sequence table
＜110〉Schering AG
Mo Fuxisi stock company
＜120〉make the mutagenic radiator binding peptide of spectral emissions characteristic of radiator
<130>
<150>DE 103 31 054.1
<151>2003-07-09
<150>US60/487,234
<151>2003-07-16
<160>40
<170>PatentIn version 3.2
<210>1
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<223>Fab fragment MOR02628 VH-CH
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Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn
20 25 30
Ser Ala Ala Trp Ser Trp Ile Arg Gln Ser Pro Gly Arg Gly Leu Glu
35 40 45
Trp Leu Gly Arg Ile Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala
50 55 60
Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn
65 70 75 80
Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Ala Arg Thr Ser Phe Tyr Gln Lys Leu Phe Phe Ile Ala
100 105 110
Phe Asp His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
115 120 125
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
130 135 140
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
145 150 155 160
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
165 170 175
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
180 185 190
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
195 200 205
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
210 215 220
Glu Pro Lys Ser Glu Phe Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
225 230 235 240
Asn Gly Ala Pro Trp Ser His Pro Gln Phe Glu Lys
245 250
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<223>Fab fragment MOR02628 VL-CL
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Asp Ile Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Gly Gly Ala Tyr
20 25 30
His Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Met Ile Tyr Gly Val Ile Tyr Arg Pro Ser Gly Val Ser Asn Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Phe Arg
85 90 95
Lys Arg Leu Asn Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110
Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu
115 120 125
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
130 135 140
Tyr Pro Gly Ala Val Thr Val Ala Trp Lys ALa Asp Ser Ser Pro Val
145 150 155 160
Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
165 170 175
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
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His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
195 200 205
Lys Thr Val Ala Pro Thr Glu Ala
210 215
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<223>DNA coding for Fab fragment MOR02628 VH-CH
<400>3
caggtgcaat tgcaacagtc tggtccgggc ctggtgaaac cgagccaaac cctgagcctg 60
acctgtgcga tttccggaga tagcgtgagc tctaattctg ctgcttggtc ttggattcgc 120
cagtctcctg ggcgtggcct cgagtggctg ggccgtatct attatcgtag caagtggtat 180
aacgattatg cggtgagcgt gaaaagccgg attaccatca acccggatac ttcgaaaaac 240
cagtttagcc tgcaactgaa cagcgtgacc ccggaagata cggccgtgta ttattgcgcg 300
cgtacttctt tttatcagaa gctttttttt attgcttttg atcattgggg ccaaggcacc 360
ctggtgacgg ttagctcagc gtcgaccaaa ggtccaagcg tgtttccgct ggctccgagc 420
agcaaaagca ccagcggcgg cacggctgcc ctgggctgcc tggttaaaga ttatttcccg 480
gaaccagtca ccgtgagctg gaacagcggg gcgctgacca gcggcgtgca tacctttccg 540
gcggtgctgc aaagcagcgg cctgtatagc ctgagcagcg ttgtgaccgt gccgagcagc 600
agcttaggca ctcagaccta tatttgcaac gtgaaccata aaccgagcaa caccaaagtg 660
gataaaaaag tggaaccgaa aagcgaattc gagcagaagc tgatctctga ggaggatctg 720
aacggcgcgc cgtggagcca cccgcagttt gaaaaatgat aa 762
<210>4
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<220>
<223>DNA coding for Fab fragment MOR02628 VL-CL
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gatatcgcac tgacccagcc agcttcagtg agcggctcac caggtcagag cattaccatc 60
tcgtgtacgg gtactagcag cgatggtggt gcttatcatt atgtgtcttg gtaccagcag 120
catcccggga aggcgccgaa acttatgatt tatggtgtta tttatcgtcc ctcaggcgtg 180
agcaaccgtt ttagcggatc caaaagcggc aacaccgcga gcctgaccat tagcggcctg 240
caagcggaag acgaagcgga ttattattgc gctgcttggg attttcgtaa gcgtcttaat 300
gtgtttggcg gcggcacgaa gttaaccgtt cttggccagc cgaaagccgc accgagtgtg 360
acgctgtttc cgccgagcag cgaagaattg caggcgaaca aagcgaccct ggtgtgcctg 420
attagcgact tttatccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480
aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540
agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600
acgcatgagg ggagcaccgt ggaaaaaacc gttgcgccga ctgaggcctg ataa 654
<210>5
<211>245
<212>PRT
<213>Artificial
<220>
<223>Fab fragment MOR02965 VH-CH
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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asn Ser Tyr
20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile His Pro Tyr Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Trp Arg Tyr Arg Trp Gly Phe Asp Ile Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Glu Phe Glu
210 215 220
Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala Pro Trp Ser His
225 230 235 240
Pro Gln Phe Glu Lys
245
<210>6
<211>214
<212>PRT
<213>Artificial
<220>
<223>Fab fragment MOR02965 VL-CL
<400>6
Asp Ile Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Tyr
20 25 30
Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Gly Asn Ser Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Trp Asp Ser Ser Phe
85 90 95
Ser Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro
100 105 110
Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu
115 120 125
Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro
130 135 140
Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala
145 150 155 160
Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala
165 170 175
Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg
180 185 190
Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr
195 200 205
Val Ala Pro Thr Glu Ala
210
<210>7
<211>741
<212>DNA
<213>Artificial
<220>
<223>DNA coding for Fab fragment MOR02965 VH-CH
<400>7
caggtgcaat tggttcagtc tggcgcggaa gtgaaaaaac cgggcagcag cgtgaaagtg 60
agctgcaaag cctccggagg cacttttaat tcttatgcta tttcttgggt gcgccaagcc 120
cctgggcagg gtctcgagtg gatgggcggt atccatccgt attttggcac tgcgaattac 180
gcgcagaagt ttcagggccg ggtgaccatt accgcggatg aaagcaccag caccgcgtat 240
atggaactga gcagcctgcg tagcgaagat acggccgtgt attattgcgc gcgtcgttgg 300
cgttatcgtt ggggttttga tatttggggc caaggcaccc tggtgacggt tagctcagcg 360
tcgaccaaag gtccaagcgt gtttccgctg gctccgagca gcaaaagcac cagcggcggc 420
acggctgccc tgggctgcct ggttaaagat tatttcccgg aaccagtcac cgtgagctgg 480
aacagcgggg cgctgaccag cggcgtgcat acctttccgg cggtgctgca aagcagcggc 540
ctgtatagcc tgagcagcgt tgtgaccgtg ccgagcagca gcttaggcac tcagacctat 600
atttgcaacg tgaaccataa accgagcaac accaaagtgg ataaaaaagt ggaaccgaaa 660
agcgaattcg agcagaagct gatctctgag gaggatctga acggcgcgcc gtggagccac 720
ccgcagtttg aaaaatgata a 741
<210>8
<211>648
<212>DNA
<213>Artificial
<220>
<223>DNA coding for Fab fragment MOR02965 VL-CL
<400>8
gatatcgtgc tgacccagcc gccttcagtg agtggcgcac caggtcagcg tgtgaccatc 60
tcgtgtagcg gcagcagcag caacattggt tcttattatg tgtattggta ccagcagttg 120
cccgggacgg cgccgaaact tctgatttat ggtaattctc agcgtccctc aggcgtgccg 180
gatcgtttta gcggatccaa aagcggcacc agcgcgagcc ttgcgattac gggcctgcaa 240
agcgaagacg aagcggatta ttattgctct tcttgggatt cttctttttc ttgggtgttt 300
ggcggcggca cgaagttaac cgttcttggc cagccgaaag ccgcaccgag tgtgacgctg 360
tttccgccga gcagcgaaga attgcaggcg aacaaagcga ccctggtgtg cctgattagc 420
gacttttatc cgggagccgt gacagtggcc tggaaggcag atagcagccc cgtcaaggcg 480
ggagtggaga ccaccacacc ctccaaacaa agcaacaaca agtacgcggc cagcagctat 540
ctgagcctga cgcctgagca gtggaagtcc cacagaagct acagctgcca ggtcacgcat 600
gaggggagca ccgtggaaaa aaccgttgcg ccgactgagg cctgataa 648
<210>9
<211>246
<212>PRT
<213>Artificial
<220>
<223>Fab fragment MOR02977 VH-CH
<400>9
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr
20 25 30
Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Asn Ile Glu Pro Tyr Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Phe Met Ser Tyr Lys His Leu Ser Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Glu Phe
210 215 220
Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala Pro Trp Ser
225 230 235 240
His Pro Gln Phe Glu Lys
245
<210>10
<211>216
<212>PRT
<213>Artificial
<220>
<223>Fab fragment MOR02977 VL-CL
<400>10
Asp Ile Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Asn
20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Met Ile Tyr Gly Gly Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Thr Thr Arg
85 90 95
Pro Leu Asn Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110
Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu
115 120 125
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
130 135 140
Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val
145 150 155 160
Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
165 170 175
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
180 185 190
His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
195 200 205
Lys Thr Val Ala Pro Thr Glu Ala
210 215
<210>11
<211>744
<212>DNA
<213>Artificial
<220>
<223>DNA coding for Fab fragment MOR02977 VH-CH
<400>11
caggtgcaat tggttcagtc tggcgcggaa gtgaaaaaac cgggcagcag cgtgaaagtg 60
agctgcaaag cctccggagg cactttttct aattatgcta ttaattgggt gcgccaagcc 120
cctgggcagg gtctcgagtg gatgggcaat atcgagccgt attttggcac tgcgaattac 180
gcgcagaagt ttcagggccg ggtgaccatt accgcggatg aaagcaccag caccgcgtat 240
atggaactga gcagcctgcg tagcgaagat acggccgtgt attattgcgc gcgttatttt 300
atgtcttata agcatctttc tgattattgg ggccaaggca ccctggtgac ggttagctca 360
gcgtcgacca aaggtccaag cgtgtttccg ctggctccga gcagcaaaag caccagcggc 420
ggcacggctg ccctgggctg cctggttaaa gattatttcc cggaaccagt caccgtgagc 480
tggaacagcg gggcgctgac cagcggcgtg catacctttc cggcggtgct gcaaagcagc 540
ggcctgtata gcctgagcag cgttgtgacc gtgccgagca gcagcttagg cactcagacc 600
tatatttgca acgtgaacca taaaccgagc aacaccaaag tggataaaaa agtggaaccg 660
aaaagcgaat tcgagcagaa gctgatctct gaggaggatc tgaacggcgc gccgtggagc 720
cacccgcagt ttgaaaaatg ataa 744
<210>12
<211>654
<212>DNA
<213>Artificial
<220>
<223>DNA coding for Fab fragment MOR02977 VL-CL
<400>12
gatatcgcac tgacccagcc agcttcagtg agcggctcac caggtcagag cattaccatc 60
tcgtgtacgg gtactagcag cgatgttggt tctaataatt atgtgtcttg gtaccagcag 120
catcccggga aggcgccgaa acttatgatt tatggtggtt ctaatcgtcc ctcaggcgtg 180
agcaaccgtt ttagcggatc caaaagcggc aacaccgcga gcctgaccat tagcggcctg 240
caagcggaag acgaagcgga ttattattgc cagtcttgga ctactcgtcc tcttaatcgt 300
gtgtttggcg gcggcacgaa gttaaccgtt cttggccagc cgaaagccgc accgagtgtg 360
acgctgtttc cgccgagcag cgaagaattg caggcgaaca aagcgaccct ggtgtgcctg 420
attagcgact tttatccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480
aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540
agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600
acgcatgagg ggagcaccgt ggaaaaaacc gttgcgccga ctgaggcctg ataa 654
<210>13
<211>241
<212>PRT
<213>Artificial
<220>
<223>Fab fragment MOR02969 VH-CH
<400>13
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp His
20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Asn Ile Ser Gly Ser Ser Ser Asn Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Tyr Gly Met Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Lys Val Glu Pro Lys Ser Glu Phe Glu Gln Lys Leu Ile
210 215 220
Ser Glu Glu Asp Leu Asn Gly Ala Pro Trp Ser His Pro Gln Phe Glu
225 230 235 240
Lys
<210>14
<211>213
<212>PRT
<213>Artificial
<220>
<223>Fab fragment MOR02969 VL-CL
<400>14
Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln
1 5 10 15
Thr Ala Arg Ile Ser Cys Ser Gly Asp Ser Ile Arg Ser Lys Tyr Val
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr
35 40 45
Arg Asp Asn Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Tyr Arg Val Gly Gly
85 90 95
Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys
100 105 110
Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln
115 120 125
Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly
130 135 140
Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly
145 150 155 160
Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala
165 170 175
Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser
180 185 190
Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val
195 200 205
Ala Pro Thr Glu Ala
210
<210>15
<211>729
<212>DNA
<213>Artificial
<220>
<223>DNA coding for Fab fragment MOR02969 VH-CH
<400>15
caggtgcaat tggtggaaag cggcggcggc ctggtgcaac cgggcggcag cctgcgtctg 60
agctgcgcgg cctccggatt taccttttct gatcattgga tgtcttgggt gcgccaagcc 120
cctgggaagg gtctcgagtg ggtgagcaat atctctggtt cttctagcaa taccaattat 180
gcggatagcg tgaaaggccg ttttaccatt tcacgtgata attcgaaaaa caccctgtat 240
ctgcaaatga acagcctgcg tgcggaagat acggccgtgt attattgcgc gcgtggttat 300
ggtatggctt attggggcca aggcaccctg gtgacggtta gctcagcgtc gaccaaaggt 360
ccaagcgtgt ttccgctggc tccgagcagc aaaagcacca gcggcggcac ggctgccctg 420
ggctgcctgg ttaaagatta tttcccggaa ccagtcaccg tgagctggaa cagcggggcg 480
ctgaccagcg gcgtgcatac ctttccggcg gtgctgcaaa gcagcggcct gtatagcctg 540
agcagcgttg tgaccgtgcc gagcagcagc ttaggcactc agacctatat ttgcaacgtg 600
aaccataaac cgagcaacac caaagtggat aaaaaagtgg aaccgaaaag cgaattcgag 660
cagaagctga tctctgagga ggatctgaac ggcgcgccgt ggagccaccc gcagtttgaa 720
aaatgataa 729
<210>16
<211>645
<212>DNA
<213>Artificial
<220>
<223>DNA coding for Fab fragment MOR02969 VL-CL
<400>16
gatatcgaac tgacccagcc gccttcagtg agcgttgcac caggtcagac cgcgcgtatc 60
tcgtgtagcg gcgattctat tcgttctaag tatgttcatt ggtaccagca gaaacccggg 120
caggcgccag ttcttgtgat ttatcgtgat aataatcgtc cctcaggcat cccggaacgc 180
tttagcggat ccaacagcgg caacaccgcg accctgacca ttagcggcac tcaggcggaa 240
gacgaagcgg attattattg ctcttcttat acttataggg ttggtggtat ggtgtttggc 300
ggcggcacga agttaaccgt tcttggccag ccgaaagccg caccgagtgt gacgctgttt 360
ccgccgagca gcgaagaatt gcaggcgaac aaagcgaccc tggtgtgcct gattagcgac 420
ttttatccgg gagccgtgac agtggcctgg aaggcagata gcagccccgt caaggcggga 480
gtggagacca ccacaccctc caaacaaagc aacaacaagt acgcggccag cagctatctg 540
agcctgacgc ctgagcagtg gaagtcccac agaagctaca gctgccaggt cacgcatgag 600
gggagcaccg tggaaaaaac cgttgcgccg actgaggcct gataa 645
<210>17
<211>214
<212>PRT
<213>Artificial
<220>
<223>Fab fragment MOR03263 VL-CL
<400>17
Asp Ile Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Tyr
20 25 30
Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Gly Asn Ser Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Trp Asp Val Ser Leu
85 90 95
Glu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro
100 105 110
Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu
115 120 125
Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro
130 135 140
Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala
145 150 155 160
Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala
165 170 175
Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg
180 185 190
Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr
195 200 205
Val Ala Pro Thr Glu Ala
210
<210>18
<211>648
<212>DNA
<213>Artificial
<220>
<223>DNA coding for Fab fragment MOR03263 VL-CL
<400>18
gatatcgtgc tgacccagcc gccttcagtg agtggcgcac caggtcagcg tgtgaccatc 60
tcgtgtagcg gcagcagcag caacattggt tcttattatg tgtattggta ccagcagttg 120
cccgggacgg cgccgaaact tctgatttat ggtaattctc agcgtccctc aggcgtgccg 180
gatcgtttta gcggatccaa aagcggcacc agcgcgagcc ttgcgattac gggcctgcaa 240
agcgaagacg aagcggatta ttattgctct tcttgggatg tttctcttga gtgggtgttt 300
ggcggcggca cgaagttaac cgttcttggc cagccgaaag ccgcaccgag tgtgacgctg 360
tttccgccga gcagcgaaga attgcaggcg aacaaagcga ccctggtgtg cctgattagc 420
gacttttatc cgggagccgt gacagtggcc tggaaggcag atagcagccc cgtcaaggcg 480
ggagtggaga ccaccacacc ctccaaacaa agcaacaaca agtacgcggc cagcagctat 540
ctgagcctga cgcctgagca gtggaagtcc cacagaagct acagctgcca ggtcacgcat 600
gaggggagca ccgtggaaaa aaccgttgcg ccgactgagg cctgataa 648
<210>19
<211>214
<212>PRT
<213>Artificial
<220>
<223>Fab fragment MOR03325 VL-CL
<400>19
Asp Ile Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Tyr
20 25 30
Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Gly Asn Ser Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Lys Ser Leu
85 90 95
Gln Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro
100 105 110
Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu
115 120 125
Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro
130 135 140
Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala
145 150 155 160
Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala
165 170 175
Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg
180 185 190
Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr
195 200 205
Val Ala Pro Thr Glu Ala
210
<210>20
<211>648
<212>DNA
<213>Artificial
<220>
<223>DNA coding for Fab fragment MOR03325 VL-CL
<400>20
gatatcgtgc tgacccagcc gccttcagtg agtggcgcac caggtcagcg tgtgaccatc 60
tcgtgtagcg gcagcagcag caacattggt tcttattatg tgtattggta ccagcagttg 120
cccgggacgg cgccgaaact tctgatttat ggtaattctc agcgtccctc aggcgtgccg 180
gatcgtttta gcggatccaa aagcggcacc agcgcgagcc ttgcgattac gggcctgcaa 240
agcgaagacg aagcggatta ttattgcgct tcttgggata agtctcttca gtgggtgttt 300
ggcggcggca cgaagttaac cgttcttggc cagccgaaag ccgcaccgag tgtgacgctg 360
tttccgccga gcagcgaaga attgcaggcg aacaaagcga ccctggtgtg cctgattagc 420
gacttttatc cgggagccgt gacagtggcc tggaaggcag atagcagccc cgtcaaggcg 480
ggagtggaga ccaccacacc ctccaaacaa agcaacaaca agtacgcggc cagcagctat 540
ctgagcctga cgcctgagca gtggaagtcc cacagaagct acagctgcca ggtcacgcat 600
gaggggagca ccgtggaaaa aaccgttgcg ccgactgagg cctgataa 648
<210>21
<211>216
<212>PRT
<213>Artificial
<220>
<223>Fab fragment MOR03201 VL-CL
<400>21
Asp Ile Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Asn
20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Met Ile Tyr Gly Gly Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Trp Thr Ser Tyr
85 90 95
Phe His Ile Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110
Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu
115 120 125
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
130 135 140
Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val
145 150 155 160
Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
165 170 175
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
180 185 190
His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
195 200 205
Lys Thr Val Ala Pro Thr Glu Ala
210 215
<210>22
<211>654
<212>DNA
<213>Artificial
<220>
<223>DNA coding for Fab fragment MOR03201 VL-CL
<400>22
gatatcgcac tgacccagcc agcttcagtg agcggctcac caggtcagag cattaccatc 60
tcgtgtacgg gtactagcag cgatgttggt tctaataatt atgtgtcttg gtaccagcag 120
catcccggga aggcgccgaa acttatgatt tatggtggtt ctaatcgtcc ctcaggcgtg 180
agcaaccgtt ttagcggatc caaaagcggc aacaccgcga gcctgaccat tagcggcctg 240
caagcggaag acgaagcgga ttattattgc tcttcttgga cttcttattt tcatattcgt 300
gtgtttggcg gcggcacgaa gttaaccgtt cttggccagc cgaaagccgc accgagtgtg 360
acgctgtttc cgccgagcag cgaagaattg caggcgaaca aagcgaccct ggtgtgcctg 420
attagcgact tttatccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480
aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540
agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600
acgcatgagg ggagcaccgt ggaaaaaacc gttgcgccga ctgaggcctg ataa 654
<210>23
<211>216
<212>PRT
<213>Artificial
<220>
<223>Fab fragment MOR03267 VL-CL
<400>23
Asp Ile Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Asn
20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Met Ile Tyr Gly Gly Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Asn
85 90 95
Phe Lys Asn Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110
Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu
115 120 125
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
130 135 140
Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val
145 150 155 160
Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
165 170 175
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
180 185 190
His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
195 200 205
Lys Thr Val Ala Pro Thr Glu Ala
210 215
<210>24
<211>654
<212>DNA
<213>Artificial
<220>
<223>DNA coding for Fab fragment MOR03267 VL-CL
<400>24
gatatcgcac tgacccagcc agcttcagtg agcggctcac caggtcagag cattaccatc 60
tcgtgtacgg gtactagcag cgatgttggt tctaataatt atgtgtcttg gtaccagcag 120
catcccggga aggcgccgaa acttatgatt tatggtggtt ctaatcgtcc ctcaggcgtg 180
agcaaccgtt ttagcggatc caaaagcggc aacaccgcga gcctgaccat tagcggcctg 240
caagcggaag acgaagcgga ttattattgc caggcttggg attctaattt taagaatcgt 300
gtgtttggcg gcggcacgaa gttaaccgtt cttggccagc cgaaagccgc accgagtgtg 360
acgctgtttc cgccgagcag cgaagaattg caggcgaaca aagcgaccct ggtgtgcctg 420
attagcgact tttatccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480
aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540
agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600
acgcatgagg ggagcaccgt ggaaaaaacc gttgcgccga ctgaggcctg ataa 654
<210>25
<211>216
<212>PRT
<213>Artificial
<220>
<223>Fab fragment MOR03268 VL-CL
<400>25
Asp Ile Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Asn
20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Met Ile Tyr Gly Gly Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Arg Ser Trp Asp Ser Asn
85 90 95
Leu Ser Tyr Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110
Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu
115 120 125
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
130 135 140
Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val
145 150 155 160
Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
165 170 175
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
180 185 190
His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
195 200 205
Lys Thr Val Ala Pro Thr Glu Ala
210 215
<210>26
<211>654
<212>DNA
<213>Artificial
<220>
<223>DNA coding for Fab fragment MOR03268 VL-CL
<400>26
gatatcgcac tgacccagcc agcttcagtg agcggctcac caggtcagag cattaccatc 60
tcgtgtacgg gtactagcag cgatgttggt tctaataatt atgtgtcttg gtaccagcag 120
catcccggga aggcgccgaa acttatgatt tatggtggtt ctaatcgtcc ctcaggcgtg 180
agcaaccgtt ttagcggatc caaaagcggc aacaccgcga gcctgaccat tagcggcctg 240
caagcggaag acgaagcgga ttattattgc cgttcttggg attctaatct ttcttattct 300
gtgtttggcg gcggcacgaa gttaaccgtt cttggccagc cgaaagccgc accgagtgtg 360
acgctgtttc cgccgagcag cgaagaattg caggcgaaca aagcgaccct ggtgtgcctg 420
attagcgact tttatccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480
aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540
agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600
acgcatgagg ggagcaccgt ggaaaaaacc gttgcgccga ctgaggcctg ataa 654
<210>27
<211>216
<212>PRT
<213>Artificial
<220>
<223>Fab fragment MOR03292 VL-CL
<400>27
Asp Ile Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Asn
20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Met Ile Tyr Gly Gly Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Ala Pro Leu
85 90 95
Phe Lys Met Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110
Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu
115 120 125
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
130 135 140
Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val
145 150 155 160
Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
165 170 175
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
180 185 190
His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
195 200 205
Lys Thr Val Ala Pro Thr Glu Ala
210 215
<210>28
<211>654
<212>DNA
<213>Artificial
<220>
<223>DNA coding for Fab fragment MOR03292 VL-CL
<400>28
gatatcgcac tgacccagcc agcttcagtg agcggctcac caggtcagag cattaccatc 60
tcgtgtacgg gtactagcag cgatgttggt tctaataatt atgtgtcttg gtaccagcag 120
catcccggga aggcgccgaa acttatgatt tatggtggtt ctaatcgtcc ctcaggcgtg 180
agcaaccgtt ttagcggatc caaaagcggc aacaccgcga gcctgaccat tagcggcctg 240
caagcggaag acgaagcgga ttattattgc cagtcttggg ctcctctttt taagatgcgt 300
gtgtttggcg gcggcacgaa gttaaccgtt cttggccagc cgaaagccgc accgagtgtg 360
acgctgtttc cgccgagcag cgaagaattg caggcgaaca aagcgaccct ggtgtgcctg 420
attagcgact tttatccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480
aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540
agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600
acgcatgagg ggagcaccgt ggaaaaaacc gttgcgccga ctgaggcctg ataa 654
<210>29
<211>216
<212>PRT
<213>Artificial
<220>
<223>Fab fragment MOR03294 VL-CL
<400>29
Asp Ile Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Asn
20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Met Ile Tyr Gly Gly Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Trp Thr Ser Ser
85 90 95
Phe Ser Ser Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110
Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu
115 120 125
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
130 135 140
Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val
145 150 155 160
Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
165 170 175
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
180 185 190
His Arg Ser Tyr Ser cys Gln Val Thr His Glu Gly Ser Thr Val Glu
195 200 205
Lys Thr Val Ala Pro Thr Glu Ala
210 215
<210>30
<211>654
<212>DNA
<213>Artificial
<220>
<223>DNA coding for Fab fragment MOR03294 VL-CL
<400>30
gatatcgcac tgacccagcc agcttcagtg agcggctcac caggtcagag cattaccatc 60
tcgtgtacgg gtactagcag cgatgttggt tctaataatt atgtgtcttg gtaccagcag 120
catcccggga aggcgccgaa acttatgatt tatggtggtt ctaatcgtcc ctcaggcgtg 180
agcaaccgtt ttagcggatc caaaagcggc aacaccgcga gcctgaccat tagcggcctg 240
caagcggaag acgaagcgga ttattattgc cagacttgga cttcttcttt ttcttctcgt 300
gtgtttggcg gcggcacgaa gttaaccgtt cttggccagc cgaaagccgc accgagtgtg 360
acgctgtttc cgccgagcag cgaagaattg caggcgaaca aagcgaccct ggtgtgcctg 420
attagcgact tttatccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480
aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540
agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600
acgcatgagg ggagcaccgt ggaaaaaacc gttgcgccga ctgaggcctg ataa 654
<210>31
<211>216
<212>PRT
<213>Artificial
<220>
<223>Fab fragment MOR03295 VL-CL
<400>31
Asp Ile Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Asn
20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Met Ile Tyr Gly Gly Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Asp Ser Ala
85 90 95
Leu Ser Asn Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110
Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu
115 120 125
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
130 135 140
Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val
145 150 155 160
Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
165 170 175
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
180 185 190
His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
195 200 205
Lys Thr Val Ala Pro Thr Glu Ala
210 215
<210>32
<211>654
<212>DNA
<213>Artificial
<220>
<223>DNA coding for Fab fragment MOR03295 VL-CL
<400>32
gatatcgcac tgacccagcc agcttcagtg agcggctcac caggtcagag cattaccatc 60
tcgtgtacgg gtactagcag cgatgttggt tctaataatt atgtgtcttg gtaccagcag 120
catcccggga aggcgccgaa acttatgatt tatggtggtt ctaatcgtcc ctcaggcgtg 180
agcaaccgtt ttagcggatc caaaagcggc aacaccgcga gcctgaccat tagcggcctg 240
caagcggaag acgaagcgga ttattattgc cagtcttggg attctgctct ttctaatcgt 300
gtgtttggcg gcggcacgaa gttaaccgtt cttggccagc cgaaagccgc accgagtgtg 360
acgctgtttc cgccgagcag cgaagaattg caggcgaaca aagcgaccct ggtgtgcctg 420
attagcgact tttatccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480
aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540
agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600
acgcatgagg ggagcaccgt ggaaaaaacc gttgcgccga ctgaggcctg ataa 654
<210>33
<211>216
<212>PRT
<213>Artificial
<220>
<223>Fab fragment MOR03309 VL-CL
<400>33
Asp Ile Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Asn
20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Met Ile Tyr Gly Gly Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Trp Asp His Gly
85 90 95
Phe Thr His Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110
Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu
115 120 125
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
130 135 140
Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val
145 150 155 160
Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
165 170 175
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
180 185 190
His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
195 200 205
Lys Thr Val Ala Pro Thr Glu Ala
210 215
<210>34
<211>654
<212>DNA
<213>Artificial
<220>
<223>DNA coding for Fab fragment MOR03309 VL-CL
<400>34
gatatcgcac tgacccagcc agcttcagtg agcggctcac caggtcagag cattaccatc 60
tcgtgtacgg gtactagcag cgatgttggt tctaataatt atgtgtcttg gtaccagcag 120
catcccggga aggcgccgaa acttatgatt tatggtggtt ctaatcgtcc ctcaggcgtg 180
agcaaccgtt ttagcggatc caaaagcggc aacaccgcga gcctgaccat tagcggcctg 240
caagcggaag acgaagcgga ttattattgc cagacttggg atcatggttt tactcatcgt 300
gtgtttggcg gcggcacgaa gttaaccgtt cttggccagc cgaaagccgc accgagtgtg 360
acgctgtttc cgccgagcag cgaagaattg caggcgaaca aagcgaccct ggtgtgcctg 420
attagcgact tttatccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480
aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540
agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600
acgcatgagg ggagcaccgt ggaaaaaacc gttgcgccga ctgaggcctg ataa 654
<210>35
<211>213
<212>PRT
<213>Artificial
<220>
<223>Fab fragment MOR03291 VL-CL
<400>35
Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln
1 5 10 15
Thr Ala Arg Ile Ser Cys Ser Gly Asp Ser Ile Arg Ser Lys Tyr Val
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr
35 40 45
Arg Asp Asn Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu ThrIle Ser Gly Thr Gln Ala Glu
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Tyr Asp Tyr Lys Ser Lys Asn
85 90 95
Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys
100 105 110
Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln
115 120 125
Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly
130 135 140
Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly
145 150 155 160
Val Glu Thr Thr Thr ProSer Lys Gln Ser Asn Asn Lys Tyr Ala Ala
165 170 175
Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser
180 185 190
Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val
195 200 205
Ala Pro Thr Glu Ala
210
<210>36
<211>645
<212>DNA
<213>Artificial
<220>
<223>DNA coding for Fab fragment MOR03291 VL-CL
<400>36
gatatcgaac tgacccagcc gccttcagtg agcgttgcac caggtcagac cgcgcgtatc 60
tcgtgtagcg gcgattctat tcgttctaag tatgttcatt ggtaccagca gaaacccggg 120
caggcgccag ttcttgtgat ttatcgtgat aataatcgtc cctcaggcat cccggaacgc 180
tttagcggat ccaacagcgg caacaccgcg accctgacca ttagcggcac tcaggcggaa 240
gacgaagcgg attattattg cgcttcttat gattataagt ctaagaatat tgtgtttggc 300
ggcggcacga agttaaccgt tcttggccag ccgaaagccg caccgagtgt gacgctgttt 360
ccgccgagca gcgaagaatt gcaggcgaac aaagcgaccc tggtgtgcct gattagcgac 420
ttttatccgg gagccgtgac agtggcctgg aaggcagata gcagccccgt caaggcggga 480
gtggagacca ccacaccctc caaacaaagc aacaacaagt acgcggccag cagctatctg 540
agcctgacgc ctgagcagtg gaagtcccac agaagctaca gctgccaggt cacgcatgag 600
gggagcaccg tggaaaaaac cgttgcgccg actgaggcct gataa 645
<210>37
<211>214
<212>PRT
<213>Artificial
<220>
<223>Fab fragment MOR03285 VL-CL
<400>37
Asp Ile Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Tyr
20 25 30
Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Gly Asn Ser Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Thr Gly Ser Tyr
85 90 95
Ala Thr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro
100 105 110
Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu
115 120 125
Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro
130 135 140
Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala
145 150 155 160
Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala
165 170 175
Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg
180 185 190
Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr
195 200 205
Val Ala Pro Thr Glu Ala
210
<210>38
<211>648
<212>DNA
<213>Artificial
<220>
<223>DNA coding for Fab fragment MOR03285 VL-CL
<400>38
gatatcgtgc tgacccagcc gccttcagtg agtggcgcac caggtcagcg tgtgaccatc 60
tcgtgtagcg gcagcagcag caacattggt tcttattatg tgtattggta ccagcagttg 120
cccgggacgg cgccgaaact tctgatttat ggtaattctc agcgtccctc aggcgtgccg 180
gatcgtttta gcggatccaa aagcggcacc agcgcgagcc ttgcgattac gggcctgcaa 240
agcgaagacg aagcggatta ttattgccag gcttggactg gttcttatgc tactgtgttt 300
ggcggcggca cgaagttaac cgttcttggc cagccgaaag ccgcaccgag tgtgacgctg 360
tttccgccga gcagcgaaga attgcaggcg aacaaagcga ccctggtgtg cctgattagc 420
gacttttatc cgggagccgt gacagtggcc tggaaggcag atagcagccc cgtcaaggcg 480
ggagtggaga ccaccacacc ctccaaacaa agcaacaaca agtacgcggc cagcagctat 540
ctgagcctga cgcctgagca gtggaagtcc cacagaagct acagctgcca ggtcacgcat 600
gaggggagca ccgtggaaaa aaccgttgcg ccgactgagg cctgataa 648
<210>39
<211>216
<212>PRT
<213>Artificial
<220>
<223>Fab fragment MOR03293VL-CL
<400>39
Asp Ile Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Asn
20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Met Ile Tyr Gly Gly Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Trp Thr Thr Ile
85 90 95
Tyr Arg Asn Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110
Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu
115 120 125
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
130 135 140
Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val
145 150 155 160
Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
165 170 175
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
180 185 190
His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
195 200 205
Lys Thr Val Ala Pro Thr Glu Ala
210 215
<210>40
<211>654
<212>DNA
<213>Artificial
<220>
<223>DNA coding for Fab fragment MOR03293 VL-CL
<400>40
gatatcgcac tgacccagcc agcttcagtg agcggctcac caggtcagag cattaccatc 60
tcgtgtacgg gtactagcag cgatgttggt tctaataatt atgtgtcttg gtaccagcag 120
catcccggga aggcgccgaa acttatgatt tatggtggtt ctaatcgtcc ctcaggcgtg 180
agcaaccgtt ttagcggatc caaaagcggc aacaccgcga gcctgaccat tagcggcctg 240
caagcggaag acgaagcgga ttattattgc tcttcttgga ctactattta tcgtaatcgt 300
gtgtttggcg gcggcacgaa gttaaccgtt cttggccagc cgaaagccgc accgagtgtg 360
acgctgtttc cgccgagcag cgaagaattg caggcgaaca aagcgaccct ggtgtgcctg 420
attagcgact tttatccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480
aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540
agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600
acgcatgagg ggagcaccgt ggaaaaaacc gttgcgccga ctgaggcctg ataa 654

Claims (34)

1. radiator binding antibody or antibody fragment, be characterised in that described antibody or antibody fragment change the spectral emissions characteristic of radiator when its antigen binding pocket and radiator interaction, wherein radiator comprises a kind of dyestuff, described dyestuff has at least one and absorbs maximum value and/or fluorescence maximum value in 700 to 1000nm spectral range, and wherein said dyestuff is selected from the polymethine dyestuff, it is right that wherein said radiator binding antibody or antibody fragment comprise a kind of VH/VL, described VH/VL to be included in following sequence to one of in: SEQ-ID No.:1+2; SEQ-ID No.:5+6; SEQ-ID No.:9+10; SEQ-ID No.:13+14; SEQ-ID No.:5+17; SEQ-ID No.:5+19; SEQ-ID No.:5+37; SEQ-ID No.:9+21; SEQ-ID No.:9+23; SEQ-ID No.:9+25; SEQ-ID No.:9+27; SEQ-ID No.:9+29; SEQ-ID No.:9+3 1; SEQ-ID No.:9+33; SEQ-ID No.:9+39; And SEQ-ID No.:13+35.
2. the radiator binding antibody or the antibody fragment of claim 1, it is selected from Fab fragment, scFv fragment, scTCR chain, single-chain antibody and composition thereof.
3. the radiator binding antibody or the antibody fragment of claim 1, wherein said dyestuff have at least one and absorb maximum value and fluorescence maximum value in 750 to 900nm spectral range.
4. the radiator binding antibody or the antibody fragment of claim 1, wherein said dyestuff is selected from dicarbocyanines, three carbonyl cyanines, indigo three carbonyl cyanines, merocyanine, styryl, squarilium and oxonol dye.
5. each radiator binding antibody or antibody fragment of claim 1-4, its for the binding affinity of radiator less than 50nm.
6. the radiator binding antibody or the antibody fragment of claim 5, its for the binding affinity of radiator less than 10nm.
7. each radiator binding antibody or antibody fragment of claim 1-4, wherein the change of radiator emission characteristic be selected from change, the phosphorescence of change, the fluorescence intensity of polarization plane change, fluorescence work-ing life change and absorb maximum value and/or the peaked red shift of fluorescence.
8. the radiator binding antibody or the antibody fragment of claim 7, the change of wherein said phosphorescence is the change of phosphorescence intensity.
9. each radiator binding antibody or antibody fragment of claim 1-4, wherein the value that moves to upper wavelength with the reagent interaction post-absorption that detects radiator and/or fluorescence maximum value is greater than 15nm.
10. the radiator binding antibody or the antibody fragment of claim 9, wherein the value that moves to upper wavelength with the reagent interaction post-absorption that detects radiator and/or fluorescence maximum value is greater than 25nm.
11. the radiator binding antibody or the antibody fragment of claim 9 are 30nm with reagent interaction post-absorption that detects radiator and/or the value that the fluorescence maximum value moves to upper wavelength wherein.
12. each radiator binding antibody or antibody fragment of claim 1-4, wherein said dyestuff comprises the cyanine dyes of general formula (I)
Figure C2004800196210003C1
Wherein D represents group (II) or (III)
Wherein the position of asterisk mark is the site that is connected with group B
And B represents group (IV), (V), (VI), (VII) or (VIII)
Figure C2004800196210004C1
R wherein 1And R 2, represent C independently of one another 1-C 4Sulfoalkyl chain, saturated or undersaturated, side chain or straight chain C 1-C 50Alkyl chain;
R 3And R 4, represent group-COOE independently of one another 1,-CONE 1E 2,-NHCOE 1,-NHCONHE 1,-NE 1E 2,-OE 1,-OSO 3E 1,-SO 3E 1,-SO 2NHE 1Or-E 1, E wherein 1And E 2Represent hydrogen atom independently of one another, C 1-C 4Sulfoalkyl chain, saturated or undersaturated, side chain or straight chain C 1-C 50Alkyl chain, R 5Represent hydrogen atom or fluorine, chlorine, bromine or iodine atom, Me, Et or Prop,
B represents numeral 2 or 3, and
X and Y represent independently O, S ,=C (CH 3) 2Or-(CH=CH)-,
And the salt of these compounds and solvate.
13. the radiator binding antibody or the antibody fragment of claim 12, wherein said R 1Or R 2By 0 to 15 Sauerstoffatom and/or at interval and/or can be replaced by 0 to 5 hydroxyl by 0 to 3 carbonyl.
14. the radiator binding antibody or the antibody fragment of claim 12, wherein said R 3Or R 4Replace at interval and/or by 0 to 5 hydroxyl by 0 to 15 Sauerstoffatom and/or by 0 to 3 carbonyl.
15. polynucleotide, it comprise coding claim 1-12 each the radiator binding antibody or the sequence of antibody fragment or its functional variant.
16. the polynucleotide of claim 15, it is DNA, RNA or PNA.
17. DNA-carrier molecule or RNA-carrier molecule, it contains the polynucleotide of at least a or multiple claim 15 and it can be expressed in cell.
18. contain the host cell of the carrier molecule of the polynucleotide of claim 15 or claim 17.
19. comprise at least a claim 1-14 each the radiator binding antibody or the antibody of antibody fragment.
20. the antibody of claim 19, it is polyclone or monoclonal antibody, people's antibody or humanized antibody, synthesizes or recombinant antibodies.
21. production claim 1-14 each the radiator binding antibody or the method for antibody fragment, comprise that described radiator comprises a kind of dyestuff with the suitable organism of a kind of radiator immunity, described dyestuff is selected from the polymethine dyestuff.
22. the method for claim 21, wherein said dyestuff are selected from dicarbocyanines, three carbonyl cyanines, indigo three carbonyl cyanines, merocyanine, styryl, squarilium and oxonol dye.
23. production claim 1-14 each the radiator binding antibody or the method for antibody fragment, comprise the reorganization and/or synthetic production of described antibody or antibody fragment.
24. each radiator binding antibody or the application that is used for the diagnostic reagent of in-vitro diagnosis in preparation of the antibody of the host cell of the carrier molecule of the polynucleotide of antibody fragment, claim 15 or 16, claim 17, claim 18 or claim 19 or 20 of claim 1-14.
25. be used for the diagnostic kit of in-vitro diagnosis, comprise be selected from claim 1-14 each the radiator binding antibody or at least a reagent of the antibody of the host cell of the carrier molecule of the polynucleotide of antibody fragment, claim 15 or 16, claim 17, claim 18 or claim 19 or 20.
26. the diagnostic kit of claim 25, it also has other adjuvant and/or guidance in common or the container that separates.
27. the method for a kind of material that external quantitative assay contains in sample comprises the steps:
A) each radiator binding antibody or antibody fragment of claim 1-14 contacted with a kind of radiator, thereby the interaction of the radiator of described conjugate and radiator binding antibody or antibody fragment produces a change in the spectral emissions characteristic of radiator, and
B) change of the spectral emissions characteristic of measurement radiator.
28. the method for the antigen detecting agent that exists in material that contains in the direct external quantitative assay sample of claim 27 or the sample, it also additionally comprises the steps,
D) the described material that contains in the quantitative sample of change of the emission characteristic by measuring radiator.
29. the method for claim 27 or 28, wherein the change of the spectral emissions characteristic of emitter portion is selected from polarization plane change, phosphorescence change, fluorescence change in work-ing life and absorbs maximum value and/or the peaked red shift of fluorescence.
30. the method for claim 29, it is the change of phosphorescence intensity that wherein said phosphorescence changes.
31. the method for claim 27 wherein contacts radiator binding antibody fragment Fab fragment, scFv fragment, scTCR chain, single-chain antibody and composition thereof with sample.
32. the method for claim 27, wherein radiator binding antibody or antibody fragment comprise each sequence of claim 1-14.
33. the method for claim 27, wherein radiator binding antibody or antibody fragment have binding affinity less than 50nm to radiator.
34. the method for claim 33, wherein radiator binding antibody or antibody fragment have binding affinity less than 10nm to radiator.
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