CA2340865A1 - High-affinity antibodies - Google Patents
High-affinity antibodies Download PDFInfo
- Publication number
- CA2340865A1 CA2340865A1 CA002340865A CA2340865A CA2340865A1 CA 2340865 A1 CA2340865 A1 CA 2340865A1 CA 002340865 A CA002340865 A CA 002340865A CA 2340865 A CA2340865 A CA 2340865A CA 2340865 A1 CA2340865 A1 CA 2340865A1
- Authority
- CA
- Canada
- Prior art keywords
- antibody
- samples
- sample
- ser
- hour
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3007—Carcino-embryonic Antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
- A61K47/6815—Enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
Abstract
High-affinity monoclonal antibodies, wherein the affinity is characterised b y: (i) incubating first and second samples of the antibody in antigen-coated microtitre plate wells at a concentration chosen to be within the linear par t of a standard curve at pH 7.2 for 1 hour at 37 ~C; (ii) removing unbound antibody from both samples; (iii) incubating the first sample with PBS at pH 7.2 for 1 hour at 37 ~C, and reducing the pH of the second sample to pH 3 or below and incubating for 1 hour at 37 ~C; (iv) removing unbound antibody fro m both samples; (v) incubating both samples with anti-antibody alkaline phosphatase-conjugate for 1 hour at 37 ~C; (vi) removing unbound conjugate from both samples; and (vii) adding PNPP substrate to the samples, measuring the absorbance of the samples at 405 nm, and determining the amount of antibody bound to antigen, wherein the amount bound in the second sample is > 50 % of that of the first sample.
Description
HIGH-AFFINITY ANTIBODIES
Field of the Invention This invention relates to antibodies and their therapeutic use.
Backctround to the Invention Antibodies have long been regarded as potentially powerful tools in the treatment of cancer and other diseases. However, although there have been some notable exceptions, this potential has not generally yet been 10 realised.
This relative lack of success may be due, at. least in part, to the u~~e of monoclonal antibodies derived from rodents, which :seldom have affinities higher than 10-9 M.
Antibodies having this level of affinity are o:E limited 15 therapeutic utility, as it has proved difficult to deliver enough antibody to the target to effect useful biological activity. Antibody binding to an antigen is reversible, and at the concentrations of antibody practical far in vivo use, dissociation will be favoured over association. In 20 principle, it i:~ possible to counter the dissociation of antigen by increasing the antibody concentration. However, this may lead to unacceptable clinical side-effects and would also increase the costs associated with the. therapy.
Summary of the Invention 25 The present invention is based on the realisation that antibodies, or fragments thereof , can be produced which are "acid-resistant" and that this property is associated with high affinity binding of an antibody for its antigen.
According t:o the present invention, a high-affinity 30 antibody has affinity characterised by:
(i) incuba.ting first and second samples of the antibody in antigen-coated microtitre plate wells at a concentration chosen to be within the linear response part of a standard curve at pH 7.2 for 1 hour at 37°C;
35 (ii) removing unbound antibody from both samples;
WO 00/12556 PC.T/GB99/02729 (iii) incubating the first sample with PBS at pH 7.2 for 1 hour at 37°C, and reducing the pH of the second sample to pH 3 or below and incubating for 1 hour at 37°C;
(iv} remov_~ng unbound antibody from both samples;
(v) incubating both samples with anti-antibody alkaline-phosphatase conjugate for 1 hour at 37°C;
(vi) removing unbound conjugate from both samples; and (vii) adding PNPP substrate to the samples, measuring absorbance of the samples at 405nm, and determining the amount of antibody bound to antigen, wherein the amount bound in the second sample is >50% of that of 'the first sample.
Preferably, the maximum pH in step (iii) is 2.5, more preferably 2Ø
Antibodies or antibody fragments with the "acid resistant" propE~rties are expected to favour association rather than dissociation and they therefore have longer localisation times at target sites, which results in a higher concentration of antibodies localised at the target sites.
In particular, this invention relates to the production of a high affinity single-chain Fv antibody fragment. This ScFv has particular advantages in that it allows better targeting to a site in vivv.
Description of the Drawincx Figure 1 illustrates the results achieved for acid-resistance of sheep and mouse monoclonal antibodies and single-chain Fvs with affinity to carcinoembryonic antigen at various pH values.
Description of the Invention The acid-resistant monoclonal antibodies according to the present im~ention may be obtained using various techniques. For' example, classical hybridoma technology can be applied, comprising the fusion of B-lymphocytes from immunised animals secreting high-affinity antibodies with an appropriate fusion partner. An alternative method is to purify the mRNA from selected lymphocytes and use the technique of PCR to amplify the antibody genes required.
Phage display technology and other techniques for the display of antibody fragments may also be used to obtain the antibody genets from naive or immunised libraries after appropriate selection procedures.
The antibody gene can be co-expressed with or otherwise chemically linked to toxins, radioisotopes or enzymes or any other desirable molecules to provide a fusion protein with strong binding characteristics. In a further alternative, the antibodies may be produced by transgenic anima7Ls as described in US-A-5770429.
The antibody may be a whole antibody, comprising heavy and light chains, and constant and variable regions.
Alternatively, the antibody is an antibody fragment, e.g.
F(ab')2, Fab, Fv or single-chain Fv fragments, provided that at least part of the variable region is present which confers the propEarty of "acid resistance". The antibody may also be an animal, chimeric or humanised antibody. A
suitable method for producing humanised antibodies is disclosed in WO-A,-92/15699.
In a preferred embodiment of the invention, the antibody is a single-chain Fv fragment. The single-chain Fv fragment comprises both heavy chain and light chain variable regions linked by a suitable peptide.
The antibodies of the present invention may be defined by their acid-resistant properties, which can be characterised by a~n acid-washed enzyme-linked immunosorbent assay (EIA), as described above. Typically the A,os value obtained by EIA will represent antibody binding of >50% for a sample at pH 3 or below, compared to the value for the sample at pH 7.2. Preferably, the A,oS value of a sample at pH 2 will represent antibody binding of >60% more preferably 70% of that obtained at pH 7.2.
The animal that is subjected to immunisation is not a rodent, but is chosen to give higher affinity antibodies.
Any large mammal may be used and suitable animals include rabbits, goats, cows and sheep.
Field of the Invention This invention relates to antibodies and their therapeutic use.
Backctround to the Invention Antibodies have long been regarded as potentially powerful tools in the treatment of cancer and other diseases. However, although there have been some notable exceptions, this potential has not generally yet been 10 realised.
This relative lack of success may be due, at. least in part, to the u~~e of monoclonal antibodies derived from rodents, which :seldom have affinities higher than 10-9 M.
Antibodies having this level of affinity are o:E limited 15 therapeutic utility, as it has proved difficult to deliver enough antibody to the target to effect useful biological activity. Antibody binding to an antigen is reversible, and at the concentrations of antibody practical far in vivo use, dissociation will be favoured over association. In 20 principle, it i:~ possible to counter the dissociation of antigen by increasing the antibody concentration. However, this may lead to unacceptable clinical side-effects and would also increase the costs associated with the. therapy.
Summary of the Invention 25 The present invention is based on the realisation that antibodies, or fragments thereof , can be produced which are "acid-resistant" and that this property is associated with high affinity binding of an antibody for its antigen.
According t:o the present invention, a high-affinity 30 antibody has affinity characterised by:
(i) incuba.ting first and second samples of the antibody in antigen-coated microtitre plate wells at a concentration chosen to be within the linear response part of a standard curve at pH 7.2 for 1 hour at 37°C;
35 (ii) removing unbound antibody from both samples;
WO 00/12556 PC.T/GB99/02729 (iii) incubating the first sample with PBS at pH 7.2 for 1 hour at 37°C, and reducing the pH of the second sample to pH 3 or below and incubating for 1 hour at 37°C;
(iv} remov_~ng unbound antibody from both samples;
(v) incubating both samples with anti-antibody alkaline-phosphatase conjugate for 1 hour at 37°C;
(vi) removing unbound conjugate from both samples; and (vii) adding PNPP substrate to the samples, measuring absorbance of the samples at 405nm, and determining the amount of antibody bound to antigen, wherein the amount bound in the second sample is >50% of that of 'the first sample.
Preferably, the maximum pH in step (iii) is 2.5, more preferably 2Ø
Antibodies or antibody fragments with the "acid resistant" propE~rties are expected to favour association rather than dissociation and they therefore have longer localisation times at target sites, which results in a higher concentration of antibodies localised at the target sites.
In particular, this invention relates to the production of a high affinity single-chain Fv antibody fragment. This ScFv has particular advantages in that it allows better targeting to a site in vivv.
Description of the Drawincx Figure 1 illustrates the results achieved for acid-resistance of sheep and mouse monoclonal antibodies and single-chain Fvs with affinity to carcinoembryonic antigen at various pH values.
Description of the Invention The acid-resistant monoclonal antibodies according to the present im~ention may be obtained using various techniques. For' example, classical hybridoma technology can be applied, comprising the fusion of B-lymphocytes from immunised animals secreting high-affinity antibodies with an appropriate fusion partner. An alternative method is to purify the mRNA from selected lymphocytes and use the technique of PCR to amplify the antibody genes required.
Phage display technology and other techniques for the display of antibody fragments may also be used to obtain the antibody genets from naive or immunised libraries after appropriate selection procedures.
The antibody gene can be co-expressed with or otherwise chemically linked to toxins, radioisotopes or enzymes or any other desirable molecules to provide a fusion protein with strong binding characteristics. In a further alternative, the antibodies may be produced by transgenic anima7Ls as described in US-A-5770429.
The antibody may be a whole antibody, comprising heavy and light chains, and constant and variable regions.
Alternatively, the antibody is an antibody fragment, e.g.
F(ab')2, Fab, Fv or single-chain Fv fragments, provided that at least part of the variable region is present which confers the propEarty of "acid resistance". The antibody may also be an animal, chimeric or humanised antibody. A
suitable method for producing humanised antibodies is disclosed in WO-A,-92/15699.
In a preferred embodiment of the invention, the antibody is a single-chain Fv fragment. The single-chain Fv fragment comprises both heavy chain and light chain variable regions linked by a suitable peptide.
The antibodies of the present invention may be defined by their acid-resistant properties, which can be characterised by a~n acid-washed enzyme-linked immunosorbent assay (EIA), as described above. Typically the A,os value obtained by EIA will represent antibody binding of >50% for a sample at pH 3 or below, compared to the value for the sample at pH 7.2. Preferably, the A,oS value of a sample at pH 2 will represent antibody binding of >60% more preferably 70% of that obtained at pH 7.2.
The animal that is subjected to immunisation is not a rodent, but is chosen to give higher affinity antibodies.
Any large mammal may be used and suitable animals include rabbits, goats, cows and sheep.
An antibody of the invention may be used in therapy and may be formulated into any suitable composition with a physi.ologically--acceptable excipient, diluent or carrier.
The following Examples illustrate the invention.
Example 1.
Sheep were immunised with carcinoembryonic antigen (CEA) in complete Freund's adjuvant, then boosted three times with antigen in incomplete Freund's adjuvant.
Animals were sacrificed after the final boost and lymph nodes removed.
The lymph nade cells were then washed and fused with sheep heteromye:loma fusion partner SFP3.2. Fused cells were plated out at a total density of approximately 106 per ml in medium containing HAT (Life Technologies). These samples were them screened for hybridomas secreting high-affinity antibodies to the specified antigen using both a normal EIA and an acid-washed EIA.
Standard EIA screening assays were carried out as follows:
Maxisorb assay plates (NUNC) were coated with CEA (0.4 ~g/ml in phosphate-buffered saline at pH 7.2), 100~C1 per well and left overnight at 4°C. The plates were then washed three times using phosphate buffered saline at pH
7.2 with 0.01% Tween 20 detergent. Any remaining reactive sites on the plates were blocked by the addition of 200,1 per well of 0.2% fat-free milk protein in PBS at pH 7.2 at 37°C for 2 haur. The plates were then washed in PBS as described above amd 45,1 of the antibody samples were added to the wells of the plates. The samples were incubated for one hour at 37°C and then washed as described previously.
Bound antibody was detected using alkaline phosphatase-conjugated donkey anti-sheep antibody (Sigma A5187 diluted 1/5000 in PBS at pH 7.2 with 1% BSA) . The plates were then washed and 1001 per well of PNPP (Sigma N2770) solution was added. Absorbance was measured using a spectrophotometer at 405nm with phosphate buffered saline as a control.
Acid-wash EIA screening assays were carried out as follows:
Coating and binding of antibody samples was as described for 'the standard EIA above. However, after 5 incubation with the antibody samples, the plates were washed and 200u:1 per well of HC1 (lOmM Stock solution) at pH 2 was added for one hour at 37°C. After three washes the antibody remaining bound to antigen was detected using alkaline phosphatase-conjugated donkey anti-sheep antibody and PNPP as described above. In order to ensure that a proper comparison was being made between antibodies at different concentrations, each sample was chosen to give an AqpS value of app>roximately 1.0 in the normal EIA (i.e. in the linear response part of the EIA curve).
Three hybridomas (1D2, 6611 and 6H9) secreted antibodies which gave a greater than 50% retention of binding in the acid washed EIA, in comparison to the binding in the non-acid washed EIA.
Examgle 2 A single-clhain Fv fragment was produced from the hybridoma 6H9 above, as follows:
mRNA was purified from the cultured hybridoma cells using oligo-dZ' cellulose. Single-stranded DNA
complementary to the mRNA (cDNA) was synthesized by reverse transcription. Universal primers, designed from the constant regions. of sheep heavy and light chain antibody genes, were u.ced in separate reverse transcription reactions to synthesise the cDNA for the antibody variable regions.
The cDNA wa.s then amplified by the polymerase chain reaction to make double-stranded DNA using primers designed from the heavy and light chain variable framework sequences. Separate polymerase chain reactions were used to amplify the heavy and light chain regions. The products were then analysed by agarose gel electrophoresis and the DNA bands equivalent to light and heavy chain genes were cut from the gel and purified.
The following Examples illustrate the invention.
Example 1.
Sheep were immunised with carcinoembryonic antigen (CEA) in complete Freund's adjuvant, then boosted three times with antigen in incomplete Freund's adjuvant.
Animals were sacrificed after the final boost and lymph nodes removed.
The lymph nade cells were then washed and fused with sheep heteromye:loma fusion partner SFP3.2. Fused cells were plated out at a total density of approximately 106 per ml in medium containing HAT (Life Technologies). These samples were them screened for hybridomas secreting high-affinity antibodies to the specified antigen using both a normal EIA and an acid-washed EIA.
Standard EIA screening assays were carried out as follows:
Maxisorb assay plates (NUNC) were coated with CEA (0.4 ~g/ml in phosphate-buffered saline at pH 7.2), 100~C1 per well and left overnight at 4°C. The plates were then washed three times using phosphate buffered saline at pH
7.2 with 0.01% Tween 20 detergent. Any remaining reactive sites on the plates were blocked by the addition of 200,1 per well of 0.2% fat-free milk protein in PBS at pH 7.2 at 37°C for 2 haur. The plates were then washed in PBS as described above amd 45,1 of the antibody samples were added to the wells of the plates. The samples were incubated for one hour at 37°C and then washed as described previously.
Bound antibody was detected using alkaline phosphatase-conjugated donkey anti-sheep antibody (Sigma A5187 diluted 1/5000 in PBS at pH 7.2 with 1% BSA) . The plates were then washed and 1001 per well of PNPP (Sigma N2770) solution was added. Absorbance was measured using a spectrophotometer at 405nm with phosphate buffered saline as a control.
Acid-wash EIA screening assays were carried out as follows:
Coating and binding of antibody samples was as described for 'the standard EIA above. However, after 5 incubation with the antibody samples, the plates were washed and 200u:1 per well of HC1 (lOmM Stock solution) at pH 2 was added for one hour at 37°C. After three washes the antibody remaining bound to antigen was detected using alkaline phosphatase-conjugated donkey anti-sheep antibody and PNPP as described above. In order to ensure that a proper comparison was being made between antibodies at different concentrations, each sample was chosen to give an AqpS value of app>roximately 1.0 in the normal EIA (i.e. in the linear response part of the EIA curve).
Three hybridomas (1D2, 6611 and 6H9) secreted antibodies which gave a greater than 50% retention of binding in the acid washed EIA, in comparison to the binding in the non-acid washed EIA.
Examgle 2 A single-clhain Fv fragment was produced from the hybridoma 6H9 above, as follows:
mRNA was purified from the cultured hybridoma cells using oligo-dZ' cellulose. Single-stranded DNA
complementary to the mRNA (cDNA) was synthesized by reverse transcription. Universal primers, designed from the constant regions. of sheep heavy and light chain antibody genes, were u.ced in separate reverse transcription reactions to synthesise the cDNA for the antibody variable regions.
The cDNA wa.s then amplified by the polymerase chain reaction to make double-stranded DNA using primers designed from the heavy and light chain variable framework sequences. Separate polymerase chain reactions were used to amplify the heavy and light chain regions. The products were then analysed by agarose gel electrophoresis and the DNA bands equivalent to light and heavy chain genes were cut from the gel and purified.
Equimolar amounts of variable heavy and Light chain DNA were mixed together with an oligonucleotide linker DNA.
The linker DNA coded for the amino acid sequence (Gly,Ser) 3 with additional nucleotides complementary to the 3' end of the heavy chain 'variable region and the 5' end of the light chain variable region. The three DNA molecules were denatured, annealed and extended in the first stage (without primers) of a two-stage PCR reaction so that the fragments were joined, thereby assembling the single-chain Fv.
The single-chain Fv DNA was amplified in the second stage of the PCR using a pair of primers derived from the heavy and light chain variable region termini with the addition of the restriction enzyme recognition sites for A1W44i and NotI., The single-chain Fv gene product was analysed by agarose gel electrophoresis and purified. The single-chain Fv was then digested with the restriction enzymes AIW44i and Notl and cloned into an expression vector. The vector was then used to transform E. coli HB
2151, and protein expression was allowed to occur. The vector was designed so as to include a hexa-histidine tag at the COOH terminus of the SFv. The single-chain Fv was purified using nickel-chelate affinity chromatography and analysed by SDS-PAGE. The amino acid sequence for the heavy chain variable region and the light chain variable region is disclosed in SEQ ID Nos. 2 and 4, respectively.
An acid-wash EI1~, was also carried out to determine the acid-resistant properties of the single-chain Fv.
Acid-wash EI:A was carried out as follows:
Carcinoembryonic antigen (CEA)-coated microtitre plates were prepa:red as described previously. Single-chain Fv samples (6H9) were diluted to a range of concentrations between lng/mI and 100ng/ml in PBS at pH 7.2 containing ix bovine serum albumin (BSA). 1001 samples were added to the mi.crotitre plate wells and incubated for 1 hour at 37°C. The platea were then washed, 200~C1 per well of citrate added, and the plates incubated for 1 hour at 37°C.
The linker DNA coded for the amino acid sequence (Gly,Ser) 3 with additional nucleotides complementary to the 3' end of the heavy chain 'variable region and the 5' end of the light chain variable region. The three DNA molecules were denatured, annealed and extended in the first stage (without primers) of a two-stage PCR reaction so that the fragments were joined, thereby assembling the single-chain Fv.
The single-chain Fv DNA was amplified in the second stage of the PCR using a pair of primers derived from the heavy and light chain variable region termini with the addition of the restriction enzyme recognition sites for A1W44i and NotI., The single-chain Fv gene product was analysed by agarose gel electrophoresis and purified. The single-chain Fv was then digested with the restriction enzymes AIW44i and Notl and cloned into an expression vector. The vector was then used to transform E. coli HB
2151, and protein expression was allowed to occur. The vector was designed so as to include a hexa-histidine tag at the COOH terminus of the SFv. The single-chain Fv was purified using nickel-chelate affinity chromatography and analysed by SDS-PAGE. The amino acid sequence for the heavy chain variable region and the light chain variable region is disclosed in SEQ ID Nos. 2 and 4, respectively.
An acid-wash EI1~, was also carried out to determine the acid-resistant properties of the single-chain Fv.
Acid-wash EI:A was carried out as follows:
Carcinoembryonic antigen (CEA)-coated microtitre plates were prepa:red as described previously. Single-chain Fv samples (6H9) were diluted to a range of concentrations between lng/mI and 100ng/ml in PBS at pH 7.2 containing ix bovine serum albumin (BSA). 1001 samples were added to the mi.crotitre plate wells and incubated for 1 hour at 37°C. The platea were then washed, 200~C1 per well of citrate added, and the plates incubated for 1 hour at 37°C.
In this case, t:he acid preparations were made using a stock solution of 100mM citrate diluted to pH values of. 4.0, 3.5, 3.0, 2.5 and 2.0 in the reaction mixture. PBS at pH 7.2 was used as a reference control. The plates were then 5 washed and 100u1 per well of mouse anti-tetra-histidine antibody (Qiage:n) (100ng/ml diluted in PBS at pH 7.2 with 1% BSA) added and incubated for 1 hour at 37°C. After plate washing t:he samples were incubated for 1 hour at 37°C
with 1001 per well of goat anti-mouse alkaline phosphatase 10 conjugate (Sigma A3688 diluted 1/1000 in PBS with 1% BSA at pH 7 . 2 ) . The plates were then washed, treated with PNPP as described previausly and the absorbance measured using a spectrophotometer at 405nm.
As a control for acid resistance, sFv samples were 15 incubated with PBS at pH 7.2 to generate an EIA response curve for the SFv samples. In the linear region, a concentration of 10-20ng/ml of the SFv sample gave an absorbance (A,oS,) of 1.0-1.5 and was therefore used to determine the amount of antibody bound in the acid washed 20 samples as a percentage of the amount bound in the reference sample.
The acid-resistant properties of the 6H9 whole antibody and th,e 6H9 single-chain Fv were compared with that for the mouse-derived anti-carcinoembryonic antigen 25 whole antibody, A5B7 and the single-chain Fv MFE. The results are shown in Figure 1, with the antigen-binding of the mouse-derived antibodies being substantially reduced at pH 3.5 and less than 5% at pH 2.5. In contrast, the 6H9 antibodies retain >70% antigen at pH 3.5, >60% at pH 2.5 30 and >50% at pH 2Ø
SEQUENCE LISTING
<110> KS Biomedix Ltd <120> ANTIBODIES
<130> rep05827wo <140>
<141>
<160> 4 <170> PatentIn Ver. 2.1 <210> 1 <211> 363 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Antibody Fragment <220>
<221> CDS
<222> (1)..(363) <400> 1 cag gtg cag ctg cag gag tcg gga ccc agc ctg gtg aag ccc tca cag 48 Gln Val Gln Leu Gln Gl.u Ser Gly Pro Ser Leu Val Lys Pro Ser Gln acc ctc tcc ctc acc tgc a cg gtc tct gga ttc tca tta acc aag tat 96 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Lys Tyr ggt gtt agt tgg gtc cgc cag get cca gga aag gcg ctt gag tgg cta 144 Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu 3.'i 40 45 ggt ggt gtg tcc agt ggt coca cta aca gcc tat aac aca gcc cta cag 192 Gly Gly Val. Ser Ser Gly Ala Leu Thr Ala Tyr Asn Thr Ala Leu Gln tcc cga ctc agc gtc acc agg gac acc tcc aag agc caa ttc tcc ctg 240 Ser Arg Leu Ser Val Thr Arg Asp Thr Ser Lys Ser Gln Phe Ser Leu tca ctg agc agc gtg act act gag gac acg gcc att tac tac tgt gcg 288 Ser Leu Ser Ser Val Thr Thr Glu Asp Thr Ala Ile Tyr Tyr Cys Ala aaa tct gtc aat ggt gac agt gtt cct tat ggt ttg gac tac tgg agc 336 Lys Ser Val Asn Gly Asp Ser Val Pro Tyr Gly Leu Asp Tyr Trp Ser 100 i05 110 cca gga ctc cta ctc acc gtc tcc tca 363 Pro Gly Leu Leu Leu Thr Val Ser Ser <210> 2 <211> 121 <212> PRT
<213> Artificial Sequence <223> Description of Artificial 5equence:Antibody Fragment <900> 2 Gln Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cy;s Thr Val Ser Gly Phe Ser Leu Thr Lys Tyr Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu Gly Gly Val Ser Ser Gly Ala Leu Tht Ala Tyr Asn Thr Ala Leu Gln Ser Arg Leu Ser Val Thr Arg Asp Thr Ser Lys Ser Gln Phe Ser Leu Ser Leu Ser Ser Val Thr Thr Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Lys Ser Val Asn Gly Asp Ser Val Pro Tyr Gly Leu Asp Tyr Trp Ser Pro Gly Leu Leu Leu Thi: Val Ser Ser 11.5 120 <210> 3 <211> 333 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Antibody Fragment <220>
<221> CDS
<222> (1)..(333) <400> 3 cag gat gtg ctg act cag ccg tcc tcc gtg tct ggg tcc ctg ggc cag 48 Gln Asp Val Leu Thr Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln agg gtc tcc atc acc tgc tct gga agc agc agc aac att gga ggt aat 96 Arg Val Ser Ile Thr Cys Ser Gly Ser Ser Ser Asn Ile Gly Gly Asn get tat gtg ggc tgg t;ac caa cag gtc cca gga tca gcc ccc aga ctc 144 Ala Tyr Val Gly Trp T:yr Gln Gln Val Pro Gly Ser Ala Pro Arg Leu ctc atc agt get aca acc gat cga gcc tcg ggg atc ccc gac cga ttc 192 Leu Ile Ser Ala Thr Thr Asp Arg Ala Ser Gly Ile Pro Asp Arg Phe tcc ggc tcc agg tct ggg aac aca gcc acc ctg acc atc agc tcg ctc 240 Ser Gly Ser Arg Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu 65 '10 75 80 cag get gag gac gag gc:c gat tat tac tgt gca tcg tat caa agt act 288 Gln Ala Glu Asp Glu Al.a Asp Tyr Tyr Cys Ala Ser Tyr Gln Ser Thr tac agt ggt gtt ttc gc~c agc ggg acc agg ctg acc gtc ctg ggt 333 Tyr Ser Gly Val Phe Gl.y Ser Gly Thr Arg Leu Thr Val Leu Gly <210> 4 <211> 111 <212> PRT
<213> Artificial Sequence <223> Description of Artificial Sequence: Antibody Fragment <400> 9 Gln Asp Val Leu Thr Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln Arg Val Ser Ile Thr Cys Ser Gly Ser Ser Ser Asn Ile Gly Gly Asn Ala Tyr Val Gly Trp Tyr Gln Gln Val Pro Gly Ser Ala Pro Arg Leu Leu Ile Ser Ala Thr T:hr Asp Arg Ala Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Arg Ser G.ly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Glu A.La Asp Tyr Tyr Cys Ala Ser Tyr Gln Ser Thr Tyr Ser Gly Val Phe G:Ly Ser Gly Thr Arg Leu Thr Val Leu Gly
with 1001 per well of goat anti-mouse alkaline phosphatase 10 conjugate (Sigma A3688 diluted 1/1000 in PBS with 1% BSA at pH 7 . 2 ) . The plates were then washed, treated with PNPP as described previausly and the absorbance measured using a spectrophotometer at 405nm.
As a control for acid resistance, sFv samples were 15 incubated with PBS at pH 7.2 to generate an EIA response curve for the SFv samples. In the linear region, a concentration of 10-20ng/ml of the SFv sample gave an absorbance (A,oS,) of 1.0-1.5 and was therefore used to determine the amount of antibody bound in the acid washed 20 samples as a percentage of the amount bound in the reference sample.
The acid-resistant properties of the 6H9 whole antibody and th,e 6H9 single-chain Fv were compared with that for the mouse-derived anti-carcinoembryonic antigen 25 whole antibody, A5B7 and the single-chain Fv MFE. The results are shown in Figure 1, with the antigen-binding of the mouse-derived antibodies being substantially reduced at pH 3.5 and less than 5% at pH 2.5. In contrast, the 6H9 antibodies retain >70% antigen at pH 3.5, >60% at pH 2.5 30 and >50% at pH 2Ø
SEQUENCE LISTING
<110> KS Biomedix Ltd <120> ANTIBODIES
<130> rep05827wo <140>
<141>
<160> 4 <170> PatentIn Ver. 2.1 <210> 1 <211> 363 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Antibody Fragment <220>
<221> CDS
<222> (1)..(363) <400> 1 cag gtg cag ctg cag gag tcg gga ccc agc ctg gtg aag ccc tca cag 48 Gln Val Gln Leu Gln Gl.u Ser Gly Pro Ser Leu Val Lys Pro Ser Gln acc ctc tcc ctc acc tgc a cg gtc tct gga ttc tca tta acc aag tat 96 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Lys Tyr ggt gtt agt tgg gtc cgc cag get cca gga aag gcg ctt gag tgg cta 144 Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu 3.'i 40 45 ggt ggt gtg tcc agt ggt coca cta aca gcc tat aac aca gcc cta cag 192 Gly Gly Val. Ser Ser Gly Ala Leu Thr Ala Tyr Asn Thr Ala Leu Gln tcc cga ctc agc gtc acc agg gac acc tcc aag agc caa ttc tcc ctg 240 Ser Arg Leu Ser Val Thr Arg Asp Thr Ser Lys Ser Gln Phe Ser Leu tca ctg agc agc gtg act act gag gac acg gcc att tac tac tgt gcg 288 Ser Leu Ser Ser Val Thr Thr Glu Asp Thr Ala Ile Tyr Tyr Cys Ala aaa tct gtc aat ggt gac agt gtt cct tat ggt ttg gac tac tgg agc 336 Lys Ser Val Asn Gly Asp Ser Val Pro Tyr Gly Leu Asp Tyr Trp Ser 100 i05 110 cca gga ctc cta ctc acc gtc tcc tca 363 Pro Gly Leu Leu Leu Thr Val Ser Ser <210> 2 <211> 121 <212> PRT
<213> Artificial Sequence <223> Description of Artificial 5equence:Antibody Fragment <900> 2 Gln Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cy;s Thr Val Ser Gly Phe Ser Leu Thr Lys Tyr Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu Gly Gly Val Ser Ser Gly Ala Leu Tht Ala Tyr Asn Thr Ala Leu Gln Ser Arg Leu Ser Val Thr Arg Asp Thr Ser Lys Ser Gln Phe Ser Leu Ser Leu Ser Ser Val Thr Thr Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Lys Ser Val Asn Gly Asp Ser Val Pro Tyr Gly Leu Asp Tyr Trp Ser Pro Gly Leu Leu Leu Thi: Val Ser Ser 11.5 120 <210> 3 <211> 333 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Antibody Fragment <220>
<221> CDS
<222> (1)..(333) <400> 3 cag gat gtg ctg act cag ccg tcc tcc gtg tct ggg tcc ctg ggc cag 48 Gln Asp Val Leu Thr Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln agg gtc tcc atc acc tgc tct gga agc agc agc aac att gga ggt aat 96 Arg Val Ser Ile Thr Cys Ser Gly Ser Ser Ser Asn Ile Gly Gly Asn get tat gtg ggc tgg t;ac caa cag gtc cca gga tca gcc ccc aga ctc 144 Ala Tyr Val Gly Trp T:yr Gln Gln Val Pro Gly Ser Ala Pro Arg Leu ctc atc agt get aca acc gat cga gcc tcg ggg atc ccc gac cga ttc 192 Leu Ile Ser Ala Thr Thr Asp Arg Ala Ser Gly Ile Pro Asp Arg Phe tcc ggc tcc agg tct ggg aac aca gcc acc ctg acc atc agc tcg ctc 240 Ser Gly Ser Arg Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu 65 '10 75 80 cag get gag gac gag gc:c gat tat tac tgt gca tcg tat caa agt act 288 Gln Ala Glu Asp Glu Al.a Asp Tyr Tyr Cys Ala Ser Tyr Gln Ser Thr tac agt ggt gtt ttc gc~c agc ggg acc agg ctg acc gtc ctg ggt 333 Tyr Ser Gly Val Phe Gl.y Ser Gly Thr Arg Leu Thr Val Leu Gly <210> 4 <211> 111 <212> PRT
<213> Artificial Sequence <223> Description of Artificial Sequence: Antibody Fragment <400> 9 Gln Asp Val Leu Thr Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln Arg Val Ser Ile Thr Cys Ser Gly Ser Ser Ser Asn Ile Gly Gly Asn Ala Tyr Val Gly Trp Tyr Gln Gln Val Pro Gly Ser Ala Pro Arg Leu Leu Ile Ser Ala Thr T:hr Asp Arg Ala Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Arg Ser G.ly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Glu A.La Asp Tyr Tyr Cys Ala Ser Tyr Gln Ser Thr Tyr Ser Gly Val Phe G:Ly Ser Gly Thr Arg Leu Thr Val Leu Gly
Claims (10)
1. A high-affinity monoclonal antibody, wherein the affinity is characterisable by:
(i) incubating first and second samples of the antibody in antigen-coated microtitre plate wells at a concentration chosen to be within the linear part of a standard curve at pH 7.2 for 1 hour at 37°C;
(ii) removing unbound antibody from both samples;
(iii) incubating the first sample with PBS at pH 7.2 for 1 hour at 37°C, and reducing the pH of the second sample to pH 3 or below and incubating for 1 hour at 37°C;
(iv) removing unbound antibody from both samples;
(v) incubating both samples with anti-antibody alkaline phosphatase-conjugate for 1 hour at 37°C;
(vi) removing unbound conjugate from both samples; and (vii) adding PNPP substrate to the samples, measuring the absorbance of the samples at 405nm, and determining the amount of antibody bound to antigen, wherein the amount bound in the second sample is >50% of that of the first sample.
(i) incubating first and second samples of the antibody in antigen-coated microtitre plate wells at a concentration chosen to be within the linear part of a standard curve at pH 7.2 for 1 hour at 37°C;
(ii) removing unbound antibody from both samples;
(iii) incubating the first sample with PBS at pH 7.2 for 1 hour at 37°C, and reducing the pH of the second sample to pH 3 or below and incubating for 1 hour at 37°C;
(iv) removing unbound antibody from both samples;
(v) incubating both samples with anti-antibody alkaline phosphatase-conjugate for 1 hour at 37°C;
(vi) removing unbound conjugate from both samples; and (vii) adding PNPP substrate to the samples, measuring the absorbance of the samples at 405nm, and determining the amount of antibody bound to antigen, wherein the amount bound in the second sample is >50% of that of the first sample.
2. An antibody according to claim 1, wherein the amount of antibody bound in the second sample is >60% of that bound in the first sample.
3. An antibody according to claim 1 or claim 2, wherein the pH in step (iii) is reduced to pH 2.5 - pH 2Ø
4. An antibody according to any preceding claim, which is non-rodent.
5. An antibody according to any preceding claim, which has affinity for a tumour-associated antigen.
6. An antibody according to claim 5, wherein the antigen is carcinoembryonic antigen.
7. An antibody according to any preceding claim, which is a single-chain Fv, F(ab1)2, Fv or fab.
8. An antibody according to claim 7 , having a heavy chain variable region comprising the amino acid sequence defined in SEQ ID NO. 2 and a light chain variable region comprising the amino acid sequence defined in SEQ ID No. 4, or a variant thereof having at least the same properties determined by the steps defined in claim 1.
9. A polynucleotide molecule encoding an antibody according to claim 8, wherein the polynucleotide comprises a nucleotide sequence defined in SEQ ID Nos. 1 and 3, or a variant thereof.
10. A cloning vehicle comprising the polynucleotide molecule according to claim 9.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9818915.2 | 1998-08-28 | ||
| GBGB9818915.2A GB9818915D0 (en) | 1998-08-28 | 1998-08-28 | Antibodies |
| PCT/GB1999/002729 WO2000012556A1 (en) | 1998-08-28 | 1999-08-20 | High-affinity antibodies |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2340865A1 true CA2340865A1 (en) | 2000-03-09 |
Family
ID=10838074
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002340865A Abandoned CA2340865A1 (en) | 1998-08-28 | 1999-08-20 | High-affinity antibodies |
Country Status (21)
| Country | Link |
|---|---|
| EP (1) | EP1107990A1 (en) |
| JP (1) | JP2002525082A (en) |
| KR (1) | KR20010089174A (en) |
| CN (1) | CN1315966A (en) |
| AU (1) | AU5435099A (en) |
| BG (1) | BG105294A (en) |
| BR (1) | BR9913303A (en) |
| CA (1) | CA2340865A1 (en) |
| EA (1) | EA200100287A1 (en) |
| GB (1) | GB9818915D0 (en) |
| HR (1) | HRP20010149A2 (en) |
| HU (1) | HUP0103233A3 (en) |
| ID (1) | ID28905A (en) |
| IL (1) | IL141523A0 (en) |
| MX (1) | MXPA01002248A (en) |
| NO (1) | NO20011006L (en) |
| PL (1) | PL346472A1 (en) |
| TR (1) | TR200100643T2 (en) |
| WO (1) | WO2000012556A1 (en) |
| YU (1) | YU15401A (en) |
| ZA (1) | ZA200101573B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0031284D0 (en) * | 2000-12-21 | 2001-01-31 | Ks Biomedix Ltd | High affinity antibodies |
| GB0112844D0 (en) * | 2001-05-25 | 2001-07-18 | Psimei Pharmaceuticals Plc | Neutron capture therapy |
| WO2003105757A2 (en) * | 2002-06-12 | 2003-12-24 | Genencor International, Inc. | Methods and compositions for milieu-dependent binding of a targeted agent to a target |
| US20030232401A1 (en) * | 2002-06-12 | 2003-12-18 | Pugia Michael J. | Bacterial test method by glycated label binding |
| EP1691763A4 (en) * | 2003-12-12 | 2008-03-12 | Genencor Int | Cab molecules |
| CN111848790B (en) * | 2020-08-07 | 2022-02-22 | 上海交通大学 | Bovine-derived single-chain antibody for resisting staphylococcus aureus and preparation and application thereof |
| CN112724255A (en) * | 2021-01-28 | 2021-04-30 | 成都金昆生物科技有限公司 | Small molecule antibodies targeting carcinoembryonic antigens |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5081235A (en) * | 1989-07-26 | 1992-01-14 | City Of Hope | Chimeric anti-cea antibody |
| GB9014932D0 (en) * | 1990-07-05 | 1990-08-22 | Celltech Ltd | Recombinant dna product and method |
-
1998
- 1998-08-28 GB GBGB9818915.2A patent/GB9818915D0/en not_active Ceased
-
1999
- 1999-08-20 JP JP2000571062A patent/JP2002525082A/en active Pending
- 1999-08-20 KR KR1020017002434A patent/KR20010089174A/en not_active Application Discontinuation
- 1999-08-20 MX MXPA01002248A patent/MXPA01002248A/en unknown
- 1999-08-20 CN CN99810367A patent/CN1315966A/en active Pending
- 1999-08-20 IL IL14152399A patent/IL141523A0/en unknown
- 1999-08-20 HU HU0103233A patent/HUP0103233A3/en unknown
- 1999-08-20 ID IDW20010714A patent/ID28905A/en unknown
- 1999-08-20 PL PL99346472A patent/PL346472A1/en not_active Application Discontinuation
- 1999-08-20 AU AU54350/99A patent/AU5435099A/en not_active Abandoned
- 1999-08-20 BR BR9913303-2A patent/BR9913303A/en not_active IP Right Cessation
- 1999-08-20 YU YU15401A patent/YU15401A/en unknown
- 1999-08-20 EP EP99940355A patent/EP1107990A1/en not_active Withdrawn
- 1999-08-20 TR TR2001/00643T patent/TR200100643T2/en unknown
- 1999-08-20 EA EA200100287A patent/EA200100287A1/en unknown
- 1999-08-20 CA CA002340865A patent/CA2340865A1/en not_active Abandoned
- 1999-08-20 WO PCT/GB1999/002729 patent/WO2000012556A1/en not_active Application Discontinuation
-
2001
- 2001-02-26 ZA ZA200101573A patent/ZA200101573B/en unknown
- 2001-02-26 BG BG105294A patent/BG105294A/en unknown
- 2001-02-27 NO NO20011006A patent/NO20011006L/en not_active Application Discontinuation
- 2001-02-28 HR HR20010149A patent/HRP20010149A2/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| IL141523A0 (en) | 2002-03-10 |
| PL346472A1 (en) | 2002-02-11 |
| ID28905A (en) | 2001-07-12 |
| BR9913303A (en) | 2001-10-09 |
| NO20011006D0 (en) | 2001-02-27 |
| WO2000012556A1 (en) | 2000-03-09 |
| HUP0103233A3 (en) | 2003-10-28 |
| AU5435099A (en) | 2000-03-21 |
| ZA200101573B (en) | 2002-02-26 |
| TR200100643T2 (en) | 2001-07-23 |
| KR20010089174A (en) | 2001-09-29 |
| HUP0103233A2 (en) | 2001-12-28 |
| EP1107990A1 (en) | 2001-06-20 |
| CN1315966A (en) | 2001-10-03 |
| GB9818915D0 (en) | 1998-10-21 |
| NO20011006L (en) | 2001-02-27 |
| MXPA01002248A (en) | 2002-05-08 |
| YU15401A (en) | 2003-07-07 |
| EA200100287A1 (en) | 2001-08-27 |
| BG105294A (en) | 2001-12-29 |
| HRP20010149A2 (en) | 2002-02-28 |
| JP2002525082A (en) | 2002-08-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Popkov et al. | Rabbit immune repertoires as sources for therapeutic monoclonal antibodies: the impact of kappa allotype-correlated variation in cysteine content on antibody libraries selected by phage display | |
| EP0894135B1 (en) | Multivalent and multispecific antigen-binding protein | |
| US6342587B1 (en) | A33 antigen specific immunoglobulin products and uses thereof | |
| JP3258630B2 (en) | Assays for measuring protein fragments in biological media | |
| Wels et al. | Construction, bacterial expression and characterization of a bifunctional single–chain antibody–phosphatase fusion protein targeted to the human ERBB–2 receptor | |
| Jespers et al. | Guiding the selection of human antibodies from phage display repertoires to a single epitope of an antigen | |
| US7122646B2 (en) | Multivalent and multispecific binding proteins, their manufacture and use | |
| US5837821A (en) | Antibody construct | |
| EP3187505B1 (en) | Production of antibody formats and immunological applications of said formats | |
| Yazaki et al. | Mammalian expression and hollow fiber bioreactor production of recombinant anti-CEA diabody and minibody for clinical applications | |
| Tillib | “Camel nanoantibody” is an efficient tool for research, diagnostics and therapy | |
| US20040220388A1 (en) | Novel heterodimeric fusion proteins | |
| JPH06510671A (en) | Production of chimeric antibodies - combinatorial approach | |
| JPH06508511A (en) | Method for producing specific binding pair members | |
| CN103201293A (en) | Methods for assessing and identifying or evolving conditionally active therapeutic proteins | |
| CN111247429A (en) | Universal reporter cell assay for specific testing of novel antigen binding modules | |
| Schiweck et al. | Sequence analysis and bacterial production of the anti‐c‐myc antibody 9E10: the VH domain has an extended CDR‐H3 and exhibits unusual solubility | |
| IL265995B2 (en) | Triple vector for expressing antibody molecules in full therapeutic format | |
| CA2340865A1 (en) | High-affinity antibodies | |
| Kim et al. | Expression and characterization of recombinant single-chain Fv and Fv fragments derived from a set of catalytic antibodies | |
| CN111492243A (en) | CAR-T cell assay for specific testing of novel antigen binding modules | |
| KR100932465B1 (en) | Humanoid Antihuman IgG Receptor Antibodies and Antibody Fragments | |
| CN110642947B (en) | Anti-human CD147 monoclonal antibody, expression vector, cell strain and application thereof | |
| US20030008355A1 (en) | High-affinity antibodies | |
| KR102511816B1 (en) | c-Myc peptide specific antibody and uses thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |