BG105294A - High affinity antibodies - Google Patents

Abstract

High-affinity monoclonal antibodies, wherein the affinity is characterised by: i) including 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 degrees C; ii) removing unbound antibody from both samples; iii) including the first sample with PBS at pH 7.2 for 1 hour at 37 degrees C, and reducing the pH of the second sample to pH 3 or below and incubating for 1 hour at 37 degrees C; iv) removing unbound antibody from both samples; v) incubating both samples with anti-antibody alkaline phosphate-conjugate for 1 hour at 37 degrees C; vi) removing unbound conjugate from both samples; and vii) adding PNPP substrate to the samples, measuring the absorbance of the sample at 405 nm, and determining the amount of antibody bound to antigen, wherein the amount bound to the second sample is >50 % of that of the first sample. 10 claims, 1 figure

Description

HIGH AFFINITY ANTIBODIES

FIELD OF THE INVENTION

The present invention relates to antibodies and their use as therapeutics.

Background of the Invention

Antibodies have long been referred to as potentially potent agents for the treatment of cancer and other diseases. However, although there are some noticeable deviations, this potential is not yet fully realized.

This relative failure may be due, at least in part, to the use of rodent-derived monoclonal antibodies that rarely have an affinity greater than 10 ' ⁹ M. Antibodies having this affinity level are of limited therapeutic utility, insofar as it has been proven difficult to deliver sufficient antibody to the target to induce useful biological activity. The binding of an antibody to an antigen is reversible, and at antibody concentrations especially for in vivo administration, dissociation will prevail over the association. In general, it is possible to enumerate the antigen dissociation by increasing the antibody concentration. However, this can lead to unacceptable clinical side effects and would also increase the cost of therapy.

General of the invention

The present invention is based on the fact that antibodies or fragments thereof that are " acid-resistant " can be produced and this property is associated with the high affinity of binding of an antibody to its antigen.

According to the present invention, a high affinity antibody has an affinity characterized by:

(i) incubating the first and second antibody samples into antigen coated wells of a microtiter plate at a concentration selected within the linear portion of a standard curve at pH 7.2 for 1 hour at 37 ° C;

(ii) removing the unbound antibody from both samples;

(iii) incubating the first sample with PBS at pH 7.2 at 37 ° C and reducing the pH of the second sample to pH 3 or lower and incubating for 1 hour at 37 ° C;

(iv) removing the unbound antibody from both samples;

(ν) incubating the two samples with an anti-antibody alkaline phosphatase conjugate for 1 hour at 37 ° C;

(vi) removing the unbound conjugate from both samples; and (vii) adding the PNPP substrate to the samples, measuring the absorbance of the samples at 405 nm, and determining the amount of antibody bound to the antigen, where 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, especially 2.0.

Antibodies or antibody fragments with "acid-resistant" properties are expected to promote association rather than dissociation and therefore have a longer time of localization at the target sites, resulting in a higher concentration of antibodies localized at these sites.

In particular, the invention relates to the production of a high-affinity fragment of a single-stranded Fv antibody. This ScFv has certain advantages with the fact that it allows better targeting of a site in vivo.

Description of the Drawing

Figure 1 illustrates the results achieved for acid resistance of sheep and mouse monoclonal antibodies and single-stranded Fvs affinity for carcinoembryonic antigen at different pH values.

Description of the invention

Acid-resistant monoclonal antibodies of the present invention can be prepared by various techniques. For example, a classic hybridoma technique may be employed that involves the fusion of B-lymphocytes from immunized animals secreting high-affinity antibodies with a suitable fusion partner. An alternative method is to purify the mRNA from selected lymphocytes and use them in the PCR technique to amplify the required antibody genes. Phage and other techniques for demonstrating antibody fragments can also be used to obtain antibody genes from natural or immunized libraries following appropriate selection procedures.

The antibody gene may also be co-expressed with or otherwise attached to toxins, radioisotopes or enzymes or any other desired molecules to provide a fusion protein with well-defined binding characteristics. In another alternative, antibodies can be produced by transgenic animals as described in US-A5770429.

The antibody may be a whole antibody comprising heavy and light chains, and constant and variable regions. Conversely, the antibody is an antibody fragment, called F (ab ') ₂ > Fab, Fv, or single-stranded Fv fragments, providing the presence of at least a portion of the variable region that provides the acid resistance property. The antibody may also be an animal, chimeric or humanized antibody. A suitable method for producing humanized antibodies is described in WO-A92 / 15699.

In a preferred embodiment of the invention, the antibody is a single-stranded Fv fragment. The single-stranded Fv fragment includes both heavy chains and light chain variable regions associated with a suitable peptide.

The antibodies of the present invention can be determined by their acid-resistant properties, which can be characterized by acid-washed enzyme-linked immunosorbent assay (EIA) as described above. Typically, the A450 value obtained by EIA will be an antibody binding of> 50% for a sample at pH 3 or lower compared to the sample value at pH 7.2. Preferably, the A4 value of ₅₀ per sample at pH 2 will represent antibody binding of > 60% and especially 70% of that obtained at pH 7.2.

The immunized animal is not a rodent, but is selected to give higher affinity antibodies. Each large mammal can be used and suitable animals include rabbits, goats, cows and sheep.

An antibody of the invention may be used for treatment and may be included in any suitable composition with a physiologically appropriate excipient, solvent or carrier.

The invention is illustrated by the following examples:

Example 1.

Sheep were immunized with carcinoembryonic antigen (CEA) in Freund's complete adjuvant and then immunized three times with antigen in Freund's incomplete adjuvant. Animals are sacrificed after final immunization and the lymph glands are removed.

The lymph gland cells were then washed and fused with sheep heteromyeloma fusion partner SFP3.2. The fused cells were seeded at a total density of approximately 10 ⁶ per ml in medium containing HAT (Life Technologies). These samples are then screened for hybridomas secreting high affinity antibodies to the specific antigen using both normal EIA and acid washed EIA.

Standard EIA studies are performed as follows:

Maxisorb research plates (NUNC) were covered with CEA (0.4 gg / ml in phosphate buffer solution at pH 7.2), 100 μΙ per well and left overnight at 4 ° C. The plates were then washed three times with 0.01% Tween 20 detergent. All other reactive sites on the plates were blocked by adding 200 μΙ per well of 0.2% fat-free milk protein in PBS at pH 7.2 at 37 ° C for% h. The plates were then washed in PBS as described above and 45 μΙ of antibody samples were added to the wells of the plates. Samples were incubated for one hour at 37 ° C and then washed as described previously. The bound antibody was then detected using an alkaline phosphatase conjugate with donkey anti-sheep antibody (Sigma D5187, diluted 1/5000 in PBS at pH 7.2 with 1% PBS). The plates were then washed and 100 μΙ added per well of PNPP solution (Sigma N2770). The absorbance was measured using a spectrophotometer at 405 nm with a phosphate buffer solution as a control.

Acid-washed EIA tests are performed as follows:

Covering and binding of antibody samples was performed as described for the standard EIA above. However, after incubation with the antibody samples, the plates were washed and 200 μΙ were added per well of HCl (10 mM Stock solution) at pH 2 for one hour at 37 ° C. After three washes, the antibody remaining bound to the antigen was detected by an alkaline phosphatase conjugate with donkey anti-sheep antibody and PNPP as described above. To ensure that a real comparison is made between antibodies at different concentrations, each sample is selected to give an A405 value of approximately 1.0 in the normal EIA (ie, in the linear portion of the EIA response curve).

Three hybridomas (1D2, 6G11 and 6H9) secrete antibodies that give more than 50% retention of binding in acid-washed EIA compared to binding in non-acid-washed EIA.

Ί

Example 2.

The single-stranded Fv fragment is produced by the 6H9 hybridoma above as follows:

The mRNA was purified from cultured hybridoma cells using oligodT cellulose. Single stranded DNA complementary to mRNA (cDNA) is synthesized by reverse transcription. Universal primers created from the constant regions of sheep heavy and light chain antibody genes are used in separate reverse transcription reactions to synthesize cDNA for antibody variable regions.

The cDNA is then amplified by the polymerase chain reaction to produce double stranded DNA using primers created from heavy and light chain variable sequence sequences. Individual polymerase chain reactions were used to amplify the heavy and light chain regions. The products were then analyzed by agarose gel electrophoresis and DNA bands equivalent to light and heavy chain genes were excised from the gel and purified.

Equimolar amounts of variable heavy and light chain DNAs are mixed together with oligonucleotide linker DNA. The linker DNA encodes the amino acid sequence (Gly ₄ 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 are denatured, despiralized and expanded in the first step (without primers) by the two-step PCR reaction, so that the fragments are joined together, thus forming a single-stranded Fv.

Single-stranded Fv DNA is amplified in the second step of

PCR using a pair of primers obtained from the end of the heavy and light chain variable regions with the addition of recognition sites by the restriction enzymes AIW441 and Notl. The single-stranded Fv gene product was analyzed by gel electrophoresis and purified. The single-stranded Fv is then digested with the restriction enzymes AIW441 and Not1 and cloned into an expression vector. The vector was then used to transform E. coli HB 2151, and the expression of the protein was allowed to flow. The wind is designed to include a hexahistidine tail at the COOH end of SFv. Single-chain Fv was purified by nickel-chelate affinity chromatography and analyzed by SDS-PAGE. The amino acid sequence for the heavy chain variable region and the light chain variable region are described in SEQ ID NOs: 2 and 4, respectively. An acid-washed EIA is also performed to determine the acid resistance property of single-stranded Fv.

The acid-washed EIA is performed as follows:

Carcinoma-embryonic antigen (CEA) coated microtiter plates were prepared as previously described. Single-stranded Fv samples (6H9) were diluted to a concentration range of 1 ng / ml to 100 ng / ml in PBS at pH 7.2 containing 1% calf serum albumin (BSA). Add 100 μΙ of the sample to the wells of the microtiter plate and incubate for 1 hour at 37 ° C. The plates were then washed, 200 μΙ of citrate per well were added and the plates were incubated for 1 hour at 37 ° C. In this case, the acidic preparations are obtained using a stock solution of 100 mM 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 control. The plates were then washed and added 100 μΙ per well of mouse anti-tetra-histidine antibody (Qiagen) (100 ng / ml diluted in PBS at pH 7.2 with 1% BSA) and incubated for 1 hour at 37 ° C. . After washing the plates, the samples are incubated for 1 hour at 37 ° C with 100 μΙ for a well of conjugate of goat anti-mouse alkaline phosphatase. (Sigma A3688, diluted 1/1000 in PBS with BSA at pH 7.2). The plates were then washed, treated with PNPP as previously described, and the absorbance measured using a spectrometer at 405 nm.

As a control for acid resistance, sFv samples were incubated with PBS at pH 7.2 to generate the EIA response curve for sFv samples. In a linear section, a concentration of 10ΣΟ ng / ml of sFv gives an absorbance (Aws) of 1-0-1.5 and is therefore used to determine the amount of antibody bound in acid-washed samples as a percentage of the amount bound in the reference sample.

The acid resistance properties of the 6H9 complete antibody and 6H9 single-stranded Fv were compared with that obtained from the murine anti-carcinoma membrane antigen, A5B7 and single-stranded Fv MFE. The results are shown in Figure 1, with antigen binding to mouse antibodies substantially reduced at pH 3.5 and less than 5% at pH 2.5. In contrast, 6H9 antibodies have> 70% antigen at pH 3.5,> 60% at pH 2.5, and> 50% at © pH 2.0.

Claims (10)

1. A high affinity monoclonal antibody, wherein the affinity is characterized by: (i) incubating the first and second antibody samples into antigen coated wells of a microtiter plate at a concentration selected within the linear portion of a standard curve at pH 7.2 for 1 hour at 37 ° C; (ii) removing the unbound antibody from both samples; (iii) incubating the first sample with PBS at pH 7.2 for one hour at 37 ° C and reducing the pH of the second sample to pH 3 or lower and incubating for 1 hour at 37 ° C; (iv) removing the unbound antibody from both samples; (v) incubating the two samples with an anti-alkaline phosphatase conjugate for 1 hour at 37 ° C; (vi) removing the unbound conjugate from both samples; and (vii) adding the PNPP substrate to the samples, measuring the absorbance of the samples at 405 nm, and determining the amount of antibody bound to the antigen, where the amount bound in the second sample is> 50% of that of the first sample. The antibody of claim 1, wherein the amount of antibody bound in the second sample is> 60% of that bound to the first sample. The antibody of claim 1 or claim 2, wherein the pH in step (iii) is reduced to pH 2.5 to pH 2.0. An antibody according to any one of the preceding claims, which is not derived from a rodent. An antibody according to any one of the preceding claims, which has an affinity for a tumor-associated antigen. The antibody of claim 5, wherein the antigen is a carcinoembryonic antigen. An antibody according to any one of the preceding claims, which is single-stranded Fv, F (ab ') 2, Fv or fab. The antibody of 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 variants thereof. ! The polynucleotide molecule encoding the antibody ί according to claim 8, wherein the polynucleotide includes the nucleotide sequence defined in SEQ ID Nos. 1 and 3 or variants thereof. j A cloning medium comprising the polynucleotide The мо molecule of claim 9.