KR20010089174A - High-affinity antibodies - Google Patents

Abstract

High affinity monochromal antibodies, wherein the affinity is characterized by (Iii) Incubate the first and second samples of antibody in antigen-coated microtiter wells at concentrations selected to be selected within the linear portion of the standard curve at pH7.2. (Ii) Remove unbound antibody from both samples. (Iii) The first sample is incubated with PBS at pH 7.2 for 1 hour at 37 ° C. (iv) Remove unbound antibody from both samples. (v) Incubate both samples with anti-antibody alkaline phosphatase-conjugate for 1 hour at 37 ° C. (vi) Remove unbound antibody from both samples. (vii) Add PNPP substrate to the sample. The absorbance of the sample is measured at 405 nm and the amount of antibody bound to the antigen is determined. Wherein the bound amount in the second sample is greater than 50% of the bound amount in the first sample.

Description

High affinity antibody {HIGH-AFFINITY ANTIBODIES}

Antibodies have long been regarded as potential potent means in the treatment of cancer or other diseases. However, with some important exceptions, this possibility is generally not yet realized.

The relatively lack of success is, at least in part, due to the use of monoclonal antibodies derived from rodents, which have little affinity greater than 10 ⁻⁹ M. Antibodies with this level of affinity are limited in therapeutic use because it is known that it is difficult to deliver sufficient antibodies to a target so that useful biological activity is performed. Antibodies bound to antigens are reversible, and dissociation is more advantageous than association at antibody concentrations practical for in vivo use. In general, increasing the antibody concentration can prevent dissociation of the antigen. However, this can cause unacceptable clinical side effects and increase the cost associated with treatment.

The present invention relates to antibodies and their therapeutic uses.

1 shows the results obtained for the acid resistance of single-chain Fvs with amounts at various pH values and affinity for mouse monoclonal antibodies and cancer embryonic antigens.

The present invention is based on the understanding that an antibody, or fragment thereof, that is "acid resistant" can be produced and that this property is related to the high affinity binding of the antibody to its antigen.

According to the present invention, high affinity antibodies have affinity characterized by the following.

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

(Ii) removing unbound antibody from both samples;

(Iii) the first sample was incubated for 1 hour at 37 ° C. in PBS at pH 7.2;

(Iii) remove unbound antibody from both samples;

(Iii) both samples were incubated with anti-antibody alkaline-phosphatase conjugates at 37 ° C. for 1 hour;

(Iii) remove the unbound conjugate in both samples; And

(Iii) add PNPP substrate to the sample, measure the absorbance of the sample at 405 nm, and determine the amount of antibody bound to the antigen, wherein the amount bound in the second sample is greater than 50% of the amount bound in the first sample do.

Preferably, the maximum pH in step (iv) is preferably 2.5, more preferably 2.0.

Antibodies or antibody fragments having “acid resistance” characteristics are expected to favor association over dissociation, and therefore they have a longer localisation time at the target site, which results in higher concentration of antibody integrated at the target site.

In particular, the present invention relates to the preparation of high affinity single-chain Fv antibody fragments. This ScFv has the particular advantage of allowing better targeting at location in vivo.

Acid resistant monoclonal antibodies according to the invention can be obtained using a variety of techniques. For example, classical cell-cell technology can be applied, which consists of fusing B-lymphocytes from an immunized animal secreting a high affinity antibody with an appropriate fusion partner. An alternative method is to purify mRNA from selected lymphocytes and use PCR to expand the required antibody genes. Phage exposure and other techniques for exposure of antibody fragments can also be used to obtain antibody genes from a library that has been saved or immunized after appropriate selection processes.

The antibody gene may be co-expressed or otherwise chemically linked with toxins, radioisotopes or enzymes or other desirable molecules to provide a fusion protein with strong binding properties. More optionally, the antibodies can be produced by transgenic animals as described in US-A-5770429.

The antibody may be a complete antibody consisting of heavy and light chains, and constant and variable regions. Optionally, the antibody is an antibody fragment, such as, for example, F (ab ') ₂ , Fab, Fv, or single chain Fv fragments, if at least some of the variable portions that provide "acid resistance" properties are present. Antibodies are animal, chimeric or humanized antibodies. Suitable methods for generating humanized antibodies are described in WO-A-92 / 15699.

In a preferred embodiment of the invention, the antibody is a single chain Fv fragment. Single chain Fv fragments include both heavy and light chain variable regions linked by suitable peptides.

Antibodies of the invention can be defined by their acid resistant properties, and acid resistance can be characterized by acid-washed enzyme-linked immunosorbent assay (EIA), as described above. Typically, the A ₄₀₅ value obtained with EIA indicates greater than 50% antibody binding for the sample at pH 3 or lower, compared to the value obtained for the sample at pH 7.2. Preferably, the sample A ₄₀₅ value at pH 2 will exhibit antibody binding greater than 60%, preferably greater than 70% of the value obtained at pH 7.2.

Immunized animals are not rodents and are selected to provide higher affinity antibodies. Any large mammal can be used and suitable animals can include rabbits, goats, cattle and sheep.

Antibodies of the invention can be used in therapy and formulated in a suitable composition with physiologically acceptable excipients, diluents or delivery agents.

The following examples illustrate the invention.

Example 1

Amounts were immunized with cancer embryonic antigens in complete Freund's supplements and then promoted three times with antigens in incomplete Freund's supplements. After promotion of final efficacy and removal of lymphadenomas, animals were sacrificed.

Lymphoma cells were then washed and fused with both xenomyeloma fusion partner SFP3.2. Fusion cells were plated in a total density of about 10 ⁶ / ml in medium containing HAT (Life Technologies). These samples were then screened for blast cells secreting high affinity antibodies against specific antigens using both normal EIA and acid-wash EIA.

Standard EIA screening was performed as follows:

MaxiZob assay plate (NUNC) was coated with 100 μl / well of CEA (0.4 μg / ml in phosphate buffered saline at pH 7.2) and left overnight at 4 ° C. The plates were then washed three times using 0.01% Tween 20 wash and pH 7 phosphate buffered saline. Reaction sites remaining on the plates were blocked by adding 200 μl / well of 0.2% fat-free milk protein in PBS, pH 7.2 for 1/2 hour at 37 ° C. It was then washed in PBS as above and 45 μl of antibody sample was added to the wells of the plate. The sample was incubated at 37 ° C. for 1 hour and then washed as above. Bound antibodies were detected using alkaline phosphatase-conjugated donkey anti-sheep antibody (Sigma A5187 diluted 1/5000 with 1% BSA in PBS at pH 7.2). The plates were then washed and 100 μl / well of PNPP (Sigma N2770) solution was added. Absorbance was measured using a spectrophotometer at 405 nm using phosphate buffered saline as a control.

Acid-wash EIA screening was performed as follows.

Antibody samples were coated and bound as described for the standard EIA above. However, after incubation with antibody samples, the plates were washed and 200 μl / well of HCl (10 mM stock) at pH 2 was added at 37 ° C. for 1 hour. After washing three times, the remaining antibody bound to the antigen was detected using alkaline phosphatase-quenched donkey anti-sheep antibody and PNPP as above. For accurate comparisons between antibodies at various concentrations, each sample was selected to have an A ₄₀₅ of about 1.0 in normal EIA (ie, in the linear response of the EIA curve).

Three apoptotic cells (1D2, 6G11 and 6H9) secreted antibodies with greater than 50% retention of binding in acid washed EIA compared to binding in non-acid washed EIA.

Example 2

Single chain Fv fragments were produced from the above-mentioned neutrophil 6H9 as follows.

mRNA was purified from oligomeric cells using oligo-dT cellulose. Single chain DNA (cDNA) complementary to mRNAd was synthesized by reverse transcription. Reverse transcription reactions were isolated to synthesize cDNA for antibody variable regions using universal primers designed from the constant regions of the positive heavy and light chain antibody genes.

The cDNA was then amplified by polymerase chain reaction to make double chain DNA using primers designed from heavy and light chain variable skeletal sequences. The heavy and light chain portions were amplified using the split polymerase chain reaction. The product was then analyzed by agarose gel electrophoresis and DNA bands equivalent to the light and heavy chain genes were cleaved from the gel and purified. Equimolar amounts of variable heavy and light chain DNA were mixed with the oligonucleotide linker DNA. Linker DNA was encoded for the amino acid sequence (Gly ₄ Ser) ₃ using additional nucleotides complementary to the 3 'end of the heavy chain variable portion and the 5' end of the light chain variable portion. Three DNA molecules were denatured, annealed and expanded in the first step of the two-step PCR reaction (without primers) to link fragments, thereby assembling single chain Fv.

Single chain Fv DNA was amplified in the second step of PCR using a pair of primers derived from the heavy and light chain variable termini with restriction enzyme recognition sites for AlW 44i and Not I. Single chain Fv gene products were analyzed and purified by agarose gel electrophoresis. The single chain Fv gene was then digested with restriction enzymes AlW 44i and Not I and cloned into expression vectors. The vector was then used to modify E. coli HB 2151 and allowed for protein expression to occur. The vector was designed to include a hexa-histidine tag at the COOH end of the SFv. Single chain Fv was purified using nickel-chelate affinity chromatography and analyzed by SDS-PAGE. The amino acid sequences for the heavy and light chain variable regions were found as SEQ ID NOs: 2 and 4, respectively. Acid-wash EIA was performed to determine the acid resistance properties of single chain Fv.

Acid-wash EIA was performed as follows.

Cancer embryonic antigen (CEA) -coated microtiter plates were prepared as described above. Single chain Fv samples (6H9) were diluted to concentrations ranging from 1 ng / ml to 100 ng / ml in PBS at pH 7.2 containing 1% bovine serum albumin (BSA). 100 μl of sample was added to the microtiter wells and incubated at 37 ° C. for 1 hour. The plate was then washed, 200 mL / well citrate was added and the plate incubated for 1 hour at 37 ° C. In this case, acids were prepared by diluting to pH values 4.0, 3.5, 3.0, 2.5 and 2.0 using 100 mM citrate stock in the reaction mixture. PBS at pH 7.2 was used as a reference control. Plates were then washed and 100 μl / well of mouse anti-tetra-histidine antibody (Qiagen) (100 ng / ml diluted in PBS at pH 7.2 with 1% BSA) was added and incubated for 1 hour at 37 ° C. After washing the plates, the samples were incubated for 1 hour at 37 ° C. with 100 μl / well of goat anti-mouse alkaline phosphatase conjugate (Sigma A3688 diluted 1/1000 in PBS with 1% BSA at pH 7.2). The plate was then washed, treated with PNPP as above, and the absorbance measured at 405 nm using a spectrophotometer.

As a control for acid resistance, sFv samples were incubated with PBS at pH 7.2 to create EIA response curves for sFv samples. In the linear section, the concentration of 10-20 ng / ml of the sFv sample was used to determine the amount of antibody bound to the acid washed sample as a percentage of the amount absorbed (A ₄₀₅ ) and 1.0 to 1.5 in the reference sample. .

The acid resistance properties of the 6H9 complete antibody and 6H9 single chain Fv were compared to those for mice induced anti-embryogenic antigen complete antibody, A5B7 and single chain Fv MFE. The results are shown in FIG. 1 with antigen-binding of antibodies induced in mice that are substantially reduced at pH 3.5 and less than 5% at pH 2.5. In contrast, 6H9 antibodies have more than 70% antigen at pH 3.5, more than 60% antibody at pH 2.5, and more than 50% antibody at pH 2.0.

Claims (10)

(Iii) incubating the first and second samples of antibody for 1 hour at 37 ° C. in antigen-coated microtiter wells at a concentration selected at pH 7.2 within the linear portion of the standard curve; (Ii) removing unbound antibody from both samples; (Iii) the first sample was incubated for 1 hour at 37 ° C. in PBS at pH 7.2; (Iii) remove unbound antibody from both samples; (Iii) both samples were incubated with anti-antibody alkaline-phosphatase conjugates at 37 ° C. for 1 hour; (Iii) remove the unbound conjugate in both samples; And (Iii) add PNPP substrate to the sample, measure the absorbance of the sample at 405 nm, and determine the amount of antibody bound to the antigen, wherein the amount bound in the second sample is 50% of the amount bound in the first sample A high affinity monochromal antibody characterized by exceeding affinity. The antibody of claim 1, wherein the amount of bound antibody in the second sample is greater than 60% of the bound amount in the first sample. The antibody of claim 1 or 2, wherein the pH is reduced from pH 2.5 to pH 2.0 in step (iii). The antibody of any one of the preceding claims, wherein the antibody is non-rodent. The antibody of claim 1, wherein the antibody has affinity for a tumor-associated antigen. The antibody of claim 5, wherein the antigen is a cancer embryonic antigen. The antibody of any one of the preceding clauses wherein the antibody is single chain Fv, F (ab ') ₂ , Fv or Fab. 8. The antibody of claim 7, wherein the heavy chain variable portion consisting of the amino acid sequence identified by SEQ ID NO: 2 and the light chain variable portion consisting of the amino acid sequence identified by SEQ ID NO: 4 or a modification thereof. A polynucleotide molecule encoding an antibody according to claim 8, wherein the polynucleotide consists of a nucleotide sequence identified by SEQ ID NOs: 1 and 3 or a modification thereof. A cloning vehicle comprising the polynucleotide molecule of claim 9.