Anti-Her2 Antibody Stability Modification And Function Optimization

Background:Antibody drug is a key area of the drug research and development. As a kind of recombinant protein, therapeutic m Abs are chemically unstable, susceptible to a variety of chemical and physical degradation reactions during manufacturing, formulation storage and in vivo usage. Asparagines(Asn) deamidation and aspartate(Asp) isomerization are the most common pathways for the chemical degradation of m Abs, which could affect the stability, biological functions, bioavailability and immunogenicity of a therapeutic m Ab. To improve the stability of an antibody is very important for their clinical application.In addition to improve the stability, optimization of antibody’s effector function is also an important research direction to enhance the clinical curative effect of an antibody. Though therapeutic antibody has strong antigen targeted ability, because of its large molecular weight, single drug is limited in the treatment of cancer, especially for the treatment of solid tumor. Cytotoxic drugs have high killing effect on cancer cells, but lack of targeted ability, often injure normal cells, thus cause serious side effects. Antibody drug conjugates(ADC) use the antibody as a carrier to deliver the "bullet" drug to the target cell, and then enter the lysosomes by endocytosis effect, release of cytotoxic drugs, so as to achieve the specific killing of tumor cells without damaging normal tissue cells. ADC, combine the target-specificity of monoclonal antibody(m Ab) and the highly active cell-killing drugs, taking advantages of the best characteristics out of both components, has become an important development direction for improving antibody effector function.Recombinant monoclonal antibody Herceptin of Genetech was the first approved m Ab drug for the treatment of HER2-overexpressing metastatic breast cancer(MBC). The 2014 global sales of Herceptin® reached $6.8 billion. Presumably, due to its chemical instability, the current marketed Herceptin® drug product is supplied in freeze-dried powder. It has been reported that the potential deamidation and isomerization in its CDR region may be the main factor affecting the stability of Herceptin®.Objective:The purpose of this study is try to create a new candidate of Herceptin® with improved stability profile without sacrificing its biological potency, so that its drug product can be delivered in liquid formulation format. On this basis, the antibody drug conjugates(ADC) were further constructed.The effect of different candidates; small molecule drugs and ADC with different conjugate mode were compared.The conclusion will highlight the development of the novel ADC drug.Methods:First, with the assistance of computer-guided modeling method, the deamidation and isomerization hotspots of Herceptin® and the mutation strategy were determined; using site directed mutagenesis, a new candidate of Herceptin® was made by replacing LC-Asn30 and HC-Asp102 with LC-Gln30 and HC-Glu102, respectively; then obtained the mutant, named as T-m Ab2, through eukaryotic expression and affinity purification, and identified the mutation sites by mass spectrometry and peptide mapping; the physicochemical properties of T-m Ab2 were analyzed comprehensively with HPLC, CE-SDS, ic IEF; further evaluated its biological activity by FACS, ELISA, in vitro cytotoxic and in vivo antitumor activity in xenograft animal model; and the long time, acceleration and high temperature stability profiles of T-m Ab2 under different p H conditions were assessed side-by-side with T-m Ab1; then the stability samples were analyzed by SEC, IEC, peptide mapping and mass spectrometry; in the end analyzed the biological activities of the ADCs which were formed by connected T-m Ab1, T-m Ab2 and T-m Ab3(De novo design and made in our lab having the same function and epitope with T-m Ab1) with the toxin molecule DM1, MMAE respectively, through the in vitro binding activity and cell cytotoxicity assay.Results:1、Based on the distance geometry, computer graphics technology, the key interaction domain between antibody T-m Ab1 and antigen HER2 was predicted with the 3-D modeling complex structure; Using the computer-guided molecular modeling and docking method; reasonably optimized and mutated the potential deamidation hotspots in the variable region of the light chain(LC-CDR1-Asn30) and isomerization hotspots in the variable region of the heavy chain(HC-CDR3-Asp102) were present; The structure of the new antibody T-m Ab2 and the complex structure of T-m Ab2 were constructed; The theoretical prediction results of T-m Ab1 and T-m Ab2 interact with HER2 model indicated that, compared with T-m Ab1, the stability of the T-m Ab2 was improved, while the binding activity with HER2 was decreased.2、Results of mass spectrometry and peptide mapping indicatied that the two degradation hotspots LC-CDR1-Asn30 and HC-CDR3-Asp102 of T-m Ab1 were successfully mutated to LC-CDR1-Gln30 and HC-CDR3-Glu102(T-m Ab2); Physical and chemical properties analysis of the antibody showed that T-m Ab2 and T-m Ab1 have similar levels of polymer, the same isoelectric variants, but charge level of T-m Ab2 was significantly lower than that in T-m Ab1;3、The in vitro Biacore affinity analysis indicated that the HER2 antigen binding rates of T-m Ab2 and T-m Ab1 were similar, but the dissociation rate of T-m Ab2 was approximately 5 times faster than T-m Ab1, leading to its affinity was weaker than that of T-m Ab1(4.7 n M vs 1 n M); In vitro binding activity(FACS, ELISA) results indicated that T-m Ab2 can specific bind to HER2 antigen, but its binding activity was weaker than T-m Ab1(about 3 times); T-m Ab2 can effectively kill breast cancer cell line BT474, but the killing activity was weaker than T-m Ab1; There were no statistically significant differences in antitumor efficacy among Herceptin, T-m Ab1 and T-m Ab2, though statistically significant tumor regression was observed when compared to the negative control group in SKOV3 Xenograft Model mice.4、Under different stressing conditions: temperature varied from 4℃ to 25℃, and p H values from 5.1, to 6.0 and 6.3, the stability profiles of T-m Ab1 and T-m Ab2 were similar; at the high temperature of 40℃, under all three p H conditions, their size heterogeneity showed no apparent differences, but there were significant differences of their charge variants; Acidic charge variant(AP2) caused by deamidation of LC-CDR1-Asn30 was not detected in T-m Ab2; while in T-m Ab1, there were obvious AP2 at p H 6.0 and 6.3, and with the increase of p H, the level of AP2 increased significantly; Acidic charge variant(AP1) existed both in T-m Ab1 and T-m Ab2, showing no significant difference, and only under p H 5.1conditon can be detected; The basic charge variant(BP1) generated by isomerization of HC-CDR3-Glu102 was not detected in T-m Ab2, and there was obvious BP1 in T-m Ab1, and with the increase of p H, the level of BP1 gradually decreased. Moreover, compared with T-m Ab1, there were no new modifications in T-m Ab2.5、There were no significant differences for the binding activity with breast cancer cells SKBR3 among the ADC formed by conjugating the naked antibody T-m Ab2 and T-m Ab3 with DM1 and MMAE; but the binding activity decreased when conjugated T-m Ab1 with MMAE, while the binding activity did not change significantly when conjugated T-m Ab1 with DM1; The binding results with gastric cancer cell N87 indicated that compared with the naked antibody, the binding activity of ADCs formed by conjugating T-m Ab1, T-m Ab2 and T-m Ab3 with the two toxin were all decreased, and T-m Ab1 decreased the most obviously;T-m Ab1-DM1, T-m Ab2-DM1 and T-m Ab3-DM1 all had significant cytotoxicity against SKOV3 and SKBR3, and there were no significant differences, but the cytotoxicity was significant higher than that of against the human gastric cancer cell line N87T-m Ab1-MMAE, T-m Ab2-MMAE and T-m Ab3-MMAE all had significant cytotoxicity against SKOV3 and SKBR3, and there were no significant differences; and the killing activity of three kinds of ADC antibody in gastric cancer cells was higher than that of SKOV3 and SKBR(about 3 times);In SKOV3 and SKBR3, the cytotoxicity of the same antibody conjugated with different toxin had no significant differences; but in N87, ADC antibody formed by conjugated with MMAE was significant higher than that of the ADC formed by conjugated with DM1, is about 12 times, 42 times and 28 times higher, respectively.Conclusion:1、After mutation of two deamidation and isomerization hotspos of Herceptin, its stability has been improved significantly, although its in vitro Her2-target binding activities were decreased slightly, its in vivo antitumor activity wasn’t affected.2、The cytotoxicity of ADC formed by antibody conjugating with small molecules DM1, MMAE had certain cell selectivity; The cytotoxicities on SKOV3 and SKBR3 of ADC formed by the three antibodies conjugating with DM1 were higher than that of on N87; whereas the cytotoxicity on N87 of ADC formed by the three antibodies conjugating with MMAE was higher than that of on SKOV3 and SKBR3; meanwhile the cytotoxicity on gastric cancer cells of ADC antibody formed by conjugating with MMAE was significant higher than that of the ADC formed by conjugating with DM1Innovation of this research:In this work, we preliminarily studied the potential strategies to improve the stability of the antibody with the help of computer virtual screening. Based on the strategy, took the HER2 targeted antibody Herceptin as the model, we created a new antibody T-m Ab2, whose stability was improved significantly meanwhile maintained the in vivo biological activity of the maternal antibody. The improved stability of the antibody is conducive to its preservationThis study also systematically evaluated the effect on the biological activity of the andibody by conjugating with different toxin. The results suggested that the biological activities of the antibody-drug conjugates(ADCs) had certain cell selectivity.