Preparation And Preliminary Study On Biological Activities Of Several Anti-CD20Antibody-Druy Conjugates

Cytotoxic anticancer drugs can not only kill tumor cells, but also kill normal cells, resulting in obvious limitations of their further application and developments. However, biotherapeutics such as antibodies against tumor-specific antigens often lack therapeutic activity. Antibody-drug conjugates (ADCs), which consist of toxic drugs and mAbs, deliver the drug to the target cells specifically. Thus, ADCs can effectively improve the drug concentration in the site of tumor, and greatly reduces the drug concentrations in other organizations so as to reduce side effects. In1958Mathe coupled anti-rat leukocyte immunoglobulin protein and methotrexate for targeted therapy for the first time, that opened the prelude of the development of antibody-drug conjugates (ADCs). But in the past few decades, none of ADCs in esearch is effective and safe enough to get FDA approved until recently. Since the US FDA approved the first ADCs, that is a humanized anti-CD33conjugated to calicheamicin (gemtuzumab ozogamicin;Mylotarg), for the treatment of acute myeloid leukemia in2000, many ADCs are being actively pursued to combat different types of cancer. A variety of antibodies such as anti-Her2mAb Herceptin, BR96, anti-CD20mAb Rituximab (RTX), anti-CD30mAb, anti-CD33mAb, anti-CD79mAb and various cytotoxic drugs such as Dolastatins(MMAE), doxorubicin (DOX),Calicheamicin, Maytansine(DMl, DM3, DM4) are used to construct different kinds of ADCs. Researches on antibody modification,development of innovative cytotoxic drugsand use of various linkers are currently under progress. Until now, more than20ADCs are in various phases of clinical trials. brentuximab vedotin and T-DM1were approved by FDA in2011, and2013, respectively.CD20is a B-cell specific surface protein expressed on mature B lymphocytes. CD20is an appealing target for monoclonal antibody (mAb) therapy, because of its overexpression on the cell membrane of most of the B-lymphocytic lymphomas. In fact, efficacy of several anti-CD20mAbs for the treatment of B-cell lymphoma has been studied for long to be achieved. The binding of mAbs to the CD20antigen on the cell surface may directly induce apoptosis, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC)However, anti-CD20mAb showed some limitations for CD20-positive B-lymphoma cell lines due to its modest efficacy:only some patients respond to the treatment, and some are initially unresponsive to it or later resist to it; Complete esponses are low; Easy to relapse. Therefore, improving the ability of anti-CD20antibody to achieve effective tumor cell killing becomes an urgent problem in clinical research, and antibody-drug conjugates seem to meet this demand. Indeed, it has been reported that conjugation of RTX with MMAE or Calicheamicin could improve the activity of RTX. However, whether this method is also applicable to many other anti-CD20antibodies is unknown to our best knowledge.To improve the activity of anti-CD20antibody and confirm the suitability of the method, we chose several anti CD20antibodies, Rituximab (RTX), Ofatumumab (OFA) and TGLA for conjugation studies. RTX (Chimeric Antibody) was the first anti-CD20mAb approved by FDA for the treatment of non-Hodgkin’s B-cell lymphomas (B-NHL).Ofatumumab (OFA) is the first fully human anti-CD20mAb for patients with chronic lymphocytic leukemia (CLL).TGLA, which shares the same epitope with RTX, exhibiting similar efficacy with rituximab on CDC, cell growth arrest and so on. We first chose TGLA and DOX, MMAE, AP3(a kind of maytansine) for the study of conjugation and activity. For the validation of the method, we also chose one drug and the two above mentioned anti-CD20Abs (RTX and OFA). Toxic drugs were conjugated to antibodies either through lysine side-chain amines or through cysteine sulfhydryl groups which were activated by reducing interchain disulfide bonds. A cysteine was then introduced by site-directed mutagenesis in the constant region of Ab for defined stoichiometry of the conjugated drugs site-specifically, so that affinity, CDC and ADCC activity is well kept. Our researches provide theoretical and experimental basis for the preclinical discovery of anti-CD20ADCs with high efficiency and low toxicity.1. Modification of anti-tumor small moleculesAt present, the drugs used in ADCs are focused on MMAE, Maytansine (AP3), Calicheamicin, doxorubicin (DOX) and so on. Before conjugation, those drugs need to be modified. We chose the toxic drugs doxorubicine and AP3which were commonly used in ADCs researches as the conjugated drugs, and modification were then performed.MC-VC-PABC-DOX uses the linker MC-VC-PABC.This linker is stable in blood and gets cleaved by lysosomal enzymes, therefore releasing active drug in target cancer cells. The PABC part could couple to-OH group or-NH2group of drugs, and the MC part could react with-SH group of an antibody. MC-VC-PABC-DOX was synthetized by a method reported by Gene M. Dubowchik et al. Fmoc-Val-OSu, L-Citrulline (L-Cit), p-aminobenzyl alcohol (PABOH),6-Maleimidohexanoic acid N-hydroxysuccinimide ester (MC-OSu) and so on were used to construct MC-VC-PABC-DOX. The reactions were monitored by MS and H-NMR and the end-product could release free drug DOX after enzymolysis.Maytansinol DM1was synthetized according to patents. Firstly, AP3was reduced to APO. After re-esterification and reduction, APO was transformed into DM1. DM1can be conjugated to Abs through several linkers.2. Synthesis and biological activity of traditional antibody-drug conjugatesAfter the modification of small molecule drugs, we chose anti-CD20antibody TGLA as the carrier,-vcDOX/-vcMMAE/DM1as the conjugated drugs to explore the conjugation conditions and cytotoxicity of conjugates.Cysteine sulfhydryl groups were activated by reducing interchain disulfide bonds of TGLA with DTT. In theory, there would be8-SH groups,2light chains each with1-SH group and2heavy chains each with3-SH groups. In this research, about6DOX were conjugated to each antibody in average after the reaction. SDS-PAGE indicated that most of the light chains had been conjugated with DOX, nevertheless, the heavy chains were separated on SDS-PAGE for the heavy molecule weight. Compared with Anti-CD20Ab, the anti-CD20-vcDOX conjugates exhibited an enhanced effect on killing the CD20-positive cells (Raji cells). Since the cytotoxicity of DOX is not potent enough, the increased cytotoxicity of ADCs is not significant.The conjugation condition for vcMMAE was the same with vcDOX, and MMAE/Ab ratio was about6.4.The ratio of DM1/TGLA for TGLA-SPDP-DM1was about3.5. The DM1was released from ADCs in the presence of DTT. The ratio of DM1/TGLA for TGLA-MCC-DM1was about2.9. MMAE and DM1ADCs significantly improved the activity of TGLA in vitro, while DOX which owns lower toxicity than MMAE or DM1only resulted in a slight increase in cytotoxicity of TGLA. This result suggested us to choose highly toxic drugs as conjugated drugs.3. Study of different anti-CD20-vcMMAE conjugatesStudies on ADCs usually focus on a selected Ab and various drugs or linkers.There is almost no report demonstrating the difference caused by different antibodies against the same antigen. Here, we synthesized three anti-CD20-vcMMAE conjugates: Ofatumumab-vcMMAE, Rituximab-vcMMAE and TGLA-vcMMAE, and then evaluated the affinity, CDC, internalization and cytotoxicity of these three types against CD20-positive human B lymphoma cells, namely, Raji, Daudi and WIL2-S cells.As a result, conjugation of anti-CD20mAbs with MMAE caused a modest decrease in the binding affinity to cell surface CD20. All anti-CD20-vcMMAE conjugates were modulated from the cell surface and subsequently entered the lysosomes through the CD20receptor-mediated endocytosis. In addition, the conjugation of MMAE significantly improved the cytotoxic activity of all anti-CD20mAbs against CD20-positive cell lines. The results of Annexin V/PI staining and WB demonstrate that treatment of CD20-positive tumor cells with anti-CD20-vcMMAE conjugates could induce apoptotic cell death through a caspase-3-like protease-dependent pathway. We also observed variable cytotoxicity between the anti-CD20mAbs (i.e., OFA, RTX and TGLA) with the same MMAE conjugated. OFA-vcMMAE exhibited great anti-tumor activity in xenograft models of CD20-positive lymphoma where OFA, equivalent free MMAE or irrelevant Herceptin-vcMMAE was ineffective. These results stimulated us to choose OFA as the carrier to carry out mutation and conjugation.4. Construction and expression of2F2and its mutant Thio-2F2In the traditional study, ADCs were synthetized by conjugating cytotoxic drugs to antibodies either through lysine side-chain amines or through cysteine sulfhydryl groups activated by reducing interchain disulfide bonds. Both procedures bring some disadvantages:1) The reaction of interchain disulfide bonds or surface amine groups may result in structural changes in Abs, sabotaging binding affinity of the antibody.2) Traditional procedures yield heterogenous products, containing a mixture of species with different molar ratios of drug to antibody. To solve the problems Jagath R Junutula et al. in Genentech have engineered reactive cysteine residues at specific sites in the constant region of antibodies to allow drugs to be conjugated with defined stoichiometry without disrupting the interchain disulfide bonds. One such ADCs exhibits high efficacy, low side effect and long half-time in vivo. Here we used the method reported by Jagath R Junutula to modify and conjugate OFA, hoping to improve the direct toxic effect under the condition of keeping the affinity, CDC and ADCC activity of antibody on tumor cells. We found DNA sequences of OFA in the US2004/0167319A15patents (namely2F2) and then construct the2F2gene carriers and stably express the Ab in CHO. Meanwhile, we performed mutation on the heavy chain constant region of2F2, that is changing an alanine (A) into a cysteine (C)(Thio-2F2). At last we proved the affinity and CDC activity of2F2and Thio-2F2preliminarily.5Synthesis and biological activity of Thio-2F2-vcNWe conjugated vcMMAE to the specific sites in the constant region of Thio-2F2with defined stoichiometry. Experiments confirmed the mutation and conjugation had little influence in affinity, CDC, ADCC activity of2F2. In addition, the conjugation of MMAE significantly improved the cytotoxic activity of Thio-2F2against CD20-positive cell lines. Thio-2F2-vcMMAE exhibited great anti-tumor activity in xenograft models of CD20-positive lymphoma at doses where primary antibody and mutant antibody were ineffective.The most important discoveries in this paper1. We synthesized various anti-CD20-drug conjugates and obtained several unreported high-activity conjugates, and demonstrated that the conjugation of high-toxic drugs could significantly improve the cytotoxic activity of anti-CD20mAbs against CD20-positive cell lines. Here, we built a technology platform of constructing anti-CD20-drug conjugates.2. We explored the mechanism of various anti-CD20-vcMMAE conjugates and found that the cytotoxicity was different between the anti-CD20mAbs (i.e., OFA, RTX and TGLA) after conjugation with the same toxic drug, broadening the design ideas of the ADCs. The new ADC OFA-vcMMAE showed excellent anti-tumor activity in vitro and in vivo. It completely inhibited the growth of two lymphoma xenografts and the recurrence rates were very low.3. We designed a2F2mutant which could conjugate drugs at specific sites and with defined stoichiometry, ensuring the homogeneity of the products; Experiments confirmed the mutation and conjugation had little influence in affinity, CDC, ADCC activity of2F2. In addition, the conjugation of MMAE significantly improved the cytotoxic activity of Thio-2F2. This method provides a reference for the designs of other antibodies or proteins-drugs conjugates.