What Is an Antibody Conjugate?

Antibody-drug conjugate (ADC) is a chemical link between a small molecule drug with a biological activity and a monoclonal antibody. The monoclonal antibody serves as a carrier to target small molecule drugs to target cells ^[1] .

Antibody drug conjugates

Research on antibody-drugconjugate (ADC) dates back to the 1980s, but it was not until 2000 that the first antibody-conjugated drug (trade name Mylotarg, developed by Pfizer) was approved by the FDA for the treatment of acute myeloid leukemia, but Due to limited coupling technology, targeting, and effectiveness, intact antibody-conjugated drugs were unstable in the blood, leading to lethal toxicity, which was withdrawn in 2010. This makes the already unknown ADC drug research even more overshadowed.

However, as Takeda / SeattleGenetics improved the original technology and developed its own new antibody coupling technology using Brentuximabvedotin (SGN-35, trade name Adcetris), a new antibody conjugate drug was approved by the FDA for use in 2011. Treatment of Hodgkin's lymphoma and systemic anaplastic large cell lymphoma. In 2013, the antibody coupling drug made another breakthrough. Ado-trastuzumabemtansine (T-DM1, trade name Kadcyla) jointly developed by Genentech / ImmunoGen was approved by the FDA for HER2-positive breast cancer. This is the first antibody coupling drug for solid tumors. . With the successful development of these two drugs, ADC drugs once again entered the field of research in a fiery state.

Antibody drug conjugates

ADC drugs use specific linkers to connect antibodies and small molecule cytotoxic drugs. Their main components include antibodies, linkers, and small molecule cytotoxic drugs (SM). Antibody molecules mainly play a targeted delivery role, and small molecule drugs work. However, some antibodies also have anti-tumor pharmacodynamic effects, such as ado-trastuzumab and maitansine (maytansine) in Kadcyla.

Due to the complex structure of ADC drugs, there are large differences between different ADC drug designs. Even for different drugs at the same target, the differences in toxicity are obvious due to the different recognition sites, linking sites, linkers, and small molecules connected. Therefore, before evaluating ADC drug toxicity, it is important to understand the design of the ADC drug. The ideal ADC drug design needs to consider antibody selection: clear targets, high expression of tumor cells, low expression of normal tissues; support drug loading, stability, and ability to internalize cells; good PK characteristics; less non-specific binding; connection Site: Generally there are lysine or cysteine residues, which can be restructured for directional coupling; Linker: stable in the circulation, can be released in the cell (such as release in lysosome digestion, or antibody Release after degradation); Cytotoxic drugs: highly pharmacodynamic effects, non-immunogenicity, and can be combined with linkers through modification, the mechanism is clear ^[2] .

Antibody drug conjugates III. Mode of action

1. Circulation, ADC drugs are administered by systematic exposure. Firstly, they enter the blood system. Since the relative molecular mass of the antibody is about 150 ku and the relative molecular mass of the cytotoxic drug is about 1 ku, the nature of the ADC drug circulation is basically the same as " "Naked" antibodies are consistent.

2. Binding antigen, the antibody recognizes specific antigenic sites, this process will determine the specificity of targeted delivery.

3. Internalization. After the antibody binds to the receptor on the cell membrane, endocytosis occurs.

4. Drug release, the linker is cleaved in the intracellular lysosome to release small molecule cytotoxic drugs.

5. Play the role of drugs, small molecule cytotoxic drugs work, kill tumor cells.

In addition to the common ADC drug designs described above, some ADC drug designs are not internalized, but are released extracellularly and function in the local microenvironment.

Antibody drug conjugates 4. Risk of toxicity

1. Antibody drug conjugates 1. antibody molecules

At present, antibody molecules for ADC drugs often use humanized monoclonal antibodies to modify the crystallizable fragment (Fc) segment to reduce antibody-dependent cell-dependent cell-mediated cytotoxicity (ADCC) and complement dependency Sexual cytotoxicity (complement dependent cytotoxicity, CDC) and so on. Firstly, antibody molecules, as biological macromolecules, have toxicity risks of general biological macromolecules, such as immunogenicity and immunotoxicity, as well as possible ADCC effects, CDC effects, and renal basement membrane immune complex deposition of monoclonal antibodies. Secondly, in ADC drugs, the most important role of antibody molecules is targeting, that is, targeted delivery of small molecule compounds to antigen-antibody binding sites. If the antibody is poorly selected or the antigen is present in normal tissues, cytotoxic drugs will be delivered to normal cells, resulting in targeted toxicity.

Because ADC drugs are highly targeted, they can cause serious toxic consequences. Third, in addition to targeted toxicity, shedding of small molecules during cycling can lead to a certain degree of off-target toxicity. If the Fc of the antibody molecule has the activity of binding to immune cell Fc receptors such as FcRs / FcRN, it is easy to bind to immune cells and cause the killing of immune cells. Finally, ADC drugs, as exogenous biological macromolecules, may also phagocytize cells in the circulation and enter the cells through cell-drinking to cause cell death.

2. Antibody drug conjugates 2. linker

Linkers commonly used in ADC drugs mainly include hydrazone bonds, disulfide bonds, and peptide bonds. The amidine bond can be hydrolyzed under acidic conditions and is a relatively unstable linker. Mylotarg uses the key as a linker, which researchers believe is an important reason for Mylotarg's failure [2]. Disulfide bonds can be hydrolyzed in a high concentration of glutathione in the cell, so it is not easy to fall off extracellularly. Peptide bonds are most tightly bound, and cleavage occurs only under the action of a lysosomal proteolytic enzyme. The stability of the linker directly affects the unintended dissociation of the cytotoxic drug. This breakage causes the exposure of small molecule cytotoxic drugs in the body, that is, off-target toxicity.

3. Antibody drug conjugates 3. Cytotoxic drugs

Cytotoxic drugs are chemotherapeutic drugs that are routinely used in clinical practice and determine the main toxic effect spectrum of ADC drugs. Since it has been widely used clinically, its toxicity characteristics are generally clear. Depending on the type of drug, such as a tubulin polymerization inhibitor or a DNA damaging agent, the risk of toxicity can be better grasped.

4. ADC Antibody drug conjugates 4. ADC molecules

After the antibodies and small molecule cytotoxic drugs are integrated into a whole through the linker, drug-antibody ratio (DAR) can significantly affect the toxicity of ADC drugs. It is generally believed that the therapeutic window for binding 2 to 4 toxic compounds to each antibody is the largest. If the DAR is too low, the efficiency of antibody carrying is low; on the other hand, if the DAR is too high, the body can easily recognize it as a foreign body and quickly clear it. The current ADC drugs are generally a mixture of different DARs. Due to the inconsistent toxicity caused by different DARs, the heterogeneity of DARs will lead to uncertainty in toxicity. There may also be cytotoxic drugs that are not bound to antibodies in this DAR mixture, causing off-target toxicity. The addition of linkers and small molecule cytotoxic drugs to antibodies may cause changes in antibody polymerization, lysis, and spatial structure, which may cause the antibodies to exhibit inconsistent characteristics with naked antibodies, resulting in unexpected toxicity.

5.ADC Antibody drug conjugates 5. ADC drug common toxicity

The FDA conducted a summary analysis of the 20 new ADC research invesligational new drugs (IND), and found that the toxicity of ADC drugs in animals is mainly hematopoietic system toxicity, liver toxicity and reproductive toxicity, and some of them are still skin Toxicity and nephrotoxicity. Among them, the toxicity of hematopoietic system, liver and reproductive system is directly related to small molecule cytotoxic drugs, and renal toxicity may be secondary damage to the immune response caused by antibodies [6]. The clinical adverse effects of approved drugs such as Adcetris and Kadcyla are mainly the bone marrow suppression-related effects (neutropenia, sepsis and bleeding), liver toxicity and renal toxicity caused by small molecule cytotoxic drugs. In addition, some patients develop injection-related toxicity, which may be related to the administration of macromolecular protein components.

Antibody drug conjugates V. Development trends

1. Antibody drug conjugates 1. Directional coupling technology

At present, ADC drugs that are at the forefront of development use traditional no-specific conjugation. The biggest disadvantage is that the obtained product is a mixture of different antibody molecules per antibody); a specific position cannot be achieved. More importantly, it is difficult to obtain uniform data (eg, PK) in clinical evaluation. In response to these shortcomings, directional coupling technology has become a hot spot for major companies to chase. Using the directional coupling technology, each antibody can carry the same number of drug molecules to obtain a homogeneous ADC drug. Conducive to the research and evaluation of pharmacodynamics. And can obtain more stable and effective results in clinical. Among them, Ambrx's UnaturalAminoacid (pAcPhe) technology is more applicable and promising.

2.ADC Antibody drug conjugates 2. Multivalent coupling ADC drugs

The development of antibody drugs and vaccines has evolved from monovalent drugs to multivalent drugs. ADC should also follow this development process, that is, linking several small molecules that cooperate with each other in the same antibody to improve the drug's efficacy. This requires more perfect even-chain technology, to the need to integrate the use of two or more technologies. But now, in Site-specific technology, the coupling of a specific number of molecules at a specific site is excessively pursued, and the diversity of coupling is ignored.

Practical traditional technology for multivalent drug coupling requires multiple drugs to be coupled to one antibody at the same time. At this time, the uniqueness of the antibody's modification of the linking group will cause a mixed product, which cannot guarantee that each antibody carries a different drug.

This problem can be solved by Site-specific technology. When performing Site-specific modification, a variety of different coupling groups can be designed. This allows a group to be used for drug coupling to linkers with corresponding groups. Link. Finally, multiple drugs are linked through linker diversification to achieve multivalent coupling of ADC drugs.

Monoclonal antibodies and their conjugates are macromolecular substances. Large drug molecules are difficult to reach through the capillary endothelial layer and through the extracellular space of the tumor to the depth of the solid tumor. The use of antibody fragments, such as Fab and Fab , to prepare conjugates with smaller molecular weights may increase the penetration into the extracellular space and increase the amount of drugs reaching deep tumor cells. "Miniaturization or moderate miniaturization is an important way to develop ADC drugs."

Antibody drug conjugates

Due to the advantages of clear targets, mature technology, and good selectivity, the study of antibody-drug conjugates is expected to continue to become a research focus in the field of anticancer in the next few years [68, 69]. To keep up with the pace of new drug research and development in the world, domestic and especially those powerful pharmaceutical companies must join the ADC research and development trend. Of course, this area has high technical requirements and a relatively narrow space for intellectual property rights, and companies such as Seattle Genetics have been operating in this area for decades. It is difficult to occupy a place without certain technical support. This is also Pfizer, Roche, Eli Lilly, etc. The root reason why international pharmaceutical giants choose to buy mature technologies or patents such as Seattle Genetics, ImmunoGen. The principle of ADC research and development is clear, but technology and experience are important, especially the design of connectors is almost an art. The opportunity to enter this huge market must have a skill.

Despite decades of R & D history, ADC still has huge room for improvement. In fact, the results of several studies show that the ratio of ADC compounds to effector molecules is much less than 1%, and the optimistic estimate is only 1.5%. Many people are disappointed with this number. Even so, the targeted drug delivery is still much higher than the traditional systemic drug delivery mode, and the incidence of adverse reactions is also significantly lower than that of traditional drugs. In turn, it leaves us more room to design a new generation of antibody-drug conjugates. Because it has been clinically proven that the current ADC drugs have brought unprecedented effects to patients, then the new generation of ADC drugs may be better.

How to enter ADC This huge market in the future requires comprehensive consideration of various factors including the financial strength, technical experience and research team of each company. For those companies with independent intellectual property rights in the field of monoclonal antibodies, the acquisition of mature effector molecules and linkers can jump into the forefront of ADC research. Having unique effector molecules is another ideal entry point into ADC research. Of course, the unique new connector will get more space. For example Mersana Therapeutics designed a new joint concept called Fleximer. The water-soluble linker uses a biodegradable polyvalent polysaccharide derivative, so that the contact point of each antibody can connect dozens of drug molecules. And because the polysaccharide is water-soluble, it avoids the self-aggregation of antibodies and other pharmacokinetic defects caused by too many drugs connected. In order to solve the confusion caused by the number of effector molecules or insufficient activity. Of course, the clinical manifestations of this Fleximer connector need further investigation.

Like the development of new connectors, any other technology that can effectively improve the characteristics of ADCs is an entry point into this field. For example, the position and coupling of effector molecules and antibodies are also critical to the stability of the ADC. The introduction of specific amino acid residues at specific positions for coupling is another entry point. Ambrx Pharmaceuticals has a place in this space. In addition to improving antibody stability, it also plays a key role in ADC homogeneity and mass production. Ambrx, Sutro Biopharma, Genentech and Pfizer all lead the field.

Finally, successful ADC drug design must not only optimize each ADC component, but also the way and details of connecting each part are equally important, including the linker and the antibody or the isolation region between the effector molecules ^[3] .