The past, present and future of therapeutic antibody engineering

Therapeutic antibodies (Abs) are customized biological agents used to diagnose and treat various diseases (such as cancer, inflammatory diseases and autoimmunity), which can activate or inhibit organisms related to specific diseases by binding to specific antigens (Ag) The process of learning, including can be used to prevent the proliferation of cancer cells
.
So far, there are about 711 therapeutic antibodies on the market, 99 of which have been approved by the U.
S.
Food and Drug Administration (FDA) and the European Medicines Agency (EMA)
.
Because therapeutic antibodies have high affinity and specificity, low immunogenicity, and ability to target a series of biomolecules, the development of a new generation of therapeutic antibodies has also received extensive attention from the outside world
.
01 Types of therapeutic antibodies According to the way antibodies are manufactured and created, therapeutic antibodies can be divided into two categories-polyclonal antibodies and monoclonal antibodies
.
Polyclonal antibodies Polyclonal antibodies (pAbs) are produced by a heterogeneous mixture of immune cells and can be obtained directly from the serum of immunized animals
.
Therefore, they can bind to different target epitopes of the same antigen
.
In this mixture, some antibodies bind to the target site, while others bind to off-target epitopes, so the performance of different batches of polyclonal antibodies against the same antigen will be variable
.
The production of polyclonal antibodies is relatively cheap and fast, but once the cells are exhausted, the same batch of antibodies cannot replicate
.
Polyclonal antibodies have high affinity, tolerance to small changes in antibodies (such as slight denaturation, polymorphism, glycosylation heterogeneity, etc.
) and robust detection capabilities, so they have been widely used in research and diagnosis Application
.
Monoclonal antibodies Monoclonal antibodies (mAbs) are purified from cultured cells, so they can specifically bind to unique antibody epitopes
.
Compared with polyclonal antibodies, monoclonal antibodies are expensive and time-consuming to produce, but they also have many advantages, such as high specificity and reproducibility..
In addition, monoclonal antibodies can be continuously produced in unlimited numbers as long as the original cell line is retained.
These characteristics also make them very useful for therapeutic purposes
.
The structure of a typical antibody molecule includes an invariable domain (C) that interacts with immune cells and a variable domain (V) that contains an antigen-binding site
.
In the past 46 years, 4 types of monoclonal antibodies have been developed on the market, and the composition of the two domains of these antibodies is also different
.
It is estimated that in clinical applications, mouse monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and human monoclonal antibodies account for 2.
8%, 12.
5%, 34.
7%, and 51% of all monoclonal antibodies, respectively.
%
.
Due to their low immunogenicity and high tolerance, human and humanized monoclonal antibodies are currently used for clinical treatment more frequently
.
02 Application of therapeutic antibodies At present, therapeutic antibodies are mainly used as research reagents, diagnostic tools and biopharmaceuticals
.
Immunoassay Immunoassay is a biochemical test and diagnostic method that uses antibodies to detect molecules (proteins, hormones, drugs, etc.
), that is, quantitative tests are performed by the binding between the antigen in the target molecule and its antibody
.
Both polyclonal antibodies and monoclonal antibodies can be used for immunoassays
.
The immunoassay methods commonly used for diagnosis are as follows
.
(1) ELISA: is a quantitative test that uses enzyme-conjugated antibodies to detect target molecules, which can be used to diagnose a variety of infectious diseases, such as AIDS
.
(2) LFT: It is a qualitative test, usually consisting of bead-conjugated antibodies of different colors on a piece of paper
.
The research sample is loaded on one end of the strip and flows laterally until it reaches the antibody end
.
This method can be used to diagnose infectious diseases (such as Covid-19), but the most common use of LFT testing is a home pregnancy test
.
(3) Westernblot: It is a semi-quantitative detection method based on gel electrophoresis to separate proteins and use specific coupled antibodies
.
It is widely used in research and is also an effective tool for early diagnosis
.
However, because its manufacturing is a sophisticated, expensive and time-consuming process, this method is not widely used in healthcare
.
(4) Flow cytometry: used to isolate and characterize the different cell types present in a population
.
Use specific fluorescent co-linked antibodies to label different types of cells, and then separate them with a laser beam
.
This method is used in cancer diagnosis to detect the presence of tumor cells in a patient's body
.
Tumors Currently, 44% of all monoclonal antibodies approved by the FDA and EMA are used to treat different types of cancer
.
Monoclonal antibodies can induce cancer cell death through different mechanisms, including neutralization, antibody-dependent cell-mediated cytotoxicity (ADDC) and complement-dependent cytotoxicity (CDC)
.
In addition, monoclonal antibodies can also be conjugated to radioisotopes, toxins, drugs, cytokines or liposomes, so that cytotoxic drugs can be delivered to the affected tissues in a higher local concentration without affecting the normal Cells cause damage
.
Recently, bispecific antibodies have shown great promise in clinical and pre-clinical development
.
They have a targeted tumor-associated antigen binding site and another site that binds to immune cell receptors, so that immune cells can get close to tumor cells and ensure that the immune response is concentrated at the site of the diseased tissue
.
Inflammation and autoimmune diseases Therapeutic monoclonal antibodies can also be used to treat inflammation and autoimmune diseases, including Crohn’s disease, rheumatoid arthritis, asthma, multiple sclerosis, psoriasis, systemic lupus erythematosus and type 1 diabetes and so on
.
So far, 36% of all monoclonal antibodies approved by the FDA and EMA are used to treat different types of inflammation and autoimmune diseases
.
Monoclonal antibodies are designed to bind and neutralize pro-inflammatory factors (such as tumor necrosis factor (TNF), interleukin receptors, integrins, cytokines, and antigens) and prevent them from exacerbating inflammation or immune responses
.
However, the etiology of most inflammatory and autoimmune diseases is complex and unclear
.
At present, most of the drugs and biopharmaceuticals for these diseases have a wide range of effects and effects, and are not disease-specific, so this therapy will cause some undesirable side effects (such as infection and tumor proliferation)
.
Further research on the complex cellular and molecular mechanisms behind these diseases will help design better therapeutic antibodies
.
Infectious diseases Antibodies used to treat infectious diseases began more than 120 years ago when serum from immunized animals was used
.
Although their high specificity makes them a valuable treatment option for many infectious diseases, the production of antibodies is expensive and the market is relatively small
.
Therefore, access to cheaper therapies (such as antiviral drugs or antibiotics) hinders their expansion in this field
.
Despite this, there are still seven monoclonal antibodies that have been approved by the FDA and EMA, one for the treatment of HIV, two for the treatment of inhaled anthrax, one for the prevention of respiratory syncytial virus, and one for the To prevent Clostridium difficile infection, 2 types are used to treat Ebola virus infection
.
Such antibodies account for 5.
6% of all antibodies approved by regulatory agencies
.
03 Antibody Engineering Technology Mouse Monoclonal Antibodies The era of therapeutic monoclonal antibodies began in 1975, when Kohler and Milstein developed hybridoma technology
.
Hybridomas are immortalized B cells formed by the fusion of immune B cells and cancer (myeloma) cells
.
In 1986, Orthoclone OKT3 (Moronoma anti-cd3), the first therapeutic antibody produced using this technology, was approved for organ transplantation
.
Unfortunately, due to high immunogenicity, the therapy was subsequently discontinued
.
Chimeric monoclonal antibody In 1984, Morrison and his colleagues developed a mouse-human chimeric monoclonal antibody using recombinant DNA technology
.
In 1994, the first chimeric therapeutic antibody ReoPro (abciximab) was approved as a platelet aggregation inhibitor
.
Man-made monoclonal antibodies At the same time, scientists are further developing humanized antibodies to overcome immunogenic reactions
.
In 1986, Jones and his colleagues successfully transplanted the antigen-binding site (called the complementarity determining region or CDR) of the antibody from the mouse antibody to the corresponding region of the human antibody
.
In this way, they increased the humanization ratio of antibodies to 85% to 90%, and further reduced the immunogenicity
.
In 1997, the first humanized therapeutic antibody Zenapax (dalizumab) was approved for organ transplantation
.
Human Monoclonal Antibodies The development of fully humanized monoclonal antibodies uses two different techniques
.
The first technique is an in vitro screening technique called phage display, which was developed by Winter and colleagues in 1990
.
By integrating human genes into bacteriophages (a virus that infects bacteria), this technology allows researchers to select monoclonal antibodies with almost any specificity
.
The first therapeutic human monoclonal antibody displayed using phage was approved in 2002, and Humira (adalimumab) is used for the treatment of autoimmune diseases
.
The second technique, developed by Green and his colleagues in 1994, involves the creation of transgenic mice expressing human antibody libraries
.
The first therapeutic monoclonal antibody derived from genetically modified humanized mice was approved in 2006.
Vectibix (panitumumab) is used to treat metastatic colorectal cancer
.
One disadvantage of this technology is that due to antigen processing and imaging of mouse genetic factors regulated by B cells, the monoclonal antibodies obtained may not be as specific as the antibodies naturally produced in humans. .
To overcome this problem, researchers have devised new methods using engineered monoclonal antibodies created by the complete human immune system
.
These technologies include human hybridoma technology and the immortalization of human B cells through Epstein-Barr virus transformation
.
However, these methods are generally not suitable for screening large antibody libraries
.
Bispecific antibodies Bispecific antibodies are designed to bind to two target targets, so they can perform two different functions
.
The first bispecific antibody was developed by Nisenoff and his colleagues in 1960
.
However, it was not until 2009 that the bispecific antibody Removab (catumasumab) was approved in Europe for the treatment of solid tumor patients with malignant ascites
.
For commercial reasons, it was recently withdrawn from the market
.
In 2014, another bispecific antibody, Blincyto (blinatumomab), was approved by the FDA for the treatment of acute lymphoblastic leukemia
.
Nanobodies Nanobodies, also known as single domain antibodies, are small single-chain antibodies from camels and sharks
.
Nanobodies are smaller and more stable than antibodies, and they are simpler and cheaper to produce
.
In 2019, the first therapeutic nanobody Cablivi (caprizumab-yhdp) was approved for the treatment of thrombotic thrombocytopenic purpura
.
Synthetic nanobody (synthetic body) is currently under development and is expected to be used to treat Covid-19 patients
.
04 Monoclonal antibody antibody production technology Hybridoma technology The production of hybridomas begins with the immunity of animals to specific antibodies, and then these B cells are collected and fused with an immortal cell line (usually myeloma cells)
.
These hybrid cells are screened and selected to produce in vitro hybridomas with unique monoclonal antibodies
.
If the cell donor is from a mouse, this technology can produce mouse, chimeric, and humanized monoclonal antibodies; if the cell donor is from a transgenic mouse or human subject expressing human genes, it can produce human monoclonal antibodies.
Cloned antibodies
.
Phage display technology This technology starts with the isolation of B cells from human blood, and then uses PCR to amplify the antibody-encoded gene and insert it into the coat protein gene of the phage
.
These genetically engineered phages display foreign protein antigens on their surfaces and are used to infect bacteria for reproduction
.
In this way, a large phage library displaying different monoclonal antibodies can be created
.
The cells are then screened and specific antigens are used to isolate antibodies
.
This technique may produce any type of antibodies, including toxic substances that cannot be used for animal immunization
.
In recent years, the research community has established other similar display systems on this basis
.
(1) Bacterial display technology: It can rely on the gene fusion of candidate antibodies and bacterial cell surface proteins
.
One advantage of this system is its fast growth rate, easy handling and cost-effectiveness
.
However, the low conversion efficiency hinders its wide applicability
.
(2) Yeast display technology: It can rely on the gene fusion of candidate antibodies and yeast cell surface proteins
.
The system is compatible with fluorescence activated cell sorting (FACS), allowing real-time analysis of candidate libraries
.
However, these libraries tend to be smaller than other display technologies
.
(3) Mammalian display technology: including the use of complex genetic engineering tools, such as CRISPR/Cas9 or transposon technology, to transfect mammalian cells with plasmids encoding candidate antibodies
.
The flow cytometer can also perform real-time analysis of the cell bank
.
One advantage of this system is that the secreted antibodies contain human PTMs, thereby minimizing potential immunogenicity issues
.
However, one limitation of this technology is that the cell growth rate is slower compared to other display systems
.
(4) Ribosome display technology: It is a cell-free in vitro technology that involves using cell extracts (prokaryotes or eukaryotes) to translate the mRNA of candidate antibodies
.
During the translation process, the mRNA remains attached to the ribosome, while the target protein is relatively protruding and folded
.
Then use the immobilized antigen to select mRNA-ribosome-antibody complexes
.
After selection, the mRNA still attached to the complex is reverse transcribed back to cDNA and amplification begins
.
One advantage of this technique is that it is not limited by the conversion efficiency of the host, allowing the generation and screening of larger libraries
.
Among all the monoclonal antibodies obtained by the above-mentioned display technology, phage display technology is still the main method
.
At the same time, several monoclonal antibodies discovered using other display methods are also in clinical trials
.
In 2018, the first monoclonal antibody obtained through yeast display technology was approved in mainland China for the treatment of Hodgkin's lymphoma
.
Genetic engineering cell culture technology In the past few decades, cell culture technology has also made significant progress.
Today, most therapeutic antibodies use genetically engineered immortalized cell lines to produce and stably express human antibody genes.
The Leizi Chinese Hamster Ovary CHO cell line is the most commonly used
.
However, one problem with using non-human cell lines is that recombinant antibodies will contain non-human PTMs that may be potentially immunogenic
.
This is why the use of human cell lines is now becoming the focus of research and development, with the goal of overcoming this shortcoming
.
Table: Comparison of the advantages and disadvantages of monoclonal antibodies produced by different technologies 05 The future and challenges of therapeutic antibodies Since the first therapeutic monoclonal antibody was approved 35 years ago, the development of monoclonal antibodies has experienced unprecedented growth
.
Currently, therapeutic antibodies have become the dominant force in the biopharmaceutical market
.
In 2020, the global monoclonal antibody market is estimated to be 143.
5 billion U.
S.
dollars, and it is expected to grow to 451.
89 billion U.
S.
dollars in 2028, with a compound annual growth rate of 14.
1%
.
However, the development and manufacture of therapeutic antibodies is still a long, difficult and expensive process
.
There are some challenges that must be overcome in this field, and stable antibody products need to be produced in large quantities at a lower cost
.
At present, pharmaceutical manufacturers are also making great efforts to strengthen stability and develop better and more flexible technologies
.
Despite many challenges, therapeutic antibodies will certainly continue to maintain a dominant therapeutic approach and market position in the foreseeable future
.