Abstract: Since the establishment of hybridoma technology in the 1980s, antibody preparation technology has made significant progress. Antibodies are a type of immunoglobulin secreted by B lymphocytes, which can produce many biological activities due to their specific binding to receptors, such as classical blocking, neutralizing activity, activation of complement, killing of target cells through Fc receptors, and regulation of immune activity. They are widely used in clinical treatment. The development of antibody technology has gone through a long process of mouse derived monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully humanized monoclonal antibodies. In the transition from mouse derived antibodies to fully human derived antibodies, various breakthroughs in biotechnology have been achieved, such as antibody library technology, humanized mouse technology, and B cell cloning technology. Nowadays, antibody drugs hold a pivotal position in the entire drug market. In the past decade (January 2011 to November 2021), 78 monoclonal antibody drugs have been approved by the FDA for market use, widely distributed in the fields of oncology, immune diseases, anti pathogen infections, nervous system and metabolic diseases. The article reviews the progress of monoclonal antibody technology and the market situation of antibody drugs, providing ideas for the preparation of new antibodies and the selection of drug targets.

As early as 1975, K ö her and Milstlein fused mouse B cells with myeloma cells to develop B lymphocyte hybridoma technology, laying the foundation for the development of monoclonal antibodies [1]. The development of monoclonal antibody technology has gone through multiple stages. Early monoclonal antibodies were all mouse derived monoclonal antibodies. As a heterologous protein, these antibodies produce human anti mouse antibodies (HAMA) in the human body, which can affect therapeutic efficacy and cause many toxic side effects, thus limiting their use [2]. Subsequently, the structure of mouse monoclonal antibodies was modified by replacing the constant region of mouse antibodies with human gene sequences, thus designing human mouse chimeric antibodies; Further use of human derived complementarity determining regions (CDRs) base sequences to replace mouse derived CDR sequences to prepare humanized antibodies. With the development of modern biotechnology and a deeper understanding of antibody structure, humanization of all base sequences has been achieved, and fully humanized monoclonal antibodies have been prepared.

1 Monoclonal antibody technology

1. 1 Mouse derived monoclonal antibody

Mouse derived monoclonal antibodies originated in 1975 when K ö hler and Milstein used Sendai virus to fuse mouse myeloma cells and mouse spleen cells to form hybridoma cells [1]. This hybridoma cell not only secretes antibodies, but also has the characteristic of immortalization, which is of milestone significance in the field of biotechnology.

1. 2 chimeric antibody

The variable region (V) of an antibody is a site that specifically recognizes antigens, while the constant region (C) is not related to antigen recognition. In the late 1980s, in order to address the toxic side effects such as HAMA caused by the use of mouse derived monoclonal antibodies in humans, scientists replaced the constant region gene sequence of mouse derived monoclonal antibodies with that of humans to prepare human mouse chimeric antibodies.

Analysis and distribution of key technologies for sterile preparations, on-site verification

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Li Gui et al. [3] reported that the use of human mouse chimeric anti-CD20 monoclonal antibody (SCT400) has achieved good results in the treatment of non Hodgkin lymphoma. Zhang et al. reported that Metuzumab has anti non-small cell lung cancer effects in mice, rats, and crab eating monkeys. Metuzumab is a human mouse chimeric IgG1 monoclonal antibody against CD147 [4].

1. 3 humanized antibodies

Although chimeric antibodies humanized the constant region sequence, they still retained approximately 30% of the mouse derived sequence. Further research on the variable region of antibodies revealed that regardless of the light or heavy chain, antigen-specific recognition is mainly determined by six CDRs, while other sequences in the variable region only serve as scaffolds and are conserved. Transplanting mouse derived CDR sequences onto the human antibody backbone results in the formation of humanized antibodies.

In a phase 1B clinical trial [5], the team led by Guo Jun from Peking University Cancer Hospital confirmed that Toripalimab combined with Axitinib has a significant effect on the treatment of metastatic mucosal melanoma. Toripalimab is a humanized anti-PD-1 IgG4 monoclonal antibody, while Axitinib is a vascular endothelial growth factor (VEGF) inhibitor. The combination of the two has achieved preliminary efficacy in the treatment of advanced melanoma. Another study confirmed that once a week subcutaneous injection of humanized bispecific antibody Emicizumab (AVE910) can significantly reduce the bleeding rate in patients with severe hemophilia A, reduce the severe burden caused by factor VIII input, and decrease the formation of anti factor VIII antibodies [6].

1.4 Whole human monoclonal antibodies

At the end of the 20th century, scientists attempted to prepare antibodies using sequences from all human sources, resulting in the birth of all human monoclonal antibodies. The current human antibody technology mainly includes antibody library technology, humanized mouse technology, and B cell cloning technology.

1. 4. Antibody library technology;    Antibody library technology refers to the amplification of the complete set of antibody variable region sequences in B cells through DNA recombinant cloning technology, and then splicing the precursor sequence of prokaryotes to the amino end of the antibody molecule variable region. Due to the similar environment of prokaryotes' periplasmic reticulum to eukaryotic cells' rough endoplasmic reticulum, antibody molecule fragments can fold in the periplasmic reticulum to form heterodimeric structures with antigen binding ability, thereby screening for antibodies with specific binding ability. After in-depth research on antibody expression systems, phage display technology, yeast display technology, ribosome display technology, and mammalian cell surface display technology have gradually emerged.

As early as 1985, Smith first inserted exogenous genes into the III gene of filamentous bacteriophage f1, creating phage display technology [7]. When screening for human epidermal growth factor receptor 2 (HER2), Yun S et al. found that by immobilizing HER2 on silica coated magnetic beads (MPs) for positive screening, the affinity of the screened HER2 was significantly improved [8]. Shukla GS et al. reported for the first time a new method for screening specific binding antibodies in stage IV cancer patients using intravenous infusion of ScFv single chain antibody phage libraries. They successfully found that clone 07-2355 has high homology with previously reported IL-17A, providing a new approach for in vivo screening of phage libraries [9].

In 1997, Boder and Wittrup established the yeast display system for the first time by recombining exogenous genes with the C-terminal coding sequence of lectins. Zhao JZ et al. [10] reported that using the variable region of anti infectious hematopoietic necrosis virus antibodies as a targeted display protein, yeast derived ScFv was displayed on the surface of yeast, and then E. coli derived ScFv was also displayed on the same yeast through artificial anchoring, which significantly increased the total ScFv level on the yeast surface.

Hanes and Pl ü ckthun first established ribosome display technology (RDT) in 1997 [11]. Compared with phage display technology and yeast display technology, ribosome display technology has the advantages of simple library construction, large library capacity, and simple screening methods. Lagaut et al. [12] successfully screened protein molecules with high affinity for CTX-M15 using ribosome display technology.

Akamatsu was the first to use mammalian cell surface display technology for screening and successfully obtained specific antibodies [13]. Bruun et al. reported the successful screening of antibodies 447-52D and HGN194 with high affinity for Env using a chimeric HIV-1 Env model library. This technique can guide the identification of Env variants with optimized antigen properties and can be used as candidate vaccines [14].

1. 4. 2-person humanized mouse technology;   Humanized mouse antibody technology refers to the use of genetic engineering techniques to replace immunoglobulin genes in animals with human immunoglobulin genes, thereby directly expressing fully humanized antibody proteins in animals. In 1983, Brinster et al. injected the rearranged functional human immunoglobulin kappa gene into mouse oocytes through microinjection to prepare transgenic mice that could specifically express immunoglobulin kappa gene in the spleen, opening up research on humanized mice in the field of immunology [15].

The first generation of humanized mice was reported by Br ü ggemann in 1989 [16], which directly transferred human immunoglobulin genes into animal bodies. Generally, only gene fragments below 5000kb can be transferred, and the regulation of the transferred gene fragments is limited, and the rearrangement diversity is also restricted [17]; The second generation of humanized mice is based on the first generation, using techniques such as embryonic stem cells and in vivo homologous recombination to transfer complete human immunoglobulin genes into the mouse body multiple times, which is beneficial for antibody rearrangement selection; Due to the influence of the transferred human gene sequence on the development of mouse B cells, antibody type conversion, and antibody affinity maturation; The third-generation humanized mice only transferred human immunoglobulin V, D, and J regions to replace mouse immunoglobulin V, D, and J regions, solving the problem of early B cell development [18]. After secondary modification, fully humanized antibodies were formed.

The current humanized mouse technology is relatively mature, and more and more fully humanized antibodies prepared using this technology are being approved for marketing. Several companies worldwide have successfully cultivated relatively mature humanized mouse strains, such as XenoMouse from Cell Genesys, UltiMab from Medarex in the United States, H2L2 and HCAB mice from Harbour, OmniMouse from OmniAb, VelocImmune mice from Regen eron in the United States, etc. [19-22].

1. 4. The preparation of antibodies by single B cell sorting using 3 B cell cloning technology refers to the use of specific antigen markers for staining from samples of human peripheral blood or bone marrow, and the use of flow cytometry sorting technology to screen out single B cells, which are then lysed, reverse transcribed, nested PCR, or 3&# 39; The RACE (Rapid amplification of cDNA ends) technique amplifies the heavy and light chain sequences of antibodies, and then recombines them into suitable vectors for expression.

Rudkin FM et al. [23] reported the preparation of monoclonal antibodies against Candida using a single human B cell, which has a protective effect against the spread of Candida. Alberto C et al. [24] reported that single B cell technology was used to amplify the heavy and light chain gene sequences of antibodies from a human survivor infected with Ebola virus (EBOV), and expressed to form a monoclonal antibody mAb114 with neutralizing ability. In 2019, novel coronavirus was reported to be prevalent among people, and has caused worldwide transmission. The receptor binding domain (RBD) of spike glycoprotein (S) on the surface of the virus can bind to human angiotensin converting enzyme 2 (ACE2), leading to serious respiratory diseases and pneumonia. Wu et al. [25] reported that using single B cell antibody amplification technology, fully human monoclonal antibodies B38 and H4 were isolated from a recovering patient, which could block the binding of virus S protein RBD and cell receptor ACE2. The antibody was confirmed in a mouse model to reduce the viral titer of infected lungs, thus having broad therapeutic prospects.

2 Approval and marketing status of two monoclonal antibody drugs

From 2011 to 2021, 78 monoclonal antibody drugs were approved for market by the US FDA in the past decade. Among them, there are 32 humanized monoclonal antibodies, accounting for 41% 03% ; 29 types of monoclonal antibodies from human sources, accounting for 37% 18% ; Five types of human mouse chimeric antibodies, accounting for 6% 41% ; In addition, there are 10 types of antibody drug conjugates (ADCs), accounting for 12.82%, and 2 types of bispecific antibodies, accounting for 2.56% (Figure 1). With the development of modern biotechnology and the in-depth research of basic medicine, the application of antibody drugs has covered the fields of tumor, immune disease, pathogen infectious diseases, metabolic disease, genetic disease and nervous system disease. More and more targets recognized by monoclonal antibody drugs have been reported, such as immune checkpoint inhibitors PD-1, PD-L1, CTLA4, tumor associated antigens GD2, HER2, VEGFR, cytokines and chemokines IL-6, IL-17A, IL-23, CD19, CCR4, etc. [26-29] (Table 1).

2.1 Tumor field

The field of tumor treatment has always been the main direction of monoclonal antibody drug development. In the past decade, 34 monoclonal antibody drugs for treating tumors have been approved for market, accounting for 43% of the total monoclonal antibody drugs on the market 59%. The types of tumors treated with monoclonal antibodies mainly focus on breast cancer, myeloma, B-cell lymphoma, bladder cancer and melanoma, in addition to neuroblastoma, squamous cell carcinoma, hairy cell leukemia, soft tissue sarcoma, non small cell lung cancer, gastric cancer, neuroblastoma, Hodgkin's lymphoma and non Hodgkin's lymphoma. Among them, the therapeutic targets of monoclonal antibodies for breast cancer are mainly HER2, including Margetu - ximab, Fam trastuzumab deruxtecan nxki, Ado trastuzumab emtansine and Pertuzumab, as well as the ADC drug Sacituzumab govitecan hziy targeting Trop-2. The types and targets of monoclonal antibodies used to treat myeloma are quite diverse, including the ADC antibody drug Belantamab mafodotin blmf targeting BCMA, the human mouse chimeric antibody Isatuximab targeting CD38, the humanized antibody Elotuzumab targeting SLAMF7, and the fully humanized antibody Daratumumab targeting CD38. Monoclonal antibodies for treating B-cell lymphoma include Tafasitamab cxix, a humanized antibody targeting CD19, Polatuzumab vedotin piiq, an ADC antibody drug targeting CD79b, and Pembrolizumab, a humanized antibody targeting PD-1. It is worth noting that on December 3, 2014, the FDA approved the antibody drug Blinatumomab for the treatment of B-cell lymphoma. This antibody drug is the first approved bispecific antibody drug that targets both CD19 and CD3 [30]. Antibody drugs for bladder cancer include the whole human antibody Durvalumab and humanized antibody Atezolizumab targeting PD-L1, and the ADC drug Enfortumab vedotin-ejfv targeting Nectin-4. Monoclonal antibody drugs for treating melanoma were launched earlier, all of which are immune checkpoint inhibitors, including the PD-1 targeted human antibody Nivolumab launched on December 22, 2014, and the CTLA-4 targeted human antibody Ipilimumab launched on March 25, 2011.

In addition, monoclonal antibody drugs for treating more than ten other types of tumors have been approved for market, targeting different therapeutic targets and antibody types. For example, the humanized antibody Naxitamab gqgk targeting GD2 (for treating neuroblastoma) and the human mouse chimeric antibody Dinutuximab (for treating neuroblastoma); Humanized antibody Moxetumumomab pasudotox tdfk targeting CD22 (for hairy cell leukemia) and ADC antibody drug Inotuzumab ozogamicin (for the treatment of acute lymphocytic leukemia); Olara tumab, a fully human antibody targeting PDGFR - α, is used for the treatment of soft tissue sarcoma; Necitu mumab, a fully human antibody targeting EGFR, is used for the treatment of non-small cell lung cancer; Ramucirumab, a fully humanized antibody targeting VEGFR2 for the treatment of non-small cell lung cancer, Brentuximab vedotin, an ADC antibody drug targeting CD30 for the treatment of Hodgkin lymphoma, and Mogamulizumab kpkc, a humanized antibody targeting CCR4 for the treatment of non Hodgkin lymphoma.

2. In the field of immune diseases

The treatment of immune diseases is the second hottest topic in the development of monoclonal antibody drugs. In the past decade, 17 monoclonal antibody drugs have been approved for market, accounting for 21% of the total number of monoclonal antibody drugs on the market 79%. The main types of monoclonal antibody therapy for immune diseases include plaque psoriasis, severe asthma, and multiple sclerosis, as well as lymphoid tissue cell proliferation, rheumatoid arthritis, allergic dermatitis, ulcerative colitis, and lupus erythematosus. Among them, monoclonal antibodies used to treat plaque psoriasis mainly target IL-23 and IL-17A, including humanized antibodies Risankizumab rzaa targeting IL-23, Tildrakizumab targeting humanized antibodies, and Guselkumab targeting whole human antibodies; Broda lumab, Ixekizumab, and Secukinumab, all human antibodies targeting IL-17A. The monoclonal antibodies used to treat asthma are humanized antibodies targeting IL-5, including Benralizumab, Reslizumab, and Mepolizumab. Monoclonal antibodies for treating multiple sclerosis include humanized antibody Ocrelizumab targeting CD20 and humanized antibody Caclizumab targeting IL-2R α.

In addition, in the field of monoclonal antibody therapy for immune diseases, there are also fully human antibodies Emapalumab lzsgemapalumab lzsg targeting INF - γ (for the treatment of lymphoid tissue cell proliferation), Sari lumab targeting IL-6 (for the treatment of rheumatoid arthritis), Dupi lumab targeting IL-4 (for the treatment of allergic dermatitis), Vedolizumab targeting α 4 β 7 integrin (for the treatment of ulcerative colitis), and Belimumab targeting BLyS (for the treatment of lupus erythematosus) that have been approved for marketing.

2.3 Pathogen infectious diseases

Monoclonal antibodies are the most direct and effective means to treat pathogen infectious diseases. In the past decade, 6 monoclonal antibody drugs have been approved for market, accounting for 7% of the total monoclonal antibody drugs on the market 69%. Monoclonal antibodies for the treatment of pathogen infectious diseases have been approved for marketing in a small number of categories, and are mainly concentrated in severe infectious diseases, including the whole human antibodies against Ebola, Ansuvimab zykl and Atolitivimab, Maftivimab, Odesivimab ebgn, which directly target Ebola virus or GP1 protein of the virus; Ibalizumab uiyk, a humanized antibody targeting HIV-1 for the treatment of HIV viral infections; Bezlotoxumab, a fully human antibody targeting C. difficile toxin B, is used to treat Clostridium difficile infection; And targeting PA components, human mouse chimeric antibody Obiltoxaximab and whole human antibody Raxi bacumab for treating anthrax.

2.4 Metabolic diseases field

Monoclonal antibody drugs are also involved in the field of metabolic diseases. In the past decade, 4 types of antibody drugs have been approved for market, accounting for 5% of the total monoclonal antibody drugs on the market 13%. The development of monoclonal antibody drugs in the treatment of metabolic diseases mainly focuses on hypercholesterolemia and thyroid eye disease. The targets for treating hypercholesterolemia are PCSK9 and ANGPTL3. The marketed antibody drugs include the fully human antibodies Evolocumab and Alirocumab targeting PCSK9, as well as the fully human antibody Evinacumab dgnb targeting ANGPTL3. The monoclonal antibody used to treat thyroid eye disease is Tepotu mumab trbw, a fully human antibody targeting IGF-1R.

2.5 Fields of neurological and genetic diseases

Monoclonal antibodies have been approved for marketing in both neurological and genetic diseases. In the past decade, 7 types of monoclonal antibodies for treating neurological diseases and 5 types of monoclonal antibody drugs for treating genetic diseases have been approved for marketing, accounting for 8% of the total monoclonal antibody drugs on the market 97% and 6 41%.

In the field of neurological diseases, monoclonal antibody drugs that have been marketed are mainly used for the treatment of migraine and optic neuritis. The drugs used to treat migraine all target CGRP, including humanized antibodies Eptinezumab jjmr, Galcane zumab gnlm, Fremanezumab vfrm, and fully humanized antibody Erenumab aooe. Monoclonal antibody drugs for treating optic neuritis and myelitis include Satralizumab mwge, a humanized antibody targeting IL-6, and Inebilizumab cdon, a humanized antibody targeting CD19.

In the field of genetic diseases, monoclonal antibody drugs that have been marketed include the humanized antibody Crizanlizumab tmca targeting P-selectin for the treatment of sickle cell anemia; Targeting blood clots, the humanized antibody Caplacizumab yhdp is used to treat thrombocytopenic purpura; Lanadelumab, a fully human antibody targeting Plasma kallikrein for the treatment of hereditary angioedema; Targeting FGF23, the fully humanized antibody Burosumab twza is used to treat hereditary rickets, while targeting Activated factor IX and X, the humanized antibody Emicizumab is used to treat hemophilia A.

2.6 novel coronavirus treatment and other fields

The global outbreak of novel coronavirus in 2019 has brought a severe test to the health and economic development of people around the world. Based on the development of antibody technology, scientists have rapidly developed therapeutic monoclonal antibodies against COVID-19, and have achieved remarkable results in clinical treatment. As of June 30, 2021, four antibodies have been urgently authorized for use by the US FDA, including: REGEN-COV (Casirivimab and Imdevimab) (recombinant human IgG1 monoclonal antibody) approved by Regeneron Pharmaceuticals on November 21, 2020, Bamlanivimab and Etesevimab (IgG1 monoclonal antibody) approved by Eli Lilly on February 9, 2021, Sotrovimab (recombinant human IgG1 kappa) approved by GSK on May 26, 2021, and Actemra (Tocilizumab) approved by Hoffmann La Roche on June 24, 2021. Recombinant human monoclonal antibody, combined with IL-6 [34].

In addition, monoclonal antibodies have been used in various types of diseases, including targeting VEGF and the humanized antibody Brolucizumab dbll for the treatment of age-related macular degeneration [35]; Targeting Sclerostin, a humanized antibody Romosozumab aqqg for the treatment of osteoporosis [36]; Targeted C5, humanized antibody Ravulizumab for the treatment of hemoglobinuria [37]; Targeting Thrombin, the humanized antibody Idarutizumab used to reverse anticoagulants [38]; And human mouse chimeric antibody Siltuximab targeting IL-6 for the treatment of Castleman's disease [39].

3 Outlook and Summary

Since K ö hler and Milstein established hybridoma cells in 1975, research on monoclonal antibodies has been initiated. In these short forty years, more and more monoclonal antibody drugs have been approved for market. In addition, a considerable number of monoclonal antibody drugs are currently in clinical trials. Monoclonal antibodies include mouse derived monoclonal antibodies, human mouse chimeric antibodies, humanized antibodies, and fully humanized monoclonal antibodies. In the past decade, 78 antibody drugs have been approved for market by the US FDA (excluding FDA emergency authorization for use), covering fields such as oncology, immune diseases, anti pathogen infections, metabolic diseases, neurological diseases, and genetic diseases. With the continuous deepening of disease research, more and more therapeutic biological targets will be revealed, and the application of monoclonal antibodies is becoming increasingly widespread. In addition, how to increase the capacity of antibody libraries, improve the efficiency of single B cell expansion, and study more stable humanized mice will become a key research direction for the development of antibody drugs in the future. Monoclonal antibody drugs will also be an important chapter in the field of drug development, attracting tens of thousands of scientists and billions of dollars in investment.

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