The potential clinical utility of antisera (a polyclonal mixture of antibodies) in the treatment of human diseases has been recognized for more than a century. However, the difficulty of isolating specific antibodies against the antigen from antisera in a reproducible manner was a major hurdle to their clinical exploitation. With the advent of murine monoclonal antibody technology, it seemed that the antibody’s therapeutic promise would be finally fulfilled. However, mouse monoclonal antibodies are foreign to human and provoke a strong immune response when administered to patients. The Human Anti-Mouse Antibody (HAMA) response limited both the dose and the number of times that could be repeated. Other important factors such as short serum half-life and poor recruitment of human immune effector functions also set back the clinical application of murine antibodies.

Therapeutic Antibody Development

The 1st attempt to engineer mouse antibodies to facilitate therapeutic use was chimerization. This involves genetically replacing mouse constant regions with the corresponding human constant regions, while retaining the mouse variable regions responsible for antigen binding. Antibody humanization (also known as CDR-grafting or reshaping) was invented as a more elegant solution to the immunogenicity problem of murine antibodies. Antibody humanization involves the design and synthesis of composite variable regions, which contain the amino acids of mouse CDRs integrated into the FWRs of a human antibody variant. The resulting antibody retains both the specificity and binding affinity of the original mouse antibody, and is sufficiently human to deceive the patient’s immune system. Both chimerization and humanization strategies have been proven successful in clinic.

A schematic diagram of mouse (top left), chimeric (top right), humanized (bottom left), and fully human (bottom right) antibodies are shown above. Human parts are shown in red, while non-human parts in blue. The International Nonproprietary Names (INN) recommends to name murine antibodies to end in “–omab”, chimeric antibodies to end in “-ximab”, humanized antibodies to end in “–zumab”, and fully human antibodies with end in “–umab”. 

There are ~300 monoclonal antibodies currently in clinic and on market for various therapeutic, diagnostic, and preventive applications. Among them,  about 40% (114) are humanized antibodies, 34% (99) are fully human antibodies, and 10% (30) are chimeric antibodies (see the pie chart below).

• Examples of approved chimeric antibodies: Basiliximab(Simulect®), Cetuximab(Erbitux®), Infliximab (Remicade®), and Rituximab (Rituxan®)
• Examples of approved humanized antibodies: Alemtuzumab (Campath®), Atlizumab (Actemra®), Bevacizumab (Avastin®), Daclizumab (Zanapax®), Natalizumab (Tysabri®), Omalizumab (Xolair®), Palivizumab (Synagis®), Pertuzumab (Omnitarg®), and Trastuzumab (Herceptin®)
• Examples of approved fully human antibodies: Adalimumab (Humira®), Belimumab (Benlysta®), Denosumab (Prolia®), Ipilimumab (Yervoy®), Ofatumumab (Arzerra®), Panitumumab(Vectibix®), and Ustekinumab (Stelara®)

Click here to see "a complete list of monoclonal antibodies for clinical use" (that includes therapeutic, diagnostic and preventive monoclonal antibodies that are approved, investigational drugs, and those withdrawn from the market.)

Antibody Humanization & Engineering

The antibody humanization process usually includes the creation of a mouse-human chimera in an initial step.  Thereafter the chimera is further humanized by the selective alteration of the sequence of amino acids in the variable region of the molecule. The process must be "selective" to retain the specificity for which the antibody was originally developed. It normally involves the design and synthesis of composite variable regions which contain mouse CDRs integrated into human framework regions, whose role is to support CDRs in the same orientation as that of a corresponding human antibody variant. Ideally a humanized antibody should be essentially identical to that of a human variant, containing the only non-human origin of CDRs responsible for antigen binding. In reality the mouse sequences make up 5-10% of the humanized antibody (for more information and examples, please visit our "Antibody Services").

The feasibility of this process is, to a large extent, due to the inter-species conserved nature of antibody variable region genes between animals and humans. Within any species, variable region gene sequences can be grouped into a number of families according to amino acid or nucleotide sequence homology. In some cases inter-species homology can be higher than intra-species homology. For example, inter-species homologies can range from 40% to 80% between mouse and human variable region sequences. Moreover the highly conserved and well defined CDR loop structures seen in mouse antibodies are also observed across the species. It is thus the sequence and structural conservation throughout the variable regions of antibodies from different species that makes antibody humanization feasible.

Success Hallmarks of Therapeutic Antibody

Over the last decade, Biotechnology & Biopharmaceutical industry has concentrated on developing new technologies for antibody production and engineering with the aims to optimize or enhance its manufacturability and therapeutic efficacy. Several key “success hallmarks” have been proposed for an effective antibody-based therapeutic for human conditions such as cancers, inflammatory and infectious diseases:

All approved antibody drugs are thought to work through a Fab-mediated or the combination of Fab- and Fc-mediated action.  However the proposed mechanisms are largely based upon data generated in vitro or in animal models. The clinical mechanisms of action of many approved antibody drugs are in actuality complicated and remain poorly understood.  In addition, antibodies frequently fail to activate ADCC or CDC and show little or no efficacy even with optimal binding to the target antigen or recruiting immune effectors.  This suggests that the clinical outcome is driven by a more complex interplay between the antibody, the cognate antigen that for example is expressed on stromal or tumor cells, and our immune system (see the figure below).  In addition there are many approaches to improving the clinical performance for antibody-based therapeutics, including antibody-drug conjugate (ADC) and bispecific antibody. 

Therapeutic Antibodies Discovery

There are technologies that completely avoid the use of mice or other animals in the discovery of antibodies for human therapeutic applications. Examples include various "display" methods that employ the selective principles of specific antibody production but exploit microorganisms (such as in phage display and yeast display) or even cell free extracts (as in ribosome display). These systems rely on the creation of antibody gene "libraries", which are usually derived from RNA of human peripheral blood. Each antibody gene is linked to a product (e.g., antibody fragment Fab or scFv) displayed by the system, allowing rapid screening for antigen-specific binders. Adalimumab (Humira) is an example of an antibody approved for human therapy that was created through phage display.

Antibody and Immunomodulation

The immune system has the intrinsic power to detect and eliminate abnormal cells, such as those derived from tumors.  This process, commonly referred to as immune surveillance, takes advantage of numerous biological features that distinguish tumor cells from their normal counterparts.  For example, tumor cells display aberrant functional behaviors and an altered surface antigen composition, typically resulting from a myriad of genetic and epigenetic changes. Abnormal cytokine and growth factor expression patterns are also common hallmarks of certain types of cancer, eliciting to either support growth or counteract local inflammation, particularly during cellular invasion and metastasis. With more advanced disease, tumor cells eventually develop active mechanisms to escape immune surveillance and induce tolerance.  There is ample evidence that B cell-driven antibody responses can trigger autologous tumor regression in animals and humans. 

Approved Antibody-based Therapeutics for Cancer and Target Antigens. 

The antigens are in black while the approved antibody therapeutics are in red.

See the list of "monoclonal antibodies for clinical use" for more details and examples.

Therapeutic Antibodies from Humans

It is possible to exploit human immune response in the discovery of truly human antibodies for therapeutic applications. Human immune response works essentially in the same way as that in a mouse. Therefore, persons experiencing a challenge to their immune system, such as an infectious virus, a passive vaccination, or abnormal tumor cells are a potential source of discovering antibodies directed against that challenge. This approach seems especially useful for the development of anti-viral and anti-cancer (of particular types) therapies that exploit the principles of passive immunity. Variants of this approach have been demonstrated with proof-of-principle in preclinical studies and several are finding way into clinical development.

Antibody R&D Capability at G&P Biosciences

We offer a range of antibody production and engineering services that may complement your research and accelerate the progress of development towards clinical use. Our services are offered as stand-alone services and also as part of a complete suite of antibody custom solution package. We offer a proprietary antibody humanization service. Using our technology, the sequences of the antibody variable domains, which determine its binding specificity, are incorporated into human donor sequences properly, thus creating a panel of humanized antibodies for expression. We also provide bundled services, starting from antigen preparation, hybridoma screening, recombinant antibody generation, affinity determination, antibody humanization and engineering to custom-scale production. We can drive your antibody R&D from any stage to the delivery of a 100% royalty free drug candidate that can be moved into clinical development rapidly (please visit our "Antibody Services" to learn more and request a quote).

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