BISPECIFIC ANTIBODY DEVELOPMENT SERVICES

ProteoGenix gives you access to the best expertise in bispecific antibody development: 3 therapeutic antibodies on the market, 300+ monoclonal antibodies successfully developed, scientists having more than 25 years experience in therapeutic antibody development. Choose the best knowledge in bispecific antibody format generation to build your blockbuster.

Our bispecific antibody development process

ProteoGenix offers the best-in-class one-stop solution for your bispecific antibody development project. Get access to:

• The expertise of renowned scientists with more than 25 years of experience in the development of therapeutic monoclonal antibodies.
• Experts which have successfully developed more than 300 monoclonal antibodies in 15 years, including 3 therapeutic monoclonal antibodies currently on the market.

Send us your requirements, our team:

• Defines the most accurate format for your application and for further pharmaceutical production (“developability”).
• Adapts the available format to your antibody sequences.
• Expresses and purify complex bispecific antibody formats.
• Optimizes and characterizes the best bispecific antibody formats.

Our complete bispecific antibody development service includes:

• Your optimized and purified bispecific antibody.
• A complete report with a description of the experimental steps, results, comments and conclusion.
• Optional: an optimized bispecific antibody production process directly transferrable to your CMO (parameters optimized includes: HC/LC ratio, expression system, linkers and many others depending on the project).
• All the property rights of the work done by ProteoGenix is transferred to the client.

Choosing ProteoGenix also means getting access to our best-in-class proprietary cell line XtenCHO™. Why best-in-class? Because it is the only cell line which has already overperformed ExpiCHO, the reference on the market, for the transient expression of therapeutic antibodies such as Pembrolizumab and many others (yields of up to 800 mg/L).

Unleash the potential of your project by choosing ProteoGenix as your privileged partner!

Bispecific antibody engineering:
get access to the largest variety of structures

Bispecific antibodies were initially produced using the quadroma method (hybrid hybridoma). This technique allows the production of antibodies in a single host cell but leads to a mixture of tailored, non-functional and homodimer compounds. Therefore, producing bispecific antibodies (BsAb) with sufficiently high yields to reach bioproduction was a real challenge due to heavy chain and light chain mispairing.

Recent engineering methods and recombinant antibody production have allowed the development of several strategies to address this problem, for example:

• The heavy chain mispairing challenge was overcome in the 90s thanks to the engineering of the CH3 domains which controls heavy chain dimerization. Several strategies were employed to force antibody heavy chain heterodimerization over homodimerization such as the well-known “knob-into-hole” approach or electrostatic steering mutations.
• To avoid light chain mispairing, several approaches have also been developed over the last decade. One of these consists in completely circumventing the issue by using identical light chains. Other examples of strategy include the separate expression of monoclonal antibodies before assembly in a single-host cell (Duobody, Genmab) or rearrangement of the heterodimerization interface by swapping between a heavy and a light chain domain (CrossMAb, Roche).

The capacity to assemble a large variety of building blocks opens the door to an infinite number of bispecific antibody formats. Controlling the various engineering technics means gaining access to the capacity to perfectly modulate the pharmacological properties of the desired compound and thus to develop your blockbuster. For this reason, choosing a company with more than 15 years of experience in antibody engineering like ProteoGenix ensures the success of your project. Our scientific experts have already overcome the most difficult engineering challenges, that’s why it’s time to put yours in safe hands!

Choosing a bispecific antibody format: a crucial step on the road to success

Recombinant bispecific antibody production technology paved the way for the generation of various bispecific antibody formats which can be classified according to their format and composition. Several differences distinguish bispecific antibodies:

• The presence or absence of a Fc region.
• Their symmetry.
• Their specificity.
• The number of binding sites.

The intensity of research activity and diversity of the bispecific antibody formats created demonstrates that a unique format is not suitable for all applications. Here are some examples of formats currently in the preclinical or Phase I clinical trial phase which demonstrate the strong potential of this class of compounds as biotherapeutics and the importance of choosing the right format: CrossMAb, DuoBody, κλ-body, SEEDbody, nanobody, BiTE, DART…

It therefore becomes particularly important to select a partner that can guide you through the difficult format selection process. With several experts having long-standing experience in the development of therapeutic monoclonal antibodies, ProteoGenix offers you the very best of their scientific knowledge in the field.

Crossmab: Bispecific Igg-like

CrossMAb is a bispecific IgG format developed by Roche to overcome the LC/HC mispairing. This technology is based on the exchanging of the light chain and heavy chain domain within a unique Fab arm to induce specific interaction while maintaining the original antigen binding affinity.

CrossMAb can be combined with the KiH approach to control both HC/HC and LC/HC pairing.

As a bispecific IgG, CrossMAb combines the advantages of IgG (long pharmacokinetic in vivo half-life, effector functions) and bispecific antibodies.

Duobody: Bispecific Igg-like

The DuoBody platform is a technology invented by Genmab based on a controlled Fab arm exchange. This technique is based on the separate expression of two IgG1, each of them containing a single matched mutation in the CH3 domain. The antibodies obtained are further mixed and separated into half molecules under reducing conditions. Single point mutations introduced in each Fab arm, coupled with oxidizing conditions, control the reassembly of half molecules leading to yields of bispecific IgG of up to 95%.

Like CrossMAb, Duobody combines the advantages of IgG and bispecific antibodies.

Duobody: Bispecific Igg-like

The DuoBody platform is a technology invented by Genmab based on a controlled Fab arm exchange. This technique is based on the separate expression of two IgG1, each of them containing a single matched mutation in the CH3 domain. The antibodies obtained are further mixed and separated into half molecules under reducing conditions. Single point mutations introduced in each Fab arm, coupled with oxidizing conditions, control the reassembly of half molecules leading to yields of bispecific IgG of up to 95%.

Like CrossMAb, Duobody combines the advantages of IgG and bispecific antibodies.

Dart: Bispecific Antibody Fragment

The DART format can be classified as a bispecific antibody fragment. DART is a diabody composed of the variable domains of two different antibodies. In this format, the variable heavy domain of the first antibody is connected via a short peptide linker to the variable light domain of the second antibody and vice versa leading to two scFv. Generation of DART is obtained by further stabilization via disulfide bond.

Like most small formats, DART are characterized by a high potency but a short serum half-life but can be further engineered to overcome this limitation, for example by fusion to a Fc region.

Nanobody: Bispecific Antibody Fragment

Nanobodies correspond to single domain antibody fragments which can be obtained from various species, such as llama or alpaca. Bispecificity can be obtained by connecting two nanobodies via short linkers.

The high-potential of nanobodies comes from their small size which permits antigen access that would not be sterically accessible to other antibodies.

Like DART, bispecific nanobodies are characterized by a high potency but a short half-life which can be overcome by fusional connection to a protein such as human serum albumin (HSA).

Dvd-ig: Appended Igg

DVD-Ig (dual variable domain-Ig) is part of the appended IgG family and was developed by Abbott. Appended IgG correspond to monoclonal IgG that have been further engineered to introduce bispecifity by addition of antigen binding sites at the N- or C- termini of either light chains or heavy chains. DVD-Ig are obtained by appending VL and VH domain of an antibody to an IgG and results in a bispecific and bivalent antibody for each antigen.

κλ-body: Bispecific Igg

κλ-body is a fully-human bispecific IgG recently developed by Novimmune to solve the LC/HC mispairing issue. This format is characterized by a common heavy chain coupled with two different light chains κ and λ governing the antigen-binding specificity. The process developed by Novimmune is based on the selection of light chains via phage display.

Seedbody: Bispecific Igg-like

SEEDbody is a bispecific antibody format developed by Merck Serono which overcomes the HC/HC mispairing issue. The SEED method is based on the alternation of human IgG and IgA sequences in the CH3 domain in order to favor the heterodimerization.

SEEDbody is a bispecific IgG and thus combines the advantages of IgG and bispecific antibodies.

Seedbody: Bispecific Igg-like

SEEDbody is a bispecific antibody format developed by Merck Serono which overcomes the HC/HC mispairing issue. The SEED method is based on the alternation of human IgG and IgA sequences in the CH3 domain in order to favor the heterodimerization.

SEEDbody is a bispecific IgG and thus combines the advantages of IgG and bispecific antibodies.

Triomab: Bispecific Igg-like

Triomab is a format developed by Trion Pharma and was the first bispecific antibody approved on the market for therapeutic application. Triomab is based on the quadroma production technique which is generally associated with HC/HC and LC/HC mispairing problems. Trion Pharma overcame both of these issues by generating a mouse/rat hybrid hybridoma.

• LC/HC mispairing was solved by combining mouse IgG2a and rat IgG2b.
• HC/HC mispairing was solved during the purification steps as mouse IgG2a have strong affinity for protein A on the contrary to rat IgG2b.

Use of bispecific antibodies as biotherapeutics

Antibodies are one of the fastest growing classes of therapeutics mainly because they have demonstrated their ability to improve the treatments of several complex diseases such as cancer or inflammatory disorders.

This success has fueled strong interest in the development and engineering of new antibody drugs including bispecific antibodies. The major advantage of bispecific antibodies over monospecific antibodies is their capacity to bind two different antigens or two epitopes of the same antigen. This specific property paves the way to a new generation of high potential biotherapeutics.

To date, three bispecific antibodies were approved for clinical use:

• Catumaxomab (REMOVAB®) is a T-cell recruiter developed by Trion Pharma which binds EpCAM and CD3. Catumaxomab is a nonhuman bispecific IgG-like rat/mouse antibody produced using the quadroma technology which was indicated as a treatment for malignant ascites (discontinued in 2014).
• Blinatumomab (BLINCYTO®) is a BiTE developed by Amgen and Micromet. It is a T-cell recruiter which targets CD3 positive T-cell and CD19 positive B-cells. Blinatumomab is indicated for the treatment of acute lymphoblastic leukemia.
• Emicizumab (HEMLIBRA®) is a therapeutic antibody developed by Roche and Chugai. This bispecific antibody induces the dimerization of its two targets (Factor IX and Factor X) in order to reactivate the latter. Emicizumab is indicated for the treatment of hemophilia A.

The most common application of bispecific antibodies relies on retargeting effector cells to tumor cells for cancer therapy. BiTE (bispecific T-cell Engager) is an example of format (developed by Micromet) based on this application. BiTE can be considered as a scFv tandem which targets a T-cell specific molecule, on the one hand, and a tumor specific antigen on the other hand, to improve patient’s immune response.

Bispecific antibodies can also overcome other limitations of monospecific antibodies. An example is their inability to cross the blood-brain barrier as the low permeability of the BBB hinders the development of antibodies which target an antigen in the CNS. A strategy currently discussed to cross the BBB consists of engineering bispecific antibodies that recognize a BBB receptor (to induce transcystosis) and the target within the CNS.

Due to their high diversity, bispecific antibodies can present many other therapeutic applications such as:

• Inhibiting two pathways at the same time.
• Enhancing antigen binding affinity.
• Increasing target specifity.
• And even more…

Bispecific antibody development services that will meet your expectations and surpass your expectations!

At ProteoGenix, we know that bispecific antibody engineering and design is only one step of the road to success. For this reason, we attach particular importance to guiding you from the development of your bispecific antibody through to its bioproduction. ProteoGenix has an extensive experience in the development of stable cell lines reaching high production yields and no unwanted side products. With ProteoGenix, you can rest assured that our scientists will be fully committed to converting your ideas into a blockbuster.