Spike & ACE2-blocking activities assay

Spike & ACE2-blocking activities assay

The Landscape of Antibody-drug Conjugate (ADC): Production, Mechanisms of Action (MOA), FDA approved-antibodies, and Functional assay

The Landscape of Antibody-drug Conjugate (ADC): Production, Mechanisms of Action (MOA), FDA approved-antibodies, and Functional assay
1.What is antibody-drug conjugate (ADC)? --Introduction
2.antibody-drug conjugate (ADC) in clinic
3.Main elements of antibody-drug conjugate (ADC)
4.Review for antibody-drug conjugate (ADC) production, quality control and functional assay
5.Our products and data of antibody-drug conjugate (ADC)
GeneMedi provides pre-made research class therapeutic Antibody-drug Conjugate (ADC) antibodies with WHO-INN name. The INN named antibodies are biosimilars expressed by mammalian cell line as a benchmark reference therapeutic antibody for cell culture, in vitro/in vivo assay development, animal model development, PK/PD model development (Pharmacokinetics & Pharmacodynamic).

Bispecific antibodies: formats, applications and products

Bispecific antibodies: formats, applications and products
1.Introduction about bispecific antibodies (BsAbs)
2.Formats of bispecific antibodies (BsAbs)
3.Peplines of bispecific antibodies (BsAbs)
4.Bispecific Antibody Development Programs Guidance for Industry by FDA
GeneMedi provides pre-made research class therapeutic Bispecific antibodies with WHO-INN name. The INN named antibodies are biosimilars expressed by mammalian cell line as a benchmark reference therapeutic antibody for cell culture, in vitro/in vivo assay development, animal model development, PK/PD model development (Pharmacokinetics & Pharmacodynamic).

The Landscape of Neutralizing antibodies (NAb): Production, Mechanisms of Action (MOA), FDA approved-antibodies, and Functional assay

The Landscape of Neutralizing antibodies (NAb): Production, Mechanisms of Action (MOA), FDA approved-antibodies, and Functional assay
1.Introduction of neutralizing antibody (NAb)
2.How to produce neutralizing antibody (NAb) (production)
3.How neutralizing antibody (NAb) work (mechanisms of action, MOA)
4.FDA approved neutralizing antibodies (NAb) and our products
5.Functional assay for neutralizing antibodies (NAb) discovery--Pseudotype virus (psedovirus, PSV) based and Neutralization Assays
6.Products Data of neutralizing antibodies (NAb)
GeneMedi provides pre-made research class therapeutic Neutralizing antibodies (NAb) with WHO-INN name. The INN named antibodies are biosimilars expressed by mammalian cell line as a benchmark reference therapeutic antibody for cell culture, in vitro/in vivo assay development, animal model development, PK/PD model development (Pharmacokinetics & Pharmacodynamic).

Virus-like particles (VLP) Platforms for Developing immunogens, vaccines and drug carriers

Virus-like particles (VLP) Platforms for Developing immunogens, vaccines and drug carriers
1.What is virus like particles (VLP)?
2.GM VLPxTM Virus-like particles development platform
3.Virus like particles (VLP) Expression platforms
4.Virus like particles (VLP) as immunogens/antigens of transmembrane protein (TM)
5.Virus like particles (VLP) for drug delivery
6.Virus like particles (VLP) as vaccine
7.Virus like particles (VLP) for drug discovery

What is antibody-drug conjugate (ADC)?

ADC consists of antibodies and payload, linker connects antibodies and small molecule drugs. After ADC drugs enter the blood, its antibody part will recognize and bind to the surface antigen of target cells. Theninternalizing ADC antigen complex into cells through endocytosis, the complex will be degraded by lysosomes and the payload will be released, so as to destroy DNA or microtubules, or exert the inhibitory effect of topoisomerase / RNA polymerase, resulting in cell death. It has the characteristics of precision and great lethality. It is a new generation of therapeutic drugs.

Endogenous coupling of amino acids and Disulfide re bridging strategy

One of the most common coupling methods is to use the lysine residue of the antibody, the amino acid nucleophilic NH2 group, to react with the electrophilic N-hydroxysuccinimide (NHS) Group on the lik payload. Although the reaction is simple, the high abundance of available lysine residues leads to the formation of uneven mixtures of many ADCs under random distribution.

Glycan coupling

Because IgG is a glycoprotein, it contains an N-glycan at n297 of CH2 domain of each heavy chain of Fc fragment. This glycosylation can be used as the attachment point of connecting payload.

Site specific biological coupling of engineered antibodies and Enzymatic method

Advances in bioorthogonal chemistry and protein engineering contribute to the generation of more uniform ADCs.

Biological coupling with engineered unnatural amino acids

In addition to thiomonoclonal antibody technology, the addition of non-standard amino acids (NCAA) provides another possibility for site-specific coupling.

Review for ADC production, quality control and functional assay

ADC drugs are essentially a mixture, which is composed of mAbs connecting different numbers of small molecule drugs. Dar represents the average number of small molecule drugs connected to each mAb. Dar directly affects the efficacy and safety of ADC drugs. In the drug development stage, the variation range of DAR value should be minimized.

Product data of ADC

In this batch, we selected 5.4 equivalent TCEP and 10.8 equivalent VCMMAE groups for DAR detection. The DAR of our sample is almost the same as that of the standard sample, but the integrity of the antibody result is not as good as that of the standard sample.

Antibody-drug conjugate (ADC) in clinical application (Approved/BLA, phaseI/II/III)

FDA approved antibody-drug conjugate (ADC) for clinical use Clinically, most of the ADC drugs approved by FDA are IgG1, and the targets are CD33, CD22, HER2 and so on. The most common payload is MMAE, also contain calicheamicin, DM1. ADC drugs are mainly used in the field of antitumor, which is one of the hot research directions in recent years. At present, 11 ADC drugs have been approved in the world, including Mylotarg (Pfizer), adcetris (Seattle genetics / Takeda), kadcyla (Roche), besponsa (Pfizer), lumoxiti (AstraZeneca), Polivy (Roche), padcev (Seattle genetics / anstelai / MSD), enhertu (AstraZeneca / first third party), trodelvy (immunomedicine), blenrep (GSK) Akalux（Rakuten Aspyrian）3. From the perspective of listed drug R & D enterprises, Pfizer, Seattle genetics, Roche and AstraZeneca have two models respectively, and the other three companies have one model respectively.

Main elements of antibody-drug conjugate (ADC)： Antibodies and their targets

Mylotarg® (gemtuzumab ozogamicin) from Wyeth/Pfizer was the first ADC to reach the market. It is composed of a recombinant humanized anti-CD33 mAb (IgG4κ antibody hP67.6) covalently attached to a calicheamicin derived payload (N-acetyl-γ-calicheamicin 1,2-dimethyl hydrazine dichloride) via a pH-sensitive hydrazone linker

Main elements of antibody-drug conjugate (ADC)：Linker (cleavable/non-cleavable, structure and mechanism)

Conjugate linker is not only the molecular part forming covalent connection between antibody and small molecule payload, but also the key element with design properties in targeted drug therapy. The addition of linkers should not induce aggregation, and it is necessary to ensure acceptable PK characteristics, limit the premature release (stability) of payloads in plasma and effectively release active molecules at targeted action sites. In the process of connection, there are many conjugate companies .Connectors are divided into two types: non-cleavable linkers and cleavable linkers.6

Main elements of antibody-drug conjugate (ADC)：Toxins/Payloads (Classification and function)

There are so many payloads, like MMAE、Calicheamicin、MMAF、DM1、SN-38 and Dxd.

Toxins/Payloads (Classification and function) of Microtubule destroying drug

There are so many payloads, like MMAE、Calicheamicin, MMAF, DM1, SN-38 and Dxd.

Toxins/Payloads (Classification and function) of DNA damage drugs

pyrrole benzodiazepines and indole chlorobenzodiazepines Pyrrolo [2,1-c] [1,4] benzodiazepine (PBD) is a natural product with antitumor activity. Their mode of action is selective alkylation in small grooves of DNA, in which the N2 of guanine forms a covalent bond with the electrophilic N10 / C11 imine on PBD

Toxins/Payloads (Classification and function) of Innovative drugs

Overexpression of anti apoptotic Bcl-2 family members (including BCL XL) is one of the mechanisms for cancer cells to obtain apoptosis resistance. Drugs that block the BH3 binding domain on BCL XL can trigger cancer cell apoptosis.

Biological coupling technology Chemical based specific in situ antibody modification

The natural structure of monoclonal antibodies provides a variety of possibilities for biological coupling. Chemical and specific natural (non engineering) antibody coupling has some advantages. It can avoid the complexity of antibody specific site mutation and the possible challenges in the amplification and optimization of cell culture.

Introduction about bispecific antibodies (BsAbs)

Bispecific antibodies (BsAbs) are designed to bind two different epitopes or antigens, whichleads to multiple mechanistic functions with synergistic effects. The attractive feature is their potential for novel functionalities, which do not exist in mixtures of the parental or reference antibodies. Till now, more than 200 BsAb-based clinical trails have been registered on clinicaltrails.org and 4 BsAbs (with one withdraw) have been granted FDA approval. The promising future makes BsAbs attracting more attentions.

Formats of bispecific antibodies (BsAbs)

Many formats have been developed for BsAb generation

Format of bispecific antibodies (BsAbs)-Tandem VHH

Since 1975, monoclonal antibodies have been successfully used for the diagnosis and therapeutics. In 1993, heavy-chain-only antibodies which bind to antigens was discovered in camelids. The variable domain of heavy chain antibody (VHH) was called as nanobodies and it was anticipated as the better therapeutic antibodies in future due to its high stability, refoldability, and manipulable characteristics. VHH has also been effectively used for the diagnosis, bioimaging, immunoassay and affinity purification. The production of nanobodies is simple and cost effective compared to the polyclonal antibodies. Besides, Nbs are stable in a wide range of temperatures and pH levels. Nanobodies are also compatible with genetic engineering methods, which allow scaffolding and alteration of amino acids to improve binding. Relating to structure, the hydrophilic side of nanobodies, that is not present in conventional antibodies, means they do not have issues with solubility and aggregation. The conventional antibodies do not bind well in grooves or cavities on the surface of the antigen. But, Nanobodies bind with the clefts of the active site of antigen.

Format of bispecific antibodies (BsAbs)-Tandem scFv

A tandem scFv links two or more scFvs with the helical peptide linkers in the orientation NH2–VL1–VH1–(linker–VL2–VH2)n–COOH, resulting in a single chain bivalent and bi-specific molecule encoded by a single gene (Figure 2). Tandem scFv can be used to target two different antigens on two different cells, two different antigens on the same cell, or two different epitopes on the same antigen with increased avidity.

Format of bispecific antibodies (BsAbs)-Dual-affinity re-targeting antibody

Dual-affinity re-targeting proteins (DARTs) encompasses of two Fv fragments, containing two single antigen-binding sites formed when two Fv fragments heterodimerize. The Fv1 contains of a VH from antibody A and a VL from antibody B, whereas Fv2 contains VH from antibody B and VL from antibody A in the order of VL (1)-VH (2) and VL (2)-VH (1) (Fig. 1). This amalgamation permits DART to mimic natural interaction within an IgG molecule. Short linker sequences between the VL and VH segments encourage a posttranslational “diabody”-type association. The peptide linkers and the covalent linkage between the two DART chains limits the freedom of the antigen binding domains, resulting in a stable association between target and effector cells. DART can be synthesized in the mammalian expression systems. DART are more stable and potent. DART does not contain Fc region and, therefore it has a short serum half-life. Several DART antibodies are under development for T-cell redirection, modulation of receptor signaling and neutralization of viruses. DART molecules were found to be more consistent than BiTE molecules in targeting and killing B-cell lymphoma.

Format of bispecific antibodies (BsAbs)-Diabody

Diabody is a noncovalent dimer of single-chain Fv (scFv) fragment that consists of the heavy chain variable (VH) and light chain variable (VL) regions connected by a small peptide linker. Another form of diabody is single-chain (Fv)2 in which two scFv fragments are covalently linked to each other. bispecific bivalent dimers are produced by using two different chains with the same orientation, the first containing the VH of Antibody 1 and the VL of Antibody 2, and the second containing the VH of Antibody 2 and the VL of Antibody 1 (Fig. 1). Varying the linker length by only one amino acid may shift the equilibrium completely to another oligomeric state with the lowest complexity being most favorable. Diabodies are one of the smallest recombinant BsAbs and the distance between the two antigen-binding sites is only 6.5 nm on average, which is less than half the distance in IgG. This compact size contribute to rapid pharmacokinetics, low immunogenicity and high tumor penetration.

Format of bispecific antibodies (BsAbs)-(scFv)2-Fab

Tribodies are multifunctional recombinant antibody derivatives. The Fab fragment serves as a specific heterodimerization signal, and the two scFv fragments are each fused to a different Fab chain. In this way we obtain a molecule of intermediate molecular weight (100 kDa) which allows incorporating three different antibody fragments (Fig. 1). This manifold, tribody, can be used to create trivalent molecules as well as bispecific molecules with bivalent binding to only one target, as trispecific molecules. These heterodimers are stable, with each of the binders retaining their specific affinities, with the bivalent tribody having higher affinity, and higher activation of T-cell proliferation and cytotoxicity in vivo. The molecule also has more favorable properties for toxic payload delivery. It can be argued that delivering a T-cell activating activity can be regarded as a toxic function. Antibodies with such a function remaining in the body for a longer time have the potency to accumulate aspecifically or form deposits in healthy tissue.

Format of bispecific antibodies (BsAbs)-Rat-mouse hybrid IgG

Rat-mouse hybrid IgG is a monoclonal antibody with binding sites for two different antigens, typically CD3 and a tumor antigen, making it a type of bispecific monoclonal antibody. In addition, its intact Fc-part can bind to an fc receptor on accessory cells like conventional monospecific antibodies. The net effect is that this type of drug links T cells (via CD3) and monocytes/macrophages, natural killer cells, dendritic cells or other Fc receptor expressing cells to the tumor cells, leading to their destruction.

Format of bispecific antibodies (BsAbs)-Hetero heavy chain, Common light chain

Since an IgG antibody has two antigen-binding fragments (Fab) both of which link to an Fc region, there had long been a concept of an asymmetric bispecific IgG antibody capable of binding to two different antigens or epitopes. The asymmetric reengineering technology immunoglobulin (ART-Ig) is a humanized asymmetric bispecific IgG antibody. An asymmetric bispecific IgG antibody typically has 2 kinds of heavy chain and 2 kinds of light chain. If the 4 chains were simultaneously expressed in a recombinant cell to produce the desired asymmetric bispecific IgG antibody, as many as 9 kinds of unwanted byproduct IgG molecules would also be assembled and secreted. A method of reducing the byproduct IgG molecules using a common light chain in the asymmetric bispecific IgG antibody and also by introducing “knobs-into-holes” mutations in its Fc region to prioritize a specific hetero-dimerization of the heavy chains.

Format of bispecific antibodies (BsAbs)-Controlled Fab arm exchange

Controlled Fab arm exchange (cFAE) has proven to be a generic and versatile technology for the efficient generation of IgG-like bispecific antibodies (DuoBodies or DBs). This involves the recombination of antigen-binding arms (heavy chain–light chain (H-L) pairs) between individually expressed antibody molecules and is termed Fab-arm exchange, can be modified to make the routine generation of bsAbs feasible. The production of DuoBodies involves (i) separate expression and purification of two parental IgG1s containing two matching point mutations, one in either IgG1, at the CH3:CH3 interface, (ii) mixing of the parental IgGs; (iii) controlled reduction of interchain disulfide bonds in vitro, allowing the recombination of H-L pairs (driven by the matching mutations); and (iv) removal of reducing agent, allowing the reoxidation of interchain disulfide bonds to occur. Fragment-based formats are compatible with well-characterized and cost-effective prokaryotic and lower eukaryotic expression systems, offering the potential advantages of high yields.

Format of bispecific antibodies (BsAbs)-Hetero H, forced HL IgG1

Hetero H, forced HL IgG1/ DuetMab replaces the native disulfide bond in the CH1-CL interface with an engineered disulfide bond (fig. 1). This enhances cognate light chain pairing. Three different positions in the CH1-CL interface are possible candidates for favoring the formation of a novel disulfide bond. An amino acid on the HC and one on the LC is replaced with cysteine in one of the Fab regions. The native disulfide bond on the other Fab region is left intact. It is advantageous that the modifications are in the CH1-CL interface and not in the variable domain, as this could have detrimental effects on antigen binding. Although, engineering in the CH1-CL interface could mean that κ and λ constant light chains would somehow affect the usefulness of this approach. However, it was shown to be compatible to both isotypes (Mazor et al. 2015). DuetMab could be generically applied to bispecific antibodies in development since the approach: (i) does not contain variable domain engineering, (ii) is compatible with both kappa and lambda isotypes and (iii) was able to induce correct heterodimerization.

Format of bispecific antibodies (BsAbs)-cH IgG1

cH IgG1 / κλ body involves the formation of in vitro display libraries with common heavy chains against two different antigens. κλ body share the same heavy chain but carry either κ or λ light chains. Three different chains (one heavy and two light) are then co-expressed in a single cell to generate a mixture containing two mAb species (one κ and one λ) and a BiAb containing a κ and λ light chain (Fig. 1.). A BiAb assembled in this manner can then be efficiently purified from the mAb species and other contaminants using highly selective affinity resins binding to either human κ or λ constant domains. A κλ-body includes a common heavy chain and two light chains (one kappa and one lambda) with distinct specificities. Meanwhile, it is noteworthy that all of these fragments are integrated without any artificial mutations or linkers. Therefore, κλ-bodies with full human IgG backbone and intact Fc have got attractive characteristics including potent functions, extended half-life, long term storage, increased stability and ease of large-scale manufacturing. Thus, κλ-bodies are considered to be a leading one among our diverse products. Antibodies in this format are ideally suited for therapeutic application in both benign and malignant diseases.

Format of bispecific antibodies (BsAbs)-Hetero H, CrossMab

CrossMab technology enforces correct light chain association based on the domain crossover of immunoglobulin domains in the Fab region of the bispecific antibody. CrossMab technology allow the generation of various bispecific antibody formats, including bi- (1+1), tri- (2+1) and tetra-(2+2) valent bispecific antibodies, as well as non-Fc tandem antigen-binding fragment (Fab)-based antibodies. These formats may be derived from any existing antibody pair using domain crossover, without the need for the identification of common light chains, post-translational processing/in vitro chemical assembly or the introduction of a set of mutations enforcing correct light chain association. The basis of the CrossMab technology is the crossover of antibody domains within one arm of a bispecific IgG antibody enabling correct chain association, whereas correct heterodimerization of the heavy chains can be achieved by the knob-into-hole technology or charge interactions. This can be achieved by exchange of either the Fab domains (in the CrossMabFab format), or only the variable VH-VL domains (CrossMabVH-VL format) or the constant CH1-CL domains (CrossMabCH1-CL format) within the Fab-fragment (Fig. 1). This class of therapeutics antibodies have novel mechanisms of actions as compared to conventional therapeutic antibodies and have a major impact on the treatment of various diseases, including oncology, infectious diseases, autoimmunity, CNS, and metabolic diseases. Taken together, CrossMab technology has proven to be very useful for the fast and straightforward generation of bispecific antibody formats to tackle novel biological challenges and help to develop novel therapeutic concepts.

Format of bispecific antibodies (BsAbs)-scFv-Fab IgG

The XmAb enables alterations with desirable effects to the Fc domain of the antibodies. The modification increases affinity to the neonatal Fc receptor which prevents the antibody from degradation. Hence, this interaction extends the antibody’s halflife of this therapeutic drug. In order to construct the XmAb format an antibody heavy and light chain and a scFv Fcfusion were subcloned into vectors. The scFv and Fc region were connected with a GS-linker. The Fc region was altered with substitutions in order to increase the differences in pI between the two heavy chains. This would increase the pI differences between homodimers and heterodimers, which would then facilitate the purification of heterodimers. For the production of the proteins, plasmids encoding all chains were co-transfected into HEK cells. The antibody was further purified using protein A chromatography and ion exchange chromatography. Due to alterations in the Fc domain, pI differences can be used in the purification. The risk of immunogenicity was also minimized by the XmAb.

Format of bispecific antibodies (BsAbs)-VH1-VH2-CH1-Fc1(G1) x VL2-VL1-CL-Fc2(G1)

CODV (cross-over dual variable domains) -Ig, contain four polypeptide chains that form two dual variable domains (four antigen binding sites) with a cross-over orientation (Figure 1), which is attained by inverting the alignment of the cognate domains on one chain. In order to adopt the correct VH/VL pairing, linker combinations were designed and optimized using a molecular modeling strategy. The overall CODV structure replicates a circular self-contained architecture (Figure 1A), with binding sites facing up to opposite sites and able to accommodate a large variety of antigen sizes while maintaining parental affinities. The CODV structure was also further developed in a trispecific format in which a single IgG Fab arm is combined with a double arm generated in the CODV structure using the KiH heterodimerization strategy (Figure 1B). A trispecific CODV molecule was successfully engineered to target three distinct epitopes on human immunodeficiency virus HIV-1 envelope, including the CD4 binding site, MPER and the V1V2 glycan site. This innovative molecule exhibited an unprecedented neutralization breadth and potency against HIV when compared to other previously described broadly neutralizing antibodies (BnAbs).

Format of bispecific antibodies (BsAbs)-VL1-CL1-VH2-CH2-Fc x VH1-CH1 x VL2-CL2

Tetravalent Fabs-In-Tandem immunoglobulins (FIT-Ig™) technology combines Fab fragments of any 2 parental mAbs create a tetravalent, dual-targeting single molecular entity, where the FabA is structurally fused to FabB in tandem at its N-terminus (Fig. 1a). A unique crisscross orientation of 2 sets of VH-CH1 and VL-CL evades any mispairing problem between two short chains and long chain. FIT-Ig have a complete Fc domain which is required for the formation of a disulfide-linked full IgG-like molecule. There is no peptide linker between two Fab moieties, no amino acid mutation in any area, and no altered Fab domain, which potentially reduce the immunogenicity risk. FIT-Ig design also provides enough flexibility allowing independent target engagement by the two antigen binding domains, as the two Fabs are connected only in one chain, instead of in both chains, therefore providing enough space for the lower domain to engage even a large antigen without any steric hindrance. FIT-Ig molecule is symmetrical and composed of long chains, 2 short chains during expression (Fig. 1b). The proper assembly is influenced by molar ratio of 3 polypeptide chains. It is known Fab domain usually have equivalent antigen specificity with mAb. FIT-Ig that utilizes 2 intact Fab domains tend to retains target binding of both parental mAbs. FIT-Ig molecule therefore can be designed based on the properties of the 2 parental mAbs.

Format of bispecific antibodies (BsAbs)-VH-1-TCR Cα x VL-1-TCR Cβ; VH-2-CH-2-Fc x VL-2-CL-2

WuXiBody, replace one parental mAb's CH1/CL region by the T cell receptor (TCR) constant domain. WuXiBody's design ensures cognate HC-LC pairing, the same goal as that being aimed by the CrossMab technology. BsAbs based on WuXiBody can adopt either asymmetric or symmetric format (Fig. 1). For asymmetric WuXiBody-based bsAbs, heterodimerization is promoted by the KiH technology. The TCR constant domain has a relatively low isoelectric point (pI) and consequently the target bsAb containing it also has a pI much lower than that of regular mAbs. In the case of asymmetric bsAbs, this feature promotes the use of ion exchange (IEX) chromatography to separate the target bsAb from potential non-TCR-containing byproducts (e.g., one type of half antibody and homodimer). Thus, introduction of TCR constant domain into WuXiBody construction not only promotes desired chain pairing but also facilitates removal of product-related impurities. Four WuXiBody-based bsAbs with different formats (two asymmetric and two symmetric ones) are show in Fig. 1.

Format of bispecific antibodies (BsAbs)-C-terminal linker of Fc

ADAPTIR-FLEX platform technologies can be used to produce monospecific, bispecific, and multispecific immunotherapeutic proteins. These protein candidates bind to one or more targets found on tumor cells, immune cells, or other cells in the body or circulation to either amplify, suppress, or regulate the body’s defense mechanisms to treat cancer and autoimmune diseases. ADAPTIR-FLEX molecules are composed of two different polypeptides that form a dimer through modifying specific sequences in the Fc region of each polypeptide. Each end of the polypeptide may contain one or two different binding domains, enabling the ADAPTIR-FLEX molecules to bind up to four targets. In addition, the ADAPTIR-FLEX platform can be used to modify the valency of binding to each target, which means it can bind a target, through one binding domain or through multiple binding domains, to a specific target to enable modifying the strength of binding to a specific target.

Format of bispecific antibodies (BsAbs)-Fc antigen binding site

The production of bispecific antibodies involves the addition of a second antigen-binding site in the Fab arms. Sometimes, these types of bispecific products have problems such as poor stability, immunogenicity and challenges in the manufacturing process. Hence, Mab2 simply replaces the Fc region of an existing antibody with an Fcab that binds to a second target of interest to create a full-length bispecific monoclonal antibody (Fig. 1a). It binds with the two different antigens at the same time with favorable pharmacokinetics, antibody-dependent cellular cytotoxicity, and excellent stability and ease of manufacturing.

Peplines of bispecific antibodies (BsAbs)

By binding with 2 antigens from 2 different cells, the BsAbs can physically link them together, thus these kinds of BsAbs are named “engagers”. The redirecting of immune cells to tumor cells by the engagers makes the immune cell activated and then eliminate the target cells. The T cell engagers are the most popular BsAbs which account for nearly half of the clinical trials aimed evaluating the BsAbs. NK cell engagers are recently developed BsAbs for NK cell-dependent tumor cell elimination.

Introduction of neutralizing antibody (NAb)

A neutralizing antibody (NAb) is an antibody that is responsible for defending cells from pathogens, which are organisms that cause disease. They are produced naturally by the body as part of its immune response, and their production is triggered by both infections and vaccinations against infections

Zaire Ebola virus neutralizing antibodies (NAb)

Zaire Ebola virus belongs to the family Filoviridae, which are highly lethal and it outbreaks since emerging in 1967, including a sustained epidemic in West Africa from 2013 to 2015. Zaire Ebolavirus (EBOV) glycoprotein (GP) is now the key target of antibodies. GP is composed of ectodomain GP1 and a trans-membrane fusion domain GP2. GP1 and GP2 helps the virus to enter host cell through attachment, uptake, and fusion. GeneMedi offers the monoclonal antibodies (MAbs) against Zaire Ebolavirus GP, and Zaire Ebolavirus GP1 for therapeutic application and development of diagnostic tests.

Influenza virus neutralizing antibodies (NAb)

Influenza is a transmissible acute respiratory disease caused by the influenza virus. The glycoprotein, hemagglutinin (HA) of influenza virus plays an important role during the viral infection. It helps the virus to bind the cell surface receptors, and transfer the genetic materials into the host cytoplasm through fusion. The neutralizing antibodies against the haemagglutinin (HA) offer the protection against influenza virus infection. GeneMedi offers the monoclonal antibodies (MAbs) against Influenza A HA, Influenza B Virus and Influenza A HA2 for therapeutic application and development of diagnostic tests.

Human cytomegalovirus (HCMV) neutralizing antibodies (NAb)

Human cytomegalovirus (HCMV) causes life-threatening illness in immunosuppressed patients. HCMV envelope proteins viz. glycoprotein B (gB), gH, gL, gO and UL128/UL130/UL131A are essential for the fusion of virus with the host plasma membranes to enter into the host cell. The gB protein is the direct mediator of cell fusion and it requires gH/gL/gO protein complex for activation. HCMV gB or gH/gL proteins provoke HCMV neutralizing antibodies to prevent the entry of virus into both fibroblasts and epithelial cells. GeneMedi offers the monoclonal antibodies (MAbs) against envelope glycoprotein H (gH) and envelope glycoprotein B (gB) for therapeutic application and development of human cytomegalovirus diagnostic tests.

Human endogenous retroviral envelope protein HERV-W-Env neutralizing antibodies (NAb)

Temelimab (formerly called GNbAC1) is an immunoglobulin (Ig) G4 monoclonal antibody that targets the human endogenous retroviral envelope protein HERV-W-Env, shown to be associated with the pathogenesis of certain autoimmune disorders such as multiple sclerosis (MS) and type 1 diabetes mellitus (T1D). By neutralizing HERV-W-Env, temelimab could act as a disease-modifying therapy for these disorders. Hence, GeneMedi offers the monoclonal antibodies against MRSV envelope protein for therapeutic application and development of diagnostic tests.

Pseudomonas aeruginosa infections neutralizing antibodies (NAb)

Pseudomonas aeruginosa is a main source of health-care related infections, including pneumonia and infections associated with urinary tract, wounds, burns, and the bloodstream. P. aeruginosa invades the eukaryotic host cells through the type III secretion system (T3SS). This T3SS helps the bacteria to translocate effector proteins directly into host cells. Mammalian immune systems produce neutralizing antibodies (Ab) against T3SS proteins during infection to inhibit the Pseudomonas aeruginosa infections. Hence, GeneMedi offers the different kind of antibodies such as pegylated, Fab, and Bispecific mAb against the PcrV type III secretion system of Pseudomonas aeruginosa.

Measles virus (MeV) neutralizing antibodies (NAb)

Measles virus (MeV) causes an extremely transmissible respiratory disease and it has also been used as an oncolytic platform. MeV enter into the cells through the cellular receptors such as SLAM (signaling lymphocyte-activating molecule) and PVRL4 (poliovirus receptor-like 4) (nectin-4). Hence, the antibodies against PVRL4 could block measles virus infections. GeneMedi offers monoclonal antibodies against the PVRL4 to prevent the MeV infection.

Anthrax protective antigen (PA) neutralizing antibodies (NAb)

Bacillus anthracis is the causative agent of anthrax. The protective antigen is an important element of the anthrax toxin. It helps bacteria to transfer the enzymatic components into the host cell, through the formation spanning pore on the membrane. antibiotics are effective at the early stages of anthrax, but antibiotics are no longer effective with the accumulation of anthrax toxin. Therefore, the anti-toxin antibody is the most promising approach to treat the anthrax. GeneMedi offers the monoclonal antibodies (MAbs) against anthrax protective antigen.

Shiga toxins (Stxs) neutralizing antibodies (NAb)

Shiga toxins (Stxs) are cytotoxic proteins secreted by Shigella dysenteriae 1 and some serogroups of Escherichia coli (called Stx1 in E. coli). Shiga toxin producing E. coli (STEC) are foodborne pathogens that may colonize and damage the human colon to access to the bloodstream and damage the organs such as kidney and brain. Presently, there are no specific protective treatment against Stx intoxication, and it based on the rehydration therapy, and dialysis. The neutralization of Stx using neutralizing antibody is one of the promising approaches. Therefore, GeneMedi offers monoclonal antibody against Shiga Toxin Type 1 and Type 2.

Toxin A neutralizing antibodies (NAb)

Clostridium difficile continues to be one of the most prevalent hospital-acquired bacterial infections in the developed world. Alternative approaches under investigation to combat the anaerobic Gram-positive bacteria include fecal transplantation therapy, vaccines, and antibody-based immunotherapies. Inhibitory antibodies that recognize the primary C. difficilevirulence factors, toxin A and toxin B, are the most popular passive immunotherapies under investigation.

FDA approved neutralizing antibodies (NAb)

Monoclonal antibodies (mAbs) against bacterial infections are more effective way to treat the antibiotic resistant strains. The antibacterial mAbs have several advantages such as reducing the antibiotic toxicity, treatment time and disease complications. there are numerous mAb products are under development for a range of infectious diseases. Currently, GeneMedi offers mAbs against gram negative bacteria, Staphylococcus epidermidis, Staphylococcus aureus and so on.

How to produce neutralizing antibody (NAb) (production)

Antibodies are made by B-cells in the bone marrow. Bone marrow has been long thought to be a hematopoietic organ. When B-cells are created, they begin to produce antibodies that will bind to specific antigens. Antigen-specific antibody producing, long-term lived plasma cells are largely found in the bone marrow, which contributes to humoral immune responses.

Functional assay for neutralizing antibodies (NAb) discovery--Pseudotype virus (psedovirus, PSV) based and Neutralization Assays

The globalization of the world’s economies, accompanied by increasing international travel, changing climates, altered human behaviour and demographics is leading to the emergence of different viral diseases, many of which are highly pathogenic and hence are considered of great public and animal health importance.

How neutralizing antibody (NAb) work (mechanisms of action, MOA)

Regarding neutralizing antibody activity, Neutralization can be achieved through four main mechanisms

FDA approved neutralizing antibodies (NAb) and our products

FDA approved neutralizing antibodies (NAb) and our products

SARS-CoV-2 neutralizing antibodies (NAb)

SARS-CoV-2, the novel coronavirus responsible for the ongoing COVID-19 pandemic, has been spreading rampantly. The global scientific community has responded rapidly to understand immune correlates of protection to develop vaccines and immunotherapeutic against the virus. The RBD of spike plays a critical role in the very first step of the virus life cycle. The neutralizing antibodies (nAbs) that target the receptor binding domain (RBD) of viral spike (S) glycoprotein[3].

Human Immunodeficiency Virus (HIV) neutralizing antibodies (NAb)

The glycoprotein (gp) 120 subunit is an important part of the envelope spikes that decorate the surface of HIV-1 and a major target for neutralizing antibodies.

Hepatitis B virus (HBV) neutralizing antibodies (NAb)

Hepatitis B virus (HBV) surface antigen (HBsAg) is considered to be the most important target for the diagnosis and immune prophylaxis of HBV infection. HBsAg-specific monoclonal antibodies (MAbs) are extensively used for studying the complex structure of the HBsAg, mapping the neutralizing epitopes and development of HBV diagnostic tests.

Rabies virus neutralizing antibodies (NAb)

Rabies virus is the causative agent of the rabies. The Rabies Virus Spike Glycoproteins are the important protein responsible for the virus entry and the major target of neutralizing antibodies. GeneMedi offers the Rabies Virus Spike Glycoprotein G, Rabies Virus Surface Glycoprotein 4 (gp4) Epitope 1, Rabies Virus Strain ERA GP Ectodomain Epitope G-II, Rabies Virus Spike Glycoprotein G, Rabies Virus Strain ERA GP Ectodomain Epitope G-III, RV Antigenic Site III, RV Antigenic Site III-specific monoclonal antibodies (MAbs) for therapeutic application and development of Rabies virus diagnostic tests.

Respiratory syncytial virus (RSV) neutralizing antibodies (NAb)

Respiratory syncytial virus (RSV) enters into the host cells through the type I fusion (F) glycoprotein. Neutralizing monoclonal antibodies, such as Suptavumab, Palivizumab, Nirsevimab, Motavizumab, Gontivimab, and felvizumab target the major antigenic sites on the RSV F glycoprotein and neutralize the entry process.

What is virus like particles (VLP)?

Virus-like particles (VLPs) are highly structured protein complexes that resemble a native virus capsid ranging 20–800 nm. It is made up of one or more different molecules with the ability to self-assemble and mimicking the form and size of a virus devoid of viral genome and are therefore non-infectious.

GM VLPxTM Virus-like particles development platform

The basic engineering procedure for a VLP construction consists of three main steps: (A) cloning and expression of VLP in the expression system, (B) purification of VLP, and (C) formulation.

Virus like particles (VLP) Expression platforms

Structure of capsid proteins is important to stimulate the immune response and it is depended on the post translational modification of different expression systems. The various expression systems such as prokaryotic (bacteria, yeast) or eukaryotic (baculovirus/insect cell, mammalian cell and plant) have been used to produce the VLP.

Virus like particles (VLP) as immunogens/antigens of transmembrane protein (TM)

A transmembrane protein (TM) is an integral membrane protein, which are embedded in the cell membrane. The transmembrane region, which directly interacts with the phospholipid bilayer and act as gateways to transport of specific substances across the membrane through signal pathways and cell metabolism.

Virus like particles (VLP) for drug delivery

VLPs are striking agent to deliver drugs, small molecules or nucleic acids due to their biocompatibility, biodegradability and targeted delivery. Different cargo-loading techniques have been used for loading either inside or outside the capsid. Different packaging system has been shown in fig. 2.

Virus like particles (VLP) as vaccine

The repeated units of protein monomer on the VLP surface are a strong immunogen. Compared to subunit peptides or proteins, epitopes on the VLPs surface have the natural structure to stimulate the B- and T-cell immune response. Moreover, repetitive epitope on the surface of VLPs stimulate the strong adaptive immunity without the help of adjuvants.

Virus like particles (VLP) for drug discovery

Drug discovery against infectious disease requires the standard biological assays. The biological assay for viral disease is depends on the infection of cultured cells with virus and observation of its effect by microscopic techniques, viral load quantification using plaque assay, or reverse transcription-PCR.

Anti-Monomethyl auristatin E (MMAE) monoclonal antibody(mAb) : Products and Application

Monomethyl auristatin E (MMAE; SGD-1010) is a synthetic derivative of dolastatin 10 and functions as a potent mitotic inhibitor by inhibiting tubulin polymerization.

Tandem VHH/ Nanobody: Structure, Production, Application and Products

Since 1975, monoclonal antibodies have been successfully used for the diagnosis and therapeutics. In 1993, heavy-chain-only antibodies which bind to antigens was discovered in camelids. The variable domain of heavy chain antibody (VHH) was called as nanobodies and it was anticipated as the better therapeutic antibodies in future due to its high stability, refoldability, and manipulable characteristics. VHH has also been effectively used for the diagnosis, bioimaging, immunoassay and affinity purification.

Tandem VHH/Nanobody (single domain antibody) - Introduction

Since 1975, monoclonal antibodies have been successfully used for the diagnosis and therapeutics. In 1993, heavy-chain-only antibodies which bind to antigens was discovered in camelids. The variable domain of heavy chain antibody (VHH) was called as nanobodies and it was anticipated as the better therapeutic antibodies in future due to its high stability, refoldability, and manipulable characteristics. VHH has also been effectively used for the diagnosis, bioimaging, immunoassay and affinity purification.

Structure and Physicochemical Features of Nanobody (single domain antibody)

Nanobody (Nb) is the smallest natural antigen-specific binding functional fragment of about 12~15 kb (Figure 2D). The molecular weight of Nb is smaller than the monoclonal antibody mAb (~150 kb), Fab fragment (~55 kb) or HcAb (~90 kb) Figure 1A–C. Former study showed that these nanobodies can be genetically engineered from the heavy-chain antibody derived from camelids or cartilaginous fish. Their immune systems have naturally evolved into high-affinity V-like domains. Nb has a stronger and faster tissue penetration ability and can reach dense tissues such as solid tumors. The half-life of Nb in the blood is also relatively shorter due to renal filtration.

Nanobody (single domain antibody) production

The production of nanobody primarily required to select a camelid animal for immunization for about 5–10 weeks. The effector B cells are extracted from the plasma peripheral lymph blood of the immunized camel to isolate the total RNA and cDNA synthesis for sequencing. Phage display technology is used to screen the specific binding ability of the nanobody. Finally, the phage vector was transferred to the Nb expression system to express the Nb proteins. Even though the Nb production technology is relatively complicated, the application of phage display technology and the screening steps of Nb are being gradually optimized.

Application of nanobodies (single domain antibodies)

Nanobodies have been used for both diagnostic and therapeutic application due to its high stability, refoldability, and manipulable characteristics. In addition, VHH has been effectively used as a research tool.

Advantages and limitation of Nanobodies (single domain antibodies)

Nanobodies are frequently compared to polyclonal, monoclonal antibodies, antibody fragments, and small molecules. The production of nanobodies is simple and cost effective compared to the polyclonal antibodies. Besides, Nbs are stable in a wide range of temperatures and pH levels. Nanobodies are also compatible with genetic engineering methods, which allow scaffolding and alteration of amino acids to improve binding. Relating to structure, the hydrophilic side of nanobodies, that is not present in conventional antibodies, means they do not have issues with solubility and aggregation. The conventional antibodies do not bind well in grooves or cavities on the surface of the antigen. But, Nanobodies bind with the clefts of the active site of antigen.

GeneMedi's products list of Nanobodies (single domain antibodies)

GeneMedi supplies products of Nanobodies.