**16. Multivalent binders**

IgG can only bind a single type of antigen. Hence it is bivalent but not bispecific. Hence, to increase the specificity, the valency of antibodies is being enhanced. Many different regions from different sources are connected together to form molecules that are multivalent and can bind to more than one type of molecules.

#### **16.1 Bispecific**

*Advances in Precision Medicine Oncology*

**13.3 Application in immunotherapy**

serum half-life.

as siRNA cannot target and have very low uptake. Coupling them with antibodies shows enhanced tumor uptake and reduced side effects [59]. An example is CXC chemokine receptor siRNA delivered by coupling with anti-HER2 peptide fusion protein e23sFv-9R is used to target breast cancer cells, having an excess of HER-2 receptors, which promotes apoptosis. Immunotoxins also can be targeted for effective therapy. Immunotoxins from plants and animals may show a rapid immunogenicity and have toxic side effects that may be harmful to humans. For example, an immunotoxin was synthesized by a combination of *Pseudomonas aeruginosa* exotoxin and short-chain variable fragment, which reduced tumor cells' reduced survival and improved the survival rate. An antibody fragment can also be attached to nanoparticles, which then enter the tumor cells via receptor-mediated endocytosis. An example is the anti-HER-2 short-chain variable fragment attached to the PEG-coated iron oxide particle, which shows a better targeting and improved

Tumor cells have barriers to protect them from the body's immune system, such as an impenetrable stroma and immunosuppressive cells in the tumor environment [60]. Tumor cells block antigen overexpression and increase regulatory-T cells and hence escape the immune system of the body. Immunotherapy overturns this mechanism and brings about the death of cancer cells. Bispecific antibodies can target two antigens at once, including an antigen on the cancer cell and a receptor on the cell of the immune system. Then the proteins in cell death signaling are activated by phosphorylation, which then initiates apoptosis. Bispecific antibodies

Chimeric antigen receptor on T cells (CAR-T) is being focused on as a target of antibodies as it helps in specific recognition of cancer cells [61]. Upon activation, it shows a high amount of cytokine release, which then kills cancer cells. This also can

For immunotherapy, a specific target is required for the immune system to attack or else; there can be severe side effects related to immunity. Also, the extent of immune response should also be considered for the efficacy of the treatment. An immune response, which is too low may not be able to kill the malignant cells, and a very strong immune response may cause cytokine storms and harm the healthy cells

IgG is the most explored antibody of choice for cancer targeting. Antigen binding if is the desired mechanism; only antigen-binding fragment can be separated and used. In such cases, the binding affinity and crystallization properties remain the same with smaller size and hybridization, which give enhanced tumor penetration. Also, modifications can be made to increase the specificity and valency of

Involves changes in the structure of IgG to alter its natural properties to obtain desired attributes. The most common structural change includes changes in antibody binding region, e.g., ranibizumab. Right now, there are many fragment-based

link two cells hence bypassing innate as well as acquired resistance.

help to circumvent tumor resistance for killer T-cells.

**86**

instead.

antibodies.

**15. IgG formats**

**14. Antibody derivatives**

This is the most used type from multispecific modified IgG. These can bind to two different antigens at once. For e.g., blinatumomab is used for some types of acute lymphocytic leukemia. It binds to CD19 protein on the cancer cell and CD3 protein on T-cell. It causes the release of cytotoxic chemicals that kill the cancer cell. Dual affinity targeting agents are made from two separate light and heavy regions linked by disulfide bonds after translation.it can be stored for a long time and should be stable for a long time [63].

#### **16.2 Multispecific and multivalent**

A combination makes multivalent antibodies of three or more antibody domains joined sequentially or in a row. These are proteins specific for two antigens containing two pairs of light and heavy variable regions connected in a single chain forming a polypeptide. By virtue of their multivalency, tandem Abs not only target tumors but also cause infiltration and destruction of tumors by killer T-cells. These have longer storage life, better pharmacokinetics, are highly potent [64].

### **17. Antibody conjugates and fusion**

Antibodies can be linked with other molecules such as proteins and peptides to form hybrids. Such combinations give altered biodistribution by targeting a different receptor, extend the half-life of formulation, or impart a new mechanism of action [65].

#### **18. Antibody-drug conjugates**

These hybrid molecules contain antibodies linked to a therapeutic agent. They combine the selective nature of antibodies with cytotoxicity of active agents [66]. It can reduce side effects to a minimum and shows maximum therapeutic success. Conjugates such as Mylotarg have been approved by the FDA. This has caused the emergence of many compounds that differ just by changing the method and site of conjugation. The drugs or linkers are attached to free lysine or cysteine, which gives different products based on the location of conjugation. The most recent trend involves forming a homogenous product by conjugation at particular sites only. It can be done by altering the structure of antibodies to incorporate non-natural amino acids, which act as a handle for attachment. Available tags have been discovered, such as sortase A tag, which attaches the drug to antibody at the glutamic acid site and attaches to therapeutic moiety through a polyglycine tag.
