User:Wang.3643/sandbox

Topic: Heterodimeric antibodies

References:

Gao, C., Mao, S., Lo, C.H.L., Wirsching, P., Lerner, R.A., and Janda, K.D. Making artificial antibodies: A format for phage display of combinatorial heterodimeric arrays. PNAS 1999 96 (11) 6025-6030. doi:10.1073/pnas.96.11.6025.

Gunasekaran K. et al. Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG. The Journal of Biological Chemistry. 2010 Jun 18; 285(25):19637-46. doi: 10.1074/jbc.M110.117382.

Müller, K.M. et al. The first constant domain (CH1 and CL) of an antibody used as heterodimerization domain for bispecific miniantibodies. FEBS Letters, Volume 422, Issue 2, 259 - 264. doi: http://dx.doi.org/10.1016/S0014-5793(98)00021-0.

Ridgway, J., Presta, L., and Carter, P. 'Knobs-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng. (1996) 9 (7): 617-621. doi:10.1093/protein/9.7.617.

Shen, J. et al. Single variable domain-IgG fusion: a novel recombinant approach to Fc domain-containing bispecific antibodies. The Journal of Biological Chemistry. 2006 Apr 21; 281, 10706-10714. doi: 10.1074/jbc.M513415200.

Oct 1 Assignment

https://en.wikipedia.org/wiki/Immunoglobulin_heavy_chain

The heavy chain doesn't always have to bind to a light chain. Pre-B lymphocytes can synthesize heavy chain in the absence of light chain, which then can allow the heavy chain to bind to a heavy-chain binding protein. [1] Reference: Haas, I.G., Wabl, M. (1983). "Immunoglobulin heavy chain binding protein". Nature: 387–389. doi:10.1038/306387a0

https://en.wikipedia.org/wiki/Heavy_chain Suggestion: This article could just be combined with the "Immunoglobulin Heavy Chain" article. Both articles are talking about the same topic and having two separate articles is redundant. This page is lacking in examples of heavy chain variants in other organisms while the "Immunoglobulin Heavy Chain" article is lacking in the specific details of what a heavy chain consists of. Or the "Immunoglobulin Heavy Chain" article could be a "Content" marker for this article since this article has a broader title.

https://en.wikipedia.org/wiki/Transfection Suggestion: This article could include a section on the uses and importance of transfection. Many scientists, especially those in the biopharmaceuticals industry use this method to see if cells will grow using DNA they subcloned in the lab. This method allows scientists to study and control gene expression, conduct mutational analyses, investigate the effects of gene expression on cell growth, etc. There isn't enough information on the usefulness of this method in the science and medical world today.

https://en.wikipedia.org/wiki/Agarose_gel_electrophoresis Suggestion: Another application for an agarose gel is to purify the DNA obtained for subcloning. This page could also include a link to the "Gel extraction" page.

Wikipedia Page Edit 11/17

FINAL DRAFT STARTS HERE

The potential to be better is always looming beyond the horizon. Humans have been progressing at exponential rates. But what’s trumping the progress is disease. To be more specific, cancer. Cancer itself is a result of a mutation in the normal cell’s growth cycle, causing those cells to grow uncontrollably and allowing natural selection to aid in the overall survival of the faulty cells as their daughter cells house the same unstable genes. This unwanted evolutionary process has caused problems for researchers because of how quickly the disease can progress. Artificial selection via cancer therapies against the more sensitive cancer cells have allowed the strongest cells to survive, further complicating the search for a cure. However, a new and innovative form of cancer therapy is currently being tested and is showing great flexibility in combating cancer. Heterodimeric antibodies make use of the body’s natural defense mechanism and allow for flexibility in the attachment of a variety of drugs to the antibody’s arms, allowing for a combined attack to greatly weaken and destroy the cancer cells in the very beginning of cancer therapy. Evolution, specifically somatic evolution, of cancer cells starts with a single cell that eventually acquires genetic variability due to some sort of mutation (Nowell, 1976). This mutation is then passed on to daughter cells and soon the inability of the cell to process its own death results in the formation of tumors. The genetic diversity in cancer also makes it difficult to cure (Merlo, 2010). With only a small amount of cells containing the mutated genes, they can quickly propagate and spread around, similar to the effect of stem cells in normal tissues. With this genetic instability and associated selective process, cancer therapies have to be tailored to be individual-specific, which is difficult to do. Another problem is that cancers are also able to evolve resistance to these therapies (Pepper et al., 2009). A lot of the resistance is due to the killing of more sensitive cancer cells while the stronger ones survive. However, creating a cocktail of drugs to kill the cancerous cells can lead to toxicity within the patient. To control cancer progression without increasing the toxic effects of a drug cocktail, antibody therapy is beginning to be included in cancer treatments. Antibody therapy is a leading form of immunotherapy because it uses the human body’s immune response to combat the formation of cancer cells. A leading form of antibody therapy is the usage of heterodimeric antibodies. Antibodies can have drugs attached to their arms, allowing those drugs to be released and distributed throughout the body. The use of heterodimeric antibodies, which are also asymmetrical antibodies, would allow for greater flexibility and new formats for attaching a variety of drugs to the antibody arms. One of the general formats for a heterodimeric antibody is the “knobs-into-holes” format (Ridgway et al., 1996). This format is specific to the heavy chain part of the constant region in antibodies. The “knobs” part is engineered by replacing a small amino acid with a larger one. It fits into the “hole”, which is engineered by replacing a large amino acid with a smaller one. What connects the “knobs” to the “holes” are the disulfide bonds between each chain. The “knobs-into-holes” shape facilitates antibody dependent cell mediated cytotoxicity. Single chain variable fragments (scFv) are connected to the variable domain of the heavy (V_H) and light chain (V_L) via a short linker peptide. The linker is rich in glycine, which gives it more flexibility, and serine/threonine, which gives it specificity. Two different scFv fragments can be connected together, via a hinge region, to the constant domain of the heavy chain (C_H) or the constant domain of the light chain (C_L) (Muller et al., 1998). This gives the antibody bispecificity, allowing for the binding specificities of two different antigens (Gunasekaran et al., 2010). One of the issues with co-expressing multiple light and heavy chains is the appearance of contaminants, thus posing a purification challenge. That’s where the heterodimer structure comes in. The mutations added to the Fc (fragment, crystallizable) region, which is composed of two heavy chains that contribute two to three constant domains, alter the charge polarity across the Fc dimer to the point that the Fc chains will support favorable heterodimer attractive interactions while the repulsive charge interactions suppress the unwanted Fc homodimer formation (Gunasekaran et al., 2010). The electrostatic steering effects can alter the rate of attraction between proteins, as well. The “knobs-into-holes” format enhances heterodimer formation but doesn’t suppress homodimer formation. The electrostatic steering further increases heterodimer formation but also has the ability to suppress homodimer formation. To further improve the function of heterodimeric antibodies, many scientists are looking towards artificial constructs. Artificial antibodies are largely diverse protein motifs that use the functional strategy of the antibody molecule, but aren’t limited by the loop and framework structural constraints of the natural antibody (Gao et al., 1999). Being able to control the combinational design of the sequence and three-dimensional space could transcend the natural design and allow for the attachment of different combinations of drugs to the arms. The primary use of bispecific antibodies is to redirect cytotoxic immune effector cells for enhanced killing of tumor cells (Shen et al., 2006). By cross-linking tumor and effector cells, bispecific antibodies bring effector cells within the proximity of the tumor cells as wells trigger their activation, leading to effective tumor cell killing. What allows heterodimeric antibodies to succeed in cancer therapies is their flexibility. They have a greater range in shapes they can take and the drugs that are attached to the arms don’t have to be the same on each arm, allowing for different combinations of drugs to be used in cancer treatment. Pharmaceuticals are able to produce highly functional bispecific, and even multispecific, antibodies. The degree to which they can function is impressive given that such a change shape from the natural form should lead to decreased functionality. What differentiates heterodimeric antibodies from other forms of cancer therapy is that it uses the body’s natural defense, antibodies, as the basis for an attack against the cancer cells. They have the ability to attack the cancer cells effectively because of the variety in drug combinations that can be attached to their arms without the risk of a toxic drug cocktail. The heterodimeric antibodies are innovative because they don’t allow the more aggressive cancer cells the chance to proliferate even more.

References Gao, C., Mao, S., Lo, C.H.L., Wirsching, P., Lerner, R.A., and Janda, K.D. Making artificial antibodies: A format for phage display of combinatorial heterodimeric arrays. PNAS. 1999; 96 (11) 6025-6030. doi:10.1073/pnas.96.11.6025.

Gunasekaran K. et al. Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG. The Journal of Biological Chemistry. 2010 Jun 18; 285(25):19637-46. doi: 10.1074/jbc.M110.117382.

Merlo, L. M., Maley, C.C. "The role of genetic diversity in cancer”. J Clin Invest. Feb 1, 2010; 120(2): 401–403. Published online Jan 25, 2010. doi: 10.1172/JCI42088

Müller, K.M. et al. The first constant domain (CH1 and CL) of an antibody used as heterodimerization domain for bispecific miniantibodies. FEBS Letters, Volume 422, Issue 2, 259 - 264. doi: http://dx.doi.org/10.1016/S0014-5793(98)00021-0.

Nowell, P. C. "The clonal evolution of tumor cell populations". Science. 1976; 194 (4260): 23–28. doi:10.1126/science.959840. .

Pepper, J.W. et al. Cancer research meets evolutionary biology. Evol. Appl. 2009; 2 (1): 62-70. doi: 10.1111/j.1752-4571.2008.00063.x.

Ridgway, J., Presta, L., and Carter, P. 'Knobs-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng. 1996; 9 (7): 617-621. doi:10.1093/protein/9.7.617.

Shen, J. et al. Single variable domain-IgG fusion: a novel recombinant approach to Fc domain-containing bispecific antibodies. The Journal of Biological Chemistry. 2006 Apr 21; 281, 10706-10714. doi: 10.1074/jbc.M513415200

Wikipedia Page Edit

https://en.wikipedia.org/wiki/Antibody#Asymmetrical_antibodies

Asymmetrical antibodies
Heterodimeric antibodies, which are also asymmetrical and antibodies, allow for greater flexibility and new formats for attaching a variety of drugs to the antibody arms. One of the general formats for a heterodimeric antibody is the “knobs-into-holes” format. This format is specific to the heavy chain part of the constant region in antibodies. The “knobs” part is engineered by replacing a small amino acid with a larger one. It fits into the “hole”, which is engineered by replacing a large amino acid with a smaller one. What connects the “knobs” to the “holes” are the disulfide bonds between each chain. The “knobs-into-holes” shape facilitates antibody dependent cell mediated cytotoxicity. Single chain variable fragments (scFv) are connected to the variable domain of the heavy and light chain via a short linker peptide. The linker is rich in glycine, which gives it more flexibility, and serine/threonine, which gives it specificity. Two different scFv fragments can be connected together, via a hinge region, to the constant domain of the heavy chain or the constant domain of the light chain. This gives the antibody bispecificity, allowing for the binding specificities of two different antigens. The “knobs-into-holes” format enhances heterodimer formation but doesn’t suppress homodimer formation.

To further improve the function of heterodimeric antibodies, many scientists are looking towards artificial constructs. Artificial antibodies are largely diverse protein motifs that use the functional strategy of the antibody molecule, but aren’t limited by the loop and framework structural constraints of the natural antibody. Being able to control the combinational design of the sequence and three-dimensional space could transcend the natural design and allow for the attachment of different combinations of drugs to the arms.

Heterodimeric antibodies have a greater range in shapes they can take and the drugs that are attached to the arms don’t have to be the same on each arm, allowing for different combinations of drugs to be used in cancer treatment. Pharmaceuticals are able to produce highly functional bispecific, and even multispecific, antibodies. The degree to which they can function is impressive given that such a change shape from the natural form should lead to decreased functionality.