Affinity exploration of a bispecific ADAPT towards CD22 for therapeutic purposes

Abstract

Leukemia is the most common type of cancer in children, with B-cell acute lymphocytic leukemia (B-ALL) being a common type in both children and adults. Different types of treatments are available, such as chemotherapy, stem cell treatment and immunotherapy. However, a recurring problem is relapses, with the survival rate after relapse being very low. In combating this issue new methods have been developed, where the usage of surface protein targeting has been a prominent feature. These new methods, such as antibody therapies and chimeric antigen receptor (CAR)-T cell therapy, targets surface proteins such as CD19 or CD22, which are exclusively expressed on the surface of B-cells. These therapies have shown great results; however, they are expensive with many patients still relapsing due to downregulation of the target protein. An alternative, which could provide cheaper production and high modularity is the usage of albumin-derived affinity proteins (ADAPT), a small triple helical scaffold protein of about 6 kDa. The cheaper production is enabled by the possibility of rapidly producing ADAPTs using a bacterial host. The high modularity means potential for multitargeting, which would be beneficial if downregulation of one targeted protein would occur. ADAPT naturally binds to human serum albumin (HSA), an ability which has been preserved to increase the serum half-life of the small ADAPT to enable therapeutic use. Moreover, other residues in the ADAPT have been found changeable in such a way that it can be modified to have an affinity towards another protein, such as for CD22, while simultaneously binding HSA. For this purpose ADAPT(ABD035)_CD22_10 has been developed. However, even though it binds in rapidly it has a high off rate, which is problematic if to be used for therapeutic purposes. In this study, 20 variants were chosen from a maturation selection with a library based on ADAPT(ABD035)_CD22_10, to investigate is any of these offered a stronger affinity. However, none of them did. Moreover, an alanine scan was executed to evaluate which positions are of great importance for the affinity to CD22 as well as for the stability of the ADAPT. Lastly, a library exploration was made to further investigate one position targeted in the alanine scan as well as a position previously unexplored. With the conclusions drawn from these results, a new design for an affinity maturation library is suggested, hopefully deriving new variants with slower off rate. This with the desire to create an ADAPT which could be used for therapeutic purposes in the future.

Popular Abstract

The goal of this project is the development of cheaper and more long-lasting alternatives for treatment of leukemia. Leukemia is the most common type of cancer in children. One type, which develops in B-cells, a type of white blood cells, is the most common type of leukemia in both children and adults. This type of cancer is called B-cell acute lymphocytic leukemia (B- ALL). The treatments used today, such as chemotherapy and stem cell therapy, usually work well in the beginning but the cancer often return, after which the chance of surviving is very low. Newer treatments, wanting to combat this issue, have been developed but are usually very expensive, and we therefore need cheaper alternatives. One such alternative could be albumin- derived affinity proteins (ADAPT). ADAPT is a tiny protein which can be modified to bind to another protein of choice, in this case a protein called CD22. CD22 is a protein on the surface of B-cells, which cannot be found in any other place of the human body. This means that if you find CD22, you have found a B-cell. Since B-ALL develops in B-cells, these are the cells we want to target. The idea is that before injecting ADAPT into the body, a drug will be attached to it, which will kill the B-cell. ADAPT therefore needs to find and bind strongly to CD22 without letting go, so that the drug has time to work. In earlier work, an ADAPT called ADAPT(ABD035)_CD22_10 has been created. It binds CD22 very fast; however, also releases within seconds. The aim of this project is to find a way for ADAPT to release more slowly.
Before continuing, let talk about some pros and cons with ADAPT and it being such a tiny protein. A con is that tiny proteins break down fast in the body, and if it breaks down fast it will not be possible to use it for treatment. To solve this problem, ADAPT can, at the same time as it binds CD22, also bind human serum albumin (HSA) which is a very common protein in the blood. Whilst ADAPT on its own is broken down within hours, HSA takes about three weeks to break down. ADAPT will bind to HSA and be able to piggy-back on its long half-life in the human body. On the other hand, a pro with ADAPTs tiny size, is that we can use bacteria to produce it. This means it can be produced at a high speed and at a low price, offering a cheaper alternative to today’s treatments.
ADAPT is built up of approximately 50 amino acids. 11 of these can be exchanged in such a way that the shape and structure of the ADAPT looks the same, but a few characteristics can be changed. These 11 amino acids are the ones thought to affect how strongly ADAPT binds to CD22. Through switching which amino acids sits where, it is possible to make ADAPT release slower from CD22. Like finding the right piece in a puzzle, where the choice of one piece could affect how well another piece fits. 20 variants, where most of these 11 amino acids had been exchanged based on earlier experiments, were examined but none bound CD22 better than the original. Instead, something called an alanine scan was made. Each of the 11 amino acids were exchanged in individual trials, one by one, to an alanine. Alanine is the smallest of all amino acids and is used as a way of mimicking complete removal of the previous amino acid. This is to see, what happens if we remove this specific amino acid? Can the ADAPT still bind to CD22? If that is the case, this position is probably open to being exchanged for other amino acids, seeing if another would suit better. Can the ADAPT suddenly not bind CD22 at all? Then the original amino acid is probably very important for the binding and should not be tampered with. The last trial of this research included a new position, in addition to the 11 positions which had previously been explored. This was made to see if it would be possible to expand the number of positions which can be exchanged to find a better match for CD22. However, this position did not have any large effect on the result. It can be changed but it will probably not matter too much. Using this information, a new framework has been designed which can be used for creating new variants of ADAPT which will hopefully stay bound to CD22 for a longer time.

• author: Jonsson Pagels, Nora
• supervisor: Solmaz Hajizadeh; Sophia Hober; Emma Larsson
• organization: Pure and Applied Biochemistry; Computational Chemistry
• course: KBKM05 20241
• year: 2024
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: cancer therapy, ADAPT, ABD, protein engineering, albumin, simultaneous bispecificity, leukemia, B-cells, CD22, applied biochemistry
• language: English
• id: 9159081
• date added to LUP: 2024-06-11 09:06:12
• date last changed: 2024-06-11 09:06:12