Skip to main content

LUP Student Papers

LUND UNIVERSITY LIBRARIES

Formulation development of a recombinant VAR2-antiCD3 protein for cancer immunotherapy

Warlin, Maja LU and Hallberg, Frida LU (2021) KLGM15 20211
Food Technology and Nutrition (M.Sc.)
Abstract
The use of immunotherapy for treating cancer has received a lot of attention in the recent years. The therapy is based on activating or deactivating the patient’s own immune system to help fight the disease. One way of doing this, is using bispecific molecules that can target both cancer cells and T-cells simultaneously.

In 2003, a researcher found that the surface protein VAR2, expressed on malaria infected erythrocytes, could also specifically bind cancer cells. An idea came to mind to combine this protein with a scFv of the antibody aCD3, for immunotherapy targeting cancer. This rVAR2-aCD3 fusion protein was the start of the company VAR2 Pharmaceuticals. Today, the product has been developed and the company is hoping to start... (More)
The use of immunotherapy for treating cancer has received a lot of attention in the recent years. The therapy is based on activating or deactivating the patient’s own immune system to help fight the disease. One way of doing this, is using bispecific molecules that can target both cancer cells and T-cells simultaneously.

In 2003, a researcher found that the surface protein VAR2, expressed on malaria infected erythrocytes, could also specifically bind cancer cells. An idea came to mind to combine this protein with a scFv of the antibody aCD3, for immunotherapy targeting cancer. This rVAR2-aCD3 fusion protein was the start of the company VAR2 Pharmaceuticals. Today, the product has been developed and the company is hoping to start clinical trials in the near future.

The aim of this master thesis was to select the best candidate for a frozen-liquid rVAR2-aCD3 buffer formulation to be used in clinical phase I studies. The method included 10 analytical techniques measuring different properties correlated to stability. SE-HPLC and SDS-PAGE were used to examine aggregation, degradation and contamination. Size distribution was studied using DLS and SEC-MALS. Furthermore, SEC-MALS and MS was used to determine the molar mass. Protein concentration was measured in NanoDrop and unfolding correlated to temperature was examined in NanoDSF. ELISA and FACS were used to investigate binding. Lastly, a fractionation of monomer and dimer was performed using NGC.

At the start of the study, the stability effect on the protein of six different buffers with pH ranging from 3-8, stored in -80°C versus 4°C respectively, were examined. A final formulation was systematically down selected through three consecutive sub studies, investigating pH dependence, tonicity and physical stress.

In the initial study it was found that low pH (3.3-4.5) seemed to destabilize the protein. Using NanoDSF, it was also found that the unfolding process related to increased temperature seemed to be pH dependent. Buffers with a pH <7 tended to unfold in two steps whereas buffers with a pH >7 unfolded in one single step. From the tonicity study, it was found that both sucrose and NaCl could work as tonicity providers to the fusion protein. However, the combination of histidine buffer and sucrose seemed to have negative effects on the desalting process. Also, it was seen that the samples stored in -80°C were more stable than the samples stored in 4°C. The formulation with PBS, tris-HCl with sucrose and histidine buffer with NaCl showed to be stable all through the 5 weeks of which the tonicity sub-study was performed.
In the final stress study, it was found that the protein was stable towards freezing and thawing up to 6 times. Furthermore, storage at room temperature and rotation caused aggregation and degradation which was found in SDS-PAGE and SE-HPLC measurements. The histidine buffer with NaCl seemed to be most stress resistant with the least aggregation and degradation products forming, keeping the highest monomer content over time and showing the highest binding towards the desired ligand decorin. Hence, histidine buffer with 150mM NaCl was chosen as the best candidate for a frozen-liquid rVAR2-aCD3 buffer formulation to be used in clinical phase I studies. (Less)
Popular Abstract (Swedish)
Varje år, varje dag, varje timme, dör någon av cancer. Cancer är den näst dödligaste sjukdomen i världen efter hjärt- och kärlsjukdomar. Dessutom drabbar sjukdomen inte bara de som dör. Många lider fysiskt och psykiskt av sin diagnos och nära och kära utsätts för stor oro för sina närstående. Den bispecifika molekylen rVAR2-aCD3 är ett nytt, lovande läkemedel mot cancer som snart kan börja prövas kliniskt.
Immunterapi för cancerbehandling har fått stor uppmärksamhet de senaste åren. Terapin går ut på att aktivera patientens eget immunförsvar för att bekämpa sjukdomen och en variant involverar bispecifika molekyler. Benämningen bispecifik innebär att molekylen innehåller två inbindningssäten, ett som binder specifikt till en cancercell och... (More)
Varje år, varje dag, varje timme, dör någon av cancer. Cancer är den näst dödligaste sjukdomen i världen efter hjärt- och kärlsjukdomar. Dessutom drabbar sjukdomen inte bara de som dör. Många lider fysiskt och psykiskt av sin diagnos och nära och kära utsätts för stor oro för sina närstående. Den bispecifika molekylen rVAR2-aCD3 är ett nytt, lovande läkemedel mot cancer som snart kan börja prövas kliniskt.
Immunterapi för cancerbehandling har fått stor uppmärksamhet de senaste åren. Terapin går ut på att aktivera patientens eget immunförsvar för att bekämpa sjukdomen och en variant involverar bispecifika molekyler. Benämningen bispecifik innebär att molekylen innehåller två inbindningssäten, ett som binder specifikt till en cancercell och ett som binder till immunförsvarets T-celler. På det viset kan läkemedlet direkt koppla samman immunförsvaret med cancertumörer.

År 2015 upptäckte forskaren Ali Salanti att proteinet VAR2 uttrycktes på ytan av malariainfekterade röda blodkroppar. Senare fann man att samma ytprotein även kunde binda specifikt till cancerceller. En idé började ta form att kombinera detta protein med en del av vårt immunförsvar, antikroppen CD3 (aCD3) som sitter på T-cellernas yta.

Denna kombination, kallad rekombinant VAR2- aCD3 (rVAR2-aCD3), blev startskottet för företaget VAR2 Pharmaceuticals som idag hoppas kunna starta kliniska prövningar på läkemedlet inom en snar framtid.
För att ett läkemedel ska hålla kvalitet och funktion hela vägen från tillverkning till patient är det viktigt att det trivs i sin miljö. Syftet med detta examensarbete var att hitta den bäst lämpade buffertlösningen till fas I kliniska studier för att hålla proteinet så stabilt som möjligt. Under arbetets gång användes 10 olika analytiska tekniker för att mäta egenskaper hos proteinet kopplade till stabilitet.
Studien innefattade att hitta rätt pH, förvaringstemperatur och hjälpämnen samt att undersöka hur proteinet påverkades av fysisk stress. Ett läkemedel kan utsättas för fysisk stress exempelvis under transport eller förvaring i rumstemperatur. Det konstaterades att lågt pH (3-4,5) tycktes destabilisera proteinet. Vi upptäckte även att både socker (sackaros) och vanligt bordssalt (NaCl) kunde fungera som stabiliserande hjälpämnen till proteinet. Under stresstudien fann man att proteinet behöll sin stabilitet trots frysning och upptining upp till sex gånger.

Slutligen utsågs histidin med salt (pH 6) till att vara den mest stabila bufferten för förvaring av proteinet i en temperatur på -80°C. Buffertlösningen gav upphov till minst antal nedbrytningsprodukter och minst antal aggregat under stress, samt påvisade god inbindning till cancercellernas bindningssäte. I framtiden är förhoppningen att rVAR2-aCD3 proteinet i histidinbuffert med salt ska kunna bli ett effektivt cancerläkemedel ute på marknaden. (Less)
Please use this url to cite or link to this publication:
author
Warlin, Maja LU and Hallberg, Frida LU
supervisor
organization
course
KLGM15 20211
year
type
H2 - Master's Degree (Two Years)
subject
keywords
immunotherapy, cancer, pharmaceuticals, proteins, analytical techniques, molecular biotechnology, formulation, life science, engineering, pharmaceutical technology
language
English
id
9043642
date added to LUP
2021-05-10 13:23:56
date last changed
2021-05-10 13:23:56
@misc{9043642,
  abstract     = {{The use of immunotherapy for treating cancer has received a lot of attention in the recent years. The therapy is based on activating or deactivating the patient’s own immune system to help fight the disease. One way of doing this, is using bispecific molecules that can target both cancer cells and T-cells simultaneously.

In 2003, a researcher found that the surface protein VAR2, expressed on malaria infected erythrocytes, could also specifically bind cancer cells. An idea came to mind to combine this protein with a scFv of the antibody aCD3, for immunotherapy targeting cancer. This rVAR2-aCD3 fusion protein was the start of the company VAR2 Pharmaceuticals. Today, the product has been developed and the company is hoping to start clinical trials in the near future.

The aim of this master thesis was to select the best candidate for a frozen-liquid rVAR2-aCD3 buffer formulation to be used in clinical phase I studies. The method included 10 analytical techniques measuring different properties correlated to stability. SE-HPLC and SDS-PAGE were used to examine aggregation, degradation and contamination. Size distribution was studied using DLS and SEC-MALS. Furthermore, SEC-MALS and MS was used to determine the molar mass. Protein concentration was measured in NanoDrop and unfolding correlated to temperature was examined in NanoDSF. ELISA and FACS were used to investigate binding. Lastly, a fractionation of monomer and dimer was performed using NGC.

At the start of the study, the stability effect on the protein of six different buffers with pH ranging from 3-8, stored in -80°C versus 4°C respectively, were examined. A final formulation was systematically down selected through three consecutive sub studies, investigating pH dependence, tonicity and physical stress.

In the initial study it was found that low pH (3.3-4.5) seemed to destabilize the protein. Using NanoDSF, it was also found that the unfolding process related to increased temperature seemed to be pH dependent. Buffers with a pH <7 tended to unfold in two steps whereas buffers with a pH >7 unfolded in one single step. From the tonicity study, it was found that both sucrose and NaCl could work as tonicity providers to the fusion protein. However, the combination of histidine buffer and sucrose seemed to have negative effects on the desalting process. Also, it was seen that the samples stored in -80°C were more stable than the samples stored in 4°C. The formulation with PBS, tris-HCl with sucrose and histidine buffer with NaCl showed to be stable all through the 5 weeks of which the tonicity sub-study was performed.
In the final stress study, it was found that the protein was stable towards freezing and thawing up to 6 times. Furthermore, storage at room temperature and rotation caused aggregation and degradation which was found in SDS-PAGE and SE-HPLC measurements. The histidine buffer with NaCl seemed to be most stress resistant with the least aggregation and degradation products forming, keeping the highest monomer content over time and showing the highest binding towards the desired ligand decorin. Hence, histidine buffer with 150mM NaCl was chosen as the best candidate for a frozen-liquid rVAR2-aCD3 buffer formulation to be used in clinical phase I studies.}},
  author       = {{Warlin, Maja and Hallberg, Frida}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Formulation development of a recombinant VAR2-antiCD3 protein for cancer immunotherapy}},
  year         = {{2021}},
}