title: The influence of constant region polymorphisms on antibody stability and structural dynamics

authors: Warrender, Annmaree Keely

subjects: Antibodies

abstract: Antibodies are highly effective mediators of the adaptive immune response. Their unique structural architecture makes them ideally suited for the specific targeting of disease-causing agents and the subsequent activation of potent effector functions upon binding to immune cells. These properties have led to the extensive use of antibodies in the biomedical industry as diagnostics and therapeutics. Notably, the IgG class of human antibodies (defined by the sequence of the constant region) has dominated the pool of antibody-based therapeutics, largely due to exceptional stability and efficient immune activation. The constant region genes of human IgG harbour a surprising amount of genetic variation in the form of allelic sequences encoding nonsynonymous mutations. Many allelic sequences have only been uncovered within the last five years, yet a handful of studies have already identified links between specific amino acid polymorphisms and improved receptor binding affinities that lead to enhanced activation of effector functions. This thesis furthers our understanding of the evolutionary pressures driving this genetic diversification by investigating the influence of IgG constant region diversity on antibody structural dynamics, including stability and flexibility.
To analyse the structural effects in detail, I expressed a large panel of 35 trastuzumab-formatted antibodies with unique constant region allele sequences from subclasses IgG1, IgG2, and IgG3. Purified antibodies were confirmed to be of high purity and the composition of appended glycans was characterised for each variant. In this thesis, I compare the thermal stability properties of the unique IgG antibodies, measured by red edge excitation shift (REES) spectroscopy. Striking stability differences were resolved both between and within IgG subclasses, leading to new insights about the effect of specific amino acid positions on antibody stabilisation. 
Following the stability analysis, I extensively evaluate the limitations of REES to measure antibody stability, with a particular emphasis on tryptophan content. Using the naturally occurring mutation of arginine to tryptophan present in three of the IgG3 alleles, I generated experimental data that demonstrates the relationship between the solvation state of local tryptophan environments and the REES effect. This analysis highlights the need for matched tryptophan content for robust comparison of antibody stability using a thermodynamic model of REES behaviour.
Finally, the effects of IgG3 hinge length polymorphisms on conformational flexibility are analysed. Here, I used small angle X-ray scattering to characterise the solution structures of allelic variants encoding three distinct hinge lengths. This work provides the first report of X-ray scattering data for naturally occurring IgG3 antibodies with hinge lengths of 32 and 47 amino acids. Comparison of the scattering behaviour and predicted ab initio model structures reveal more compact conformations with reduced space between the Fab domains and the Fc domain when the hinge is truncated, thereby resulting in reduced overall flexibility of the full-length structure. This study highlights dynamic differences between the IgG3 variants tested, which are likely to translate into differences in biological functions.
Taken together, this data provides an important basis for future investigations into the functional roles of IgG allelic diversity. Moreover, the experimental schemes optimised throughout this thesis will enable the high-throughput screening of antibody constant region variants in the future, particularly as more genetic diversity comes to light.

text: The influence of constant region polymorphisms on antibody stability and structural dynamics Antibodies are highly effective mediators of the adaptive immune response. Their unique structural architecture makes them ideally suited for the specific targeting of disease-causing agents and the subsequent activation of potent effector functions upon binding to immune cells. These properties have led to the extensive use of antibodies in the biomedical industry as diagnostics and therapeutics. Notably, the IgG class of human antibodies (defined by the sequence of the constant region) has dominated the pool of antibody-based therapeutics, largely due to exceptional stability and efficient immune activation. The constant region genes of human IgG harbour a surprising amount of genetic variation in the form of allelic sequences encoding nonsynonymous mutations. Many allelic sequences have only been uncovered within the last five years, yet a handful of studies have already identified links between specific amino acid polymorphisms and improved receptor binding affinities that lead to enhanced activation of effector functions. This thesis furthers our understanding of the evolutionary pressures driving this genetic diversification by investigating the influence of IgG constant region diversity on antibody structural dynamics, including stability and flexibility.
To analyse the structural effects in detail, I expressed a large panel of 35 trastuzumab-formatted antibodies with unique constant region allele sequences from subclasses IgG1, IgG2, and IgG3. Purified antibodies were confirmed to be of high purity and the composition of appended glycans was characterised for each variant. In this thesis, I compare the thermal stability properties of the unique IgG antibodies, measured by red edge excitation shift (REES) spectroscopy. Striking stability differences were resolved both between and within IgG subclasses, leading to new insights about the effect of specific amino acid positions on antibody stabilisation. 
Following the stability analysis, I extensively evaluate the limitations of REES to measure antibody stability, with a particular emphasis on tryptophan content. Using the naturally occurring mutation of arginine to tryptophan present in three of the IgG3 alleles, I generated experimental data that demonstrates the relationship between the solvation state of local tryptophan environments and the REES effect. This analysis highlights the need for matched tryptophan content for robust comparison of antibody stability using a thermodynamic model of REES behaviour.
Finally, the effects of IgG3 hinge length polymorphisms on conformational flexibility are analysed. Here, I used small angle X-ray scattering to characterise the solution structures of allelic variants encoding three distinct hinge lengths. This work provides the first report of X-ray scattering data for naturally occurring IgG3 antibodies with hinge lengths of 32 and 47 amino acids. Comparison of the scattering behaviour and predicted ab initio model structures reveal more compact conformations with reduced space between the Fab domains and the Fc domain when the hinge is truncated, thereby resulting in reduced overall flexibility of the full-length structure. This study highlights dynamic differences between the IgG3 variants tested, which are likely to translate into differences in biological functions.
Taken together, this data provides an important basis for future investigations into the functional roles of IgG allelic diversity. Moreover, the experimental schemes optimised throughout this thesis will enable the high-throughput screening of antibody constant region variants in the future, particularly as more genetic diversity comes to light.

year: 2023

title: Unimodal relationship between biomass and species richness of deep-sea nematodes: implications for the link between productivity and diversity

authors: Leduc, Daniel

subjects: Biomass

abstract: Describing large-scale patterns of biological diversity is a first step towards understanding the mechanisms that generate and maintain diversity. The highly diverse deep-sea floor is the largest ecosystem on Earth, but the productivity-diversity relationship in this biome is not well characterized. We investigated this relationship by using biomass of nematodes as a proxy for productivity (particulate organic carbon flux to the seabed). We used sample data collected from the New Zealand and Antarctic regions and combined these with published data from around the globe for broader analyses. There was a significant unimodal relationship between nematode biomass and diversity, i.e. expected number of species, ES(51) both within the New Zealand region and across ocean basins. This relationship remained significant after accounting for the effects of both water depth and nematode abundance. These findings support earlier suggestions of a unimodal productivity-diversity relationship in the deep sea that were based on other proxies (e.g. water depth, modelled particulate organic carbon flux). We argue that the 'productivity context' is of primary importance when determining the strength and nature of the relationship between other environmental factors and diversity. Studies that include either or both extremes of the productivity scale are likely to find that productivity is the main factor limiting deep-sea diversity, whereas those focusing on the intermediate productivity range are more likely to find that other factors (e.g. disturbance, habitat heterogeneity) play a role

text: Unimodal relationship between biomass and species richness of deep-sea nematodes: implications for the link between productivity and diversity Describing large-scale patterns of biological diversity is a first step towards understanding the mechanisms that generate and maintain diversity. The highly diverse deep-sea floor is the largest ecosystem on Earth, but the productivity-diversity relationship in this biome is not well characterized. We investigated this relationship by using biomass of nematodes as a proxy for productivity (particulate organic carbon flux to the seabed). We used sample data collected from the New Zealand and Antarctic regions and combined these with published data from around the globe for broader analyses. There was a significant unimodal relationship between nematode biomass and diversity, i.e. expected number of species, ES(51) both within the New Zealand region and across ocean basins. This relationship remained significant after accounting for the effects of both water depth and nematode abundance. These findings support earlier suggestions of a unimodal productivity-diversity relationship in the deep sea that were based on other proxies (e.g. water depth, modelled particulate organic carbon flux). We argue that the 'productivity context' is of primary importance when determining the strength and nature of the relationship between other environmental factors and diversity. Studies that include either or both extremes of the productivity scale are likely to find that productivity is the main factor limiting deep-sea diversity, whereas those focusing on the intermediate productivity range are more likely to find that other factors (e.g. disturbance, habitat heterogeneity) play a role

year: 2012

title: Positive Ageing in Place: Older Māori in Traditional and Non-traditional Place

authors: Williams, Cassandra

subjects: Māori

abstract: This thesis examines the role of place in experiences of ageing for older Māori, and the extent to which places of choice facilitate a sense of positive ageing. Particular attention is given to multiple experiences of home and the complexities surrounding Māori affiliations to place. Participants were older whānau members over the age of 55 who whakapapa to, and have lived, or currently live in Manaia in the Coromandel. Two participants still reside in Manaia to this day, and two have moved into urban areas in Paeroa and Taupo. Two semi-structured interviews were conducted with participants in their own homes. The first interview focused on housing experiences, personal background and support. The second interview incorporated Māori relationship principles relating to the importance of turangawaewae, whānaungatanga, wairua and manaakitanga. My analysis was informed by a narrative approach, and the relevance of Māori principles to participants’ positive ageing experiences.

I found that Māori relationships with places of ancestral connection are complex and do not exclude other places becoming equally, if not as important to individuals. Experiences of home for participants were multiple and extended to places with which they had forged memories, relationships and positive attachments. Findings indicate that the ability to age positively within particular places is largely reliant on the supports available for older Māori. Wairua was acknowledged by participants as being significant in aiding their positive ageing experiences. Strong connections to non-living objects such as places, nature and tupuna were expressed as being a crucial part of their philosophies on life and death. This feeling of knowing ancestors were part of everyday life alleviated feelings of loneliness and reiterated the never-ending connections between whānau. Everyday contact with whānau provided practical social support as well as a chance for reciprocal relationships to be fostered. When whānau support was not readily available due to geographical distances, technological means of relationship building were used to enable ageing at a distance, allowing home to be played out across physical localities. Health issues were expressed by participants, both historical and reoccurring, and appropriate health care services were regarded as playing an active role in participants’ ageing experiences. Three out of four participants accessed a Māori health provider. By accessing a health care service that was financially inexpensive, where the doctors built a rapport with patients and took advice, participants felt culturally safe and reported positive relationships with their provider. Finances were reported as being a support that needed to be strengthened and a lack of income had a negative effect on participants ageing experiences. Participants’ everyday lives were impacted by financial instability, with a lack of finances standing in the way of fully interacting with the community, negotiating place and being able to age in places of choice.
This thesis argues that ageing in place facilitates positive ageing by allowing older Māori to remain in their places of choice with firm attachments where they can maintain control over their affairs and maintain a sense of continuity. Māori ageing needs to be viewed within a history of colonisation, urbanisation, experiences, relationships and spiritual connectedness to human and non-human entities. It is within this context that ageing in place can be truly explored. Recommendations are made to aid positive ageing in place for older Māori. These include further research into supports, a review of the age of eligibility for state pensions, subsidised transport initiatives, and making Māori health services more accessible.

text: Positive Ageing in Place: Older Māori in Traditional and Non-traditional Place This thesis examines the role of place in experiences of ageing for older Māori, and the extent to which places of choice facilitate a sense of positive ageing. Particular attention is given to multiple experiences of home and the complexities surrounding Māori affiliations to place. Participants were older whānau members over the age of 55 who whakapapa to, and have lived, or currently live in Manaia in the Coromandel. Two participants still reside in Manaia to this day, and two have moved into urban areas in Paeroa and Taupo. Two semi-structured interviews were conducted with participants in their own homes. The first interview focused on housing experiences, personal background and support. The second interview incorporated Māori relationship principles relating to the importance of turangawaewae, whānaungatanga, wairua and manaakitanga. My analysis was informed by a narrative approach, and the relevance of Māori principles to participants’ positive ageing experiences.

I found that Māori relationships with places of ancestral connection are complex and do not exclude other places becoming equally, if not as important to individuals. Experiences of home for participants were multiple and extended to places with which they had forged memories, relationships and positive attachments. Findings indicate that the ability to age positively within particular places is largely reliant on the supports available for older Māori. Wairua was acknowledged by participants as being significant in aiding their positive ageing experiences. Strong connections to non-living objects such as places, nature and tupuna were expressed as being a crucial part of their philosophies on life and death. This feeling of knowing ancestors were part of everyday life alleviated feelings of loneliness and reiterated the never-ending connections between whānau. Everyday contact with whānau provided practical social support as well as a chance for reciprocal relationships to be fostered. When whānau support was not readily available due to geographical distances, technological means of relationship building were used to enable ageing at a distance, allowing home to be played out across physical localities. Health issues were expressed by participants, both historical and reoccurring, and appropriate health care services were regarded as playing an active role in participants’ ageing experiences. Three out of four participants accessed a Māori health provider. By accessing a health care service that was financially inexpensive, where the doctors built a rapport with patients and took advice, participants felt culturally safe and reported positive relationships with their provider. Finances were reported as being a support that needed to be strengthened and a lack of income had a negative effect on participants ageing experiences. Participants’ everyday lives were impacted by financial instability, with a lack of finances standing in the way of fully interacting with the community, negotiating place and being able to age in places of choice.
This thesis argues that ageing in place facilitates positive ageing by allowing older Māori to remain in their places of choice with firm attachments where they can maintain control over their affairs and maintain a sense of continuity. Māori ageing needs to be viewed within a history of colonisation, urbanisation, experiences, relationships and spiritual connectedness to human and non-human entities. It is within this context that ageing in place can be truly explored. Recommendations are made to aid positive ageing in place for older Māori. These include further research into supports, a review of the age of eligibility for state pensions, subsidised transport initiatives, and making Māori health services more accessible.

year: 2012