title: The effects of allelic hinge variation in IgG3 antibodies on protein dynamics and effector functions

authors: Kuplich Barcellos, Marina

abstract: Antibodies play a crucial role in eliminating infections by recognising foreign antigens with high specificity and affinity. If a target is not directly neutralised, antibodies can mediate potent effector function responses by coordinating the recruitment of immune cells through their constant regions. These powerful immune functions are being widely explored in the form of IgG-based monoclonal antibody therapies for the treatment of infections, cancer, and autoimmune diseases. However, the antibody constant region exhibits significant genetic polymorphism, and new variants are rapidly being discovered by advances in genomic sequencing techniques. The documented effects of this diversity include differences in structural dynamics, thermal stability, and Fc-mediated effector functions among a number of antibody subclasses, including IgG1, IgG2, and IgG3. Some variants are reported to be a contributing factor to variability in the outcome of disease and responsiveness to vaccines in humans. The IgG3 subclass, in particular, displays striking differences in the antibody hinge region – responsible for connecting target binding with constant-region-mediated immune function. Here, exon duplications mean the hinge region can vary by as many as 45 amino acids in length. This work seeks to better understand some of the functional consequences of this unusual IgG3 variation. 

To assess the effects of allelic hinge variation on protein dynamics and effector functions, we have expressed a panel of seven anti-CD20 antibodies with unique constant region allele sequences from subclasses IgG1, IgG2, and IgG3. Sequence verified plasmids were used to express full-length antibodies in HEK293 cells at high purity. We first used small angle X-ray scattering (SAXS) to assess the variability in size and structural flexibility between the allelic variants and compared our data with previous research on the same variant panel with antiHER2 variable domain sequences. Following this structural analysis, we next undertook ADCC (antibody-dependent cellular cytotoxicity) and ADCP (antibody dependent cellular phagocytosis) assays to determine if differences in structural dynamics between the polymorphic variants could be linked to differences in effector function. These assays specifically used genome-engineered reporter cell lines to evaluate the ability of allelic variants to engage constant region binding FcyRIIIa and FcyRIIa Fc receptors, typically found on peripheral blood mononuclear cells. Activation was measured by quantifying the levels of luciferase expressed as a reporter.  

The findings provided in this study demonstrate that allelic variants with longer hinges have larger and more flexible structures and tend to present weaker ADCC and ADCP responses. The data presented here is the basis for future work on the influence of IgG constant region diversity on antibody structural dynamics and effector functions, which could contribute to improvements in the design of therapeutic antibodies.

text: The effects of allelic hinge variation in IgG3 antibodies on protein dynamics and effector functions Antibodies play a crucial role in eliminating infections by recognising foreign antigens with high specificity and affinity. If a target is not directly neutralised, antibodies can mediate potent effector function responses by coordinating the recruitment of immune cells through their constant regions. These powerful immune functions are being widely explored in the form of IgG-based monoclonal antibody therapies for the treatment of infections, cancer, and autoimmune diseases. However, the antibody constant region exhibits significant genetic polymorphism, and new variants are rapidly being discovered by advances in genomic sequencing techniques. The documented effects of this diversity include differences in structural dynamics, thermal stability, and Fc-mediated effector functions among a number of antibody subclasses, including IgG1, IgG2, and IgG3. Some variants are reported to be a contributing factor to variability in the outcome of disease and responsiveness to vaccines in humans. The IgG3 subclass, in particular, displays striking differences in the antibody hinge region – responsible for connecting target binding with constant-region-mediated immune function. Here, exon duplications mean the hinge region can vary by as many as 45 amino acids in length. This work seeks to better understand some of the functional consequences of this unusual IgG3 variation. 

To assess the effects of allelic hinge variation on protein dynamics and effector functions, we have expressed a panel of seven anti-CD20 antibodies with unique constant region allele sequences from subclasses IgG1, IgG2, and IgG3. Sequence verified plasmids were used to express full-length antibodies in HEK293 cells at high purity. We first used small angle X-ray scattering (SAXS) to assess the variability in size and structural flexibility between the allelic variants and compared our data with previous research on the same variant panel with antiHER2 variable domain sequences. Following this structural analysis, we next undertook ADCC (antibody-dependent cellular cytotoxicity) and ADCP (antibody dependent cellular phagocytosis) assays to determine if differences in structural dynamics between the polymorphic variants could be linked to differences in effector function. These assays specifically used genome-engineered reporter cell lines to evaluate the ability of allelic variants to engage constant region binding FcyRIIIa and FcyRIIa Fc receptors, typically found on peripheral blood mononuclear cells. Activation was measured by quantifying the levels of luciferase expressed as a reporter.  

The findings provided in this study demonstrate that allelic variants with longer hinges have larger and more flexible structures and tend to present weaker ADCC and ADCP responses. The data presented here is the basis for future work on the influence of IgG constant region diversity on antibody structural dynamics and effector functions, which could contribute to improvements in the design of therapeutic antibodies.

year: 2024

title: Sex differences in outcomes from mild traumatic brain injury eight years post-injury.

authors: Starkey, Nicola J.

subjects: Adult

abstract: The long-term effects of mild TBI (mTBI) are not well understood, and there is an ongoing debate about whether there are sex differences in outcomes following mTBI. This study examined i) symptom burden and functional outcomes at 8-years post-injury in males and females following mTBI; ii) sex differences in outcomes at 8-years post-injury for those aged <45 years and ≥45 years and; iii) sex differences in outcomes for single and repetitive TBI. Adults (≥16 years at injury) identified as part of a population-based TBI incidence study (BIONIC) who experienced mTBI 8-years ago (N = 151) and a TBI-free sample (N = 151) completed self-report measures of symptoms and symptom burden (Rivermead Post-Concussion Symptom Questionnaire, Hospital Anxiety and Depression Scale, Post-traumatic Stress Disorder Checklist), and functional outcomes (Participation Assessments with Recombined Tools, Work Limitations Questionnaire). The mTBI group reported significantly greater post-concussion symptoms compared to the TBI-free group (F(1,298) = 26.84, p<.01, ηp2 = .08). Females with mTBI were twice as likely to exceed clinical cut-offs for post-concussive (X2 (1)>5.2, p<.05, V>.19) and PTSD symptoms (X2(1) = 6.10, p = .014, V = .20) compared to the other groups, and reported their health had the greatest impact on time-related work demands (F(1,171) = 4.36, p = .04, ηp2 = .03. There was no interaction between sex and age on outcomes. The repetitive mTBI group reported significantly greater post-concussion symptoms (F(1,147) = 9.80, p<.01, ηp2 = .06) compared to the single mTBI group. Twice the proportion of women with repetitive mTBI exceeded the clinical cut-offs for post-concussive (X2(1)>6.90, p<.01, V>.30), anxiety (X2(1)>3.95, p<.05, V>.23) and PTSD symptoms (X2(1)>5.11, p<.02, V>.26) compared with males with repetitive TBI or women with single TBI. Thus, at 8-years post-mTBI, people continued to report a high symptom burden. Women with mTBI, particularly those with a history of repetitive mTBI, had the greatest symptom burden and were most likely to have symptoms of clinical significance. When treating mTBI it is important to assess TBI history, particularly in women. This may help identify those at greatest risk of poor long-term outcomes to direct early treatment and intervention.

text: Sex differences in outcomes from mild traumatic brain injury eight years post-injury. The long-term effects of mild TBI (mTBI) are not well understood, and there is an ongoing debate about whether there are sex differences in outcomes following mTBI. This study examined i) symptom burden and functional outcomes at 8-years post-injury in males and females following mTBI; ii) sex differences in outcomes at 8-years post-injury for those aged <45 years and ≥45 years and; iii) sex differences in outcomes for single and repetitive TBI. Adults (≥16 years at injury) identified as part of a population-based TBI incidence study (BIONIC) who experienced mTBI 8-years ago (N = 151) and a TBI-free sample (N = 151) completed self-report measures of symptoms and symptom burden (Rivermead Post-Concussion Symptom Questionnaire, Hospital Anxiety and Depression Scale, Post-traumatic Stress Disorder Checklist), and functional outcomes (Participation Assessments with Recombined Tools, Work Limitations Questionnaire). The mTBI group reported significantly greater post-concussion symptoms compared to the TBI-free group (F(1,298) = 26.84, p<.01, ηp2 = .08). Females with mTBI were twice as likely to exceed clinical cut-offs for post-concussive (X2 (1)>5.2, p<.05, V>.19) and PTSD symptoms (X2(1) = 6.10, p = .014, V = .20) compared to the other groups, and reported their health had the greatest impact on time-related work demands (F(1,171) = 4.36, p = .04, ηp2 = .03. There was no interaction between sex and age on outcomes. The repetitive mTBI group reported significantly greater post-concussion symptoms (F(1,147) = 9.80, p<.01, ηp2 = .06) compared to the single mTBI group. Twice the proportion of women with repetitive mTBI exceeded the clinical cut-offs for post-concussive (X2(1)>6.90, p<.01, V>.30), anxiety (X2(1)>3.95, p<.05, V>.23) and PTSD symptoms (X2(1)>5.11, p<.02, V>.26) compared with males with repetitive TBI or women with single TBI. Thus, at 8-years post-mTBI, people continued to report a high symptom burden. Women with mTBI, particularly those with a history of repetitive mTBI, had the greatest symptom burden and were most likely to have symptoms of clinical significance. When treating mTBI it is important to assess TBI history, particularly in women. This may help identify those at greatest risk of poor long-term outcomes to direct early treatment and intervention.

year: 2022

title: Ati Ue: Te Aitanga a Tuoro

authors: James, Steven (Tipene) Robert

subjects: Ngati Whakaue

abstract: This thesis is based on the manuscripts of my tupuna Tuoro Akapita Pango that are held in a treasured whānau box. The manuscripts contain history and kōrero of Te Arawa written by multiple generations of the whānau Pango. Within this thesis I explore the value of inter-generational knowledge through considering what lies within these whānau manuscripts and taking the opportunity to discover more history about Ngāti Whakaue through a whānau lens. Researching these manuscripts enables reconnection of the past and the present; the reconnection between forgotten knowledge and whānau. 
Specifically, this thesis proposes a Ngāti Whakaue approach to engaging historical materials and research. It centres the voices of the people, such as the many family members involved within the process, as well as the box of writings and the many gems that hide within. Also, it asks what the manuscripts mean for the current descendants involved in the research. 
The research has been a means of reconnecting with the past to give relevance and context to the present. The objective is demonstrating the value of iwi-specific research methods so that whānau feel encouraged to embark on their own journey.

text: Ati Ue: Te Aitanga a Tuoro This thesis is based on the manuscripts of my tupuna Tuoro Akapita Pango that are held in a treasured whānau box. The manuscripts contain history and kōrero of Te Arawa written by multiple generations of the whānau Pango. Within this thesis I explore the value of inter-generational knowledge through considering what lies within these whānau manuscripts and taking the opportunity to discover more history about Ngāti Whakaue through a whānau lens. Researching these manuscripts enables reconnection of the past and the present; the reconnection between forgotten knowledge and whānau. 
Specifically, this thesis proposes a Ngāti Whakaue approach to engaging historical materials and research. It centres the voices of the people, such as the many family members involved within the process, as well as the box of writings and the many gems that hide within. Also, it asks what the manuscripts mean for the current descendants involved in the research. 
The research has been a means of reconnecting with the past to give relevance and context to the present. The objective is demonstrating the value of iwi-specific research methods so that whānau feel encouraged to embark on their own journey.

year: 2021