Germinal center guided affinity maturation of B cells
NTS Identifier
NTS-NO-997306 v.1, 25-01-2024
NTS National Identifier
Field will not be published.
Country
Norway
Language
en
EU Submission
Field will not be published.
Yes
Project duration expressed in months.
47
Keywords
animal model
antibody
B cells
Immunologi
immunotherapy
Purpose(s) of the project
Translational and applied research: Human Immune Disorders
Objectives and predicted benefits of the project
Describe the objectives of the project (for example, addressing certain scientific unknowns, or scientific or clinical needs).
Therapeutic antibodies (Abs) have an undisputed bright future (https://www.antibodysociety.org/resources/approved-antibodies/). Achieving optimal affinity of monoclonal antibodies (mAbs) is essential for developing candidates with the highest likelihood of clinical success. The current methods for affinity optimization, carry the risk of compromising other vital features of mAbs, such as stability, specificity, and solubility. In addition, current in vivo models are labor intensive and require the use of many mice to obtain lead specificities. In contrast, we have preliminary data supporting a method that seems to overcome these challenges by producing superior affinity mAbs ex vivo rapidly and cost-effectively, without compromising other essential characteristics. Our strategy enables ex vivo development of therapeutic monoclonal antibodies, based on selected mutations obtained from the germinal center reaction.
Antibodies are secreted by B cells, and their quality is determined in the germinal center (GC) reaction in the lymph nodes. This project aims to use the GC reaction in the lymph nodes of mice to produce somatic hypermutations from which we are able to select only the affinity enhancing mutations. To this end, the PI has developed B cell receptor knock-in mice of fixed specificities that will serve as the templates for introducing mutations in the GC reaction. The PI has extensive experience using such KI mice. Some strains have been published (PMID: 30181412) and some are unpublished (publication will be finalized with the completion of this project. We have developed a computational software that helps us extract affinity enhancing mutations rapidly. The current project aims to establish a final set of rules enabling rapid affinity enhancement ex vivo. This will be done for multiple specificities targeting pathogens that are of major health concerns.
We will use multiple lines of B cell receptor knock-in mice, with specifies towards SARS-CoV2 and Influenza and analyse the mutations these accrue in the Germinal Center to make. Mutation patterns will be analyzed with our custom software, to tailor superior affinity mAbs ex vivo. We will also analyse mutations from memory responses by using genetic fate mapping (S1PRT2 CreERT). Analysis of mutation patterns in memory B cells will be incorporated into our bioinformatic software. The applicant and PI has over a decade of experience with all experiments listed herein.
What are the potential benefits likely to derive from this project? Explain how science could be advanced, or humans, animals or environment may ultimately benefit from the project. Where applicable, differentiate between short-term benefits (within the duration of the project) and long-term benefits (which may accrue after the project is finished).
mAbs can be employed both as prophylactic and therapeutic agents against various pathogens, including respiratory syncytial virus (RSV), SARS-CoV-2, Influenza, HIV, and Ebola, among others.
The versatility of mAbs allows them to offer protection against pathogens for which effective immunization strategies are challenging to develop. Furthermore, they can serve as a crucial first line of defense against emerging pathogens, especially for individuals with compromised immune systems or those unable to tolerate vaccines. In addition to their significance in infectious diseases, mAbs have revolutionized targeted therapy in various medical conditions such as breast cancer, leukemia, asthma, macular degeneration, arthritis, Crohn´s disease, and transplant procedures.
Our approach shows great promise in enhancing the affinity of mAbs for any target. This implies that we can target specificities that currently are not possible to generate mAbs towards. This is uniquely done by passing B cells through the germinal center reaction.
Predicted harms
In what procedures will the animals typically be used (for example, injections, surgical procedures)? Indicate the number and duration of these procedures.
See attachemnt "procedures", exceeding word limit.
See also "timeline" for the general overview of experiments and groups.
What are the expected impacts/adverse effects on the animals, for example pain, weight loss, inactivity/reduced mobility, stress, abnormal behaviour, and the duration of those effects?
Mice will not become sick, the endpoint are determined by the mutational load/B cell subset we are expecting at the different timepoints. The influenza A virus strain PR8 is a low-pathogenic mouse-adapted RNA virus. In the C57BL/6 mouse model, intranasally injected IAV viruses are cleared 10 days after infection (J Immunol. 1990;144:3980–6). Regardless, we will adopt the precautionary principle and check for weight loss and any signs of cachexia throughout the whole experiment. However we do not expect mortality or the need of euthanasia to prevent pain and distress. During oral gavage, mice may experience minor discomfort due to the insertion of the gavage needle, which can cause a temporary sensation of pressure and stress. mRNA vaccination in the flank muscle induces only a minor sensation of pain from the needle insertion, lasting no longer than a couple of seconds. Likewise Retro-orbital injection in mice is a technique that allows for rapid administration of substances directly into the venous sinus, reducing the time used for injection and stressful preparation of the mice in comparison to tail vein injection. The procedure itself is done under isoflurane and takes no more than 4-5 seconds to perform. This is thus a procedure only inducing momentary and very minor discomfort. Also base of tail and footpad injections are rapid and induce only minor discomfort induced by the needle prick. As we use alum imject which only causes minor and temporary inflammation (in addition in very small volumes), our footpad injections cannot be compared to the often cited CFA injections which cause long lasting and major inflammation. In summary all of the listed techniques only cause minor and temporary pain sensations, limited in time to the period for sample administration.
What species and numbers of animals are expected to be used? What are the expected severities and the numbers of animals in each severity category (per species)?
Species
Total number
Estimated numbers per severity
Non recovery
Mild
Moderate
Severe
Mice (Mus musculus)
2800
0
0
2800
0
Mice (Mus musculus)
350
0
350
0
0
Mice (Mus musculus)
250
0
250
0
0
Mice (Mus musculus)
250
0
250
0
0
Mice (Mus musculus)
750
0
750
0
0
Mice (Mus musculus)
750
0
750
0
0
What will happen to the animals kept alive at the end of the procedure?
Species
Estimated numbers of animals to be reused, to be returned to habitat/husbandry system or to be rehomed
Reused
Returned
Rehomed
Please provide reasons for the planned fate of the animals after the procedure.
Germinal center B cells cannot be extracted and analysed without euthanising the mice.
Application of the Three Rs
1. Replacement
State which non-animal alternatives are available in this field and why they cannot be used for the purposes of the project.
We are studying the germinal center reaction. This is a highly specialized reaction that only occurs in the peripheral lymph nodes of mammals. We cannot obtain the germinal center somatic hypermutations without using a mouse model. The process of affinity enhancing somatic hypermutations does not occus anywhere other than the germinal center reaction. This process is only found in model organisms. Our software is tailored to extract affinity enhancing mutations from the germinal center reaction. Our efficient way of extracting affinity enhancing mutations for ex vivo production of mAbs, will replace the use of a great number of mouse experiments. Currently humanized mice are used in the production pipeline for therapeutic mAbs. This is an inefficient process that in addition does not guarantee the sought after specificity. Our method will replace these experiments and thus represents a major refinement of current methodology.
See attachement "footpad s.c immunization" for details on thje procedure.
2. Reduction
Explain how the numbers of animals for this project were determined. Describe steps that have been taken to reduce the number of animals to be used, and principles used to design studies. Where applicable, describe practices that will be used throughout the project to minimise the number of animals used consistent with scientific objectives. Those practices may include e.g. pilot studies, computer modelling, sharing of tissue and reuse.
We have estimated the minimal number of mice necessary to obtain a large enough dataset to assess affinity enhancing mutations.
3. Refinement
Give examples of the specific measures (e.g., increased monitoring, post-operative care, pain management, training of animals) to be taken, in relation to the procedures, to minimise welfare costs (harms) to the animals. Describe the mechanisms to take up emerging refinement techniques during the lifetime of the project.
Minimal pain will be experienced in the mice, only from the process of a subcutaneous injection of immunogens. The process takes as little as 3-5 seconds.
As for PR8 infection, the mice should not become sick. However to monitor the situation, we will use a scoring sheet provided as a supplement.
Explain the choice of species and the related life stages.
This project uses B cell receptor knock-in mice that have been generated specifically for the project. The B cell receptor specificities are either for Influenza or SARS-CoV2. We use the germinal center reaction in the lymph nodes to obtain affinity enhancing mutations. The B cell receptor knock-in mice will additionally have the inducible S1PRT2 reporter, enabling analysis of memory B cells and plasma cells, also with the aim to find affinity enhancing mutations. Recipients will be congenically marked (CD45.1 vs CD45.2) WT mice. All mice will be adult mice. Experimentally we can use either male or female mice, as the load of somatic hypermutations does not vary between the two.
Project selected for Retrospective Assessment
Project selected for RA?
No
Deadline for RA
Reasons for retrospective assessment
Contains severe procedures
Uses non-human primates
Other reason
Explanation of the other reason for retrospective assessment
Additional fields
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National field 2
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National field 3
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ICD code 2
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ICD code 3
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Link to the previous NTS version outside the EC system