Pre clinical models Flashcards
what is rituximab?
Human CD20 mAb - the first monoclonal approved for treatment of cancer (B cell malignancies)
Targets CD20
Since treated millions, generating 6-7billion dollars a year
now used to treat and expanding number of autoimmune disorders
patients can develop resistance
what is the most important method of killing by rituximab?
Ab- mediated killing
what are the activating Fc receptor names? in mice
FcγR1, FcγR3, FcγR4
what is the inhibitory Fc receptor name? in mice
FcγR2B
what are the activating Fc receptors in humans
FcγR1, FcγR2A, FcγR2C, FcγR3A, FcγR3B
what antibody subclass is the basis for rituximab?
IgG
how do Fc receptors contribute to the tumour microenvironment?
Footnote
depening on the balance of activating and inhibiting Fc receptors present on myeloid cells there will be a skew towards proinflammatory (activating) or anti-inflammatory (inhibiting). When balanced there is homeostasis
Anti inflammatory microenvironment is suppressive and seen in the tumour
by immunoreceptor tyrosine based activator/inhibitor motif signalling
what is the mechanims of action of rituximab?
Rituximab mAB binds CD20 on the tumour cells and attracts effector cells like macrophages and natural killer cells which bind via the Fc part of the antibody which subsequently activates them to kill the tumour via phagocytosis or release of perforin (respectively)
what is the mechanism of resistance to rituximab?
Rituximab mAB binds CD20, but then flips back and binds to the inhibitory receptor Fcgamma2B on the tumor cell (by cis binding). This causes internalisation of the receptor antibody complex and subsequent degradation before it can activate effector cells
what combination therapy helps treat rituximab-resistant patients?
Rituximab (anti-hCD20) and anti-hCD32 combination
what relavent preclinical model was needed for the Rituximab (anti-hCD20) and anti-hCD32 combination therapy trial?
mice that expressed human cd20 and cd32b and lack the mice genes
what pharmacodynamics/kinetics were performed on antiCD32B mAb?
half life at different dosages, % circulating B cells, % splenic B cells,
what factors were part of the safety profile for antiCD32B mAb
mouse weight, cytokine concentration
what are patient-derived xenograft (PDX) models?
Patient-derived xenograft (PDX) models are models of cancer where the tissue or cells from a patient’s tumor are implanted into an immunedeficient or humanized mouse. PDX models simulate human tumor biology allowing for natural cancer progression, and offer the most translational research model for evaluating efficacy.
how do you create a PDX model?
take tumour from a patient, inject a small portion into immunocompromised mice (inject into subcutaneous or mammary fat and (depending on original tissue the tumour was taken from)
flow cytometry to assess status and phenotype of the cell
what stage of development are the antiCD32b mAb at?
phase 1/2 clinical trials in UK and USA
describe how humanised mice are made
how did scienctists overcome the tumour resistance to rituximab?
by inhibiting the inhibitory receptor
unfortunately they couldn’t target the entire receptor due to 98% homology of the extracellular domain of CD32B (FcR2B) and CD32A (FcR2A) which is an activator receptor.
So instead they identified unique epilogs that could be targeted to areas specific to CD32B and not present in CD32A and bound by antibodies
how do you create a humanised mouse model?
take a sample of peripheral blood, cord blood or fetal liver and idolate CD34+ Haematopoietic stem cell
what mouse model was used in the research fro antiCD32B antibodies?
A mouse that lacked the mice genes for Fc but has the human Fc genes specifically human CD20 and CD32A and B
describe the research that investigated rituximab (anti-hCD20) and anti-hCD32 combination therapy in vivo
Southampton Project started in 2009/10
Teamed up with company.
To identify which antibodies were successfull in overcoming the resistance to rituximab
Relevant pre clinical models needed
Used mice that expressed human cd20 and cd32b and lack the mice genes for Fc but have the human Fcs
Monotherapy, Each independently then combine together
Depletion of B cells is extended by two weeks when the therapies were combined
Then the pharmacodynamics
Then measured the depletion of B cells- dose dependent over time
Also analysed safety, kept an eye on general health and weight
what is patient matched co-engraftment
when part of the tumor AND the other cells (eg CAFs, immune infiltrate) are injected into the mouse
what is a common method to assess status and phenotype of cells within a model system?
Flow cytometry
what are the benefits of immunodeficient mouse hosts for research
- enables long term experiments
superior ability to be humanised: excellent engraftment and differentiation of human hematopoietic stem cells into mature human lymphoid and myeloid cells
superior for HIV and other infectious disease research: can essentially be engrafted with a human immune system
what are the major factors that need to be considered when choosing an immunodeficient mouse model?
background features
Functionality of various endogenous immune system components
Leakiness (tendency to produce some function B and T cells as they age)
Lifespan
Radiosensitivity
Breeding performance
Gene features
Mutant gene effects
availability
husbandry (held in specific pathogen-free environments?)
Research type
beta 2 microglobulin and perforin mutations lower the activity of what cell type?
lower NK cell activity
what mutation do ‘Nude’ mice have?
Foxn1^nu
what is beta 2 microglobulin required for?
B2m is required for normal expression of major histocompatibility class I proteins (displaying viral and self antigens to potentially responsive T cells) and for CD8+ T cell maturation and NK cell development
what are the effects on a mouse with homozygous Foxn1^nu mutation?
Homozygous mutants lack a thymus and therefore are T cell deficient; they respond very poorly to thymus-dependent antigens, are unable to reject allogeneic and xenogeneic grafts, and have greatly increased susceptibility to infection.
Footnote
What is the function of Rag1?
Recombination activating gene 1
Rag1 is essential for the V(D)J gene rearrangements that generate functional antigen receptors in T and B cells;
Rag1^tm1Mom homozygous mutations have what effect on the mouse?
homozygous Rag1tm1Mom mutants have no mature, functional T and B cells. The Rag1tm1Mom mutation on the NOD background renders NOD mice diabetes-free. Aging NOD.129S7(B6)-Rag1tm1Mom/J mice develop B cell lymphomas at a high frequency
Footnote
what is Prkdc normally involved with?
Protein kinase DNA activated catalytic polypeptide
Prkdc is instrumental in repairing double-stranded DNA breaks and in recombining the variable (V), diversity (D), and joining (J) segments of immunoglobulin and T-cell receptor genes.
Prkdc^scid homozygous mutants have what effect
Homozygous mutants have no mature T and B cells, cannot mount cell-mediated and humoral adaptive immune responses, do not reject allogeneic and xenogeneic grafts, and are useful cancer research models. The scid mutation renders NOD mice diabetes-free and thereby makes them useful for adoptive transfer of diabetes by T cells.
what does the scid mutation in the Prkdc gene stand for
severe combined immunodeficient
give two examples of severe combined immunodeficient mice
(Prkdc^scid and Rag1^null)
what three ways can we model human pathogenesis in mice?
that can be used for predictive testing
Use human IPS/ES cells
Use (human) more mature sepcialised stem cells type like HSC
Use human differentiated cells
and inject into mouse along with the pathogen to recapitualte the pathology
why may some infectious disease not be reproducible in wild type mice and how do we solve this problem?
some pathogens that infect human cells do not effect mouse cells (eg HIV, plasmodium)
to solve this we create chimeric mice that possess human cells of a particular type so that when we add the pathogen it models human pathogenesis for predictive preclinical testing
how did we model COVID-19 infection & pathology in humanised mice?
to infect cells, covid requires the ACE2 protein
generation of humanised mouse (neonatal mice were infected with human stem cells)
one they grew to adulthood, then intrathecal injection of AAV was used to express ACE2 in the lung
then infect with virus to evaluate and monitor development and therapy in mice
only mice with human cells, hACE2 and infection with COVID shoed a weight cahnge signifying the model was successful
how can canger be generated de novo in mice?
Before injecting stem cells, they transferred human oncogenic genes into them via virus. Attached to a promoter only present in B cells.
Have developed a mouse with a matching immune system
healthy cells and malignant cells all human – to test immune drugs or cancer development
what are HSC-engrafted mice and what is the main advantage?
HSC-engrafted mice develop human lymphocytes in the peripheral blood, bone marrow, thymus, and spleen by 12-16 weeks post-engraftment.
The main advantage of this type of engraftment is that both B and T cells develop from human stem cells in the mouse host and subsequently undergo negative selection, resulting in immune cells that are not self-reactive and tolerant of the host.
To engraft an immunodeficient mouse with a human immune system
what two ways can we engraft immunodeficient mice wiht a human immune system?
either human hematopoietic stem cells (HSCs) or peripheral blood mononuclear cells (PBMCs) are used
Because PBMCs are mature human cells, Graft-vs. Host Disease (GvHD) begins soon after engraftment. These models are, therefore, useful for shorter studies that can take place immediately.
what are benefits and downsides of using PBMCs to engraft an immunodefient mouse wiht a human immune system
it can be a valuable model of dysfunctional T cells for autoimmune research.
PBMC-engrafted mice have also been used to demonstrate the efficacy of T cell modulating drugs in vivo
PBMCs are mature human cells, Graft-vs. Host Disease (GvHD) begins soon after engraftment. These models are, therefore, useful for shorter studies that can take place immediately.
GvHD development can limit the application of PBMC-engrafted mice to immuno-oncology studies
what three ways can we generate humanised mice?
Humanised genetically engineered models (GEMs) - contain one or more human gene through transgenesis or targetted gene replacement (knock ins)
Human immune system engraftment
Human microbiome mice - transferring fecal microbiota from a human donor into a germ-free mouse
what are humanised microbiome mice?
Fecal microbiota transplants (FMT) from human donors are orally-gavaged into germ-free mice, resulting in the establishment of a human-derived microbiome in the mouse. The immune systems in the resulting mice and their offspring develop in the context of the human gut microbiome and may be more translational.
what are humanised ‘HIS’ mice?
intrahepatic transplantation of CD34+ haematopoietic stem cells into new born mice
derive from mobilised peripheral blood, cord blood o fetal liver
what are ‘BLT’ mice
bacon lettuce tomato
what are ‘BLT’ mice
mice transplanted with stem cells derived from fetal bone marrow, liver or thymus
CD34+ HSC intravenous injection into adult mouse
fetal thymus and liver combined producing BLT organoid and transplanted under the renal capsul of mice
what are huPBL-Scid mice?
severe combined immunodeficient adult mice transplanted intraperitoneally with peripheral blood myeloid cells
