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Pre clinical models Flashcards

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1
Q

what is rituximab?

A

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

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2
Q

what is the most important method of killing by rituximab?

A

Ab- mediated killing

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3
Q

what are the activating Fc receptor names? in mice

A

FcγR1, FcγR3, FcγR4

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4
Q

what is the inhibitory Fc receptor name? in mice

A

FcγR2B

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5
Q

what are the activating Fc receptors in humans

A

FcγR1, FcγR2A, FcγR2C, FcγR3A, FcγR3B

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6
Q

what antibody subclass is the basis for rituximab?

A

IgG

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7
Q

how do Fc receptors contribute to the tumour microenvironment?

A

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

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8
Q

what is the mechanims of action of rituximab?

A

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)

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9
Q

what is the mechanism of resistance to rituximab?

A

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

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10
Q

what combination therapy helps treat rituximab-resistant patients?

A

Rituximab (anti-hCD20) and anti-hCD32 combination

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11
Q

what relavent preclinical model was needed for the Rituximab (anti-hCD20) and anti-hCD32 combination therapy trial?

A

mice that expressed human cd20 and cd32b and lack the mice genes

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12
Q

what pharmacodynamics/kinetics were performed on antiCD32B mAb?

A

half life at different dosages, % circulating B cells, % splenic B cells,

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13
Q

what factors were part of the safety profile for antiCD32B mAb

A

mouse weight, cytokine concentration

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14
Q

what are patient-derived xenograft (PDX) models?

A

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.

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15
Q

how do you create a PDX model?

A

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

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16
Q

what stage of development are the antiCD32b mAb at?

A

phase 1/2 clinical trials in UK and USA

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17
Q

describe how humanised mice are made

A
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18
Q

how did scienctists overcome the tumour resistance to rituximab?

A

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

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19
Q

how do you create a humanised mouse model?

A

take a sample of peripheral blood, cord blood or fetal liver and idolate CD34+ Haematopoietic stem cell

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20
Q

what mouse model was used in the research fro antiCD32B antibodies?

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A

A mouse that lacked the mice genes for Fc but has the human Fc genes specifically human CD20 and CD32A and B

21
Q

describe the research that investigated rituximab (anti-hCD20) and anti-hCD32 combination therapy in vivo

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A

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

22
Q

what is patient matched co-engraftment

Study These Flashcards
A

when part of the tumor AND the other cells (eg CAFs, immune infiltrate) are injected into the mouse

23
Q

what is a common method to assess status and phenotype of cells within a model system?

Study These Flashcards
A

Flow cytometry

24
Q

what are the benefits of immunodeficient mouse hosts for research

Study These Flashcards
A
  • 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
25
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
26
beta 2 microglobulin and perforin mutations lower the activity of what cell type?
lower NK cell activity
27
what mutation do 'Nude' mice have?
Foxn1^nu
28
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
29
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.
30
What is the function of Rag1? # Footnote Recombination activating gene 1
Rag1 is essential for the V(D)J gene rearrangements that generate functional antigen receptors in T and B cells;
31
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
32
what is Prkdc normally involved with? # Footnote 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.
33
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.
34
what does the scid mutation in the Prkdc gene stand for
severe combined immunodeficient
35
give two examples of severe combined immunodeficient mice
(Prkdc^scid and Rag1^null)
36
what three ways can we model human pathogenesis in mice? ## Footnote 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
37
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
38
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 ## Footnote only mice with human cells, hACE2 and infection with COVID shoed a weight cahnge signifying the model was successful
39
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. ## Footnote Have developed a mouse with a matching immune system healthy cells and malignant cells all human – to test immune drugs or cancer development
40
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. ## Footnote To engraft an immunodeficient mouse with a human immune system
41
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 ## Footnote 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.
42
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
43
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
44
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.
45
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
46
what are 'BLT' mice
bacon lettuce tomato
47
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
48
what are huPBL-Scid mice?
severe combined immunodeficient adult mice transplanted intraperitoneally with peripheral blood myeloid cells

Biomedical technology (9 decks)

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