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Cellular Basis of Immunity > Lectures 13 & 14 - Monoclonal antibodies, cancer immunotherapy and molecular imaging > Flashcards

Lectures 13 & 14 - Monoclonal antibodies, cancer immunotherapy and molecular imaging Flashcards

1
Q

Describe a polyclonal response to antigens

A

Injection with a single antigen will produce a complex mixture of antibodies, each made by a different clone of B cells

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2
Q

What are monoclonal antibodies?

A

Antibodies that bind to epitopes specific to individual antigens

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3
Q

What are the two features on the outside of malignant cells you can look for to target in the production of MABs?

A

Epitopes unique to the malignant or faulty cell

Conserved antigens that are present both on healthy and malignant cells, but in the malignant cell the epitope has a mutation

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4
Q

What did the work of George Kohler and Cesar Milstein achieve between 1975 and 1984?

A

The development of hybridoma technology to produce monoclonal antibodies

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5
Q

How are hybridoma cells produced?

A

A single clone of cells secreting a single
antibody is made by fusing a B cell (Ig+ splenocyte
with finite lifespan) with a myeloma cell (cancerous
Ig- B cell with infinite lifespan).
This results in hybrid cells (hybridomas) that are both immortal and produce a specific monoclonal AB

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6
Q

How are MABs produced in hybridomas lab?

A
  1. Immunise mouse injecting into peritoneum
  2. Antigen goes into blood stream and activates b cells within spleen and lymphatics
  3. Activation of low affinity pentameric IgM
  4. Booster injections given over time every 2 weeks
    Switching from multivalent IgM to bivalent IgG
  5. Aim to get IgG as downstream these are the easiest to work with in terms of therapy and diagnostics
  6. Test serum from tail vein bleed for response to target antigen
  7. If its responded give booster injection and then euthanise mouse and remove mouse spleen and make hybrid cells
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7
Q

How are hybridomas produced in lab?

A
  1. Immunise mouse or rabbit with antigen of choice
  2. Remove the spleen from the immunised animal to gather B cells secreting antigen specific ABs
  3. Screen myeloma cells from culture for those that are unable to make antibody and cannot survive in HAT medium
  4. Fuse splenocytic b cells and myeloma cells to produce hybridoma
  5. Transfer cells to HAT medium, where unfocused B cells will die naturally and immortal hybridomas will survive
  6. Screen remaining cells for specific antibody producing clones, isolating these
  7. Grow the single clone in tissue culture medium, these cells will produce monoclonal antibodies against antigen
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8
Q

How does selection of cells through the use of HAT media work?

A

Aminopterin blocks the main biosynthetic pathways for nucleic acid, so stops production of RNA and DNA
Within the cells there is a salvage pathway and this relies on 2 enzymes – thymidine kinase and HGPRT
If you give exogenous H and T in the HAT medium, they can salvage nucleic acid biosynthesis
Both cell types have thymidine kinase so can make DNA but only those with the HGPRT gene can make RNA through the salvage pathway
Splenocytes are HGPRT+ but aren’t able to grow in long term culture
Myeloma cells are HGPRT- therefore aren’t able to grow in HAT medium
Hybredoma cells has HGPRT+ gene from splenocyte and immortality from myeloma so can grow in both HAT medium and long term culture
Exposing the cells to HAT medium in long term culture ensures only the hybredoma cells survive

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9
Q

Which assay is used to identify the well containing the clone which produces the AB of interest?

A

ELISA

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10
Q

What is phage display?

A

A technique in which peptides or proteins (e.g.
antibody fragments) are expressed as a fusion
with a coat protein of a bacteriophage

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11
Q

How are phages made?

A

Take B cell source and make combinatorial library:
1. Source can be an immunised b cell source/ naïve animal/ human b cells

  1. Take mRNA from these cells and turn into complementary dna
  2. Amplify v gene family using PCR. This will amplify variable heavy and light chains encoded within the complementary DNA
  3. Reassemble to make combinations of vl and vh that may not even be in the animal in the first place
  4. Put into phage which then expresses this info on cell surface as AB fragments
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12
Q

How can phage display be used to produce MABs?

A

Many phages are produced with different gene combinations for Vh and Vl genes (these determine the heavy and light chain fragments of the antibody)
Each phage will therefore produce different subsets of antibodies, which by use of an ELSIA can be screened for their ability to bind the specific antigen of interest.
The population of phages producing the specific MAB we are interested in is gradually enriched by challenge and washing of those that don’t bind. (Biopanning)

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13
Q

What are the steps in the process of phage selection?

A
  1. Build a phage antibody library
  2. Biopanning and washing to remove phages that don’t bind strongly to antigen
  3. Shadow stick selection uses UV light to destroy phages that aren’t required
  4. Reinfection of phages with e.coli in order to bulk up phage production of antibody of interest.
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14
Q

What are the widespread applications of MABs in science and diagnostic medicine?

A 
Diagnostic pathology (cytology and histology)
In vitro diagnostic assays based on ELISA/ lateral flow/ proximity ligation assays for pathogens and biomarkers of disease
Affinity purification and characterisation of antigens
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15
Q

Why do MABs need to be humanised?

A
  1. Immunogenicity - Most MABs are of mouse origin and are therefore xenogeneic, resulting in HAMA relations, causing their rejection and consequential destruction
  2. Origin - Their mouse origin diminishes their ability to elicit effector mechanisms such as complement mediated lysis or ADCC
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16
Q

What strategies have been adopted in order to humanise xenoantibodies?

A
  1. Production of human hybridomas from human B lymphocytes, this however has very low efficiency and there are some ethical issues surrounding it
  2. Replacement of the constant regions of mouse mAbs with those of human antibodies yielding chimeric ABs - this leaves the important heavy and light chain of Fab fragment, this however will still be recognised by the human immune system as foreign eventually and rejected.
  3. Replacement of the complementarity determining regions (CDRs) of a human antibody with those of a mouse antibody via CDR grafting to make a humanised antibody
  4. Production of fully humanised mAbs in transgenic mice - this involves taking a mouse and knocking out their entire genetic mechanism to make human abs in mice and don’t express any mouse ab
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17
Q

What are chimeric antibodies?

A

Antibodies that are engineered to contain both mouse and human regions.
The variable heavy and light chains on the human antibody are replaced with that of a mouse, so these regions can bind specifically to the antigen of interest.
The fixed regions of the human AB remain and these are able to mediate ADCC and CDC which a mouse AB would be unable to do alone.

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18
Q

What is CDR grafting?

A

Replacement of the hyper variable loops within the CDRs (variable heavy and light chains) in a human AB with that of a mouse, in order to increase their binding specificity

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19
Q

How is CDR grafting achieved in vitro?

A
  1. Take a mouse MAB cell line and extract the mRNA that encodes the variable AB domains
  2. Clone this mRNA into a mammalian expression vector, which will produce a full length human AB from the genetic information
  3. This vector is then transvected into CHO (Chinese hamster ovary) cells
  4. CHO then produces human ABs with the CDRs of the original mouse MAB.
20
Q

What are the 3 phases of cancer immune surveillance?

A
  1. Elimination - Innate and adaptive immune response active against tumor
  2. Equilibrium - Tumor is contained by the immune system but not completely destroyed
  3. Escape - Tumor antigen editing via genomic instability causes immune evasion and tutor microenvironment becomes immunosuppressive
21
Q

Which phase of cancer immune surveillance is the target of cancer immunotherapy?

A
  1. Escape
22
Q

In cancer immunology, what is the role of Calreticulin?

A

Within the cancer cell, calreticulin binds unfolded abnormal proteins and shifts them to be exposed on the plasma membrane of the cell.
Calreticulin is also a chaperone protein that activates MHCI but its primary role is translocation of unfolded proteins o the cell surface.
This signals to the macrophages that the cancerous cell must be destroyed

23
Q

Which types of treatment harness the immune system to target cancer?

A

Passive vaccination - Use of therapeutic MABs
Immune checkpoint modulation - Alter how T cells interact with costimulatory pairs on APC and cancer cell
Immune cell therapy - Take cells out of the body, prime them and alter them transgenically before re-infusion back into the human

24
Q

What is the mechanism of action of Rituximab?

A

Naked AB which switches on ADCC or CMC brining in cells of the immune system using their Fc domain

25
Q

What is the mechanism of action of Herceptin?

A

AB blocks certain proteins interacting with receptors that have mutated on the outside of the cell and which when bound to a protein activate the cancer cell to divide

26
Q

What are radioimmunoconjugate antibodies?

A

ABs that carry a radio nucleotide that can ‘batter’ the cancer cell in situ, as it releases energy to damage the DNA within the cancer cell. This happens very locally.

27
Q

What are immunocytokine antibodies?

A

ABs that carry particular cytokines and when they bind, signal to the immune system

28
Q

What are Immunotoxin antibodies?

A

ABs that carry immunotoxins, which are targeted to kill specific cancer cells. These must be high affinity and specificity in order to ensure immunotoxins are targeted to the correct cells

29
Q

What are immunoconjugates?

A

Conjugate an enzyme to an antibody and this enzyme is capable of causing conversion of a prodrug to drug upon binding, leading to targeted drug dispersal

30
Q

What are immunoliposomes?

A

ABs that carry liposomes. These liposomes carry non soluble toxic compounds, that when the AB binds, disperses and releases drug or toxin into the local area

31
Q

What are bispecific mAbs?

A

ABs that bind to markers on the outside of tutor cells or protein marker on outside of NK cells.
They drag NK cells in and cause them to go after and destroy the tutor cell.

32
Q

What mAb therapy can be used to treat Non Hodgkins Lymphoma?

A

Rituximab

33
Q

What is Rituxan?

A

a chimeric human-mouse mAb targeted against CD20,
an antigen present on the surface of neoplastic B cells.
It is able to lyse CD20+ cells using CDC or ADCC

34
Q

What can mAb therapy can be used to treat Breast Cancer?

A

Herceptin

35
Q

What are the two common gene mutations in Breast Cancer?

A

BRCA1 and BRCA2
BRCA1 common in Ashkenazi jews
BRCA2 tumor suppressor gene

36
Q

Which protein expressed by breast cancer cells are recognised by herceptin?

A

HER2 (human epidermal growth factor causes breast cancer cell growth)

37
Q

How can mAbs be used to treat alzheimers disease?

A

mAbs bind the amyloid beta peptides preventing plaque formation

38
Q

How does the mAb therapy for rheumatoid arthritis work?

A

Tocilizumab binds IL-6 receptor preventing IL-6 binding and dampening down the inflammatory response

39
Q

Which costimulator pair down regulates T cells?

A

PD1 and CTLA4

40
Q

How does adoptive immune cell therapy with normal TILs for the treatment of cancer work?

A

Tumor infiltrating lymphocytes are taken from around the cancer cells and grow in culture in a lab

The cells that show the greatest recognition of the patients tutor are then selected from these

Cells are then expanded and activated with cytokines before being re-infused back into the patient

41
Q

How does adoptive immune cell therapy with transgenic TILs for the treatment of cancer work?

A

T cells are engineered to have a specific transgenic TCR which we know has high affinity to a certain tumour antigen such as CD28

42
Q

How does adoptive immune cell therapy with CAR-T cells for the treatment of cancer work?

A
  1. A patients isolated T cells are collected from the blood and genetically modified to express protein known as CAR
  2. CARs allow modified T cells to attach to specific proteins on the surface of cancer cells.
  3. Once bund to cancer cells, the modified T cells become activated and attack the cancer cells
  4. The modified cells are grown in the lab to produce large populations of the cells
  5. CAR-T cells are then infused into the patient
43
Q

What does CAR-T cells stand for?

A

Chimeric antigen receptor modified T cells

44
Q

What are CARs?

A

CARs are modified forms of a T cell receptor, which is expressed on the surface of T cells.

45
Q

What are radio labelled antibodies used for in nuclear medicine?

A

To locate cancer cells or infections using CT/MRI/PET to pick up the rate of decay of the isotope

Cellular Basis of Immunity (13 decks)

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