At the beginning they show you the two topics (only XLA and inflammation):
Part Halin
XLA: -Describe the mechanism (Bcells cannot produce antibodies, there’s a defect in the rearrangement process in the bone marrow: surrogate light chain is not replaced after rearrangement of the heavy chain, Btk tyrosine kinase (this is defective) should transmit the signal that heavy chain is rearranged and to rearrange the light chain) -What is the effect of it ? (no antibodies can be produced, as with surrogate light chain they’re not functional, she also added that they can’t survive and go into adoptosis) -what’s the role of ABs, what do the patients suffer from (they’re prone to infections: they activate complement, neutralize/immobilize, enhance opsonisation, also maybe cancer but B cell are not that important in immunosurveillance) -other immunodeficiencies, which part of the immune system is impaired (SCID, ADA, innate immune system phagocytes, complement) -compare severity to the others (in general B cell deficiencies are not that severe than Tcell deficiencies -> Tcells are also needed for B cell activation, innate immune system deficiencies can be as severe.) -how would you treat it (polyclonal Abs from non-diseased blood samples) -are there other conditions where you use polyclonal Abs? (don’t remember this one)
Part Neri
Inflammation
-which cytokines are blocked (Il-1b, Il-6, IL-17/23, TNF-a) -what’s the mechanism of action of the antibodies (neutralize them, crosslink them, form immunocomplexes -> that’s important otherwise could increase t_0.5 of cytokines) -discuss their role in diseases (went for IL-17/23 in psoriasis) -explain their role in the disease (this one was difficult, but he was nice and helped finding the answer: he wanted to hear that Th17 cells (against extracellular aggressors) produce it, IL-17 leads to production of CSF in endothelial cells (?not sure here) which then leads to production and rectrution of neutrophils from the bone marrow. (I guess its better if you go for TNF-a and explain its role with endothelial activation as IL-17 is not really covered in the lecture) -how effective is it? how is efficacy measured? (didn’t know for IL-17 but said for TNF-a effectiveness is measured with the ACR score and ACR20 is about 50 in rheumatoid arth. and guessed that cytokine blockade is similarly effective in psoriasis i.e., quite good effective -> he added that in psoriasis it’s even better like 90%)
In general, they are nice and the exam is fair. The part with Neri was a bit messy but he helps you stay on track.
They show the two topics: serum sickness and production systems
Halin: What do you understand under serum sickness? -I started with the historical part, how they transferred serum between animals and then from horses to humans (diphtheria), it is classified as a type III hypersensitivity reaction (mediated by IgG antibodies). What else in the serum (other than horse antibodies) could we make antibodies against? -proteins present in the blood, the most abundant is albumin Time course of the immune reaction? -BCR binds the antibody, internalizes it, presents a peptide on MHC II, activates T cell (CD4), T cell activates B cells, polyclonal response (different B cells make antibodies with different specificities against the same antibody), it takes about a week to develop antibodies; the own antibodies bind the horse antibodies and create a big immune complex How is this immune complex cleared? -complement activation; transfer to spleen and phagocytosis (she wanted a quite precise answer here and helped me get to it) What are the symptoms of serum sickness? (where do the immune complexes go/accumulate?) -she helped me with the comparison to SLE: rash, painful and inflamed joints, kidney damage (checked by urine test - what do you check for? - proteins and blood) Compare/contrast with contact hypersensitivity? -priming stage: antigen is present at low doses, taken up into APCs, goes to lymph nodes, presentation to T cells -> by the time the T cells get to the contact site, the antigen may not be present anymore (short exposure, unlike with IgGs which stay in blood for a long time); response to the antigen only happens at a later exposure
Neri: Name the most important production systems: -E.coli, yeast, mammalian cells Production in E. coli: what do you need for it? -non-pathogenic strain, plasmid - gene of interest, downstream of a promoter e.g. lac, IPTG can be used to switch on production, antibiotic resistance gene What products can you produce in E.coli and which can you not? -in general: good for simple folding/structure, non-glycosylated, e.g. insulin; not good for mAb (complex folding, glycosylation) We saw that insulin has the A and B chain - how do you produce it in E.coli? -either you produce the 2 chains individually and then the disulfide bonds are formed (in the periplasmic space!) or you produce a precursor and cleave parts of it Stable vs. transient expression? -stable: integrated into chromosomal DNA, not lost after proliferation, large yields, low flexibility, used in industry ... ; transient: gene stays extrachromosomally, lost after cell divisions (he stopped me before I listed more) When you stably integrate a gene, can you just use it to produce your product or what do you need to do before? -you have to show that the cells can produce the protein, use chemically defined media (was not sure what exactly he meant, so I just started listing what I remembered, he interrupted me for the next question) Are the cells really monoclonal? -mammalian cells are not, they can have different karyotypes (he said yes, and also can produce different yields - but the product can be the same)
I think this is everything. They are very nice, Halin gave hints to help me to get to the answers she wanted to hear, and Neri interrupted me when he has heard what he wanted, when he wanted to ask a follow-up question or move on to the next one.
H: Was ist Immunodefficiency? A: Immunsystem kann nicht richtig auf Pathogene reagieren. H: primary vs aquired ID A: primary genetisch, merkt man schon früh als kind, aquired durch HIV, chemotherapie. H: was ist bei chemo das problem? A: killing of Neurtophils, B and T cell is not that bad H: Why? A: Neutrophils are part of innate IS, needed as first-line defense. H: What else? A: TH17 defense. H: Was für genetische ID gibt es? A: zBsp. betreffend AK, XLA, habe XLA erklärt H: Was haben Betroffene für Probleme? A: Opportunistische Infekte, Bakterien, Viren H: Warum Viren? A: AK gebraucht um zBsp andocken von Virus an Zelle zu unterbinden (SARS-CoV-2 und Spike) H: ist XLA schlimm? A: gibt schlimmere, XSCID, weil pathogen-frei und brauchen Knochenmark-Transplantation.
N: What does immunogenity mean? A: Answer of IS to drug, production of AB N: How would you detect AB against IFN-beta? A: ELISA, explain how it works N: Would that also work for Humira? A: No, because Humira and Humira-AB are both fully human -> rabbit-anti-human-AB would bind to both N: What are they doing instead? A: Biacore N: No, but that would have been a much better idea A: Displacement assay with rabbit-AB N: What is the problem with this method? A: didnt know, said something wrong N: Well, what part of AB is most immunogenic? A: variable region N: Explained to me why this matters, but I didnt understand N: Do you know a product on the market which is modified and is still not immunogenic? A: Insulin N: What do you do when the patient makes Anti-Humira-ABs? A: Switch to a different product.
02.02.2023, 14.45 - 15.00 Topics: Autoimmunity and Viral Quality Control
Halin: Autoimmunity Define autoimmunity? (Immune response against self antigens, represents breakdown in self-tolerance mechanisms) What self-tolerance mechanisms are in place? (wanted to know more about peripheral anergy and how that works, so I talked about co-stimulatory molecules CD80/86, how they are low and not upregulated when we are presenting self antigens, how CD80 and CD86 can bind to both stimulatory receptors [CD28] or inhibitory ones [CTLA-4]; induction of Tregs and activation of T cells; upon CD28 binding: IL-2 as the survival signal) In the context of IL-2: Is there a drug that one can use in the context of transplant rejection that directly or indirectly targets this? (Cyclosporin and explained the mechanism of action; she then also mentioned Basiliximab and I said that blocks binding of IL-2 to the IL-2R) Do you know of any genetic mutations that can cause autoimmunity? (Many, I just went for my favourite - AIRE => APECED, explained negative selection in thymus, what AIRE does and what happens if it doesn't function properly).
Neri: Viral quality control What do we do with the cell culture supernatant? (TEM: talked about how that is the only method where we don't rely on any hypotheses/assumptions as to which viruses might already be present; also mentioned endogenous retroviruses) MAP, HAP, RAP and inapparent virus assay (just explained everything from the slides and mentioned again the fact that for the ELISA in MAP/HAP/RAP, one needs to already suspect certain viruses and use those antigens to coat the wells) Downstream processing: We miniaturize our system and perform certain tests with it, can you tell me which? (Viral reduction factor; explained the process, also mentioned that one can simply add up the log-numbers for the 3 orthogonal methods which he liked very much :)) Last question: GMP facilities. What role do they play for the control of viral contamination? (This took me a bit by surprise, but I just said that it is not just about the production process itself, the facility also plays a role, mentioned that for some parenteral drugs we do sterilization or produce them under aseptic conditions, that the generation of MCB has to take place in a GMP facility; he added that there are these classes (Reinraumklassen A-D, flashback to Qualitätsmanagement from 6. semester BSc ^^) but he already seemed content with the answer so he probably didn't expect you to elaborate that much on this.
I think my exam ended after about 10 mins. They were really nice, encouraging, nodding and smiling all the time. Some people have mentioned that they prefer short answers, but I found that they never interrupted me when I elaborated on a topic as long as that was still relevant to the questions they asked.
Here are some of the topics asked by Prof. Halin & Prof. Neri. In the beginning they show you the two topics (on the screen). They explained to me that they have this "exam book" (with two topics per page) and I observed that usually they asked two consecutive people about the same two topics.
immunodeficiencies & glycosylation
XLA and inflammation & cytokines
Lupus & arthritis (humira dosier)
tetanus vaccination & bispecific antibodies
Hypersensitivity reactions & halflife of therapeutic proteins
cancer antigens & immunogenicity in cancer therapy
vaccinations & bioassays
Blood groups (inkl. Rhesus) & protein engeneering
AIDS & GMP
Autoimmunity & humanization/generation of igG
vaccine conjugates & Fc Fusion
Atopy & Glycoenginieering
Tumor immunology & Anti-drug conjugates
Contact hypersensitivity & quality control
Coagulation product & graft rejection
Allergy & hystory of therapeutic proteins
Adaptive immunity against Bacteria & Pegylated proteins
Pharmaforum APV
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31.01.2023 08:15
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