Lecture CAR-T cells PDF

**Lecture CAR-T cells** B cell immunity: B cells make antibodies \--\> there are surface antigens on the tumor and antibodies bind on these surface antigens \--\> this causes killer cells to be guided and bind to the antibodies to kill the tumor cells. T cell immunity: T cells recognize peptides p...

**Lecture CAR-T cells** B cell immunity: B cells make antibodies \--\> there are surface antigens on the tumor and antibodies bind on these surface antigens \--\> this causes killer cells to be guided and bind to the antibodies to kill the tumor cells. T cell immunity: T cells recognize peptides presented on HLA molecules and through TCR they mediated lysis of the tumor cell after binding. - Adoptive T cell immunotherapy: since we recognize that immune effector cells can recognize tumors and kill them, this immunotherapy is made. Immunotherapy therapy releases the T cells of the body to recognize tumor and kill it. In adoptive T cell immunotherapy, T cells are taken out of the body and given back to the patients after renewal. History on immunotherapy - In the past: In bone marrow transplantation you get rid of the hematopoietic system in one patient and give the donor bone marrow to this patient. these new immune cells are from the donor. If there is a relapse of the leukemia, the immune cell transplants recognize these and kill this, being curative. - Then: Tumor was cut into fragments and cultured in order to find which were the activated cells. Hypothesis was that the active cells were specific for the tumor. These cells were isolated and grown into higher amounts \--\> given back to patients. This gave huge reduction of tumors. - In the 90s: The function of the activation of T cells were studied with at the same time another researcher finding the 1^st^ generation of chimeric antigen receptors (CAR). Different groups did different research and all together they produced chimeric molecule that can mediate T cell activation and stimulation = second generation CARS CAR-T cells: There are surface antigens on the tumor which bind the antibody and the HLA molecules bind to T cell receptors which are activated. CAR is a chimeric molecule: it borrows binding domain of the antibody. - T cell receptors can recognize extra -and intracellular antigens but they are restricted to HLA specificity so we cannot use cells from one individual to treat another individua with another HLA molecule composition. Only the patients which have this specific HLA allele can be treated - HLA downregulation is how the tumor cell escapes immune system: if this happens then the TCR is useless - Chimeric antigen receptors (CARs) borrows pieces of the TCR and the costimulatory domains (CD28 and 4-1BB): they can only recognize extracellular antigens and because of that there is not peptide processing required and no restriction of HLA. Hence, fi this is down regulated by tumor, the CARs can still recognize. These CARs can also be customized. This gives flexibility. There have been several generation of CARs throughout the years - 1^st^ generation - 2^nd^ generation: costimulatory domain is added for example 4-1BB or OX-4O - 3^rd^ generation: included two co-stimulatory domains and an activation domain all in 1 line. While there were studies showing that this generation could have some better signaling strength, in the clinic most of the results were less good than 2^nd^. The reason can be that this molecule is much bigger and the second and third domain are too far from the membrane: physiologically all domains are clustered together in the synapse, close to the cell membrane which is the optimal way - 4^th^ generation CARs co-expressed with other molecules that can enhance the activity of CARs ad eventually enhance the antigen effect - CARs are synthetic immunology (engineering): I have a clinical problem I want to solve go back to T cell biology make the CAR Building CARs  Design has to go into DNA molecule cloning into a viral vector infect our T cells which then express our CAR on the surface The first clinical case was done with cars targeting CD19. This is being expressed on B cell malignancies only. The cells expand and a cytokine storm is started at the beginning. Challenges of CARs - Biological challenges of CARs - There were relapses by antigen escape - There was on-target and off-target toxicity - Intrinsic CAR T cell dysfunction - Suppressive tumor microenvironment - Manufacturing challenges: the making of CAR T cells is very complicated, therefore the cost increases. Moreover, there are manufacturing time delays. Solving the biological problems: we know that every part of CAR can provide different functionality. We want to change the CAR in these specific parts to answer different clinical challenges. Some cases - On target tumor toxicity: CD19 is very specific so giving CAR T cells to these patients will kill the tumor but also the B cells. This is fixed easily. However CEA is an antigen expressed in other cancers. When we use CARs against CEA we also kill healthy cells of the colon which express lower levels of CEA \--\> causing severe colitis. To overcome this we can change the injection site, going from systemic injection to local injection. Another way is to do and mRNA transfer causing only a 2 week window for the CARs to be expressed, however toxicity to vital organs are not fixed. Lastly the binding strength (affinity of antibody to antigen) can be changed. If this binds with less strength then it may distinguish between tumor cells with high antigen levels and healthy cells expressing it at lower levels. Split CARs: instead of having one CAR, we make another CAR that binds so week that even if it binds antigen, it does not activate TCR. We rely on another receptor on the CARs that needs to bind to the antigen in order for it to activate TCR Inhibitory CARs: recognition of both receptors on the CARs, the CARs is not activated. Tumor cells only have one of the antibody to bind therefore still activating the TCR killing tumor cell. - Using an inhibitory car for the off-target cells, the off-target cells remain there but the target cells are eliminated. If we do not have an inhibitory CAR, everything is eliminated - Overactivation CAR T cells causes cytokine release syndrome and neurotoxicity. How to control CAR T cell function - By making it dependent on a drug that you want to give: this is an intermediate between the two receptors. - You can have a dimerization agent. - Drugs that are inhibitors of known kinase stopping the intracellular signaling of CARs - Antigen escape: - Transduce T cells so it has two receptors - one receptor with two binding domains - a mixture of CAR T cells of which one binds to one antigen and the other to another antigen - Universal CARs: CARs work with adaptors. By Changin adopters you can change antigen targeting, causing one CAR T cells to target more antigen types. - CAR-T cell persistence is needed (at least for leukemias) in order to sustain the removal of tumor. Relapses were related with lack of detection of CAR T cells. - The best CAR T cell phenotypes are the T~SCM~ CAR T cells. Next generation manufacturing reaches for these populations: - Growing cells in specific cytokine cocktails to protect and maintain this population of stem cell memory cells - Inhibitors of T cell signaling so it blocks the maturing of the cells outside the patients Overcoming the immunosuppressive tumor microenvironment - PD-L1 / PD-1 interaction: - we can have a receptor that binds to PD-1 which is a dominant negative it has a mutation that does not mediate signaling, so it is not inhibitory. This receptor can compete with the endogenous receptor and binds to higher affinity to PD-L1, not allowing PD01 to bind - Switch receptor with PD-L1 on the outside but something else on the inside. It causes it to be a positive signal to the cell after binding PD-1 - Armored CAR T cells secreting various cytokines or chemokines to alter the inflammatory milieu in TME and support CAR T cell function Evaluation of CAR T cell efficacy - Traditional ways - By flow cytometry to detect and phenotype of T cells - qPCRs to know how many CAR T cells are in the blood: - genome sequencing for vector integration sites to see if there are specific clones of the CAR T cells that are expanding. - New - Mass cytometry: with this we can evaluate more markers than flow cytometry which gives the opportunity to see immune cell populations which we otherwise could not be able to detect. - Single cell transcriptomics: this way the faith of specific T cell clones can be tracked in the CARs. This way we can create biomarkers **Lecture Translating Novel Immunotherapies into a successful spin-off** Angiogenesis is the formation of blood vessels - Tissue is oxygen depleted: this is the resting endothelial cells -\> hypoxia - Hypoxia is a transcription factor for VEGF which is the growth factor that can activate endothelial cell sin a preexisting blood vessel to form a new vasculature. - Activation endothelial cells to make proteases to degrade the matrix. This extracellular matrix is stiff and tight and endothelial cells want to growth in it, hence it is degraded. Endothelial cells start migrating and proliferating. In the end you get sprouts that anastomose circulation. This circulation brings oxygen to the cells and the (tumor) cells can growth. This vasculature can easily allow the cells to go into the blood vessels and cause metastasis. - Hypothesis was: If you can intervene with one of these steps, you have the tool to treat cancer. Angiogenesis inhibition does to meet its expectation: research was done by making a monoclonal antibody that neutralizes VEGF. This antibody was successful in the clinic for colorectal cancer in combination with chemotherapy. The survival increased for about 3-4 months as resistance happened quickly. This drug showed limited efficacy with in most cases only active in combination therapy. Moreover, it has serious side effects seen such as blood clotting and proteinuria (destroying your kidneys). - Blocking VEGF must cause the tumor to not grow however this does not happen because you do not treat your vasculature at all, you treat the tumor cells (the tumor cells make VEGF). The tumor cells start to mutate and there will be a growth advantage of cells that are dependent on other growth factors. There are also tyrosine kinase inhibitors developed that block function of the intracellular part of the VEGF receptor. All these VEGF blockers suffer from limited efficacy, serious side effects, and fast resistance. - Good side effect of VEGF inhibitors is that it stimulates immunity because VEGF has at least 25 immunosuppressive function such as inhibition of CD8 cells. Suppressed ICAM in tumor endothelium: For leukocytes to go into the tumor the first thing that should happen is the leukocytes need to leave the blood vessel and go through the endothelial cells. The molecule involved in this is ICAM1. In the vasculature it is required and sufficient for leukocytes to go outside the blood vessel (there are other molecules but ICAM1 is the most important one). - Research (Griffioen et al 1996): Single cell suspension and staining for ICAM1 and endothelial cells (CD34): Low expression of ICAM1 in the vasculature of the tumor when compared to the normal tissue of the same patient/tissue. This means that the tumor can protect itself by down regulating ICAM1. The reason for this is while you stimulate proliferation of cells, you loose the expression of ICAM1. This also has functional effects: when putting inflammatory cytokines to stimulate the ICAM1, adhesion increases. Angiogenesis makes a tumor unsusceptible for immunity. - Can we block this?: *This is Tumor endothelial cell anergy* Angiogenesis inhibition overcomes tumor endothelial cell anergy: mouse were injected with a tumor and when injecting this with an angiogenesis inhibitor the growth goes down. You also stimulate the number of leukocytes going in at the same time: especially increased CD8 cells. Anginex is the synthetic peptide based on the structure of PF4: this is used to inhibit angiogenesis. First there is more ICAM, more leukocytes and then in later stages we get less blood vessels. - In human patients we saw that if you treat with evastin or sunitinib that there is more leukocytes in the tumor when compared to control tumor (Nowak-sliwinska et al angiogenesis, 2023) - It is not only VEGF targeted therapy that causes this but also when we target the EGF receptor. Hence, all angiogenesis inhibitors do this. Combination drug causes a better response than only one drug. For example combining avelumab with axitinib causes better treatment of advanced renal cell carcinoma (Dudley and Griffioen, angiogenesis 2023). - Endothelial cell anergy is nothing else than a vasculature immune checkpoints which can be overcome with a inhibitor. T cells can kill tumor cells but very often this does not happen due to checkpoint molecules that inhibit T cells. You can use immune checkpoint inhibitors (antibody) to prevent this interaction causing the tumor cell to die. Moreover, tumor cells will not die because they inhibited ICAM1. By simply making a drug that causes this molecule to be expressed again, we can fix this. This means that it is still necessary to develop better angiogenesis inhibitors (even when at the start this gave not so good outcomes). - Making therapies based on the targets (instead of VEGF): we want to target a molecule that is not expressed anywhere else in the body. We have 15 genes in our body that we do not use anymore from our developmental stage but which are still present in the vasculature of the tumors. These genes are found by deep sequencing. - Example: Vimentin is a cytoskeletal protein to keep the organelles In place. It is known that this is expressed in the cytosol of the cells. Research showed that when the cells were not permeabilized, vimentin is spit out by the endothelial cells in order to make a deposit of stuff on which they can easily migrate and form blood vessels. In normal tissues this molecule cannot go out only by endothelial cells in the tumor. - A monoclonal antibody is difficult to make and the production is very expensive therefore a vaccine is a better option against the developmental stage genes - Vaccinations create polyclonal antibodies that are better in neutralizing stuff - It is less invasive: it is an injection in the arm which protects you for a long period of time (1-1.5 yrs) - Vaccination Is less expensive: you need less protein than making the monoclonal antibody - Myltiple targeting possible - Problem the immune system will not make antibodies for a self antigen (the embryonic genes). Fixing this by using conjugate vaccinations: coupling the self to non self (bacterial protein for example) causes the immune system to think that this is non-self. And makes the monoclonal antibody. You create an auto immune reaction but this is not dangerous because the gene is no where else anymore. - iBoost technology. The TRXtr (the foreign non-self antibody conjugated to self) part is taken from the self-antigen and is shortened into an antigen that causes a very subtle immune response so that the immune response for your self antigen becomes a lot better. Research with the conjugated vaccine - CVx1 vaccine in colorectal carcinoma in healthy mice tumor injected all mice make antibodies but the body weight did not differ (probably not toxic). - Research done in dogs with bladder cancer: 4 vaccinations with 2 eeks intervals and every couple of months maintenance antibodies are given all dogs made antibodies (Engbersen et al cancers 2023) and the survival was doubled. - Clinical study in dogs with osteosarcoma: In the past, doing a surgery stretched the survival to 72 days and chemotherapy with the surgery stretched with 300 days. In this new trial dogs were given the vaccine: this vaccine stretched the survival as far as the chemotherapy. **Lecture monitoring-imaging of immune targets in rheumatic diseases** PET allows for in vivo visualization of receptors and immune cells in vivo. A PET tracer consists of a compound that is capable of binding to immune targets and by coupling to a radionucleotide we can visualize these targets. Several radionucleotides can be coupled to one target: - the half life of these can differ: if you want to visualize something big you use a large half life but if we want to visualize a small peptides we need a nucleotide with smaller half life. - Radiation burden should be kept within safe limits in the body - When you inject a pet tracer consisting of positron emitters comes into tissue and binds to negative electron. The gamma rays are detected b the circulate camera and because these are in the opposite directions it can localize where the signal is coming from in the body 1. Pet tracer injected intravenously. It is distributed cross tissues 2. Patient positioned in scanner 3. Scans - Advantage = the whole bod can be visualized in one imaging session. - At the molecular level: injected into the blood vessels and at the site of inflammation it penetrates tissue easily and binds to its specific receptors. Potential clinical application of PET - Early diagnosis of rheumatic diseases: before the clinician can diagnose a disease. - Example: when patient has pain in the joint but there is no swelling yet but it is possible that it will develop to RA, PET can help to visualize and we can intervein early on \--\> preventive strategies - Early therapy monitoring to predict outcome of treatment: currently we treat on a trial/ error based with treatment. Usually we need 3-6 months whether this works and if it does not work it is switched to another drug, taking a few years for the right medication. This needs to be prevented - Not every disease is homogenous: each treatment is based on the different types of immunotypes. With PET we can develop personalized treatment strategies - Imaging can be used to see where the pathology is located in the body and with imagen we can guide biopsies in these areas and learn what is going on there - We can also label the drug and do kinetic studies to see where it goes to, to help in drug development *Imaging data of rheumatic immune targets* Macrophages - Normally there is a very thin layer of synovial tissue (few cell layers) and there is no fluid present. In RA (pathological) there is a lot of fluid , the joint is swollen, and the synovial tissue layer is increased - The macrophages play a central role in RA. It produces several proinflammatory cytokines and thought that it is interacting with T/B cells and osteoclasts to damage the joints. - Not treated RA causes full deformation of the joints which is irreversible - Macrophages are important because - They are present early on in development and hence provides possibilities for early diagnosis - Relates to therapy efficacy - RA is a systemic auto immune disease so it can effect several organs in the body - Examples of tracers - FDG is a glucose compound that can accumulate in any metabolic active tissue and since inflammation is metabolically active, it will accumulate FDG - FDG causes black spots that corresponds to inflammation in joints - Very sensitive tracers but not specific so any metabolic active process will accumulate FDG, which is less useful for rheumatology - PK-11195 is a protein from the TSO class and it is expressed on activated macrophages. It came from the neurology field because it was used as tracer for neuro inflammation. - Uptake of this tracer is happening in inflamed joints. To prove whether this is mediated through macrophage, a staining for different macrophages are done in vivo. - Early diagnosis in patients with painful joint without swelling: Almost all PET positive patients developed RA in about one year. However some patients were missed. This was related to only imaging hands of the study and the ones that were missed developed Rain feet and the tracer has quite some background activity in muscles/soft tissue which limited specificity. - Another study for early treatment monitoring: imaging at baseline showed inflammation and after 2 weeks with the anti-inflammatory treatment we could see the visual change and quantification of the signals also showed decrease. - Folate receptor is an important receptor on macrophages: it is expressed on activated macrophages - This folate receptor beta is a target for macrophage imaging in RA. - Previously shown in ovarian cancer patients that folate imaging also detected arthritis, hence they thought this folate might be useful in arthritis monitoring. - Research to make the new tracer - In synovial fluid, the folate receptor expression was quantified: higher in RA patients - Stained for the folate receptor showed positive staining and the PEGylated folate receptor was develop din two steps. The affinity plots of the PEGylated compound (fluoro-PEG-folate ) showed: - Food folyc acid is rapidly methylated which has less affinity to binding to the F-PEG-folate receptor, so little competition is seen. - Animal models with the new compound; - Arthritis model: one knee has arthritis and the other is a control - The uptake of the fluoro-PEG-folate is seen in the inflamed joints but not in the control, hence specifically targeting the arthritis. - In humans: it was seen that there is a clear uptake of the folate marker in the inflamed joints. Advantage is that It is very rapidly cleared from blood so less background. This compound can be used for early diagnostics and hence early treatment. B cells - Bb cells produces auto antibody directed against the own tissue of the body. - The most important one in clinic: IGM RF and Anti-CCP - Are produced by the plasma cells which are developed in the maturation process of the B cells - CD20+ B cells are often in lymphocyte aggregates in synovial tissue. When giving an anti CD20 antibody (rituximab) a decrease is seen in inflammation when taking biopsies. But this is very invasive so an alternative might be imaging. PET scans are used to quantify the expression of CD20+ B cells in the joints and this worked - If this is a B cell driven disease we can use this as monitoring Fibronectin - Associated with angiogenesis and is expressed in RA synovium as a target - A construct of a small antibody fragment (D8) was made and coupled this to IL10 to develop targeted delivery of this anti inflammatory cytokine in the inflamed joints. The hypothesis was that targeting delivery would be better than giving it systemically because there was evidence that systemically was not Woking good enough. After imaging of whole body, in R patients this compound reached the joints and there was high accumulation in liver and spleen. Why: Compared healthy rats to rat model with arthritis: found that the fibronectin expression was increased significantly in the arthritic rats in the spleen/liver when compared to healthy rats. Which meant that a lot of this drug would go to the liver/spleen, causing huge amounts of side effects. Currently this drug is being changed to decrease this. Spondylarthritis is mainly effecting the spine and pelvis and it is usually happening in young people before 45 years. Here there is inflammation in spine which is evolving into new bone formation. There can be new bone formation between vertebrae which can cause bamboo spine formation where all the vertebrae are interconnected by new bone formation causing a curve in the spine of these patients. - Diagnosing this disease very early on Looking when the new bone formation is happening because it is most likely happening early in disease but if we use regular X-ray, we can see this around 3-5 years after formation (even diagnostic CT-scans cannot find it in early stages). Looking at molecular levels using PET and fluorite that enters he new bone formation process might help to diagnose it early on: Research found that F-18-fluoride PET-CT found early bone formation. - Pet scans to use guided biopsies in this disease from the vertebrae form control vs pathological areas: they could visualize new bone formation in the body - Before vs 12 week after anti-TNF treatment showed reduction of the new formed bone marrow **Lecture clinical aspects of immunotherapy side effects - the presentation itself contains all the information needed.** There are 4 groups of antibodies that are targeting 4 specific targets as checkpoint inhibitors: the targets are PD1, CTLA4, PDL-, LAG3. Checkpoint inhibitors have improved survival of patients with stage IV solid tumors including melanoma, renal, lung, and bladder cancer but there is till room for improvement. The is an increasing number of indications: The application of ICI is rapidly increasing. We see more indication, different tumor types and more patient subgroups. The application is also used in combination. Not only in stage 4 but also Early stages where patients have their tumors removed such as stage 3, we treat with immunotherapy. Even with melanoma in stage 2 some drug is more effective than later stages. These patients live longer when treated at early stage with les side effects. Side effects of ICI can occurat any time but mostly in the first 3 months. The side effects can occur in many organs at the same time but mostly thyroid abnormalities colitis, diarrhea, hepatitis, pain in the joints, and fatigue. Some of them are more rare such as adrenal problems - Ipilimumab based therapy kickstarts immune response but also increases the side-effect chance a lot (very toxic) This table shows any grade at a specific number of patient and grade 3-4 adverse events happening in the patients (lower percentage) How can you approve a drug that causes 3-4 adverse effects in 60 percent of patients (IPI NIVO) Increasing toxicity incidence and prevalence - ICIC at stage IV: Patients live longer with ICI but also live longer with ICI side effects: the chronic and late-onset side effects are highly relevant: - ICI in earlier stages - Stage III : adjuvant anti-PD1 given. This reduces the chance of recurrence but It is not shown that the patients live longer yet in melanoma. About 40% of these patients will have chronic adverse effects - Stage II: adjuvant pembrolizumab. The prognosis is relatively good: the pembro approved survival but still in the patient population 60% have grade 3-4 adverse effects and 3\# get related endocrinopathies. Is there a relation between side-effects and efficacy: patients with melanoma because of the shared antigens between the pigments of the skin and the melanoma appearing.., tend to have a good outcome when they have skin toxicity. If patient comes with adverse event, we have to grade this on a severity scale. We have a grade 1 (mild) to 5(patient dies due to toxicity). There are no specific gradings for immune related side effects, this table is just general. For example if you look at the grade for arthritis : grade 1 is mild pain, grade 2 is moderate pain, grade 3 is severe pain limiting safe care. You can imagine that for rheumatologists this is very vague: every patient has different skin problems - Grade 1: no drug, monitoring - Grade 2: monitor but you may interrupt if there is a persistent grade 2 - Grade 3: More severe, start treatment which is generally high dose steroid treatment - This is based on ESMO guidelines but there is no randomizes studies that tell us how many treatment is better: if the side-effect is severe we give 2kg per kilo of steroids or give high dose of pulses and if it is not that severe we give 1kg per kilo. If this is not enough you may add other immunosuppressants. Case 1: sander, 44 - Two years ago he presented with melanoma - Increased LDH indicates slightly worse prognosis - After 4 cycles with IPI-NIVO, he was admitted with severe colitis (diarrhea) : grade 3 colitis (irAEs) - Endoscopy showed bowel problems - Some time he was giving infliximab: normally this drug is given for a very long time but in these type of patients it takes one to two treatment of this drug to get rid of diarrhea - He has a response already (toxicity) is it useful to give immunotherapy? Yes if It is NIVO caused toxicity it is good to give the immunosuppressant anti-PDL1 - After a month he god bijniefinsufficientie which let them to think that we need to continue with anti-PDL1 - But after he got pancreatitis they stopped the treatment Impact immune suppressants (IS) on outcome - Corticosteroids will likely not take the response away, once there was a response this will be durable (taken from literature) - Second line immunosuppressants - DMTR shows shorter survival in patients with grade 3 side effects if treated with steroids and ICI c Lecture multiplexed IHC IHC and cancer immune monitoring - Monitoring happens over time - IHC is done on biopsies, but this is invasive so often researchers only take pre clinical biopsies to check for biomarkers in order to diagnose and sometimes also to check what treatment is best for these people - After treatment biopsies are often not done - Cancer immunoscore is a biomarker to check for the amount of lymphocytes - You stain for CD3 and CD8 and check for the lymphocytes in the tumor center CT and invasive margin IM Higher immunoscore correlates with better survival and therefore respond better to standard chemotherapy More novel treatment approaches are targeting the TME. - Can we predict responses to pd1/pdl1 inhibitors? - Mainly patients are treated in clinical trials and either respond or not respond and then we look back whether there are biomarkers that could have predicted this - Mainly single markers are explored but in research are also using more advanced technologies like multiplex IHC - The markers that are most predictive for responses are derived from profiling cells that are directly associated with tumors so IHC is very good technique!! (to see their interaction with TME) - Biomarkers pd1/pdl1 - PDL-1 WAS PREDICTIVE IN LOW AMOUNT of cases because the PDL-1/PD-1 is very heterogenous. It is still used often but not the best biomarkers - CD8+ T cell infiltrate as marker for PD-L1 blockage: patient that have lot of CD8 T cells are responding better to PD-L1 blockade. If you give immune checkpoint modulation, these cd8 t cells can become active again - What do you need to activate T cell: effective antigen presentation by MHC class I/II. However, a lot oft humors downregulate MHC expression \--\> even though CD8 T cells are present, they cannot be activated. - MHC class I expression can be predictive for responses to checkpoint inhibition in some cancer and MHC class II for melanoma and Hodgkin lymphoma - These are all single markers so this is not sufficient. You need to do a integrated high-dimensional analysis of the tumor and immune profile based on multiple technological approaches including (multiplex) IHC. Look at: - Lymphocyte score - Mutational load - Is PD-L1 present or other immune checkpoints - Immune cell infiltration: intratumorally T cells - Absence of soluble inhibitors - Metabolism? Multiplex IHC has a higher performance than single staining DAB and other biomarker's - We can look at co-expression of markers on cells and look at spatial distribution of the cell types - Examples of publications that used multiplex IHC for responsive monitoring - Checked at immunoscore \--\> then looked at PD-1expression on cytotoxic CD8 T cells used multiplex IHC - Found that the pd1 expression was mainly found in the CD8 T cells - People with already CD8 T cells had better response to PD1 blockage - In non small cell lung cancer showed different patterns of tumor infiltrating lymphocytes: - 3 different tumor infiltatin lymphocyte phenotypes - Type 1: no t cells - Type 2: dormant tumor infiltrating lymphocyte patterns: a lot of cd3 but low effector and proliferative capacity - Type 3: high cd3 positive and high effector/proliferative capacity - Treating patients with pd-1 blockage: type one do not do well with this, type three do a bit better but the dormant phenotype do best: so this means that these dormant T cells are probably blocked by pd-1 and if you block this pd-1 they can get activated - Type 3 not good maybe because there is no reactivation of immune cells because they are already active. These already active immune cells do not help with the cancer so there is another problem. The problem is not that they are not active! - Looked at how pd1 and pd-l1 are coexpressed: patients with high pd1/pd-l1 coexpression do best in pd-1 blockade - Looked at individual biomarkers not associated with how well patients do with pd-1 blockade so you really need to use multiplex IHC - Melanoma cells looking into the types of T cells - Combination of two checkpoint inhibitors took responders and non responders - Sox10 = tumor cells - Exhausted cells are the cells that are likel dormant and these can be activated again upon pd-1 blockage - Neighboring cells - Treatment with pd-1 blockade found neighborhoods - Patient that responded best where patients with highest amount of PD-1 + T cells Conclusion - Multiplex IHC has a lot of potential because you can see co-expression of markers and look at spatial arrangement of cell types - Multiplex IHC can be used to predict responses to immunotherapy Mainly shown in PD-1 here but can be others - Single stain IHC is always the reference and starting point for multiplex IHC - Other emerging methods - Conjugate primary antibodies to DNA barcodes - Cytof - Combination of biomarker approaches are used to really predict responses in patients so not only multiplex but also single cell ihc and rna sequencing and so on