Hybridoma Cell Creation and Monoclonal Antibody Production for Cancer Treatment, Schemi e mappe concettuali di Biotecnologie Mediche

Scarica Hybridoma Cell Creation and Monoclonal Antibody Production for Cancer Treatment e più Schemi e mappe concettuali in PDF di Biotecnologie Mediche solo su Docsity! 1 Biotechnological applications Therapeutic Antibodies - Considered as “magic bullet” to cure human disease → Paul Ehrlich (histologist), 1906 - Creation of a “monoclonal antibodies” by using hybridoma technology → Milstein and Köhlor, 1975 1986 → first approved clinical use for a monoclonal antibody 1997 → rituximab, the first Mab with blockbuster sales In this table, mAbs for infective disease are not present but they could be used also as anti-infection such as in the COVID-19 (temporary until the vaccine’s creation). Paper → Continuous cultures of fused cells secreting antibody of predefined specificity, G. Kohler and C. Milstein, 1975 Nature. Aim: creation of a new cell line made by fusing together 2 cells type → mouse myeloma and mouse spleen cells from an immunized donor. They immunized the mouse and isolated the spleen. 1st myeloma cells → [mature plasma cells function: produce massive amount of Abs that have recognized the Ag on cell surface, in normal condition they don’t grow particularly well in culture] those cells are plasma cells that become cancerous and produces large amounts of a single Abs because they undergo unregulated cell division. Idea to create a compound that targets and destroys a disease-causing organism Growth of the clinical application of this technology By doing electrophoretogram in a healthy donor serum there are a lot of different Abs, instead in a person with multiple melanoma there’s a single clonal population. 2 Problem: these multiple melanoma cells make the Ab, but it is no what we want → so what we can do? Immunization of the animal with a selected Ag. Spleen → function: filter the blood. It is an immune organ because there are small lymph nodes. When the spleen is taken from mouse → almost 108 cells, 15% of this are lymphocytes, very good source of B cells than peripheral blood. B lymphocytes in the spleen are used in creating HYBRIDOMAS for mAb production: immunize mouse with the Ag of interest → get the B cells from the spleen because some of them will express the Ab against this Ag → fuse those B cells with myeloma cells → creation of small factory for the production of Abs against the specific Ag. THYMYDLATE SYNTHESIS: THYMIDINE → one of the four bases specific for DNA, derived from uracil though a methylation reaction (addition of CH3 at 5 position) done by the enzyme thymidylate synthase. Remember that the carbons and hydrogens of the methyl group come from the tetrahydrofolate (=reduced form of folic acid). The THF becomes oxidized in dihydrofolate and the CH3 is transferred for the formation of dTMP. Dihydrofolate → don’t have any functional properties, so we must recycle it back to THF (functional form) thanks to the dihydrofolate reductase enzyme. 5 To analyze results, authors used isoelectric focusing. The authors took their hybridoma cells (selected before in HAT media), grew up in culture and added radiolabeled lysine to the culture media. At this point those cells were analyzed by isoelectric focusing → Abs produced are IgG. To determine the presence of specific Ab-producing cells, the authors did a plaque assay by using SRBC as immunogen. The hybrid cells were cloned in soft agar and clones producing antibodies were detected by an overlay of SRBC and complement → hybrids capable of lysing SRBC were those that were able to produce the specific Ab. Conclusion of the paper → melanoma cells were capable making Abs, but hybrids were making mixture of Abs which is not what we want, what is preferred is the production of a single specific Ab provided by the B cells. Such cultures could be valuable for medical and industrial use. To create a mAb-treating cancer is needed an antigen (TAA: tumor-associated antigen) that should have the following properties: - expression on all tumor cells including cancer stem cell - expression on tumor cells surface - functions in tumor cell survival → if this Ag is not required for the tumor cell to survive, it will stop producing that Ag so it will be not target anymore by the mAb - lack of expression on normal tissues → just on the cancer cells Unfortunately, none of TAAs have all these IDEAL characteristics! → technique for separating different molecules by differences in their isoelectric point (each aa has an isoelectric point where the charge is 0) 6 RITUXIMAB Functions: • complement-dependent cytotoxicity → Fc portion of the mAb binds the C1 component of the complement, leading to the activation of the complement cascade and further cell lysis though the formation of MAC; • antibody-dependent cell-mediated cytotoxicity (ADCC) → Fc portion of the mAb binds the Fcγ receptor of effector cells (NK, macrophages), leading to the release of perforin from these cells that induce cell lysis; • direct cytotoxicity → by binding CD20 on B cells, it will activate downstream pathways that will cause antiproliferative effects or cell death (it may involve apoptosis or other cell-death pathways) Rituximab: - chimeric Ab → both mouse and human sequences - It will also destroy normal B cells, but it will not target lymphoid progenitor because CD20 is not expressed → after the treatment the B cell compartment is restored - specific for CD20 Paper → Screening hybridomas for specific monoclonal antibodies, Reff et all (1994) Blood Aim: mice immunization with a human lymphoblastoid cell line (SB) that express CD20. They monitored the mouse serum for the Ab against CD20. Once they had the spleens the cells were harvested and those were fused with myeloma cells to create hybridomas. To screen hybridoma cells that were making α-CD20 Ab, a radiolabeled iodine was used in order to track the Ab. For the screening: Characteristics: - 35 kDa integral membrane protein of cell surface (so it is accessible) - involved in calcium transport and required for B lymphocyte proliferation - does not internalize after binding antibody γ emitter, it is damaging DNA because emits strong radiations. Problems for the thyroid By mixing SB cells and labeled antibody, the Ab will bind the CD20 on the cell surface. 7 Two conditions: ➢ If hybridoma cell were making antibodies against CD20 → Ab binds CD20 on SB cells → prevent the radiolabeled antibody from binding → radiolabeled Ab will be found in the supernatant ➢ If hybridoma cell does not make α-CD20 Ab → 125Iodine-antiCD20 binds CD20 on SB cells Southern Blotting → DNA detection Northern Blotting → RNA detection PCR → DNA amplification ELISA → antibody screening ▪ Enzyme-linked ImmunoSorbant Assay ▪ Direct assay or Indirect Assay Horseradish peroxidase → enzyme usually used for ELISA assay, to measure the presence of the Ab we need to give a substrate (luminol) and the enzyme will break it down which then it will decay and emits light. ▪ Direct assay not the best way to screen culture supernatant from hybridomas → not practical ▪ Indirect assay → Ag linked to a solid surface which is then bound by a primary Ab, then a secondary conjugate Ab (e.g. goat anti-mouse) Mouse mAb used as a starting point for making chimeric Ab → recombinant technology by using vectors Indication that this hybridoma is making α-CD20 Ab Gene encoded for β-lactamase: needed for antibiotic selection → resistant to ampicillin and able to grow in culture → ampicillin: inhibits bacterial cell wall synthesis, is analog of D-Ala-D-Ala that is necessary for the cross binding of bacterial wall. Ampicillin competes for the biding and the cell wall has less structural integrity, leading to cell lysis/death. 10 Different assays to observe cell lysis: Additional studies on antibody function in non-human primate model → this is important before testing on humans - Clonus monkeys were humanized with the Ab → weekly injection for 4 weeks - Aim: analyzing if the monkeys still have B cells in the circulation after treatment → results: depletion of bone marrow/lymph node Antibody selectivity → analysis of T lymphocytes 36 days after last injection Results: high number in order to compensate the disruption of the B cells All these preclinical tests lead to this conclusion: this Ab may be effective, B cell depleted, and T cell not affected → going on with human clinical tests B cell lymphoma → most prevalent in US is DLBL (Diffuse large B cell lymphoma) that express CD20 Rituximab Clinical Trials CHOP → four drug chemotherapeutic cocktail (not specific) typically used to treat B cell lymphoma. Could be the therapy more effective by adding Rituximab? CDCC – complement dependent cell lysis *Chromo release → just if there’s the cell lysis in the extracellular milieu Humanized mAb can direct cellular lysis This because effector cells cannot recognize mouse constant region C: cyclophosphamide → H: hydroxy-daunomycin → O: oncovin (vincristine sulfate) P: prednisone → steroid, immunosuppressor ONCOVIN STRUCTURE → anticancer drug, interacts with microtubules (structure needed for MITOSIS for segregating chromosome into 2 cells) → natural product, from a plant → lot of side effects because it isn’t specific Microtubules can be altered by: ▪ Destabilizers: disruption ▪ Stabilizers: alter its dynamicity 11 → It is referred to as a nitrogen mustard (= chemical warfare agents) Carbon ions are highly reactive Sulfur gas loses its Cl → forms carbonium ion that immediate attaches the sulfur forming a sulfonium ion → this will react with water and any other nucleophiles → in biological system there are known as blistering agents → they burn and blister the skin or any other part of the body they contact → they are very useful for their extreme degree of reactivity; they are too reactive to be used, for this reason there are nitrogen mustards. • less reactive for its chemistry (for the ring) and for this reason this drug can be given orally • it’s a prodrug, in this state is not effective at all • bioactivated in the human body which occurs in the liver though p450 enzyme; it will remove the ring by releasing then acrolein (extremely reactive, ends up in the blatter causing damaging → for this reason when the patient is treated with cyclophosphamide, it is also given MESNA which reacts with the acrolein and detoxifies it) • phospharamide mustard: reactive nitrogen mustard which can react with two Guanine (G) because is a bifunctional crosslinking reagent, interfere with DNA replication, cell division, leading to tumor cell death • Anthracyclines which forms complex with DNA causing: - Inhibition of topoisomerases ability, leading to single and double-stranded breaks in DNA (highly toxic to cells) - Replication interfering Side effects of Oncovin: - Peripheral neuropathy - Vomiting - Nausea - Suppression of bone marrow activity CYCLOPHOSPHAMIDE Drug derived from an agent that was used to kill people because its high degree of toxicity → SS John Harvey, Bari Italy, December 1943: German air force bombed the ships in the harbor but one of them contained 100 tons of sulfur mustard gas bombs → after the explosion the gasses flowed into the city → people ingested it and dying for bone marrow failure → this chemical was killing the dividing cells → began clinical trial for the use of mustard gasses in treating leukemia Sulfur mustards HYDROXY-DAUNOMYCIN 12 - ROS generation • Side effects → specific heart problem which can show up immediately upon taken the drugs, shortly after or years after. This led to the creation of a new field “oncologic cardiology”: very important aspect because usually chemo drugs can induce also other problems not related to cancer, so there’s the need of experts to resolve those problems. NB. All these drugs that affect dividing cell will have side effects → bone marrow suppression because there’s a high rate of naturally division (reduce number of RBC, megakaryocytes → the individual will bleed more, WBC → risk of infection) Vinca plant→ was used to vomiting The aim is to improve the drugs with few side effects! Clinical Trials → adding Rituximab to CHOP can increase the benefit Standard of care for B cell lymphoma was CHOP (potential toxic if we increase the dosage) until early 2000 Paper → CHOP Chemotherapy plus Rituximab compared with CHOP alone in Elderly patients with Diffuse large-B-cell lymphoma, N. Engl. J Med, 2002 Aim: comparison between CHOP and R-CHOP in randomized people with DLBL RESULTS → after 24 months follow up Events: what is happening to the lymphoma Responsiveness to the treatment Progression after stable disease: initial good response but then the cancer came back Death without progression: death for other reason (comorbidities due to their age) Rituximab reduced the relative risk of an event by 42% Cancer increases especially after 60 years old, the problem with those patients is that they have also other problems due to their age (comorbidities such as diabetes, so on) → in the paper there are some who were above 75 y → uniform distribution of the groups Performance status: are the people in general healthy (additional disabilities)? Stage of the disease: from 1 to 4, what is size of the tumor, metastasize, etc. B symptoms: typical symptoms that are seen with lymphoma such as weight loss, fever Besides DLBL, in the clinical trial were included also other lymphomas in a very small number. Prognostic score: score that shows which is the risk of death, combination of disease stage, performance status and LDH levels 15 Humanizing mAb → all the structure will be human, except of the hypervariable regions To measure the affinity between Ab and Ag → measuring the Kd of interaction using Biacore technology measure the “on rate”: time necessary to make a complex between antigen and antibody “off rate”: how quickly they dissociate If the off rate is very small, there a very tight interaction: it will be a very small number, and this means that the Kd will be very low. If it is low, the affinity will be very high. Three-dimensional structure of the variable regions of heavy and light chains → 2 separate regions within the variable regions → red box: made up of beta-sheets, known as platform which is holding the loops to form the antigen binding site. In the humanized Ab the platform is human, and the hypervariable regions are from mouse (→ chimeric Ab has both variable regions from mouse, instead the constant is from humans). First of all they started with a chimeric Ab → then the variable regions where replaced with human sequences → become a fully human but the antibody is not directed to the antigen of interest → what they did: straight CDR swap = changing the hypervariable residues from human to mouse, now this should direct the humanized Ab to the Ag (AS YOU CAN SEE IN THE TABLE: this didn’t work!!!! 1350-fold increases in Kd, the affinity of the antibody for the antigen dropped, not effective) By trying to humanize the Antibody from a chimeric to a more human ab there was incompatibility between sequences in the platform and sequences within the CDR region → understand where the major incompatibility was → made 2 new antibodies. 16 F(ab)-2 → chimera light chain → the affinity (˃145) means that the Ab still don’t recognize the Ag very well but seems that the major problem was the heavy chain (incompatibility in the sequences with the heavy chain) F(ab)-3 → has a chimera heavy chain → the affinity is within the range (2.6) → this means that the compatibility was in the heavy chain They understand that the Arginine in position H71 was somehow interfering with the complementary determine region either H1 and H2 regions → maybe it is a hydrophobic region in the mouse region → if we have this hydrophobic region next to the arginine (polar?) there could be incompatibility, it can push the H1 loop away → alter the arrangements → no binding of the antigen They had to change eight different position in the human platform (most in the heavy chain) → in order to get no interference between the human sequences in the platform and the ability of the hypervariable sequences to bind the antigen → conclusion: they not only have to change the hypervariable sequences using mutagenesis from human to mouse, but they had to change also platform sequences to make sure that the mouse sequences that they introduce were compatible with the human framework → lot of mutational changes F(ab)-12 → final modified humanized Ab which has 1.6 Kd, very similar to the original chimera → kept the Ag-biding affinity of the mouse sequences but making the antibody much more human. → humanized antibody against VEGF (Vascular Endothelial growth factor): BEVACIZUMAB • Good half-life in humans: this is very good because is usually injected • No evidence of anti-drug antibodies: this because is more humanized • Used to treat cancer in a different way from Rituximab • Targeting angiogenesis in cancer Remember that cells undergo an epithelial to mesenchymal transition (EMT) to promote metastasis Vitamin C is required for the activity of hydroxylases involved in oxygen sensing. Hydroxylation of HIF1alpha is a critical part of the oxygen sensing mechanism. Necrotic core in the tumor → this happens because some cells are not getting enough nutrients from the blood (anoxia) → to resolve the problem VEGF is activated to get new vessels Proposed by Folkman in 1971 → the idea was to target the blood vessels because tumor needs glucose, nutrients to grow Every time that there’s a solid tumor growing → neovascularization Targeting normal cells supporting tumor cell growth may be more efficacious than targeting genetically unstable tumor cells → also because in this way the drug-resistance mechanisms of tumor cells would be not significant Target for anti-angiogenesis therapy → VEGF 17 under normal circumstances we have the “normal” design → creation of capillaries too during tumor growth → abnormal vasculature, describes as tortuous, no arterial, venous marcation due to the constitutive activation of pro-angiogenic signals, the vasculature is also leaky and often insufficient bc they need a lot of nutrients. This is very important also bc this structure impede drug delivery to tumor. Folkman idea →by targeting angiogenesis we can reduce the blood supply of the tumor, no nutrients to survive → tumor shrinking In reality by using Bevacizumab normalize (not perfectly) the tumor vasculature, looking like normal one → this is helpful for drug delivery Which is NOT a current approach to inhibit angiogenesis? Alkylating agent Two strategies for Anti-angiogenesis therapy: 1) SORAFENIB → Tyrosine kinase inhibitor, small molecule inhibitor of VEGF2 2) BEVACIZUMAB Humanized mAb that will bind and mask VEGF → doesn’t allow VEGF to interact with its receptor and promote angiogenesis - Approved to treat glioblastoma → not so affected - Used in combination with other drugs to metastatic colorectal cancer, some non-small cell lung cancer and metastatic renal cell cancer Benefits of this drug: 1) Vessels are less leaky → difficult then for the tumor to metastasize 2) Normalization of blood vessels → improve drug delivery Paper → A Bevacizumab “success” story, J. Clin., 2007 Aim: clinical trial of 829 patients with metastatic colon cancer, comparison between two drugs: standard therapy and standard therapy + bevacizumab mab → antibody-based therapy – chimera umab → humanized or human nib → small molecule kinase inhibitors tinib → small molecule Y kinase inhibitors SUFFIX 20 mAb used beyond cancer → such as those used in autoimmune disorders, chronic inflammatory diseases in which patients take long life the drugs. Fully human Ab → less immunogenic than a chimeric Ab Etanercept → human constant region used for stability, instead the binding site is the human TNFr2, so this means that doesn’t have any variable sequence → better than Adalimumab Clinical trial for Infliximab (chimeric drugs) → looking for benefits in RA *CRP: C reactive protein, marker for inflammation After treatment → joint swollen and CRP goes down → good results HUMA: human anti-mouse antibodies → immunogenicity against the mouse variable region With repetitive injection of Infliximab → no longer works effectively, there’s a initial response but then decrease. Every time you injected a patient with these drugs, there’s the risk of immunizing with the drug. Infliximab + MTX → reduce the possibility to create Ab against this type of drug (MTX targets tumor dividing cells but, in this case, it will reduce B cell expansion and Ab production) → strong therapy In Crohn’s Disease → 60% patients raise Ab to the drug by the 5th injection HUMA responses to different anti-TNF drugs: - Infliximab → 51% of patients raised Ab in response to 1mg/kg dosing - Adalimumab → 6% less immunogenic at lowest dose → bc the hypervariable region will always have the potential to become immunogenic for the patient → antibodies against these regions are called anti-idiotype Ab Solution = use human/humanized Ab 21 - Etanercept → 2% less immunogenic at the standard dose → best one that is very used, very affective therapy Side effects of Etanercept: - Increase risk for infections (sepsis, tuberculosis) that may lead to hospitalization or death - Possible reactivation of tuberculosis - Invasive fungal infections - Malignancies → lymphoma and other in higher rate than others Patients take the drugs even if there are a lot of adverse effect because they are very effective drugs. 2) Cholesterol and Cardiovascular Disease mAb cannot target the enzyme bc is inside the cell LDLr → major player for mAb target LDL → bad cholesterol, it is cleared from the circulation though LDLr that is found on liver → used then for bile acid synthesis and secretion Familial Hypercholesterolemia → individuals that have extremely high levels of LDL cholesterol in their serum Caused by: - LDL receptor (95%) → major player in lowering cholesterol serum LDL and to prevent cardiovascular disease - Mutations in ApoB (4%) → constitutive protein of LDL, this polymorphism interfere with the recognition of LDL by LDLr - PCSK9 protein (1%) → gene mutation, gain of function that lead to increase LDL serum Combination treatment between statin and mAb: Statin → reduce hepatic cholesterol level → increase LDLr expression in order to take more LDL from the serum Drug NOT approved for lowering cholesterol → Praluent (alirocumab) How statin works → inhibit hydroxylmethylglutaryl CoA reductase PCSK9: made in the liver, downregulated LDLr on the liver surface if the liver has enough cholesterol The protein is expressed by liver cells, secreted from the cell and binds to LDLr → LDL and LDLr are internalized by endocytosis → the protein causes LDLr and LDL degradation in lysosome → lower LDLr on surface. 22 There’s a mAb against PCSK9 → binds to the protein and prevent the interaction with LDLr → the receptor will be internalized with the LDL → LDL will be released and degraded in lysosome → LDLr will be recycled The combination will lead to LDLr increase on the cell surface → more LDL will be taken out from the serum → reducing the risk of cardiovascular disease Treatment decreases cholesterol to very low levels These mAb are very expensive → they are just used in some patients 3) Migraine headaches Risk factor of this global problem → women men ratio 2:1, Caucasian race, genetic factors, old age Factors that can influence this problem→ caffeine use, bad lifestyle, spleen disorders Therapy → calcitonin-gene related protein Erenumab → mAb that targets the receptor and interfere with CGRP binding, cost a lot. Clinical trials → placebo vs Galcanezumab (mAb against this headaches) Conclusion: patients receiving the mAb will have decrease pain, low frequency episodic migraine → favor the mAb treating. mAb → stable in the serum → you have to take this injection once a month instead of taking a lot of drugs during the days . Side effects → doesn’t matter if the headache is so bad 4) Hemophilia Defects in factor: - VII → very rare, autosomal recessive disease that lead to excessive bleeding disorder - VIII → Hemophilia A - IX → Hemophilia B, known also as Christmas disease Protein first characterized in 1983, vasodilate protein which increase during acute migraine headaches attacks The CGRP is released from the trigeminus system → vasodilation, sensing pain The use of mAb prevent the binding between the CGRP and its receptor and having its biological effects. 25 Benefit: phages are small so we can use a lot of them to create proteins. Phage particles expressed on the surface → now we can do a binding assay → bind our phage on the epitope of interest, wash away all the particles that weren’t able to bound → elute → re-infect the bacteria and re-expand the population → cycle Selection of the phage particles → isolate the DNA and sequence the H and L chains → creation of Ab Involvement in a lot of studies of single variable fragments → use the human framework in which we have the CDR → at least for one of this CDR, you will put on a randomized library of tri-nucleotides (tri because of the genetic code) → result: on of this CDR have a high degree of aa polymorphism within it. You can do that with all 6 CDR but this increase the complexity, not so useful. CDR3 of the heavy chain → at least one of these residues is always blocked when an Ag binds. Phage display → used to create variable region in order to not involve mouse → making “fully human Ab” 2) Transgenic mice - Putting the human Ab into a mouse → this Ab are large (1.3 mb for H; 1.8 mb for K L chain) bc they have a lot of cassette 1 mb= 1000000 bases They took mouse EC cells and knock out the H and K L chain site → immunocompromised mouse Making homozygous H knock out in one mouse and K light chain in another → crossed together → creation of homozygous mouse that is knock out for both locus On the other hand they inserted in the human heavy and K light chain → creation of mouse which have both chain HuAb x DI → XenoMouse that has the mouse heavy chain and K light chain genes knocked out and they have human heavy chain and k light chain knock in. Screening strategies - Uses low concentration of Ag for the phage binding - Yes extensive washing after the bound, slow off rate - Short incubation of phage with Ag, faster on rate 26 Lambda chain are not manipulated → expressed just in low levels XenoMouse → capable of producing high affinity fully human Abs, one of the uses of this mouse was to create a fully human Ab against CD20 Ag → Ofatumumab Rituximab actions: - Complement-mediated lysis: constant region of the Ab that directing complement biding and cell lysis - ADCC: FcγRIIIa which is the receptor of effector cells that is recognizing the constant region of our mAb. Interaction between this receptor and the therapeutic Ab → there are polymorphism (at position 158 – can be either Val or Phe, both are hydrophobic residues, but they will have distinctive characteristics) in the constant region (Fc) of this receptor that can influence the activity of the drug. → they looked for FCGR3A gene (encodes for FcγRIIIa) of immune cells in patients with B cell follicular lymphoma. Results: those people that were heterozygous for the polymorphism in constant regions had bad prognosis, instead homozygous V polymorphism less bad. Interaction of Ig with FcγRIIIa receptor (represented by the green one, has 2 domains and one of this set in the hinge region of the Ig, the major interaction is with the Fc domain) Ig are subject of N-linked glycosylation → CH2 region Turns out that both FcγRIIIa and the FC region of the Ig are subject of glycosylation (hinge region) In this process carbohydrates are linked to a specific aa → Asparagine Glycosylation occupies space nearby where the receptor interacts with the Fc of the Ab → By manipulating can enhance effector functions? Can we make the Ab better? • Fully human Ab produced in transgenic mice immunized with CD20 → goal: replacing Rituximab (1st blockbuster drug) • Phase 3 clinical trials comparing these two drugs → there’s no statistically significant difference, they have the same result So the question is: Is there anything out there that can beat Rituximab in a head to head trial? 27 Signal peptide → direct the Ig towards the ER, at the end this gets cleaved Oligosaccharide transferase → involved in the glycosylation, recognize the Asn-X-Ser or Asn-X-Thr. Two general classes of N-linked Glycosylation: 1) High mannose → some peripheral Mannose are still there 2) Complex → to recognize it we have NANA Starting from ER to Golgi we have the addition and processing of the core oligosaccharide → high mannose class stops adding sugars, instead the complex one keeps trimming and adds more sugar (see the arrow) Ab are glycoproteins and glycosylation can influence effector function? [Fucose residues added on GlcNAc linked to the Asn] Cells normally used to make mAb don’t express glycosyltransferase 1,4-N- acetylglucosaminyiltransferase III (GnT-III) and Man-2 (for Mannose addition) → by transfecting cells with those enzymes, the addition of GnT-III interfere with the recognition of fucose residue and we will have a bisected mannose. By changing the glycosylation pattern of the immunoglobulin and preventing the fucose addition → no longer the stereo clash, greater ability of the receptor to bind to the Ig Fc region GA101 (Obinutuzumab) → glycoengineered Ab without the glycosylation pattern (no fucose residue), enhanced ability to be recognized by the Fc receptor and be more effective in mediating the destruction of target cells → much more effective cytotoxicity than Rituximab (yes fucose residue). In a clinical trial in CLL patients there’s prolonged progression free survival better than Rituximab Can we use therapeutic Ab for infection diseases? COVID-19 → COronaVIrus Disease 2019, SARS-CoV-2 (Severe acute respiratory syndrome) Symptoms: fever, cough, some have additional one such as loss of taste and smell correlated with loss of appetite and fatigue. Alveoli → field with fluids, inflammatory cells are “bad players” that induce to cell destruction (such as in RA) SARS-CoV → severe acute in 2003, related with this new virus SARS-CoV-2 MERS-CoV → middle east 2012 Transmission: droplets → social distancing Dies quickly on surfaces Replication number → R0 Fatty acids (soap) → will destroy the phospholipid membrane of the virus 30 T CELLS and Immunomodulatory therapy Genetically engineered T cells → against cancer The opportunity for toxicity becomes grater if we are activating many T cells. Idea: use the IS to target tumor cells which are instable, direct the T cells against the protein produced by tumors. Transfection of the autologous T cells that are engineered (also we have the selection of T cells with high affinity receptors) → promote T cell proliferation CAR-T : Chimeric Antigen Receptor T cells Structure: variable and constant domain, 2 chains (α/β) in the TCR, CD3 complex with different chains (zeta more important) for the effective signal transduction once TCR binds. LAT: linker of activated T cells CAR-T: much more range of interaction with different types of Ag than TCR. External part is the ligand biding domain that is linked to signaling domains → usually used by T cell accessory protein. Depending on the CAR we can have also a second signaling domain. Very dangerous T cell → to activate those cells there’s the need of multiple costimulatory signaling (also to not target self Ag). 1. TCR- MHC complex with peptide Ag → this biding alone is insufficient to start the transduction, this will provide specificity 2. CD28-CD80 of APC → necessary for the full activation of T cells, amplifies intracellular signals 3. Cell-cell adhesion proteins 31 Because of the toxicity they cannot be used so much for solid tumors → they are able to recognize the Ag on different cells Initial CAR-T studies → B cell leukemia and lymphomas → e.g. these T cells are activated just by one Ab, in this case CD19 Methodology: All this process is performed just in 10 days → T cells are taken from the patients → selection of T with sorting →T transduction (CAR) → T cell expansion though IL-2 (specific for T cell)→ autologous transplant CAR-T’s FACS in vitro → Expression of both CD8 and Anti-Fab Study: patients in advance state of disease → treated with different therapies, tested with CAR-T → outcome: high toxicities such as fever, confusion, coma, not predictable → this TOXICITY is caused by the cytokine storm activated by the CAR-T infusion Another thought experiment: Target the tumor in a more specific without the collateral damages of CAR-T - More selectivity for the tumor - Less toxicity Try to solve it with the use of mAb 32 But this process already is present in our body when we have cancer → there’s are some lymphocytes infiltrated in the tumor, but the problem is that initially they are not enough! Here there’s just the expansion of these T cells. No long-term engraftment!!! In solid tumor the effectivity is limited because there’s an immunosuppressive environment that limit the expansion and activation of T cells. Immunoregulation of T cells: CTLA-4 → member of the CD28 family of co-receptors but it inhibits T cells by interacting B7 (→ prima interagiva con CD28) activated after 48-72h, prevents also autoimmune disorders. By making mAb against these proteins in order to activate the T cell in tumor environment → immunomodulatory theory IPILIMUMAB 1. Clinical trial for metastatic melanoma → mAb vs vaccine Results: with Ipi we have an increase of survival in the patients, not so good 2. Clinical trial → Ipi vs Ipi + standard chemotherapy for melanoma (dacarbazine) Results: improved outcomes for Ipi + SC, for overall survival we see some benefits, but they are disappointing What does this mean? There’s more to the tumor immunosuppressive environment than the downregulation of T cells by CTLA-4. Another protein involved → PD-1 and PD-1L that reduce T cell signaling, cytokines production, cytotoxicity of the cells just though this biding (bc there’s the dephosphorylation of CD3). Usually PD-1L is expressed by APC but also tumor cells. mAb against PD-1 → Nivolumab Melanoma: high degree of genetic instability, side effects with this new immune-modulatory agent: autoimmunity in which patients T cells are attacking the body. There are other types of solid tumors that are responding well? Yes → Lynch syndrome: hereditary nonpolyposis colorectal cancer caused by the deficiency of DNA repair system, high rate of mutations → treated with anti-PD1 Pembrolizumab → great response in tumor with high genetic instability