Tecniche immunochimiche ✓Principi generali: immunità umorale ✓Immunoglobuline: struttura, produzione anticorpi policlonali, monoclonali ✓Applicazioni: ▪ Immunoprecipitazione, immunodifusione, immunoelettroforesi, Co-IP, test di agglutinazione ▪ Dosaggi RIA, ELISA ▪ Immunoblotting: Western Blot ▪ Immunoistochimica (IHC) Microscopia fluorescenza, elettronica ▪ Citofluorimetria a flusso ▪ Impiego in terapia Determination of Ab affinity (Ka) by equilibrium dialysis.

Semipermeable membrane

A radioactively labeled ligand that is small to pass through (haptens, oligopeptides ) Plot of concentration of ligand in each compartment with time

The difference in the two compartments Sensitivity of various immunoassays

* * * * * * *

* **

* * Determinanti antigenici/epitopi

Complessi antigene- anticorpo Precipitation reactions in fluids yield a precipitiation curve

❖ Sensitivity ❖ Specificity Effect of Ab/Ag ratios on the formation of Ab-Ag precipitates Immunoprecipitation Immunoprecipitation (IP) is a technique to isolate a specific protein out of a solution using an antibody that binds to it. Antibody-protein complexes are removed from the solution with the addition of an insoluble form of an antibody binding protein, such as Protein A or Protein G Immunoprecipitation*

*antibody conjugated to agarose or Magnetic Beads. Immunoprecipitation Immunoprecipitation assays detect the interaction of a target protein with other proteins or nucleic acids

•Co-Immunoprecipitation (Co-IP) is a powerful method that is most widely used by researchers to analyze protein–protein interactions. This process provides a rapid and simple method to separate a specific protein from a sample containing thousands of different proteins, such as serum, cell lysate, homogenized tissue or conditioned media. Principle and method of co-immunoprecipitation (Co-IP)

In Co-IP, complexes of two (or more) proteins are isolated using a procedure similar to the IP procedure. Co-IP is often used for the analysis of interactions of multiple proteins and their functions. The principle of Co-IP is the same as IP, except that associated proteins are precipitated. Immunoprecipitation Immunoprecipitation assays detect the interaction of a target protein with other proteins or nucleic acids. Assay examples include Co-IP, ChIP, RIP.

•Chromatin Immunoprecipitation (ChIP) is a type of immunoprecipitation used to investigate regions of genome associated with a target DNA-binding protein, or conversely to identify specific proteins associated with a particular region of the genome. •RNA Immunoprecipitation (RIP) is performed with an antibody that targets a specific RNA-binding protein. By isolating the protein, the RNA bound to it is also isolated. The RNA-protein complexes are separated by RNA extraction. The RNA can be analyzed by cDNA sequencing or RT-PCR. ChIP assays Protein and associated chromatin in living cells or tissues are cross-linked using formaldehyde. The cross-linked DNA–protein complexes (chromatin- protein) are then sheared into ∼500 bp DNA fragments using either enzymatic digestion or physical shearing by sonication. The DNA-protein complexes are then immunoprecipitated by an appropriate protein-specific antibody. After the cross-links are reversed, the associated DNA fragments are eluted, which is followed by immunoprecipitation of the cross-linked complexes and analysis of the resultant DNA by endpoint or quantitative polymerase chain reaction (qPCR), or next-generation sequencing (ChIP-seq). ChIP assays can be used for the following studies:

❖DNA sequences occupied by multiple specific protein targets ❖Binding sites and distribution of a particular protein, such as a transcription factors, transcription co- factors, DNA replication factors and DNA repair proteins throughout the entire genome, under specified cellular conditions ❖Gene transcription and polymerase activity ❖Complex DNA/protein interactions underlying disease phenotypes ❖Modifications to proteins, such as histones, that may influence chromatin structure and gene expression ❖Nucleosome architecture and regulation of chromosomal maintenance Immunodiffusione in gel

Diffusione radiale semplice di Mancini

Radial immunodiffusion in the Ab-containing semisolid medium

-> The area is proportional to the conc. of Ag The region of equivalence

Diagrammatic representation of radial & double immunodiffusion. precipitation reactions in gels yield visible precipitin lines; no visible precipitate forms in regions of Ab or Ag excess. Immunodiffusione Doppia Immunoelettroforesi

HS = proteine del siero umano HSA = albumina serica umana Immunoelectrophoresis

an antigen mixture is first electrophoresed to separate its components by charge - diffusion & producing lines of precipitation. Immunoelectrophoresis Immunoelettroforesi bidimensionale

Risultato dell’immunoelettroforesi bidimensionale di siero umano (HS): (a) con agar contenente anticorpi anti-HS nella seconda dimensione; (b) con agar contenente anticorpi anti IGM umana nella seconda dimensione Rocket Immunoelettroforesi Visualizzazione bande di precipitazione Test di fissazione del complemento

Rivela la capacità di un siero di attivare la cascata del complemento in presenza di un antigene specifico per l’eventuale anticorpo del paziente Test di Agglutinazione su particelle di Lattice Anticorpi specifici verso un particolare antigene microbico vengono adsorbiti in fase solida (su particelle di lattice). Se l’antigene è presente nel campione, questo si lega all’anticorpo determinandola formazione di un agglutinato. Reazione di agglutinazione positiva.

Sferette di polistirene del diametro di 1 micron rivestite di anticorpi monoclonali su un comune supporto bianco per agglutinazione poste a contatto con campioni contaminati da micoplasmi. Mediante la reazione antigene- anticorpo, in pochi minuti attorno alle sferette si generano agglomerati di micoplasmi visibili a occhio nudo. Agglutination Reactions -The original pregnancy test kit employed hapten inhibition (agglutina tion inhibition) to determine the presence or absence of human chorionic gonadotropin (HCG) -The kits currently on the market use ELISA-based assays. -Also used to determine the use of illegal drugs, & immunity (Ab) to virus (rubella).

Agglutination Reactions -visible clumping by interaction between Ab & a particulate antigen such as RBC, latex beads. -depend on the crosslinking of polyvalent antigens, similar to precipitation reactions (lgM is a good agglutinin) -provide a way to type bacteria with a panel of typing antisera. -routinely performed to type RBCs for blood transfusion.

+ + + (control) Demonstration of humaglutination using Ab against sheep red blood cells (SRBCs): a constant # of SRBCs plus serial two-fold dilutions of anti-SRBC serum If sufficient antibody is present to agglutinate and form cross-linking with the antigen, the antibody-antigen complex forms a mat at the bottom of the well. If insufficient antibody is present, the cells roll down the sloping sides of the well to form a red pellet or "button" at the bottom of the well. Western blot Trasferimento su membrana* electroblotting

*PVDF nitrocellulosa Trasferimento su membrana: esempio di Western blot

Prima del trasferimento Dopo il trasferimento

Gel E Membrana

Il trasferimento delle proteine su di una membrana viene fatto avvenire mediante applicazione di una differenza di potenziale.

Le proteine migrano in una direzione che dipende dalla loro carica. Nel caso del trasferimento di proteine separate mediante SDS-PAGE la loro migrazione è anodica dal momento che recano una carica netta negativa data dall’SDS. Perché è utile avere delle proteine immobilizzate su di una membrana?

Proteine immobilizzate sulla SUPERFICIE di un supporto solido => DISPONIBILI per una serie di operazioni

• Riconoscimento anticorpale • Interazioni proteina/proteina

Western blot Workflow Semidry Nitrocellulosa 0.22 mm PAGE Colorazione Saturazione Utilizzo Wet PVDF 0.45 mm Western blotting (blotting di proteine) Rivelazione enzimatica di antigeni separati elettroforeticamente

Anche : Comunemente: Anticorpi secondari marcati con 125I, con Anticorpi secondari coniugati ad un enzima fluoresceina, oro, biotinilati. Gli enzimi più utilizzati (con prodotti precibitabili) : AP (Alkaline Phosphatase) HRP (Horse Radish Peroxidase) BCIP/NBT 4-cloro-1-naftolo, 5-Bromo-4-Chloro-3’-Indolyphosphate p- Toluidine Salt / Nitro-Blue Tetrazolium Chloride oppure luminolo

Enhanced Chemi Luminescence (ECL) SDS PAGE and Western Blot to detect a protein of interest

Enhanced Chemi Luminescence (ECL) Effect of oxidative agents on the Prx expression in S. solfataricus

Western blot

H2O2 Paraquat tert-butyl hydroperoxide

0 30 60 120 0 30 60 120 0 30 60 120 Prx

The peroxiredoxin is up regulated by the different stressors Ascaris suum cytochrome b5, an adult-specific secretory protein reducing oxygen-avid ferric hemoglobin Hashimoto M. et al. Archives of Biochemistry and Biophysics 471 (2008)) 42–49 Western blot

➢ Nell’ adulto di Ascaris suum il citocromo b5 è espresso solo nella forma matura e solo nella frazione citosolica e nel fluido perienterico

➢ Contrariamente a quanto precedentemente ipotizzato è assente nei mitocondri e nei microsomi Una applicazione clinica del Western Blot

Viral Proteins

HIV, like any other virus, is composed of a number of different proteins. The Western Blot positive control lane contains proteins from patient sera as well as HIV proteins. HIV positivity can therefore only be confirmed by the presence of the following types of proteins:

gp160 viral envelope precursor (env) gp120 viral envelope protein (env) binds to CD4 p24 viral core protein (gag) p31 Reverse Transcriptase (pol)

Band pattern interpretation

In 1987 the Centers for Disease Control along with several other organizations established criteria for serologic interpretation of HIV Western blot tests. The criteria are listed below. Lane 1, HIV+ serum No bands present → Negative (positive control) Lane 2, HIV- serum Bands at either p31 OR p24 AND (negative control) bands present at either gp160 OR gp120 → Positive Lane A, Patient A Lane B, Patient B Bands present, but pattern does not meet criteria for Lane C, Patient C positivity → Indeterminate Ready-to-use clinically defined tissue lysates

Analysis of HID5 in Ductal Carcinoma/ Normal Adjacent Tissue Lysates

Western blot analysis of HID5. MCF-7 breast carcinoma cells were Tumor tissue specimens are cut and used as a positive control for detecting HID5 (~11 kDa). HID5 processed using precisely defined was screened in five patient sets of protocols to maximize selection of living ductal carcinoma/normal adjacent tumor tissue while avoiding necrotic and tissue lysates, and detected in the normal tissue. Normal adjacent tumor sample of patient #4 (T4). specimens are cut from tissue distal to The higher molecular weight band the tumor, an area grossly free of detected by the antibody in the tumor. All protocols used are IRB & tissue samples is uncharacterized. HIPAA approved T = ductal carcinoma tumor lysate. N = normal adjacent lysate The principle of radioimmunoassay (RIA) fluorescent immunoassay (FIA) - One of the most sensitive technique for measuring hormones, drugs, & vitamins at conc. of 10-12 M first discovered by Rosalyn Yalow and Solomon Aaron Berson in the 1950s. - The principle involves competitive binding of radiolabeled Ag and unlabeled Ag to the limited supply of a high affinity Ab. The principle of radioimmunoassay (RIA)

A radioimmunoassay for - One of the most sensitive adrenocorticotropic hormone technique for measuring (ACTH; called corticotropin) hormones, drugs, & vitamins at conc. of 10-12 M first discovered by Rosalyn Yalow and Solomon Aaron Berson in the 1950s. - The principle involves competitive binding of radiolabeled Ag and unlabeled Ag to the limited supply of a high affinity Ab. The Nobel Prize in Physiology or Medicine 1977

The Nobel Prize in Physiology or Medicine 1977 was divided, one half jointly to Roger Guillemin and Andrew V. Schally "for their discoveries concerning the peptide hormone production of the brain" and the other half to Rosalyn Yalow "for the development of radioimmunoassays of peptide hormones."

Radioimmunoassay: A Probe for Fine Structure of Biological Systems A solid-phase radioimmunoassay (RIA) to detect hepatitis B A standard curve to virus in blood samples determine the conc. of HBsAg in unknown serum. Enzyme-Linked Immunosorbant Assay (ELISA)

Used for Ag detection Ab-enzyme • Immobilize Ag conjugate X • Add antibody-enzyme conjugate Y • Amount of antibody-enzyme conjugate bound is proportional Immobilized Ag to amount of Ag in the sample

• Add substrate of enzyme • Amount of color is proportional to amount of Ag in patient’s sample Enzyme-Linked Immunosorbant Assay (ELISA)

Used for Ab detection Ab- • Immobilize Ag enzyme conjugate • Incubate with patient sample Ab in X • Add antibody and patient’s antibody-enzyme conjugate sample Y • Amount of antibody-enzyme conjugate bound is proportional Immobilized Ag to amount of Ab in the sample

• Add substrate of enzyme • Amount of color is proportional to amount of Ab in patient’s sample An example of enzyme-linked immunosorbent assay

Antigene lavaggio +AB primario lavaggio

+Ab secondario lavaggio sviluppo ELISA Dilutions of patient sample are placed in

adjacent wells of microtiter plate

1/8

1/4

1/2

1/16

1/64

1/32

1/256 1/512 Patient # 1/128 1 2 + Control − Control

More intense color = more Ab present Enzyme-Linked ImmunoSorbent Assay (ELISA)

Partially purified, inactivated HIV antigens pre-coated onto an ELISA plate

Patient serum which contains antibodies. If the patient is HIV+, then this serum will contain antibodies to HIV, and those antibodies will bind to the HIV antigens on the plate.

Anti-human immunoglobulin coupled to an enzyme. This is the second antibody, and it binds to human antibodies.

Chromogen or substrate which changes color when cleaved by the enzyme attached to the second antibody.

Positive ELISA Test Negative ELISA Test to detect Ab (HIV, HCV)

to detect Ag

to detect Ag

Variations in the enzyme-linked immunosorbent assay (ELISA) technique, similar to RIA except using an Enzyme (alkaline ⓟ, horseradish peroxidase, & β- galactosidase): safer & less costly. Enzyme-Linked Immunosorbent Assay ELISA

immunofluorescence

Direct

Indirect Amplification of signal with polyclonal secondary antibodies labeled with a fluorophore

Amplification of signal by a secondary antibody conjugated with multiple fluorophore– protein complexes Amplification of signal with polyclonal secondary antibodies conjugated with multiple fluorophore–protein complexes Interazione del coniugato biotina-IgG con il tetramero di avidina

The avidin fold

Tetramero di avidina con le quattro molecole di biotina Microspettrofluorimetria • Si possono “marcare” cellule con anticorpi fluorescenti • PIP3 rivelato con anti- PIP3 in cellule stimolate con insulina

PIP3=phosphatidylinositol 3,4,5-trisphosphate

Neuroni di cervelletto in coltura in cui è stata colorata una proteina del citoscheletro. è stato utilizzato un anticorpo specifico per la beta3-tubulina Marcatura del citoscheletro e del nucleo con anticorpi fluorescenti Fluorochromes -Rhodamine (515→546nm)

mIgM-producing B cells indirectly stained with rhodamine-conjugated secondary Ab under a fluorescence microscope. The three types of cytoskeletal filaments: actin filaments, microtubules, and intermediate filaments (labeled with an antibody)

fluorescence microscope

Endothelial cells from the bovine pulmonary artery.

Bundles of actin filaments called “stress fibers” are stained red; microtubules, radiating from the cell center, are stained green; chromosomes (in the nucleus) are stained blue. - Ag-Ab attached to a synthetic bead complex - labeling Ag with radiolabeled leucine, cysteine, or methionine → A radiolabeled Ag-Ab complex → SDS•PAGE → autoradiography - Ag-Ab complex + 2nd Ab attatched to a synthetic bead or magnetic beads > magnet

EM showing a cell with magnetic beads attached to its surface via antibodies.

Immunoprecipitates can be collected using magnetic beads coupled to a secondary antibody. electron-dense labels Absorbs electrons.

An immunoelectronmicrograph of the surface of a B-cell lymphoma was stained with two antibodies (Ab against class II MHC labeled with 30nm gold particles, & another Ab against class I MHC w/ 15nm gold particles. (The density of class I exceeds that of class II) - Electron-dense label (ferritin or colloidal gold) is conjugated to the Fc portion. flow cytometer

labeled with fluorescein (green) labeled with rhodamine (red)

(exciting the fluorochrome) ↓ each droplet (cell) emits the fluorescence each dot (small electrical change) represents a cell

Separation of fluorochrome-labeled cells with the flow cytometer which uses a laser beam & light detector. different Ag in different cells / different levels of Ag in the same type of cell → fluorescence intensity / the size of cells. Applications for monoclonal and polyclonal antibodies • Medical diagnostics • Basic material for therapeutic antibodies:

1. are capable of tracking down specific cells, such as cancer cells, and destroying them, without many of the debilitating toxic side effects of chemotherapy and radiation. 2. that target a variety of inflammatory and immunologic diseases, such as allergic asthma, inflammatory bowel disease, ecc… Antibody-dependent cellular cytotoxicity ADCC

MAbs bind to antigen on the tumor cell surface, providing the target for Fc receptors on the surface of natural killer (NK) cells triggering release of perforin and granzymes that lyse the tumor cell (a). (b−d) Cell debris taken by antigen- presenting cells (b), present the tumor antigens to B cells, triggering the release of antibodies with specificities for numerous epitopes on the target antigens (c) and cytotoxic T lymphocytes (CTLs) that are capable of recognizing and killing cells that express the target antigen (d).

Complement-directed cytotoxicity CDC Binding of mAbs to antigen on the cell surface (a) exposes binding site on mAbs for proteins that initiate the complement cascade (b), triggering the release of chemotactic factors (c) and formation of the membrane attack complex (d), promoting target-cell lysis. Examples of antibody-mediated signaling inhibition

Binding of ligand to a growth factor receptor triggers a dimerization event and activation of a signaling cascade, leading to cellular proliferation and resistance to cytotoxic agents. MAb-based signaling inhibition can occur by blocking the dimerization event or by interfering with ligand binding

Antibody-directed enzyme prodrug therapy

(a) The mAb-enzyme conjugate binds to tumor cell- surface antigen. (b−d) After unbound mAb-enzyme is actively or passively cleared from circulation, the cytotoxic agent is administered in an inactive (prodrug) form (b), which is selectively bound by the mAb-enzyme conjugate on the tumor cell surface (c), cleaving the inactivating sequence from the prodrug and releasing multiple copies of active drug in the tumor microenvironment (d). Antibody fusion proteins

❖ Antibodies and antibody fragments are frequently used to target effector molecules to specific sites or cells within the body. The entity to be delivered to the target site may be linked to the antibody by synthesis as a fusion protein.

❖ Antibody fusion proteins may also be utilised in in vitro diagnostic assays.

❖ Commonly delivered entities include toxins, enzymes, viral particles, lipids, radioisotopes and further antibodies in a ‘bispecific’ format. Single Chain variable Fragment single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids.

linker • limited therapeutic value primarily due to the patients' allergic response to them. • researchers have used recombinant DNA technology to create "humanised" antibodies lowering the risk of allergic response and making the drugs safer and more effective. Nature Reviews Cancer 1, 118-129 (2001); doi:10.1038/35101072 IMPROVING THE EFFICACY OF ANTIBODY-BASED CANCER THERAPIES • Chimeric antibodies: the constant domains of the original antibody have been replaced with human- derived sequence retain the entire variable domains of the original antibody’s heavy and light chains, and can lead to an immune response in patients.

• Obtained by joining the antigen-binding variable domains of a mouse monoclonal antibody (mAb) to human constant domains: mouse VL to human CL and mouse VH to human CH1–CH2–CH3 for light and heavy chains, respectively. ►Humanised antibodies entirely human-derived sequence, including largely human derived variable domain sequence, retaining the minimum non-human sequence to retain the binding properties. ► All parts of an antibody except for the CDRs can be replaced to generate a humanised antibody. ►In the simplest case, these are created by grafting the antigen-binding loops, (CDRs), from a mouse mAb into a human IgG. Humanized antibodies require the transfer of one or more additional residues from the so-called framework regions (FRs) of the mouse parent mAb.

Information on this website should be attributed to The Antibody Society (antibodysociety.org)

International non- proprietary name, US product Brand name Target; Format Indication first approved or First EU First US identifier reviewed approval year approval Estimated PDUFA year date

Muromonab- CD3 Orthoclone CD3; Murine IgG2a Reversal of kidney transplant Okt3 rejection mAbs 1986* 1986# Efalizumab Raptiva CD11a; Humanized IgG1 Psoriasis 2004# 2003# Tositumomab-I131 Bexxar CD20; Murine IgG2a Non-Hodgkin lymphoma NA 2003# 122 Nebacumab Centoxin Endotoxin; Human IgM Gram-negative sepsis 1991*# NA Edrecolomab Panorex EpCAM; Murine IgG2a Colon cancer 1995*# NA Catumaxomab Removab EPCAM/CD3; Rat/mouse bispecific mAb Malignant ascites 2009# NA Daclizumab Zinbryta; IL-2R; Humanized IgG1 Multiple sclerosis#; prevention of Zenapax kidney transplant rejection# 2016#; 2016#; 1999# 1997# Abciximab Reopro GPIIb/IIIa; Chimeric IgG1 Fab Prevention of blood clots in angioplasty 1995* 1994 Rituximab MabThera, CD20; Chimeric IgG1 Non-Hodgkin lymphoma Rituxan 1998 1997 Basiliximab Simulect IL-2R; Chimeric IgG1 Prevention of kidney transplant rejection 1998 1998 Palivizumab Synagis RSV; Humanized IgG1 Prevention of respiratory syncytial virus infection 1999 1998 Infliximab Remicade TNF; Chimeric IgG1 Crohn disease 1999 1998 Trastuzumab Herceptin HER2; Humanized IgG1 Breast cancer 2000 1998 Adalimumab Humira TNF; Human IgG1 Rheumatoid arthritis 2003 2002 Ibritumomab tiuxetan Zevalin CD20; Murine IgG1 Non-Hodgkin lymphoma 2004 2002 Omalizumab Xolair IgE; Humanized IgG1 Asthma 2005 2003 Cetuximab Erbitux EGFR; Chimeric IgG1 Colorectal cancer 2004 2004 Bevacizumab Avastin VEGF; Humanized IgG1 Colorectal cancer 2005 2004 Natalizumab Tysabri a4 integrin; Humanized IgG4 Multiple sclerosis 2006 2004 Panitumumab Vectibix EGFR; Human IgG2 Colorectal cancer 2007 2006 Ranibizumab Lucentis VEGF; Humanized IgG1 Fab Macular degeneration 2007 2006 Eculizumab Soliris C5; Humanized IgG2/4 Paroxysmal nocturnal hemoglobinuria 2007 2007 Certolizumab pegol Cimzia TNF; Humanized Fab, pegylated Crohn disease 2009 2008 Ustekinumab Stelara IL-12/23; Human IgG1 Psoriasis 2009 2009 Canakinumab Ilaris IL-1β; Human IgG1 Muckle-Wells syndrome 2009 2009 Golimumab Simponi TNF; Human IgG1 Rheumatoid and psoriatic arthritis, ankylosing spondylitis

2009 2009 Ofatumumab Arzerra CD20; Human IgG1 Chronic lymphocytic leukemia

2010 2009 Tocilizumab RoActemra, IL-6R; Humanized IgG1 Rheumatoid arthritis Actemra 2009 2010 Denosumab Prolia RANK-L; Human IgG2 Bone Loss 2010 2010 Belimumab Benlysta BLyS; Human IgG1 Systemic lupus erythematosus

2011 2011 Ipilimumab Yervoy CTLA-4; Human IgG1 Metastatic melanoma 2011 2011 Brentuximab vedotin Adcetris CD30; Chimeric IgG1; ADC Hodgkin lymphoma, systemic anaplastic large cell lymphoma

2012 2011 Pertuzumab Perjeta HER2; humanized IgG1 Breast Cancer 2013 2012 Ado-trastuzumab emtansine Kadcyla HER2; humanized IgG1; ADC Breast cancer 2013 2012 Raxibacumab (Pending) B. anthrasis PA; Human IgG1 Anthrax infection NA 2012 Obinutuzumab Gazyva, CD20; Humanized IgG1 Glycoengineered Chronic lymphocytic leukemia Gazyvaro 2014 2013 Siltuximab Sylvant IL-6; Chimeric IgG1 Castleman disease 2014 2014 Ramucirumab Cyramza VEGFR2; Human IgG1 Gastric cancer 2014 2014 Vedolizumab Entyvio α4β7 integrin; humanized IgG1 Ulcerative colitis, Crohn disease

2014 2014 Nivolumab Opdivo PD1; Human IgG4 Melanoma, non-small cell lung cancer 2015 2014 Pembrolizumab Keytruda PD1; Humanized IgG4 Melanoma 2015 2014 Blinatumomab Blincyto CD19, CD3; Murine bispecific tandem scFv Acute lymphoblastic leukemia

2015 2014 Alemtuzumab Lemtrada; CD52; Humanized IgG1 Multiple sclerosis; chronic MabCampath, myeloid leukemia# Campath-1H

2014; 2013; 2001# 2001# Evolocumab Repatha PCSK9; Human IgG2 High cholesterol 2015 2015 Idarucizumab Praxbind Dabigatran; Humanized Fab Reversal of dabigatran-induced anticoagulation 2015 2015 Necitumumab Portrazza EGFR; Human IgG1 Non-small cell lung cancer 2015 2015 Dinutuximab Unituxin GD2; Chimeric IgG1 Neuroblastoma 2015 2015 Secukinumab Cosentyx IL-17a; Human IgG1 Psoriasis 2015 2015 Mepolizumab Nucala IL-5; Humanized IgG1 Severe eosinophilic asthma

2015 2015 Alirocumab Praluent PCSK9; Human IgG1 High cholesterol 2015 2015 Daratumumab Darzalex CD38; Human IgG1 Multiple myeloma 2016 2015 Elotuzumab Empliciti SLAMF7; Humanized IgG1 Multiple myeloma 2016 2015 Ixekizumab Taltz IL-17a; Humanized IgG4 Psoriasis 2016 2016 Reslizumab Cinqaero, IL-5; Humanized IgG4 Asthma Cinqair 2016 2016 Olaratumab Lartruvo PDGFRα; Human IgG1 Soft tissue sarcoma 2016 2016 Bezlotoxumab Zinplava Clostridium difficile enterotoxin B; Human IgG1 Prevention of Clostridium difficile infection recurrence

2017 2016 Atezolizumab Tecentriq PD-L1; Humanized IgG1 Bladder cancer 2017 2016 Obiltoxaximab Anthim B. anthrasis PA; Chimeric IgG1 Prevention of inhalational anthrax

2020 2016 Brodalumab Siliq, LUMICEF IL-17R; Human IgG2 Plaque psoriasis

2017 2017 Dupilumab Dupixent IL-4R α; Human IgG4 Atopic dermatitis 2017 2017 Inotuzumab ozogamicin

BESPONSA CD22; Humanized IgG4; ADC Acute lymphoblastic leukemia 2017 2017 Guselkumab TREMFYA IL-23 p19; Human IgG1 Plaque psoriasis 2017 2017 Sarilumab Kevzara IL-6R; Human IgG1 Rheumatoid arthritis 2017 2017 Avelumab Bavencio PD-L1; Human IgG1 Merkel cell carcinoma 2017 2017 Emicizumab

Hemlibra Factor Ixa, X; Humanized IgG4, bispecific Hemophilia A 2018 2017 Ocrelizumab OCREVUS CD20; Humanized IgG1 Multiple sclerosis 2018 2017 Benralizumab Fasenra IL-5R α; Humanized IgG1 Asthma 2018 2017 Durvalumab IMFINZI PD-L1; Human IgG1 Bladder cancer 2018 2017 Gemtuzumab ozogamicin

Mylotarg CD33; Humanized IgG4; ADC Acute myeloid leukemia 2018 2017; 2000# Erenumab, erenumab-aooe Aimovig CGRP receptor; Human IgG2 Migraine prevention 2018 2018 Galcanezumab, galcanezumab-gnlm Emgality CGRP; Humanized IgG4 Migraine prevention 2018 2018 Burosumab, burosumab-twza

Crysvita FGF23; Human IgG1 X-linked hypophosphatemia 2018 2018 Lanadelumab, lanadelumab-flyo

Takhzyro Plasma kallikrelin; Human IgG1 Hereditary angioedema attacks 2018 2018

Mycosis fungoides or Sézary Mogamulizumab, mogamulizumab-kpkc Poteligeo CCR4; Humanized IgG1 syndrome 2018 2018 Tildrakizumab; tildrakizumab-asmn Ilumya IL-23 p19; Humanized IgG1 Plaque psoriasis 2018 2018 Fremanezumab, fremanezumab-vfrm Ajovy CGRP; Humanized IgG2 Migraine prevention 2019 2018

Ravulizumab, ravulizumab-cwvz Ultomiris C5; Humanized IgG2/4 Paroxysmal nocturnal hemoglobinuria 2019 2018 Cemiplimab, cemiplimab-rwlc

Libtayo PD-1; Human mAb IgG4 Cutaneous squamous cell carcinoma 2019 2018 Ibalizumab, ibalizumab-uiyk Trogarzo CD4; Humanized IgG4 HIV infection 2019 2018

Primary hemophagocytic Emapalumab, emapalumab-lzsg Gamifant IFNg; Human IgG1 lymphohistiocytosis NA 2018 Moxetumomab pasudotox, moxetumomab pasudotox-tdfk Lumoxiti CD22; Murine IgG1 dsFv immunotoxin Hairy cell leukemia 2021 2018

von Willebrand factor; Humanized Acquired thrombotic Caplacizumab, caplacizumab-yhdp Cablivi Nanobody thrombocytopenic purpura 2018 2019 Risankizumab, risankizumab-rzaa Skyrizi IL-23 p19; Humanized IgG1 Plaque psoriasis 2019 2019

Polatuzumab vedotin, polatuzumab vedotin-piiq Polivy CD79b; Humanized IgG1 ADC Diffuse large B-cell lymphoma 2020 2019 Romosozumab, romosozumab-aqqg

Osteoporosis in postmenopausal women at increased risk of Evenity Sclerostin; Humanized IgG2 fracture 2019 2019

Neovascular age- Brolucizumab, brolucizumab-dbll Beovu VEGF-A; Humanized scFv related macular degeneration 2020 2019

Crizanlizumab; crizanlizumab-tmca Adakveo CD62 (aka P-selectin); Humanized IgG2 Sickle cell disease 2020 2019

Enfortumab vedotin, enfortumab vedotin-ejfv Padcev Nectin-4; Human IgG1 ADC Urothelial cancer In review 2019

[fam-]trastuzumab deruxtecan, fam-trastuzumab deruxtecan-nxki Enhertu HER2; Humanized IgG1 ADC HER2+ metastatic breast cancer 2021 2019 Teprotumumab, teprotumumab-trbw Tepezza IGF-1R; Human IgG1 Thyroid eye disease NA 2020

Eptinezumab, eptinezumab-jjmr VYEPTI CGRP; Humanized IgG1 Migraine prevention In review 2020 Isatuximab, isatuximab-irfc Sarclisa CD38; Chimeric IgG1 Multiple myeloma 2020 2020

Sacituzumab govitecan; sacituzumab govitecan- hziy TRODELVY TROP-2; Humanized IgG1 ADC Triple-neg. breast cancer In review 2020

Neuromyelitis optica spectrum Inebilizumab, inebilizumab-cdon Uplizna CD19; Humanized IgG1 disorders In review 2020

Tafasitamab, tafasitamab-cxix Monjuvi CD19; Humanized IgG1 Diffuse large B-cell lymphoma In review 2020 Belantamab mafodotin, belantamab mafodotin- blmf B-cell maturation antigen; Humanized IgG1 BLENREP ADC Multiple myeloma 2020 2020

Neuromyelitis optica spectrum EC decision Satralizumab, satralizumab-mwge Enspryng IL-6R; Humanized IgG2 disorder pending 2020

Atoltivimab, maftivimab, and odesivimab-ebgn Inmazeb Ebola virus; mixture of 3 human IgG1 Ebola virus infection NA 2020 Naxitamab-gqgk GD2; Humanized IgG1 High-risk neuroblastoma and refractory osteomedullary disease DANYELZA NA 2020

Margetuximab-cmkb MARGENZA HER2; Chimeric IgG1 HER2+ metastatic breast cancer NA 2020

Ansuvimab-zykl Ebanga Ebola virus glycoprotein; Human IgG1 Ebola virus infection NA 2020

Homozygous familial Evinacumab Evkeeza Angiopoietin-like 3; Human IgG4 hypercholesterolemia In review 2021

EC decision Dostarlimab, dostarlimab-gxly Jemperli PD-1; Humanized IgG4 Endometrial cancer pending 2021 CD19; Humanized IgG1 ADC Diffuse large B-cell lymphoma Loncastuximab tesirine, loncastuximab tesirine- lpyl Zynlonta NA 2021 Nerve growth factor; Humanized IgG2 Pain due to osteoarthritis of knee or hip Tanezumab (Pending) In review In review Aducanumab (Pending) Amyloid beta; Human IgG1 Alzheimer's disease In review In review 07/06/2021 Tralokinumab IL-13; Human IgG4 Atopic dermatitis EC decision Q2 2021 (Pending) pending In review Teplizumab (Pending) CD3; Humanized IgG1 Type 1 diabetes NA In review 02/07/2021

Hematopoietic stem cell 17/07/2021 transplant-associated thrombotic Narsoplimab (Pending) MASP-2, Human IgG4 microangiopathies NA In review Retifanlimab PD-1; Humanized IgG4 Carcinoma of the anal canal 25/07/2021 (Pending) In review In review Oportuzumab monatox EpCAM; Humanized scFv immunotoxin Bladder cancer 8/18/2021; Rolling (Pending) NA In review BLA

Anifrolumab (Pending) IFNAR1; human IgG1 Systemic lupus erythematosus In review In review 30/09/2021

NA; Real-Time Oncology Review Inolimomb (Pending) CD25; Muine IgG1 Graft vs. host disease NA In review Bimekizumab (Pending) IL-17A, F; Humanized IgG1 Psoriasis In review In review NA Balstilimab (Pending) PD-1; Human IgG4 Cervical cancer NA In review NA; Rolling BLA Sutimlimab (BIVV009) (Pending) C1s; Humanized IgG4 Cold agglutinin disease NA In review NA

Rolling BLA Ublituximab (Pending) CD20; Chimeric IgG1 Chronic lymphocytic leukemia NA In review Amivantamab EGFR, cMET; Human bispecific IgG1 NSCLC w/ EGFR exon 20 insertion mutations NA (Pending) In review In review Tisotumab vedotin (Pending) Tissue factor; Human IgG1 ADC Cervical cancer NA In review NA PD-1; Humanized IgG4 NA Toripalimab Tuoyi Nasopharyngeal carcinoma NA In review Omburtamab B7-H3; Murine IgG1 CNS/leptomeningeal metastasis from neuroblastoma NA (Pending) In review NA Balstilimab (Pending) PD-1; Human IgG4 Cervical cancer NA In review NA