Anticorpi.

Presentazione sul tema: "Anticorpi."— Transcript della presentazione:

1 Anticorpi

2 Antigen-combining sites are comprised of the variable-heavy and variable-light chains, and germline antibody gene rearrangements are supplemented by somatic mutations in the hypervariable regions of these chains (for example, the complementarity-determining regions; CDRs) to define the antigen specificity of each antibody-producing B-cell clone. The Fc domain of the antibody is exclusively comprised of constant regions, which contain specialized binding domains that are responsible for complement fixation, binding to the neonatal Fc receptor (to regulate circulating IgG levels) and to cellular Fc receptors.

3 Antibody effector functions
Antibody effector functions.   Human antibodies, particularly IgG1 and IgG3, can potentially direct the killing of tumour cells by antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC)42. ADCC is triggered by an interaction between the Fc region of an antibody that has bound, through its antigen-binding region, to a tumour cell and the Fc receptors (Fc Rs), particularly Fc RI and Fc RIII, on immune effector cells such as neutrophils, macrophages and natural killer cells. The tumour cell is eliminated by phagocytosis or lysis, depending on the type of mediating effector cell. A prerequisite first step for CDC is recruitment of the complement component C1q by IgG bound to the tumour cell surface. This triggers a proteolytic cascade to activate complement. This can lead to the formation of a membrane attack complex that kills the target cell by disrupting its cell membrane. Alternatively, tumour-cell-bound C1q can bind to complement receptors, such as C1qR, CR1 (CD35) and CR3 (CD11b/CD18), on effector cells, such as neutrophils, macrophages and natural killer cells. This can trigger cell-mediated tumour-cell lysis or phagocytosis, depending on the type of effector cell.

4 A monoclonal antibody specific for a tumour-associated antigen allows the enrichment of cytokines in the tumour microenvironment. In the case of interleukin-2 (IL-2) it enhances antibody-dependent cellular cytoxicity mediated by Fc-receptor positive effector cells such as natural killer cells. In addition, tumour-targeted IL-2 stimulates T cells to expand and attack the tumour. High concentrations of plasmin at the tumour site enable the cleavage of IL-2 from the fusion protein through the plasmin-cutting site within the linker (depicted in the figure by scissors). Download file

5

6

7 FATTORI CHE DETEMINANO L’EFFICACIA IN VIVO DELLA TERAPIA CON ANTICORPI
Modalità di somministrazione Caratteristiche del bersaglio

8 CARATTERISTICHE DEL BERSAGLIO
Natura del tumore Neoplasia solida/ematologica Dimensioni Localizzazione Vascolarizzazione Antigene bersaglio Livello di espressione Specificità Rilascio Internalizzazione

9 FATTORI CHE DETEMINANO L’EFFICACIA IN VIVO DELLA TERAPIA CON ANTICORPI
Modalità di somministrazione Caratteristiche del bersaglio Caratteristiche dell’anticorpo

10 CARATTERISTICHE DELL’ANTICORPO
SPECIE DI ORIGINE, DIMENSIONI, AFFINITÀ Clearance in vivo Immunogenicità Penetrazione e uptake nel tumore

11 OKT®3 (muromonab-CD3): primo anticorpo (murino) approvato per l’uso terapeutico nell’uomo

12 PRINCIPALI LIMITAZIONI ALL’USO DI ANTICORPI MONOCLONALI MURINI NELL’UOMO
Inefficienza delle funzioni effettrici Breve vita media in circolo (t1/ h) Sviluppo di risposta HAMA

13 CONSEGUENZE DELLA RISPOSTA HAMA
Clearance accelerata Ridotta efficacia Preclusione di somministrazioni ripetute

14

15

16

17 In vitro and in vivo human antibody techniques exemplified by phage display and transgenic mouse technologies.   a | The in vitro process is based on panning the library of antibodies against an immobilized target. The non-binding phage antibodies are washed away and the recovered antibodies are amplified by infection in Escherichia coli. The selection rounds are subsequently repeated until the desired specificity is obtained. The antibody format for screening is either Fab or single-chain Fv. The expression of antibodies in E. coli and recent developments in screening technologies77 have made it possible to screen tens of thousands of clones for specificity. The antibody fragments themselves can be used as therapeutic agents as discussed in this review, but they can also be converted into intact immunoglobulins by the cloning of the variable genes into plasmids incorporating the constant-region genes of immunoglobulins. The genes are transfected into cell lines and therefore produce fully human immunoglobulins. b | The in vivo process is based on the immunization of a transgenic mouse. The mouse has been genetically engineered and bred for the expression of human immunoglobulins. The B cells harvested after immunization can be immortalized by fusion with a myeloma cell line, as in traditional hybridoma technology. The hybridomas can then be screened for specific antibodies. Phage Display Library - Antibody Production This technique is used  for producing antibody-like molecules.  Gene segments encoding the antigen-binding variable of V domains of antibodies are fused to genes encoding the coat protein of a bacteriophage.  Bacteriophage containing such gene fusions are used to infect bacteria, and the resulting phage particles have coats that express the antibody-like fusion protein, with the antigen-binding domain displayed on the outside of the bateriophage.  A collection of recombinant phage, each displaying a different antigen-binding domain on its surface, is known as a phage display library.  In much the same way that antibodies specific for a particular antigen can be isolated from a complex mixture by affinity chromatography, phage expressing antigen-binding domains specific for a particular antigen can be isolated by selecting the phage in the library for binding to that antigen.  The phage particles that bind are recovered and used to infect fresh bacteria.  Each phage isolated in this way will produce a monoclonal antigen-binding particle analogous to a monoclonal antibody. The genes encoding the antigen binding site, which are unique to each phage, can then be recovered from the phage DNA and used to construct genes for a complete antibody molecule by joining then to parts of immunoglobulin genes that encode the invariant parts of the an antibody.  When these reconstructed antibody genes are introduced into a suitable host cell line, the transfected cells can secrete antibodies with all the desirable characteristics of monoclonal antibodies of the suitable host cell line. In much the same way that a collection of phage can display a wide variety of potential antigen-binding sites, the phage can also be engineered to display a wide variety of antigens; such a library is known as an antigen display library.  In such cases, the antigen displayed are often short peptides encoded by chemically synthesized DNA sequences that have mixtures of all four nucleotides in some positions, so that all possible amino acids are incorporated.  It is not usual for every position in a peptide to be allowed to vary in this way, since the number of variable positions, there are over 2 x 1010 possible sequences of eight amino acids.

18 LIBRERIE ANTICORPALI ESPRESSE SULLA SUPERFICIE DI FAGI FILAMENTOSI

19 LIBRERIA ANTICORPALE Le variabili anticorpali vengono isolate più facilmente da linfociti di sangue periferico di donatore o di paziente ma potrebbero essere isolate anche dalla milza dal midollo osseo o da un linfonodo

20 PROCESSO DI SELEZIONE FAGICA (panning)

21

22 DATI CLINICI DI IMMUNOGENICITÀ E FARMACOCINETICA DI ANTICORPI MONOCLONALI
HAMA Anti idiotipo t1/2 β MURINO 60% 30% 20-60h CHIMERICO 6-30% 0-6% h UMANIZZATO 0-1% 2-3 settimane

23 BIODISTRIBUZIONE DI ANTICORPI MONOCLONALI
RIDOTTO ACCUMULO NEL TUMORE POSSIBILI SOLUZIONI: Somministrazione loco-regionale Uso di frammenti anticorpali

24 Antibodies and antibody fragments
Antibodies and antibody fragments.   Targeting antibodies are normally monoclonal immunoglobulin G (IgG) (Aa) or IgG fragments (B–D). F(ab')2 (B) or Fab' (C) fragments can be made by enzymatic cleavage of the whole monoclonal antibody (mAb) (Aa) or by molecular biological techniques — for example, Fab' (C), scFV (Da), bivalent (Db) or recombinant fragments (Dc). mAbs that are made from the traditional hybridoma technique are murine in origin. Recent developments have led to improved techniques for the production of chimeric, humanized or fully human antibodies or fragments (Ab–d). VH, variable heavy chain; VL, variable light chain.

25 ANTICORPI “NUDI” IN USO CLINICO
TRASTUZUMAB (Herceptin): MAb umanizzato vs. ErbB2/HER2/Neu RITUXIMAB (Rituxan): MAb chimerico vs. CD20

26 CD20 è una fosfoproteina transmembrana
espressa nella maggior parte dei linfomi a cellule B , assente nella maggior parte delle altre cellule dell’organismo espressa nella maggior parte dei B linfociti, tranne che nelle cellule precursore immature e nelle plasmacellule non viene dispersa nel plasma, né viene internalizzata dopo il legame con l’anticorpo è essenziale nel differenziamento e nella proliferazione dei B-linfociti

27 ANTICORPI “NUDI” IN USO CLINICO
RITUXIMAB (Rituxan): MAb chimerico vs. CD20 TRASTUZUMAB (Herceptin): MAb umanizzato vs. ErbB2/HER2/Neu ALEMTUZUMAB (Campath): MAb umanizzato vs. CD52 CETUXIMAB (Erbitux): MAb chimerico vs. EGFr BEVACIZUMAB (Avastin): MAb umanizzato vs. VEGF

28 STRATEGIE PER L’ARMAMENTO
DEGLI ANTICORPI IMMUNOCONIUGATI

29 To regain their cytotoxic activity, the cytotoxic agent has to be cleaved from the chemo-immunoconjugate. Uptake of antibodies predominantly occurs via the clathrin-mediated endocytosis pathway. After binding the respective antigen associated with coated pits, antibody–drug conjugates will be readily endocytosed, from where they transit through several stages of transport and endosomal vesicles and finally end up in a lysosome. There, linkers and antibody will be cleaved releasing the cytotoxic agent which — after exit from the lysosomal compartment — exerts its cytotoxic effect.

30 STRATEGIE PER L’ARMAMENTO
DEGLI ANTICORPI IMMUNOCONIUGATI IMMUNOTOSSINE Uso di tossine a basso peso molecolare (p.e. calicheamicina) IgG4 κ anti CD-33 + calicheamicina MYLOTARG® (Gemtuzumab ozogamicina)

31 STRATEGIE PER L’ARMAMENTO
DEGLI ANTICORPI IMMUNOCONIUGATI IMMUNOTOSSINE Uso di tossine a basso peso molecolare Uso di tossine proteiche

32 TOSSINE UTILIZZATE PER LA COSTRUZIONE DI IMMUNOTOSSINE
Esotossina A di Pseudomonas aeruginosa (PE, 613 aa) Tossina difterica, prodotta da Corynebacterium diphtheriae (DT, 580 aa) Dominio III Dominio II Dominio I Legame alla cellula Traslocazione Blocco ADP-ribosilazione di EF2 BLOCCO DELLA SINTESI PROTEICA

33 TOSSINE UTILIZZATE PER LA COSTRUZIONE DI IMMUNOTOSSINE
Tossine di origine vegetale: ricina, gelonina, saporina ATTACCO DIRETTO ALLA SUBUNITÀ 28S DEL RIBOSOMA BLOCCO DELLA SINTESI PROTEICA

34 ESEMPIO: BL22 Pastan et al. Nature Reviews Cancer 6, 559–565 (July 2006) | doi: /nrc1891

35 STRATEGIE PER L’ARMAMENTO
DEGLI ANTICORPI IMMUNOCONIUGATI IMMUNOTOSSINE Uso di tossine a basso peso molecolare Uso di tossine proteiche IMMUNOCITOCHINE

36 Strategies for enhancing the potency of antitumour antibodies
Strategies for enhancing the potency of antitumour antibodies.   Numerous strategies for improving the efficacy of antitumour antibodies are now being tested, including the representative examples shown here and described in Box 2. a | Enhancing effector functions involve improving antibody-dependent cellular cytotoxicity and/or complement-dependent cytotoxicity by means of site-directed mutations or manipulation of antibody glycosylation. b | Direct arming of antibodies entails their covalent linkage to killing machinery, such as radionuclides or toxins (for example, small molecules or proteins). Alternatively, arming antibodies with cytokines is intended to create high intratumour concentrations of cytokines to stimulate the antitumour immune response (T cells, B cells or natural killer cells), while avoiding the toxicities associated with systemic cytokine delivery. c | Indirect arming of antibodies can be achieved by attaching engineered antibody fragments to the surface of liposomes loaded with drugs or toxins for tumour-specific delivery. Bispecific antibodies that bind to two different antigens can be preloaded with the cytotoxic machinery before administration (indirect arming) or alternatively pre-targeted to the tumour before delivery of the cytotoxic payload. d | Pre-targeting strategies aim for the selective delivery of radionuclides to tumours or selective intratumour activation of prodrugs, thereby diminishing the systemic toxicities of these cytotoxic agents. For prodrug pre-targeting, an antibody-fragment–enzyme fusion protein is typically allowed to localize to a tumour and be cleared from the system. A prodrug is then administered and ideally converted to an active drug solely within the tumour. For radionuclide pre-targeting, an antibody–streptavidin conjugate is allowed to accrue within a tumour and is then used to capture a biotin–chelator–radionuclide complex. scFv, single-chain variable fragment.

37 Comparison of path lengths and emission tracks of - and --particle emissions used in antibody-targeted radiation therapy.   The --particle emissions occur in a spectrum of path lengths directly related to particle energy. The sparse energy track from these emissions is deposited over many cell diameters some distance from the decay event. The -particle emissions occur at a discrete energy and path length, resulting in a high linear energy transfer. The dense energy track from these emissions is deposited directly from the decay event over only a few cell diameters, 50–80 m in tissues.

38 general view of the conjugation of a bifunctional chelating agent to a monoclonal antibody.   Specifically, a bifunctional chelating agent possesses two functionalities. One portion binds (crab = chelos = chelate) metallic radionuclides, and the other portion bearing a reactive functional group (X) reacts and covalently binds to N-terminal and -amines from lysines on the antibody. In general, the metallic radionuclide is added last in this sequence, before purification of the final product; however, variations in which a pre-formed radiometal complex is conjugated to the monoclonal antibody are known.

39 VANTAGGI LEGATI ALL’USO DI ANTICORPI CONIUGATI
CON RADIONUCLIDI bystander effect le radiazioni raggiungono regioni del tumore che l’anticorpo di per sé non raggiungerebbe l’effetto prescinde dall’intervento del sistema immunitario dell’ospite

40 Strategies for enhancing the potency of antitumour antibodies
Strategies for enhancing the potency of antitumour antibodies.   Numerous strategies for improving the efficacy of antitumour antibodies are now being tested, including the representative examples shown here and described in Box 2. a | Enhancing effector functions involve improving antibody-dependent cellular cytotoxicity and/or complement-dependent cytotoxicity by means of site-directed mutations or manipulation of antibody glycosylation. b | Direct arming of antibodies entails their covalent linkage to killing machinery, such as radionuclides or toxins (for example, small molecules or proteins). Alternatively, arming antibodies with cytokines is intended to create high intratumour concentrations of cytokines to stimulate the antitumour immune response (T cells, B cells or natural killer cells), while avoiding the toxicities associated with systemic cytokine delivery. c | Indirect arming of antibodies can be achieved by attaching engineered antibody fragments to the surface of liposomes loaded with drugs or toxins for tumour-specific delivery. Bispecific antibodies that bind to two different antigens can be preloaded with the cytotoxic machinery before administration (indirect arming) or alternatively pre-targeted to the tumour before delivery of the cytotoxic payload. d | Pre-targeting strategies aim for the selective delivery of radionuclides to tumours or selective intratumour activation of prodrugs, thereby diminishing the systemic toxicities of these cytotoxic agents. For prodrug pre-targeting, an antibody-fragment–enzyme fusion protein is typically allowed to localize to a tumour and be cleared from the system. A prodrug is then administered and ideally converted to an active drug solely within the tumour. For radionuclide pre-targeting, an antibody–streptavidin conjugate is allowed to accrue within a tumour and is then used to capture a biotin–chelator–radionuclide complex. scFv, single-chain variable fragment.

41 Examples of the main classes of LTT
Examples of the main classes of LTT.   a | Immunoconstructs are formed by the linking of antibodies, antibody fragments or non-antibody ligands to therapeutic molecules, such as toxins (immunotoxins), radioisotopes (radioimmunotherapy), drugs (immunoconjugates) or enzymes (ADEPT). Drug release, if required (immunotoxins and immunoconjugates), occurs through intracellular degradation of the peptide linker. b | Immunoliposomes are formed by the attachment of multivalent arrays of antibodies, antibody fragments or non-antibody ligands to the liposome surface or, as in the example, to the terminus of hydrophilic polymers, such as polyethylene glycol (PEG), which are grafted at the liposome surface. The liposomes contain up to several million molecules of the therapeutic and release of the therapeutic occurs gradually by diffusion down its concentration gradient. c | Immunopolymers are formed by linking both therapeutic agents and targeting ligands to separate sites on water-soluble, biodegradable polymers, such as hydroxypropylmethacrylamine (HPMA), with the use of appropriate degradable spacers to allow for drug release. ADEPT, antibody-directed enzyme–prodrug therapy; LTT, ligand-targeted therapeutic.

42 Strategies for enhancing the potency of antitumour antibodies
Strategies for enhancing the potency of antitumour antibodies.   Numerous strategies for improving the efficacy of antitumour antibodies are now being tested, including the representative examples shown here and described in Box 2. a | Enhancing effector functions involve improving antibody-dependent cellular cytotoxicity and/or complement-dependent cytotoxicity by means of site-directed mutations or manipulation of antibody glycosylation. b | Direct arming of antibodies entails their covalent linkage to killing machinery, such as radionuclides or toxins (for example, small molecules or proteins). Alternatively, arming antibodies with cytokines is intended to create high intratumour concentrations of cytokines to stimulate the antitumour immune response (T cells, B cells or natural killer cells), while avoiding the toxicities associated with systemic cytokine delivery. c | Indirect arming of antibodies can be achieved by attaching engineered antibody fragments to the surface of liposomes loaded with drugs or toxins for tumour-specific delivery. Bispecific antibodies that bind to two different antigens can be preloaded with the cytotoxic machinery before administration (indirect arming) or alternatively pre-targeted to the tumour before delivery of the cytotoxic payload. d | Pre-targeting strategies aim for the selective delivery of radionuclides to tumours or selective intratumour activation of prodrugs, thereby diminishing the systemic toxicities of these cytotoxic agents. For prodrug pre-targeting, an antibody-fragment–enzyme fusion protein is typically allowed to localize to a tumour and be cleared from the system. A prodrug is then administered and ideally converted to an active drug solely within the tumour. For radionuclide pre-targeting, an antibody–streptavidin conjugate is allowed to accrue within a tumour and is then used to capture a biotin–chelator–radionuclide complex. scFv, single-chain variable fragment.

43 PRE-TARGETING: ADEPT (ANTIBODY–DIRECTED ENZYME PRODRUG THERAPY )
Somministrazione i.v.o intratumorale di anticorpi coniugati o fusi con enzimi FASE I distribuzione degli anticorpi e eliminazione della quota non legata al bersaglio FASE II Somministrazione sistemica del profarmaco FASE III

44 PRE-TARGETED RADIOIMMUNOTHERAPY
Complessi biotina-chelante-radionuclide Streptavidina