The generation of cytotoxic T cell epitopes and their generation for cancer immunotherapy Kessler, J.

Hele tekst

(1)The generation of cytotoxic T cell epitopes and their generation for cancer immunotherapy Kessler, J.. Citation Kessler, J. (2009, October 27). The generation of cytotoxic T cell epitopes and their generation for cancer immunotherapy. Retrieved from https://hdl.handle.net/1887/14260 Version:. Corrected Publisher’s Version. License:. Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden. Downloaded from:. https://hdl.handle.net/1887/14260. Note: To cite this publication please use the final published version (if applicable)..

(2) CHAPTER 1.

(3) 1 RETPAHC. Published (in part) in Leukemia 21:1859-1874, 2007.

(4) Background and scope of the thesis.

(5) . .

(6) !" #"$ $% " &'! (

(7) ! ) ( $ *(+ ,- . + / . +" 0 &2 % / 0 + +$ % 0 3 ) " ) ) " ) 0

(8) 4 ) 0 4 ) 0 4 ) 5 ) 5 ( ) 5

(9) ( 6 ) " ) 6 )!7 !7 ) 6

(10) )!7 $ 6

(11) 6

(12)

(13) !7 $ ) 3 6

(14) 4 6 )'73 6 )'73 '73 $ 8 ) # " "$ 9 . Chapter 1 – Background and scope of the thesis | 11.

(15) 1. Prologue More than three decades after the discovery &'! EF+ ) &'! E

(16) F &'!" EF+6 $ ) &'!" ) $ EF ) ) " )G E/+0F+ H ertheless, trials in cancer patients have not shown consistent and high percentages of E5"8F+ + G E9F+ =2 > Although the T cell arm of the immune sys G% % infected cells, the similar use of T cells for the = > G ) $ G ? cumulating understanding of the mechanisms % " tumor immunity in patients is currently being. @ notherapeutic treatments that will likely raise I @ crucially important that both an optimal immunostimulatory context is realized and that !,6+ cytotoxic T lympho =!7>!,+ => " =33> G ) The studies in this thesis address the epitopes K $!7# generation and presentation and, based on + ) " ) $ 12 | Chapter 1 – Background and scope of the thesis. ductory chapter will therefore especially review current knowledge regarding T cell immunity in cancer, its induction by immunotherapy, the identity of TAA, and the generation, prediction ) Because two of the studies in this thesis are at the basis of some reports in the literature, the outline and scope of each chapter is integrated, as boxed intermezzo, at its appropriate place in the text.. 2. Innate and adaptive immunity work together and are linked % =% > $ The innate system, composed of primarily + =,!>+ cytes, natural killer cells and the complement system, is evolutionary much older and con ) present at the start of the immune response and immediately interacts with pathogens, foreign antigens, cells sensed as abnormal and conserved structures shared by large groups " =" " >? hand, the adaptive response, executed by B- and T lymphocytes, before it can exert its @ +) ) and selection for which it uses clonal receptors ) $ $+ the adaptive response, unlike the innate re + is only since the last decade that the prescient $! D 868 innate immune system is driving the adaptive.

(17) EF$ ) E

(18) F I + adaptive immune response regulation has. /9 ( "H =(H(> + 8/8$N & + ) at the level of the lymphocyte by the non-self K = so-called signal one is the self-nonself dis >* of accumulating incompatible observations G+ (H( $ ) 859+ = $ > EF+ and later the focus shifted to the stimulator = P3!> + G " ) + second costimulatory signal to the lymphocyte EF +860+) $D (KE/F 868+! D K $ 3!) to be induced through ligation of so-called pat =--> 3!$ conserved pathogen-associated molecular pat =3&>$ E0F+ $ + -- 3! $ " " " + model has been coined either the infectiousH +--" ( " E5+6F 885+ ) =7-> ) E8F-- $ ( ous TLR that recognize a variety of conserved microbial-associated products, like lipopolysa =7(>+ $ ) E

(19) 9F+ -. by further unravelling the linkage between & + 88+&K " , = >E

(20) F This theory, initially purely theoretical, made ‘danger’ caused to cells and tissues the central concept and added still an extra level of cells 3! activation could also be induced by danger/ 2 = > cells, thus including endogenous non-foreign 3!" 3&K stated herself, “the danger theory, may seem to propose just one more step down the path of slowly increasingly complex cellular interac+ @@+ . @ + ‘foreignness’ of a pathogen is not the important feature that triggers a response, and ‘selfness’ UE

(21)

(22) F , $ K $D co-worker Medzhitov, because of its “inherent U3 = >#V = > is induced by a danger signal, but the danger ) 2 $ UE

(23) F? + + W theory, being above all its conceptual emptiness and vagueness when the concept ‘danger’ ) K E

(24) F!

(25) 998+ studies have revealed several endogenous nonforeign alarm signals, like heat shock proteins, "X= IHX>+ "Y= 7"Y> !,9" =!,/>E

(26) /FI +7- have been found to engage not only pathogenic components but also those from endogenous + , model is the most comprehensive theory be 3!"! LN O .

(27) ' + derestimate the importance of the exogenous D ) + ) identify, the important linkage between the innate and adaptive immune responses inducing 3! As the immune system is a diverse collection of mechanisms that have come together during the course of evolution, it is impossible to explain its complexity by a too much restricted = ( > to do so by a too much generalized metaphori + , > + &K E

(28) 0+

(29) 5F E

(30) 6F $ + together innate and adaptive immune mechanisms can counteract the attack of in principle all pathogens ranging from viruses and bacteria " . 3. The T cell response in a nutshell [ N " $ = $ N receptors) that recognize pathogen-derived native proteins, polysaccharides and lipids, T lymphocytes by their cell surface expressed T =!-> ) K % + + G Upon proper activation, B- and T cells divide, G $ + G @ + Two major subsets of T cells collaborate to @ #!,6+ G =!7> K ) =6L

(31) > $'73 surface and CD4+ = > K ) =/L

(32) 9> $'73 14 | Chapter 1 – Background and scope of the thesis. the activation and regulation of B cells, CTL 3! surface expressed molecules like CD40-ligand =!,/>

(33) =

(34) > N => cells accomplish CTL activation via their stim @ 3!$ & + !,+ = > G" + preventing autoimmunity but also suppressing " E

(35) 8F!7 cells that lyse target cells expressing their cognate class I-presented peptide by perforin and/ I" =) > is presented in an appropriate costimulatory G$ 3!+ ,! This second signal can be provided by any of N5"E9F HI =HI->"EF Crucially important costimulatory molecules N5" !,69=N5>!,60 =N5

(36) > !,

(37) 6 E

(38) F- +!,69q60 T cell-expressed counter-regulatory receptor CTLA-4, whose expression is upregulated after EF+ $ $$I @ + + % HI-" $ tial T cell activation to further costimulate and !,

(39) 5EF+"NN =!,5>E/F+?{9=!,>. -+ G = > + !,59+"NN" +?{9" . -" + G $ 3!EF3 molecules like intercellular adhesion molecule = !3&"> 7I3" $ E0F3 from cell surface receptors that mediate co-.

(40) stimulation, the cytokine milieu composed of 7"

(41) = $ >E5F+ 7"5 E6F+ 7"

(42) = $,!>E8"F+ 7"/ E

(43) +F$ $ + H G positive signalling, negative regulation of T cell $ ," = $ !,

(44) 6" G >N5" $ ," =,"7N5"'>+ G E9FN5" $ !?(" =N5> !?( G EF Th1-responses through the induction of IL-10 E/F + negative signals coming together in the immu $ 3!+ ,!+ E0F + + @ ? + % + !,0

(45) 7E5F !!-5E6F+ from the lymph node via the blood into the Thus, to accomplish Th and CTL activation, ,! => 7- $3&(= P7(>+q G !,9$!,9" = G >E8"/

(46) F

(47) >!,9 be seen as a master switch for T cell costimulation because of its ability to induce B7-family HI ,!E+/F?,! $W =HI>X+ = IH>Y+ HI~ 7"YE/F ,!+ tissues – particularly in barrier organs such as $ L$ $E//F+. are dedicated to capturing antigens, mostly by ,! 7-+ E/0F+ ,! E/5+/6F % specialized at presenting antigens and stimulating T cells through enhanced expression !,69q60 7"

(48) + !7 @ E+/8F,! $ $ of immunity and tolerance dependent on their E09F? + outlined, when in an immunogenic context DC activate naive anti-foreign T cells and on the other hand, when DC have an immature phenotype, they are capable of tolerizing autoreactive T cells – which have escaped the process of L = K>E0F( subsets of DC exist in vivo with distinct roles in immunity to infection and maintenance of self @ cation and intrinsic abilities to capture, process E0

(49) F. 4. Protein degradation pathways and the generation of T cell epitopes '73 $ surface of virtually all nucleated cells, whereas '73 G $3!+$ $W $'73 class I and class II molecules are produced through proteolysis in one of the three major intracellular protein degradation and antigen G#=>*$% =*$>" ceeds via the proteasome, which is called the. $% " =*(>P=

(50) > tophagy, which is an intracellular degradation system that delivers cytoplasmic constituents ! LN O /.

(51) @ E0F# " +autophagy, and macroautophagy, the latter $ $ K P=> " diated lysosomal degradation of extracellular $ The classical but outdated doctrine holds that class I presented peptides are derived from $*(" radation, whereas class II presented peptides result from degradation of exogenous proteins ' + in table 1, current knowledge reveals exceptions to these rules and interconnections be-. 4.1. The autophagic pathway of antigen processing Autophagy is in principle a nonselective process involved in removal of damaged or surplus organelles, turnover of long lived proteins, production of amino acids in nutrient + E5F In autophagy part of the cytoplasm becomes $ $ I $ " autophagosome with a lysosomal vesicle results in degradation of enclosed cytoplasmic ) $ *(+$ . Table 1. Common and uncommon antigen processing pathways for the generation of MHC class I and class II presented peptides. Class II presentation. Class I presentation a. common (cytosolic and nuclear antigens). UPS pathway. exception (cytosolic antigens). Autophagic pathway. common (endogenous, often foreign, antigens). Endocytic pathway. common (exogenous antigens). b. b. exception (endogenous foreign antigens). c. cross-presentation (exogenous antigens). d. a *($ $ -. E0"00F+ G 05 b The balance between the autophagic and endocytic pathways for class II presentation is not yet fully. E06F c Until now this pathway has been reported only twice, in conjunction with proteasomal degradation E08+59F d " 2 *(+$ 3" " E5F 3 $ " E5

(52) F. $ exception is the cross-presentation pathway that enables DC and macrophages to pres G $ $ for class I presentation by the proteasomal pathway because of its relevance for this thesis =I >I+ + cross-presentation pathways are addressed $ W=I > 16 | Chapter 1 – Background and scope of the thesis. protein degradation is estimated to be as large *(E5F7 + attracted new research and its emerging roles in innate and adaptive immune responses are $ E5/+50F agy eliminating intracellular pathogens, this process has been shown to deliver viral genetic material to endosomal TLRs in plasmacytoid ,!+ $ IHX E55F + % .

(53) &'! tion to CD4+ E56F class II presentation is even considered to de ) " =" > E58F3 = $ endosomes where class II loading occurs) it is easier to understand the role of autophagy in class II peptide-generation, autophagic degradation may also be involved in class I antigen processing, for instance by its clearance of. $% E69F- + ) volvement of autophagy in class I presentation, intricately linked to the proteasomal route, has been demonstrated in macrophages for endogenous antigens from herpes simplex E59F $ &'! 4.2. The endocytic pathway of antigen processing 3! K of, dying virally infected cells and dying tumor cells into the endocytic pathway to provide a representation of the protein environment The antigens can be endocytosed by a variety ,! cient in all forms of endocytosis, being phago=$ + phagosome), marcro- and micro-pinocytosis and receptor-mediated uptake mechanisms E09F, plished in diverse endosomal-lysosomal compartments by a large collection of proteases $ $ ) $ ' % E6F& In a late endosomal, early lysosomal compart-. &'! =& !> class II molecules and are loaded in the class II binding groove through exchange with the =" !7 > E6

(54) F* G + DC will be activated and migrate to T cell ar E6F + + pathogen-derived peptides produced in the endocytic pathway will allow the initiation of a CD4+ 4.3. Cross-presentation: cross-talk between antigen processing pathways I + !,6+ T cells must $ $ = &'!" complex) together with costimulation pro $ 3!+ ,!3,! are mostly not infected themselves by viruses = >+ % G tissues and display it through a process termed " !,6+ T cells in the E6F +" $ !,6+ T cell response can be initiated towards viral infections or mutations that exclusively occur in parenchymal E6/F,! with the capacity to cross-present exogenous + antigenic material is transferred from the endosomal-lysosomal pathway into the cytosol $ *(" &'! -G antigenic material can, therefore, end up stimulating either the CD4+ T cell response and/ !,6+ T cell response, dependent on the to the cross-presentation pathway can occur already directly at the moment of endocytosis, as has been observed for antigen uptake via the E60F+ Chapter 1 – Background and scope of the thesis | 17.

(55) Bacterium Peptide - MHC class I complex. Peptide - MHC class II complex Plasma membrane Early phagosome. Late phagosome. C. Vesicular transport. 7. Endolysomal tubule. Golgi Phagolysosome Endosome. B. Lysosome. 7 Autophagosome. Damaged organelles, macromolecules and intracellular pathogens. Lysosome. MHC class II. Peptide - class II complex. degradation. A. 5. Error. 1. TAP. Amino-peptidases & Endo-peptidases. 2. Ubiquitin. ER. Amino-peptidases. ?. 4. 3. 6 2m MHC class I. Proteasome. Peptide. Peptide. ?. Figure 1. Overview of the common antigen processing pathways for MHC class I and II presentation. A. *()$ $% $

(56) 0( = > ) $ =

(57) >$ $ = > ! =>+ ( 3 - = >H" =/> =0> $ " G I+ G . =5> $ I + autophagic pathway (B) and the endocytic pathway (C)+ G G$ &'! + Figure adapted from reference 68.. from extracellular sources in the endocytic route to the cytosol may rely on diverse, as yet + E06F The delivery of proteins or peptides through a $ + " -" $ 7 + - $ G E65+66F Alternatively, peptides and/or proteins may leak from the phagosomes into the cytosol or 6 O ! LN . $ Moreover, evidence exist for loading of class I molecules in the endo-lysosomal compart E68F " tation independent of cytosolic transit and E5F( separation of cross-presentation and endogenous class I presentation may have the advantages of speed and absence of competition E89F + pathway has been implicated in cross-presen-.

(58) E5

(59) F => % for cross-presentation by DC in vitro and in 3 $ 3 ) " =" > =" E6F> $ E8F+ K major mechanism by which the immune system monitors the presence of foreign antigen E8

(60) F 4.4. The ubiquitin-proteasome pathway for MHC class I antigen processing $&'! class I molecules is mainly achieved during the continuous turnover of endogenous proteins Apart from non-selective lysosome-mediated degradation, it has become clear since the 859 3" $ $% " =*( E8F> % + $mal and damaged proteins, but also in protein E8F substrates that are involved in many cellular E8/F+ E80+85F++ $E86+88F ( $ ) ) $% 3" $ +

(61) + K E8F $% $

(62) 0( K 3" E99F Although the majority of proteasome-mediated protein degradation is considered to start $ $% + $% ". independent degradation can be accomplished $$

(63) 9(

(64) 0( $ E9+9

(65) F 4.4.1. Structure of the proteasome G " $ + $% G $ $ ( eral types of proteasomes exist which share a +

(66) 9( E9F Y" $ =Y"5> $ $ @ X" $ =X"5> of each subunit two copies are present in the

(67) 9( ranted proteolysis of cellular proteins and its access is restricted to unfolded proteins or E9F3

(68) 9( 3" independent manner, which can be used in vitro for the assessment of proteasomal cleavages in model polypeptides, it can not actively.

(69) 0(

(70) 9( . L $ L 8( GE9F 8(E9/F K " $% + $ $3" + $% + a passageway for threading unfolded proteins

(71) 9( G$ of the channel that is otherwise blocked by H" X" $

(72) 6=3

(73) 6>E9/+90F G+ 8( cap, can associate with one or both ends of the

(74) 9( + $ E95+96F- + Chapter 1 – Background and scope of the thesis | 19.

(75) ) $ @ Y/" $ E98F )! % + the class I-presented peptide repertoire in the $ )@ could be important for proper positive selec!,6+ E9F

(76) proteasome The catalytic activities of the proteasome re Y" $ +

(77) 9( Y" $ sponsible for the so-called caspase-like activity, cleaving after acidic or small hydrophobic resi +Y

(78) $$= " > Y/" $ $ $ = " >EF! % + @ $ + $ )ity with the capacity to cut in principle after or $ E

(79) "F' + = >$ % $ $ I instance the proteasome does not readily cleave E/F3 $= >+ H" !" W + up to eight residues, contribute to the propen K ) $! % + @ "W viral proteins could lead to abrogation of CTL E0+5F(+$Gterminal liberation of a CTL epitope from hepatitis C virus was impaired due to a mutation W E6F $ ) G $ 20 | Chapter 1 – Background and scope of the thesis. reliably predict proteasomal cleavages in silico = $ > E8F+ $ leaves the proteasome before the next one en E

(80) F + amide bond may be either cleaved or not in different copies of the same protein, mostly leading to a cleavage pattern with partially overlap @ $ @ % $ of proteasome degradation fragments varies $

(81) + 58 / E

(82) +F * W HI"~ + cells of lymphoid organs, proteasomes exchange the normal, so-called constitutive, $ =Y+Y

(83) Y/> @ $ + =>" $ Y= 7&

(84) >+Y

(85) =&!7>Y/ =7&5>+ " E

(86) 9"

(87)

(88) F! ) " $ % + % +@ E

(89) F! % +@ have been found in cells expressing either of $ ( !7 " $ E

(90) "

(91) 0F G $ E

(92) 5F ( HI"~ 3

(93) 6 3

(94) 6+ the immuno-subunits, is up-regulated in DC. E

(95) 6F+ @ proteasome composition in DC is only very moderate due to low turnover of proteasomes E

(96) 8F 3

(97) 6 $ reportedly enhanced the presentation of some.

(98) E

(99) 0+9F$ W on antigen processing is not yet completely 4.4.3. Substrates for the UPS, rapid protein turnover and the DRiP model In 1996, it was proposed by Yewdell that a sig) $ originate from so-called defective ribosomal =,->EF G observation that after viral infection, epitopes derived from long-lived viral proteins are rap+ + E

(100) +F,- include all proteins that fail to achieve a stable conformation due to defects in transcription, +") EFI ,- $ E/+0F+$ $ E+5+6F =>$ G&'! -+ E6F=

(101) > W K 3$ production and degradation of viral proteins E5FN &'! K linkage of translation and antigen presentation would make perfect sense for immunity to acute virus infections, in which speed is of extreme importance to minimize viral replicaE8F3$ ,- $ ) E9F+ ) ,-" + G $ + $ E8+F An alternative model proposes that a subset of nascent polypeptides is stochastically delivered

(102) 9( $ protein folding machinery, which would also G E0F . mature DC, in which antigen processing and K +,- to be stored rapidly in intracellular aggregates $ ,37 (= " >E

(103) F ,37 ( ,- $ $% EF +,! ,-$ ,37 ( Another special source of polypeptides for cytosolic proteolysis are the products of crypEF+ encoded by introns, intron/exon junctions, /"" + E/F polypeptides with no biological function may $ ,-EF( eral CTL epitopes have been demonstrated to E0"8F 4.4.4. Generation of class I ligands, overview &'! process that can be divided in the three most ) # -+ =->+ $ G In principle all proteins that are tagged for destruction are prone to be degraded into single H G ) $ the proteasome, cytosol-resident aminopeptidases and endopeptidases accomplish further protein degradation of proteasome-degrada =

(104) L

(105)

(106) + 5"8 EF>! been implicated are puromycine sensitive =(3>E/9F+$ =N'>E/9F =73>E/F on proteasomal products and contributing to protein degradation are tripeptidyl peptidase II Chapter 1 – Background and scope of the thesis | 21.

(107) = >E/

(108) F =?> E/"//F4 = > + E/0F+ E/5F+ K = ,>E/6F+ G the cytosol, but their involvement in protein $ The generation of immunogenic peptides presented by class I molecules can be considered as a by-product of protein degradation, because upon partial degradation a small proportion = 6L0> E/8F$ - is accomplished by the transporter associated =3>+ = 6L0 E09F>3" = 0> -+ $ H" $ -" -3q-33E0

(109) +0F+ $ + $ &'! =Y

(110) " $ > G anism assisted by the chaperones calnexin, cal /5 = 0> ) )$ % $( 869 peptide function as anchors, of which the side chains bind in pockets of the class I binding groove, enabling peptides to bind with high EF $ = position two and the C-terminal position for the human class I molecules) and secondary % $ ) $ &'!q'73 + which also allows the in silico prediction of. 22 | Chapter 1 – Background and scope of the thesis. peptide binding for any peptide with the ap E0/F G '73 ecules – each individual expresses up to six '73 + E00F>L$ '73 E05F overlapping binding motifs, but each molecule ) ) 4.4.5. Generation of class I ligands, postproteasomal processing I $ + ) =6L

(111) +89> ( $ C-terminal excision of model CTL epitopes in "$ E06"59F with a failure to detect C-terminal trimming -E/+/8F+ led to the current notion that the proteasome liberates the exact C-terminus of the vast ma2 E+5F Although some CTL epitopes are directly $ + )tion of class I ligands is made as N-terminally G $ eration of the amino-terminus of these class I ligands is accomplished by aminopeptidases =(3+N'+ 73> -=-33q-3>- in the function of these N-terminal trimming K E5

(112) FI + ( HI7 $ =?43> 73 E/F 73" E5

(113) F $ -$ -33q-3 % E5"5/F+ $ => $-33E5/+50F!.

(114) results have been reported as to which extent -33q-3W "!,6+ E5/+55FI + - $ => $ -33q-3 $ E56F Cytosolic endopeptidases of which the role in class I antigen processing has been studied are ? " /L0&, $ 6, K 9 dimers each that form a twisted, spindle shape E9+58+69F ) = > tripeptides from the substrate’s N-terminus E69F +H" $ !7 =-*> =2 (3>E6F $ impaired proteasome function when cells are cultured under prolonged periods with protea $E6

(115) "6/F ) to exert a relatively low endoproteolytic activity of the trypsin-like type next to its amino-pep E6F3 +!7 ' 4H $ $ at its N-terminus and its C-terminus in an $ E60F+ !7 W K $ " E65+66F ' 4H ) epitope of which the C-terminus is known to $ 3 $ $ degradation of the majority of cytosolic poly =/>E/

(116) F+ that this enzyme may be necessary for the post ' + $ % E68"8F $ % . =!" > !7 = 8

(117) > ? $% G tallopeptidase of which the crystal structure revealed a deep substrate-binding channel E8F? W G$ $ E8+8/F+ L/ !" $ 0L5E80F + ? ity to either destroy or generate class I ligands ) $ = a minimal epitope or a C-terminal extended " >3 @ ? overexpression on the presentation of a spe)!7 =0/M. tuberculosis) $ E85F& + + @ ? on the production of class I presented peptides E/+//+86F? G ?=/0" fold the physiological level) reduced total class I expression, and RNAi-mediated silencing of ?= > G E//F3 ) + $? = ( HI7>E//F3 + $ ?$ antigen destruction and antigen generation, because, using a biochemical approach, in the cytosol both the substrates and products of ? E88F 4.4.6. Major unresolved questions in class I antigen processing ( processing of endogenous proteins are still $ $ I+ discussed before, the precise origin of class I presented peptides has not yet been unrav 3 sentation of epitopes derived from long lived ! LN O

(118) .

(119) proteins occurs, it has not yet been established ,-E9Fq $ E/F G 2 3 account for rapid antigenic presentation by E0F ( + 2 some in antigen processing is undisputed for the majority of class I ligands, a vast number of studies have indicated that there might $ ) " that is generated independently – or partially independently – of the proteasome by other N E

(120) 99F$ that proteasome inhibitor insensitivity was " ) '73"3"3 + ed were not resistant to inhibitor treatment, suggesting that a non-proteasomal protease or peptidase may preferentially generate peptides with basic C-termini binding in the acidic I" '73"3q3"$ 7 +7 $ $ tance to proteasome inhibition of cell surface G E

(121) 9F They also observed relatively high levels of reexpression of class I molecules accommodat $!" ='73"3+"306+"N

(122) 59/>+ but several other alleles, in particular those that bind a broad array of C-termini, displayed $ +ison by mass spectrometry of the cell surface '73"N

(123) 59/" " der conditions with and without proteasome inhibition revealed that the repertoire was @ '73"N

(124) 59/$ !" = $> " = > $ ) E

(125) 9

(126) F E8F $$ 24 | Chapter 1 – Background and scope of the thesis. @ @ ) E65+66+

(127) 9F cells with proteasome inhibitors even led to ) E

(128) 9"

(129) 96F + strongly suggest the existence of endoproteolytic activities that complement the proteasome in the generation of the C-terminus of ' +$ tool proteasome inhibitors that are known to be leaky, especially for the tryptic-activity EF+ proof the existence of proteasome-indepen ? ) ' 4H $ " $ E60F' + $ % broad role of this enzyme in the generation of E8

(130) F + G of non-proteasomal processing and the identity of the alternative endopeptidases that are $ G A third open issue is the extent to which cytosolic peptides are protected by chaperones E+

(131) 98F ("teasomal N-terminally extended variants of the ?43( HI7 $ -!E

(132) 9F+ + showed that both N-terminal and C-terminal pre-proteasomal processing-intermediates ( HI7" 89X E

(133) F ) % N only one model CTL epitope was studied, the prevalence of the association of processingintermediates with chaperones is not clear, and the nature of the pre-proteasomal intermedi E

(134) 98F.

(135) Scope of the thesis, mechanisms of antigen processing: In chapter 6, the non-proteasomal processing leading to class I presentation is elabo 3 that liberate the C-terminus of CTL epitopes ) H ) time implicated in the C- and N-terminal ) !7 ? is shown to function as a C-terminal trimming enzyme, generating a CTL epitope by )!" both enzymes in class I antigen processing . 5. Immunity to cancer and tumor immunoediting I + munity to malignancies is deeply rooted in the E

(136)

(137) FN observation in a patient that recovered from sarcoma after he had developed severe erysipelas, at the end of the 19th century, the New [! cancer patients with bacterial vaccines result E

(138) F much later that the underlying mechanisms, inducing innate immunity, were explained at the molecular level by the discovery of bacte GE

(139) F E

(140) F 8/9+ E

(141) /F ) G " )tigens and, thus, recognisability of tumors by " ) N E

(142) 0+

(143) 5F E

(144) 6F theory of immunosurveillance of cancer, speculating that spontaneously arising cancer cells. are often destroyed or kept in check by the 3 $ ) "859$ ' that showed absence of immunogenicity of E

(145) 8F' + several years later the group of Boon found that spontaneous murine leukemia cells possessed weak antigens that only led to rejection after the immune system was challenged with related more immunogenic tumor cells E

(146)

(147) 9FN $ insights in the basics of antigen presentation E+

(148) F+ 889 ) $$ ) E

(149)

(150) F E/+0F !7 ( + ) " ) and insights in immunity to cancer in general Current knowledge tells that by the time cancer is clinically detectable, it likely has already been adapted to the host immune recognition + @ surveillance – which has been doubted for a $ @ development were found between athymic = pletely lack functional T cells) and syngeneic " E

(151)

(152)

(153) +

(154)

(155) FL$ $ E

(156)

(157) +

(158)

(159) /FI + ) IH~ $ity to both chemically induced and spontane E

(160)

(161) 0F genetic trait was serendipitously found that conferred resistance to a highly aggressive E

(162)

(163) 5F ) killer cells, macrophages, and neutrophils that E

(164)

(165) 6F Immunosurveillance is now seen as a phase ! LN O

(166) /.

(167) in a broader evolutionary process of the tumor as reaction to immune pressure, called E

(168)

(169) 8F ranges from tumor recognition and elimina= > =$ > Both innate and adaptive cellular immunity takes part in tumor suppression and tumor G W $ multiple variables such as the tumor’s type, anatomic location, stromal response, cytokine ) E

(170)

(171) F +$ + % $ phase is envisioned in which occult tumors are kept in check by the immune system during a period of latency, previously also called tumor E

(172) 9F3 rence of metastatic melanoma in two allograft recipients that had received kidneys from the @ melanoma 16 years before her death, indeed % $ E

(173) F Often – it is not known how often – tumors escape from naturally induced immune pres ?$ + cumstances of non-optimal therapeutically H +@ of tumor and immune system, are known that contribute to escape from natural or thera " =

(174)

(175) > The tumor’s inherent low capacity to appropriately stimulate the immune system, primarily $ $ W GE

(176) F+ $ $ = " )>&'!" G + T cell ignorance, anergy or deletion, together E

(177) F. 26 | Chapter 1 – Background and scope of the thesis. Cross-presentation of tumor antigens and $ % + robust tumor immunity, will fail when a too $ = $E

(178) /F > ,! E

(179) 0F3 = > ently express danger signals, such as uric acid E

(180) 5F+$ that mostly the tumor microenvironment $ = $ .I"Y IL-10) and passively suppresses the induction =

(181) 6>pecially the maturation of DC is mostly lacking or incomplete in the tumor environment E

(182) 8+

(183) 9F( $ ,! KE

(184) 0+

(185) F induce regulatory T cells that often play an immune suppressive role in cancer immunity E

(186) 8+

(187)

(188) F3 complished by tumors that express high levels ,"7 $N5" $ ," G E

(189) F& + + caused by natural immunity or by therapeutically induced immunity, may result in loss of E

(190)

(191) +

(192) F$ E

(193) /F+ I+ antigen processing and presentation pathways, 3 + occur in tumors, highly likely as a result of im E9/+

(194) 0F. 6. T cell mediated immunotherapy for cancer, modalities, and basic requirements Immunotherapy of cancer by T cells can be divided in passive adoptive T cell transfer and active immunostimulatory vaccination strate-.

(195) =

(196) 5"

(197) 8>( $ % + adoptive transfer and active vaccination strate $ K "" ) q " )

(198) The only routine immunotherapy for cancer in the clinic to date is the infusion of donor lymphocytes after allogeneic stem cell transplanta ) E9F broad donor-derived CD4+!,6+ T cell ) = > G - $ were observed in metastatic melanoma patients after adoptive transfer of autologous " )) = 7> that were ex vivo expanded to high numbers E

(199) /9F " $ regime in this trial may have contributed to the further expansion in vivo of the adoptively transferred T cells, by making space and also by the depletion of negative regulatory CD4+ T cells or so-called myeloid derived suppres E

(200) /+

(201) /

(202) FI + !,+ T cell component in the transferred TILs has !,6+ T cell populaE

(203) /9FN G " )!7 $ G+ @ " ) =!-> $ =N7> E

(204) /F reported in patients with metastatic mela N7 !- ) &3-"=

(205) 5"/>'73"3

(206) " E

(207) /F!- vantage that the problem of expanding enough " ) $ ' +. targeting a single epitope may lead to antigen + N7 @ !- !7 @ 33 G antigens Irradiated autologous tumor cells or allogeneic '73" ) G .&"!(I+ 7"

(208) kines or costimulatory molecules have been. In various clinical trials this type of vaccine has E

(209) //F $ E

(210) /0"

(211) /8F'ever, several disadvantages are connected to this strategy like the suboptimal direct antigen presenting capacity of tumor cells, absence '73 + " presentation, and often the lack of autologous tumor samples needed for preparation of the ? ) = ,!E

(212) 09F>+ ='(> from the tumor and DC transfected with am) -H3E

(213) 0+

(214) 0

(215) F vantage of vaccination with autologous tumor + +'(-H3+ ) of relevant tumor antigens, including those % autologous tumor material are all personalized non-standardized vaccines that have to be pro pies aim to induce T cell responses against as $ = ) > " ) '73 = '73 > . Chapter 1 – Background and scope of the thesis | 27.

(216) length tumor associated antigens 4 $ ,H3 ) length tumor associated antigens and vaccination with recombinant tumor proteins themselves have been applied in vaccines aiming to raise humoral and T cell responses against G 7 + DC electroporated with mRNA encoding full length TAA are currently being optimized for E

(217) 0F4 aiming to raise immunity to a full length an '73type of the individual patient does not need to $ ? + from the problems related to each mode of de = +,H3+-H3+ P

(218) 5

(219) 6>+ whole antigens has the important drawback that vaccine induced immune pressure may induce escape through antigen loss variants of $ vented by vaccination with multiple full length ) = ,H3+ -H3E

(220) 0F > ! cell epitope containing synthetic peptides ( ) ) ) " )!7 E0F+ of immunizing cancer patients with synthetic $ $ H ous clinical peptide vaccine trials have been The relatively poor immunogenicity of pep % $ 2 together with adjuvants or loaded on DC =

(221) 0

(222) 0/>I mization of the peptide vaccination strategy E

(223) 00F ) $ $ !,6+ T cell re

(224) 6 O ! LN . sponses a concomitant CD4+ T helper response E/

(225) +

(226) 05"

(227) 08F +'73 " ) ably incorporated in peptide vaccines to pro !7 Important advantages of peptide vaccination ) synthesize peptides by good manufacturing =.&>+ $ $ "$ @ I +) increasing the immunostimulatory context of the vaccine – like conjugation with synthetic =7-> E

(228) 59FL $ K = >!7 => induce outgrowth of antigen loss variants of + $ '73 !7 @ " ) '73 !,+ = 9" > " % $ $ =,!> $ $ )E

(229) 5"

(230) 5F. 7. Tumor associated antigens and. I important criteria to classify tumor associated =33> #=>$ G = $ q >+ =

(231) > )=$ G >=> TAA in the oncogenic process and/or cancer 3+=>$ turnover kinetics of the TAA are important to E

(232) 5F+ /E

(233) 5/F.

(234) [ $ G + ) % gens that are restricted to only an individual tumor in one patient – which for obvious reasons restricts their immunotherapeutic applicability – and the antigens that are shared be [ $ " )+ ) *% " ) from mutations occurring in a single tumor ) G % point-mutation was found in the melanoma as L=&*&> E

(235) 50F = G +

(236) 55> ( " )@ gens are expressed in both the tumor and its G q =&3-"q& "3+ 99+ +-

No results found