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The Black Widow Protocol for Coordinated Cancer Immunotherapy Co-editing cancer cells and T-cells for minimal immunotherapeutic side effects 12

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Eric Werner at Oxford Advanced Research Foundation
Eric Werner
  • Oxford Advanced Research Foundation
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Abstract and Figures

One of the main problems with immunotherapy of cancer is the possibility of severe life threatening side effects. Under a new paradigm (the Cancer Network Paradigm) of how cancer works side effects can be avoided by two coordinated protocols that combine cancer network editing with T-cell network editing. A third meta-protocol integrates the two protocols. Together these protocols produce an engineered cooperative multi-cellular communication system between designed T-cells and edited cancer cells. The result is that edited cancer cells emit unique “kill me!” signals to obliging T-cells. Side effects are eliminated because the “kill me!” signal is unique to cancer cells and the designed T-cells only react to this one unique signal ignoring all other cells. The combination of the three protocols is called the Black Widow Immunotherapy Protocol (BWIP) or simply the Black Widow Protocol. The result of the coordinated protocols is a marriage between T-cells and cancer cells after which the T-cells destroy their partner cancer cells. Using cancer network editing, a gene activation link is inserted into the cancer network such that it produces a unique external signal to T-cells to indicate the cell is a cancer cell. In coordination, T-cells are edited to only attack the cells that emit the unique cancer signal. Black Widow coordinated cancer network immunotherapy forces cancer cells to emit unique signals that their co-engineered T-cells can recognize. Under this method transformed cancer cells cooperate with co-adapted T-cells to insure the cancer cells’ destruction. Unlike normal T-cell immunotherapy, there should be no side effects since co-engineered T-cells only kill co-edited cancer cells leaving all other cells alone. The Black Widow Protocol can be transformed to co-edited cancer cells and oncolytic viruses creating Viral Black Widow Protocols.
Protocol 2: Genome editing of T-cell to recognize coordinately edited cancer cells Protocol 2. In coordination with Protocol 1: Co-edit T-cells to recognize the unique identifier signal of Protocol 1 1. Sequence the T-cell genome mNTG to get the digital version dNTG 2. Translate the digital natural genome dNTG into the CancerCAD artificial genome dATG 3. Remove all or inactivate all receptor genes in given T-cells using CRISPR-like genome editing 4. Insert digital artificial code for the receptor gene dAtrg* into the dATG network. The code dAtrg* generates the virtual receptor vTR*. It transforms the artificial T-cell genome dATG into dATG* 5. Reverse Translate dAtrg* into the digital natural encoding of the gene receptor dNtrg*. 6. Synthesize the natural molecular gene receptor DNA code mNtrg* using the digital natural code dNtrg* as a template 7. Use CRISPR to co-edit T-cells: Insert and link the synthesized DNA receptor gene mNtrg* into the natural Tcell genome to give a transformed T*-cell with genome mNTG*. The transformed genome mNTG* expresses the receptor mTR* that recognize the cancer signal mCs* from co-edited cancer cells 8. Result: The T-cell has been transformed into a T*-cell that has the unique receptor mTR* for the unique cancer signal mCs* expressed by the co-edited cancer cells (using Protocol 1). The edited T*-cells kill only cells expressing the designed cancer signal mCs*. Protocol 2.2. Instead of Protocol 2, one could introduce a much simpler protocol as is done for designed T-cells now. First, remove all receptors. Second, insert the unique cancer signal receptor gene into the T-cell. This would avoid having to solve the decoding steps 2 and 5 in protocol 2. Protocol 2.2 is technologically feasible today(8, 20, 24, 40, 47).
Protocol 2: Genome editing of T-cell to recognize coordinately edited cancer cells Protocol 2. In coordination with Protocol 1: Co-edit T-cells to recognize the unique identifier signal of Protocol 1 1. Sequence the T-cell genome mNTG to get the digital version dNTG 2. Translate the digital natural genome dNTG into the CancerCAD artificial genome dATG 3. Remove all or inactivate all receptor genes in given T-cells using CRISPR-like genome editing 4. Insert digital artificial code for the receptor gene dAtrg* into the dATG network. The code dAtrg* generates the virtual receptor vTR*. It transforms the artificial T-cell genome dATG into dATG* 5. Reverse Translate dAtrg* into the digital natural encoding of the gene receptor dNtrg*. 6. Synthesize the natural molecular gene receptor DNA code mNtrg* using the digital natural code dNtrg* as a template 7. Use CRISPR to co-edit T-cells: Insert and link the synthesized DNA receptor gene mNtrg* into the natural Tcell genome to give a transformed T*-cell with genome mNTG*. The transformed genome mNTG* expresses the receptor mTR* that recognize the cancer signal mCs* from co-edited cancer cells 8. Result: The T-cell has been transformed into a T*-cell that has the unique receptor mTR* for the unique cancer signal mCs* expressed by the co-edited cancer cells (using Protocol 1). The edited T*-cells kill only cells expressing the designed cancer signal mCs*. Protocol 2.2. Instead of Protocol 2, one could introduce a much simpler protocol as is done for designed T-cells now. First, remove all receptors. Second, insert the unique cancer signal receptor gene into the T-cell. This would avoid having to solve the decoding steps 2 and 5 in protocol 2. Protocol 2.2 is technologically feasible today(8, 20, 24, 40, 47).
… 
Black Widow Protocol Result: The co-edited cancer network in the left cancer cell C* (in Red ) sends unique cancer signals mCs* to the receptors mTR* (in Green) of the co-edited T*-cells (in Blue) on the right. Result: Co-edited cancer cells are destroyed by co-edited T*-cells. Meta-Protocol 3: 1. Apply Protocol 1 to one or more cancer cells. a. Result: the cell expresses a unique identifier signal mCs* on its cell surface 2. Apply Protocol 2 (or 2.2) to design co-adapted T-cells to interact with cells transformed by Protocol 1. a. Result: Protocol 2 T-cells have the designed receptor mTR* that recognizes the designed signal mCs* of Protocol 1 transformed cancer cells 3. Deliver Protocol 2 T-cells (T*-cells) to Protocol 1 cancer cells (C*-cells). a. Result: T*-cells kill C*-cells and only C*-cells. 4. Result: Cancer cells with co-edited cancer C*-cells with the modified cancer networks emit signals mCs* are now recognized by the receptor mTR* of co-adapted T*-cells such that these co-edited T*-cells kill the co-edited cancer C*-cells. 5. End Result: Cancer are destroyed by co-adapted T*-cells that kill all co-edited cancer cells because they recognize the unique identifying signals expressed by co-adapted cancer cells. There should be no side effects since the cancer signal mCs* expressed by cancer cells is unique and T*-cells kill only cells that express this unique signal.
Black Widow Protocol Result: The co-edited cancer network in the left cancer cell C* (in Red ) sends unique cancer signals mCs* to the receptors mTR* (in Green) of the co-edited T*-cells (in Blue) on the right. Result: Co-edited cancer cells are destroyed by co-edited T*-cells. Meta-Protocol 3: 1. Apply Protocol 1 to one or more cancer cells. a. Result: the cell expresses a unique identifier signal mCs* on its cell surface 2. Apply Protocol 2 (or 2.2) to design co-adapted T-cells to interact with cells transformed by Protocol 1. a. Result: Protocol 2 T-cells have the designed receptor mTR* that recognizes the designed signal mCs* of Protocol 1 transformed cancer cells 3. Deliver Protocol 2 T-cells (T*-cells) to Protocol 1 cancer cells (C*-cells). a. Result: T*-cells kill C*-cells and only C*-cells. 4. Result: Cancer cells with co-edited cancer C*-cells with the modified cancer networks emit signals mCs* are now recognized by the receptor mTR* of co-adapted T*-cells such that these co-edited T*-cells kill the co-edited cancer C*-cells. 5. End Result: Cancer are destroyed by co-adapted T*-cells that kill all co-edited cancer cells because they recognize the unique identifying signals expressed by co-adapted cancer cells. There should be no side effects since the cancer signal mCs* expressed by cancer cells is unique and T*-cells kill only cells that express this unique signal.
… 
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All content in this area was uploaded by Eric Werner on Jul 27, 2018
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Key Words: Black Widow Protocol, cancer, cancer networks, cancer therapy, caner cell signaling, T-cell receptor design,
cancer cure protocols, immunotherapy, cell signaling, multi-cellular cell communication systems, communication system
networks, genome editing, genome design, gene synthesis, genome synthesis, CRISPR, cancer CAD software, cancer
network editing, T-cell editing and synthesis, synthetic T-cells, cancer cell “kill me!” signals, CAR T-cells, adoptive CAR T-
cell transfer, antigen, ligand, .EC"*93$;;<'".E]"*93$;;<'"*93$;;"3+$3U7-0&2"0&+0R02-%<'"382-U0&$<'"-&3-;8203"N0%6<'"^0%1;"Z;13U"
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Introduction%%
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Background%
Immunotherapy of cancer offers genuine new hopes and challenges for previously difficult to manage
cancers(1-53), including %$36%%$&2":;0-R;1<2-51(54, 55). However, all current approaches to
immunotherapy including adoptive T-cell therapy(38), checkpoint inhibitors(34), cytokines(7, 19, 49, 50,
52), and oncolytic viruses(1, 2, 15, 48, 56, 57) can have toxic side effects. While these side effects can
be reasonably managed for some immunotherapies for some of cancers, in general the severity of side
effects is a major hindrance to the general applicability of immunotherapy(2, 3, 8, 13, 15, 18, 19, 23, 31,
32, 36, 37, 42, 52, 57). A new paradigm offers a deeper understanding of how cancer is controlled,
called the Cancer Network Paradigm(58-60). This new paradigm is the basis of novel protocols that may
lead to a roadmap to cure cancer.
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/-50&1&2"%-;$"2+$8"+1/"0&"2+$"71<2)""K-5$"2%1&<3%0720-&",132-%<"518"0&02012$"2+$"$L$3620-&"-,"1"
/$N$;-75$&21;"&$2P-%U"90&3;6/0&:"1"31&3$%"&$2P-%U)""b-P$N$%'"5-<2"7%-2$0&"3-/0&:":$&$<'"
0&3;6/0&:"2%1&<3%0720-&",132-%<'"1%$"0&<2$1/"3-&2%-;;$/"R8"&-&97%-2$0&"3-/0&:"/$N$;-75$&21;"
&$2P-%U<)""*+6<'"6&/$%"2+$"&$P"71%1/0:5'"2+$<$"&-&97%-2$0&"3-/0&:"31&3$%"&$2P-%U<"3-&2%-;"
31&3$%"3$;;<)""*-"36%$"31&3$%"2+$<$"31&3$%"&$2P-%U<"56<2"R$"/$3-/$/"1&/"2+$&"$/02$/)"*+$"
3-5R0&120-&"-,".O4K=O":$&-5$"$/020&:"1&/"2+$"31&3$%"&$2P-%U"71%1/0:5",-%5"1"7-P$%,6;"
R0-$&:0&$$%0&:"2$3+&-;-:8"2-"057;$5$&2"1"&$P"%-1/517"2-"36%$"31&3$%>8,&20,&24,&40,&47,&61-67,&69,&
80-89D)"""
*+$"gene-centered&paradigm"-,"/$N$;-75$&2"1&/"31&3$%"+1<"@A'AAA"<-5$"7%-R;$5<"2-"<-;N$"
R$,-%$"02<"/$/0312$/"%$<$1%3+$%<"31&",6;;8"6&/$%<21&/"+-P"/$N$;-75$&2"1&/"31&3$%"P-%U)"M//"2-"
2+12"12";$1<2"2P-"-%"5-%$"0&2$%1320-&<"R$2P$$&"2+-<$"@A'AAA":$&$<"1&/"8-6"+1N$"1&"1<2%-&-5031;"
&65R$%"-,"7%-R;$5<"2-"<-;N$)""*+$"network&paradigm":%1&2<"2+$",6;;"3-57;$L028"-,"2+$"3$;;'"R62"
563+";0U$"1&"10%7;1&$"-%"31%'"3-&2%-;"-,"1"31%\<"-%"1"3$;;\<"1320-&<"518"R$"<057;$"1&/"7%$<$&2"1"563+"
$1<0$%"7%-R;$5"2-"<-;N$"2+1&"6&/$%<21&/0&:"P+12"2+$"0&&1%/<"-,"2+$"31%"-%"3$;;"/-"2-"13+0$N$"2+-<$"
1320-&<)""#$"<20;;"/-&\2"6&/$%<21&/"2+$"V61&265"<212$"-,"1"R%03U'"R62"P$"1%$"1R;$"2-"6<$"R%03U<"$N$%8"
/18"2-"R60;/"+-6<$<)"42"518"R$"3$&26%0$<"R$,-%$"P$"6&/$%<21&/"P+12"1;;"2+-<$"@A'AAA":$&$<"0&"ETM"
/-'"R62"P$"518"R$"1R;$"2-"3-&2%-;"2+$"/$N$;-75$&2"-,"$5R%8-<"1&/"31&3$%"0&"2+$"&-2"2--"/0<21&2"
,626%$)""
Single-edit%versus%dual-protocol%immunotherapy%
=$%<-&1;0[$/"0556&-2+$%178"0<"1"single-edit&protocol"P+$%$"2+$"7120$&2\<"31&3$%"3$;;<"1%$"<3%$$&$/"
,-%"51%U$%<"1&/"2+$"*93$;;"0<"$&:0&$$%$/"2-"+1N$"%$3$72-%<",-%"2+-<$"51%U$%<>8,&20,&24,&40,&47D)""
b-P$N$%'"2+$"31&3$%"3$;;<"2+$5<$;N$<"1%$"&-2"$/02$/)"4&"3-&2%1<2'"Z;13U"#0/-P"31&3$%"&$2P-%U"
0556&-2+$%178"0<"1"hybrid"dual-edit&immunotherapy"2+12"$/02<"&-2"c6<2"*93$;;<"R62"1;<-"2+$"31&3$%"
&$2P-%U<"0&"31&3$%"3$;;<)""4&"+8R%0/"/61;9$/02"0556&-2+$%178"31&3$%"3$;;<"1%$"2%1&<,-%5$/"R8"
31&3$%"&$2P-%U"$/020&:"2-"3--7$%12$"0&"2+$0%"-P&"/$<2%6320-&"R8"2+$"3-9/$<0:&$/"*93$;;<)"""
(%03"#$%&$%'"*+$"Z;13U"#0/-P"=%-2-3-;",-%".1&3$%"4556&-2+$%178""
Cite%As!"#$%&$%'"()'"*+$"Z;13U"#0/-P"=%-2-3-;",-%".--%/0&12$/".1&3$%"4556&-2+$%178!".-9$/020&:"31&3$%"3$;;<"1&/"*9
3$;;<",-%"50&051;"0556&-2+$%17$6203"<0/$"$,,$32<)"=%$7%0&2">?6;8"@ABCD"EF4!"G4&<$%2"+$%$"2+$"DOI"12"2-7"-,"/-365$&2H"
"
"
]"
Disadvantages%with%single-edit%immunotherapy""
B) Consistency!and!Completeness:"M"31&3$%"U0;;"<8<2$5"0<"consistent"0,"02"U0;;<"only"31&3$%"3$;;<)"
42"0<"complete"0,"02"U0;;<"all"31&3$%"3$;;<)"4,"2+$"*93$;;<"/$<0:&"0<"&-2"<7$30,03"$&-6:+'"2+$&"*9
3$;;<"P0;;"12213U"5-%$"2+1&"c6<2"31&3$%"3$;;<"1&/"2+$"<8<2$5"0<"inconsistent)""M&/"02",10;<"0,"2+$"
*93$;;"/$<0:&"0<"&-&"0&3;6<0N$"$&-6:+'"P+$%$"*93$;;<"/-&\2"/$<2%-8"$N$%8"31&3$%"3$;;)"*+$&"
2+$"U0;;"<8<2$5"0<"incomplete"1&/"2+$"31&3$%"31&"3-5$"R13U",6;;",-%3$)""
@) T-cell!only!edits!are!inconsistent!and!incomplete!"4&"<0&:;$9$/02"0556&-2+$%178"only&the&
T-cell&genome&is&edited)""*+$"31&3$%"3$;;<"1%$";$,2"1<"0<)""4,"2+$"0556&-2+$%178",10;<"2+$"31&3$%"
3$;;<"3-&20&6$"2-"7%-;0,$%12$)""
d) Side!effects!"E6$"2-"02<"0&3-&<0<2$&38"single-edit&immunotherapy"+1<"<0:&0,031&2"2-L03028"
3+1;;$&:$<"P02+"severe,&even&deadly&side&effec2<)".1&3$%"3$;;<"$L7%$<<"51&8"-,"2+$"51%U$%<"-%"
<0:&1;<"2+12"<-5$"&-%51;"3$;;<"$L7%$<<)""b$&3$'"$N$&"1"/$<0:&$/"*93$;;"518"U0;;"3$;;<"-,"
&-%51;"3$;;<"0,"2+$8"$L7%$<<"<-5$"-,"2+$"51%U$%<"2+12"31&3$%"3$;;<"$L7%$<<)""*+0<"31&";$1/"2-"
<6R2;$"-%":%-<<"<0/$"$,,$32<"<63+"1<"-%:1&";-<<"-%";-<<"-,"3$;;<"P02+"$<<$&201;",6&320-&<">2,&3,&
14,&19,&31,&36,&37,&42D)""
]) Off-target!effects:&.1&3$%"3$;;"<0:&1;<"1%$"&-2"6&0V6$"2-"31&3$%"3$;;<)"*+$%$"518"R$"off-
target&effects'"0)$)'"2-L03028"P02+"/$<2%6320-&"-,"&-%51;"3$;;<'"P02+"$N$&"2+$"5-<2"31%$,6;;8"
/$<0:&$/"*93$;;<"R$316<$"2+$8"6<$"2+$":$&$93$&2$%$/"5-/$;"-,"+-P"31&3$%"P-%U<)""*+$%$"0<"
&-":61%1&2$$"2+12"<0:&1;0&:":$&$<"$L7%$<<$/"R8"31&3$%"3$;;<"P0;;"&-2"1;<-"R$"$L7%$<<$/"R8"
&-%51;"3$;;<"<-5$P+$%$"0&"/$N$;-75$&2"1&/",6&320-&0&:>42D)""
g) Cold!tumors:"".-;/"265-%<"/-"&-2"$502"<0:&1;<"1&/_-%"/-"&-2"+1N$"<6,,030$&2"562120-&1;"
R6%/$&"2-"$502"&-N$;"31&3$%"<0:&1;<)""b$&3$'"2+$8"%$<0<2"0556&-2+$%178)""
h) !Expensive!""*+$"51%U$%<"-%"<0:&1;<"$L7%$<<$/"R8"2+$"31&3$%"3$;;<"56<2"R$",-6&/"1&/"
7$%<-&1;0[$/"*93$;;<"56<2"R$"/$<0:&$/",-%"$13+"7120$&2)"""""
i) External!approach!""K0&:;$9$/02"0556&-2+$%178"0<"1&"external&approach)"42"/-$<"&-2"12213U"
2+$"%--2"316<$"-,"31&3$%)"42<"12213U<"%$;8"-&"/0,,$%$&20120&:"2+$"$L2$%&1;"<0:&1;<"31&3$%"3$;;<"
:$&$%12$",%-5"2+-<$":$&$%12$/"R8"&-%51;"3$;;<)""42";$1N$<"2+$"0&2$%&1;"31&3$%9316<0&:"
&$2P-%U"0&"31&3$%"3$;;<"6&2-63+$/)""*+$"R1<03"/%0N$%"-,"31&3$%"%$510&<"0&2132)""
"
To%solve%the%present%problems%of%immunotherapy!""4"/$N$;-7$/"1"7%-2-3-;"31;;$/"2+$"Black&
Widow&Protocol."".1&3$%"3$;;<"3--7$%12$"P02+"*93$;;<"$&/0&:"0&"*93$;;<"U0;;0&:"2+$0%"3--7$%120N$"
71%2&$%)"f8"7%-2-3-;"$/02<"&-2"c6<2"*93$;;<"R62"1;<-"2+$"31&3$%"3$;;<)"#$"$/02"31&3$%"3$;;<"2-"$502"1"
6&0V6$"31&3$%"<0:&1;"2+12"&-"-2+$%"3$;;"0&"2+$"+651&"R-/8"31&"$502)"4&"3--%/0&120-&'"P$"/$<0:&"1"*9
3$;;"2+12"-&;8"%$<7-&/<"2-"2+$"6&0V6$"31&3$%"<0:&1;"1&/"U0;;<"2+$"31&3$%)"M<"1"%$<6;2"2+$"/$<0:&$/"*9
3$;;"-&;8"U0;;<"31&3$%"3$;;<"1&/";$1N$<"&-%51;"3$;;<"1;-&$)"b$&3$'"&-"<0/$"$,,$32<)"M//020-&1;;8'"<-"
51&8"31&3$%<"2+12"$N1/$"7%$<$&2"0556&-2+$%178"<6//$&;8"R$3-5$"2%$121R;$)"I-%"$L157;$'"1;;"
287$<"-,"3-;/"265-%<"R$3-5$"2%$121R;$)""
(%03"#$%&$%'"*+$"Z;13U"#0/-P"=%-2-3-;",-%".1&3$%"4556&-2+$%178""
Cite%As!"#$%&$%'"()'"*+$"Z;13U"#0/-P"=%-2-3-;",-%".--%/0&12$/".1&3$%"4556&-2+$%178!".-9$/020&:"31&3$%"3$;;<"1&/"*9
3$;;<",-%"50&051;"0556&-2+$%17$6203"<0/$"$,,$32<)"=%$7%0&2">?6;8"@ABCD"EF4!"G4&<$%2"+$%$"2+$"DOI"12"2-7"-,"/-365$&2H"
"
"
g"
Protocol 1. Edit cancer cell networks to send a unique signal
recognized by co-edited T-cells
"
Figure%1%Protocol%1%Edit%cancer%cells%to%cooperate%with%coordinated%edited%T-cells
"
Protocol!1.!!In!coordination!with!Protocol!2:!Co-edit!cancer!cells!to!send!a!unique!signal!recognized!by!T*-cells!
edited!by!Protocol!2.!
1. Sequence&the&molecular&natural&genome&(mNG)&of&the&cancer&cell.&&Output:&A&digital&natural&genome&(dNG)&
2. Translate&the&digital&natural&DNA&code&dNG&into&digital&artificial&DNA&code&(dAG)&of&the&Cancer-CAD&software&&
3. Analyze,!simulate,!search!and!edit&the&artificial&genome&network&dAG&
a. Find!and!Analyze&the&cancer&causing&networks&in&dAG&
b. Design!a!unique!identifier!signal!gene(dAsg*)&that&expresses&a&unique&cancer&signal&(vCs*)&to&T-cells&
c. Edit&the&cancer&networks&within&dAG&by&inserting&and&linking&in&the&signal&gene&dAsg*&link&into&one&or&
more&of&the&cancer&loops&in&dAG.&&
d. Result:!&The&artificial&cancerous&genome&dAG&has&been&transformed&in&dAG*&that&express&vCs*&&
4. Reverse!Translate&the&artificial&signal&gene&dAsg*&into&the&natural&code&dNsg*&&
5. Insert!and!link!the&digital&natural&signal&code&dNsg*&into&the&digital&natural&genome&dNG&to&create&the&
transformed&digital&genome&dNG*&that&expresses&the&natural&version&of&the&cancer&signal&vNCs*&&
6. Synthesize!the&molecular&signal-code&mNsg*&using&the&natural&digital&natural&signal&code&dNsg*&as&a&template&&
7. Use!CRISPR!to!insert!and!link!in&the&molecular&signal-code&mNsg*&into&the&corresponding&cancer&network&
within&the&natural&molecular&genome&mNG&to&transform&it&into&the&genome&mNG*&
8. Result&:&The&molecular&natural&genome&mNG&of&the&cancer&cell&has&been&transformed&into&a&genome&mNG*&where&
the&cancer&network&now&activates&the&synthesized&signal&code&mNsg*&which&in&turn&expresses&the&unique&
molecular&cancer&signal&mCs*&on&the&cell&surface&of&the&cancer&cell.&&The&coordinately&edited&T*-cell&(by&Protocol&2&
below)&can&now&recognize&the&cancer&C*-cell&and&kill&it.&&
!
(%03"#$%&$%'"*+$"Z;13U"#0/-P"=%-2-3-;",-%".1&3$%"4556&-2+$%178""
Cite%As!"#$%&$%'"()'"*+$"Z;13U"#0/-P"=%-2-3-;",-%".--%/0&12$/".1&3$%"4556&-2+$%178!".-9$/020&:"31&3$%"3$;;<"1&/"*9
3$;;<",-%"50&051;"0556&-2+$%17$6203"<0/$"$,,$32<)"=%$7%0&2">?6;8"@ABCD"EF4!"G4&<$%2"+$%$"2+$"DOI"12"2-7"-,"/-365$&2H"
"
"
h"
Protocol 2. Edit T-cell networks to express a unique receptor
that recognizes co-edited cancer cell signals
"
"
Figure%2%Protocol%2:%%Genome%editing%of%T-cell%to%recognize%coordinately%edited%cancer%cells&
!
Protocol!2.!In!coordination!with!Protocol!1:!Co-edit!T-cells!to!recognize!the!unique!identifier!signal!of!Protocol!1&
1. Sequence!the&T-cell&genome&mNTG&to&get&the&digital&version&dNTG!
2. Translate!the&digital&natural&genome&dNTG&into&the&CancerCAD&artificial&genome&dATG&&
3. Remove!all&or&inactivate&all&receptor&genes&in&given&T-cells&using&CRISPR-like&genome&editing!
4. Insert!digital&artificial&code&for&the&receptor&gene&dAtrg*&into&the&dATG&network.&The&code&dAtrg*&generates&the&
virtual&receptor&vTR*.&It&transforms&the&artificial&T-cell&genome&dATG&into&dATG*&
5. Reverse!Translate!dAtrg*&into&the&digital&natural&encoding&of&the&gene&receptor&dNtrg*.&
6. Synthesize!the&natural&molecular&gene&receptor&DNA&code&mNtrg*&using&the&digital&natural&code&dNtrg*&as&a&
template&
7. Use!CRISPR&to&co-edit!T-cells:!!Insert!and!link&the&synthesized&DNA&receptor&gene!mNtrg*&into&the&natural&T-
cell&genome&to&give&a&transformed&T*-cell&with&genome&mNTG*.&The&transformed&genome&mNTG*&expresses&the&
receptor&mTR*&that&recognize&the&cancer&signal&mCs*&from&co-edited&cancer&cells&&
8. Result:!!The&T-cell&has&been&transformed&into&a&T*-cell&that&has&the&unique&receptor&mTR*&for&the&unique&cancer&
signal&mCs*&expressed&by&the&co-edited&cancer&cells&(using&Protocol&1).&&The&edited&T*-cells&kill&only&cells&
expressing&the&designed&cancer&signal&mCs*.&&
&
Protocol!2.2.!!4&<2$1/"-,"=%-2-3-;"@'"-&$"3-6;/"0&2%-/63$"1"563+"<057;$%"7%-2-3-;"1<"0<"/-&$",-%"/$<0:&$/"*93$;;<"&-P)"
I0%<2'"%$5-N$"1;;"%$3$72-%<)"K$3-&/'"0&<$%2"2+$"6&0V6$"31&3$%"<0:&1;"%$3$72-%":$&$"0&2-"2+$"*93$;;)"*+0<"P-6;/"1N-0/"
+1N0&:"2-"<-;N$"2+$"/$3-/0&:"<2$7<"@"1&/"g"0&"7%-2-3-;"@)""=%-2-3-;"@)@"0<"2$3+&-;-:031;;8",$1<0R;$"2-/18>8,&20,&24,&40,&
47D)""
"(%03"#$%&$%'"*+$"Z;13U"#0/-P"=%-2-3-;",-%".1&3$%"4556&-2+$%178"" " "
"
Cite%As!"#$%&$%'"()'"*+$"Z;13U"#0/-P"=%-2-3-;",-%".--%/0&12$/".1&3$%"4556&-2+$%178!".-9$/020&:"31&3$%"3$;;<"1&/"*9
3$;;<",-%"50&051;"0556&-2+$%17$6203"<0/$"$,,$32<)"=%$7%0&2">?6;8"@ABCD"EF4!"G4&<$%2"+$%$"2+$"DOI"12"2-7"-,"/-365$&2H"
"
"
i"
&
Meta-Protocol 3. Black Widow Coordinated Network-
Immunotherapy: Combine Protocol 1 and Protocol 2
"
"
Figure%3.%%Black%Widow%Protocol%Result:%The%co-edited%cancer%network%in%the%left%cancer%cell%C*%(in%Red%)%sends%
unique%cancer%signals%mCs*%to%the%receptors%mTR*%(in%Green)%of%the%co-edited%T*-cells%(in%Blue)%on%the%right.%%
Result:%%Co-edited%cancer%cells%are%destroyed%by%co-edited%T*-cells.%&
!
Meta-Protocol!3:!!!
1. Apply!Protocol&1&to&one&or&more&cancer&cells.&&
a. Result:&the&cell&expresses&a&unique&identifier&signal&mCs*&on&its&cell&surface&
2. Apply!Protocol!2!(or!2.2)&to&design&co-adapted&T-cells&to&interact&with&cells&transformed&by&Protocol&1.&&
a. Result:!Protocol&2&T-cells&have&the&designed&receptor&mTR*&that&recognizes&the&designed&signal&mCs*&of&
Protocol&1&transformed&cancer&cells&
3. Deliver&Protocol&2&T-cells&(T*-cells)&to&Protocol&1&cancer&cells&(C*-cells).&&
a. Result:&T*-cells&kill&C*-cells&and&only&C*-cells.&&
4. Result:&&Cancer!cells&with&co-edited&cancer&C*-cells&with&the&modified&cancer&networks&emit&signals&mCs*&are&
now&recognized&by&the&receptor&mTR*&of&co-adapted&T*-cells&such&that&these&co-edited&T*-cells&kill&the&co-edited&
cancer&C*-cells.&&
5. End!Result:&Cancer!are!destroyed!by&co-adapted&T*-cells&that&kill&all&co-edited&cancer&cells&because&they&
recognize&the&unique&identifying&signals&expressed&by&co-adapted&cancer&cells.&There&should&be&no&side&effects&
since&the&cancer&signal&mCs*&expressed&by&cancer&cells&is&unique&and&T*-cells&kill&only&cells&that&express&this&
unique&signal.&&
"
(%03"#$%&$%'"*+$"Z;13U"#0/-P"=%-2-3-;",-%".1&3$%"4556&-2+$%178""
Cite%As!"#$%&$%'"()'"*+$"Z;13U"#0/-P"=%-2-3-;",-%".--%/0&12$/".1&3$%"4556&-2+$%178!".-9$/020&:"31&3$%"3$;;<"1&/"*9
3$;;<",-%"50&051;"0556&-2+$%17$6203"<0/$"$,,$32<)"=%$7%0&2">?6;8"@ABCD"EF4!"G4&<$%2"+$%$"2+$"DOI"12"2-7"-,"/-365$&2H"
"
C"
Discussion!""
"
*+$"Z;13U"#0/-P"=%-2-3-;"0<"1"5$2+-/"2-"0&/63$"3-556&03120N$"3--7$%120-&"R$2P$$&"31&3$%"3$;;<"
1&/"*93$;;<)""42"R60;/<"1"communication&or&signaling"protocol]"R$2P$$&"2P-"287$<"-,"3$;;<>70,&71D)""
*+$"<21&/1%/"0556&-2+$%178"1;<-"3+1&:$<"2+$"&-%51;"3-556&03120-&"7%-2-3-;"R$2P$$&"*93$;;<"
1&/"31&3$%"3$;;<"R8"0&<$%20&:"$L2%1"%$3$72-%<",-%"2+$"<0:&1;<"<$&2"-62"R8"31&3$%"3$;;<)"Z62"02"/-$<"&-2"
5-/0,8"2+$"3-556&03120-&"<2%12$:8"-,"2+$"31&3$%"3$;;<)"b$&3$'"02"0<"1"-&$9<0/$/"$/02"-,"*93$;;<"1&/"
&-2"31&3$%"3$;;<)"*+$"31&3$%"3-&20&6$<"2-",6&320-&"1<"R$,-%$)"*+$"7%-R;$5"P02+"-&$9<0/$/"$/02<"-,"
3-556&03120-&"<2%12$:0$<"0<"2+12"2+$"31&3$%"3$;;"$502<"<0:&1;<"2+12"518"1;<-"R$"$5022$/"R8"&-%51;"
3$;;<)"b$&3$'"2+$"/$<0:&$/'"$/02$/"*93$;;<"518"U0;;"&-%51;"3$;;<"$<<$&201;",-%"N1%0-6<",6&320-&<)"
b$&3$'"2+$"7-<<0R0;028"<$N$%$'";0,$"2+%$12$&0&:"<0/$"$,,$32<)""Z62"2+$"-N$%1;;"7-0&2"0<"2+12"P$"1%$"
5-/0,80&:"3$;;6;1%"3-556&03120-&"<2%12$:0$<"R$2P$$&"3$;;<)"""
.1&3$%9.ME"<-,2P1%$"1;;-P<"6<"2-"5-/$;"1&/"<056;12$"2+$"$,,$32<"-,"<63+"<0:&1;0&:"7%-2-3-;<"-&"
3$;;6;1%"R$+1N0-%)""*+0<"$&1R;$<"6<"2-"2$<2"5$2+-/<'"<63+"1<"2+$"Black&Widow&Protocol'"2+$"Cell&
Suicide&Protocol>69D"1&/"2+$"Cancer&Cure&Protocol"-%"Roadmap&Protocol>67D"1<"7-2$&201;"31&3$%"
2+$%17$6203<'"R8"$/020&:"N0%261;"31&3$%"1&/_-%"N0%261;"*93$;;<"3$;;<"-&"2+$"3-5762$%"1&/"-R<$%N0&:"
2+$"%$<6;20&:"]9/05$&<0-&1;"<713$9205$"56;2093$;;6;1%"0&2$%1320-&<"P02+0&"2+$"31&3$%9.ME"<-,2P1%$"
7%-:%15)""#+0;$"<63+"177%-13+$<"-&;8"177%-L0512$"%$1;028'"2+$8"/-":0N$"$<<$&201;"0&<0:+2<"0&2-"
+-P"31&3$%"3$;;<"P-%U'"+-P"2+$8"1%$"3-&2%-;;$/"1&/"+-P"2+$8"0&2$%132"P02+"-2+$%"3$;;<)"*+0<"31&"
;$1/"2-"&$P",626%$"2+$%17$6203"177%-13+$<"2-"31&3$%"1<"P$;;"1<"<+-P0&:"7-2$&201;"7%-R;$5<"1&/"
<0/$"$,,$32<"-,"31&3$%"2+$%17$6203"7%-2-3-;<)"*+6<"7%-1320N$;8"<1N0&:"7120$&2<",%-5"+1%5)"""
Z$316<$"-,"2+$"6&0V6$"/$<0:&$/"<0:&1;9%$3$72-%"3-5R0&120-&'"2+$%$"<+-6;/"R$"&-"
0556&-2+$%17$6203"<0/$"$,,$32<"1<";-&:"1<"-&;8"31&3$%"3$;;<"1&/"*e93$;;<"1%$"3-9$/02$/)"Z62"<$$"2+$"
/0<36<<0-&"-,"3-57;$2$"$%1/03120-&"-,"31&3$%"3$;;<"1&/"2+$"$,,$32<"-,"3+$3U7-0&2"0&+0R02-%<"R$;-P)""
Advantages%of%Black%Widow%hybrid%dual-protocol%immunotherapy"
B) *+$"Black!Widow!Protocol!is%consistent!and!complete!"M77;0$/"3-%%$32;8"02"<+-6;/"-&;8"
U0;;"31&3$%"3$;;<">consistencyD"1&/"02"<+-6;/"U0;;"1;;"31&3$%"3$;;<">completenessD"2+12"$502"2+$"
6&0V6$;8"/$<0:&$/"31&3$%"<0:&1;",-%"2+$"6&0V6$;8"/$<0:&$/"*93$;;"%$3$72-%)"
@) No!off-target!effects!"b8R%0/"/61;97%-2-3-;"0556&-2+$%178"<-;N$<"2+$"<0/$9$,,$32<"7%-R;$5"
R8"0&<$%20&:"1"6&0V6$"0/$&20,80&:"<0:&1;"0&2-"2+$"31&3$%"&$2P-%U"-,"2+$"31&3$%"3$;;)"*+$"
31&3$%"3$;;"2+$&"<$&/<"1"<0:&1;"6&0V6$"2-"2+$"+651&"R-/8)"*+$"3-9/$<0:&$/"*93$;;"%$3$72-%<"
-&;8"%$3-:&0[$"2+$"6&0V6$"<0:&1;"<$&2"R8"2+$"3-9$/02$/"31&3$%"3$;;<)"""
""""""""""""""""""""""""""""""""""""""""""""""""""""""""
]"*+0<"6<$"-,"2+$"P-%/"j7%-2-3-;\"+1<"/0,,$%$&2"5$1&0&:<)"4&"2+$"Black&Widow&Protocol"02"%$,$%<"2-"1"
step-by-step&method"6<$/"R8"<30$&20<2<"-%"5$/031;"<7$301;0<2<"2-"$;050&12$"31&3$%"3$;;<"0&"1"7120$&2)"
4&"3-&2%1<2'"1"communication&protocol"0<"1"<2%12$:8"-,"3-556&03120N$"0&2$%1320-&"R$2P$$&"2P-"-%"
5-%$"1:$&2<'"0&"2+0<"31<$'"3$;;<)">K$$"58"P-%U"-&"3-556&03120-&"2+$-%8"iA)"()"#$%&$%'"*-P1%/"1"
2+$-%8"-,"3-556&03120-&"1&/"3--7$%120-&",-%"56;201:$&2"7;1&&0&:)"Theoretical&Aspects&of&
Reasoning&About&Knowledge:&Proceedings&of&the&Second&Conference'"B@k9B]d">BkCCDl"iB)"()"
#$%&$%'"0&"Foundations&of&Distributed&Artificial&Intelligence,"m)"F`b1%$'"1&/"T)"?$&&0&:<'"(/)">#0;$8'"
BkkhD)D"
(%03"#$%&$%'"*+$"Z;13U"#0/-P"=%-2-3-;",-%".1&3$%"4556&-2+$%178""
Cite%As!"#$%&$%'"()'"*+$"Z;13U"#0/-P"=%-2-3-;",-%".--%/0&12$/".1&3$%"4556&-2+$%178!".-9$/020&:"31&3$%"3$;;<"1&/"*9
3$;;<",-%"50&051;"0556&-2+$%17$6203"<0/$"$,,$32<)"=%$7%0&2">?6;8"@ABCD"EF4!"G4&<$%2"+$%$"2+$"DOI"12"2-7"-,"/-365$&2H"
"
k"
d) *+$"unique!identifier!signal":$&$">.1&3$%"K0:&1;"m$&$"mCs*&1R-N$D"+1<"2-"R$"designed%
only!once"-%"1",6&320-&1;"<0:&1;0&:":$&$"3+-<$&",%-5"1&-2+$%"<7$30$<"<63+"2+12"+651&"3$;;<"
/-"&-2":$&$%12$"2+12"<0:&1;0&:":$&$"7%-/632)"K-"2--"0&"3--%/0&120-&"P02+"/$<0:&$/"31&3$%"
<0:&1;"/.<":$&$'"2+$"6&0V6$"%$3$72-%":$&$">/*OD"2+12":$&$%12$<"2+$"5-;$36;1%"%$3$72-%"
5*Oe",-%"2+$"5T<e"5-;$36;1%"<0:&1;"-&;8"+1<"2-"R$"/$<0:&$/"-%"3+-<$&"-&3$)"""
"
]) Cold!tumors!become!treatable:%".-;/"265-%<'"P+03+"1%$"R8"&126%$"%$<0<21&2"2-"
0556&-2+$%178"/6$"2-";13U"-,"31&3$%"2-"*93$;;"<0:&1;0&:'"R$3-5$"2%$121R;$"-&3$"2+$0%"
31&3$%"&$2P-%U<"1%$"$/02$/)"*+$8"1%$"-7$&"2-"0556&-2+$%178"R8"3-9$/02$/"*e93$;;<)""*+0<"
50:+2"0&3;6/$"R%$1<2"31&3$%"1&/"-2+$%"31&3$%<'"P+03+"1%$"%$<0<21&2"2-"0556&-2+$%178)""
"
g) More!efficient!and!less!costly!""Z-2+"2+$"<0:&1;":$&$",-%"31&3$%"3$;;<"1&/"2+$"%$3$72-%":$&$"
,-%"/$<0:&$/"*93$;;<"1%$"/$<0:&$/"-&;8"-&3$)"*+$8"1%$"6&0N$%<1;",-%"1;;"7120$&2<)"""4&"3-&2%1<2'"
2+$"*93$;;<"-,"<0&:;$9$/02"0556&-2+$%178"+1N$"2-"R$"/$<0:&$/",-%"$13+"7120$&2)"b$&3$'"2+$"
;1R$;"7$%<-&1;0[$/"5$/030&$)""
h) Network!paradigm!of!cancer!""*+$"Z;13U"#0/-P"0<"1"+8R%0/"/61;"7%-2-3-;"0556&-2+$%178"
2+12"0<"R1<$/"-&"2+$"5-%$"1/N1&3$/"&$2P-%U"71%1/0:5"-,"+-P"31&3$%"P-%U<>58D)"42":0N$<"
2+$",0%<2",6;;"2+$-%8"+-P"31&3$%<"1%$"3-&2%-;;$/"R8"2+$0%"31&3$%"&$2P-%U<)""42"$L7;10&<"31&3$%"
1&/"$5R%8-&03"7+$&-5$&1"2+12"2+$":$&$93$&2$%$/"71%1/0:5"31&&-2"$N$&",-%56;12$)""""
i) Complimentary!to!the!Cancer!Network!Protocols>67-69D!"42"518"R$"$1<0$%"2-"+1N$"1"
3-57;$L"31&3$%"3$;;"$L7%$<<"1"6&0V6$"<0:&1;"R8"0&<$%20&:"2+$"3-/$",-%"2+12"<0:&1;"P02+0&"-&$"
-,"2+$"31&3$%";--7<'"6<0&:"2+$"Black&Widow&Protocol'"2+1&"2-"3-%%$32"1":$&-5$"2+12"518"
+1N$"R$$&"/0<2-%2$/"-N$%"205$"/6$"2-"2+$"/8&1503<"-,"31&3$%"&$2P-%U"3-&2%-;)"*+6<'"P+$&"
/0%$32"2%1&<,-%5120-&"-,"31&3$%"&$2P-%U<">Cancer&Network&Protocols>67-69DD"+1<";0502$/"
177;031R0;028"/6$"2-"/$;0N$%8"7%-R;$5<"1&/"3-57;$L"3+%-5-<-51;"0%%$:6;1%020$<"/6$"0&"71%2"
2-"2+$"31&3$%"&$2P-%U"/8&1503<'"2+$"Black&Widow&Protocol"31&"3-57;05$&2"<21&/1%/"
0556&-2+$%178"1&/"2+$"Cancer&Network&Protocols>67-69D)"*+$"Black&Widow&Protocol"1&/"
Cancer&Network&Protocols"3-57;05$&2"$13+"-2+$%"R$316<$"+8R%0/"/61;97%-2-3-;"
0556&-2+$%178"0<"R1<$/"-&"2+$"<15$"31&3$%"&$2P-%U"71%1/0:5)"*+$"Black&Widow&dual-
protocol&immunotherapy&31&"U0;;"31&3$%"3$;;<"50<<$/"R8"Cancer&Network&Protocols>67-69D"0,"
2+$"7120$&2"+1<"56;207;$'"3-57;$L"31&3$%<"<056;21&$-6<;8)"""
Cancer&Network&Protocols"1%$"<20;;"&$$/$/"2-"7%-1320N$;8"3-%%$32"0&+$%02$/"31&3$%"&$2P-%U<)""
M//020-&1;;8'"Cancer&Network&Protocols"31&"R$"6<$/"2-"3-%%$32"2+$"&$2P-%U<"-,"13V60%$/"
31&3$%<"R$,-%$"2+$8"0&/63$"/0<2-%20-&"0&"1"31&3$%"3$;;\<":$&-5$)"""
C) Internal!and!external!approach!"4&"3-&2%1<2"2-"external&approaches"-,"<0&:;$9$/02"*93$;;"
0556&-2+$%178'"<21&/1%/"0556&-2+$%178'"<6%:$%8'"3+$5-2+$%178'"1&/"%1/0120-&"2+$%178'"
2+$"network&paradigm&of&cancer"0<"1&"internal&approach)""*+$"31&3$%"&$2P-%U"71%1/0:5"
:0N$<"133$<<"2-"2+$"0&2$%&1;"3-&2%-;"<8<2$5"-,"31&3$%"3$;;<)""*+$"31&3$%"$/02"7%-2-3-;"
/$<3%0R$/"+$%$"0<"1&"0&2$%&1;"177%-13+)""42"/0%$32;8"2%1&<,-%5<"2+$"31&3$%"&$2P-%U<"2-"<$&/"
6&0V6$"<0:&1;<"2-"3-9$/02$/"*93$;;<)""#+0;$"2+$"*93$;;<"<20;;"U0;;"31&3$%"3$;;<"$L2$%&1;;8"R1<$/"
-&"2+$"<0:&1;"$5022$/"R8"3-9$/02$/"31&3$%"3$;;<'"2+$"12213U<"1%$"+0:+;8"<7$30,03"/6$"2-"2+$"
$/020&:"-,"0&2$%&1;"31&3$%"&$2P-%U<)""
(%03"#$%&$%'"*+$"Z;13U"#0/-P"=%-2-3-;",-%".1&3$%"4556&-2+$%178""
Cite%As!"#$%&$%'"()'"*+$"Z;13U"#0/-P"=%-2-3-;",-%".--%/0&12$/".1&3$%"4556&-2+$%178!".-9$/020&:"31&3$%"3$;;<"1&/"*9
3$;;<",-%"50&051;"0556&-2+$%17$6203"<0/$"$,,$32<)"=%$7%0&2">?6;8"@ABCD"EF4!"G4&<$%2"+$%$"2+$"DOI"12"2-7"-,"/-365$&2H"
"
BA"
Overview!of!Cancer!Network!Editing!Methods!
4&"2+0<"1&/"7%$N0-6<"76R;03120-&<"2+%$$"R%-1/"287$"-,"31&3$%"36%$"7%-2-3-;<"+1N$"R$$&"0&2%-/63$/"
R1<$/"-&"1"3-5R0&120-&"-,"31&3$%9.ME"$/020&:"-,"&126%1;"1&/"1%20,0301;"ETM"0&"3-5R0&120-&"P02+"
.O4K=O9:$&-5$"$/020&:"-,"5-;$36;1%"ETM)""
B) Eliminative!cancer!network!editing(67)!"
1) (/02";0&U<"-,"31&3$%"&$2P-%U"R8"2%1&<,-%50&:"2+$"31&3$%"&$2P-%U"2-"1"+$1;2+8"
&$2P-%U"-%"
R) E$;$2$"<$320-&<"-,"2+$"&$2P-%U"<63+"1<"&-/$<"1&/_-%";0&U<"2-"51U$"2+$"&$2P-%U"
0&1320N$"
@) Cell!suicide!protocol(69)!"4&<$%2"1&"17-72-<0<";0&U"0&2-"2+$"31&3$%"&$2P-%U"
3. Black!Widow!Immunotherapy!Protocols:!M"3--%/0&12$/"31&3$%"&$2P-%U"0556&-2+$%178.!
4&<$%2"6&0V6$"31&3$%"<0:&1;":$&$"2-"R$"%$3-:&0[$/"R8"3-9$/02$/"*e93$;;"P02+"1"%$3$72-%",-%"
2+12"6&0V6$"<0:&1;)!
4. Viral!Black!Widow!Immunotherapeutic!Protocols:!Coordinated&editing&of&cancer&networks&
and&oncolytic&viruses.!4,"Protocol!2"0<"%$7;13$/"P02+"1"7%-2-3-;"2-":$&$%12$"1&"-&3-;8203"N0%6<"
2+12"%$3-:&0[$<"2+$"6&0V6$"31&3$%"<0:&1;"5.<e"2+$&"P$"+1N$"3%$12$/"virus-based"Black&
Widow&Protocol)""4&<$%2"1"6&0V6$"31&3$%"<0:&1;":$&$"2-"R$"%$3-:&0[$/"R8"2+$"3-9$/02$/"
-&3-;8203"N0%6<"P02+"1"%$3$72-%",-%"2+12"6&0V6$"<0:&1;)""K0&3$"2+$"N0%6<"R1<$/"Z;13U"#0/-P"
=%-2-3-;"1&/"2+$"*93$;;"Z;13U"#0/-P"=%-2-3-;"31&"6<$"2+$"<15$"6&0V6$"/$<0:&$/"31&3$%"
<0:&1;'"2+$"2P-"2+$%170$<"3-6;/"R$"6<$/"0&"3-5R0&120-&)"K$$">90D)!
!
Eliminative!cancer!network!editing!versus!Black!Widow!Protocol!immunotherapy%
*+$%$"518"R$"31&3$%"&$2P-%U<"2+12"1%$"/0,,036;2"2-"$;050&12$"R8"$;050&120N$"31&3$%"&$2P-%U"$/020&:"
>67-69D"1;-&$"/6$"2-"2+$0%"3-57;$L028)"4&"<63+"31<$<"Z;13U"#0/-P"&$2P-%U90556&-2+$%178"518"
R$"1&"$1<0$%"177%-13+"<0&3$"02"0&N-;N$<"0&<$%20&:"1"6&0V6$"*e93$;;"%$3-:&0[1R;$"<0:&1;"0&2-"c6<2"-&$"
-,"2+$"1320N$"31&3$%"&$2P-%U";--7<"%12+$%"2+1&"P+12"518"-2+$%P0<$"0&N-;N$"56;207;$"&$2P-%U"
$/02<)"
*+6<'"Z;13U"#0/-P"=%-2-3-;"P-6;/"1;;-P"/0%$32$/"*e93$;;"/$<2%6320-&"-,"3$;;<"50<<$/"R8"2+$"
$;050&120N$"31&3$%"&$2P-%U"$/020&:)""
"
Eliminative&Cancer&Network&Protocols"1%$"<20;;"&$$/$/"2-"7%-1320N$;8"3-%%$32"0&+$%02$/"31&3$%"
&$2P-%U<)""M//020-&1;;8'"Cancer&Network&Protocols"31&"R$"6<$/"2-"3-%%$32"2+$"&$2P-%U<"-,"13V60%$/"
31&3$%<"R$,-%$"2+$8"0&/63$"/0<2-%20-&"0&"1"31&3$%"3$;;\<":$&-5$)"""
Challenges%and%Problems%
*+$%$"1%$"<20;;":%$12"challenges"2+12"+1N$"2-"R$"<-;N$/"1&/"<-5$"7%-R;$5<"2+12"518"0&"7%0&307;$"
&$N$%"R$"<-;N$/"R8"*93$;;"0556&-2+$%178)""
B) Precise&cancer&cell&editing!"4,"2+$"6&0V6$"31&3$%"<0:&1;":$&$"3-/$"/T<:e"-%"02<"5-;$36;1%"
<8&2+$<0[$/"N$%<0-&"5T<:e"0<"0&<$%2$/"0&"2+$"-62<0/$"2+$"31&3$%"3$;;"&$2P-%U"2+$&"2+$"<0:&1;"
P-6;/"0&"5-<2"31<$<"&-2"R$"$L7%$<<$/)"M&/"2+$"31&3$%"3$;;<"P-6;/"3-&20&6$"2-"7%-;0,$%12$)""
(%03"#$%&$%'"*+$"Z;13U"#0/-P"=%-2-3-;",-%".1&3$%"4556&-2+$%178""
Cite%As!"#$%&$%'"()'"*+$"Z;13U"#0/-P"=%-2-3-;",-%".--%/0&12$/".1&3$%"4556&-2+$%178!".-9$/020&:"31&3$%"3$;;<"1&/"*9
3$;;<",-%"50&051;"0556&-2+$%17$6203"<0/$"$,,$32<)"=%$7%0&2">?6;8"@ABCD"EF4!"G4&<$%2"+$%$"2+$"DOI"12"2-7"-,"/-365$&2H"
"
BB"
@) Misplaced&edits&in&non&cancer&cells!"4,"2+$"31&3$%"<0:&1;":$&$"3-/$"/T<:e"-%"02<"5-;$36;1%"
<8&2+$<0[$/"N$%<0-&"5T<:e"0<"0&<$%2$/"0&2-"&-&931&3$%"3$;;<'"2+$&"<63+"3$;;<"518"$L7%$<<"
2+$",1;<$"31&3$%"<0:&1;)""*+$&"2+$"3-9/$<0:&$/"*e93$;;<"518"U0;;"2+-<$"50<$/02$/"3$;;<)"*+$"
<$N$%028"-,"<0/$"$,,$32<"/$7$&/<"-&"+-P"51&8"&-&931&3$%"3$;;<"1%$"$/02$/'"2+$";-3120-&<"-,"
2+$"0&3-%%$32"$/02<"1&/"-2+$%",132-%<"<63+"1<"3+$3U"7-0&2"<0:&1;0&:)"""
d) Completeness&cancer&cell&eradication!"*+$"/$:%$$"-,"$,,03138"-,"2+$"Z;13U"#0/-P"
4556&-2+$%178"=%-2-3-;"/$7$&/<"-&"-2+$%"3$;;"3-556&03120-&"7%-2-3-;<"<63+"1<"
3+$3U7-0&2"0&+0R02-%<g)""
]) Other&T-cell&receptors!!"4,"2+$"/$<0:&$/"*e93$;;<"<20;;"+1N$"%$3$72-%<",-%"-2+$%"-2+$%"3$;;"
<0:&1;<"/0,,$%$&2",%-5"2+$"6&0V6$"/$<0:&$/"<0:&1;"5.<e"2+$&"<0/$"$,,$32"1%$"<20;;"7-<<0R;$"
<0&3$"2+$<$"*e93$;;<"3-6;/"<20;;"%$3-:&0[$"&-&931&3$%-6<"3$;;<"1&/"7-2$&201;;"U0;;"2+$5)"4,"&-2"
1;;"-2+$%"*93$;;"%$3$72-%<"1%$"U&-3U$/"-62'"2+$&"2+$"3-9$/02$/"*93$;;<"518"12213U"-2+$%"
20<<6$)""b$&3$'"02"0<"R$<2"2-"%$5-N$"1;;"<63+"%$3$72-%<",%-5"2+$"*e93$;;)"""O$5-N0&:"1;;"<63+"*9
3$;;"%$3$72-%<"518"R$"3+1;;$&:0&:)""
g) Residual&check&point&receptors!"4,"&-2"1;;"3+$3U"7-0&2"%$3$72-%<"1%$"U&-3U$/"-62"2+$&"2+$"3-9
$/02$/"*93$;;"518"&-2"U0;;"31&3$%"3$;;<"2+12"$502"3+$3U"7-0&2"<0:&1;<)""
h) Mutations&in&the&inserted&receptor!"4,"2+$"&-N$;"6&0V6$"0&<$%2$/"*93$;;"%$3$72-%"0<"56212$/"
2+$&"02"518"%$3-:&0[$"-2+$%"<0:&1;<"R$<0/$<"2+$"/$<0:&$/"5.<e"31&3$%"<0:&1;"316<0&:"<0/$"
$,,$32<)"
i) Poorly&designed&receptors!"4,"2+$"%$3$72-%"7--%;8"/$<0:&$/"02"518"%$3-:&0[$"5-%$"2+1&"-&$"
<0:&1;"0&3;6/0&:"<0:&1;<",%-5"&-&"31&3$%"3$;;<)"b$&3$'"7-<<0R;$"<0/$"$,,$32<)""
C) Structually&ambigous&receptors!"(N$&"0,"2+$"&-N$;"0&<$%2$/"*93$;;"%$3$72-%"0<"5-;$36;1%;8"
6&0V6$"02"518"+1N$"<2%626%1;"$V60N1;$&2<"2-"&126%1;"*93$;;"%$3$72-%<)""*+$&"2+$"%$3$72-%"
518"%$3-:&0[$"&126%1;"<0:&1;<",%-5"&-&"31&3$%"3$;;<"316<0&:"7-<<0R;$"<0/$"$,,$32<)"
k) Decoding!"*+$"31&3$%"&$2P-%U<"0&"31&3$%"3$;;<"+1N$"2-"R$",-6&/"2-"R$"$/02$/)"*+12"5$1&<"
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BA) Delivery&Challenges!"""
1) All"31&3$%"3$;;<"&$$/"2-"R$"$/02$/"2-"3-&N$%2"2+$5"2-"31&3$%".e93$;;<"2+12"$502"2+$"
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"
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3$;;<",-%"50&051;"0556&-2+$%17$6203"<0/$"$,,$32<)"=%$7%0&2">?6;8"@ABCD"EF4!"G4&<$%2"+$%$"2+$"DOI"12"2-7"-,"/-365$&2H"
"
B@"
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4,"2+$%$"1%$"T"31&3$%"3$;;<"1&/"T"*93$;;<"<63+"2+12"2+$"T"*93$;;<"U0;;"T"31&3$%"3$;;<"12"$13+"
<2$7'"0,"2+$8"/-"<-"R$,-%$"2+$"31&3$%"3$;;<"/0N0/$"2+$&"2+$"31&3$%"0<"<2-77$/)"4,"2+$8"/-"<-"
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1,2$%"$13+"><8&3+%-&-6<D"31&3$%"3$;;"/0N0<0-&"2+$&"T"31&3$%"3$;;<"%$510&"1/"0&,0&0265)""
4,"<-5$+-P"1"31&3$%"3$;;"$<317$<"1&/"7%-/63$<"@"<6%N0N0&:"31&3$%"3$;;<"<-"2+12"2+$%$"1%$"
TnB"31&3$%"3$;;<"2+$&"2+12"-&$"$L2%1"31&3$%"3$;;"P0;;"V603U;8"-62713$"1;;"122$572<"R8"T"*9
3$;;<"2-"1;;"/$<2%-8"31&3$%"3$;;<)"*+$"31&3$%"P0;;":%-P"$L7-&$&201;"1<"<--&"1<"2+$%$"-&$"$L2%1"
31&3$%"3$;;"R$8-&/"2+$"T"*93$;;<)"*+$%$,-%$'"*93$;;<"P-6;/"+1N$"2-"7%-;0,$%12$"$L7-&$&201;;8"
2-"U$$7"67"2+$"2+$"$L7-&$&201;;8"7%-;0,$%120&:"31&3$%"3$;;<)""*+$"-&;8"+$/:$"0<"0,"2+$"31&3$%"
3$;;"/0N0<0-&"0<"/$7$&/$&2"-&"<0:&1;<",%-5"&$0:+R-%0&:"3$;;<)"*+$&"$N$&"$L7-&$&201;"31&3$%"
&$2P-%U<"518"-&;8"%$<6;2"0&";0&$1%":%-P2+">I-%"1&"$L7;1&120-&'"<$$"<0:&1;0&:"31&3$%<"0&"58"
.1&3$%"T$2P-%U"717$%>58DD)""
B@) I0&1;;8,&Cells&are&alive)""K0&3$"P$"1%$"/$1;0&:"P02+";0N0&:"3$;;<"2+12"1/172"1&/"$N-;N$"2+$%$"
P0;;"R$";0U$;8"6&,-%<$$&"3+1;;$&:$<"2-"1&8".O4K=O"R1<$/":$&-5$"$/020&:"177%-13+)"""
Conclusion"
b$%$"4"7%$<$&2$/"1"5$2+-/",-%"U0;;0&:"31&3$%"3$;;<"6<0&:"2+$"Z;13U"#0/-P"=%-2-3-;'"1"3--%/0&12$/"
:$&-5$"$/020&:"<2%12$:8">f$219=%-2-3-;"dD"2+12"3-5R0&$<"2P-"$/020&:"7%-2-3-;<">=%-2-3-;<"B"1&/"@"
-%"@)@D)""*+$",0%<2"7%-2-3-;"0<"6<$/"2-"$/02"2+$"31&3$%"&$2P-%U"0&"2+$"31&3$%"3$;;"2-",-%3$"02"<$&/"1"
6&0V6$"<0:&1;"2+12"0<"%$3-:&0[$/"R8"1"3--%/0&12$;8"$/02$/"*93$;;)""*+$"<$3-&/"7%-2-3-;"0<"6<$/"2-"3-9
$/02"2+$"*93$;;"2-"3%$12$"%$3$72-%<"2+12"%$3-:&0[$"2+$"6&0V6$"<0:&1;"<$&2"R8"2+$"$/02$/"31&3$%"3$;;)""
*+$"5$2197%-2-3-;">=%-2-3-;"dD"3-5R0&$<"7%-2-3-;<"1&/"B"1&/"@"-%"@)@)""*+$"%$<6;2"0<"2+12"2+$"
0556&-2+$%17$6203"<0/$9$,,$32<"1%$"$;050&12$/)""b-P$N$%'"$N$&"0,"1;;"2+$"/$;0N$%8"3+1;;$&:$<"1%$"
P-%U$/"-62'"2+$%$"518"<20;;"R$"7%-R;$5<"P02+"2%$120&:"$L7-&$&201;"31&3$%<"R8"76%$"*93$;;"
0556&-2+$%178)""4&"<702$"-,"2+$"3+1;;$&:$<'"2+$"Black&Widow&Protocol&"0<"0&"7%0&307;$"R$22$%"2+1&"
2+$"7%$<$&2"<21&/1%/"*93$;;"R1<$/"0556&-2+$%178"P02+"02<"*93$;;"-&;8"$/020&:'"R$316<$"2+$"Black&
Widow&Protocol&"0<"/61;9$/02"7%-2-3-1;;"2+12"0&2$%1&;;8"$/02<"31&3$%"3$;;"71%2&$%)""
*+$"Black&Widow&Protocol&0<"R1<$/"-&"2+$"cancer&network&paradigm'"P+03+"<212$<"2+12"
developmental&networks&control&cancer&cells"1&/"not"<-931;;$/"oncogenes'"P+03+"1%$"R1<$/"-&"2+$"
:$&$93$&2$%$/"2+$-%8"-,"+-P"31&3$%"0<"P-%U<)""#+0;$"-&3-:$&$<"518"<-5$205$<"0&02012$"2+$"
$L$3620-&"-,"1"31&3$%"&$2P-%U'"2+$8"/-"&-2"3-&2%-;"31&3$%)"
*+$"P+-;$"7%$<$&2"gene-centered&paradigm"-,"+-P"31&3$%"P-%U<"762<"1",6&/15$&21;"%-1/R;-3U"2-"
1&8",626%$"%-1/517<"2-"36%$"31&3$%)"O$<$1%3+$%<"&$$/"2-"<P023+"2-"2+$"31&3$%"&$2P-%U"
71%1/0:5>58,&60,&72,&73,&75-77D"2-"$,,$320N$;8"12213U"31&3$%)"I-%"2+12"2-"+177$&"2+$%$"+1<"2-"R$"1"
,6&/15$&21;"%$N-;620-&"2+$0%"6&/$%<21&/0&:"-,"+-P"31&3$%"P-%U<>68D)"""
&
(%03"#$%&$%'"*+$"Z;13U"#0/-P"=%-2-3-;",-%".1&3$%"4556&-2+$%178""
Cite%As!"#$%&$%'"()'"*+$"Z;13U"#0/-P"=%-2-3-;",-%".--%/0&12$/".1&3$%"4556&-2+$%178!".-9$/020&:"31&3$%"3$;;<"1&/"*9
3$;;<",-%"50&051;"0556&-2+$%17$6203"<0/$"$,,$32<)"=%$7%0&2">?6;8"@ABCD"EF4!"G4&<$%2"+$%$"2+$"DOI"12"2-7"-,"/-365$&2H"
"
Bd"
References%
%
B)".)"M3+1%/&et&al.'"J0:+20&:"1"I0%$"0&"2+$"*65-%"f03%-$&N0%-&5$&2"S<0&:"F&3-;8203"4556&-2+$%178)"EBioMedicine"31'"Bi9@]"
>@ABCD)"
@)"*)"M;;$&'"J)"m6&/%1c1U67715'"M"%-;$"-,"0556&-2+$%178"0&"5$21<21203"51;0:&1&2"5$;1&-51)"Cent&Nerv&Syst&Agents&Med&
Chem"12'"BC@9BCC">@AB@D)"
d)"E)"M)"M&26&$<&et&al.'"4&2$%7%$20&:"*9.$;;".%-<<9%$1320N028"2+%-6:+"K2%6326%$!"457;03120-&<",-%"*.O9Z1<$/".1&3$%"
4556&-2+$%178)"Front&Immunol"8'"B@BA">@ABiD)"
])"f)"Z1;c$N03'"K)"M)"b-;<2$0&'"=%$<$&2"1&/"I626%$"-,"4556&-2+$%178"0&"2+$"f1&1:$5$&2"-,"f6;207;$"f8$;-51)"J&Oncol&Pract"
14'"]Ad9]BA">@ABCD)"
g)"Z)"Z1%1/1%1&"T-N$0%8&et&al.'"K7$30,03"0556&-2+$%178"0&"+$712-3$;;6;1%"31&3$%!"M"<8<2$51203"%$N0$P)"J&Gastroenterol&Hepatol"
32'"ddk9dgB">@ABiD)"
h)"f)"*)"Z$2+6&$'"M)"^)"?-:;$U1%'"=$%<-&1;0[$/"*"3$;;95$/012$/"31&3$%"0556&-2+$%178!"7%-:%$<<"1&/"3+1;;$&:$<)"Curr&Opin&
Biotechnol"48'"B]@9Bg@">@ABiD)"
i)"T)"o)"Z%-3UP$;;'"Z)"K)"=1%U$%'"*65-%"0&+$%$&2"0&2$%,$%-&<!"457132"-&"0556&$"%$1320N028"1&/"0556&-2+$%178)"Cytokine'""
>@ABCD)"
C)"E)".+1U%1N1%20'"?)"b)".+-'"Z)"b)"#$0&R$%:'"T)"f)"#-&:'"#)"#)"#-&:'"K8&2+$203"R0-;-:8"177%-13+$<"0&"31&3$%"
0556&-2+$%178'":$&$203"&$2P-%U"$&:0&$$%0&:'"1&/":$&-5$"$/020&:)"Integr&Biol&(Camb)"8'"gA]9gBi">@ABhD)"
k)"K)".+16/+6%0&et&al.'"*BB*K"0556&-2+$%178"%$710%<"=4do9Mo*"<0:&1;0&:"0&"*93$;;<!".;6$<"2-P1%/"$&+1&3$/"*93$;;"<6%N0N1;"0&"
%12":;0-51"5-/$;)"J&Cell&Physiol"233'"igk9iiA">@ABCD)"
BA)"T)".+$&'"p)"J0'"T)"o)".+0&21;1'"q)"()"*1&-'"=)"K)"M/6<650;;0'"E%0N0&:".MO<"-&"2+$"6&$N$&"%-1/"-,"1&20:$&"+$2$%-:$&$028"0&"<-;0/"
265-%<)"Curr&Opin&Immunol"51'"BAd9BBA">@ABCD)"
BB)"?)"()".+$-&:'"J)"K6&'"*1%:$20&:"2+$"4EFB_*EF@9orT9M+O"=12+P18",-%".1&3$%"4556&-2+$%178"9".+1;;$&:$<"1&/"
F77-%26&020$<)"Trends&Pharmacol&Sci"39'"dAi9d@g">@ABCD)"
B@)".)".+$<2$%'"f)"I)"K1&515$/'"?)"#1&:'"4)"f$;$%-'"4556&-2+$%178"21%:$20&:"]9BZZ!"5$3+1&0<203"%120-&1;$'"3;0&031;"%$<6;2<'"
1&/",626%$"<2%12$:0$<)"Blood"131'"]k9gi">@ABCD)"
Bd)"M)"E1+51&0'"?)"K)"E$;0<;$'"*mI9R$21"0&"*".$;;"Z0-;-:8!"457;03120-&<",-%".1&3$%"4556&-2+$%178)"Cancers&(Basel)"10'"">@ABCD)"
B])"*)".)"m1&:1/+1%'"O)"b)"^-&/$%+$0/$'"f020:120&:"2+$"2-L03"$,,$32<"-,"1&2031&3$%"0556&-2+$%178)"Nat&Rev&Clin&Oncol"11'"kB9kk"
>@AB]D)"
Bg)"Z)"b$0&%03+'".)".[16/$%&1'"?)"S)"f1%V61%/2'"4556&-2+$%178"-,"b$712-3$;;6;1%".1%30&-51)"Oncol&Res&Treat"41'"@k@9@ki"
>@ABCD)"
Bh)"r)"b6'"q)"m)"*01&'".)"q+1&:'".+05$%03"1&20:$&"%$3$72-%">.MOD92%1&</63$/"&126%1;"U0;;$%"3$;;<"0&"265-%"0556&-2+$%178)"Acta&
Pharmacol&Sin"39'"Bhi9Bih">@ABCD)"
Bi)"J)"b61&:'"b)"p6'"m)"=$&:'"*JO95$/012$/"5$21R-;03"%$7%-:%1550&:"0&"2+$"265-%"503%-$&N0%-&5$&2!"7-2$&201;"&-N$;"
<2%12$:0$<",-%"31&3$%"0556&-2+$%178)"Cell&Mol&Immunol'"">@ABCD)"
BC)"b)"?01&et&al.'"4556&-2+$%178",-%"2%07;$9&$:120N$"R%$1<2"31&3$%!"(L0<20&:"3+1;;$&:$<"1&/"$L3020&:"7%-<7$32<)"Drug&Resist&
Updat"32'"B9Bg">@ABiD)"
Bk)"I)"o%-<3+0&<U8&et&al.'"T$P"/%6:<'"&$P"2-L03020$<!"<$N$%$"<0/$"$,,$32<"-,"5-/$%&"21%:$2$/"1&/"0556&-2+$%178"-,"31&3$%"1&/"
2+$0%"51&1:$5$&2)"Crit&Care"21'"Ck">@ABiD)"
@A)"f)"J$:62'"m)"E-;2-&'"M)"M)"f01&'"F)"m)"F2251&&'"M)"o)"K$P$;;'".O4K=O95$/012$/"*.O"%$7;13$5$&2":$&$%12$<"<67$%0-%"
1&2031&3$%"2%1&<:$&03"*"3$;;<)"Blood"131'"dBB9d@@">@ABCD)"
@B)"f)"J$:62'"M)"o)"K$P$;;'"E$<0:&$%"*93$;;<"1&/"*93$;;"%$3$72-%<",-%"36<2-50[$/"31&3$%"0556&-2+$%170$<)"Curr&Opin&Pharmacol"
41'"kh9BAd">@ABCD)"
@@)"O)"E)"J$-&$'"J)"M)"(5$&<'"*1%:$20&:"1/$&-<0&$",-%"31&3$%"0556&-2+$%178)"J&Immunother&Cancer"6'"gi">@ABCD)"
@d)"K)"*)"J06&et&al.'"*+$".6%%$&2"K2126<"1&/"I626%$"O-;$"-,"2+$"=+-<7+-0&-<020/$"d"o0&1<$_Mo*"K0:&1;0&:"=12+P18"0&"S%-2+$;01;"
.1&3$%!"M&"F;/"=12+P18"0&"2+$"T$P"4556&-2+$%178"(%1)"Clin&Genitourin&Cancer"16'"$@hk9$@ih">@ABCD)"
@])"M)"T)"f0;0-2-6'"J)".)"=171/-7-6;-6'".MO"*93$;;"*+$%178!"M"T$P"(%1"0&".1&3$%"4556&-2+$%178)"Curr&Pharm&Biotechnol"19'"
g9BC">@ABCD)"
@g)"^)"=)"f-6%02<'"?)".)"#0cU51&<'"J)"M)"?--<2$&'"f)"m)"T$2$1'"*%10&$/"0556&028"1<"1"&-N$;"2+$%17$6203"<2%12$:8)"Curr&Opin&
Pharmacol"41'"g@9gC">@ABCD)"
@h)"r)"f6%121'"r)"K102-'"*)"o-21&0'"*)"f12-[1U0'".E]i9<0:&1;"%$:6;12-%8"7%-2$0&"1;7+1"<0:&1;0&:"<8<2$5"1&/"02<"177;03120-&"2-"
31&3$%"0556&-2+$%178)"Cancer&Sci'"">@ABCD)"
@i)"m)"()"T1-65'"f)"f-%U-<'"Z)"o05'"#)"M%1,12'"T-N$;"21%:$2$/"2+$%170$<"1&/"0556&-2+$%178",-%"1/N1&3$/"2+8%-0/"31&3$%<)"
Mol&Cancer"17'"gB">@ABCD)"
@C)"K)"T08-&:$%$'"M)"K1;2-<'"?)"()"m%18'"4556&-2+$%178"3-5R0&120-&"<2%12$:0$<">&-&93+$5-2+$%178D"0&"&-&9<51;;"3$;;";6&:"
31&3$%)"J&Thorac&Dis"10'"K]dd9K]gA">@ABCD)"
@k)"?)"K)"F`E-&&$;;'"E)"f1<<0'"f)"#)"J)"*$&:'"f)"f1&/1;1'"=4do9Mo*95*FO"0&+0R020-&"0&"31&3$%"0556&-2+$%178'"%$/6L)"Semin&
Cancer&Biol"48'"kB9BAd">@ABCD)"
dA)"?)"=-;0NU1'"?%)&et&al.'"M/N1&3$<"0&"(L7$%05$&21;"*1%:$2$/"*+$%178"1&/"4556&-2+$%178",-%"=120$&2<"P02+"m;0-R;1<2-51"
f6;20,-%5$)"Anticancer&Res"37'"@B9dd">@ABiD)"
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3$;;<",-%"50&051;"0556&-2+$%17$6203"<0/$"$,,$32<)"=%$7%0&2">?6;8"@ABCD"EF4!"G4&<$%2"+$%$"2+$"DOI"12"2-7"-,"/-365$&2H"
"
B]"
dB)"K)"=-;8[-0/0<&et&al.'"M320N$"/$&/%0203"3$;;"0556&-2+$%178",-%":;0-R;1<2-51!".6%%$&2"<2126<"1&/"3+1;;$&:$<)"Br&J&Neurosurg"
29'"Bki9@Ag">@ABgD)"
d@)"o)"=%1c17120'".)"=$%$['"J)"Z)"=)"O-c1<'"Z)"Z6%U$'"?)"M)"m6$N1%19=120&-'"I6&320-&<"-,"Tom@E"0&".EC>nD"*"3$;;<!"1&"-77-%26&028"
,-%"0556&-2+$%178)"Cell&Mol&Immunol'"">@ABCD)"
dd)"I)"O1/-:&1'"f)"E0$/$%03+'"K2%$<<90&/63$/"3$;;6;1%"%$<7-&<$<"0&"0556&-:$&03"3$;;"/$12+!"457;03120-&<",-%"31&3$%"
0556&-2+$%178)"Biochem&Pharmacol"153'"B@9@d">@ABCD)"
d])"M)"O0R1<'"?)"E)"#-;3+-U'".1&3$%"0556&-2+$%178"6<0&:"3+$3U7-0&2"R;-3U1/$)"Science"359'"BdgA9Bdgg">@ABCD)"
dg)"?)"O0$2+'"K)"K6R%151&01&'"f$3+1&0<5<"-,"4&2%0&<03"*65-%"O$<0<21&3$"2-"4556&-2+$%178)"Int&J&Mol&Sci"19'"">@ABCD)"
dh)"b)"b)"K+1&et&al.'".+051$%03"1&20:$&"%$3$72-%"*93$;;"2+$%178",-%"265-6%"0556&-2+$%178)"Biosci&Rep"37'"">@ABiD)"
di)"f)"O)"K+$$&'"K)"I0$%0&:'"4&"<026"N1330&120-&!"b1%N$<20&:";-P"+1&:0&:",%602"-&"2+$"31&3$%"0556&-2+$%178"2%$$)"Wiley&
Interdiscip&Rev&Nanomed&Nanobiotechnol'"$Bg@]">@ABCD)"
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... In short it allows a person with less than a high school education to edit genomes of any animal or plant. Indeed, high school students are now using this technology to do experiments that previously research scientists could only dream about [6,15,4,8] 2 CRISPR for curing cancer and for creating a cancer bomb My research has focused on the wonderful potential of applying CRISPR-like genome network editing to cure cancer [23,22,25,26,24]. It is based on the new cancer network paradigm [20] which goes to the core of how cancer cells are controlled. ...
The Coming CRISPR Wars: Or why genome editing can be more dangerous than nuclear weapons
Preprint
Full-text available
  • Jan 2019
While CRISPR-Cas genome editing technology has been heralded as a great advancement for science, human health, bioengineering and medicine, there is a potential dark side of CRISPR genome editing that may be a greater danger to humanity than nuclear weapons. We present some of the more obvious potential military and bioterrorism applications of CRISPR and related genome editing technology. CRISPR weapons have significant advantages over conventional nuclear weapons. The balance of power based on mutual assured destruction cannot be maintained once CRISPR weapons enter the international arena. Disturbingly, because of the easy entrance to the technology, it opens the door for small organizations and countries to enter the international arms arena. The simplicity and ease of development of such weapons makes it essential that CRISPR genome editing technology be carefully applied and regulated.
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The Internet of Life Chapter 1: Universal nonrandom network protocols govern development and evolution of all bilaterians
Preprint
Full-text available
  • Feb 2020
All diploid sexual organisms have two distinct haploid genomes, one from each parent. There is the male derived haploid genome and the female derived haploid genome. Each genome contains a distinct developmental control network that directs the development of the embryo to an adult. Run separately, independent of the influence of the other network, the each haploid genome produces a morphologically different organism. The interrelationship of the male and female haploid genome networks is governed by an interaction protocol that determines which parental network is in control in any given cell at any given point in development. The protocol consists of two interacting half-protocols, one for each parental hap-loid genome. The full interaction protocol is itself a higher-level, meta-network, or internetwork between the two lower-level, parental developmental control networks. Computer simulations show that if the interaction protocol is random then there is a loss of bilateral symmetry in the generated organism. Therefore, for all bilaterally symmetric organisms, the interaction protocol between the two parental genomes cannot be random. This implies that a nonrandom ur-protocol must have evolved with the first diploid bilaterians in the Precambrian more than 570 million years ago. Nonrandom protocols partition the embryo and adult into dynamic sections that are variably controlled by one or the other parental haploid genome network. Developmental networks and their meta-network protocols provide fundamentally new insights into embryonic and post-embryonic development, developmental pathologies, animal and plant hybrids, heterosis, and evolutionary dynamics.
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Human enteroviruses exhibit selective oncolytic activity in the model of human glioblastoma multiforme xenografts in immunodeficient mice
Article
Full-text available
  • Jan 2018
Stem cells that penetrated deeply into the brain tissue are the main reason behind the relapses of glioblastoma multiforme after surgery. Finding new approaches to counter such relapses, including those that make use of oncolytic viruses, is a pressing issue. This study aimed to determine the sensitivity of cells of human glioblastoma multiforme to non-pathogenic enteroviruses, in vitro and in vivo (mice xenografts model). Glioblastoma tumor cells were exposed to type 1 poliovirus (Sabin vaccine strain), Coxsackie virus A7 (strain LEV8), Coxsackie virus A9 (strain LEV9) and Coxsackie virus B5 (strain LEV14). The virus reproduction intensity and cytolytic activity were assessed through infection of monolayered glioblastoma cell cultures. The ability of glialoblastoma cell cultures (enriched with tumor stem cells) to build subcutaneous tumors in immunodeficient mice after those cultures were exposed to viruses signaled the effectiveness of glioblastoma stem cells destruction. The study revealed that Coxsackie virus A7 and type 1 poliovirus possess the most pronounced oncolytic and replicative properties when tested on gliblastoma cells infected with viruses in vitro and on subcutaneous tumor xenografts in immunodeficient mice (in vivo). Type 1 poliovirus and Coxsackie virus A7 virus prevented development of tumors when glioblastoma neurospheric cell cultures were preincubated with viruses before subcutaneous implantation. Coxsackie virus B5 only managed to reduce the number of tumors developed, and Coxsackie virus A9 did not affect the tumor development at all. Thus, a number of non-pathogenic enteroviruses strains can destroy glioblastoma's stem cells, i.e. they show promise in the context of development of therapeutic agents for relapse-free treatment of glioblastomas. © 2018 Pirogov Russian National Research Medical University. All rights reserved.
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Application of iNKT Cell-targeted Active Immunotherapy in Cancer Treatment
Article
Full-text available
  • Jul 2018
In tumor immunity, invariant natural killer T (iNKT) cells play a pivotal role as a link between the innate and adaptive immune systems. With a precisely regulated activation mechanism, iNKT cells have the ability to respond quickly to antigenic stimulation and rapidly produce cytokines and chemokines, and subsequently an effective antitumor immune response. The development of iNKT cell-targeted active immunotherapy enables, not only an antitumor immune response through innate and acquired immunity, but also the conversion of an immunosuppressive into an immunogenic microenvironment. This review is focused on the activation mechanism and the role of iNKT cells after therapeutic active immunization. The therapeutic strategy targeting iNKT cells is expected to be applied to clinical practice in combination with surgery and chemotherapy.
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Applications and advances of CRISPR-Cas9 in cancer immunotherapy
Article
Full-text available
  • Jul 2018
Immunotherapy has emerged as one of the most promising therapeutic strategies in cancer. The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR-Cas9) system, as an RNA-guided genome editing technology, is triggering a revolutionary change in cancer immunotherapy. With its versatility and ease of use, CRISPR-Cas9 can be implemented to fuel the production of therapeutic immune cells, such as construction of chimeric antigen receptor T (CAR-T) cells and programmed cell death protein 1 knockout. Therefore, CRISPR-Cas9 technology holds great promise in cancer immunotherapy. In this review, we will introduce the origin, development and mechanism of CRISPR-Cas9. Also, we will focus on its various applications in cancer immunotherapy, especially CAR-T cell-based immunotherapy, and discuss the potential challenges it faces.
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A Cancer Cell Suicide Protocol -Using cancer-CAD and CRISPR to edit cancer cell networks to induce cancer cell apoptosis
Preprint
Full-text available
  • Jul 2018
Genome editing technology(1-6) combined with Werner's network theory of cancer(7) opens up a new pathway to cure cancer by induced cancer cell suicide. We adapt the Cancer Cure Protocol(8-13) to design a different protocol that stops cancer by inserting into or modifying links in the cancer network that activate the cell death (apoptosis) pathway. The edit to stop cancer growth depends on the architecture of the cancer network. Different network link transformations have different effects depending on the cancer network link architecture. The ability to visualize the cancer network and simulate the dynamic effects of network edits in virtual space-time helps tremendously in understanding how the cancer cell is controlled and how to stop its proliferation. In cancer-CAD software, aspects of these edits can be automated with algorithms that suggest therapeutic network link edits automatically. The user can choose between various possible network edits by observing their effects on tumor growth in the simulation. Once an optimal network transformation is found, the edits can be synthesized and implemented in live cancer cells using CRISPR or analogous molecular genome editing technologies.
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Cancer Immunotherapy and the Immune Response in Follicular Lymphoma
Article
Full-text available
  • Jun 2018
Follicular lymphoma (FL) is the most frequent indolent lymphoma in the Western world and is characterized in almost all cases by the t(14;18) translocation that results in overexpression of BCL2, an anti-apoptotic protein. The entity includes a spectrum of subentities that differ from an indolent to a very aggressive growth pattern. As a consequence, treatment can include watch & wait up to intensive chemotherapy including allogeneic stem cell transplantation. The immune cell microenvironment has been recognized as a major driver of outcome of FL patients and gene expression profiling has identified a clinically relevant gene expression signature that classifies an immune response to the lymphoma cells. It is known for some time that the immune cell composition of the lymphoma microenvironment is important because high numbers of tissue-infiltrating macrophages correlate with poor outcome in patients receiving chemotherapy but not in patients receiving the combination of chemotherapy and CD20-specific monoclonal antibody rituximab. In addition, TCR signaling of tumor-infiltrating lymphocytes is dysfunctional leading to an impaired capacity to form an intact immunologic synapse. Approaches restoring local T cell function, e.g., by usage of checkpoint inhibitors has demonstrated clinical activity (ORR 40%) and can achieve long-term remissions. Ongoing trials with re-programmed autologous CART cells achieve response rates in approximately 50% of FL patients with relapsed and even refractory disease. Responses lasting for more than 6 months might be durable, indicative for a successful restoration of a functional immune system. In summary, FL is a malignant disease where the control by the immune system ultimately decides about progression and transformation rate. The advent of monoclonal antibodies has changed the way we treat FL and new approaches restoring the individual immune control will hopefully improve results further.
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Targeting adenosine for cancer immunotherapy
Article
Full-text available
  • Jun 2018
Immune checkpoint antagonists (CTLA-4 and PD-1/PD-L1) and CAR T-cell therapies generate unparalleled durable responses in several cancers and have firmly established immunotherapy as a new pillar of cancer therapy. To extend the impact of immunotherapy to more patients and a broader range of cancers, targeting additional mechanisms of tumor immune evasion will be critical. Adenosine signaling has emerged as a key metabolic pathway that regulates tumor immunity. Adenosine is an immunosuppressive metabolite produced at high levels within the tumor microenvironment. Hypoxia, high cell turnover, and expression of CD39 and CD73 are important factors in adenosine production. Adenosine signaling through the A2a receptor expressed on immune cells potently dampens immune responses in inflamed tissues. In this article, we will describe the role of adenosine signaling in regulating tumor immunity, highlighting potential therapeutic targets in the pathway. We will also review preclinical data for each target and provide an update of current clinical activity within the field. Together, current data suggest that rational combination immunotherapy strategies that incorporate inhibitors of the hypoxia-CD39-CD73-A2aR pathway have great promise for further improving clinical outcomes in cancer patients.
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The CD47-SIRPα signaling system and its application to cancer immunotherapy
Article
Full-text available
  • Jun 2018
Tumor cells evade immune surveillance through direct or indirect interactions with various types of immune cell, with much recent attention having focused on modifying immune cell responses as the basis for the development of new cancer treatments. Signal regulatory protein α (SIRPα) and CD47 are both transmembrane proteins that interact with each other and constitute a cell‐cell communication system. SIRPα is particularly abundant in myeloid cells such as macrophages and dendritic cells, whereas CD47 is expressed ubiquitously and its expression level is elevated in cancer cells. Recent studies have shown that blockade of the CD47‐SIRPα interaction enhances the phagocytic activity of phagocytes such as macrophages toward tumor cells in vitro as well as results in the efficient eradication of tumor cells in a variety of xenograft or syngeneic mouse models of cancer. Moreover, CD47 blockade has been shown to promote the stimulation of tumor‐specific cytotoxic T cells by macrophages or dendritic cells. Biological agents, such as Abs and recombinant proteins, that target human CD47 or SIRPα have been developed and are being tested in preclinical models of human cancer or in clinical trials with cancer patients. Preclinical studies have also suggested that CD47 or SIRPα blockade may have a synergistic antitumor effect in combination with immune checkpoint inhibitors that target the adaptive immune system. Targeting of the CD47‐SIRPα signaling system is thus a promising strategy for cancer treatment based on modulation of both innate and acquired immune responses to tumor cells. This article is protected by copyright. All rights reserved.
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Combination and inducible adjuvants targeting nucleic acid sensors
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Full-text available
  • Jun 2018
Innate immune sensing of nucleic acids derived from invading pathogens or tumor cells via pattern recognition receptors is crucial for mounting protective immune responses against infectious disease and cancer. Recently, discovery of tremendous amounts of nucleic acid sensors as well as identification of natural and synthetic ligands for these receptors revealed the potential of adjuvants targeting nucleic acid sensing pathways for designing efficacious vaccines. Especially, current data indicated that unique adjuvants targeting TLR9 and stimulator of interferon genes (STING)-dependent cytosolic nucleic acid sensing pathways along with the combinations of already existing adjuvants are promising candidates for this purpose. Here, we review current vaccine adjuvants targeting nucleic acid sensors and their modes of action.
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Present and Future of Immunotherapy in the Management of Multiple Myeloma
Article
  • Jul 2018
Multiple myeloma (MM) is the second most common hematologic malignancy with an increasing incidence and prevalence. The wide array of effective antimyeloma agents have transformed MM into a chronic condition for some patients, requiring long-term management planning. Immunomodulatory drugs and proteasome inhibitors have played a pivotal role in defining the most effective regimens for both transplantation-eligible and transplantation-ineligible subgroups. Nevertheless, recent approvals of immunotherapies in MM such as daratumumab have added another important component to combination treatments for both relapsed or refractory and newly diagnosed disease. Evolving novel therapies such as chimeric antigen receptor T cells are poised to raise the bar even further, holding a promise of effective treatment option for patients who would otherwise have limited treatment alternatives. As we continue to therapeutically exploit the essential roles of cell-mediated immune surveillance, antigen presentation, and modulation of inhibitory surface signaling, we are rapidly establishing the cornerstone role of immunotherapies in the management of all phases of MM. In this review, we will cover the spectrum of available immunotherapies approved for clinical use in MM, as well as briefly describe those in early- and late-phase development, with the focus of raising the awareness of the expanding immuno-oncology armamentarium in MM.
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Recurrent Glioblastoma Treated with Recombinant Poliovirus
Article
  • Jun 2018
  • NEW ENGL J MED
Background The prognosis of patients with recurrent World Health Organization (WHO) grade IV malignant glioma is dismal, and there is currently no effective therapy. We conducted a dose-finding and toxicity study in this population of patients, evaluating convection-enhanced, intratumoral delivery of the recombinant nonpathogenic polio–rhinovirus chimera (PVSRIPO). PVSRIPO recognizes the poliovirus receptor CD155, which is widely expressed in neoplastic cells of solid tumors and in major components of the tumor microenvironment. Methods We enrolled consecutive adult patients who had recurrent supratentorial WHO grade IV malignant glioma, confirmed on histopathological testing, with measurable disease (contrast-enhancing tumor of ≥1 cm and ≤5.5 cm in the greatest dimension). The study evaluated seven doses, ranging between 10⁷ and 10¹⁰ 50% tissue-culture infectious doses (TCID50), first in a dose-escalation phase and then in a dose-expansion phase. Results From May 2012 through May 2017, a total of 61 patients were enrolled and received a dose of PVSRIPO. Dose level −1 (5.0×10⁷ TCID50) was identified as the phase 2 dose. One dose-limiting toxic effect was observed; a patient in whom dose level 5 (10¹⁰ TCID50) was administered had a grade 4 intracranial hemorrhage immediately after the catheter was removed. To mitigate locoregional inflammation of the infused tumor with prolonged glucocorticoid use, dose level 5 was deescalated to reach the phase 2 dose. In the dose-expansion phase, 19% of the patients had a PVSRIPO-related adverse event of grade 3 or higher. Overall survival among the patients who received PVSRIPO reached a plateau of 21% (95% confidence interval, 11 to 33) at 24 months that was sustained at 36 months. Conclusions Intratumoral infusion of PVSRIPO in patients with recurrent WHO grade IV malignant glioma confirmed the absence of neurovirulent potential. The survival rate among patients who received PVSRIPO immunotherapy was higher at 24 and 36 months than the rate among historical controls. (Funded by the Brain Tumor Research Charity and others; ClinicalTrials.gov number, NCT01491893.)
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