US 2017.007 3423A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0073423 A1 JUILLERAT et al. (43) Pub. Date: Mar. 16, 2017

(54) METHOD OF ENGINEERING MULTI-INPUT Publication Classification SIGNAL SENSITIVET CELL FOR (51) Int. Cl. MMUNOTHERAPY C07K 6/30 (2006.01) C07K 6/28 (2006.01) (71) Applicant: CELLECTIS, Paris (FR) C07K I4/47 (2006.01) (72) Inventors: Alexandre JUILLERAT, New York, (52) U.S. Cl. NY (US); Claudia BERTONATI, Saint CPC ...... C07K 16/30 (2013.01); C07K 14/47 Louis (FR); Julien VALTON, New (2013.01); C07K 16/2863 (2013.01); C07K York, NY (US); Philippe 2319/03 (2013.01); C07K 2319/30 (2013.01); DUCHATEAU, Draveil (FR); Laurent C07K 2317/622 (2013.01) POIROT, Paris (FR) (57) ABSTRACT (73) Assignee. CELLECTIS, Paris (FR) The present invention relates to a method to engineer (21) Appl. No.: 15/106.783 immune cell for immunotherapy. In particular said immune cells are engineered with chimeric antigen receptors, which (22) PCT Fed: Dec. 19, 2014 be activated by the combination of hypoxia and ligand extracellular binding as input signals. The invention also (86) PCT No.: PCT/EP2014/078876 relates to new designed chimeric antigen receptors which are S 371 (c)(1), able to redirect immune cell specificity and reactivity toward (2) Date: Jun. 20, 2016 a selected target exploiting the ligand-binding domain prop erties and the hypoxia condition. The present invention also (30) Foreign Application Priority Data relates to cells obtained by the present method, in particular T-cells, comprising said chimeric antigen receptors for use Dec. 20, 2013 (DK) ...... PA 2013 TO806 in cancer treatments. Patent Application Publication Mar. 16, 2017. Sheet 1 of 38 US 2017/0073423 A1

AND logic gate

Input 1. Output Input 2

Input 1 Input 2 Output

Fig. 1 Patent Application Publication Mar. 16, 2017. Sheet 2 of 38 US 2017/0073423 A1

RTK-based CAR architecture

Tumor cell Tumor cell

Ligand induced dimerization of engineered RTKc is

O O Tumor cell ligand Transmitter "active' (, S domainExtracellular ligand-binding

Half transmitter "inactive' D i Half transmitter autoinhibition domains Patent Application Publication Mar. 16, 2017. Sheet 3 of 38 US 2017/0073423 A1

Tumor cell Tumor cell

No signal - Signal

Prior gene of interest inactivated cell

O O Tumor cell ligand (, S extracellular ligand-binding domain

Transmitter domains

GO Gene of interest Fig. 3 Patent Application Publication Mar. 16, 2017. Sheet 4 of 38 US 2017/0073423 A1

Tumor

Cell

O O Tumor cell ligand Protease target sequence t ) extracellular ligand-binding domain Signaling protein

O Protease

Fig. 4 Patent Application Publication Mar. 16, 2017. Sheet 5 of 38 US 2017/0073423 A1

Tumor cell

O Tumor cell

O O Tumor cell ligand Reconstituted split-intein "active' extracellular ligand-binding domain

N- and C-terminal domains Reconstituted signaling protein of a split intein 'active' O Fragmented signaling protein "inactive'

Fig. 5 Patent Application Publication Mar. 16, 2017. Sheet 6 of 38 US 2017/0073423 A1

Tumor Cell C Tumor cell

O Tumor cell ligand 3. Reconstituted split-intein 'active' extracellular ligand binding domain

N- and C-terminal domains Reconstituted transmitter of a split intein "active' Fragmented transmitter "inactive' Fig. 6 Patent Application Publication Mar. 16, 2017. Sheet 7 of 38 US 2017/0073423 A1

Tumor Cell O Tumor cell

O

Reconstituted split-kinase O O Tumor cell ligand "active'

extracellular ligand binding domain Phosphorylated signaling Protein "active' N- and C-terminal domains of a split kinase Non-phosphorylated signaling protein "inactive' Fig. 7 Patent Application Publication Mar. 16, 2017. Sheet 8 of 38 US 2017/0073423 A1

Tumor cell Tumor cell

2 2

O O Tumor cell ligand Reconstituted split-kinase "active" extracellular ligand binding domain O Non-Phosphorylated tSignaling protein N- and C-terminal domains Inactive of a split kinase ub Phosphorylated signaling Signaling protein protein "active' binding region Fig. 8 Patent Application Publication Mar. 16, 2017. Sheet 9 of 38 US 2017/0073423 A1

Tumor cell Tumor cell Tumor cell

O O Tumor cell ligand t S Extracellular ligand-binding domain

A N- and C-terminal domains Signaling protein of a split protease S. Protease target Sequence Reconstituted protease D "active'

Fig. 9 Patent Application Publication Mar. 16, 2017. Sheet 10 of 38 US 2017/0073423 A1

1||30.Joun1]||30.Jouun

||30.Jouun1 Patent Application Publication Mar. 16, 2017. Sheet 11 of 38 US 2017/0073423 A1

Tumor cell

Tumor cell

Activated conformatio Inactive conformatio

O O Tumor cell ligand O V tallar binding extracellular ligand-binding domain Scaffolding protein Scaffolding protein transmitter transmitter "active' "inactive'

Fig.11 Patent Application Publication Mar. 16, 2017. Sheet 12 of 38

||30.Joun.1

Jouun1||30.Jouun1||30.

Patent Application Publication Mar. 16, 2017. Sheet 13 of 38 US 2017/0073423 A1

Tumor cell

O Tumor cell

O O Tumor cell ligand High affinity binding region s extracellular ligand-binding domain d Transmitter "active' Transmitter "inactive' O Transmitter activator Autoinhibition domain

Fig. 13 Patent Application Publication Mar. 16, 2017. Sheet 14 of 38

||30.Jouun1||30.1

||30.JouunL

Patent Application Publication Mar. 16, 2017. Sheet 15 of 38 US 2017/0073423 A1

||30.Jouun1

||30.Joun.1

||30.Jouun1 Patent Application Publication Mar. 16, 2017. Sheet 16 of 38 US 2017/0073423 A1

Hypoxia dependant expression of CAR specific for Antigen 1

O Antigen 1 Q scFV anti-Antigen 1

Engineered T cell

CoStimulator domains

Activation domains

OxiTF (TAL effector as non limiting example) ONOO Hypoxia sensitive domai cytoplasm

ON nucleus

TALE binding site n TALE CAR ORF inding site

Fig. 16 Patent Application Publication Mar. 16, 2017. Sheet 17 of 38 US 2017/0073423 A1

Constitutive expression of CAR specific for Antigen 1 AND hypoxia dependent expression of CAR II specific Antigen 2

C Antigen 2 O Antigen 1 scFW anti ScFW anti Antigen 2 ( Antigen 1

Engineered T cell 8. suboptimalCAR Costimulatory domains Activation domains

OxiTF (TAL Effectors as non limiting example) ONOO Hypoxia sensitive cytoplasm domain

ON nucleus in TALE TALE binding site 2. S CAR ORF

Fig. 17 Patent Application Publication Mar. 16, 2017. Sheet 18 of 38 US 2017/0073423 A1

Tumor cell Tumor Cell

No signal

O O Tumor cell ligand

Extracellular ligand-binding domain "inactive"

Extracellular ligand-binding domain "active'

Transmitter domain

Fig. 18 Patent Application Publication Mar. 16, 2017. Sheet 19 of 38 US 2017/0073423 A1

AND logic gate

Input 1 Input 2 input 3–G>o- Output

Input 1 Input 2 input 3 Outpu

O O 1. O O 1. O O 1. 1. 1. O O 1. 1. 1.

Fig. 19 Patent Application Publication Mar. 16, 2017. Sheet 20 of 38 US 2017/0073423 A1

Antigen l Antigen 2 healthy tissue) O O cancer cell)

scFW 1 arti S cFW 2 aftanti AntigenX 2 Antigen

Co-inhibitory Co-stimulatory dorrain Activaction domain

Engineered T cell

Fig. 20 Patent Application Publication Mar. 16, 2017. Sheet 21 of 38 US 2017/0073423 A1

Izººl Patent Application Publication Mar. 16, 2017. Sheet 22 of 38 US 2017/0073423 A1

A. nucleus High O, Low O. normoxia / \ hypoxia

degradation

Fig. 22 Patent Application Publication Mar. 16, 2017. Sheet 23 of 38 US 2017/0073423 A1

Antigen targeting domain

Hinge domain Transmembrane domain l Oxygen sensitive domain

Activation and Co la Stimulation domains Oxygen sensitive domain Activation domains Co-stimulation domains

Single-chain CAR Multi-chain CAR

Fig. 23 Patent Application Publication Mar. 16, 2017. Sheet 24 of 38 US 2017/0073423 A1

o o R.A, APCA

1o R1-A, APC-A

sample conditions. Histogram

a by Normoxia Dotted line a-HiFew Normoxia Dotted line-Filled a-HIFay Hyp Patent Application Publication Mar. 16, 2017. Sheet 25 of 38 US 2017/0073423 A1

e x E ix. i. s 8: s e

2x e

-10° a 10° o to' o Arc

N

s

s s

Sis s as

2 s . CAR Expression

sample Target cell killing

Control mcCAR Patent Application Publication Mar. 16, 2017. Sheet 26 of 38 US 2017/0073423 A1

?p 53 I

1. R1A: APCA

O R1A APA (The legends are inserted in the figures description) Patent Application Publication Mar. 16, 2017. Sheet 27 of 38 US 2017/0073423 A1

11

?p 3 I

?p 8 I

3. d APC-A

(The legends are inserted in the figures description) Patent Application Publication Mar. 16, 2017. Sheet 28 of 38 US 2017/0073423 A1

Gate Gate Target Target receptor receptor tumor cell ell 1. 2

Engineered Engineered T-cell T-cell

Fig. 27 Patent Application Publication Mar. 16, 2017. Sheet 29 of 38 US 2017/0073423 A1

Gate Gate receptor receptor 1. 2

Antigen specific targeting domain

extracellular spacer domain (Hinge) Transmembrane domain

Structural or functional linker domain

Interacting partner domain

Transmitter transcription factor

Fig. 28 Patent Application Publication Mar. 16, 2017. Sheet 30 of 38 US 2017/0073423 A1

position atgg atgg Antiger Antiger targeting targeting tCCC tCCC 2 Hinge Hinge gata gata 3. transmenarmer brane transmembrane

4. linker gagc gagc

5 tgga gaat

6

7 gaat

Fig. 29 Patent Application Publication Mar. 16, 2017. Sheet 31 of 38 US 2017/0073423 A1

Membrane protein Surface partner Name expression

GG83 H GG111 H GG121 H GG152 H GG153 H GG155 H GG155 H GG158 H

Fig. 30 Patent Application Publication Mar. 16, 2017. Sheet 32 of 38 US 2017/0073423 A1

Reporter Gal4 TetO

5 Gal4 NF-Kb GAL4 VP64 H TetR_NF-Kb H t TetR VP64 H

Fig. 31 Patent Application Publication Mar. 16, 2017. Sheet 33 of 38 US 2017/0073423 A1

y s:C SS. Av SS. SY SS. 4NS S S. y S. St S S.

s S Sis SSf Ss S5 Ss S5 700 pb 500 pb -

200 pb - 150 pb &

100 pb 75 pb

50 pb <-->PCR <-->PR <-->R ATF2/LATR2 Lck2F/LcK2R ZAP70-2F/ZAP70-2R

Fig. 32A Patent Application Publication Mar. 16, 2017. Sheet 34 of 38 US 2017/0073423 A1

O S in y S sy S sy So S S is S C Se Se Se stS SS SS SS SS SS SSf

8 700 pb 500 pb 400 pb 300 pb

200 pb 150 pb

100 pb

PCR PCR PCR LFA11 F1/LFA11 R2 LATF1/LATR1 LcK1F/LcK1R

Fig. 32B Patent Application Publication Mar. 16, 2017. Sheet 35 of 38 US 2017/0073423 A1

700 pb 500 pb - 400 pb 300 pb

200 pb 150 pb

100 pb

at is see e PCR PCR PCR CD28 Exon F1 TRAT1 Exon F2 TRAT1 Exon F2 CD28 Exon R1 TRAT1 Exon R2 TRAT1 Exon R2 uter

Fig. 32C Patent Application Publication Mar. 16, 2017 Sheet 36 of 38 US 2017/0073423 A1

KOZAP70

KOZAP70. Untreated Ko ZAP70 PMA Iono 5

4. A. O 1. 3. t : 2. 2 o s O O1 - It is re-er-ri et at re-ret to r 5K OK 5, 2OK 250 O 5OK 100K 150K 200K 250K FSC-A :: FSC-A FSC-A : FSC-A

Ko ZAP70 PHA KO ZAP70 CD3/CD28

5OK 100K 150K 200K 250K O 5OK OK 15OK 200K 25OK FSC-A :: FSCA FSC-A : FSC-A s Figure 33 Patent Application Publication Mar. 16, 2017. Sheet 37 of 38 US 2017/0073423 A1

NOKO LT Untreated LT PMA. ONO s i

1 o r. If i-perspirits to re-ror it retire for . . . 15OK 200K 250K 5K O. 5 OO 250 FSC-A :: FSC-A FSC-A :: FSC-A

LT PHA LT cD3tcD28

o sok 1 ook 150K, 200K 250K 1. 15OK 20ok 25OK FSC-A :: FSC-A FSC-A :: FSC-A

Figure 33 (cont.) Patent Application Publication Mar. 16, 2017. Sheet 38 of 38 US 2017/0073423 A1

H?HORIVO snapnu

SHÀHOHVO snapnu ) a 88$$$$$.88: S-INORIVO snapnu

Fig. 34 US 2017/0073423 A1 Mar. 16, 2017

METHOD OF ENGINEERING MULTI-INPUT purposes. Bispecific tandem CAR has already been SIGNAL SENSITIVET CELL FOR described (International application: WO2013123061, US. IMMUNOTHERAPY Patent application: US20130280220). However, in this design the bispecific chimeric antigen receptor comprises (a) FIELD OF THE DESCRIPTION at least two antigen-specific targeting regions, (b) an extra cellular spacer domain, (c) a transmembrane domain, (d) at 0001. The present invention relates to a method to engi least one co-stimulatory domain and (e) an intracellular neer T cell for immunotherapy. In particular said T cells are signaling domain, wherein each antigen-specific targeting engineered in order to be activated by the combination of region comprises an antigen-specific single chain FV (ScPV) input signals. The present invention relates to new designed fragment, and binds a different antigen. Such design may chimeric antigen receptors which are able to redirect theoretically still lead to the T-cell activation independently immune cell specificity and reactivity toward a selected to the recognition and binding of both antigens as one cannot target exploiting the ligand-binding domain properties. The exclude that the binding of one single chain Fv may trigger present invention also relates to cells obtained by the present activation. Kloss, Condomines et al. 2013 described another method, preferably comprising said chimeric antigen recep combinatorial antigen recognition approach. A CAR com tors for use in therapeutic or prophylactic treatment. prising a signaling domain mediated the recognition of one antigen and another receptor comprising a co-stimulatory BACKGROUND OF THE INVENTION domain specific for a second antigen are expressed at the 0002 Adoptive immunotherapy, which involves the Surface of a T cell. This dual targeting approach facilitates transfer of autologous antigen-specific T cells generated ex augmented T cell reactivity against tumor positive for two Vivo, is a promising strategy to treat viral infections and antigens. However this approach alone fails to prevent T cell cancer. The T cells used for adoptive immunotherapy can be reactivity to single-positive tumors. To remedy this failure, generated either by expansion of antigen-specific T cells or search of adapted configuration of CAR are required. redirection of T cells through genetic engineering (Park, 0004 To avoid the tuning of CAR used for the combi Rosenberg et al. 2011). Transfer of viral antigen specific T natorial antigen recognition, the inventors developed a sys cells is a well-established procedure used for the treatment tem wherein activation of T cell is only induced through the of transplant associated viral infections and rare viral-related combination of at least two signals. Each input signal alone malignancies. Similarly, isolation and transfer of tumor does not induce the activation of T cell. Environmental specific T cells has been shown to be successful in treating signal integration by a modular AND gate within a CAR melanoma. design may provide the ultimate strategy to insure safety and expand the number of Surface antigens available for thera 0003 Novel specificities in T cells have been success peutic purposes. fully generated through the genetic transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) (Jena, 0005 Logic gates are the basic building blocks in elec Dottietal. 2010). CARs are synthetic receptors consisting of tronic circuits that perform logical operations. These have a targeting moiety that is associated with one or more input and output signals in the form of 0s and 1's: '0' signaling domains in a single fusion molecule. In general, signifies the absence of signal while 1 signifies its pres the binding moiety of a CAR consists of an antigen-binding ence. Similar to the electronic logic gates, cellular signals domain of a single-chain antibody (ScPV), comprising the can serve as logic gates. light and heavy variable fragments of a monoclonal antibody 0006 Synthetic biology applies many of the principles of joined by a flexible linker. Binding moieties based on engineering to the field of biology in order to create bio receptor or ligand domains have also been used Successfully. logical devices which can ultimately be integrated into The signaling domains for first generation CARS are derived increasingly complex systems. These principles include from the cytoplasmic region of the CD37eta or the Fc standardization of parts, modularity, abstraction, reliability, receptor gamma chains. First generation CARS have been predictability, and uniformity (Andrianantoandro, Basu et al. shown to successfully redirect T cell cytotoxicity, however, 2006). The application of engineering principles to biology they failed to provide prolonged expansion and anti-tumor is complicated by the inability to predict the functions of activity in vivo. Signaling domains from co-stimulatory even simple devices and modules within the cellular envi molecules including CD28, OX-40 (CD134), ICOS and ronment. Some of the confounding factors are gene expres 4-1BB (CD137) have been added alone (second generation) sion noise, mutation, cell death, undefined and changing or in combination (third generation) to enhance Survival and extracellular environments, and interactions with the cellular increase proliferation of CAR modified T cells. CARs have context (Andrianantoandro, Basu et al. 2006). Thus, while Successfully allowed T cells to be redirected againstantigens synthetic biology offers much promise in developing sys expressed at the Surface of tumor cells from various malig tems to address challenges faced in the fields of manufac nancies including lymphomas and Solid tumors (Jena, Dotti turing, environment and Sustainability, and health and medi et al. 2010). However, for example, cancer cells are unstable cine, the realization of this potential is currently limited by and some cells may no longer possess the target antigen. the diversity of available parts and effective design frame These cells, referred to as antigen loss escape variants, works (Wang, Wei et al. 2013). escape destruction by the therapy and may continue to grow and spread unchecked. Cancer and healthy cells may express SUMMARY OF THE INVENTION the same antigen although at different levels. In Such case, 0007. The present invention is drawn to apply synthetic having the possibility to combine at least two antigens in biology principles such as logic “AND GATE' to immune order for the engineered T cell to discriminate between cell technology in order for the cells to be stimulated and/or healthy tissue and cancer cells would present extremely activated only by the combinations of at least two input valuable advantage over actual technology for therapeutic signals (FIG. 1). In particular, the present invention relates US 2017/0073423 A1 Mar. 16, 2017

to a method of engineering immune cell for immunotherapy activation derives from the co-localization of the two CARs by render them sensitive to the combination of at least two dues to the presence of both tumor ligand cells. The co input signals. Said input signals can be external stimuli Such localization of the two CARs allows their activation medi as hypoxia or the recognition of a ligand, preferably via the ated by the cleavage of the target sequence protease and the expression at the surface of the cell of a specific chimeric following release of the signaling protein. antigen receptor capable of recognizing said ligand. Accord (0012 FIG. 5: AND GATE: Split protein system. The ing to the present invention, the recognition of the input simultaneous presence of two tumor cell ligands will acti signals allow the combination of at least two transmitter vate the two CARs. The intracellular domain of the first domains which activate immune cell response, preferably CAR comprehends a fragment of the 'signaling domain via signaling protein. Each transmitter protein is indepen and C or N domain of the intein. The cytoplasmatic domain dently inactive and thus does not activate immune cell of the second CAR is harboring the complementary intein response. Only the combination of these two transmitter domain plus the complementary signaling domain fragment. domains allows T cell activation. The transmitter domains Each CAR independently is not activated by the presence of can be as non limiting examples, protease and an anchored the single tumor ligand cell, the activation derives from the membrane Substrate domain comprising a protease cleavage co-localization of the two CARs dues to the presence of both site linked to a signaling protein, split proteins, scaffolding tumor ligand cells. The co-localization of the two CARs proteins, domains capable of dimerizing, autoinhibited pro allows their activation through the reconstitution of the full tein with compound able to retrieve the inhibition, comple active split intein driving the reconstitution of the complete mentation of a prior inactivated gene. The present invention active form of the signaling protein which could initiate also relates to new design of chimeric antigen receptors, different pathways of activation of the T cell. Examples of cells comprising said chimeric antigen receptors or obtained signaling proteins are ZAP70, SH2 domains, and kinase by the method of the invention, and therapeutic treatment domain. using said engineered immune cell. (0013 FIG. 6: AND GATE: Split protein system and release of the signaling protein. The simultaneous presence BRIEF DESCRIPTION OF THE FIGURES AND of two tumor cell ligands will activate the two CARs. The TABLES intracellular domain of the first CAR comprehends the C 0008 FIG. 1: Logic “AND GATE synthetic biology terminal inactive fragment of the "signaling protein’ and C principle. Input (1.2) can be antigens expressed by tumor or N domain of the intein. The intracellular domain of the cells (and/or healthy cells) and/or tumor microenvironments. second CAR is harboring a dimerization domain which Output corresponds to the resulting activation of the immune could homodimerize with an additional multi-domain. This cell. multi-domain is constituted by the second intein domain and 0009 FIG. 2: AND GATE: Tumor antigen-driven the N domain of the signaling protein fragment. Each CAR dimerization and activation of receptor tyrosine kinase independently is not activated by the presence of the single (RTK)-based chimeric antigen receptors. The simultaneous tumor ligand cell, the activation derives from the co-local presence of two tumor cell ligands co localized at the tumor ization of the two CARs dues to the presence of both tumor cell surface will drive the dimerization of two heterodimeric ligand cells. The co-localization of the two CARs allows receptor tyrosine kinase-based chimeric antigen receptors, their activation through the reconstitution of the full active and lead to their activation via transphosphorylation. On split intein driving the reconstitution of the complete active both CARS, the transmitter domains are maintained in an form of the signaling protein, which could be released into inactive state by autoinhibition (e.g.: the kinase active site is the cytoplasm to initiate the activation of the T cell. masked by autoinhibitory domain). The presence of two (0014 FIG. 7: AND GATE: Kinase based split protein tumor cell ligands colocalized at the tumor cell Surface system. The simultaneous presence of two tumor cell ligands enable driving dimerization of the two CARs resulting in the will activate the two CARs. The intracellular domain of the relief of kinase autoinhibition and enabling activation of first CAR comprehends a signaling protein binding region transmitter domain via transphosphorylation or its interac and C or N domain of a split kinase. The intracellular tion with other molecules as non limiting examples. domain of the second CAR is harboring the complementary 0010 FIG. 3: AND GATE: prior inactivated gene kinase domain. Each CAR independently is not activated by complementation: in a simplified example two different the presence of the single tumor ligand cell, the activation tumor cell ligands can be recognized by two different CARs derives from the co-localization of the two CARs dues to the whose cytoplasmatic domains comprehend two different presence of both tumor ligand cells. The co-localization of domains. Simultaneously the knock out of a key gene (GOI) the two CARs allows their activation through the reconsti in the signal pathway of the T cell has been performed. Upon tution of the full active kinase which could be phosphory the co-localization of the two CARs followed the recogni lated hence start the activation of the T cell. Example of split tion of the tumor ligand cells the first CAR can activate a kinase could be LCK. factor which will enable the reactivation of the GOI neces (0015 FIG. 8: AND GATE: Kinase based split protein sary to the transmission of the signal mediated by the second system activation in trans. The simultaneous presence of two CAR. tumor cell ligands will activate the two CARs. The intrac 0011 FIG. 4: AND GATE: Protease system. The simul ellular domain of the first CAR comprehends a signaling taneous presence of two tumor cell ligands will activate the binding region and C or N domain of a split kinase. The two CARs. The intracellular domain of the first CAR intracellular domain of the second CAR is harboring the comprehends a protease target sequence linked to a signal complementary kinase domain. Each CAR independently is ing protein; the intracellular domain of the second CAR is not activated by the presence of the single tumor ligand cell; harboring a protease. Each CAR independently is not acti the activation derives from the co-localization of the two vated by the presence of the single tumor ligand cell, the CARs dues to the presence of both tumor ligand cells. US 2017/0073423 A1 Mar. 16, 2017

0016. The co-localization of the two CARs allows their (0021 FIG. 13: AND GATE: Autoinhibition system: activation through the reconstitution of the full active kinase induced activation upon competitive binding. The simulta which could cause a conformational modification on the neous presence of two tumor cell ligands will activate the signaling protein binding region allowing the binding of the transmitter. On the first CAR, the transmitter domain is signaling protein which could be activated by a phosphory maintained in an inactive state by autoinhibition (e.g.: by lation in trans. interaction with a "shielding protein or antibody). The 0017 FIG. 9: AND GATE: Protease based split system colocalization of the second CAR upon binding to its ligand and re-localization of the signaling protein. The simultane will displace the shielding molecule to itself on a domain of ous presence of two tumor cell ligands will activate the two higher affinity (intermolecular displacement). The CARs. The intracellular domain of the first CAR compre “unshielded transmitter can then be activated (e.g.: by hends the C or N domain of a split protease, a protease target post-translational modifications or interaction with other sequence and the signaling protein. The intracellular domain molecules). of the second CAR is harboring the complementary split (0022 FIG. 14: AND GATE: Autoinhibition system: protease domain. Each CAR independently is not activated induced activation upon enzymatic cleavage of the inhibi by the presence of the single tumor ligand cell; the activation tion domain. The simultaneous presence of two tumor cell derives from the co-localization of the two CARs dues to the ligands will activate the transmitter. On the first CAR, the presence of both tumor ligand cells. The co-localization of transmitter domain is maintained in an inactive state by the two CARs allows their activation through the reconsti autoinhibition (e.g.: by interaction with a "shielding protein tution of the full active protease which could cleave the or antibody). The colocalization of the second CAR upon protease target sequence and cause the release of the sig binding to its ligand will bring a protease domain in close naling protein. proximity of a protease target sequence present on the first 0018 FIG. 10: AND GATE: Protease based split system car and thus allow to displace the shielding molecule. The using three CARs. The simultaneous presence of three tumor “unshielded transmitter can then be activated (e.g.: by cell ligands will activate the CARs. The intracellular domain post-translational modifications or interaction with other of the first CAR comprehends the protease target sequence molecules). and the signaling protein. The intracellular domains of the (0023 FIG. 15: AND GATE: Receptor binding and exter second and third CARs are constituted by the two comple nal stimuli to induce activation of the transmitter protein. mentary split protease domains. Each CAR independently is The simultaneous binding of a CAR to its tumor cell ligand not activated by the presence of the single tumor ligand cell; and the exposition of the engineered T cell to a tumor cell the activation derives from the co-localization of three extracellular stimulus will activate the transmitter. External CARs dues to the presence of the three tumor ligand cells. stimuli encompass variation in concentration of metabolites, The co-localization of three CARs allows their activation Small molecules, peptide, Small proteins (chemokines, through the reconstitution of the full active protease which cytokines) and physico/chemical conditions (pH, hypoxia, could cleave the protease target sequence and cause the redox potential). release of the signaling protein. (0024 FIG. 16: AND GATE: Hypoxia dependent activa 0019 FIG. 11: AND GATE: Scaffolding protease based tion system in the presence of one tumor antigen. system. The simultaneous presence of two tumor cell ligands 0025. The simultaneous presence of engineered T cells will activate the two CARs. The intracellular domain of the with one tumor cell ligand in an oxygen depleted environ first CAR comprehends a first protein domain. The intrac ment, triggers T cells activation. To enable Such logical AND ellular domain of the second CAR is harboring a second gate activation system, T cell are engineered to harbor an protein domain. Each CAR independently is not activated by oxygen-inducible synthetic activation pathway. Such syn the presence of the single tumor ligand cell, the activation thetic pathway is made of three different elements including derives from the co-localization of the two CARs dues to the an engineered transcription factor sensitive to oxygen con presence of both tumor ligand cells. The co-localization of centration (OxiTF), a synthetic promoter specific for the the two CARs allows their activation through the binding of OxiTF driving the expression of the third element, a chi the protein domain 1 and 2 to the inactive scaffolding meric antigen receptor (CAR I). The OxiTF is design to protein. Upon the complex binding the active form of the activate a synthetic genetic element encoding a CAR spe scaffolding protein is reconstituted and the T cell can be cific for tumor antigen within engineered T cells. Upon solid activated. Examples of Scaffolding proteins are Carmal, tumor encounter, engineered T cells detect oxygen depletion SP76, hemITAM, DLG1, KSR. and trigger CAR I production. Cell surface exposure of 0020 FIG. 12: AND GATE: Based on double activation CARI enables the recognition of tumor antigen that even of engineered heterodimeric domains. The simultaneous tually triggers T cells activation and proliferation via the presence of two tumor cell ligands will activate the two activation and co-stimulatory domains present within CARI. CARs. The intracellular domain of the first CAR compre (0026 FIG. 17: AND GATE: Hypoxia dependent activa hends a first transmitter binding domain. The intracellular tion system in the presence of two tumor antigen. domain of the second CAR is harboring a second transmitter 0027. The simultaneous presence of engineered T cells binding domain. Each Car independently is not activated by with two tumor cell ligands in an oxygen depleted environ the presence of the single tumor ligand cell, the activation ment, triggers T cells activation. To enable Such logical AND derives from the co-localization of the two CARs dues to the gate activation system, T cells are engineered to harbor an presence of both tumor ligand cells. The co-localization of oxygen-inducible synthetic activation pathway. Such syn the two CARs allows the activation of the two transmitter thetic pathway is made of four different elements including binding domains (e.g. phosphorylation and post-translation an engineered transcription factor sensitive to oxygen con modifications) which can trigger the recruitment of a trans centration (OxiTF), a synthetic promoter specific for the mitter which can activate the T cell. OxiTF driving the expression of the third element, a chi US 2017/0073423 A1 Mar. 16, 2017 meric antigen receptor (CAR II) specific for tumor antigen 0033 FIG. 23. Different chimeric antigen receptor II. The system is completed with a fourth element, consisting (CAR) architectures carrying oxygen-sensitive domain Such in a constitutively expressed CARI, specific for tumor as HIFC. At left, a single-chain CAR (scCAR) carries on the antigen I. The OxiTF is design to activate a synthetic genetic same and unique chain the extracellular binding domain element encoding a CARII within engineered T cells. Upon (here schv), the oxygen domain (ex: HIF1) and the activa Solid tumor encounter, engineered T cells detect oxygen tion and co-activation domains. At right is shown as an depletion and trigger CARI production. Cell Surface expo exemplary conformation a multi-chain CAR (mcCAR) sure of CARII along with CARI enables the recognition of wherein the C-chain carries the Sclv and the oxygen tumor antigen II in addition to the pre-existing CARI-tumor domain; the B-chain carries the co-stimulatory domain and antigen I complex. Simultaneous presence of both CAR/ the Y-chain carries the activation domain(s). Tumor antigen complexes eventually triggers T cells acti 0034 FIG. 24. A) Surface presentation of chain-HIF1 Vation and proliferation via the activation and co-stimulatory (a.a. 380-630) versus C.-chain WT C-in normoxia or domains present within CARI and II. hypoxia. The surface exposition of CAR T-cells having 0028 FIG. 18: AND GATE: Illustration of the AND HIF1 in hypoxia is similar to the one of control CART-cells GATE principle applied to the T cell system activation. The (without C.-HIF1), whereas the surface exposition is much extracellular of a CAR contains two ligand-binding domains reduced in normoxia condition, showing a good expression that that exist under two conformations (“active' and “inac of CAR-OHIF1. tive”). In absence of the two tumor cell ligands, the equi 0035 (B) Surface presentation of the C-chain-HIF1 (a.a. librium is strongly displaced towards the “inactive form'. 380-630) versus C-chain WT after return from hypoxia to Only the simultaneous binding of the two ligand-binding normoxia. The expression of CAR CHIF1 drops from domains to their respective tumor cell ligands (two inputs) hypoxia to normoxia condition. This is a reversible and will trigger a positive signal to the intracellular domain of dynamic system: in normoxia condition, the CAR expres the CAR (output). sion is inhibited by the degradation of C-HIF1 and C-chain 0029 FIG. 19: AND NOT GATE: General schema. Illus polypeptides in a temporally manner, and in hypoxia con tration of the AND NOT GATE principle applied to the T dition (i.e. tumor environment), the C-HIF1 and C-chain cell system activation. The simultaneous presence of two polypeptides are expressed. tumor cell ligands and the absence of an healthy cell ligands 0036 FIG. 25. (A) Surface detection of C-HIF mcCAR will trigger a positive output. The input 1 and 2 corresponds versus control CAR in normoxia or hypoxia. In this experi to the presence of a tumor cell ligand while the third input ment, less total RNA is used, the results obtained are similar should not be a healthy cell ligand. The first and the second to those of FIG. 23; CAR have a co-stimulatory cytoplasmatic domains while the 0037 (B) Induced cytotoxicity in normoxia. The control third CAR is harboring an inhibitory domain whose inhibitor multi-chain CAR (without C.-HIF1) shows a high target cell effect will be blocked in case of non recognition of the killing, whereas the latter is null for the HIF-mcCAR in healthy cell ligand. normoxia. In view of these results of cytotoxicity, as well as those of surface exposition, this indicates that the HIF 0030 FIG. 20. Generation of two types of LCKs to system is fully functional within a chimericantigen receptor. inhibit and to stimulate the T cell signaling cascade The first 0038 FIG. 26. Surface presentation of various C-chain CAR will recognize an antigen of an healthy cell with an HIF versus WTOL-chain in normoxia or hypoxia. (A) HIF1 inhibitory domain which will stimulate the transcription of mcCAR (a.a. 380-630) construct with the -EA-linker; (B) a form constitutively negatively regulated of LCK. This HIF1-mcCAR (a.a. 344-417); (C) HIF3-mcCAR (a.a. 480 first CAR will be coupled with a second one which contains 571); (D-E-F): same as for (A-B-C) but return from hypoxia a co-stimulatory domains which will activate the transcrip to normoxia. tion of LCK" form, producing an high level of activation of 0039 All the results obtained here by lentiviral delivery the T cell. demonstrate that both HIF1 and HIF3 systems are functional 0031 FIG. 21. Control of T cell activation through CAR and behave similarly. Also, it is shown that different parts of mediated regulation of CARMA 1 protein. The stimulation the HIF protein can be used with or without linker. of TCR after recognizing the antigen is linked to the 0040 Legends: recruitment of CD28 which leads to the activation of PKC0, which in turn phosphorylates and activates CARMA1. CARMA1 constitutes a crucial signalosome for the trans Sample Conditions Histogram mission of the T cell Receptor (TCR) signaling and in Isotype control Hypoxia solide line-Filled general for T cell activation. CARMA1 recruits different C. By Normoxia Dotted line proteins forming a multi-protein complexes that finally can C. By Hypoxia Dashed line activate two different signaling cascades: NF-KB and c-jun C-HIF By Normoxia Dotted line-Filled N-terminal kinase (JNK). C-HIF By Hypoxia Dashed line-Filled 0032 FIG. 22. Functioning of the HIF hypoxia System. In normoxia (high O), HIFC. is hydroxylated by HIFC.- 0041 FIG. 27 (A) Schematic representation of the dual specific prolyl hydroxylases (PHD1-3) which are oxygen receptors logic AND gate. (B) Interaction of both membrane sensing. Hydroxylation triggers poly-ubiquitylation of HIFC. protein partners with their target ligand will trigger the and targets the latter for proteosomal degradation by an E3 colocalization of the intracellular interacting domains. (C) ubiquitin ligase. In hypoxia (low O), occur an inhibition of Release of the transmitter domain is triggering the output hydroxylation via TCA cycle intermediates, a stabilization signal. of the HIFC. protein and an impairment of HIF transcrip 0042 FIG. 28 Schematic representation of the composi tional activity. tion of both gate receptors. US 2017/0073423 A1 Mar. 16, 2017

0043 FIG. 29 Schematic representation of the molecular the HIF-1C. Subunit, upregulation of several genes to pro assembly strategy of the components for both gates, wherein mote Survival in low-oxygen conditions, and ultimately the spacers are indicated. binding of HIF-1 to HIF-responsive elements (HREs). 0044 FIG. 30 Surface expression of 7 membrane protein 0055. By “activation of immune cell, it is meant that the partners. GG83, GG111, GG121, GG152, GG153, GG155, combination of two inputs signals triggers (directly or GG156 and GG 158 were tested; intensity of the signal (++: indirectly) the combination of two transmitters domains, very good, +: good). which in its turn generates a signal (positive or negative) to 004.5 FIG. 31 Expression of the lentiviral delivered the immune cell carrying the CAR, preferably by transduc RQR8 cassette by mRNA transfection of different transac tion means. tivators. These constructions are composed of a DNA bind 0056 Consequently, a signal is emitted for the CAR ing domain (TetO or GalA) and a transcription activation expression and finally the lysis of the tumoral cells may domain (VP64 or NF-kB), are transfected and are tested. The happen. data obtained clearly indicated the expression of the lenti viral delivered RQR8 cassette by mRNA transfection of the Methods of Engineering Immune Cells adequate transactivator. 0046 FIG. 32 T7 endonuclease assay demonstrating tar 0057 The inventors developed methods of engineering geted mutagenesis at the endogenous loci using the designed Such immune cells based on the rational combination of TALEN: all the 3 panels A, B, C depict the knock-out (KO) regulatory modules in artificial circuits for performing tasks, of enzymes involved in T-cell signaling and/or functioning, including complex binary computation operations based on such as LAT, LCK, ZAP70, LFA, TRAT or CD28. The data logic gates. The term "gate' is used to refer to a device or obtained clearly indicate a high level of targeted mutagen molecular mechanism that produces a particular (predeter esis at all targeted loci using the designed TALEN. mined) output in response to two or more input signals. 0047 FIG. 33 Degranulation experiments following the According to the present invention, the logical AND gate Knock-outs of ZAP70. The data obtained clearly indicate a refers to the immune cell activation, in particular T cell strong staining decrease for the knock-out engineered T-cell cytotoxicity against a target cell through the combination of relative to WT T-cells different transmitter domains and activation of specific pro 0048 FIG. 34 Schematic representation of bi-specific teins (signaling proteins) resulting from the combination of CAR (biCAR) functioning. The bi-specific CAR (biCAR) is at least two input signals. As non limiting examples, each composed of two CARs (biCAR1 and biCAR2) whose schv signal can act together or separately to activate protein have specific affinity to two different target cell antigens. function or to remove an inhibiting protein. In another When only one of these scFc binds to its specific antigen, particular embodiment, said input signal can be the output there is no activation of the CAR and therefore no killing of signal resulting from prior input signals. In particular, the the cell. When both scFv bind to their specific antigens, an present invention relates to a method of engineering an activation of the CAR occurs and the target cell killed. immune cell for immunotherapy, in particular, method of 0049 Table 1: Proteins that interact with the CARMA1 engineering an immune cell for targeting specifically a cell SignaloSome comprising: 0050 Table 2: CARMA1 Phosphorylation Sites 0.058 (a) Providing an immune cell; 0059 (b) Engineering said immune cell to render said DETAILED DESCRIPTION OF THE cell sensitive to at least two input signals such that the INVENTION combination of input signals induces combination of at least two transmitter domains which results in activa 0051. The ability to control functional responses in adop tion of said immune cell, wherein each transmitter tive T cell therapy is a key issue. In Such therapeutic domain alone does not activate said immune cell. strategies, T cells are engineered by expressing Surface 0060. This method is different from the combinatorial exposed chimeric antigen receptor (CAR) that achieves high antigen recognition system described in (Kloss, Condomines tumor specific target cell recognition. However, to control et al. 2013) wherein said transmitter domains are signaling and minimize potential toxic off-target effects, design of domain and co-stimulatory domains of a chimeric antigen multi-input systems is highly desirable. receptor as described below, said signaling domain alone can 0052 Depending of the type of “input signal”, “the activate T cell activation. combination of at least two transmitter domains' can be performed by direct or indirect means. Input Signal: Recognition of a Ligand by Immune Cell 0053 For instance in case of the split-ubiquitin system, the combination of two input signals, which are the recog 0061. In a particular embodiment, said input signal can nition of two different cell target ligands by the scFv from be the recognition of a ligand by said engineered immune the 2 CARs, makes possible the co-localization of the two cell, in particular by chimeric antigen receptor expressed at transmitter domains, i.e. the C- and N-terminal parts of the the Surface of said engineered immune cell. ubiquitin enzyme, therefore the activity of the latter can 0062. The chimeric antigen receptor (CAR) according to occur and a signal is produced. the present invention comprises an extracellular ligand 0054 The hypoxia HIFalpha system functions in a more binding domain and an intracellular domain, more particu indirect manner especially in regard to “the combination of larly, an extracellular ligand binding domain, a transmem transmitters’. The combination of two input signals occurs brane domain and an intracellular domain. between one hypoxia external signal and the other from the 0063. The term “extracellular ligand-binding domain” as cell target ligand recognition by scFv of the CAR. At this used herein is defined as an oligo- or polypeptide that is stage, occurs a cascade of reactions such as inhibition of capable of binding a ligand. Preferably, the domain will be phosphorylation of HIF prolyl-hydroxylase, stabilization of capable of interacting with a cell surface molecule. For US 2017/0073423 A1 Mar. 16, 2017 example, the extracellular ligand-binding domain may be prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, chosen to recognize a ligand that acts as a cell Surface LAGA-1a, p53, prostein, PSMA, surviving and telomerase, marker on target cells associated with a particular disease prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, state. Thus examples of cell Surface markers that may act as ELF2M, neutrophil elastase, ephrin B2, CD22, insulin ligands include those associated with viral, bacterial and growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelin, a parasitic infections, autoimmune disease and cancer cells. In major histocompatibility complex (MHC) molecule present particular, the extracellular ligand-binding domain can com ing a tumor-specific peptide epitope, 5T4, ROR1, Nkp30, prise an antigen binding domain derived from an antibody NKG2D, tumor stromal antigens, the extra domain A (EDA) against an antigen of the target. As non limiting examples, and extra domain B (EDB) of fibronectin and the A1 domain the antigen of the target can be a tumor-associated Surface of tenascin-C (TnC A1) and fibroblast associated protein antigen as described above. (fap). LRP6, melanoma-associated Chondroitin Sulfate Pro 0064. The extracellular ligand-binding domain can also teoglycan (MCSP), CD38/CS1, MART1, WT1, MUC1, comprise a peptide binding an antigen of the target, a peptide LMP2, Idiotype, NY-ESO-1, Ras mutant, gp100, proteinase or a protein binding an antibody that binds an antigen of the 3, bcr-abl, tyrosinase, hTERT, EphA2, ML-TAP ERG, target, a peptide or a protein ligand Such as a growth factor, NA17, PAX3, ALK, Androgen receptor; a lineage-specific a cytokine or a hormone as non limiting examples binding or tissue specific antigen such as CD3, CD4, CD8, CD24, a receptor on the target, or a domain derived from a receptor CD25, CD33, CD34, CD79, CD116, CD117, CD135, Such as a growth factor receptor, a cytokine receptor or a CD123, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 hormone receptor as non limiting examples, binding a (CD86), endoglin, a major histocompatibility complex peptide or a protein ligand on the target. Preferably the target (MHC) molecule, BCMA (CD269, TNFRSF17), or a virus is a cell or a virus. specific Surface antigen such as an HIV-specific antigen 0065. In a preferred embodiment, said extracellular (such as HIV gp120); an EBV-specific antigen, a CMV ligand-binding domain is a single chain antibody fragment specific antigen, a HPV-specific antigen, a Lasse Virus (scFV) comprising the light (V) and the heavy (V) variable specific antigen, an Influenza Virus-specific antigen as well fragment of a target antigen specific monoclonal antibody as any derivate or variant of these surface markers. In joined by a flexible linker. Other binding domain than scFv specific cases, the ligand that the chimeric antigen receptor can also be used for predefined targeting of lymphocytes, recognizes is present on the Surface of a target cell, particu Such as camelid single-domain antibody fragments, receptor larly cancer cell or viral cell. In some embodiments, the ligands like a vascular endothelial growth factor polypep ligand that the chimeric antigen receptor recognizes is tide, an integrin-binding peptide, heregulin or an IL-13 present in a tumor microenvironment. In some aspects of the mutein, antibody binding domains, antibody hyperVariable invention, the ligand that the chimeric antigen receptor loops or CDRS as non limiting examples. recognizes is a growth factor. 0066. In another preferred embodiment, said extracellu 0068. In a preferred embodiment, said input signals can lar binding domain can be a DARPin (designed ankyrin be the recognition of at least two different ligands by repeat protein). DARPins are genetically engineered anti chimeric antigen receptors expressed at the Surface of engi body mimetic proteins typically exhibiting highly specific neered immune cells. Thus, the immune cell of the present and high-affinity target protein binding. They are derived method can be engineered by expressing at the Surface of from natural ankyrin proteins and comprise at least three, said immune cell at least two Chimeric Antigen Receptors usually four or five repeat motifs of these proteins. DARPins (CAR) each comprising an extracellular domain capable of are Small, single domain proteins which can be selected to recognizing different ligands and an intracellular domain bind any given target protein with high affinity and speci comprising transmitter domain. The combination of at least ficity (Epa, Dolezal et al. 2013; Friedrich, Hanauer et al. the two input signals corresponding to the recognition of 2013; Jost, Schilling et al. 2013). According to the present different ligands by each extracellular domains of said CARs invention, DARPins can be engineered to comprise multiple allows the combination of at least two transmitter domains antigen recognition sites. Thus, said DARPins can be used and thus activation of said immune cell. to recognize a series of consecutive different antigens as well 0069. In a particular embodiment, the present method as a unique antigen. Thus, the present invention relates to a comprises the expression of at least two CARS comprising method comprising providing an immune cell, and express an extracellular ligand binding domain capable of recogniz ing at the Surface of said immune cell chimeric antigen ing the combination of several ligands such as non limiting receptor which comprises a designed ankyrin repeat protein examples the combination of HER2, MUC1, CD44, CD49f capable of recognizing at least one specific ligand, prefer and/or epCAM to target breast cancer, the combination of ably at two specific ligands. mesothelin, folate receptor-alpha, CD44 and/or CD133 to 0067. As non limiting example, the ligand of the target target ovarian cancer cells, the combination of HER2 and can be a tumor-associated Surface antigen, such as ErbB2 IL13R-alpha2 for the treatment of glioblastoma, CD19 and (HER2/neu), carcinoembryonic antigen (CEA), epithelial CD20, Cd19 and CD22, CD20 and LI-CAM, LI-CAM and cell adhesion molecule (EpCAM), epidermal growth factor GD2, EGFR and LICAM, EGFR and C-MAT, EGFR and receptor (EGFR), EGFR variant III (EGFRVIII), CD19, HER2, C-MET and HER2, EGFR and ROR1. In specific CD20, CD30, CD40, disialoganglioside GD2, GD3, C-type cases, at least one of the ligand that the chimeric antigen lectin-like molecule-1 (CLL-1), ductal-epithelial mucine, receptor recognizes is present on the Surface of a target cell, gp36, TAG-72, glycosphingolipids, glioma-associated anti particularly cancer cell. In some embodiments, at least one gen, B-human chorionic gonadotropin, alphafetoprotein of the ligand that the chimeric antigen receptor recognizes is (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN present in a tumor microenvironment. In some aspects of the CAIX, human telomerase reverse transcriptase, RU 1, RU2 invention, at least one of the ligand that the chimeric antigen (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, receptor recognizes is a growth factor. In some embodi US 2017/0073423 A1 Mar. 16, 2017 ments, the first ligand is specific for an antigen present on a preferred embodiment, said CAR can comprise the extra cancer cell Surface and the second ligand is present in a cellular domain, transmembrane, and/or the intracellular tumor microenvironment. domain of a receptor tyrosine kinase, preferably selected 0070 The CAR according to the present invention is from the group consisting of Trk A. c-Kit, FGFR and EGFR/ expressed on the surface membrane of the cell. Thus, the Erb. Said tyrosine kinase transmembrane domain and/or CAR comprises a transmembrane domain. The distinguish intracellular domain can be linked to an extracellular ligand ing features of appropriate transmembrane domains com binding domain and intracellular domain according to the prise the ability to be expressed at the surface of a cell, present invention (FIG. 2). In particular embodiment, said preferably in the present invention an immune cell, in engineered cells comprise different CARS comprising dif particular lymphocyte cells or Natural killer (NK) cells, and ferent transmembrane domains. to interact together for directing cellular response of immune 0073 Said transmembrane domain can also be an integ cell against a predefined target cell. The transmembrane rin. Integrins are heterodimeric integral membrane proteins domain can be derived either from a natural or from a composed of a C. and B chains which combined together synthetic source. form the LFA-1 (integrin lymphocyte function-associated 0071. The transmembrane domain can be derived from antigen-1) which is expressed on all leukocytes. LFA-1 any membrane-bound or transmembrane protein. As non plays a central role in leukocyte intercellular adhesion limiting examples, the transmembrane polypeptide can be a through interactions with its ligand, ICAMs 1-3 (intercellu subunit of the T cell receptor such as C. B. Y or ö, polypeptide lar adhesion molecules 1 through 3), and also it has an constituting CD3 complex, IL2 receptor p55 (O. chain), p75 important role in lymphocyte co-stimulatory signaling (B chain) or Y chain, Subunit chain of Fc receptors, in (Chen and Flies 2013). The molecular details of the binding particular Fcy receptor III or CD proteins. Alternatively the of LAF-1 to its immunoglobulin ICAM-1 are quite known transmembrane domain can be synthetic and can comprise allowing a careful engineering of LAF-1 binding site. The predominantly hydrophobic residues such as leucine and affinity of C, domain for ICAM-1 is regulated by the valine. In a preferred embodiment said transmembrane displacement of its C-terminal helix which is conformational domain is derived from the human CD8 alpha chain (e.g. linked to alterations of specific loops in LAF-1. The active NP 001139345.1). Said transmembrane domain can also be and low conformations differ of 500 and 10,000 folds. It is a CD8 transmembrane domain (alpha and beta chains). Said also interesting to note that two types of antagonists are Transmembrane domain can be engineered to create obli known for LFA-1 and their mechanism of action is known. gated hetero or homodimers. In particular embodiment said Integrin cell surface adhesion receptors can transmit a signal CARS can comprise transmembrane domains or intracellular from the outside to inside but also viceversa. There are domains which can only dimerize after ligand recognition. cytoskeletal proteins as Talin which binds to the integrin tail Another example of transmembrane domain can be LFA-1 to transfer a message from inside to outside. NKG2-D receptor. NKG2D (natural killer cell group 2D) is 0074 Integrins are part of the immunological synapse a C-type lectin-like receptor expressed on NK cells, Yö-TcR" and their spatial/location in the synapse seems to be strate T cells, and CD8"CfB-TcPR' T cells (Bauer, Groh et al. 1999). gically to the creation of an effective response to the T cell NKG2D is associated with the transmembrane adapter pro stimulation caused by the recognition of exposed antigens tein DAP10 (Wu, Song et al. 1999), whose cytoplasmic on the antigen presenting cells (Singleton, Roybal et al. domain binds to the p 85 subunit of the PI-3 kinase. In a 2009). preferred embodiment, two complementary architectures of 0075 Indeed here we expose the idea to use the integrin CAR recognizing two different ligands can be expressed at scaffold to modulate the response of T cell exposing CAR. the Surface of the immune cell, a first CAR comprising an The integrin can be used to boost the activity of CAR ITAM motif as described below and a second CAR com engineered T cell enhancing its natural role of adhesion prising NKG2-D triggering an alternative signaling path between the T cell and the tumoral cell allowing for a higher way. concentration of perforin and granzyme at the immunologi 0072 Another example of transmembrane domain can be cal synapse. More we can imagine to use the integrin to a receptor tyrosine kinase. Receptor tyrosine kinase are cell create a new generation of CAR whose scaffold could be a surface receptors involved in different critical cellular regu fusion between the integrin scaffold (i.e. the alpha and the latory process including cell proliferation, cell differentia beta chains but also other chains) and ScFV domains (or any tion, cell Survival, cell migration, as well as cell cycle other type of antigen receptors). The possibility to modulate control. Receptor tyrosine kinase comprises an extracellular the 3D conformation of the integrins respects with the domain, a single transmembrane helix and an intracellular presence of Small molecules in the cytoplasm creates domain comprising tyrosine kinase function that is most of remarkably opportunities. Indeed the integrin is naturally time autoregulated by additional carboxy-terminal and jux present in two forms: one low affinity form which hinders tamembrane domains. Activation of receptor tyrosine kinase the active domains (the one responsible for the binding of is generally elicited by ligand-mediated dimerization. the natural ligand i.e. ICAM) at the membrane surface and Thanks to their bivalence, growth hormone ligand has the one active form with very high affinity for the natural ligand capacity to simultaneously interact with two receptor mono which is exposing the active domains in the extracellular mers and promotes dimerization. Such dimerization induces milieu. the activation of intracellular kinase domains through con 0076. The transmembrane domain can further comprise a formational changes followed by trans-phosphorylation of stalk region between said extracellular ligand-binding different tyrosines located within their intracellular domain. domain and said transmembrane domain. The term 'stalk The different phosphotyrosines generated eventually serve region' (also named hinge region) used herein generally as docking site for the recruitment of downstream signaling means any oligo- or polypeptide that functions to link the partners that activate the cellular regulatory pathways. In a transmembrane domain to the extracellular ligand-binding US 2017/0073423 A1 Mar. 16, 2017 domain. In particular, stalk region are used to provide more alpha, and have an identity of over 80%, preferably 90% or flexibility and accessibility for the extracellular ligand more preferably 95% with respectively SEQ ID NO:22, 23, binding domain. A stalk region may comprise up to 300 85, and SEQ ID NO:26, 27. amino acids, preferably 10 to 100 amino acids and most I0088 According to another embodiment, said intracellu preferably 25 to 50 amino acids. Stalk region may be derived lar domain contains a linker chosen amongst CD37eta, from all or part of naturally occurring molecules, such as FceRIg, CD28, 4-1 BB, OX40, DAP10, CD28, CD275, from all or part of the extracellular region of CD8, CD4. HVEM, LIGHT, CD40L, GITR, TIM1, SLAM, CD2, TLT CD28 or RTK, or from all or part of an antibody constant 2, LAG3, DAP12, CD84, CD244, CD229, LTBR and region. Alternatively the stalk region may be a synthetic CD278, and have an identity of over 80%, preferably 90% sequence that corresponds to a naturally occurring stalk or more preferably 95% with respectively SEQID NO.47 to sequence, or may be an entirely synthetic stalk sequence. 70. 0077. The intracellular domain of the CAR according to I0089. According to another embodiment, said activation the present invention comprises a transmitter domain. domain is CD37eta, and said activation domain is chosen Indeed, according to the present invention, the input signals between 4-1BB or CD28. induce the combination of the transmitter domains leading to immune cell activation. In a particular embodiment, said Complementation of a Prior Inactivated Gene transmitter domain is a signaling protein, and the combina tion of signaling protein function induces immune cell 0090 The transmitter proteins can also complement a activation. In another particular embodiment, said transmit prior inactivated gene or activate a gene in the nucleus to ter domains are at least two molecules which can interact complement a prior inactivated gene. Thus, following com together, and the interaction induce immune cell activation. bination of input signals, the combination of two transmitter 0078. In a particular embodiment, said CAR can be a signals allows the complementation of the inactivated gene multi-chain CAR comprising at least a transmembrane poly and thus the activation of the T cell. peptide which comprises at least one extracellular ligand 0091 Domains involved in the formation of the immune binding domain; and a transmembrane polypeptide compris synapse can be used as target for gene inactivation, and thus ing at least one transmitter domain such that said polypep for complementation of this gene. Said immune cell inacti tides assemble together to form a multi-chain Chimeric vated for this gene can be used to engineer cell according to Antigen Receptor (PCT/US2013/058005). Said multi-chain the present invention. Thus, following combination of input CAR can comprise several extracellular ligand binding signals, the combination of transmitter domains induces the domains, to simultaneously bind different ligands. In par expression of a gene capable of complementing said inac ticular, said different extracellular ligand-binding domains tivating gene. As nonlimiting examples, said domains which can be placed on different transmembrane polypeptides participate to the formation of the immunological synapse or composing the multi-chain CAR. In another embodiment, to the transfer of the signal include as non-limiting the present invention relates to a population of multi-chain examples: LCK, ZAP70. Itk, LAT, SLP76, GADS, GRB2, CARS comprising each one different extracellular ligand PLC-y1, or VAV1. Other examples can be DOK1 and DOK2 binding domains. proteins which negatively control the T cell receptor signal 0079. In a particular embodiment, said chimeric antigen ing by recruiting other negative regulators as RAS GTP. receptor comprises at least: SHIP1 and CSK. Transcription factors modulated by the 0080 an extracellular ligand binding domain capable immunological synapse domains can also be activate to of recognizing said specific ligand; complement inactivated cell. Said transcription factors 0081 a transmembrane domain; include as non-limiting examples: NFAT (nuclear factor of I0082 an intracellular domain comprising at least an activated T cells). NE-Kb (nuclear factor kappa-light chain activation and co-activation domains and an oxygen enhancer of activated B cells), mTOR, AP1/2, ERK1/2, sensitive domain. C-MAE. For example, ZAP-70 (Zeta-chain-associated pro 0083. According to another embodiment, said extracel tein kinase 70) is a protein normally expressed near the lular domain contains additionally a hinge. surface membrane of T cells. It is part of the T cell receptor, 0084. According to another embodiment, the scFv con and plays a critical role in T cell signaling. Following tained in said extracellular binding domain are directed to antigen recognition by CAR comprising the CD3 Zeta sig the CD19, 5T4, ROR1, CD123 or CD33 cell target antigens, naling domain in immune cell, ZAP70 binds to the CD3 Zeta and have respectively at least and identity of over 80%, domain inducing activation of immune cell response. Thus, preferably 90% or more preferably 95% with SEQ ID NO: in inactivated ZAP70 gene T cell, the antigen recognition of 32,35,38: SEQID NO:33: SEQID NO:34: SEQID NO:36 only one antigen by CAR comprising CD3 Zeta domain does and SEQ ID NO:37. not induce T cell activation. However, the recognition of a 0085. According to another embodiment, said hinge is second ligand by another CAR comprising an intracellular chosen from CD8a, IgG1 or EpoR-D2, and have an identity ZAP70 domain can complement the prior inactivated of over 80%, preferably 90% or more preferably 95% with ZAP70 gene and thus allows the activation of the T cell via respectively SEQ ID NO:39, 40 and 41. CD3 Zeta (FIG. 3). I0086 According to another embodiment, said transmem brane domain is chosen from CD8a, 4-1 BB, DAP10, CD28 Protease System or FceRIalpha, and have an identity of over 80%, preferably 0092. Transmitter domains according to the present 90% or more preferably 95% with respectively SEQ ID invention can be a protease and a Substrate protein compris NO:42, 43, 44, 45 and 46. ing a signaling protein linked to a membrane anchoring 0087. According to another embodiment, said oxygen domain via a protease cleavage site. The combination of the sensitive domain is chosen between HIF1 alpha or HIF3 two transmitter domains results in activation of the immune US 2017/0073423 A1 Mar. 16, 2017 cell. Indeed, cleavage of the substrate protein by the protease specificity and efficiency by increasing the proximity and results in the release of signaling protein and thus in immune effective concentration of components in the scaffold com cell activation (FIG. 4). Said membrane anchoring domain plex resulting in the activation of the immune cell. As non can be a terminal extension which anchors the Substrate limiting example, a scaffold protein can bind a protein protein to the membrane of the cell. In particular embodi kinase and its substrate thereby ensuring specific kinase ment, said Substrate protein is a part of the intracellular phosphorylation or said scaffold protein can result in allos domain of a chimeric antigen receptor. Said protease can be teric changes of the signaling members. Said scaffold protein as non-limiting examples: TEV protease, Factor Xa, throm can regulate signal transduction, can help localize pathway bin, engineered viral proteases, enterokinase and HRV3C. components (organized in complexes) to specific areas of the cell Such as the plasma membrane, can coordinate Split-Protein Based System signaling feedbacks, or can protect activated molecules from inactivation. Said Scaffold protein according to the present 0093. In another embodiment, the transmitter domains invention can be as non-limiting example SH2 domain as in are split proteins. This system is based on protein comple SYK tyrosine kinase or ZAP70 which can recognize and mentation assays wherein a functional molecule is dissected bind different ITAM domains (transmitter domains) as into two non-functional fragments. Functionality is restored described for C-type lectin and hemlTAM (FIG. 11) or when the fragments are reassembled by attached protein CARMA-1 as described in example 2. protein interaction domains. The functional molecule used in the protein complementation assays can be an active enzyme, or a signaling protein. Said split proteins encom Double Activation System pass, as non-limiting examples, split kinases, split proteases and split inteins. 0.098 Transmitter domains can also be a homo or het 0094. In a particular embodiment, said split proteins are erodimeric proteins, in particular an intracellular domain of split inteins which can reassemble together and restore the a receptor which can dimerize with another transmitter functionality of the intein. Inteins are internal protein domain such as another intracellular domain of a receptor or sequences that catalyze a protein-splicing reaction, which a cytosolic protein. The dimerization of these transmitter precisely excise the intein sequence and join the flanking domains transduces a signal downstream. One example of sequence with a peptide bond. Split intein is any intein in signaling proteins involving homo- or hetero-dimerization which the N-terminal domain of the intein and the C-termi of proteins can be a tyrosine kinase receptor as described nal domain of the intein are not directly linked via a peptide above involving the JAK/Stat signaling pathway. Activation bond. Natural split inteins have been identified in cyano of such components is generally elicited by ligand-mediated bacteria and archaea, but split inteins can also be created dimerization. Said transmitter domains that homodimerize artificially by separating an intein sequence into two frag can be engineered to form obligated heterodimer. In a ments (International application WO2013/045632). Accord particular embodiment, said CARS can comprise the trans ing to the present invention, the protein splicing reaction membrane, and optionally the intracellular domains of a precisely excises the intein sequence and joins the flanking receptor tyrosine kinase, preferably selected from the group sequence to reconstitute a signaling protein which induces consisting of Trk A. c-Kit, FGFR and EGFR/Erb. The rec immune cell activation (FIG. 5). In a particular embodiment, ognition of the ligands induces the dimerization of the said signaling protein can be released upon spilt intein receptor and thus activation of the signaling protein resulting reassembly (FIG. 6). in immune cell activation (FIG. 12). 0095. In another particular embodiment, said split pro teins can be split kinases which can assemble together to Autoinhibited System reconstitute a functional kinase (FIGS. 7 and 8). Said kinase can phosphorylate a signaling protein to induce immune cell 0099 Transmitter domains can also be a non-activate activation. In a particular embodiment, said kinases can be form of an autoinhibited molecule. Autoinhibited com as non limiting examples: CaMKII, Lck, PKCd, HPK1, pounds may exist in an autoinhibited State or an active state. PK0, IKKB, CK1C. which will phosphorylate the serine The autoinhibited state results in perturbed catalytic function residues on the linker region of the CARMA 1 protein of the protein, or perturbs the ability of the protein to interact inducing NF-KB and JNK signaling pathway. with another ligand. An autoinhibited State typically occurs 0096. In another particular embodiment, said split pro in the absence of phosphorylation of the kinase. Activation teins can be split-protease which can interact together to of Such autoinhibited protein can involve a conformational form a functional protease as described above (FIGS. 9 and change of the compound. This conformational change can be 10). Said protease can interact with a substrate protein and the consequence of the interaction with another compound. cleave the target protease site to release the signaling pro Said inhibitory compounds can be allosteric inhibitory com tein. pounds. Allosteric inhibitory compounds bind and form a specific association with an autoinhibited compound so as to Scaffolding Systems preserve a conserve non activated conformational state of the autoinhibited compound. Autoinhibition can be relieved 0097 Scaffold proteins are crucial regulators of many by interacting with another transmitter domain which can key signaling pathways. By Scaffold protein, it is meant a have a higher affinity binding (FIG. 13) or which can induce protein able to interact and/or bind with multiple members for examples covalent modifications (e.g. de-phosphory of a signaling pathway, tethering them into complexes. In the lation) of the interaction region or proteolysis (FIG. 14). As present invention, the transmitter domains can be members non limiting examples, said inhibitors can be class I and II of the signaling pathway which can recruit a scaffold pro of p21 activated kinase (pak) inhibitor, Rho activated protein tein. This assembly may be able to enhance signaling inhibitors, autoinhibites non receptor serine/threonine US 2017/0073423 A1 Mar. 16, 2017

kinase inhibitors, phosphatase inhibitor and autoinhibited cytoplasmic sequences of the T cell receptor and co-recep small GTPase effector inhibitors. tors that act in concert to initiate signal transduction follow 0100. The combination of transmitter domains according ing antigen receptor engagement, as well as any derivate or to the present invention is responsible for intracellular variant of these sequences and any synthetic sequence that signaling following the binding of extracellular ligand bind has the same functional capability. Signal transduction ing domain and results in the activation of the immune cell domain comprises two distinct classes of cytoplasmic sig and immune response. In other words, the signaling protein naling sequence, those that initiate antigen-dependent pri is responsible for the activation of at least one of the normal mary activation, and those that act in an antigen-independent functions of the engineered immune cell. For example, the manner to provide a secondary or co-stimulatory signal. function of a T cell can be a cytolytic activity or helper Primary cytoplasmic signaling sequence can comprise sig activity including the secretion of cytokines. Thus, the term naling motifs which are known as immunoreceptor tyrosine “signaling protein’ refers to a protein which transduces the based activation motifs of ITAMs. ITAMs are well defined transmitter domain function signal and directs the cell to signaling motifs found in the intracytoplasmic tail of a perform a specialized function. In a particular embodiment, variety of receptors that serve as binding sites for syk/zap70 said transmitter domain can be a signaling protein. Trans class tyrosine kinases or Ick. Examples of ITAM used in the mission of the signals can result from: protein/protein inter invention can include as non limiting examples those actions, protein/DNA interaction, protein/RNA interaction, derived from TCRZeta, FcRgamma, FcRbeta, FcRepsilon, protein/small molecule interaction, post translational protein CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, modification, conformational change, Subcellular relocaliza CD79b and CD66d. tion. 0106. In particular embodiment the signal transduction 0101. In particular, the signaling protein can complement domain of the CAR of the present invention comprises a a prior inactivated gene or activate a gene in the nucleus to co-stimulatory signal molecule. A co-stimulatory molecule complement a prior inactivated gene. Domains involved in is a cell Surface molecule other than an antigen receptor or the formation of the immune synapse can be used as target their ligands that is required for an efficient immune for gene inactivation, and thus for complementation of this response. “Co-stimulatory ligand’ refers to a molecule on an gene. Said immune cell inactivated for this gene can be used antigen presenting cell that specifically binds a cognate to engineer cell according to the present invention. Thus, co-stimulatory molecule on a T cell, thereby providing a following combination of input signals, the combination of signal which, in addition to the primary signal provided by, transmitter domains induces the expression of a gene for instance, binding of a TCR/CD3 complex with an MHC capable of complementing said inactivating gene. molecule loaded with peptide, mediates a T cell response, 0102. In another particular embodiment, the signaling including, but not limited to, proliferation activation, differ protein can activate a gene in the nucleus. Examples of entiation and the like. A co-stimulatory ligand can include signaling protein can be members of NFAT transcription but is not limited to CD7. B7-1 (CD80), B7-2 (CD86), factor family which are inducible factor that could bind the PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory interleukin-2 promoter in activated T cells. The regulation of ligand (ICOS-L), intercellular adhesion molecule (ICAM, NFAT proteins involves metabolites and proteins such as CD3OL, CD40, CD70, CD83, HLA-G, MICA, M1CB, calcium, calcineurin and Homer scaffolding proteins. Said HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, an signaling protein can be an activated engineered form of agonist or antibody that binds Toll ligand receptor and a NFAT avoiding regulation by calcineurin and Homer pro ligand that specifically binds with B7-H3. A “co-stimulatory teins. Said signaling protein can be a NF-KB engineered to molecule' refers to the cognate binding partner on a T cell avoid sequestration in the cytoplasm by IKb allowing acti that specifically binds with a co-stimulatory ligand, thereby Vation of T cells. Said signaling protein can also be the mediating a co-stimulatory response by the cell. Such as, but expression of the three IKK subunits (IKKO., IKKB, IKKY). not limited to proliferation. Co-stimulatory molecules Reconstituted IKK complex activated NF-kB pathway, by include, but are not limited to an MHC class I molecule, triggering the ubiquitination of the IKB. Also the activation BTLA and Toll ligand receptor. Examples of costimulatory of the JNK signaling could be triggered through the direct molecules include CD27, CD28, CD8, 4-1BB (CD137), expression of signaling protein AP-1 (transcription factor). OX40, CD30, CD40, PD-1, ICOS, lymphocyte function In another particular embodiment, said signaling protein can associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, be an engineered transcription activator like effector (TALE) B7-H3 and a ligand that specifically binds with CD83 and binding domain that will specifically target and activate the like. transcription of the same gene as for the NFAT and NF-kb. 0103) In another particular embodiment, said signaling External Stimuli protein can inhibit a signaling pathway through protein protein interaction or can activate a gene in the nucleus to 0107. In another aspect of the invention, the input signal inhibit a signaling pathway. can be an external stimulus (FIG. 15). Said external stimuli 0104 Said signaling protein can be vaccinia H1 related encompass as non limiting examples variation upon pres proteins (VHR) a member of the mitogen-activated protein ence of a tumor cell in the microenvironment of the engi kinase phosphatases (MKPs) family which dephosphory neered T cell of Small molecules, peptide, Small proteins, lates and inactivates an extracellular signal regulated kinases (chemokines, cytokines) and physicochemical conditions (ERK) signaling proteins. Such as pH, hypoxia, redox potential. Redox regulatory 0105. According to the present invention, said transmitter elements can be oxygen or nitrogen Such as reactive oxygen domains or signaling protein induced by transmitter domains or nitrogen specied (NS and RNS) as non limiting examples. can be a signal transducing domain. Preferred examples of 0108. In a particular embodiment, said external stimulus signal transducing domain for use in a CAR can be the can be hypoxic environment. US 2017/0073423 A1 Mar. 16, 2017

0109. In a preferred embodiment, the response to said sitive to oxygen (oxiTF) and incorporating within the cell a hypoxia condition is triggered by the alpha hypoxia induc synthetic promoter specific for the OxiTF driving the ible factor 1 (HIF-1C...) or by the alpha hypoxia inducible expression of a transmitter domain (FIG. 16). Said OxiTF factor 3 (HIF-3C). can be an engineered transcription factor Such as TAL 0110. In a more preferred embodiment, the said HIF-1C. effector, Zinc finger effector, CRISP effector, as non limiting polypeptide sequence has over 80% identity, preferably 90% examples, fused to an HIF1 alpha C-terminal domain. Under identity or more preferably 95% identity with SEQ ID NO. hypoxia, engineered immune cells sense oxygen depletion, 5 or to SEQ ID NO. 22-23, or the said HIF-3a polypeptide in particular in tumor environment, and trigger the expres sequence has over 80% identity, preferably 90% identity or sion of a transmitter domain which induces immune cell more preferably 95% identity with SEQ ID NO. 26-27. activation. Said transmitter domain can be a transmitter 0111 Indeed, local tissue hypoxia is associated with domain or more particularly a chimeric antigen receptor many different disease states including certain tumors, cer comprising transmitter domains as described above. Said tain inflammatory processes and conditions of neovascular chimeric antigen receptor comprises an extracellular ligand ization. Solid tumours, in particular, show relatively aberrant binding domain capable of recognizing a specific ligand and vascularization that causes intermittent/absent perfusion the recognition of said ligand by said CAR is the second leading to hypoxia. A multifaceted adaptive response to input signal. Said method of engineering immune cells can hypoxia is facilitated by the stabilization and accumulation further comprise the step of expressing at the Surface of said of the alpha subunit of hypoxia inducible factor 1 (HIF-1). immune cell another chimeric antigen receptor. In a more Under normoxia, HIF1C. is inhibited through the hydroxy preferred embodiment. Such engineered cell can comprised lation of specific proline residues located in its C-terminal a first CAR constitutively expressed at the surface of the region. Such hydroxylation is known to promote recruitment immune cell and a second CAR expressed only in hypoxic of VHL, an E3 ubiquitin ligase that triggers ubiquitinylation conditions, the first and second CARS comprising extracel of HIF1C. and its degradation by proteasome. In hypoxic lular binding domains capable of recognizing two different conditions, HIF-1C. forms a complex with its binding part ligands (FIG. 17). ner, aryl hydrocarbon receptor nuclear translocator (ARNT), 0114. In another particular embodiment, the present as well as the p300/CBP transcriptional coactivators that invention encompasses a method of engineering an immune bind to the hypoxia response element (HRE) in the untrans cell wherein the chimeric antigen receptor is a multi-chain lated regions of hypoxically regulated genes. This complex CAR. In a preferred embodiment, C, B and Y chains of said induces the transcription of genes that serve to maintain multi-chain CAR have respectively an identity of over 80%, cellular homeostasis in the face of hypoxic conditions. For preferably 90% identity or more preferably 95% identity example, the HIF-1/p300/CBP complex plays a role in with SEQ ID NO. 7, 3 and 4. inducing expression of genes such as those encoding eryth 0.115. In another particular embodiment, the present ropoietin, which leads to erythropoiesis; vascular endothe invention relates to a method of engineering an immune cell lial cell growth factor (VEGF), which is a primary mediator comprising a step of expressing a CAR which comprises at of angiogenesis; iNOS and heme oxygenase, which play least two extracellular ligand binding domains. Bispecific roles in vasodilation; and the glucose transporter and gly tandem CAR previously described (International applica colytic enzymes, which play roles in anaerobic metabolism. tion: WO2013123061, US. Patent application: Beside HIF-1, the HIF-3 C. system (ref Uniprot: Q9Y2N7 for US20130280220), may theoretically still lead to the T cell human sequence) is also contemplated in the present inven activation independently to the recognition and binding of tion. Also involved in adaptive response to hypoxia, its both antigen as one cannot exclude that the binding of one action is known to Suppress hypoxia-inducible expression of single chain Fv may trigger activation. So to avoid these HIF1A and EPAS, and to binds to core DNA sequence drawbacks, the inventors sought to design new bispecific 5'-TACGTG-3' within the hypoxia response element (HRE) CAR comprising at least two extracellular ligand binding of target gene promoters. Its expression and characterization domains, which cannot induce T cell activation by the in human kidney are presented in Hara et al. (2001). binding of only one ligand. Said CAR can comprise another 0112. In a particular aspect of the invention, said input domain which perturbs the ability the signaling function of signal can be the hypoxic microenvironment which is the CAR, or perturbs the ability of the protein to interact detected by the engineered cell. Thus, according to the with another ligand. The recognition of at least two ligands method of the present invention, the immune cell can be by the extracellular ligand binding domains can involve a engineered to be sensitive to hypoxic environment. Said conformational change of the CAR and thus the transduction immune cell is engineered to trigger cytotoxicity via acti of the signal of the CAR. The conformational change can Vation of synthetic hypoxia dependent activation pathways. occur for example by the interaction with a ligand which can In particular, said immune cell can be engineered to induce have a higher affinity binding, such idiotypic antibody (FIG. expression of a transmitter domain under hypoxia inducible 18). promoter. Said hypoxia inducible promoter can be com posed of HRE. The consensus sequence of HRE is (G/C/T) Other AND Logic Gate System ACGTGC(G/C). Usually, multiple copies of HREs appear in 0116. In another aspect of the invention, the inventors a hypoxia inducible promoter. In a preferred embodiment, also developed methods of engineering Such immune cells said hypoxia inducible promoter is composed of HREs and based on logic gates, wherein only the presence of one a basal promoter such as SV40 promoter. For example, said specific input signal and not the other produces a particular multiple copies of HRE can be derived from the PGK-1 output in response (FIG. 19). According to the present promoter, the EPO, GAPDH, VEGF and survivin promoters. invention, the immune cell activation, in particular T cell 0113. In another particular embodiment, said immune cytotoxicity against a target cell is induced following the cell is engineered by expressing a transcription factor sen recognition of one of several input signals, in particular the US 2017/0073423 A1 Mar. 16, 2017 recognition of a ligand on a cancer cell and not the recog binding domain and an intracellular domain comprising a nition of a ligand present on the healthy cells. In particular, transmitter domain as described above. In particular, said the present invention relates to a method of engineering an transmitter domain is selecting from the group consisting of immune cell for immunotherapy, in particular, method of protease, split protein, members of signaling pathway engineering an immune cell for targeting specifically a cell recruiting a scaffold protein, one monomer of the dimeric comprising: domain, an autoinhibited compound. 0117 (a) Providing an immune cell; I0123. According to one embodiment, the chimeric anti 0118 (b) Engineering said immune cell to render said gen receptor comprises: cell sensitive to at least one among several input signals 0.124 the alpha-chain comprises extracellularly the Such that only the specific input signal induces activa CD8 hinge, FcRC. as transmembrane domain, and intra tion of said immune cell response. cellularly a part of FcRC. combined with HIF1alpha or 0119. In a particular embodiment, the present invention HIF3 alpha subunit; relates to a method of engineering immune cell by express 0.125 the beta-chain comprises the FcRB as extracel ing at the surface of the cell at least one first CAR com lular and transmembrane domain, and AITAM-41BBas prising an extracellular ligand binding domain capable of intracellular co-stimulation domain; recognizing a ligand at the Surface of a tumor cell and 0.126 the gamma-chain comprises the FcRY as trans another CAR comprising an extracellular ligand binding membrane domain, and AITAM-CD3 as intracellular domain capable of recognizing a ligand at the Surface of activation domain. healthy cell. Such that the recognition of the ligand at the I0127. The present invention also relates to polynucle surface of a healthy cell inhibits the activation of immune otides, vectors encoding the above described CAR accord cell via an inhibitory transmitter domain, while the recog ing to the invention. The polynucleotide may consist in an nition of the ligand at the Surface of the target cell induces expression cassette or expression vector (e.g. a plasmid for the activation of the immune cell via transmitter domain. introduction into a bacterial host cell, or a viral vector such Inhibitory or activation transmitter domain can be derived as a baculovirus vector for transfection of an insect host cell, from the SRC family kinase (SFK) member LCK. In a more or a plasmid or viral vector Such as a lentivirus for trans preferred embodiment, inhibitory transmitter domain is a fection of a mammalian host cell). constitutively negatively regulated LCK, preferably com I0128. In a particular embodiment, the different nucleic prising a mutation at the position Y394 (NCBI Reference acid sequences can be included in one polynucleotide or Sequence: NP 005347.3) and activation domain is a con Vector which comprises a nucleic acid sequence encoding stitutively active LCK form, preferably which comprises a ribosomal skip sequence Such as a sequence encoding a 2A mutation at position Y505 (NCBI Reference Sequence: peptide. 2A peptides, which were identified in the Aphtho NP 005347.3) (see example 2 and FIG. 20). virus Subgroup of picornaviruses, causes a ribosomal "skip' from one codon to the next without the formation of a Delivery Methods peptide bond between the two amino acids encoded by the 0120. The different methods described above involve codons (see (Doronina, Wu et al. 2008). By “codon’ is expressing CAR at the Surface of a cell. As non-limiting meant three nucleotides on an mRNA (or on the sense strand example, said CAR can be expressed by introducing CAR of a DNA molecule) that are translated by a ribosome into into a cell. CAR can be introduced as transgene encoded by one amino acid residue. Thus, two polypeptides can be one plasmidic vector. Said plasmid vector can also contain synthesized from a single, contiguous open reading frame a selection marker which provides for identification and/or within an mRNA when the polypeptides are separated by a selection of cells which received said vector. 2A oligopeptide sequence that is in frame. Such ribosomal 0121 Polypeptides may be synthesized in situ in the cell skip mechanisms are well known in the art and are known as a result of the introduction of polynucleotides encoding to be used by several vectors for the expression of several said polypeptides into the cell. Alternatively, said polypep proteins encoded by a single messenger RNA. tides could be produced outside the cell and then introduced I0129. To direct, transmembrane polypeptide into the thereto. Methods for introducing a polynucleotide construct secretory pathway of a host cell, a secretory signal sequence into cells are known in the art and including as non limiting (also known as a leader sequence, prepro sequence or pre examples stable transformation methods wherein the poly sequence) is provided in polynucleotide sequence or vector nucleotide construct is integrated into the genome of the cell, sequence. The secretory signal sequence is operably linked transient transformation methods wherein the polynucle to the transmembrane nucleic acid sequence, i.e., the two otide construct is not integrated into the genome of the cell sequences are joined in the correct reading frame and and virus mediated methods. Said polynucleotides may be positioned to direct the newly synthesized polypeptide into introduced into a cell by for example, recombinant viral the secretory pathway of the host cell. Secretory signal vectors (e.g. retroviruses, adenoviruses), liposome and the sequences are commonly positioned 5' to the nucleic acid like. For example, transient transformation methods include sequence encoding the polypeptide of interest, although for example microinjection, electroporation or particle bom certain secretory signal sequences may be positioned else bardment. Said polynucleotides may be included in vectors, where in the nucleic acid sequence of interest (see, e.g., more particularly plasmids or virus, in view of being Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. expressed in cells. Pat. No. 5,143,830). 0.130 Those skilled in the art will recognize that, in view Chimeric Antigen Receptors, Polynucleotides and Vectors of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules. 0122) The present invention also relates to a chimeric Preferably, the nucleic acid sequences of the present inven antigen receptor which comprises an extracellular ligand tion are codon-optimized for expression in mammalian cells, US 2017/0073423 A1 Mar. 16, 2017

preferably for expression in human cells. Codon-optimiza cell according to the invention or cell line derived from said tion refers to the exchange in a sequence of interest of isolated cell can be used in the manufacture of a medicament codons that are generally rare in highly expressed genes of for treatment of a cancer, viral infection or autoimmune a given species by codons that are generally frequent in disease in a patient in need thereof. highly expressed genes of Such species, such codons encod I0135) In another aspect, the present invention relies on ing the amino acids as the codons that are being exchanged. methods for treating patients in need thereof, said method comprising at least one of the following steps: Engineered Immune Cells 0.136 (a) providing an immune-cell obtainable by any 0131 The present invention also relates to isolated cells one of the methods previously described: or cell lines susceptible to be obtained by said method to 0.137 (b) Administrating said transformed immune engineer cells. In particular said isolated cell comprises at cells to said patient, least one CAR as described above. In a preferred embodi 0.138. On one embodiment, said T cells of the invention ment, said isolated cell comprises a population of CARS can undergo robust in vivo T cell expansion and can persist each one comprising different extracellular ligand binding for an extended amount of time. domains. In particular, said isolated cell comprises exog 0.139 Said treatment can be ameliorating, curative or enous polynucleotide sequence encoding CAR. prophylactic. It may be either part of an autologous immu 0.132. In the scope of the present invention is also encom notherapy or part of an allogenic immunotherapy treatment. passed an isolated immune cell, preferably a T cell obtained By autologous, it is meant that cells, cell line or population according to any one of the methods previously described. of cells used for treating patients are originating from said Said immune cell refers to a cell of hematopoietic origin patient or from a Human Leucocyte Antigen (HLA) com functionally involved in the initiation and/or execution of patible donor. By allogeneic is meant that the cells or innate and/or adaptive immune response. Said immune cell population of cells used for treating patients are not origi according to the present invention can be derived from a nating from said patient but from a donor. stem cell. The stem cells can be adult stem cells, non-human 0140 Cells that can be used with the disclosed methods embryonic stem cells, more particularly non-human stem are described in the previous section. Said treatment can be cells, cord blood stem cells, progenitor cells, bone marrow used to treat patients diagnosed with cancer, viral infection, stem cells, induced pluripotent stem cells, totipotent stem autoimmune disorders or Graft versus Host Disease cells or hematopoietic stem cells. Representative human (GvHD). Cancers that may be treated include tumors that are cells are CD34+ cells. Said isolated cell can also be a not vascularized, or not yet substantially vascularized, as dendritic cell, killer dendritic cell, a mast cell, a NK-cell, a well as vascularized tumors. The cancers may comprise non B-cell or a T cell selected from the group consisting of Solid tumors (such as hematological tumors, for example, inflammatory T-lymphocytes, cytotoxic T-lymphocytes, leukemias and lymphomas) or may comprise Solid tumors. regulatory T-lymphocytes or helper T-lymphocytes. In Types of cancers to be treated with the multi-chain CARs of another embodiment, said cell can be derived from the group the invention include, but are not limited to, carcinoma, consisting of CD4+ T-lymphocytes and CD8+ T-lympho blastoma, and sarcoma, and certain leukemia or lymphoid cytes. Prior to expansion and genetic modification of the malignancies, benign and malignant tumors, and malignan cells of the invention, a source of cells can be obtained from cies e.g., sarcomas, carcinomas, and melanomas. Adult a Subject through a variety of non-limiting methods. Cells tumors/cancers and pediatric tumors/cancers are also can be obtained from a number of non-limiting sources, included. including peripheral blood mononuclear cells, bone marrow, 0.141. It can be a treatment in combination with one or lymph node tissue, cord blood, thymus tissue, tissue from a more therapies against cancer selected from the group of site of infection, ascites, pleural effusion, spleen tissue, and antibodies therapy, chemotherapy, cytokines therapy, den tumors. In certain embodiments of the present invention, any dritic cell therapy, gene therapy, hormone therapy, laser light number of T cell lines available and known to those skilled therapy and radiation therapy. in the art, may be used. In another embodiment, said cell can 0142. The administration of the cells or population of be derived from a healthy donor, from a patient diagnosed cells according to the present invention may be carried out with cancer or from a patient diagnosed with an infection. In in any convenient manner, including by aerosol inhalation, another embodiment, said cell is part of a mixed population injection, ingestion, transfusion, implantation or transplan of cells which present different phenotypic characteristics. tation. The compositions described herein may be adminis In the scope of the present invention is also encompassed a tered to a patient Subcutaneously, intradermally, intratumor cell line obtained from a transformed T-cell according to the ally, intranodally, intramedullary, intramuscularly, by method previously described. intravenous or intralymphatic injection, or intraperitoneally. 0133. In another embodiment, said isolated cell accord In one embodiment, the cell compositions of the present ing to the present invention comprises a polynucleotide invention are preferably administered by intravenous injec encoding CAR. tion. 0143. The administration of the cells or population of cells can consist of the administration of 10-10 cells per kg Therapeutic Applications body weight, preferably 10 to 10° cells/kg body weight 0134. In another embodiment, isolated cell obtained by including all integer values of cell numbers within those the different methods or cell line derived from said isolated ranges. The cells or population of cells can be administrated cell as previously described can be used as a medicament. In in one or more doses. In another embodiment, said effective another embodiment, said medicament can be used for amount of cells are administrated as a single dose. In another treating cancer, autoimmune disease or infections in a embodiment, said effective amount of cells are adminis patient in need thereof. In another embodiment, said isolated trated as more than one dose over a period time. Timing of US 2017/0073423 A1 Mar. 16, 2017 administration is within the judgment of managing physi For the degenerated nucleotides, r represents g or a cian and depends on the clinical condition of the patient. The (purine nucleotides), k represents g or t, S represents g cells or population of cells may be obtained from any source, or c, w represents a or t, m represents a or c, y such as a blood bank or a donor. While individual needs represents t or c (pyrimidine nucleotides), d represents vary, determination of optimal ranges of effective amounts g, a or t, V represents g, a or c, b represents g. t or c, h of a given cell type for a particular disease or conditions represents a, t or c, and n represents g, a, t or c. within the skill of the art. An effective amount means an 0.148 “As used herein, “nucleic acid” or “polynucle amount which provides a therapeutic or prophylactic benefit. otides' refers to nucleotides and/or polynucleotides, The dosage administrated will be dependent upon the age, such as deoxyribonucleic acid (DNA) or ribonucleic health and weight of the recipient, kind of concurrent acid (RNA), oligonucleotides, fragments generated by treatment, if any, frequency of treatment and the nature of the polymerase chain reaction (PCR), and fragments the effect desired. generated by any of ligation, Scission, endonuclease 0144. In another embodiment, said effective amount of action, and exonuclease action. Nucleic acid molecules cells or composition comprising those cells are adminis can be composed of monomers that are naturally trated parenterally. Said administration can be an intrave occurring nucleotides (such as DNA and RNA), or nous administration. Said administration can be directly analogs of naturally-occurring nucleotides (e.g., done by injection within a tumor. enantiomeric forms of naturally-occurring nucleo 0145. In certain embodiments of the present invention, tides), or a combination of both. Modified nucleotides cells are administered to a patient in conjunction with (e.g., can have alterations in Sugar moieties and/or in pyrimi before, simultaneously or following) any number of relevant dine or purine base moieties. Sugar modifications treatment modalities, including but not limited to treatment include, for example, replacement of one or more with agents such as antiviral therapy, cidofovir and inter hydroxyl groups with halogens, alkyl groups, amines, leukin-2, Cytarabine (also known as ARA-C) or nataliz and azido groups, or Sugars can be functionalized as imab treatment for MS patients or efaliztimab treatment for ethers or esters. Moreover, the entire Sugar moiety can psoriasis patients or other treatments for PML patients. In be replaced with sterically and electronically similar further embodiments, the T cells of the invention may be structures, such as aza-Sugars and carbocyclic Sugar used in combination with chemotherapy, radiation, immu analogs. Examples of modifications in a base moiety nosuppressive agents, such as cyclosporin, azathioprine, include alkylated purines and pyrimidines, acylated methotrexate, mycophenolate, and FK506, antibodies, or purines or pyrimidines, or other well-known heterocy other immunoablative agents such as CAMPATH, anti-CD3 clic substitutes. Nucleic acid monomers can be linked antibodies or other antibody therapies, cytoxin, fludaribine, by phosphodiester bonds or analogs of Such linkages. cyclosporin, FK506, rapamycin, mycoplienolic acid, Ste Nucleic acids can be either single stranded or double roids, FR901228, cytokines, and irradiation. These drugs stranded. inhibit either the calcium dependent phosphatase calcineurin 0149. By chimeric antigen receptor (CAR) is intended (cyclosporine and FK506) or inhibit the p70S6 kinase that is molecules that combine a binding domain against a important for growth factor induced signaling (rapamycin) component present on the target cell, for example an (Henderson, Naya et al. 1991; Liu, Albers et al. 1992: Bierer, antibody-based specificity for a desired antigen (e.g., Hollander et al. 1993). In a further embodiment, the cell tumor antigen) with a T cell receptor-activating intra compositions of the present invention are administered to a cellular domain to generate a chimeric protein that patient in conjunction with (e.g., before, simultaneously or exhibits a specific anti-target cellular immune activity. following) bone marrow transplantation, T cell ablative Generally, CAR consists of an extracellular single therapy using either chemotherapy agents such as, fludara chain antibody (scFv) fused to the intracellular signal bine, external-beam radiation therapy (XRT), cyclophosph ing domain of the T cell antigen receptor complex Zeta amide, or antibodies such as OKT3 or CAMPATH, In chain (sclv:) and have the ability, when expressed in another embodiment, the cell compositions of the present T cells, to redirect antigen recognition based on the invention are administered following B-cell ablative therapy monoclonal antibody's specificity. Such as agents that react with CD20, e.g., Rituxan. For 0150. By “delivery vector” or “delivery vectors” is example, in one embodiment, Subjects may undergo stan intended any delivery vector which can be used in the dard treatment with high dose chemotherapy followed by present invention to put into cell contact (i.e “contact peripheral blood stem cell transplantation. In certain ing') or deliver inside cells or subcellular compart embodiments, following the transplant, Subjects receive an ments (i.e “introducing) agents/chemicals and mol infusion of the expanded immune cells of the present ecules (proteins or nucleic acids) needed in the present invention. In an additional embodiment, expanded cells are invention. It includes, but is not limited to liposomal administered before or following Surgery. delivery vectors, viral delivery vectors, drug delivery vectors, chemical carriers, polymeric carriers, lipo OTHER DEFINITIONS plexes, polyplexes, dendrimers, microbubbles (ultra 0146 Amino acid residues in a polypeptide sequence Sound contrast agents), nanoparticles, emulsions or are designated herein according to the one-letter code, other appropriate transfer vectors. These delivery vec in which, for example, Q means Glin or Glutamine tors allow delivery of molecules, chemicals, macromol residue, R means Arg or Arginine residue and D means ecules (genes, proteins), or other vectors such as plas Asp or Aspartic acid residue. mids, peptides developed by Diatos. In these cases, 0147 Nucleotides are designated as follows: one-letter delivery vectors are molecule carriers. By “delivery code is used for designating the base of a nucleoside: a vector” or “delivery vectors' is also intended delivery is adenine, t is thymine, c is cytosine, and g is guanine. methods to perform transfection. US 2017/0073423 A1 Mar. 16, 2017 15

0151. The terms “vector or “vectors' refer to a can affect the coding sequence of a gene or its regula nucleic acid molecule capable of transporting another tory sequence. It may also affect the structure of the nucleic acid to which it has been linked. A “vector” in genomic sequence or the structure/stability of the the present invention includes, but is not limited to, a encoded mRNA. viral vector, a plasmid, a RNA vector or a linear or 0156 by “functional variant' is intended a catalyti circular DNA or RNA molecule which may consists of cally active mutant of a protein or a protein domain; a chromosomal, non chromosomal, semi-synthetic or Such mutant may have the same activity compared to its synthetic nucleic acids. Preferred vectors are those parent protein or protein domain or additional proper capable of autonomous replication (episomal vector) ties, or higher or lower activity. and/or expression of nucleic acids to which they are (O157 “identity” refers to sequence identity between linked (expression vectors). Large numbers of Suitable two nucleic acid molecules or polypeptides. Identity vectors are known to those of skill in the art and can be determined by comparing a position in each commercially available. sequence which may be aligned for purposes of com 0152 Viral vectors include retrovirus, adenovirus, par parison. When a position in the compared sequence is vovirus (e. g. adenoassociated viruses), coronavirus, nega occupied by the same base, then the molecules are tive Strand RNA viruses such as orthomyxovirus (e. g., identical at that position. A degree of similarity or influenza virus), rhabdovirus (e. g., rabies and vesicular identity between nucleic acid or amino acid sequences stomatitis virus), paramyxovirus (e.g. measles and Sendai), is a function of the number of identical or matching positive Strand RNA viruses such as picornavirus and nucleotides at positions shared by the nucleic acid alphavirus, and double-stranded DNA viruses including sequences. Various alignment algorithms and/or pro adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 grams may be used to calculate the identity between and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus two sequences, including FASTA, or BLAST which are (e. g., vaccinia, fowlpox and canarypox). Other viruses available as a part of the GCG sequence analysis include Norwalk virus, togavirus, flavivirus, reoviruses, package (University of Wisconsin, Madison, Wis.), and papovavirus, hepadnavirus, and hepatitis virus, for example. can be used with, e.g., default setting. For example, Examples of retroviruses include: avian leukosis-sarcoma, polypeptides having at least 70%, 85%, 90%, 95%, mammalian C-type, B-type viruses, D type viruses, HTLV 98% or 99% identity to specific polypeptides described BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviri herein and preferably exhibiting substantially the same dae: The viruses and their replication. In Fundamental functions, as well as polynucleotide encoding such Virology, Third Edition, B. N. Fields, et al., Eds. Lippincott polypeptides, are contemplated. Raven Publishers, Philadelphia, 1996). 0158. The term “subject” or “patient” as used herein 0153. By “lentiviral vector” is meant HIV-Based len includes all members of the animal kingdom including tiviral vectors that are very promising for gene delivery non-human primates and humans. because of their relatively large packaging capacity, 0159. The term “Hypoxia” refers to a condition reduced immunogenicity and their ability to stably wherein the oxygen concentration is below normal transduce with high efficiency a large range of different levels for a particular tissue (such as a tumor). Hypoxia cell types. Lentiviral vectors are usually generated in a particular tissue as, compared to Surrounding following transient transfection of three (packaging, tissue, is referred to as relative tissue hypoxia. An envelope and transfer) or more plasmids into producer example of relative tissue hypoxia is tumor hypoxia in cells. Like HIV, lentiviral vectors enter the target cell which a tumor has lower levels of p0, than that of through the interaction of viral Surface glycoproteins Surrounding non-tumor tissue. In some examples of the with receptors on the cell surface. On entry, the viral disclosed methods, the level of oxygen is for example RNA undergoes reverse transcription, which is medi 10% or less (for example, 9%, 8%, 7%, 6%. 5%, 4%, ated by the viral reverse transcriptase complex. The 3%, 2%, or 1%), or for example 50 mmHg or less (for product of reverse transcription is a double-stranded example, 45 mmHg, 40 mmHg, 35 mmHg, 30 mmHg, linear viral DNA, which is the substrate for viral 25 mmHg, 20 mmHg, 15 mmHg, 10 mmHg, 5 mmHg, integration in the DNA of infected cells. By “integra 4 mmHg, 3 mmHg, 2 mmHg, or 1 mmHg). The body tive lentiviral vectors (or LV), is meant such vectors as as a whole (generalized hypoxia) or a region of the nonlimiting example, that are able to integrate the body (tissue hypoxia) may be deprived of adequate genome of a target cell. At the opposite by “non oxygen. Those of skill in the art would be familiar with integrative lentiviral vectors (or NILV) is meant effi the measurement of oxygen levels in biological systems cient gene delivery vectors that do not integrate the and that oxygen measurements may be expressed in genome of a target cell through the action of the virus “mmHg,” wherein, for example, 10% 0, is equal to 76 integrase. mmHg and 1% 0 is equal to 7.6 mmHg. 0154 Delivery vectors and vectors can be associated (0160. By “Transcription Activator like Effector or combined with any cellular permeabilization tech (TALE) it is meant a binding domain protein wherein niques such as Sonoporation or electroporation or sequence specificity is driven by a series of 33-35 derivatives of these techniques. amino acids repeats originating from Xanthomonas or 0155 by “mutation' is intended the substitution, dele Ralstonia bacterial proteins. These repeats differ essen tion, insertion of up to one, two, three, four, five, six, tially by two amino acids positions that specify an seven, eight, nine, ten, eleven, twelve, thirteen, four interaction with a base pair (Boch, Scholze et al. 2009; teen, fifteen, twenty, twenty five, thirty, forty, fifty, or Moscou and Bogdanove 2009). Each base pair in the more nucleotides/amino acids in a polynucleotide DNA target is contacted by a single repeat, with the (cDNA, gene) or a polypeptide sequence. The mutation specificity resulting from the two variant amino acids US 2017/0073423 A1 Mar. 16, 2017 16

of the repeat (the so-called repeat variable dipeptide, cific ligands and intracellular domains comprising RVD). TALE binding domains may further comprise an transmitter domains capable of activating immune cell N-terminal translocation domain responsible for the in combination. requirement of a first thymine base (T) of the targeted (0167 4. The method according to any one of claims 1 sequence and a C-terminal domain that containing a to 3 wherein said input signal is an external stimulus nuclear localization signals (NLS). A TALE nucleic Selected from the group consisting of the presence of acid binding domain generally corresponds to an engi particular Small molecules, chemokines, cytokines, neered core TALE scaffold comprising a plurality of physicochemical conditions, hypoxia. TALE repeat sequences, each repeat comprising a RVD (0168 5. The method of claim 4 wherein external specific to each nucleotides base of a TALE recognition stimulus is hypoxia and wherein said immune cell is site. In the present invention, each TALE repeat engineered to express at least one transmitter domain sequence of said core scaffold is made of 30 to 42 under the control of a hypoxia inducible promoter. amino acids, more preferably 33 or 34 wherein two (0169 6. The method of claim 4 wherein external critical amino acids (the so-called repeat variable stimulus is hypoxia and wherein said immune cell is dipeptide, RVD) located at positions 12 and 13 medi engineered to express at least one chimeric antigen ates the recognition of one nucleotide of said TALE receptor comprising a transmitter domain under the binding site sequence; equivalent two critical amino control of a hypoxia inducible promoter and wherein acids can be located at positions other than 12 and 13 the recognition of a ligand of said chimeric antigen specially in TALE repeat sequence taller than 33 or 34 receptor is another input signal. amino acids long. Preferably, RVDs associated with 0170 7. The method according to any one of claims 1 recognition of the different nucleotides are HD for to 6 wherein said transmitter domains are a protease recognizing C, NG for recognizing T, NI for recogniz and a Substrate protein comprising a signaling protein ing A, NN for recognizing G or A. In another embodi linked to a membrane anchoring domain via a protease ment, critical amino acids 12 and 13 can be mutated cleavage site such that said protease cleavage induces towards other amino acid residues in order to modulate the release of the signaling protein capable of activating their specificity towards nucleotides A, T, C and G and said immune cell. in particular to enhance this specificity. ATALE nucleic 0171 8. The method of claim 7 wherein said protease acid binding domain usually comprises between 8 and is selected from the group consisting of TEV protease, 30 TALE repeat sequences. More preferably, said core Factor Xa, thrombin, engineered viral proteases. scaffold of the present invention comprises between 8 0172 9. The method according to any one of claims 1 and 20 TALE repeat sequences; again more preferably to 8 wherein said transmitter domains are split proteins 15 TALE repeat sequences. It can also comprise an which assemble to reconstitute a signaling protein additional single truncated TALE repeat sequence capable of activating said immune cell. made of 20 amino acids located at the C-terminus of 0173 10. The method of claim 9 wherein said split said set of TALE repeat sequences, i.e. an additional proteins are split proteases which assemble together to C-terminal half-TALE repeat sequence. reconstitute a protease capable of cleaving a Substrate (0161 The invention is more particularly related to the protein and releasing a signaling protein capable of following objects: activating said immune cell. 0162. 1. A method of engineering an immune cell for 0174 11. The method of claim 9 wherein said split specifically targeting a cell comprising: proteins are split kinases which assemble together to 0163 (a) Providing an immune cell; reconstitute a kinase which activates a signaling protein 0164 (b) Engineering said immune cell to render capable of activating said immune cell. said cell sensitive to at least two input signals such 0175 12. The method of claim 9 wherein said split that the combination of input signals induces com proteins are a split-inteins which assemble together to bination of at least two transmitter domains which reconstitute an intein which excises the intein sequence results in activation of said immune cell, wherein and join with a peptide bond flanking signaling protein each transmitter domain alone does not activate said sequences capable of activating said immune cell. immune cell. (0176) 13. The method according to any one of claims 0.165 2. The method of claim 1 wherein at least one 1 to 8 wherein said transmitter domains are members of said input signal is the recognition of a specific ligand a signaling pathway which can recruit a scaffold pro by said immune cell which is engineered by expressing tein. at the Surface of the cell a chimeric antigen receptor 0.177 14. The method of claim 13 wherein said scaf (CAR) comprising an extracellular ligand binding fold protein is SYK tyrosine kinase or ZAP70. domain capable of recognizing said specific ligand and 0178 15. The method according to any one of claims an intracellular domain comprising a transmitter 1 to 8 wherein said transmitter domains are dimeric domain capable of activating said immune cell in proteins. combination with another transmitter domain option 0179 16. The method according to any one of claims ally via a signaling protein. 1 to 8 wherein said transmitter domain is an autoin (0166 3. The method of claim 1 wherein at least said hibited compound. two input signals are the recognition of different spe 0180 17. The method according to any one of claims cific ligands by said immune cell which is engineered 1 to 8 wherein said engineered immune cell is initially by expressing at the Surface of the cell chimeric antigen inactivating for a gene and wherein said combination of receptors (CARS) comprising extracellular ligand bind transmitter domains is capable of complementing said ing domains capable of recognizing said different spe inactivated gene. US 2017/0073423 A1 Mar. 16, 2017 17

0181 18. A chimeric antigen receptor comprising an coiled-coil domain which is responsible for the oligomer extracellular ligand binding domain and an intracellular ization state of CARMA1 and can regulate the binding of domain which comprise at least one transmitter this last to the mucosa-associated lymphoma translocation domain. gene1 (MALTA1) (essential for the activation of NF-kB). 0182 19. The chimeric antigen receptor of claim 18 Finally the linker region between the coiled-coil domain and wherein said transmitter domains are selected from the the MAGUK domain appears to play an important role in group consisting of a protease, split protein, dimeric restraining the conformation of CARMA 1 in a “closed protein, member of a signaling pathway which can (inactive) form. On the contrary the phosphorylation of the recruit a scaffold protein and autoinhibited compound. 0183 20. A polynucleotide encoding a chimeric anti serine residues of this Zone produced by PKC0 (and other gen receptor of claim 18 or 19. kinases see Table 1) promotes a high level of CARM1 0.184 21. An isolated immune cell which comprises a activation with the following boost of the NF-kB signaling chimeric antigen receptor of claim 18 or 19. pathway. The negatively regulation of this pathway is oper 0185. 22. An isolated immune cell obtained by any one ated by PP2A, which remove the phosphorylation of the method according to any one of claims 1 to 17. CARMA1 at the specific residue 5645. 0186 23. An isolated immune cell of claim 21 or 22 for 0193 The stimulation of TCR after recognizing the anti its use as a medicament. gen is linked to the recruitment of CD28 which leads to the 0187. 24. An isolated immune cell according to any of activation of PKC0, which in turn phosphorylates and claims 21 to 23 for treating a cancer, an autoimmune activates CARMA1. Once activated CARMA1 binds to condition or an infection by a pathogen. Bcl10 via CARD-CARD interactions; this binary complex 0188 25. A method of treating a subject in need thereof comprising: recruits MALT1 to form a ternary complex: CARMA 1 0189 (a) Providing a immune cell according to Bcl10-MALT1 (CBM). The CBM complex is necessary for claim 21 or 22, the activation of the NF-kB and JNK. All the proteins 0.190 (b) Administrating said immune cells to said reported in Table 1 have been characterized for their inter patient. actions with CARMA1 at different level and in different roles (see FIG. 21). EXAMPLES TABLE 1. Example 1 Proteins that Interact with the CARMA1 signalosome Control of T Cell Activation Through CAR-Mediated Regulation of CARMA1 Protein KINASES UBIQUITIN LIGASES OTHER 0191 The scaffold protein caspase recruitment domain containing membrane-associated guanylate kinase protein-1 PKCd UBC13-UEV1A Bcl10 (CARMA1) is a member of the MAGUK family of kinases IKK Complex TRAF6 MALT1 (Roche, Ramadas et al. 2013). CARMA1 constitutes a PDK1 TRAF2 ADAP crucial signaloSome for the transmission of the T cell Receptor (TCR) signaling and in general for T cell activa CaMKII cIAP2 Caspase 8 tion. The intracellular CARMA1 concentration is a key HPK1 NEDD4. Net1 element in the regulation of its activity. An enhancement of CK1a. ITCH the CARMA1 signaling has been observed at low and Akt CBL-b moderate concentrations while a decrease of the activity has TAK1 COP9 been reported at high concentration dues to the sequestration of the different components away form each other (biphasic RIP2 STUB1 response). Following TCR engagement, CARMA1 recruits MKK7 (JNK activation) CYLD different proteins forming a multi-protein complexes that (de-ubiquitylating enzyme) finally can activate two different signaling cascade: NF-kB Calcineurin (phosphatase) A2O and c-jun N-terminal kinase (JNK) (Blonska and Lin 2009). (de-ubiquitylating enzyme) 0.192 CARMA1 is composed by five structural domains PP2A (phosphatase) connected by linker regions. Among these five domains three constitute the membrane guanilate kinase domain (MAGUK): a PDZ homology domain (postsynaptic density protein), a SRC homology domain (SH3) and a guanylate kinase domain (GUK). The MAGUK domain is necessary Split-Protein Based Systems for cellular adhesion, formation of multi-domain complexes and signal transduction, thus this region is essential for 0194 In a first example, the inventor plan to use one of CARMA1 regulating its localization at the membrane and its the kinases listed in Table 2 as system of split proteins which state of oligomerization. The N-terminal domain of once reconstituted (after co-localization of two CARs) will CARMA1 is responsible for the activation and recruitment phosphorylate the serine residues on the linker region of the (CARD) of different proteins. Indeed the CARD domain is CARMA 1 protein giving the start signal to the NF-KB and responsible for the interaction with B-cell CLL-lymphoma JNK. The generation of a split kinase has been already 10 (Bcl10) which per se mediates activation of NF-kB and reported with Success in the case of a thymidine kinase JNK. The N-terminal domain is structurally followed by a (Massoud, Paulmurugan et al. 2010). US 2017/0073423 A1 Mar. 16, 2017

TABLE 2 CARMA 1 Phosphorylation Sites (Thome, Charton et al. 2010). Human CARMA1 S109 T110 S551 S552 S555 S565 S608 S637 S645 Kinase CaMKII PKCd HPK1 PKC0 IKKB ? CK1C PKC PKC8 Effect of Mutation

NF-kB Normal JNK Normal 2

Scaffolding Protein Systems a constitutively LCK" (Y505->X505+Y394. On the con (0195 In addition to its carboxy-terminal MAGUK-like trary we could create a constitutively negatively regulated features, CARMA1 contains a CARD motif and a coiled coil LCK if we mutate the Y394 to any other residues (Y394 domain that are functionally crucial. CARD motifs are >X394 and Y505). This mutation should avoid the phos protein-protein interaction domains that can mediate homo phorylation of this position and the consequently it should typic CARD/CARD interactions between two, or possibly create a LCK. even three CARD containing binding partners. The CARD 0199 Thus we can plan a schema in which a first CAR of CARMA1 mediates homotypic interaction with the adap will recognize an antigen of an healthy cell with an inhibi tor Bcl10 which contains an amino-terminal CARD motif tory domain which will stimulate the transcription of a form and a Ser/Thr-rich carboxyl terminus of unknown structure. constitutively negatively regulated of LCK. This first Bcl10 constitutively forms a complex with MALT1 neces CAR will be coupled with a second one which contains a sary for the activation of JNK and NF-kB (see FIG. 21). co-stimulatory domains which will activate the transcription Hence a fully reconstituted CARMA1 can be obtained after of LCK" form, producing an high level of activation of the co-localization of two CARs once harboring the carboxy T cell (FIG. 20). terminal MAGUK-like features and a second one the CARD Example 3 motif and the coiled coil domain; the reconstitution of the CARMA1 will allow the assembly of Bcl10 and MALT1 Use of Environmental Condition (Hypoxia) to with the consequent activation of the two endogenous path Control HIF1C. (a.a. 380-603) mcCAR Fusion ways JNK and NF-kB. Surface Presentation mRNA Delivery Example 2 0200. The schematic functioning of the HIFO-system is depicted in FIG. 22 at both conditions (normoxia and Generation of Two Types of LCKs to Inhibit and to hypoxia). In FIG. 23 is shown different CAR architectures Stimulate the Signaling Cascade (single-chain and multi-chain). In the following experi (0196) LCK (NCBI Reference Sequence: NP 005347.3) ments, the multi-chain CAR conformation was used. is one of the first molecules to be activated following TCR Constructs and mRNA Preparation engagement (Borger, Filby et al. 2013; Borger, Zamoyska et 0201 All constructs originated from the pCLS24707 al. 2013; Brownlie and Zamoyska 2013). LCK is constitu (SEQID NO: 1) which encode the C-chain (SEQID NO: 2), tively active in T cell maintaining a low level of phospho |B-chain (SEQID NO:3) and Y-chain (SEQID NO: 4) of the rylation of the chain of CD3 associated to TCR. LCK binds multichain CAR (mcCAR). The sequence coding for the to the cytoplasmatic domain of CD8 following the interac amino acids 380 to 603 of the Hypoxia-inducible factor tion between TCR and peptide-MHC, the co-receptor CD8 1-alpha (HIF1 accession number Q16665, (SEQ ID NO. 5) drives LCK in proximity of the chain of CD3 associated was synthesized, in two parts, de novo (GeneCust) and to TCR. The targets of LCK are the tyrosine residues on the cloned, using classical molecular biology technics, down ITAM of the TCR associated chain of CD3 but also CD38 stream the C-chain, using a short -GS-linker (SEQ ID NO: chain, CD3e chain and ZAP70. The phosphorylation of 6) leading to pCLS26580 (SEQ ID NO: 7). ZAP70 promotes a conformational change which actives its 0202 All individual chains were amplified by PCR using kinase activity leading to the phosphorylation of LAT (adap oligo pairs C-chain-F/O-chain-R, 3-chain-F/B-chain-R, tor molecule linker for activation T cells). In turn Lat will Y-chain-F/y-chain-R and C.-chain-F/o-chain-HIF-R (SEQID recruit multiple downstream adaptors and signaling mol NO: 8 to 9) prior to mRNA synthesis. mRNA encoding the ecules. C-chain, B-chain, Y-chain or C-chain-HIF1 were in vitro 0.197 LCK is positively regulates by phosphorylation of transcribed from the PCR product and polyadenylated using an active tyrosine (394 aa) in the catalytic centre which the mMessage mMachine T7 Ultra kit (Life technologies) stabilizes an active conformation. On the contrary LCK is following the manufacturer's instructions. RNAs were puri also negatively regulated by phosphorylation of a tyrosine in fied with RNeasy columns (Qiagen), eluted in cytoporation its C-terminal domain (505 aa). The activating tyrosine medium T and quantified by measuring absorbance at 260 residue is auto phosphorylated by LCK and dephosphory nm using a Nanodrop ND-1000 spectrophotometer. Quality lated by CD45 and other phosphatase (such as PTPN6. of the RNA was verified on a denaturing formaldehyde/ PTPN22). The negatively regulating tyrosine is phosphory MOPS agarose gel. lated by CSK and dephosphorylated by CD45. 0198 The possibility to create a mutant of LCK which Transfection will be not phosphorylated on the C-terminal tyrosine (505 0203 T lymphocytes were transfected by electrontransfer aa) gives the possibility to engineered LCK in a way to have of messenger RNA using an AgilePulse MAX system (Har US 2017/0073423 A1 Mar. 16, 2017 vard Apparatus) 3 to 6 days after activation. Following Example 5 removal of activation beads, cells were pelleted, resus pended in cytoporation medium T at >28x106 cells/ml. Use of Environmental Condition (Hypoxia) to 5x106 cells were mixed with 27.5 ug total RNA (10 ug C. Control HIF1C. (a.a. 380-603) mcCAR Fusion chain, 7.5 ug B chain and 10 ugly chain) or with 32.5ug total Surface Presentation and Cytotoxicity—Lentiviral RNA (15ug C. chain-HIF1, 7.5ug B chain and 10 ugly chain) Delivery into a 0.4 cm cuvette. The electroporation consisted of two 0.1 ms pulses at 1200 V followed by four 0.2 ms pulses at 0208. The alpha-HIF, beta, gamma chains were amplified 130V. Following electroporation, cells were diluted into 2 by PCR, using oligo pairs GAC-chain-F/GAC.-chain-HIF-R, mL culture medium and incubated either at 37° C./5% CO GAB-chain-F/GAB-chain-R, GAY-chain-F/GAY-chain-R (referred as normoxia) or at 37°C. with low O. concentra respectively (SEQ ID NO: 15 to 20). The three chains were tion (referred as hypoxia) for 17 h. Hypoxic conditions were assembled, using the Gibbson assembly protocol (New created using an atmosphere generation system (2.5 L England Biolabs) in a lentiviral plasmid under the control of AnaeroJAR assembly, Anaerogen 2.5 L, Anaerobic indicator an SFFV promoter leading to pCLS26949 (SEQID NO: 21). Viral vectors were produced by GIGA-viral vectors (Bel BR0055 Oxoid) as described by the manufacturer. A fraction gium) from pCLS26949 (SEQ ID NO: 21) and pCLS24707 of the cells from the hypoxia condition were kept and (SEQ ID NO: 1) encoding the alpha chain without the HIF incubated at 37° C./5% CO (normoxia) for 4-6 h. domain. Flow Cytometry Surface Labelling 0204 First labelling for the detection of the C-chain was performed with anti-Fab'2-Biotin (goat anti-mouse IgG, 0209 Following lentiviral transduction, cells were incu Fab'2 fragment specific, 115-066-072, Jackson Immunore bated at 37° C./5% CO, (referred as normoxia). 3 to 10 days search) in PBSSVF2%, EDTA2 mM, azide 0.1% for 20 min post transduction, engineered T-cells were incubated either at 4°C. followed by a washing step with PBSSVF2% EDTA at 37° C./5% CO (referred as normoxia) or at 37° C. with 2 mM azide 0.1%. Second labelling was performed with low O. concentration (referred as hypoxia) for various time Streptavidin-APC in PBS SVF2% EDTA 2 mM azide 0.1% periods (1-24 hours). Hypoxic conditions were created as for 20 min at 4°C. followed by a washing step in PBS. Cell described by the manufacturers using either an atmosphere viability was monitored using the efluor450 (ebioscience generation system (2.5 L AnaeroJAR assembly, Anaerogen 65-0863-14) in PBS for 20 min 4°C., followed by a washing 2.5 L, Anaerobic indicator BR0055 Oxoid) or the Oxyrase step with PBS SVF2% EDTA 2 mM azide 0.1%. Flow Enzyme System (EC-Oxyrase) or combination of the two cytometry was performed using the MACSQUANT (Milte methods. Detection of surface presentation of the CAR was nyi Biotec) and data analysis was performed with the performed as described in Example 3. FlowJo software. 0205 The data obtained clearly indicated an improved Induced Cytotoxicity Surface exposition in hypoxic condition (VS normoxia) when 0210. The cytolytic activity and specificity of engineered the O.-chain was fused the HIF1C. fragment (FIG. 24). T-cells was assessed using a flow cytometry-based cytotox icity assay in hypoxia or normoxia. In this assay target cells Example 4 presenting the CAR target antigen (target--) and target cells not presenting the CAR target antigen (target-) are labelled Use of Environmental Condition (Hypoxia) to with either CelTraceTM CFSE or CellTraceTM Violet. The Prevent Cytotoxicity Induced by HIF1C. (a.a. mixed target cell populations (1:1 ratio) was co-incubate at 380-603) mcCAR Fusion 37°C. with various ratio of engineered effector CART cells (Effector/Target ratio of 10:1 to 1:1) in a final volume in 0206 Transfection of T-cells was performed as in X-Vivo-15 media, for various time periods (4 h to 24 h). example 1 with 2 ug total RNA (0.94 ug O. chain, 0.47 ug B 0211. The whole cell population was recovered and chain and 0.62 ugly chain). Surface detection was performed labeled with eFluor780 viability marker before being fixed in normoxia and hypoxia as described in Example 3 (FIG. by 4% PFA. Fixed cells were analyzed by flow cytometry to 25A). determine their viability (target--, target- and effector 0207. The cytolytic activity and specificity of engineered T-cells). Flow cytometry and data analysis were performed T-cells was assessed (1 day post transfection) using a flow as described in Example 3. cytometry-based cytotoxicity assay in normoxia. In this assay target cells presenting the CAR target antigen (target--) Example 6 and target cells not presenting the CAR target antigen (target-) are labelled with either CellTraceTM CFSE or Use of Environmental Condition (Hypoxia) to CellTraceTM violet. The mixed target cell populations (1:1 Control mcCAR Surface Presentation by ratio) was co-incubate at 37° C. with various ratio of Alternative HIF1C or HIF3C. Domains engineered effector CAR T cells (Effector/Target ratio of 10:1) in a final volume in X-Vivo-15 media, for a 4 h time 0212 All constructs originated from the pCLS24707 periods. The whole cell population was recovered and (SEQID NO: 1) which encode the C-chain (SEQID NO: 2), labeled with eFluor780 viability marker before being fixed |B-chain (SEQID NO:3) and Y-chain (SEQID NO: 4) of the by 4% PFA. Fixed cells were analyzed by flow cytometry to multichain CAR (mcCAR). The sequence coding for the determine their viability (target--, target- and effector amino acids 344 to 417 (SEQ ID NO:22) or 530-652 (SEQ T-cells). Flow cytometry and data analysis were performed ID NO. 23) of the Hypoxia-inducible factor 1-alpha (HIF1 as described in Example 3 (FIG. 25B). accession number Q16665) were assembled and cloned from US 2017/0073423 A1 Mar. 16, 2017 20 de novo synthesized genes (GeneCust) as in Example 1, 0221 For the first membrane protein partner: atgg-(a)- leading to pCLS26959 and pCLS26960 (SEQ ID NO: 24 to tecc: tecc-(b)-gata; gata-(c)caga; caga-(d)-gagc; gagc-(e1)- 25) respectively. gaat (FIG. 29). 0213. The sequence coding for the amino acids 480 to 0222 For the second membrane protein partner: atgg-(a)- 571 (SEQ ID NO: 26) or 466-571 (SEQ ID NO: 27) of the tecc: tecc-(b)-gata; gata-(c)caga; caga-(d)-gagc; gagc-(e2)- Hypoxia-inducible factor 3-alpha (HIF3 accession number tgga; tgga-(f)-tage; tagc-(g)-gaat (FIG. 29). Q9Y2N7) were assembled and cloned from de novo syn 0223) The different blocs are either synthesized de novo thesized genes (GeneCust) as in Example 3, leading to (GeneCust), assembled from oligos or amplified by PCR and pCLS26961 and pCLS26962 (SEQ ID NO: 28 to 29) inserted in either a puC57 or pET cloning vectors using respectively. classical molecular biology technics. Inserts containing the 0214. The sequence coding for the amino acids 380 to bloc of interest are amplified from the puC57 or plET by 630 of the Hypoxia-inducible factor 1-alpha (HIF1 acces PCR with oligonucleotides located a few dozen base pairs sion number Q16665, (SEQ ID NO: 5) was assembled and upstream and downstream the cloned position (SEQID NO: cloned from de novo synthesized genes (GeneCust), using 86 to 89). The PCR products are gel-purified and diluted to classical molecular biology technics, downstream the "/40 of their size in base pairs (ng/ul). The sequences coding C-chain, using a short-EA-linker (SEQ ID NO:30) leading for the membrane protein partners are assembled in a to pCLS26784 (SEQ ID NO:31). one-pot reaction by iterative rounds of restriction and liga 0215 Synthesis of mRNA, transfection, normoxia or tion (1 cycle: 37°C., 5 min, 45 cycles: 2 min at 37°C., 5 min hypoxia conditions, and flow cytometry in were generated at 16° C., 1 cycle: 5 min at 37° C., 1 cycle: 10 min at 80° and performed as described in Example 3. C. and 1 cycle: 2 min at 25° C.) in presence of an pre 0216. The data obtained clearly indicated an improved digested receiving plasmid (2 Jul of each bloc, 1 ul receiving Surface exposition in hypoxic condition (vs normoxia) with vector at 10 ng/ul, 1 ul ATP at 50 mM, 1 ul BbsI NEB, 1 uL the different fusions of HIF1C. and HIF3C fragment to the T4 Ligase (5 U/ul), 2 LLT4 Ligase Buffer 10x, total volume alpha chain (FIG. 26 A-F). 20 ul) (SEQID NO: 90) containing a kanamycin resistance gene marker. Examples of assembled membrane protein Example 7 partners are given (SEQ ID NO: 92 to 147). mRNA Preparation Design of a Dual Receptors Gate 0224. The sequence coding for the assembled membrane protein partners are sub-cloned (NcoI and HindIII) in a 0217. In a general aspect, the system is composed of two plasmid under the control of a T7 promoter (SEQ ID NO: membrane protein partners that are interacting upon co 202) using classical molecular biology technics. Alterna localization (triggered by the binding to the two target tively, the sequence coding for the assembled membrane antigens) and releasing a transmitter protein (FIG. 27). protein partners are amplified by PCR using oligonucle otides pairs bringing a T7 promoter (SEQ ID NO: 149 to Assembly of the Membrane Protein Partners 151) using classical molecular biology technics. Addition 0218. The first membrane protein partner is composed of ally, for the mcCAR-based membrane protein partner, the different blocs (from the N- to the C-termini): (a) a signal beta and gamma chains are amplified from pCLS24707 sequence for the addressing to the membrane and an anti (SEQ ID NO: 152) using oligonucleotides B-chain-F/B- gen-specific targeting regions (SEQID NO:32 to 38), (b) an chain-R and Y-chain-F/y-chain-R (SEQ ID NO: 153 to 156). extracellular spacer domain (so-called hinge) (SEQ ID NO: 0225 mRNA encoding the membrane protein partners 39 to 41), (c) a transmembrane domain (SEQ ID NO: 42 to are in vitro transcribed from the PCR product and polyade 46), and (d) an intracellular structural and/or signaling linker nylated using the mMessage mMachine T7 Ultra kit (Life domain (SEQ ID NO: 47 to 70) and (e1) one of the technologies) following the manufacturers instructions. interacting partner domains (SEQ ID NO: 71 to 77) (FIG. RNAs are purified with RNeasy columns (Qiagen), eluted in 28). cytoporation medium T and quantified by measuring absor 0219. The second membrane protein partner is composed bance at 260 nm using a Nanodrop ND-1000 spectropho of different blocs (from the N- to the C-termini): (a) a signal tometer. Quality of the RNA is verified on a denaturing sequence for the addressing to the membrane and an anti formaldehyde/MOPS agarose gel. gen-specific targeting regions (SEQID NO:32 to 38), (b) an Transfection extracellular spacer domain (so-called hinge) (SEQ ID NO: 39 to 41), (c) a transmembrane domain (SEQ ID NO: 42 to 0226 T lymphocytes are transfected by electrontransfer 46), (d) an intracellular structural and/or signaling linker of messenger RNA using an AgilePulse MAX system (Har domain (SEQID NO: 47 to 70), (e2) the second interacting vard Apparatus) 3 to 6 days after activation. Following partner domain (SEQ ID NO: 78 to 79), (f) a transcription removal of activation beads, cells are pelleted, resuspended factor composed of a DNA binding domain (SEQID NO: 80 in cytoporation medium T at >28x106 cells/ml. 5x106 cells to 81) and (g) a transactivation domain (SEQID NO: 82 to are mixed with 1 to 30 ug total RNA into a 0.4 cm cuvette. 82) (FIG. 28). The 2 parts of the split-ubiquitin system is the The electroporation consisted of two 0.1 ms pulses at 1200 Nub/Cub in human is depicted by SEQ ID NO:72-77 and V followed by four 0.2 ms pulses at 130V. Following NO:79 respectively. The 2 interactor domains TEV of pro electroporation, cells are diluted into 2 ml culture medium tease system from Tobacco Etch virus are depicted by SEQ and incubated either at 37° C./5% CO. ID NO:71 and 78. 0220. The blocs are designed to incorporate at each Flow Cytometry extremity a type IIs restriction sites (BbsI) that allows 0227 First labelling for the detection of the membrane enzymatic creation of unique overhangs for each position: protein partners is performed with anti-Fab'2-Biotin conju US 2017/0073423 A1 Mar. 16, 2017 gated (goat anti-mouse IgG, Fab'2 fragment specific, 115 tein partners, (ii) only one interacting membrane protein 066-072, Jackson Immunoresearch) in PBS SVF2%, EDTA partner and (iii) none of the interacting membrane protein 2 mM, azide 0.1% for 20 min at 4°C. followed by a washing partners. step with PBS SVF2% EDTA 2 mM azide 0.1%. Second labelling is performed with Streptavidin-APC in PBS Example 9 SVF2% EDTA 2 mM azide 0.1% for 20 min at 4° C. followed by a washing step in PBS. Cell viability is moni Knock-Out of Proteins Involved in the TCR tored using the efluor450 (ebioscience 65-0863-14) in PBS Signaling Pathway for 20 min 4° C., followed by a washing step with PBS SVF2% EDTA 2 mM azide 0.1%. Flow cytometry is per 0234. To create a T-cell custom readout system for the formed using the MACSQUANT (Miltenyi Biotec) and data dual membrane protein partners strategy, knock-outs of analysis is performed with the FlowJo software. genes coding for proteins involved in the TCR pathway (SEQ ID NO: 166 to 174) are realized using TALEN (SEQ 0228. Examples of surface exposition of different mem ID NO: 175 to 192). mRNA preparation and transfection is brane protein partners (SEQID NO: 96, 106, 110, 125, 126, performed as described in Example 7. TALEN activity in 128, 129 and 131) are given in FIG. 30. T-cells is monitored at the endogenous locus using the enzymatic T7 assay using conventional protocols. The data Example 8 obtained clearly indicated a high level of targeted mutagen esis at all targeted loci using the designed TALEN (FIG. 32). Dual Receptors Gate Readouts—Lentiviral Delivery 0235. The effect of the knock-out on the induced degranulation capacity of the engineered T-cell is assessed. 0229. To demonstrate the possibilities of the dual mem Engineered T-cells are cultured in 96-well plates (80,000 brane protein partners strategy, readouts based on the cells/well) in a final volume of 100 ul of X-VivoTM-15 expression of a reporter gene are built. These reporter medium (Lonza) for 6 hours at 37° C. with 5% CO. Cell systems are composed of several repeats of either the TetO stimulation is performed with either Human T-Activator (7x) or the Ga14 (5x) operator sequence that are placed CDP/CD28 beads (Life Technologies, #11132D) or PMA upstream of a minimal CMV promoter, allowing expression (20 ng/ml) and ionomycin (1 uM) or PHA (1.5 g/mL). of an RQR8 or a renilla reporter gene placed downstream of CD107a staining was done during cell stimulation, by the this artificial promoter leading to pCLS26301, pCLS26303, addition of an APC-conjugated anti-CD107a antibody (BD pCLS27049 and pCLS27050 (SEQ ID NO: 157 to 160). Biosciences) together with 1 g/ml of anti-CD49d (BD These construct are cloned in a lentiviral expression vector. Biosciences), 1 ug/ml of anti-CD28 (Miltenyi), and 1 x Viral vectors are prepared using the commercially available Monensin solution (eBioscience). After the 6 h incubation lentiviral expression systems according to the manufacturer period, cells were stained with a fixable viability dye (eBio protocols. science) and PE-conjugated anti-CD8 (Miltenyi) and ana 0230. To evaluate the possibility to monitor the expres lyzed by flow cytometry. The data obtained clearly indicated sion of the RQR8 gene, transactivators composed of a DNA a strong staining decrease for the knock-out engineered binding domain (TetO or Ga14) and a transcription activa T-cell relative to WT T-cells (FIG. 33). tion domain (VP64 or NF-kB) are constructed (SEQ ID NO: 161 to 164). Corresponding mRNAs are produced as Example 10 described in Example 7 and T-cells previously transduced with the reporter systems (readouts) are transfected with Complementation of Knock-Outs Using the these mRNAs coding for the transactivators. Membrane Protein Partners Strategy 0231. The data obtained clearly indicated the expression 0236. The gene coding for the KO protein (e.g. ZAP70) of the lentiviral delivered RQR8 cassette by mRNA trans (SEQ ID NO: 193) are cloned in the readout systems fection of the adequate transactivator (FIG. 31). described in Example 8 in place of the RQR8 or renilla 0232. The membrane protein partners are sub-cloned in a genes. Alternatively, target DNA sequences (SEQ ID NO: lentiviral plasmid under the control of an SFFV promoter 247) of human transcription factors (e.g.: HNF1B and (SEQ ID NO: 165). Alternatively, the assembled membrane HNF1A) (SEQ ID NO: 195 and 196) are cloned to replace protein partners are sub-cloned in a lentiviral production the TetO or the Ga14 operator sequence in the readouts. plasmid (under the control of an SFFV promoter (SEQ ID DNA sequences coding for these human transcription factors NO: 165)) upstream a 2A cis-acting hydrolase element (e.g.: HNF1B and HNF1A) are synthesized de novo to followed by a reporter marker (e.g. fluorescent proteins). create blocs (SEQ ID NO: 197 to 198) compatible with the Standard molecular biology technics such as PCR, enzy assembly process of membrane protein partners described in matic restriction digestion and ligation are applied to create Example 7. Design of TALEN used to perform knock-out of all constructions. Viral vectors are either obtained from genes (e.g. ZAP70), lentiviral vector production, mRNA commercial providers or prepared using commercially avail preparation, T-cell transfection or transduction of the read able lentiviral expression systems according to the manu outs and membrane protein partners is done as described in facturer protocols. Examples 7, 8 and 9. The complementation of the knock-out 0233. The two interacting membrane protein partners are is monitored using either the degranulation assay or a then delivered in T-cell previously transduced with the flow-based cytotoxicity assay in presence of target cells reporter systems (readouts) as either mRNA (Example 7) or presenting antigens for (i) both interacting membrane pro lentiviral vectors or combination of the two. Expression of tein partners, (ii) only one interacting membrane protein the reporter system is recorded in presence of target cells partner and (iii) none of the interacting membrane protein presenting antigens for (i) both interacting membrane pro partners. US 2017/0073423 A1 Mar. 16, 2017 22

Example 11 0248. The identity of both CARs is then confirmed by sequencing (or deep sequencing in case of libraries) using Design of a Bispecific CAR (biCAR) Gate standard molecular biology procedure. 0237 Assembly of the biCAR Partners REFERENCES 0238. The biCAR partners partner are composed of dif ferent blocs (from the N- to the C-termini): (a) a signal 0249 Andrianantoandro, E. S. Basu, et al. (2006). “Syn sequence for the addressing to the membrane and an anti thetic biology: new engineering rules for an emerging gen-specific targeting domain (b) an extracellular spacer discipline.” Mol Syst Biol 2: 2006 0028. domain (so-called hinge), (c) a transmembrane domain and (0250 Bauer, S. V. Groh, et al. (1999). “Activation of NK (d) an intracellular activation and/or costimulatory domain cells and T cells by NKG2D, a receptor for stress (FIG. 23). inducible MICA.” Science 285(5428): 727-9. 0239. The functioning of such biCAR gates is shown in (0251 Bierer, B. E., G. Hollander, et al. (1993). Figure “Cyclosporin A and FK506: molecular mechanisms of immunosuppression and probes for transplantation biol 0240 Antigen-specific targeting domains are selected ogy.” Curr Opin Immunol 5(5): 763-73. from pools of candidates either based on biochemical cri (0252) Blonska, M. and X. Lin (2009). “CARMA 1-medi teria (e.g. equilibrium dissociation constants (K), on- and ated NF-kappaB and JNK activation in lymphocytes.” off-rates (k, and k) or randomly as collections or libraries. Immunol Rev 228(1): 199-211. 0241 The biCARs are either synthesized de novo or (0253 Boch, J., H. Scholze, et al. (2009). “Breaking the assembled as in previous examples. mRNA preparation, code of DNA binding specificity of TAL-type III effec transfection and flow cytometry experiments are performed tors.” Science 326(5959): 1509-12. as in previous examples according to the manufacturer (0254 Borger, J. G. A. Filby, et al. (2013). “Differential recommendations. polarization of C-terminal Src kinase between naive and 0242. The biCAR partners are sub-cloned in a lentiviral antigen-experienced CD8+ T cells.' J Immunol 190(7): plasmid either under the control of the adequate promoter or 3O89-99. under the adequate promoter upstream a 2A cis-acting (0255 Borger, J. G., R. Zamoyska, et al. (2013). “Prox hydrolase element followed by a different reporter marker imity of TCR and its CD8 coreceptor controls sensitivity (e.g. fluorescent proteins) for each biCAR partners (one of T cells.” Immunol Lett 157(1-2): 16-22. reporter marker per library). Standard molecular biology (0256 Brownlie, R. J. and R. Zamoyska (2013). “T cell technics Such as PCR, enzymatic restriction digestion and receptor signalling networks: branched, diversified and ligation are applied to create all constructions. bounded.” Nat Rev Immunol 13(4): 257-69. 0243 Viral vectors of individual biCARs, collection of (0257 Chen, L. and D. B. Flies (2013). “Molecular biCARs or libraries of biCARs are either obtained from mechanisms of T cell co-stimulation and co-inhibition.” commercial providers or prepared using commercially avail Nat Rev Immunol 13(4): 227-42. able lentiviral expression systems according to the manu (0258 Doronina, V.A., C. Wu, et al. (2008). “Site-specific facturer protocols. release of nascent chains from ribosomes at a sense codon.” Mol Cell Biol 28(13): 4227-39. Example 12 (0259 Epa, V. C., O. Dolezal, et al. (2013). “Structural model for the interaction of a designed Ankyrin Repeat Characterization of biCAR Gate System in Protein with the human epidermal growth factor receptor Immortalized or in Primary T-Cells 2. PLoS One 8(3): e59163. 0260 Friedrich, K., J. R. Hanauer, et al. (2013). “DAR 0244 Both biCAR partners composing the biCAR gate Pin-targeting of measles virus: unique bispecificity, effec are delivered in immortalized human T-cells (Jurkat) or in tive oncolysis, and enhanced safety.” Mol Ther 21 (4): primary T-cells as lentiviral vectors either individually or as 849-59. libraries. 0261 Hara, S., Hamada J., Kobayashi C. Kondo Y., 0245. The transduced T-cells are purified for double Imura N (2001). “Expression and characterization of positive surface biCAR expression or double positive hypoxia-inducible factor (HIF)-3alpha in human kidney: reporter marker expression using bulk FACS sorting or suppression of HIF-mediated gene expression by HIF magnetic separation. 3alpha.” Biochem Biophys Res Commun.: 287(4):808-13 0246 The whole bulk double positive biCAR transduced 0262 Henderson, D. J. I. Naya, et al. (1991). “Compari population is then assessed for target cell driven activation son of the effects of FK-506, cyclosporin A and rapamycin (degranulation) using (i) a model cell line expressing only on IL-2 production.” Immunology 73(3): 316-21. the 1 CAR target antigens and (ii) a model cell line 0263 Jena, B., G. Dotti, et al. (2010). “Redirecting T-cell expressing only the 2" CAR target antigen. Populations that specificity by introducing a tumor-specific chimeric anti present no or weak activation induced by targets cells gen receptor.” Blood 116(7): 1035-44. presenting only one antigen are isolated bulk using FACS 0264. Jost, C. J. Schilling, et al. (2013). “Structural Basis Sorting or magnetic separation. for Eliciting a Cytotoxic Effect in HER2-Overexpressing 0247 These populations are then assessed for target cell Cancer Cells via Binding to the Extracellular Domain of driven activation (degranulation) using a model cell line HER2.' Structure 21 (11): 1979-91. expressing both CAR target antigens. Populations that pres 0265 Kloss, C. C., M. Condomines, et al. (2013). “Com ent medium or strong activation induced by targets cells binatorial antigen recognition with balanced signaling presenting both CAR antigens are isolated bulk using FACS promotes selective tumor eradication by engineered T Sorting or magnetic separation. cells.” Nat Biotechnol 31(1): 71-5. US 2017/0073423 A1 Mar. 16, 2017 23

0266 Liu, J., M. W. Albers, et al. (1992). “Inhibition of (0270 Roche, M. I., R. A. Ramadas, et al. (2013). “The T cell signaling by immunophilin-ligand complexes cor role of CARMA1 in T cells.” Crit Rev Immunol 33(3): relates with loss of calcineurin phosphatase activity.” 219-43. Biochemistry 31(16): 3896-901. (0271 Singleton, K. L., K. T. Roybal, et al. (2009). "Spatiotemporal patterning during T cell activation is 0267 Massoud, T. F., R. Paulmurugan, et al. (2010). “A highly diverse.” Sci Signal 2065): ra15. molecularly engineered split reporter for imaging protein (0272 Thome, M., J. E. Charton, et al. (2010). “Antigen protein interactions with positron emission tomography.” receptor signaling to NF-kappaB via CARMA1, BCL10, Nat Med 16(8): 921-6. and MALT1. " Cold Spring Harb Perspect Biol. 2(9): 0268 Moscou, M. J. and A. J. Bogdanove (2009). “A aOO3OO4. simple cipher governs DNA recognition by TAL effectors. (0273 Wang, Y. H. K. Y. Wei, et al. (2013). “Synthetic biology: advancing the design of diverse genetic sys Science 326(5959): 1501. tems.” Annu Rev Chem Biomol Eng 4: 69-102. 0269 Park, T. S., S.A. Rosenberg, et al. (2011). “Treating (0274 Wu, J. Y. Song, et al. (1999). “An activating cancer with genetically engineered T cells.' Trends Bio immunoreceptor complex formed by NKG2D and technol 29(11): 550-7. DAP10. "Science 285(5428): 730-2.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 198

SEO ID NO 1 LENGTH: 2541 TYPE: DNA ORGANISM: artificial sequence FEATURE; OTHER INFORMATION: pCLS 24707 <4 OOs SEQUENCE: 1 atgat Cocag cc.gtggit cot gctgctgctg. Ctgctggtgg agcaggcagc tigcactggga 60

gaaccc.cagc tigtgctacat cotggacgcc attctgttcc td tacggcat tdtgctgaca 12O Ctgctgtatt gtaggctgaa gatcCaggit C ccaaag.ccg ct attactt C at atgagaag 18O

agcc.gcgtga agttcagc.cg at CCCtgac go accagcat accagc aggg acagaaccag 24 O

Ctgtataacg agctgaatct gggacggaga gaggaatacg acgt.cctgga talagaggcgc 3 OO

ggCagggat C ctgaaatggg cqggaagcct cacggaaaa acccacagga ggggctgtac 360

aatgaactgc agaaggacaa aatggctgag gCatatagtgaaatcggaat galagggcgag 42O agaaggcgcg ggaaaggaca catggcctg taccaggggc tigtccaccgc cacaaaagac 48O

act tatgatg cactgcatat gcaggcc ctg. CCCC citcgcg gCagcggggc caccaact tc 54 O to cct gctga agcaggctgg agacgtggag gaaaatc.ccg gC cc tatggc tic cc.gcaatg 6 OO gag tocc ct a cactgctgtg cgtgg cactg. Ctgttctittg caccagatgg cqtgctggca 660 gaggit coagc tigcagcagtic aggaccagaa citgatcaaac ccggagcatc titgaaaatg 72O

agttgta agg cctoaggct a tactitt Cacc tottacgtga tigcactgggit Caagcagaaa 78O

CctggaCagg gcctggagtg gatcggctat attaatc.cat acaacgacgg gaccalagtac 84 O

aacgaaaagt ttaaaggcaa ggcaa.cactg act agtgata agagct cotc tactgcttac 9 OO atggagctga gttcactgac cagogalagac to cqctgtgt act attgcgc aagaggaacc 96.O tact attacg gCtct agggit gttcgattac toggggcagg galaccacact gacagt cagc 102O tc.cggaggag gaggat.ccgg aggaggaggg totggaggcg ggggaagttga catcgtgatg 108O

acacaggc.cg ct cotagcat tccagtgact cocggcgagt cagt cagoat ct cotgtcgg 114 O

totagtaaga gcctgctgaa ct coaatgga aacacat at C titactggitt totgcagagg 12 OO

Cctggcc agt ccc.cacagct gctgatctat cqcatgtcta acctggc.cag togcgtgc cc 1260

gat.cggttct ctggcagtgg gt caggalacc gcct ttacac taggattag cc.gcgt.cgag 132O

US 2017/0073423 A1 Mar. 16, 2017 25

- Continued Gly Ser Arg Val Phe Asp Tyr Trp Gly Glin Gly Thr Thr Lieu. Thr Val 13 O 135 14 O Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Asp Ile Val Met Thr Glin Ala Ala Pro Ser Ile Pro Val Thr Pro 1.65 17O 17s Gly Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Lieu. Lieu. Asn 18O 185 19 O Ser Asn Gly Asn Thr Tyr Lieu. Tyr Trp Phe Leu Glin Arg Pro Gly Glin 195 2OO 2O5 Ser Pro Glin Lieu. Lieu. Ile Tyr Arg Met Ser Asn Lieu Ala Ser Gly Val 21 O 215 22O Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Lieu. Arg 225 23 O 235 24 O Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Glin 245 250 255 His Leu Glu Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Lieu. Glu Lieu. 26 O 265 27 O Lys Arg Ala Asp Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 27s 28O 285 Pro Thir Ile Ala Ser Glin Pro Lieu. Ser Lieu. Arg Pro Glu Ala Cys Arg 29 O 295 3 OO Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Lieu. Asp Phe Ala Cys 3. OS 310 315 32O Asp Phe Phe Ile Pro Leu Lleu Val Val Ile Leu Phe Ala Val Asp Thr 3.25 330 335 Gly Leu Phe Ile Ser Thr Glin Glin Glin Val Thr Phe Lieu. Leu Lys Ile 34 O 345 35. O Lys Arg Thr Arg Lys Gly Phe Arg Lieu. Lieu. Asn. Pro His Pro Llys Pro 355 360 365

Asn Pro Lys Asn. Asn 37 O

<210s, SEQ ID NO 3 &211s LENGTH: 257 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: beta-chain

<4 OOs, SEQUENCE: 3 Met Asp Thr Glu Ser Asn Arg Arg Ala Asn Lieu Ala Lieu Pro Glin Glu 1. 5 1O 15

Pro Ser Ser Wall Pro Ala Phe Glu Wall Lieu. Glu Ile Ser Pro Glin Glu 2O 25 3O Val Ser Ser Gly Arg Lieu Lleu Lys Ser Ala Ser Ser Pro Pro Lieu. His 35 4 O 45

Thir Trp Lieu. Thr Val Lieu Lys Lys Glu Glin Glu Phe Leu Gly Val Thr SO 55 6 O

Glin Ile Lieu. Thir Ala Met Ile Cys Lieu. Cys Phe Gly Thr Val Val Cys 65 70 7s 8O Ser Val Lieu. Asp Ile Ser His Ile Glu Gly Asp Ile Phe Ser Ser Phe 85 90 95 US 2017/0073423 A1 Mar. 16, 2017 26

- Continued Lys Ala Gly Tyr Pro Phe Trp Gly Ala Ile Phe Phe Ser Ile Ser Gly 1OO 105 11 O Met Lieu. Ser Ile Ile Ser Glu Arg Arg Asn Ala Thr Tyr Lieu Val Arg 115 12 O 125 Gly Ser Lieu. Gly Ala Asn. Thir Ala Ser Ser Ile Ala Gly Gly Thr Gly 13 O 135 14 O Ile Thir Ile Lieu. Ile Ile Asn Lieu Lys Llys Ser Lieu Ala Tyr Ile His 145 150 155 160 Ile His Ser Cys Glin Llys Phe Phe Glu Thir Lys Cys Phe Met Ala Ser 1.65 17O 17s Phe Ser Thr Glu Ile Val Val Met Met Leu Phe Lieu. Thir Ile Leu Gly 18O 185 19 O Lieu. Gly Ser Ala Val Ser Lieu. Thir Ile Cys Gly Ala Gly Glu Glu Lieu. 195 2OO 2O5 Lys Gly Asn Llys Val Pro Glu Lys Arg Gly Arg Llys Llys Lieu. Lieu. Tyr 21 O 215 22O Ile Phe Lys Gln Pro Phe Met Arg Pro Val Glin Thr Thr Glin Glu Glu 225 23 O 235 24 O Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu 245 250 255

Lell

<210 SEQ ID NO 4 &211s LENGTH: 173 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: gamma-chain

<4 OOs, SEQUENCE: 4

Met Ile Pro Ala Wal Wall Lieu. Lieu. Lieu. Lieu. Lieu. Lieu Val Glu Glin Ala 1. 5 1O 15 Ala Ala Lieu. Gly Glu Pro Glin Lieu. Cys Tyr Ile Lieu. Asp Ala Ile Lieu 2O 25 3O Phe Lieu. Tyr Gly Ile Val Lieu. Thir Lieu. Lieu. Tyr Cys Arg Lieu Lys Ile 35 4 O 45 Glin Val Arg Lys Ala Ala Ile Thir Ser Tyr Glu Lys Ser Arg Val Lys SO 55 6 O Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Glin Glin Gly Glin Asn Glin 65 70 7s 8O Lieu. Tyr Asn. Glu Lieu. Asn Lieu. Gly Arg Arg Glu Glu Tyr Asp Val Lieu. 85 90 95 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Llys Pro Arg Arg 1OO 105 11 O

Lys Asn Pro Glin Glu Gly Lieu. Tyr Asn. Glu Lieu. Glin Lys Asp Llys Met 115 12 O 125

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 13 O 135 14 O Lys Gly His Asp Gly Lieu. Tyr Glin Gly Lieu. Ser Thr Ala Thir Lys Asp 145 150 155 160

Thir Tyr Asp Ala Lieu. His Met Glin Ala Lieu Pro Pro Arg 1.65 17O US 2017/0073423 A1 Mar. 16, 2017 27

- Continued

<210s, SEQ ID NO 5 &211s LENGTH: 224 212. TYPE: PRT <213> ORGANISM: homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: Hypoxia-inducible factor 1-alpha 380 - 603 <4 OOs, SEQUENCE: 5 Ser Glu Asp Thir Ser Ser Lieu. Phe Asp Llys Lieu Lys Lys Glu Pro Asp 1. 5 1O 15 Ala Lieu. Thir Lieu. Lieu Ala Pro Ala Ala Gly Asp Thir Ile Ile Ser Lieu 2O 25 3O Asp Phe Gly Ser Asn Asp Thr Glu Thir Asp Asp Glin Gln Lieu. Glu Glu 35 4 O 45 Val Pro Lieu. Tyr Asn Asp Wal Met Lieu Pro Ser Pro Asn. Glu Lys Lieu SO 55 6 O

Glin Asn. Ile Asn. Leu Ala Met Ser Pro Leu Pro Thir Ala Glu. Thir Pro 65 70 7s 8O Llys Pro Lieu. Arg Ser Ser Ala Asp Pro Ala Lieu. Asn Glin Glu Val Ala 85 90 95 Lieu Lys Lieu. Glu Pro Asn Pro Glu Ser Leu Glu Lieu Ser Phe Thr Met 1OO 105 11 O Pro Glin Ile Glin Asp Gln Thr Pro Ser Pro Ser Asp Gly Ser Thr Arg 115 12 O 125 Gln Ser Ser Pro Glu Pro Asn Ser Pro Ser Glu Tyr Cys Phe Tyr Val 13 O 135 14 O Asp Ser Asp Met Val Asn. Glu Phe Llys Lieu. Glu Lieu Val Glu Lys Lieu 145 150 155 160 Phe Ala Glu Asp Thr Glu Ala Lys Asn Pro Phe Ser Thr Glin Asp Thr 1.65 17O 17s Asp Lieu. Asp Lieu. Glu Met Lieu Ala Pro Tyr Ile Pro Met Asp Asp Asp 18O 185 19 O Phe Gln Leu Arg Ser Phe Asp Gln Leu Ser Pro Leu Glu Ser Ser Ser 195 2OO 2O5

Ala Ser Pro Glu Ser Ala Ser Pro Glin Ser Thir Wall. Thir Wall Phe Glin 21 O 215 22O

<210s, SEQ ID NO 6 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: - GS- linker

<4 OOs, SEQUENCE: 6 Ser Ser Gly Ser Gly Ser Gly Ser 1. 5

<210s, SEQ ID NO 7 &211s LENGTH: 605 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: pCLS2658O

<4 OO > SEQUENCE: 7 Met Ala Pro Ala Met Glu Ser Pro Thir Lieu. Lieu. Cys Val Ala Lieu. Lieu. 1. 5 1O 15 US 2017/0073423 A1 Mar. 16, 2017 28

- Continued

Phe Phe Ala Pro Asp Gly Val Lieu Ala Glu Val Glin Lieu. Glin Glin Ser 2O 25 3O Gly Pro Glu Lieu. Ile Llys Pro Gly Ala Ser Val Llys Met Ser Cys Llys 35 4 O 45 Ala Ser Gly Tyr Thr Phe Thir Ser Tyr Val Met His Trp Val Lys Glin SO 55 6 O Llys Pro Gly Glin Gly Lieu. Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn 65 70 7s 8O Asp Gly. Thir Lys Tyr Asn. Glu Lys Phe Lys Gly Lys Ala Thr Lieu. Thr 85 90 95 Ser Asp Llys Ser Ser Ser Thr Ala Tyr Met Glu Lieu Ser Ser Lieu. Thr 1OO 105 11 O Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Thr Tyr Tyr Tyr 115 12 O 125 Gly Ser Arg Val Phe Asp Tyr Trp Gly Glin Gly Thr Thr Lieu. Thr Val 13 O 135 14 O Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Asp Ile Val Met Thr Glin Ala Ala Pro Ser Ile Pro Val Thr Pro 1.65 17O 17s Gly Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Lieu. Lieu. Asn 18O 185 19 O Ser Asn Gly Asn Thr Tyr Lieu. Tyr Trp Phe Leu Glin Arg Pro Gly Glin 195 2OO 2O5 Ser Pro Glin Lieu. Lieu. Ile Tyr Arg Met Ser Asn Lieu Ala Ser Gly Val 21 O 215 22O Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Lieu. Arg 225 23 O 235 24 O Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Glin 245 250 255 His Leu Glu Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Lieu. Glu Lieu. 26 O 265 27 O Lys Arg Ala Asp Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 27s 28O 285 Pro Thir Ile Ala Ser Glin Pro Lieu. Ser Lieu. Arg Pro Glu Ala Cys Arg 29 O 295 3 OO Pro Ala Ala Gly Gly Ala Wal His Thr Arg Gly Lieu. Asp Phe Ala Cys 3. OS 310 315 32O Asp Phe Phe Ile Pro Leu Lleu Val Val Ile Leu Phe Ala Val Asp Thr 3.25 330 335 Gly Leu Phe Ile Ser Thr Glin Glin Glin Val Thr Phe Lieu. Leu Lys Ile 34 O 345 35. O Lys Arg Thr Arg Lys Gly Phe Arg Lieu. Lieu. Asn. Pro His Pro Llys Pro 355 360 365

Asn Pro Lys Asn. Asn. Ser Ser Gly Ser Gly Ser Gly Ser Ser Glu Asp 37 O 375 38O

Thir Ser Ser Lieu. Phe Asp Llys Lieu Lys Lys Glu Pro Asp Ala Lieu. Thir 385 390 395 4 OO

Lieu. Lieu Ala Pro Ala Ala Gly Asp Thir Ile Ile Ser Lieu. Asp Phe Gly 4 OS 41O 415 US 2017/0073423 A1 Mar. 16, 2017 29

- Continued

Ser Asn Asp Thr Glu Thir Asp Asp Glin Glin Lieu. Glu Glu Val Pro Lieu. 42O 425 43 O Tyr Asn Asp Wal Met Lieu Pro Ser Pro Asn. Glu Lys Lieu. Glin Asn. Ile 435 44 O 445 Asn Lieu Ala Met Ser Pro Leu Pro Thr Ala Glu Thr Pro Llys Pro Leu 450 45.5 460 Arg Ser Ser Ala Asp Pro Ala Lieu. Asn Glin Glu Val Ala Lieu Lys Lieu 465 470 47s 48O

Glu Pro Asn. Pro Glu Ser Lieu. Glu Lieu. Ser Phe Thir Met Pro Glin Ile 485 490 495 Gln Asp Gln Thr Pro Ser Pro Ser Asp Gly Ser Thr Arg Glin Ser Ser SOO 505 51O Pro Glu Pro Asn Ser Pro Ser Glu Tyr Cys Phe Tyr Val Asp Ser Asp 515 52O 525 Met Val Asn. Glu Phe Llys Lieu. Glu Lieu Val Glu Lys Lieu. Phe Ala Glu 53 O 535 54 O Asp Thr Glu Ala Lys Asn Pro Phe Ser Thr Glin Asp Thr Asp Lieu. Asp 5.45 550 555 560 Lieu. Glu Met Lieu Ala Pro Tyr Ile Pro Met Asp Asp Asp Phe Glin Lieu. 565 st O sts Arg Ser Phe Asp Gln Leu Ser Pro Leu Glu Ser Ser Ser Ala Ser Pro 58O 585 59 O

Gil Ser Ala Ser Pro Glin Ser Thr Wall. Thir Wall Phe Glin 595 6OO 605

<210s, SEQ ID NO 8 &211s LENGTH: 55 &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: alpha-chain-F

<4 OOs, SEQUENCE: 8 gcatcgtaat acgact cact at agggcagg ccaccatggc ticcc.gcaatig gag to 55

<210s, SEQ ID NO 9 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: alpha-chain-R <4 OOs, SEQUENCE: 9 t caattgttc ttggggtttg gt 22

<210s, SEQ ID NO 10 &211s LENGTH: 54 &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: beta-chain-F

<4 OOs, SEQUENCE: 10 gcatcgtaat acgact cact at agggcagg ccaccatgga cactgagtct aacc 54

<210s, SEQ ID NO 11 &211s LENGTH: 2O &212s. TYPE: DNA US 2017/0073423 A1 Mar. 16, 2017 30

- Continued ORGANISM: artificial sequence FEATURE: OTHER INFORMATION: beta-chain-R

SEQUENCE: 11 t cacagctica cagcct cott

SEQ ID NO 12 LENGTH: 55 TYPE: DNA ORGANISM: artificial sequence FEATURE: OTHER INFORMATION: gamma-chain-F

SEQUENCE: 12 gcatcgtaat acgact cact at agggcagg ccaccatgat CCC agcc.gtg gtc.ct 55

SEQ ID NO 13 LENGTH: 2O TYPE: DNA ORGANISM: artificial sequence FEATURE: OTHER INFORMATION: gamma-chain-R

SEQUENCE: 13 tCag.cgaggg ggCagggcct

SEQ ID NO 14 LENGTH: 44 TYPE: DNA ORGANISM: artificial sequence FEATURE: OTHER INFORMATION: alpha-chain-HIF-R

SEQUENCE: 14 acgtaagctt C9gcc.gct at Cactgaaa.ca cagtgacggit tac 44

SEO ID NO 15 LENGTH: 22 TYPE: DNA ORGANISM: artificial sequence FEATURE: OTHER INFORMATION: GAalpha-chain-F

SEQUENCE: 15 atggct cocg caatggagtic cc 22

SEQ ID NO 16 LENGTH: 46 TYPE: DNA ORGANISM: artificial sequence FEATURE: OTHER INFORMATION: GAalpha-chain-HIF-R

SEQUENCE: 16 ggat.cct cqt ccct ct cogc titc cctdaaa cacagtgacg gttgac 46

SEO ID NO 17 LENGTH: 24 TYPE: DNA ORGANISM: artificial sequence FEATURE: OTHER INFORMATION: GAbeta-chain-F

US 2017/0073423 A1 Mar. 16, 2017 33

- Continued

Ctagtgggcg cctgctgaag agtgccticaa goc CCCCtct gcacacttgg Ctgaccgtgc 312 O tgaagaaaga gcaggaattic ctgggagt ca CCC agat cot gacagctatg atttgcctgt 318O gttittggcac agtggtctgc agtgtgctgg acatctgaca tattgagggg gat at Cttct 324 O cct cittittaa gogotggg tac cctttittggg gag caat citt ctittagcatt to cqgaatgc 33 OO tgtcaatcat tagcgaaagg cqCaacgcaa Cat atctggit gcgaggaagc Ctgggcgcaa. 3360 at actgc.cag ttcaatcgcc ggcgggacag gcatc act at tctgat catt aacctgaaga 342O aaagcc tiggc tita catccac att cattcct gcc agaagtt ctittgagact aaatgtttca 3480 tggcct Cttt tagtaccgaa atcgtggit ca tatgctgtt cct gaccatt Ctggggctgg 354 O gatcc.gc.cgt gtc.tctgaca atctg.cggcg Ctggggagga actgaagggc aacaaggtoc 36OO cagaga agcg agggcggaag aaactgctgt at attittcaa acagcct titt atgagaccag 366 O tgcagaccac acaggaggaa gatggctgct cct gtaggitt toccgaggala gaggaaggag 372 O gctgtgagct gtga 3734

<210s, SEQ ID NO 22 &211s LENGTH: 74 212. TYPE: PRT <213> ORGANISM: homo sapiens 22 Os. FEATURE: 223 OTHER INFORMATION: 344-417 HIF1

<4 OOs, SEQUENCE: 22 Gly Ile Ile Gln His Asp Lieu. Ile Phe Ser Leu Gln Gln Thr Glu. Cys 1. 5 1O 15 Val Lieu Lys Pro Val Glu Ser Ser Asp Met Lys Met Thr Gln Leu Phe 2O 25 3O Thir Lys Val Glu Ser Glu Asp Thir Ser Ser Lieu. Phe Asp Llys Lieu Lys 35 4 O 45 Lys Glu Pro Asp Ala Lieu. Thir Lieu. Lieu Ala Pro Ala Ala Gly Asp Thr SO 55 6 O Ile Ile Ser Lieu. Asp Phe Gly Ser Asn Asp 65 70

<210s, SEQ ID NO 23 &211s LENGTH: 123 212. TYPE: PRT <213> ORGANISM: homo sapiens 22 Os. FEATURE: 223 OTHER INFORMATION: 53 O-652 HIF1

<4 OOs, SEQUENCE: 23 Glu Phe Llys Lieu. Glu Lieu Val Glu Lys Lieu. Phe Ala Glu Asp Thr Glu 1. 5 1O 15

Ala Lys Asn. Pro Phe Ser Thr Glin Asp Thr Asp Lieu. Asp Lieu. Glu Met 2O 25 3O

Lieu Ala Pro Tyr Ile Pro Met Asp Asp Asp Phe Glin Lieu. Arg Ser Phe 35 4 O 45

Asp Gln Lieu. Ser Pro Lieu. Glu Ser Ser Ser Ala Ser Pro Glu Ser Ala SO 55 6 O

Ser Pro Glin Ser Thir Wall. Thir Wall Phe Glin Glin. Thir Glin Ile Glin Glu 65 70 7s 8O

Pro Thr Ala Asn Ala Thir Thr Thr Thr Ala Thr Thr Asp Glu Lieu Lys 85 90 95 US 2017/0073423 A1 Mar. 16, 2017 34

- Continued

Thr Val Thir Lys Asp Arg Met Glu Asp Ile Lys Ile Lieu. Ile Ala Ser 1OO 105 11 O Pro Ser Pro Thr His Ile His Lys Glu Thir Thr 115 12 O

<210s, SEQ ID NO 24 &211s LENGTH: 45.5 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: pCLS2.6959 <4 OOs, SEQUENCE: 24 Met Ala Pro Ala Met Glu Ser Pro Thir Lieu. Lieu. Cys Val Ala Lieu. Lieu. 1. 5 1O 15 Phe Phe Ala Pro Asp Gly Val Lieu Ala Glu Val Glin Lieu. Glin Glin Ser 2O 25 3O Gly Pro Glu Lieu. Ile Llys Pro Gly Ala Ser Val Llys Met Ser Cys Llys 35 4 O 45 Ala Ser Gly Tyr Thr Phe Thir Ser Tyr Val Met His Trp Val Lys Glin SO 55 6 O Llys Pro Gly Glin Gly Lieu. Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn 65 70 7s 8O Asp Gly. Thir Lys Tyr Asn. Glu Lys Phe Lys Gly Lys Ala Thr Lieu. Thr 85 90 95 Ser Asp Llys Ser Ser Ser Thr Ala Tyr Met Glu Lieu Ser Ser Lieu. Thr 1OO 105 11 O Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Thr Tyr Tyr Tyr 115 12 O 125 Gly Ser Arg Val Phe Asp Tyr Trp Gly Glin Gly Thr Thr Lieu. Thr Val 13 O 135 14 O Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Asp Ile Val Met Thr Glin Ala Ala Pro Ser Ile Pro Val Thr Pro 1.65 17O 17s Gly Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Lieu. Lieu. Asn 18O 185 19 O Ser Asn Gly Asn Thr Tyr Lieu. Tyr Trp Phe Leu Glin Arg Pro Gly Glin 195 2OO 2O5 Ser Pro Glin Lieu. Lieu. Ile Tyr Arg Met Ser Asn Lieu Ala Ser Gly Val 21 O 215 22O Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Lieu. Arg 225 23 O 235 24 O

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Glin 245 250 255

His Leu Glu Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Lieu. Glu Lieu. 26 O 265 27 O

Lys Arg Ala Asp Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 27s 28O 285

Pro Thir Ile Ala Ser Glin Pro Lieu. Ser Lieu. Arg Pro Glu Ala Cys Arg 29 O 295 3 OO Pro Ala Ala Gly Gly Ala Wal His Thr Arg Gly Lieu. Asp Phe Ala Cys 3. OS 310 315 32O US 2017/0073423 A1 Mar. 16, 2017 35

- Continued

Asp Phe Phe Ile Pro Leu Lleu Val Val Ile Leu Phe Ala Val Asp Thr 3.25 330 335 Gly Leu Phe Ile Ser Thr Glin Glin Glin Val Thr Phe Lieu. Leu Lys Ile 34 O 345 35. O Lys Arg Thr Arg Lys Gly Phe Arg Lieu. Lieu. Asn. Pro His Pro Llys Pro 355 360 365 Asn Pro Lys Asn Asn Ser Ser Gly Ser Gly Ser Gly Ser Gly Ile Ile 37 O 375 38O Glin His Asp Lieu. Ile Phe Ser Lieu. Glin Glin Thr Glu. Cys Val Lieu Lys 385 390 395 4 OO Pro Val Glu Ser Ser Asp Met Lys Met Thr Gln Leu Phe Thr Llys Val 4 OS 41O 415 Glu Ser Glu Asp Thir Ser Ser Lieu. Phe Asp Llys Lieu Lys Lys Glu Pro 42O 425 43 O Asp Ala Lieu. Thir Lieu. Lieu Ala Pro Ala Ala Gly Asp Thir Ile Ile Ser 435 44 O 445 Lieu. Asp Phe Gly Ser Asn Asp 450 45.5

<210s, SEQ ID NO 25 &211s LENGTH: 504 212. TYPE: PRT <213> ORGANISM: artificial sequence & 22 O FEATURE; <223> OTHER INFORMATION: pCLS26960

<4 OOs, SEQUENCE: 25 Met Ala Pro Ala Met Glu Ser Pro Thir Lieu. Lieu. Cys Val Ala Lieu. Lieu. 1. 5 1O 15 Phe Phe Ala Pro Asp Gly Val Lieu Ala Glu Val Glin Lieu. Glin Glin Ser 2O 25 3O Gly Pro Glu Lieu. Ile Llys Pro Gly Ala Ser Val Llys Met Ser Cys Llys 35 4 O 45 Ala Ser Gly Tyr Thr Phe Thir Ser Tyr Val Met His Trp Val Lys Glin SO 55 6 O Llys Pro Gly Glin Gly Lieu. Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn 65 70 7s 8O Asp Gly. Thir Lys Tyr Asn. Glu Lys Phe Lys Gly Lys Ala Thr Lieu. Thr 85 90 95 Ser Asp Llys Ser Ser Ser Thr Ala Tyr Met Glu Lieu Ser Ser Lieu. Thr 1OO 105 11 O Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Thr Tyr Tyr Tyr 115 12 O 125 Gly Ser Arg Val Phe Asp Tyr Trp Gly Glin Gly Thr Thr Lieu. Thr Val 13 O 135 14 O Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160

Ser Asp Ile Val Met Thr Glin Ala Ala Pro Ser Ile Pro Val Thr Pro 1.65 17O 17s

Gly Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Lieu. Lieu. Asn 18O 185 19 O

Ser Asn Gly Asn Thr Tyr Lieu. Tyr Trp Phe Leu Glin Arg Pro Gly Glin 195 2OO 2O5 US 2017/0073423 A1 Mar. 16, 2017 36

- Continued

Ser Pro Glin Lieu. Lieu. Ile Tyr Arg Met Ser Asn Lieu Ala Ser Gly Val 21 O 215 22O Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Lieu. Arg 225 23 O 235 24 O Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Glin 245 250 255 His Leu Glu Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Lieu. Glu Lieu. 26 O 265 27 O Lys Arg Ala Asp Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 27s 28O 285 Pro Thir Ile Ala Ser Glin Pro Lieu. Ser Lieu. Arg Pro Glu Ala Cys Arg 29 O 295 3 OO Pro Ala Ala Gly Gly Ala Wal His Thr Arg Gly Lieu. Asp Phe Ala Cys 3. OS 310 315 32O Asp Phe Phe Ile Pro Leu Lleu Val Val Ile Leu Phe Ala Val Asp Thr 3.25 330 335 Gly Leu Phe Ile Ser Thr Glin Glin Glin Val Thr Phe Lieu. Leu Lys Ile 34 O 345 35. O Lys Arg Thr Arg Lys Gly Phe Arg Lieu. Lieu. Asn. Pro His Pro Llys Pro 355 360 365 Asn Pro Lys Asn Asn Ser Ser Gly Ser Gly Ser Gly Ser Glu Phe Lys 37 O 375 38O Lieu. Glu Lieu Val Glu Lys Lieu. Phe Ala Glu Asp Thr Glu Ala Lys Asn 385 390 395 4 OO Pro Phe Ser Thr Glin Asp Thr Asp Lieu. Asp Lieu. Glu Met Lieu Ala Pro 4 OS 41O 415 Tyr Ile Pro Met Asp Asp Asp Phe Glin Lieu. Arg Ser Phe Asp Glin Lieu. 42O 425 43 O

Ser Pro Lieu. Glu Ser Ser Ser Ala Ser Pro Glu Ser Ala Ser Pro Glin 435 44 O 445

Ser Thir Wall. Thir Wall Phe Glin Glin. Thir Glin Ile Glin Glu Pro Thir Ala 450 45.5 460 Asn Ala Thir Thr Thr Thr Ala Thr Thr Asp Glu Lieu Lys Thr Val Thr 465 470 47s 48O Lys Asp Arg Met Glu Asp Ile Lys Ile Lieu. Ile Ala Ser Pro Ser Pro 485 490 495 Thr His Ile His Lys Glu Thir Thr SOO

<210s, SEQ ID NO 26 &211s LENGTH: 92 212. TYPE: PRT <213> ORGANISM: homo sapiens 22 Os. FEATURE: 223 OTHER INFORMATION: 480-571 HIF3

<4 OOs, SEQUENCE: 26 Glin Asp Ala Asp Ala Lieu. Asp Lieu. Glu Met Lieu Ala Pro Tyr Ile Ser 1. 5 1O 15

Met Asp Asp Asp Phe Glin Lieu. Asn Ala Ser Glu Glin Lieu Pro Arg Ala 2O 25 3O Tyr His Arg Pro Lieu. Gly Ala Val Pro Arg Pro Arg Ala Arg Ser Phe 35 4 O 45 US 2017/0073423 A1 Mar. 16, 2017 37

- Continued

His Gly Lieu. Ser Pro Pro Ala Lieu. Glu Pro Ser Lieu. Lieu Pro Arg Trp SO 55 6 O Gly Ser Asp Pro Arg Lieu. Ser Cys Ser Ser Pro Ser Arg Gly Asp Pro 65 70 7s 8O Ser Ala Ser Ser Pro Met Ala Gly Ala Arg Lys Arg 85 90

<210s, SEQ ID NO 27 &211s LENGTH: 106 212. TYPE: PRT <213> ORGANISM: homo sapiens 22 Os. FEATURE: 223 OTHER INFORMATION: 466 - 571 HIF3

<4 OOs, SEQUENCE: 27 Gly Lys Asp Thr Glu Ala Val Glu Thir Asp Lieu. Asp Ile Ala Glin Asp 1. 5 1O 15 Ala Asp Ala Lieu. Asp Lieu. Glu Met Lieu Ala Pro Tyr Ile Ser Met Asp 2O 25 3O Asp Asp Phe Glin Lieu. Asn Ala Ser Glu Glin Lieu Pro Arg Ala Tyr His 35 4 O 45 Arg Pro Lieu. Gly Ala Val Pro Arg Pro Arg Ala Arg Ser Phe His Gly SO 55 6 O Lieu. Ser Pro Pro Ala Lieu. Glu Pro Ser Leu Lleu Pro Arg Trp Gly Ser 65 70 75 8O Asp Pro Arg Lieu. Ser Cys Ser Ser Pro Ser Arg Gly Asp Pro Ser Ala 85 90 95 Ser Ser Pro Met Ala Gly Ala Arg Lys Arg 1OO 105

<210s, SEQ ID NO 28 &211s LENGTH: 473 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: pCLS26961 <4 OOs, SEQUENCE: 28 Met Ala Pro Ala Met Glu Ser Pro Thir Lieu. Lieu. Cys Val Ala Lieu. Lieu. 1. 5 1O 15 Phe Phe Ala Pro Asp Gly Val Lieu Ala Glu Val Glin Lieu. Glin Glin Ser 2O 25 3O Gly Pro Glu Lieu. Ile Llys Pro Gly Ala Ser Val Llys Met Ser Cys Llys 35 4 O 45 Ala Ser Gly Tyr Thr Phe Thir Ser Tyr Val Met His Trp Val Lys Glin SO 55 6 O

Llys Pro Gly Glin Gly Lieu. Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn 65 70 7s 8O

Asp Gly. Thir Lys Tyr Asn. Glu Lys Phe Lys Gly Lys Ala Thr Lieu. Thr 85 90 95

Ser Asp Llys Ser Ser Ser Thr Ala Tyr Met Glu Lieu Ser Ser Lieu. Thr 1OO 105 11 O

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Thr Tyr Tyr Tyr 115 12 O 125

Gly Ser Arg Val Phe Asp Tyr Trp Gly Glin Gly Thr Thr Lieu. Thr Val US 2017/0073423 A1 Mar. 16, 2017 38

- Continued

13 O 135 14 O Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Asp Ile Val Met Thr Glin Ala Ala Pro Ser Ile Pro Val Thr Pro 1.65 17O 17s Gly Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Lieu. Lieu. Asn 18O 185 19 O Ser Asn Gly Asn Thr Tyr Lieu. Tyr Trp Phe Leu Glin Arg Pro Gly Glin 195 2OO 2O5 Ser Pro Glin Lieu. Lieu. Ile Tyr Arg Met Ser Asn Lieu Ala Ser Gly Val 21 O 215 22O Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Lieu. Arg 225 23 O 235 24 O Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Glin 245 250 255 His Leu Glu Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Lieu. Glu Lieu. 26 O 265 27 O Lys Arg Ala Asp Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 27s 28O 285 Pro Thir Ile Ala Ser Glin Pro Lieu. Ser Lieu. Arg Pro Glu Ala Cys Arg 29 O 295 3 OO Pro Ala Ala Gly Gly Ala Wal His Thr Arg Gly Lieu. Asp Phe Ala Cys 305 310 315 32O Asp Phe Phe Ile Pro Leu Lleu Val Val Ile Leu Phe Ala Val Asp Thr 3.25 330 335 Gly Leu Phe Ile Ser Thr Glin Glin Glin Val Thr Phe Lieu. Leu Lys Ile 34 O 345 35. O Lys Arg Thr Arg Lys Gly Phe Arg Lieu. Lieu. Asn. Pro His Pro Llys Pro 355 360 365 Asn Pro Lys Asn. Asn. Ser Ser Gly Ser Gly Ser Gly Ser Glin Asp Ala 37 O 375 38O Asp Ala Lieu. Asp Lieu. Glu Met Lieu Ala Pro Tyr Ile Ser Met Asp Asp 385 390 395 4 OO Asp Phe Glin Lieu. Asn Ala Ser Glu Gln Lieu Pro Arg Ala Tyr His Arg 4 OS 41O 415 Pro Lieu. Gly Ala Val Pro Arg Pro Arg Ala Arg Ser Phe His Gly Lieu. 42O 425 43 O Ser Pro Pro Ala Lieu. Glu Pro Ser Lieu. Lieu Pro Arg Trp Gly Ser Asp 435 44 O 445 Pro Arg Lieu Ser Cys Ser Ser Pro Ser Arg Gly Asp Pro Ser Ala Ser 450 45.5 460 Ser Pro Met Ala Gly Ala Arg Lys Arg 465 470

<210s, SEQ ID NO 29 &211s LENGTH: 487 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: pCLS26962

<4 OOs, SEQUENCE: 29 Met Ala Pro Ala Met Glu Ser Pro Thir Lieu. Lieu. Cys Val Ala Lieu. Lieu. US 2017/0073423 A1 Mar. 16, 2017 39

- Continued

1. 5 1O 15 Phe Phe Ala Pro Asp Gly Val Lieu Ala Glu Val Glin Lieu. Glin Glin Ser 2O 25 3O Gly Pro Glu Lieu. Ile Llys Pro Gly Ala Ser Val Llys Met Ser Cys Llys 35 4 O 45 Ala Ser Gly Tyr Thr Phe Thir Ser Tyr Val Met His Trp Val Lys Glin SO 55 6 O Llys Pro Gly Glin Gly Lieu. Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn 65 70 7s 8O Asp Gly. Thir Lys Tyr Asn. Glu Lys Phe Lys Gly Lys Ala Thr Lieu. Thr 85 90 95 Ser Asp Llys Ser Ser Ser Thr Ala Tyr Met Glu Lieu Ser Ser Lieu. Thr 1OO 105 11 O Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Thr Tyr Tyr Tyr 115 12 O 125 Gly Ser Arg Val Phe Asp Tyr Trp Gly Glin Gly Thr Thr Lieu. Thr Val 13 O 135 14 O Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Asp Ile Val Met Thr Glin Ala Ala Pro Ser Ile Pro Val Thr Pro 1.65 17O 17s Gly Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Lieu. Lieu. Asn 18O 185 19 O Ser Asn Gly Asn Thr Tyr Lieu. Tyr Trp Phe Leu Glin Arg Pro Gly Glin 195 2OO 2O5 Ser Pro Glin Lieu. Lieu. Ile Tyr Arg Met Ser Asn Lieu Ala Ser Gly Val 21 O 215 22O Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Lieu. Arg 225 23 O 235 24 O Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Glin 245 250 255 His Leu Glu Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Lieu. Glu Lieu. 26 O 265 27 O Lys Arg Ala Asp Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 27s 28O 285 Pro Thir Ile Ala Ser Glin Pro Lieu. Ser Lieu. Arg Pro Glu Ala Cys Arg 29 O 295 3 OO Pro Ala Ala Gly Gly Ala Wal His Thr Arg Gly Lieu. Asp Phe Ala Cys 3. OS 310 315 32O Asp Phe Phe Ile Pro Leu Lleu Val Val Ile Leu Phe Ala Val Asp Thr 3.25 330 335

Gly Leu Phe Ile Ser Thr Glin Glin Glin Val Thr Phe Lieu. Leu Lys Ile 34 O 345 35. O Lys Arg Thr Arg Lys Gly Phe Arg Lieu. Lieu. Asn. Pro His Pro Llys Pro 355 360 365

Asn Pro Lys Asn. Asn. Ser Ser Gly Ser Gly Ser Gly Ser Gly Lys Asp 37 O 375 38O

Thr Glu Ala Val Glu Thir Asp Lieu. Asp Ile Ala Glin Asp Ala Asp Ala 385 390 395 4 OO

Lieu. Asp Lieu. Glu Met Lieu Ala Pro Tyr Ile Ser Met Asp Asp Asp Phe 4 OS 41O 415 US 2017/0073423 A1 Mar. 16, 2017 40

- Continued

Glin Luell Asn Ala Ser Glu Glin Luell Pro Arg Ala Tyr His Arg Pro Luell 425 43 O

Gly Ala Wall Pro Arg Pro Arg Ala Arg Ser Phe His Gly Luell Ser Pro 435 44 O 445

Pro Ala Luell Glu Pro Ser Lell Luell Pro Arg Trp Gly Ser Asp Pro Arg 450 45.5 460

Lell Ser Ser Ser Pro Ser Arg Gly Asp Pro Ser Ala Ser Ser Pro 465 470 47s 48O

Met Ala Gly Ala Arg Lys Arg 485

SEQ ID NO 3 O LENGTH: TYPE : PRT ORGANISM: artificial sequence FEATURE: OTHER INFORMATION: -EA linker

<4 OOs, SEQUENCE: 3 O Glu Ala Ala Ala Arg Glu Ala Ala Ala Arg 1. 5 1O

SEQ ID NO 31 LENGTH: 609 TYPE : PRT ORGANISM; artificial sequence FEATURE: OTHER INFORMATION: pCLS26784

SEQUENCE: 31

Met Ala Pro Ala Met Glu Ser Pro Thir Luell Luell Wall Ala Luell Luell 1. 5 15

Phe Phe Ala Pro Asp Gly Wall Luell Ala Glu Wall Glin Lell Glin Glin Ser 2O 25

Gly Pro Glu Luell Ile Lys Pro Gly Ala Ser Wall Met Ser 35 4 O 45

Ala Ser Gly Thir Phe Thir Ser Wall Met His Trp Wall Glin SO 55 6 O

Lys Pro Gly Glin Gly Lell Glu Trp Ile Gly Tyr Ile Asn Pro Asn 65 70 8O

Asp Gly Thir Tyr Asn Glu Phe Lys Gly Ala Thir Luell Thir 85 90 95

Ser Asp Ser Ser Ser Thir Ala Tyr Met Glu Lell Ser Ser Luell Thir 1OO 105 11 O

Ser Glu Asp Ser Ala Wall Tyr Ala Arg Gly Thir 115 12 O 125

Gly Ser Arg Wall Phe Asp Tyr Trp Gly Glin Gly Thir Thir Luell Thir Wall 13 O 135 14 O

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160

Ser Asp Ile Wall Met Thir Glin Ala Ala Pro Ser Ile Pro Wall Thir Pro 1.65 17O 17s

Gly Glu Ser Wall Ser Ile Ser Arg Ser Ser Ser Luell Luell Asn 18O 185 19 O

Ser Asn Gly Asn Thir Tyr Lell Trp Phe Luell Glin Arg Pro Gly Glin US 2017/0073423 A1 Mar. 16, 2017 41

- Continued

195 2OO 2O5 Ser Pro Glin Lieu. Lieu. Ile Tyr Arg Met Ser Asn Lieu Ala Ser Gly Val 21 O 215 22O Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Lieu. Arg 225 23 O 235 24 O Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Glin 245 250 255 His Leu Glu Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Lieu. Glu Lieu. 26 O 265 27 O Lys Arg Ala Asp Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 27s 28O 285 Pro Thir Ile Ala Ser Glin Pro Lieu. Ser Lieu. Arg Pro Glu Ala Cys Arg 29 O 295 3 OO Pro Ala Ala Gly Gly Ala Wal His Thr Arg Gly Lieu. Asp Phe Ala Cys 3. OS 310 315 32O Asp Phe Phe Ile Pro Leu Lleu Val Val Ile Leu Phe Ala Val Asp Thr 3.25 330 335 Gly Leu Phe Ile Ser Thr Glin Glin Glin Val Thr Phe Lieu. Leu Lys Ile 34 O 345 35. O Lys Arg Thr Arg Lys Gly Phe Arg Lieu. Lieu. Asn. Pro His Pro Llys Pro 355 360 365 Asn Pro Lys Asn. Asn. Ser Arg Glu Ala Ala Ala Arg Glu Ala Ala Ala 370 375 380 Arg Ser Glu Asp Thir Ser Ser Lieu. Phe Asp Llys Lieu Lys Lys Glu Pro 385 390 395 4 OO Asp Ala Lieu. Thir Lieu. Lieu Ala Pro Ala Ala Gly Asp Thir Ile Ile Ser 4 OS 41O 415 Lieu. Asp Phe Gly Ser Asn Asp Thr Glu Thir Asp Asp Glin Glin Lieu. Glu 42O 425 43 O Glu Val Pro Leu Tyr Asn Asp Val Met Leu Pro Ser Pro Asn Glu Lys 435 44 O 445

Leul Glin Asn. Ile Asn. Leu Ala Met Ser Pro Leu Pro Thir Ala Glu Thir 450 45.5 460 Pro Llys Pro Lieu. Arg Ser Ser Ala Asp Pro Ala Lieu. Asn Glin Glu Val 465 470 47s 48O Ala Lieu Lys Lieu. Glu Pro Asn Pro Glu Ser Lieu. Glu Lieu. Ser Phe Thr 485 490 495 Met Pro Glin Ile Glin Asp Gln Thr Pro Ser Pro Ser Asp Gly Ser Thr SOO 505 51O Arg Glin Ser Ser Pro Glu Pro Asn Ser Pro Ser Glu Tyr Cys Phe Tyr 515 52O 525

Val Asp Ser Asp Met Val Asn. Glu Phe Llys Lieu. Glu Lieu Val Glu Lys 53 O 535 54 O

Lieu. Phe Ala Glu Asp Thr Glu Ala Lys Asn Pro Phe Ser Thr Glin Asp 5.45 550 555 560

Thir Asp Lieu. Asp Lieu. Glu Met Lieu Ala Pro Tyr Ile Pro Met Asp Asp 565 st O sts

Asp Phe Glin Lieu. Arg Ser Phe Asp Gln Lieu. Ser Pro Lieu. Glu Ser Ser 58O 585 59 O

Ser Ala Ser Pro Glu Ser Ala Ser Pro Glin Ser Thir Wall. Thir Wall Phe 595 6OO 605 US 2017/0073423 A1 Mar. 16, 2017 42

- Continued

Glin

<210s, SEQ ID NO 32 &211s LENGTH: 273 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223 OTHER INFORMATION: CD19 - ScFW - a

<4 OOs, SEQUENCE: 32

Met Ala Lieu. Pro Wall. Thir Ala Lieu. Lieu. Lieu Pro Leu Ala Lieu. Lieu. Lieu. 1. 5 1O 15 His Ala Ala Arg Pro Glu Val Glin Lieu. Glin Glin Ser Gly Pro Glu Lieu. 2O 25 3O Ile Llys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr 35 4 O 45 Thr Phe Thr Ser Tyr Val Met His Trp Val Lys Gln Lys Pro Gly Glin SO 55 6 O Gly Lieu. Glu Trp Ile Gly Tyr Ile ASn Pro Tyr Asn Asp Gly. Thir Lys 65 70 7s 8O Tyr Asn. Glu Lys Phe Lys Gly Lys Ala Thir Lieu. Thir Ser Asp Llys Ser 85 90 95 Ser Ser Thr Ala Tyr Met Glu Lieu Ser Ser Lieu. Thir Ser Glu Asp Ser 1OO 105 11 O Ala Val Tyr Tyr Cys Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Arg Val 115 12 O 125 Phe Asp Tyr Trp Gly Glin Gly Thr Thr Lieu. Thr Val Ser Ser Gly Gly 13 O 135 14 O Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val 145 150 155 160 Met Thr Glin Ala Ala Pro Ser Ile Pro Val Thr Pro Gly Glu Ser Val 1.65 17O 17s Ser Ile Ser Cys Arg Ser Ser Lys Ser Lieu. Lieu. Asn. Ser Asn Gly Asn 18O 185 19 O Thr Tyr Lieu. Tyr Trp Phe Leu Glin Arg Pro Gly Glin Ser Pro Gln Leu 195 2OO 2O5 Lieu. Ile Tyr Arg Met Ser Asn Lieu Ala Ser Gly Val Pro Asp Arg Phe 21 O 215 22O Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Lieu. Arg Ile Ser Arg Val 225 23 O 235 24 O Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His Lieu. Glu Tyr 245 250 255 Pro Phe Thr Phe Gly Ala Gly. Thir Lys Lieu. Glu Lieu Lys Arg Ser Asp 26 O 265 27 O

Pro

<210s, SEQ ID NO 33 &211s LENGTH: 267 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223 OTHER INFORMATION: ST4 - ScFW

<4 OOs, SEQUENCE: 33 US 2017/0073423 A1 Mar. 16, 2017 43

- Continued

Met Ala Lieu. Pro Wall. Thir Ala Lieu. Lieu. Lieu Pro Leu Ala Lieu. Lieu. Lieu. 1. 5 1O 15 His Ala Ala Arg Pro Glu Val Glin Lieu. Glin Glin Ser Gly Pro Asp Lieu. 2O 25 3O Val Llys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr 35 4 O 45 Ser Phe Thr Gly Tyr Tyr Met His Trp Val Lys Glin Ser His Gly Lys SO 55 6 O Ser Lieu. Glu Trp Ile Gly Arg Ile Asn Pro Asn. Asn Gly Val Thir Lieu. 65 70 7s 8O Tyr Asn Glin Llys Phe Lys Asp Lys Ala Ile Lieu. Thr Val Asp Llys Ser 85 90 95 Ser Thir Thr Ala Tyr Met Glu Lieu. Arg Ser Lieu. Thir Ser Glu Asp Ser 1OO 105 11 O Ala Val Tyr Tyr Cys Ala Arg Ser Thr Met Ile Thr Asn Tyr Val Met 115 12 O 125 Asp Tyr Trp Gly Glin Val Thir Ser Val Thr Val Ser Ser Gly Gly Gly 13 O 135 14 O Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ile Val Met 145 150 155 160 Thr Glin Thr Pro Thr Phe Lieu. Leu Val Ser Ala Gly Asp Arg Val Thr 1.65 17O 17s Ile Thr Cys Lys Ala Ser Glin Ser Val Ser ASn Asp Val Ala Trp Tyr 18O 185 19 O Gln Glin Llys Pro Gly Glin Ser Pro Thr Lieu. Lieu. Ile Ser Tyr Thr Ser 195 2OO 2O5 Ser Arg Tyr Ala Gly Val Pro Asp Arg Phe Ile Gly Ser Gly Tyr Gly 21 O 215 22O Thr Asp Phe Thr Phe Thir Ile Ser Thr Lieu. Glin Ala Glu Asp Leu Ala 225 23 O 235 24 O Val Tyr Phe Cys Glin Glin Asp Tyr Asn Ser Pro Pro Thr Phe Gly Gly 245 250 255 Gly. Thir Lys Lieu. Glu Ile Lys Arg Ser Asp Pro 26 O 265

<210s, SEQ ID NO 34 &211s LENGTH: 265 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223 OTHER INFORMATION: RORI- ScFW

<4 OOs, SEQUENCE: 34

Met Ala Lieu. Pro Wall. Thir Ala Lieu. Lieu. Lieu Pro Leu Ala Lieu. Lieu. Lieu. 1. 5 1O 15 His Ala Ala Arg Pro Glin Val Glin Lieu. Glin Glin Ser Gly Ala Glu Lieu. 2O 25 3O

Val Arg Pro Gly Ala Ser Val Thir Lieu. Ser Cys Lys Ala Ser Gly Tyr 35 4 O 45 Thr Phe Ser Asp Tyr Glu Met His Trp Val Ile Glin Thr Pro Val His SO 55 6 O

Gly Lieu. Glu Trp Ile Gly Ala Ile Asp Pro Glu Thr Gly Gly Thr Ala 65 70 7s 8O US 2017/0073423 A1 Mar. 16, 2017 44

- Continued Tyr Asn Glin Llys Phe Lys Gly Lys Ala Ile Lieu. Thir Ala Asp Llys Ser 85 90 95 Ser Ser Thr Ala Tyr Met Glu Lieu. Arg Ser Lieu. Thir Ser Glu Asp Ser 1OO 105 11 O Ala Val Tyr Tyr Cys Thr Gly Tyr Tyr Asp Tyr Asp Ser Phe Thr Tyr 115 12 O 125 Trp Gly Glin Gly Thr Lieu Val Thr Val Ser Ala Gly Gly Gly Gly Ser 13 O 135 14 O Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Glin 145 150 155 160 Ser Gln Lys Ile Met Ser Thr Thr Val Gly Asp Arg Val Ser Ile Thr 1.65 17O 17s Cys Lys Ala Ser Glin Asn. Wall Asp Ala Ala Val Ala Trp Tyr Glin Glin 18O 185 19 O Llys Pro Gly Glin Ser Pro Llys Lieu. Lieu. Ile Tyr Ser Ala Ser Asn Arg 195 2OO 2O5 Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 21 O 215 22O Phe Thr Lieu. Thir Ile Ser Asn Met Glin Ser Glu Asp Leu Ala Asp Tyr 225 23 O 235 24 O Phe Cys Glin Glin Tyr Asp Ile Tyr Pro Tyr Thr Phe Gly Gly Gly Thr 245 250 255 Llys Lieu. Glu Ile Lys Arg Ser Asp Pro 26 O 265

<210s, SEQ ID NO 35 &211s LENGTH: 270 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223 OTHER INFORMATION: CD19 - ScFw -

<4 OOs, SEQUENCE: 35

Met Ala Lieu. Pro Wall. Thir Ala Lieu. Lieu. Lieu Pro Leu Ala Lieu. Lieu. Lieu. 1. 5 1O 15 His Ala Ala Arg Pro Glu Wall Lys Lieu. Glin Glu Ser Gly Pro Gly Lieu. 2O 25 3O Val Ala Pro Ser Glin Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val 35 4 O 45 Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys SO 55 6 O Gly Lieu. Glu Trp Lieu. Gly Val Ile Trp Gly Ser Glu Thir Thr Tyr Tyr 65 70 7s 8O Asn Ser Ala Lieu Lys Ser Arg Lieu. Thir Ile Ile Lys Asp Asn. Ser Lys 85 90 95

Ser Glin Val Phe Lieu Lys Met Asn. Ser Lieu. Glin Thr Asp Asp Thir Ala 1OO 105 11 O Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met 115 12 O 125

Asp Tyr Trp Gly Glin Gly. Thir Ser Val Thr Val Ser Ser Gly Gly Gly 13 O 135 14 O Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glin Met 145 150 155 160 US 2017/0073423 A1 Mar. 16, 2017 45

- Continued

Thr Glin Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr 1.65 17O 17s Ile Ser Cys Arg Ala Ser Glin Asp Ile Ser Lys Tyr Lieu. Asn Trp Tyr 18O 185 19 O Gln Glin Llys Pro Asp Gly Thr Val Lys Lieu. Lieu. Ile Tyr His Thr Ser 195 2OO 2O5 Arg Lieu. His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 21 O 215 22O Thir Asp Tyr Ser Lieu. Thir Ile Ser Asn Lieu. Glu Glin Glu Asp Ile Ala 225 23 O 235 24 O Thr Tyr Phe Cys Glin Glin Gly Asn Thr Lieu Pro Tyr Thr Phe Gly Gly 245 250 255 Gly Thr Lys Lieu. Glu Ile Thr Arg Ser Asp Pro Thr Ser Ser 26 O 265 27 O

<210s, SEQ ID NO 36 &211s LENGTH: 282 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223 OTHER INFORMATION: CD123 - ScFW

<4 OOs, SEQUENCE: 36

Met Ala Lieu. Pro Wall. Thir Ala Lieu. Lieu. Lieu Pro Leu Ala Lieu. Lieu. Lieu. 1. 5 1O 15 His Ala Ala Arg Pro Glu Wall Lys Lieu Val Glu Ser Gly Gly Gly Lieu. 2O 25 3O Val Glin Pro Gly Gly Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Phe 35 4 O 45 Thr Phe Thr Asp Tyr Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys SO 55 6 O Ala Lieu. Glu Trp Lieu Ala Lieu. Ile Arg Ser Lys Ala Asp Gly Tyr Thr 65 70 7s 8O Thr Glu Tyr Ser Ala Ser Wall Lys Gly Arg Phe Thr Lieu. Ser Arg Asp 85 90 95 Asp Ser Glin Ser Ile Lieu. Tyr Lieu Gln Met Asn Ala Lieu. Arg Pro Glu 1OO 105 11 O Asp Ser Ala Thr Tyr Tyr Cys Ala Arg Asp Ala Ala Tyr Tyr Ser Tyr 115 12 O 125 Tyr Ser Pro Glu Gly Ala Met Asp Tyr Trp Gly Glin Gly Thr Ser Val 13 O 135 14 O Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 145 150 155 160 Gly Gly Ser Met Ala Asp Tyr Lys Asp Ile Val Met Thr Glin Ser His 1.65 17O 17s

Llys Phe Met Ser Thr Ser Val Gly Asp Arg Val Asn Ile Thr Cys Lys 18O 185 19 O

Ala Ser Glin Asn Val Asp Ser Ala Val Ala Trp Tyr Glin Glin Llys Pro 195 2OO 2O5

Gly Glin Ser Pro Lys Ala Lieu. Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser 21 O 215 22O

Gly Val Pro Asp Arg Phe Thr Gly Arg Gly Ser Gly Thr Asp Phe Thr 225 23 O 235 24 O US 2017/0073423 A1 Mar. 16, 2017 46

- Continued

Lieu. Thir Ile Ser Ser Val Glin Ala Glu Asp Lieu Ala Val Tyr Tyr Cys 245 250 255 Gln Glin Tyr Tyr Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Lieu. 26 O 265 27 O Glu Ile Lys Arg Ser Asp Pro Thr Ser Ser 27s 28O

<210s, SEQ ID NO 37 &211s LENGTH: 267 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223 OTHER INFORMATION: CD33 - ScFW

<4 OO > SEQUENCE: 37

Met Ala Lieu. Pro Wall. Thir Ala Lieu. Lieu. Lieu Pro Leu Ala Lieu. Lieu. Lieu. 1. 5 1O 15 His Ala Ala Arg Pro Glu Val Glin Lieu. Glin Glin Ser Gly Pro Glu Lieu. 2O 25 3O Val Llys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr 35 4 O 45 Thr Phe Thr Asp Tyr Asn Met His Trp Val Lys Glin Ser His Gly Lys SO 55 6 O Ser Lieu. Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly 65 70 7s 8O Tyr Asn Glin Llys Phe Llys Ser Lys Ala Thir Lieu. Thr Val Asp Asn. Ser 85 90 95 Ser Ser Thr Ala Tyr Met Asp Val Arg Ser Lieu. Thir Ser Glu Asp Ser 1OO 105 11 O Ala Val Tyr Tyr Cys Ala Arg Gly Arg Pro Ala Met Asp Tyr Trp Gly 115 12 O 125 Gln Gly. Thir Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 13 O 135 14 O Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Lieu. Thr Glin Ser Pro 145 150 155 160 Ala Ser Lieu Ala Val Ser Lieu. Gly Glin Arg Ala Thir Ile Ser Cys Arg 1.65 17O 17s Ala Ser Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe 18O 185 19 O Glin Glin Llys Pro Gly Glin Pro Pro Llys Lieu. Lieu. Ile Tyr Ala Ala Ser 195 2OO 2O5 Asn Glin Gly Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly 21 O 215 22O Thir Asp Phe Ser Lieu. Asn. Ile His Pro Met Glu Glu Asp Asp Thir Ala 225 23 O 235 24 O

Met Tyr Phe Cys Glin Glin Ser Lys Glu Val Pro Trp Thr Phe Gly Gly 245 250 255

Gly. Thir Lys Lieu. Glu Ile Lys Arg Ser Asp Pro 26 O 265

<210s, SEQ ID NO 38 &211s LENGTH: 277 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: US 2017/0073423 A1 Mar. 16, 2017 47

- Continued

223 OTHER INFORMATION: CD19 - ScFW - c.

<4 OOs, SEQUENCE: 38 Met Ala Pro Ala Met Glu Ser Pro Thir Lieu. Lieu. Cys Val Ala Lieu. Lieu. 1. 5 1O 15 Phe Phe Ala Pro Asp Gly Val Lieu Ala Glu Val Glin Lieu. Glin Glin Ser 2O 25 3O Gly Pro Glu Lieu. Ile Llys Pro Gly Ala Ser Val Llys Met Ser Cys Llys 35 4 O 45 Ala Ser Gly Tyr Thr Phe Thir Ser Tyr Val Met His Trp Val Lys Glin SO 55 6 O Llys Pro Gly Glin Gly Lieu. Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn 65 70 7s 8O Asp Gly. Thir Lys Tyr Asn. Glu Lys Phe Lys Gly Lys Ala Thr Lieu. Thr 85 90 95 Ser Asp Llys Ser Ser Ser Thr Ala Tyr Met Glu Lieu Ser Ser Lieu. Thr 1OO 105 11 O Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Thr Tyr Tyr Tyr 115 12 O 125 Gly Ser Arg Val Phe Asp Tyr Trp Gly Glin Gly Thr Thr Lieu. Thr Val 13 O 135 14 O Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Asp Ile Val Met Thr Glin Ala Ala Pro Ser Ile Pro Val Thr Pro 1.65 17O 17s Gly Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Lieu. Lieu. Asn 18O 185 19 O Ser Asn Gly Asn Thr Tyr Lieu. Tyr Trp Phe Leu Glin Arg Pro Gly Glin 195 2OO 2O5 Ser Pro Glin Lieu. Lieu. Ile Tyr Arg Met Ser Asn Lieu Ala Ser Gly Val 21 O 215 22O Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Lieu. Arg 225 23 O 235 24 O Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Glin 245 250 255 His Leu Glu Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Lieu. Glu Lieu. 26 O 265 27 O Lys Arg Ser Asp Pro 27s

<210s, SEQ ID NO 39 &211s LENGTH: 45 212. TYPE: PRT <213> ORGANISM: homo sapiens 22 Os. FEATURE: 223 OTHER INFORMATION: CD8a TM

<4 OOs, SEQUENCE: 39 Thir Thir Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1. 5 1O 15

Ser Glin Pro Lieu. Ser Lieu. Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 2O 25 3O Gly Ala Wal His Thr Arg Gly Lieu. Asp Phe Ala Cys Asp 35 4 O 45 US 2017/0073423 A1 Mar. 16, 2017 48

- Continued

<210s, SEQ ID NO 4 O &211s LENGTH: 233 212. TYPE: PRT <213> ORGANISM: homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: IgG1 TM <4 OOs, SEQUENCE: 4 O Glu Pro Llys Ser Pro Asp Llys Thr His Thr Cys Pro Pro Cys Pro Ala 1. 5 1O 15 Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Llys Pro Llys 2O 25 3O Asp Thr Lieu Met Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val 35 4 O 45 Asp Wal Ser His Glu Asp Pro Glu Val Llys Phe Asn Trp Tyr Val Asp SO 55 6 O Gly Val Glu Val His Asn Ala Lys Thr Llys Pro Arg Glu Glu Glin Tyr 65 70 7s 8O Asn Ser Thr Tyr Arg Val Val Ser Val Lieu. Thr Val Lieu. His Glin Asp 85 90 95 Trp Lieu. Asn Gly Lys Glu Tyr Lys Cys Llys Val Ser Asn Lys Ala Lieu. 1OO 105 11 O Pro Ala Pro Ile Glu Lys Thir Ile Ser Lys Ala Lys Gly Glin Pro Arg 115 120 125 Glu Pro Glin Val Tyr Thr Lieu Pro Pro Ser Arg Asp Glu Lieu. Thir Lys 13 O 135 14 O Asn Glin Val Ser Lieu. Thr Cys Lieu Val Lys Gly Phe Tyr Pro Ser Asp 145 150 155 160 Ile Ala Val Glu Trp Glu Ser Asn Gly Glin Pro Glu Asn. Asn Tyr Lys 1.65 17O 17s Thir Thr Pro Pro Val Lieu. Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 18O 185 19 O Llys Lieu. Thr Val Asp Llys Ser Arg Trp Glin Glin Gly Asn Val Phe Ser 195 2OO 2O5 Cys Ser Val Met His Glu Ala Lieu. His Asn His Tyr Thr Glin Lys Ser 21 O 215 22O Lieu. Ser Lieu. Ser Pro Gly Llys Lys Asp 225 23 O

<210s, SEQ ID NO 41 &211s LENGTH: 101 212. TYPE: PRT <213> ORGANISM: homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: EpoR D2 TM

<4 OOs, SEQUENCE: 41 Ala Pro Val Gly Lieu Val Ala Arg Lieu Ala Asp Glu Ser Gly. His Val 1. 5 1O 15

Val Lieu. Arg Trp Leu Pro Pro Pro Glu Thr Pro Met Thr Ser His Ile 2O 25 3O

Arg Tyr Glu Val Asp Val Ser Ala Gly Asin Gly Ala Gly Ser Val Glin 35 4 O 45

Arg Val Glu Ile Lieu. Glu Gly Arg Thr Glu. CyS Val Lieu. Ser Asn Lieu US 2017/0073423 A1 Mar. 16, 2017 49

- Continued

SO 55 6 O Arg Gly Arg Thr Arg Tyr Thr Phe Ala Val Arg Ala Arg Met Ala Glu 65 70 7s 8O Pro Ser Phe Gly Gly Phe Trp Ser Ala Trp Ser Glu Pro Val Ser Lieu. 85 90 95 Lieu. Thr Pro Ser Asp 1OO

<210s, SEQ ID NO 42 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: homo sapiens 22 Os. FEATURE: 223 OTHER INFORMATION: CD8a TM

<4 OOs, SEQUENCE: 42 Ile Tyr Ile Trp Ala Pro Lieu Ala Gly Thr Cys Gly Val Lieu. Lieu. Lieu. 1. 5 1O 15 Ser Leu Val Ile Thr Lieu. Tyr Cys Arg 2O 25

<210s, SEQ ID NO 43 &211s LENGTH: 31 212. TYPE: PRT <213> ORGANISM: homo sapiens 22 Os. FEATURE: & 223 OTHER INFORMATION: 4 - 1BB TM

<4 OOs, SEQUENCE: 43

Ile Ile Ser Phe Phe Leu Ala Lieu. Thir Ser Thir Ala Leu Lleu Phe Lieu. 1. 5 1O 15 Lieu. Phe Phe Lieu. Thir Lieu. Arg Phe Ser Val Val Lys Arg Gly Arg 2O 25 3O

<210s, SEQ ID NO 44 &211s LENGTH: 27 212. TYPE: PRT <213> ORGANISM: homo sapiens 22 Os. FEATURE: 223 OTHER INFORMATION: DAP10 TM

<4 OOs, SEQUENCE: 44 Ile Lieu. Lieu Ala Gly Lieu Val Ala Ala Asp Ala Val Ala Ser Lieu. Lieu. 1. 5 1O 15 Ile Val Gly Ala Val Phe Lieu. Cys Ala Arg Arg 2O 25

<210s, SEQ ID NO 45 &211s LENGTH: 33 212. TYPE: PRT <213> ORGANISM: homo sapiens 22 Os. FEATURE: 223 OTHER INFORMATION: CD28 TM

<4 OOs, SEQUENCE: 45 Ile Phe Trp Val Lieu Val Val Val Gly Gly Val Lieu Ala Cys Tyr Ser 1. 5 1O 15

Lieu. Lieu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg 2O 25 3O

Arg US 2017/0073423 A1 Mar. 16, 2017 50

- Continued

SEQ ID NO 46 LENGTH: 54 TYPE : PRT ORGANISM: homo sapiens FEATURE: OTHER INFORMATION: FceRIa TM

<4 OOs, SEQUENCE: 46 Asp Ile Phe Ile Pro Lieu. Lieu Val Wall Ile Lieu. Phe Ala Val Asp Thir 1. 5 1O 15

Gly Leu Phe Ile Ser Thr Glin Glin Glin Wall. Thir Phe Lell Lieu Lys Ile 2O 25 3O

Lys Arg Thr Arg Lys Gly Phe Arg Lieu. Luell ASn Pro His Pro Llys Pro 35 4 O 45

Asn Pro Lys Asn. Asn Arg SO

<210s, SEQ ID NO 47 &211s LENGTH: 119 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223s OTHER INFORMATION: CD32 linker

<4 OOs, SEQUENCE: 47 Val Llys Phe Ser Arg Ser Ala Asp Ala Pro Ala Gln Gln Gly Gln 1. 5 1O 15

Asn Glin Lieu. Tyr Asn. Glu Lieu. Asn Lieu. Gly Arg Arg Glu Glu Tyr Asp 2O 25 3O

Val Lieu. Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 35 4 O 45

Arg Arg Lys Asn Pro Glin Glu Gly Lieu. Tyr ASn Glu Lell Gln Lys Asp SO 55 6 O

Llys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Gly Glu Arg Arg 65 70

Arg Gly Lys Gly His Asp Gly Lieu. Tyr Glin Gly Lell Ser Thir Ala Thir 85 90 95

Lys Asp Thr Tyr Asp Ala Lieu. His Met Glin Ala Lell Pro Pro Arg Gly 1OO 105 11 O Ser Gly Ser Gly Ser Gly Ser 115

<210s, SEQ ID NO 48 &211s LENGTH: 49 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: FoeRIg linker

<4 OOs, SEQUENCE: 48

Lieu Lys Ile Glin Val Arg Lys Ala Ala Ile Thir Ser Tyr Glu Lys Ser 1. 5 15

Asp Gly Val Tyr Thr Gly Lieu Ser Thr Arg Asn Gln Glu Thr Tyr Glu 25 3O

Thr Lieu Lys His Glu Lys Pro Pro Glin Gly Ser Gly Ser Gly Ser Gly 35 4 O 45 US 2017/0073423 A1 Mar. 16, 2017 51

- Continued

Ser

<210s, SEQ ID NO 49 &211s LENGTH: 45 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223s OTHER INFORMATION: CD28 linker

<4 OOs, SEQUENCE: 49 Ser Arg Lieu. Lieu. His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro 1. 5 1O 15 Gly Pro Thr Arg Llys His Tyr Glin Pro Tyr Ala Pro Pro Arg Asp Phe 2O 25 3O Ala Ala Tyr Arg Ser Gly Ser Gly Ser Gly Ser Gly Ser 35 4 O 45

<210s, SEQ ID NO 50 &211s LENGTH: 48 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: 4-1BB linker

<4 OOs, SEQUENCE: 50 Gly Arg Llys Llys Lieu. Lieu. Tyr Ile Phe Lys Glin Pro Phe Met Arg Pro 1. 5 1O 15 Val Glin Thir Thr Glin Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 2O 25 3O Glu Glu Glu Gly Gly Cys Glu Lieu. Gly Ser Gly Ser Gly Ser Gly Ser 35 4 O 45

<210s, SEQ ID NO 51 &211s LENGTH: 50 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223s OTHER INFORMATION: OX4 O linker

<4 OOs, SEQUENCE: 51 Ala Lieu. Tyr Lieu. Lieu. Arg Arg Asp Glin Arg Lieu Pro Pro Asp Ala His 1. 5 1O 15 Llys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Glin Glu Glu Gln 2O 25 3O Ala Asp Ala His Ser Thr Lieu Ala Lys Ile Gly Ser Gly Ser Gly Ser 35 4 O 45 Gly Ser SO

<210s, SEQ ID NO 52 &211s LENGTH: 28 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223s OTHER INFORMATION: DAP1O linker

<4 OOs, SEQUENCE: 52 Pro Arg Arg Ser Pro Ala Glin Glu Asp Gly Llys Val Tyr Ile Asn Met 1. 5 1O 15

Pro Gly Arg Gly Gly Ser Gly Ser Gly Ser Gly Ser US 2017/0073423 A1 Mar. 16, 2017 52

- Continued

2O 25

<210s, SEQ ID NO 53 &211s LENGTH: 54 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223s OTHER INFORMATION: CD18 linker

<4 OOs, SEQUENCE: 53 Lys Ala Lieu. Ile His Lieu. Ser Asp Lieu. Arg Glu Tyr Arg Arg Phe Glu 1. 5 1O 15 Lys Glu Lys Lieu Lys Ser Glin Trp Asn. Asn Asp ASn Pro Lieu. Phe Lys 2O 25 3O Ser Ala Thr Thr Thr Val Met Asn Pro Llys Phe Ala Glu Ser Gly Ser 35 4 O 45 Gly Ser Gly Ser Gly Ser SO

<210s, SEQ ID NO 54 &211s LENGTH: 49 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223s OTHER INFORMATION: CD28 linker

<4 OOs, SEQUENCE: 54 Arg Ser Lys Arg Ser Arg Lieu. Lieu. His Ser Asp Tyr Met Asn Met Thr 1. 5 1O 15 Pro Arg Arg Pro Gly Pro Thr Arg Llys His Tyr Gln Pro Tyr Ala Pro 2O 25 3O Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gly Ser Gly Ser Gly Ser Gly 35 4 O 45

Ser

<210s, SEQ ID NO 55 &211s LENGTH: 33 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223s OTHER INFORMATION: CD275 linker

<4 OO > SEQUENCE: 55 Arg Asp Arg Cys Lieu Gln His Ser Tyr Ala Gly Ala Trp Ala Val Ser 1. 5 1O 15 Pro Glu Thr Glu Lieu. Thr Gly His Val Gly Ser Gly Ser Gly Ser Gly 2O 25 3O

Ser

<210s, SEQ ID NO 56 &211s LENGTH: 68 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223s OTHER INFORMATION: HVEM linker

<4 OOs, SEQUENCE: 56 Cys Val Lys Arg Arg Llys Pro Arg Gly Asp Val Val Llys Val Ile Val 1. 5 1O 15 US 2017/0073423 A1 Mar. 16, 2017 53

- Continued Ser Val Glin Arg Lys Arg Glin Glu Ala Glu Gly Glu Ala Thr Val Ile 2O 25 3O Glu Ala Lieu. Glin Ala Pro Pro Asp Val Thir Thr Val Ala Val Glu Glu 35 4 O 45 Thir Ile Pro Ser Phe Thr Gly Arg Ser Pro Asn His Gly Ser Gly Ser SO 55 6 O Gly Ser Gly Ser 65

<210s, SEQ ID NO 57 &211s LENGTH: 45 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223s OTHER INFORMATION: LIGHT linker

<4 OO > SEQUENCE: 57 Met Glu Glu Ser Val Val Arg Pro Ser Val Phe Val Val Asp Gly Glin 1. 5 1O 15 Thir Asp Ile Pro Phe Thr Arg Lieu. Gly Arg Ser His Arg Arg Glin Ser 2O 25 3O Cys Ser Val Ala Arg Gly Ser Gly Ser Gly Ser Gly Ser 35 4 O 45

<210s, SEQ ID NO 58 & 211 LENGTH: 30 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223s OTHER INFORMATION: CD4 OL linker

<4 OOs, SEQUENCE: 58 Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly 1. 5 1O 15 Lieu Pro Ile Ser Met Lys Gly Ser Gly Ser Gly Ser Gly Ser 2O 25 3O

<210s, SEQ ID NO 59 &211s LENGTH: 66 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223s OTHER INFORMATION: GITR linker

<4 OO > SEQUENCE: 59 Gln Leu Gly Lieu. His Ile Trp Gln Leu Arg Ser Glin Cys Met Trp Pro 1. 5 1O 15 Arg Glu Thr Glin Lieu. Lieu. Lieu. Glu Val Pro Pro Ser Thr Glu Asp Ala 2O 25 3O

Arg Ser Cys Glin Phe Pro Glu Glu Glu Arg Gly Glu Arg Ser Ala Glu 35 4 O 45

Glu Lys Gly Arg Lieu. Gly Asp Lieu. Trp Val Gly Ser Gly Ser Gly Ser SO 55 6 O

Gly Ser 65

<210s, SEQ ID NO 60 &211s LENGTH: 56 212. TYPE: PRT US 2017/0073423 A1 Mar. 16, 2017 54

- Continued <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223s OTHER INFORMATION: TIM1 linker

<4 OOs, SEQUENCE: 60 Lys Llys Tyr Phe Phe Lys Lys Glu Val Glin Gln Leu Ser Val Ser Phe 1. 5 1O 15 Ser Ser Lieu. Glin Ile Lys Ala Lieu. Glin Asn Ala Val Glu Lys Glu Val 2O 25 3O Glin Ala Glu Asp Asn. Ile Tyr Ile Glu Asn. Ser Lieu. Tyr Ala Thr Asp 35 4 O 45 Gly Ser Gly Ser Gly Ser Gly Ser SO 55

<210s, SEQ ID NO 61 &211s LENGTH: 85 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223s OTHER INFORMATION: SLAM linker

<4 OOs, SEQUENCE: 61 Glin Lieu. Arg Arg Arg Gly Llys Thr Asn His Tyr Glin Thir Thr Val Glu 1. 5 1O 15 Llys Llys Ser Lieu. Thir Ile Tyr Ala Glin Val Glin Llys Pro Gly Pro Lieu. 2O 25 3O Gln Lys Llys Lieu. Asp Ser Phe Pro Ala Glin Asp Pro Cys Thr Thr Ile 35 4 O 45 Tyr Val Ala Ala Thr Glu Pro Val Pro Glu Ser Val Glin Glu. Thir Asn SO 55 6 O Ser Ile Thr Val Tyr Ala Ser Val Thr Lieu Pro Glu Ser Gly Ser Gly 65 70 7s 8O Ser Gly Ser Gly Ser 85

<210s, SEQ ID NO 62 &211s LENGTH: 124 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223s OTHER INFORMATION: CD2 linker

<4 OOs, SEQUENCE: 62 Lys Arg Llys Lys Glin Arg Ser Arg Arg Asn Asp Glu Glu Lieu. Glu Thir 1. 5 1O 15 Arg Ala His Arg Val Ala Thr Glu Glu Arg Gly Arg Llys Pro His Glin 2O 25 3O

Ile Pro Ala Ser Thr Pro Glin ASn Pro Ala Thir Ser Glin His Pro Pro 35 4 O 45

Pro Pro Pro Gly His Arg Ser Glin Ala Pro Ser His Arg Pro Pro Pro SO 55 6 O

Pro Gly His Arg Val Gln His Gln Pro Glin Lys Arg Pro Pro Ala Pro 65 70 7s 8O

Ser Gly Thr Glin Val His Glin Gln Lys Gly Pro Pro Leu Pro Arg Pro 85 90 95

Arg Val Glin Pro Llys Pro Pro His Gly Ala Ala Glu Asn. Ser Lieu. Ser 1OO 105 11 O US 2017/0073423 A1 Mar. 16, 2017 55

- Continued

Pro Ser Ser Asn Gly Ser Gly Ser Gly Ser Gly Ser 115 12 O

<210s, SEQ ID NO 63 &211s LENGTH: 4 O 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223s OTHER INFORMATION: TLT-2 linker

<4 OOs, SEQUENCE: 63 Lys Lys Arg His Met Ala Ser Tyr Ser Met Cys Ser Asp Pro Ser Thr 1. 5 1O 15 Arg Asp Pro Pro Gly Arg Pro Glu Pro Tyr Val Glu Val Tyr Lieu. Ile 2O 25 3O Gly Ser Gly Ser Gly Ser Gly Ser 35 4 O

<210s, SEQ ID NO 64 &211s LENGTH: 62 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: LAG3 linker

<4 OOs, SEQUENCE: 64 His Lieu. Trp Arg Arg Gln Trp Arg Pro Arg Arg Phe Ser Ala Lieu. Glu 1. 5 1O 15 Glin Gly Ile His Pro Pro Glin Ala Glin Ser Lys Ile Glu Glu Lieu. Glu 2O 25 3O

Glin Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu 35 4 O 45 Pro Glu Pro Glu Gln Leu Gly Ser Gly Ser Gly Ser Gly Ser SO 55 6 O

<210s, SEQ ID NO 65 &211s LENGTH: 60 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223s OTHER INFORMATION: DAP12 linker

<4 OOs, SEQUENCE: 65 Tyr Phe Lieu. Gly Arg Lieu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala 1. 5 1O 15 Ala Thr Arg Lys Glin Arg Ile Thr Glu Thr Glu Ser Pro Tyr Glin Glu 2O 25 3O Lieu. Glin Gly Glin Arg Ser Asp Val Tyr Ser Asp Lieu. Asn Thr Glin Arg 35 4 O 45 Pro Tyr Tyr Lys Gly Ser Gly Ser Gly Ser Gly Ser SO 55 6 O

<210s, SEQ ID NO 66 &211s LENGTH: 107 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223s OTHER INFORMATION: CD84 linker

<4 OOs, SEQUENCE: 66 US 2017/0073423 A1 Mar. 16, 2017 56

- Continued

Arg Lieu. Phe Lys Arg Arg Glin Gly Arg Ile Phe Pro Glu Gly Ser Cys 1. 5 1O 15 Lieu. Asn Thr Phe Thr Lys Asn Pro Tyr Ala Ala Ser Lys Llys Thir Ile 2O 25 3O Tyr Thr Tyr Ile Met Ala Ser Arg Asn Thr Gln Pro Ala Glu Ser Arg 35 4 O 45 Ile Tyr Asp Glu Ile Lieu. Glin Ser Llys Val Lieu Pro Ser Lys Glu Glu SO 55 6 O Pro Val Asn Thr Val Tyr Ser Glu Val Glin Phe Ala Asp Llys Met Gly 65 70 7s 8O Lys Ala Ser Thr Glin Asp Ser Llys Pro Pro Gly Thr Ser Ser Tyr Glu 85 90 95 Ile Val Ile Gly Ser Gly Ser Gly Ser Gly Ser 1OO 105

<210s, SEQ ID NO 67 &211s LENGTH: 113 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223s OTHER INFORMATION: CD244 linker

<4 OO > SEQUENCE: 67 Glu Phe Lieu. Thir Ile Tyr Glu Asp Wall Lys Asp Lieu Lys Thr Arg Arg 1. 5 1O 15 Asn His Glu Gln Glu Gln Thr Phe Pro Gly Gly Gly Ser Thr Ile Tyr 2O 25 3O

Ser Met Ile Glin Ser Glin Ser Ser Ala Pro Thir Ser Glin Glu Pro Ala 35 4 O 45 Tyr Thr Lieu. Tyr Ser Lieu. Ile Glin Pro Ser Arg Lys Ser Gly Ser Arg SO 55 6 O Lys Arg Asn His Ser Pro Ser Phe Asin Ser Thr Ile Tyr Glu Val Ile 65 70 7s 8O Gly Lys Ser Glin Pro Lys Ala Glin Asn Pro Ala Arg Lieu. Ser Arg Llys 85 90 95 Glu Lieu. Glu Asn Phe Asp Val Tyr Ser Gly Ser Gly Ser Gly Ser Gly 1OO 105 11 O

Ser

<210s, SEQ ID NO 68 &211s LENGTH: 157 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223s OTHER INFORMATION: CD229 linker

<4 OOs, SEQUENCE: 68 Lieu. Tyr Ser Val Lieu. Ser Glin Gly Tyr Glu Lys Lieu. Asp Thr Pro Lieu. 1. 5 1O 15

Arg Pro Ala Arg Glin Gln Pro Thr Pro Thr Ser Asp Ser Ser Ser Asp 2O 25 3O

Ser Asn Lieu. Thir Thr Glu Glu Asp Glu Asp Arg Pro Glu Val His Lys 35 4 O 45

Pro Ile Ser Gly Arg Tyr Glu Val Phe Asp Glin Val Thr Glin Glu Gly SO 55 6 O US 2017/0073423 A1 Mar. 16, 2017 57

- Continued

Ala Gly His Asp Pro Ala Pro Glu Gly Glin Ala Asp Tyr Asp Pro Val 65 70 7s 8O Thr Pro Tyr Val Thr Glu Val Glu Ser Val Val Gly Glu Asn Thr Met 85 90 95 Tyr Ala Glin Val Phe Asn Lieu. Glin Gly Lys Thr Pro Val Ser Gln Lys 1OO 105 11 O Glu Glu Ser Ser Ala Thr Ile Tyr Cys Ser Ile Arg Llys Pro Glin Val 115 12 O 125 Val Pro Pro Pro Glin Glin Asn Asp Leu Glu Ile Pro Glu Ser Pro Thr 13 O 135 14 O Tyr Glu Asn Phe Thr Gly Ser Gly Ser Gly Ser Gly Ser 145 150 155

<210s, SEQ ID NO 69 &211s LENGTH: 151 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223s OTHER INFORMATION: LTBR linker

<4 OOs, SEQUENCE: 69 Lys Ala His Pro Tyr Phe Pro Asp Leu Val Glin Pro Leu Lleu Pro Ile 1. 5 1O 15 Ser Gly Asp Val Ser Pro Val Ser Thr Gly Lieu Pro Ala Ala Pro Val 2O 25 30 Lieu. Glu Ala Gly Val Pro Glin Glin Glin Ser Pro Lieu. Asp Lieu. Thir Arg 35 4 O 45 Glu Pro Gln Leu Glu Pro Gly Glu Glin Ser Glin Val Ala His Gly Thr SO 55 6 O Asn Gly Ile His Val Thr Gly Gly Ser Met Thir Ile Thr Gly Asn Ile 65 70 7s 8O Tyr Ile Tyr Asn Gly Pro Val Lieu. Gly Gly Pro Pro Gly Pro Gly Asp 85 90 95 Lieu Pro Ala Thr Pro Glu Pro Pro Tyr Pro Ile Pro Glu Glu Gly Asp 1OO 105 11 O Pro Gly Pro Pro Gly Lieu Ser Thr Pro His Glin Glu Asp Gly Lys Ala 115 12 O 125 Trp. His Leu Ala Glu Thr Glu. His Cys Gly Ala Thr Pro Ser Asn Gly 13 O 135 14 O Ser Gly Ser Gly Ser Gly Ser 145 150

<210s, SEQ ID NO 70 &211s LENGTH: 46 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223s OTHER INFORMATION: CD278 linker

<4 OO > SEQUENCE: 7 O Cys Trp Lieu. Thir Lys Llys Llys Tyr Ser Ser Ser Val His Asp Pro Asn 1. 5 1O 15

Gly Glu Tyr Met Phe Met Arg Ala Val Asn. Thir Ala Lys Llys Ser Arg 2O 25 3O

Lieu. Thir Asp Val Thr Lieu. Gly Ser Gly Ser Gly Ser Gly Ser US 2017/0073423 A1 Mar. 16, 2017 58

- Continued

35 4 O 45

<210s, SEQ ID NO 71 &211s LENGTH: 249 212. TYPE: PRT <213> ORGANISM: Tobacco etch virus 22 Os. FEATURE: 223 OTHER INFORMATION : TEW Interactor 1.

<4 OOs, SEQUENCE: 71 Ser Gly Ser Gly Ser Gly Gly Glu Ser Lieu. Phe Lys Gly Pro Arg Asp 1. 5 1O 15 Tyr Asn Pro Ile Ser Ser Thr Ile Cys His Lieu. Thr Asn Glu Ser Asp 2O 25 3O Gly His Thir Thr Ser Lieu. Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile 35 4 O 45 Thir Asn Llys His Lieu. Phe Arg Arg Asn. Asn Gly Thr Lieu. Lieu Val Glin SO 55 6 O Ser Lieu. His Gly Val Phe Llys Val Lys Asn Thr Thr Thr Lieu. Glin Glin 65 70 7s 8O His Lieu. Ile Asp Gly Arg Asp Met Ile Ile Ile Arg Met Pro Lys Asp 85 90 95 Phe Pro Pro Phe Pro Gln Lys Lieu Lys Phe Arg Glu Pro Glin Arg Glu 1OO 105 11 O Glu Arg Ile Cys Lieu Val Thir Thr ASn Phe Gln Thr Llys Ser Met Ser 115 12 O 125 Ser Met Val Ser Asp Thir Ser Cys Thr Phe Pro Ser Ser Asp Gly Ile 13 O 135 14 O Phe Trp Llys His Trp Ile Glin Thr Lys Asp Gly Glin Cys Gly Ser Pro 145 150 155 160 Lieu Val Ser Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser Ala Ser 1.65 17O 17s Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe 18O 185 19 O Met Glu Lieu. Lieu. Thr Asn Glin Glu Ala Glin Gln Trp Val Ser Gly Trp 195 2OO 2O5 Arg Lieu. Asn Ala Asp Ser Val Lieu. Trp Gly Gly His Llys Val Phe Met 21 O 215 22O Ser Llys Pro Glu Glu Pro Phe Glin Pro Val Lys Glu Ala Thr Gln Leu 225 23 O 235 24 O Met Asin Glu Lieu Val Tyr Ser Glu Glu 245

<210s, SEQ ID NO 72 &211s LENGTH: 4 O 212. TYPE: PRT <213> ORGANISM: homo sapiens 22 Os. FEATURE: <223 is OTHER INFORMATION: Nuo Human Interactor 1

<4 OOs, SEQUENCE: 72 Ser Gly Ser Gly Ser Gly Met Glin Ile Phe Val Lys Thr Lieu. Thr Gly 1. 5 1O 15

Lys. Thir Ile Thr Lieu. Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val 2O 25 3O US 2017/0073423 A1 Mar. 16, 2017 59

- Continued Lys Ala Lys Ile Glin Asp Llys Glu 35 4 O

<210s, SEQ ID NO 73 &211s LENGTH: 4 O 212. TYPE: PRT <213> ORGANISM: homo sapiens 22 Os. FEATURE: <223 is OTHER INFORMATION: Nuo-I13A. Human Interactor 1

<4 OO > SEQUENCE: 73 Ser Gly Ser Gly Ser Gly Met Glin Ile Phe Val Lys Thr Lieu. Thr Gly 1. 5 1O 15 Lys Thr Ala Thr Lieu. Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val 2O 25 3O Lys Ala Lys Ile Glin Asp Llys Glu 35 4 O

<210s, SEQ ID NO 74 &211s LENGTH: 4 O 212. TYPE: PRT <213> ORGANISM: homo sapiens 22 Os. FEATURE: <223 is OTHER INFORMATION: Nuo-I13 G. Human Interactor 1

<4 OOs, SEQUENCE: 74 Ser Gly Ser Gly Ser Gly Met Glin Ile Phe Val Lys Thr Lieu. Thr Gly 1. 5 1O 15 Lys Thr Gly Thr Lieu. Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val 2O 25 3O Lys Ala Lys Ile Glin Asp Llys Glu 35 4 O

<210s, SEQ ID NO 75 &211s LENGTH: 4 O 212. TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae 22 Os. FEATURE: <223 is OTHER INFORMATION: Nuo Yeast Interactor 1

<4 OO > SEQUENCE: 75 Ser Gly Ser Gly Ser Gly Met Glin Ile Phe Val Lys Thr Lieu. Thr Gly 1. 5 1O 15 Llys Thir Ile Thir Lieu. Glu Val Glu Ser Ser Asp Thir Ile Asp Asn. Wall 2O 25 3O Llys Ser Lys Ile Glin Asp Llys Glu 35 4 O

<210s, SEQ ID NO 76 &211s LENGTH: 4 O 212. TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae 22 Os. FEATURE: <223 is OTHER INFORMATION: Nuo-I13A Yeast Interactor 1

<4 OO > SEQUENCE: 76 Ser Gly Ser Gly Ser Gly Met Glin Ile Phe Val Lys Thr Lieu. Thr Gly 1. 5 1O 15

Llys Thr Ala Thir Lieu. Glu Val Glu Ser Ser Asp Thir Ile Asp Asn. Wall 2O 25 3O US 2017/0073423 A1 Mar. 16, 2017 60

- Continued Llys Ser Lys Ile Glin Asp Lys Glu 35 4 O

<210s, SEQ ID NO 77 &211s LENGTH: 4 O 212. TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae 22 Os. FEATURE: <223 is OTHER INFORMATION: Nuo-I13G Yeast Interactor 1

<4 OO > SEQUENCE: 77 Ser Gly Ser Gly Ser Gly Met Glin Ile Phe Val Lys Thr Lieu. Thr Gly 1. 5 15 Lys Thr Gly Thr Lieu. Glu Val Glu Ser Ser Asp Thir Ile Asp Asin Val 2O 25 Llys Ser Lys Ile Glin Asp Lys Glu 35 4 O

<210s, SEQ ID NO 78 &211s LENGTH: 2O 212. TYPE: PRT <213s ORGANISM: Tobacco etch virus 22 Os. FEATURE: 223 OTHER INFORMATION: TEV cleavage site Interactor 2 <4 OO > SEQUENCE: 78 Ser Gly Ser Gly Ser Gly Glu Asn Lieu. Tyr Phe Glin Ser Gly Ser Gly 1. 5 15 Ser Gly Ser Gly 2O

<210s, SEQ ID NO 79 &211s LENGTH: 61 212. TYPE: PRT <213> ORGANISM: homo sapiens 22 Os. FEATURE: <223 is OTHER INFORMATION: Cuo Human Interactor 2

<4 OO > SEQUENCE: 79 Ser Gly Ser Gly Ser Gly Gly Ile Pro Pro Asp Gln Glin Arg Lieu. Ile 1. 5 1O 15 Phe Ala Gly Lys Glin Lieu. Glu Asp Gly Arg Thr Lieu. Ser Asp Tyr Asn 2O 25 3O

Ile Gln Lys Glu Ser Thr Lieu. His Lieu Val Lieu. Arg Lieu. Arg Gly Gly 35 4 O 45 Met His Arg Ser Ala Cys Gly Arg Met Ala Gly Ser Gly SO 55 6 O

<210s, SEQ ID NO 8O &211s LENGTH: 2O7 212. TYPE: PRT <213> ORGANISM: homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: tetR DNA binding

<4 OOs, SEQUENCE: 80 Met Ser Arg Lieu. Asp Llys Ser Llys Val Ile Asn. Ser Ala Lieu. Glu Lieu. 1. 5 1O 15

Lieu. Asn. Glu Val Gly Ile Glu Gly Lieu. Thir Thr Arg Llys Lieu Ala Glin 2O 25 3O US 2017/0073423 A1 Mar. 16, 2017 61

- Continued Llys Lieu. Gly Val Glu Glin Pro Thir Lieu. Tyr Trp His Val Lys Asn Lys 35 4 O 45 Arg Ala Lieu. Lieu. Asp Ala Lieu Ala Ile Glu Met Lieu. Asp Arg His His SO 55 6 O Thr His Phe Cys Pro Leu Glu Gly Glu Ser Trp Glin Asp Phe Leu Arg 65 70 7s 8O Asn Asn Ala Lys Ser Phe Arg Cys Ala Lieu. Lieu. Ser His Arg Asp Gly 85 90 95 Ala Lys Val His Leu Gly Thr Arg Pro Thr Glu Lys Glin Tyr Glu Thr 1OO 105 11 O Lieu. Glu Asn Glin Lieu Ala Phe Lieu. Cys Glin Glin Gly Phe Ser Lieu. Glu 115 12 O 125 Asn Ala Lieu. Tyr Ala Lieu. Ser Ala Val Gly His Phe Thr Lieu. Gly Cys 13 O 135 14 O Val Lieu. Glu Asp Glin Glu. His Glin Val Ala Lys Glu Glu Arg Glu Thr 145 150 155 160 Pro Thir Thr Asp Ser Met Pro Pro Leu Lieu. Arg Glin Ala Ile Glu Lieu. 1.65 17O 17s Phe Asp His Glin Gly Ala Glu Pro Ala Phe Lieu. Phe Gly Lieu. Glu Lieu. 18O 185 19 O Ile Ile Cys Gly Lieu. Glu Lys Glin Lieu Lys Cys Glu Ser Gly Ser 195 2OO 2O5

<210s, SEQ ID NO 81 &211s LENGTH: 147 212. TYPE: PRT <213> ORGANISM: homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: GalA DNA binding <4 OOs, SEQUENCE: 81 Met Lys Lieu. Lieu. Ser Ser Ile Glu Glin Ala Cys Asp Ile Cys Arg Lieu. 1. 5 1O 15 Llys Llys Lieu Lys Cys Ser Lys Glu Lys Pro Llys Cys Ala Lys Cys Lieu. 2O 25 3O Lys Asn. Asn Trp Glu. Cys Arg Tyr Ser Pro Llys Thr Lys Arg Ser Pro 35 4 O 45 Lieu. Thir Arg Ala His Lieu. Thr Glu Val Glu Ser Arg Lieu. Glu Arg Lieu. SO 55 6 O Glu Gln Lieu. Phe Lieu. Lieu. Ile Phe Pro Arg Glu Asp Lieu. Asp Met Ile 65 70 7s 8O Lieu Lys Met Asp Ser Lieu. Glin Asp Ile Lys Ala Lieu. Lieu. Thr Gly Lieu. 85 90 95 Phe Val Glin Asp Asn Val Asn Lys Asp Ala Val Thr Asp Arg Lieu Ala 1OO 105 11 O Ser Val Glu Thir Asp Met Pro Leu. Thir Lieu. Arg Gln His Arg Ile Ser 115 12 O 125

Ala Thir Ser Ser Ser Glu Glu Ser Ser Asn Lys Gly Glin Arg Glin Lieu 13 O 135 14 O

Thir Wal Ser 145

<210s, SEQ ID NO 82 &211s LENGTH: 128