INHIBITORY CHIMERIC RECEPTOR ARCHITECTURES

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said Sequence Listing XML, created on Sep. 1, 2022, is named STB-021WOC1_SL.xml, and is 126,885 bytes in size.

BACKGROUND

Chimeric antigen receptors (CARs) enable targeted in vivo activation of immunomodulatory cells, such as T cells. These recombinant membrane receptors have an antigen-binding domain and one or more signaling domains (e.g., T cell activation domains). These special receptors allow the T cells to recognize a specific protein antigen on tumor cells and induce T cell activation and signaling pathways. Recent results of clinical trials with chimeric receptor-expressing T cells have provided compelling support of their utility as agents for cancer immunotherapy. However, despite these promising results, a number of side effects associated the CAR T-cell therapeutics were identified, raising significant safety concerns. One side effect is “on-target but off-tissue” adverse events from TCR and CAR engineered T cells, in which a CAR T cell binds to its ligand outside of the target tumor tissue and induces an immune response. Therefore, the ability to identify appropriate CAR targets is important to effectively targeting and treating the tumor without damaging normal cells that express the same target antigen.

Inhibitory chimeric antigen receptors (also known as iCARs) are protein constructions that inhibit or reduce immunomodulatory cell activity after binding their cognate ligands on a target cell. Current iCAR designs leverage PD-1 intracellular domains for inhibition, but have proven difficult to reproduce. Thus, alternative inhibitory domains for use in iCARs are needed.

SUMMARY

Provided herein are chimeric inhibitory receptors comprising: an extracellular protein-binding domain; a transmembrane domain, wherein the transmembrane domain is operably linked to the extracellular protein-binding domain; and one or more intracellular signaling domains, wherein the one or more intracellular signaling domains are operably linked to the transmembrane domain, and wherein at least one of the one or more intracellular signaling domain is capable of preventing, attenuating, or inhibiting activation of a tumor-targeting chimeric receptor expressed on an immunomodulatory cell.

In some aspects, the one or more intracellular signaling domains are each derived from a protein selected from the group consisting of: SLAP1, SLAP2, Dok-1, Dok-2, LAIR1, GRB-2, CD200R, SIRPα, HAVR, GITR, PD-L1, KIR2DL1, KIR2DL2, KIR2DL3KIR3DL2, CD94, KLRG-1, CEACAM1, LIR2, LIR3, LIR5, SIGLEC-2, and SIGLEC-10.

In some aspects, the transmembrane domain is derived from the same protein as one of the one or more intracellular signaling domains.

In some aspects, the transmembrane domain further comprises at least a portion of an extracellular domain of the same protein.

In some aspects, the transmembrane domain is derived from a first protein and the one or more intracellular signaling domains are derived from a second protein that are distinct from the first protein.

In some aspects, one of the one or more intracellular signaling domains are derived from SLAP1.

In some aspects, the intracellular signaling domain comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to

(SEQ ID NO: 4)

PAPAERPLPNPEGLDSDFLAVLSDYPSPDISPPIF

RRGEKLRVISDEGGWWKAISLSTGRESYIPGICVA

RVYHGWLFEGLGRDKAEELLQLPDTKVGSFMIRES

ETKKGFYSLSVRHRQVKHYRIFRLPNNWYYISPRL

TFQCLEDLVNHYSEVADGLCCVLTTPCLTQSTAAP

AVRASSSPVTLRQKTVDWRRVSRLQEDPEGTENPL

GVDESLFSYGLRESIASYLSLTSEDNTSFDRKKKS

ISLMYGGSKRKSSFFSSPPYFED,

or

(SEQ ID NO: 5)

PAPAERPLPNPEGLDSDFLA

VLSDYPSPDISPPIFRRGEKLRVISDEGGWWKAIS

LSTGRESYIPGICVARVYHGWLFEGLGRDKAEELL

QLPDTKVGSFMIRESETKKGFYSLSVRHRQVKHYR

IFRLPNNWYYISPRLTFQCLEDLVNHYSEVADGLC

CVLTTPCLTQSTAAPAVRASSSPVTLRQKTVDWRR

VSRLQEDPEGTENPLGVDESLFSYGLRESIASYLS

LTSEDNTSF.

In some aspects, the intracellular signaling domain comprises the amino acid sequence of

(SEQ ID NO: 4)

PAPAERPLPNPEGLDSDFLAVLSDYPSPDISPPIF

RRGEKLRVISDEGGWWKAISLSTGRESYIPGICVA

RVYHGWLFEGLGRDKAEELLQLPDTKVGSFMIRES

ETKKGFYSLSVRHRQVKHYRIFRLPNNWYYISPRL

TFQCLEDLVNHYSEVADGLCCVLTTPCLTQSTAAP

AVRASSSPVTLRQKTVDWRRVSRLQEDPEGTENPL

GVDESLFSYGLRESIASYLSLTSEDNTSFDRKKKS

ISLMYGGSKRKSSFFSSPPYFED,

or

(SEQ ID NO: 5)

PAPAERPLPNPEGLDSDFLAV

LSDYPSPDISPPIFRRGEKLRVISDEGGWWKAISL

STGRESYIPGICVARVYHGWLFEGLGRDKAEELLQ

LPDTKVGSFMIRESETKKGFYSLSVRHRQVKHYRI

FRLPNNWYYISPRLTFQCLEDLVNHYSEVADGLCC

VLTTPCLTQSTAAPAVRASSSPVTLRQKTVDWRRV

SRLQEDPEGTENPLGVDESLFSYGLRESIASYLSL

TSEDNTSF.

In some aspects, one of the one or more intracellular signaling domains is derived from SLAP2.

(SEQ ID NO: 6)

RKSLPSPSLSSSVQGQGPVTMEAERSKATAVALGS

FPAGGPAELSLRLGEPLTIVSEDGDWWTVLSEVSG

REYNIPSVHVAKVSHGWLYEGLSREKAEELLLLPG

NPGGAFLIRESQTRRGSYSLSVRLSRPASWDRIRH

YRIHCLDNGWLYISPRLTFPSLQALVDHYSELADD

ICCLLKEPCVLQRAGPLPGKDIPLPVTVQRTPLNW

KELDSSLLFSEAATGEESLLSEGLRESLSFYISLN

DEAVSLDDA.

In some aspects, the intracellular signaling domain comprises the amino acid sequence of

In some aspects, one of the one or more intracellular signaling domains is derived from KIR2DL1.

(SEQ ID NO: 60)

HRWCSNKKNAAVMDQESAGNRTANSEDSDEQDPQE

VTYTQLNHCVFTQRKITRPSQRPKTPPTDIIVYTE

LPNAESRSKVVSCP.

In some aspects, the intracellular signaling domain comprises the amino acid sequence of

(SEQ ID NO: 60)

HRWCSNKKNAAVMDQESAGNRTANSEDSDEQDPQE

VTYTQLNHCVFTQRKITRPSQRPKTPPTDIIVYTE

LPNAESRSKVVSCP.

In some aspects, one of the one or more intracellular signaling domains is derived from KLRG-1.

In some aspects, the intracellular signaling domain comprises the amino acid sequence of MTDSVIYSMLELPTATQAQNDYGPQQKSSSSRPSCSCLGSG (SEQ ID NO: 61).

In some aspects, one of the one or more intracellular signaling domains is derived from LAIR1.

(SEQ ID NO: 62)

HRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTA

DKATVNGLPEKDRETDTSALAAGSSQEVTYAQLDH

WALTQRTARAVSPQSTKPMAESITYAAVARH.

In some aspects, the intracellular signaling domain comprises the amino acid sequence of

(SEQ ID NO: 62)

HRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTA

DKATVNGLPEKDRETDTSALAAGSSQEVTYAQLDH

WALTQRTARAVSPQSTKPMAESITYAAVARH.

In some aspects, one of the one or more intracellular signaling domains is derived from LIR2.

(SEQ ID NO: 63)

LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGL

QWRSSPAADAQEENLYAAVKDTQPEDGVEMDTRAA

ASEAPQDVTYAQLHSLTLRRKATEPPPSQEREPPA

EPSIYATLAIH.

In some aspects, the intracellular signaling domain comprises the amino acid sequence of

(SEQ ID NO: 63)

LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGL

QWRSSPAADAQEENLYAAVKDTQPEDGVEMDTRAA

ASEAPQDVTYAQLHSLTLRRKATEPPPSQEREPPA

EPSIYATLAIH.

In some aspects, one of the one or more intracellular signaling domains is derived from LIR3.

(SEQ ID NO: 64)

RRQRHSKHRTSDQRKTDFQRPAGAAETEPKDRGLLR

RSSPAADVQEENLYAAVKDTQSEDRVELDSQSPHD

EDPQAVTYAPVKHSSPRREMASPPSSLSGEFLDTK

DRQVEEDRQMDTEAAASEASQDVTYAQLHSLTLRR

KATEPPPSQEGEPPAEPSIYATLAIH.

In some aspects, the intracellular signaling domain comprises the amino acid sequence of

(SEQ ID NO: 64)

RRQRHSKHRTSDQRKTDFQRPAGAAETEPKDRGLL

RRSSPAADVQEENLYAAVKDTQSEDRVELDSQSPH

DEDPQAVTYAPVKHSSPRREMASPPSSLSGEFLDT

KDRQVEEDRQMDTEAAASEASQDVTYAQLHSLTLR

RKATEPPPSQEGEPPAEPSIYATLAIH.

In some aspects, wherein one of the one or more intracellular signaling domains is derived from LIR5.

(SEQ ID NO: 65)

QHWRQGKHRTLAQRQADFQRPPGAAEPEPKDGGLQ

RRSSPAADVQGENFCAAVKNTQPEDGVEMDTRQSP

HDEDPQAVTYAKVKHSRPRREMASPPSPLSGEFLD

TKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSFTL

RQKATEPPPSQEGASPAEPSVYATLAIH.

In some aspects, the intracellular signaling domain comprises the amino acid sequence of

(SEQ ID NO: 65)

QHWRQGKHRTLAQRQADFQRPPGAAEPEPKDGGLQ

RRSSPAADVQGENFCAAVKNTQPEDGVEMDTRQSP

HDEDPQAVTYAKVKHSRPRREMASPPSPLSGEFLD

TKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSFTL

RQKATEPPPSQEGASPAEPSVYATLAIH.

In some aspects, one of the one or more intracellular signaling domains is derived from SIGLEC-2.

(SEQ ID NO: 66)

KLQRRWKRTQSQQGLQENSSGQSFFVRNKKVRRAP

LSEGPHSLGCYNPMMEDGISYTTLRFPEMNIPRTG

DAESSEMQRPPPDCDDTVTYSALHKRQVGDYENVI

PDFPEDEGIHYSELIQFGVGERPQAQENVDYVILK

H.

In some aspects, the intracellular signaling domain comprises the amino acid sequence of

(SEQ ID NO: 66)

KLQRRWKRTQSQQGLQENSSGQSFFVRNKKVRRAP

LSEGPHSLGCYNPMMEDGISYTTLRFPEMNIPRTG

DAESSEMQRPPPDCDDTVTYSALHKRQVGDYENVI

PDFPEDEGIHYSELIQFGVGERPQAQENVDYVILK

H.

In some aspects, one of the one or more intracellular signaling domains is derived from SIGLEC-10.

(SEQ ID NO: 67)

KILPKRRTQTETPRPRFSRHSTILDYINVVPTAGP

LAQKRNQKATPNSPRTPLPPGAPSPESKKNQKKQY

QLPSFPEPKSSTQAPESQESQEELHYATLNFPGVR

PRPEARMPKGTQADYAEVKFQ.

In some aspects, the intracellular signaling domain comprises the amino acid sequence of

(SEQ ID NO: 67)

KILPKRRTQTETPRPRFSRHSTILDYINVVPTAGPLAQKRNQKATPNSPR

TPLPPGAPSPESKKNQKKQYQLPSFPEPKSSTQAPESQESQEELHYATLN

FPGVRPRPEARMPKGTQADYAEVKFQ.

In some aspects, the transmembrane domain is derived from a protein selected from the group consisting of: CD8, CD28, CD3ζ, CD4, 4-IBB, OX40, ICOS, 2B4, CD25, CD7, LAX, LAT, LAIR1, GRB-2, Dok-1, Dok-2, SLAP1, SLAP2, CD200R, SIRPα, HAVR, GITR, PD-L1, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, CD94, KLRG-1, CEACAM1, LIR2, LIR3, LIR5, SIGLEC-2, and SIGLEC-10.

In some aspects, the chimeric inhibitory receptor comprises a transmembrane domain derived from CD28.

In some aspects, the transmembrane domain comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to

(SEQ ID NO: 20)

FWVLVVVGGVLACYSLLVTVAFIIFWV.

In some aspects, the transmembrane domain comprises the amino acid sequence of

(SEQ ID NO: 20)

FWVLVVVGGVLACYSLLVTVAFIIFWV.

In some aspects, the chimeric inhibitory receptor comprises a transmembrane domain derived from KIR2DL1.

(SEQ ID NO: 76)

ILIGTSVVIILFILLFFLL.

In some aspects, the transmembrane domain comprises the amino acid sequence of

(SEQ ID NO: 76)

ILIGTSVVIILFILLFFLL.

In some aspects, the chimeric inhibitory receptor comprises a transmembrane domain derived from KLRG-1.

(SEQ ID NO: 78)

VAIALGLLTAVLLSVLLYQWI.

In some aspects, the transmembrane domain comprises the amino acid sequence of

(SEQ ID NO: 78)

VAIALGLLTAVLLSVLLYQWI.

In some aspects, the chimeric inhibitory receptor comprises a transmembrane domain derived from LAIR1.

(SEQ ID NO: 79)

ILIGVSVVFLFCLLLLVLFCL.

In some aspects, the transmembrane domain comprises the amino acid sequence of

(SEQ ID NO: 79)

ILIGVSVVFLFCLLLLVLFCL.

In some aspects, the chimeric inhibitory receptor comprises a transmembrane domain derived from LIR2.

(SEQ ID NO: 80)

VIGILVAVVLLLLLLLLLFLI.

In some aspects, the transmembrane domain comprises the amino acid sequence of

(SEQ ID NO: 80)

VIGILVAVVLLLLLLLLLFLI.

In some aspects, the chimeric inhibitory receptor comprises a transmembrane domain derived from LIR3.

(SEQ ID NO: 81)

VLIGVSVAFVLLLFLLLFLLL.

In some aspects, the transmembrane domain comprises the amino acid sequence of

(SEQ ID NO: 81)

VLIGVSVAFVLLLFLLLFLLL.

In some aspects, the chimeric inhibitory receptor comprises a transmembrane domain derived from LIR5.

(SEQ ID NO: 82)

VLIGVLVVSILLLSLLLFLLL.

In some aspects, the transmembrane domain comprises the amino acid sequence of

(SEQ ID NO: 82)

VLIGVLVVSILLLSLLLFLLL.

In some aspects, the chimeric inhibitory receptor comprises a transmembrane domain derived from SIGLEC-2.

(SEQ ID NO: 83)

VAVGLGSCLAILILAICGL.

In some aspects, the transmembrane domain comprises the amino acid sequence of

(SEQ ID NO: 83)

VAVGLGSCLAILILAICGL.

In some aspects, the chimeric inhibitory receptor comprises a transmembrane domain derived from SIGLEC-10.

(SEQ ID NO: 84)

GAFLGIGITALLFLCLALIIM.

In some aspects, the transmembrane domain comprises the amino acid sequence of

(SEQ ID NO: 84)

GAFLGIGITALLFLCLALIIM.

In some aspects, the one or more intracellular signaling domains are two intracellular signaling domains.

In some aspects, the chimeric inhibitory receptor comprises a first intracellular signaling domain derived from KIR2DL1 and a second intracellular signaling domain derived from LIR2.

In some aspects, the chimeric inhibitory receptor comprises a first intracellular signaling domain derived from KIR2DL1 and a second intracellular signaling domain derived from LIR3.

In some aspects, the chimeric inhibitory receptor comprises a first intracellular signaling domain derived from KIR2DL1 and a second intracellular signaling domain derived from LIR5.

In some aspects, the first intracellular signaling domain further comprises a transmembrane domain derived from KIR2DL1.

In some aspects, the chimeric inhibitory receptor comprises a first intracellular signaling domain derived from LIR2 and a second intracellular signaling domain derived from KIR2DL1.

In some aspects, first intracellular signaling domain further comprises a transmembrane domain derived from LIR2.

In some aspects, the chimeric inhibitory receptor comprises a first intracellular signaling domain derived from LIR3 and a second intracellular signaling domain derived from KIR2DL1.

In some aspects, the first intracellular signaling domain further comprises a transmembrane domain derived from LIR3.

In some aspects, the chimeric inhibitory receptor comprises a first intracellular signaling domain derived from LIR5 and a second intracellular signaling domain derived from KIR2DL1.

In some aspects, the first intracellular signaling domain further comprises a transmembrane domain derived from LIR5.

In some aspects, the protein is not expressed on the target tumor.

In some aspects, the protein is expressed on a non-tumor cell.

In some aspects, the protein is expressed on a non-tumor cell derived from a tissue selected from the group consisting of brain, neuronal tissue, endocrine, endothelial, bone, bone marrow, immune system, muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, urinary bladder, male reproductive organs, female reproductive organs, adipose, soft tissue, and skin.

In some aspects, the extracellular protein binding domain comprises a ligand-binding domain.

In some aspects, the extracellular protein binding domain comprises a receptor-binding domain.

In some aspects, the extracellular protein binding domain comprises an antigen-binding domain.

In some aspects, the antigen-binding domain comprises an antibody, an antigen-binding fragment of an antibody, a F(ab) fragment, a F(ab′) fragment, a single chain variable fragment (scFv), or a single-domain antibody (sdAb).

In some aspects, the antigen-binding domain comprises a single chain variable fragment (scFv).

In some aspects, each scFv comprises a heavy chain variable domain (VH) and a light chain variable domain (VL).

In some aspects, the VH and VL are separated by a peptide linker.

In some aspects, the peptide linker comprises an amino acid sequence selected from the group consisting of: GGS (SEQ ID NO: 23), GGSGGS (SEQ ID NO: 24), GGSGGSGGS (SEQ ID NO: 25), GGSGGSGGSGGS (SEQ ID NO: 26), GGSGGSGGSGGSGGS (SEQ ID NO: 27), GGGS (SEQ ID NO: 28), GGGSGGGS (SEQ ID NO: 29), GGGSGGGSGGGS (SEQ ID NO: 30), GGGSGGGSGGGSGGGS (SEQ ID NO: 31), GGGSGGGSGGGSGGGSGGGS (SEQ ID NO: 32), GGGGS (SEQ ID NO: 33), GGGGSGGGGS (SEQ ID NO: 34), GGGGSGGGGSGGGGS (SEQ ID NO: 35), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 36), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 37), and TTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACDQTTPGERSSLPAFY PGTSGSCSGCGSLSLP (SEQ ID NO: 94).

In some aspects, the scFv comprises the structure VH-L-VL or VL-L-VH, wherein VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain.

In some aspects, the transmembrane domain is physically linked to the extracellular protein-binding domain.

In some aspects, one of the one or more intracellular signaling domain is physically linked to the transmembrane domain.

In some aspects, the transmembrane domain is physically linked to the extracellular protein binding domain and one of the one or more intracellular signaling domains is physically linked to the transmembrane domain.

In some aspects, the extracellular protein binding has a high binding affinity.

In some aspects, the extracellular protein binding has a low binding affinity.

In some aspects, the chimeric inhibitory receptor is capable of suppressing cytokine production from an activated immunomodulatory cell.

In some aspects, the chimeric inhibitory receptor is capable of suppressing a cell-mediated immune response to a target cell, wherein the immune response is induced by activation of the immunomodulatory cell.

In some aspects, the target cell is a tumor cell.

In some aspects, the one or more intracellular signaling domains comprises one or more modifications.

In some aspects, the one or more modifications modulate sensitivity of the chimeric inhibitory receptor relative to the otherwise identical, unmodified receptor.

In some aspects, the one or more modifications increase sensitivity of the chimeric inhibitory receptor relative to the otherwise identical, unmodified receptor.

In some aspects, the one or more modifications reduce sensitivity of the chimeric inhibitory receptor relative to the otherwise identical, unmodified receptor.

In some aspects, the one or more modifications modulate potency of the chimeric inhibitory receptor relative to the otherwise identical, unmodified receptor.

In some aspects, the one or more modifications increase potency of the chimeric inhibitory receptor relative to the otherwise identical, unmodified receptor.

In some aspects, the one or more modifications reduce potency of the chimeric inhibitory receptor relative to the otherwise identical, unmodified receptor.

In some aspects, the one or more modifications modulate basal prevention, attenuation, or inhibition of activation of the tumor-targeting chimeric receptor when expressed on an immunomodulatory cell relative to the otherwise identical, unmodified receptor.

In some aspects, the one or more modifications reduce basal prevention, attenuation, or inhibition relative to the otherwise identical, unmodified receptor.

In some aspects, the one or more modifications increase basal prevention, attenuation, or inhibition relative to the otherwise identical, unmodified receptor.

In some aspects, the chimeric inhibitory receptor further comprises a spacer region positioned between the extracellular protein binding domain and the transmembrane domain and operably linked to each of the extracellular protein-binding domain and the transmembrane domain.

In some aspects, the chimeric inhibitory receptor further comprises a spacer region positioned between the extracellular protein binding domain and the transmembrane domain and physically linked to each of the extracellular protein binding domain and the transmembrane domain.

In some aspects, the spacer region is derived from a protein selected from the group consisting of: CD8α, CD4, CD7, CD28, IgG1, IgG4, FcγRIIIα, LNGFR, and PDGFR.

In some aspects, the spacer region comprises an amino acid sequence selected from the group consisting of:

(SEQ ID NO: 39)

AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP,

(SEQ ID NO: 40)

ESKYGPPCPSCP,

(SEQ ID NO: 41)

ESKYGPPAPSAP,

(SEQ ID NO: 42)

ESKYGPPCPPCP,

(SEQ ID NO: 43)

EPKSCDKTHTCP,

(SEQ ID NO: 44)

AAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV

HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN,

(SEQ ID NO: 46)

ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVS

ATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACR

VCEAGSGLVFSCQDKQNTVCEECPDGTYSDEADAEC,

(SEQ ID NO: 47)

ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVC,

and

(SEQ ID NO: 48)

AVGQDTQEVIVVPHSLPFKV.

In some aspects, the spacer region modulates sensitivity of the chimeric inhibitory receptor relative to an otherwise identical chimeric inhibitory receptor lacking the spacer region.

In some aspects, the spacer region increases sensitivity of the chimeric inhibitory receptor relative to an otherwise identical chimeric inhibitory receptor lacking the spacer region.

In some aspects, the spacer region reduces sensitivity of the chimeric inhibitory receptor relative to an otherwise identical chimeric inhibitory receptor lacking the spacer region.

In some aspects, the spacer region modulates potency of the chimeric inhibitory receptor relative to an otherwise identical chimeric inhibitory receptor lacking the spacer region.

In some aspects, the spacer region increases potency of the chimeric inhibitory receptor relative to an otherwise identical chimeric inhibitory receptor lacking the spacer region.

In some aspects, the spacer region reduces potency of the chimeric inhibitory receptor relative to an otherwise identical chimeric inhibitory receptor lacking the spacer region.

In some aspects, the spacer region modulates basal prevention, attenuation, or inhibition of activation of the tumor-targeting chimeric receptor when expressed on an immunomodulatory cell relative to an otherwise identical chimeric inhibitory receptor lacking the spacer region.

In some aspects, the spacer region reduces basal prevention, attenuation, or inhibition relative to an otherwise identical chimeric inhibitory receptor lacking the spacer region.

In some aspects, the spacer region increases basal prevention, attenuation, or inhibition relative to an otherwise identical chimeric inhibitory receptor lacking the spacer region.

In some aspects, the chimeric inhibitory receptor further comprises an intracellular spacer region positioned between the transmembrane domain and one of the one or more intracellular signaling domains and operably linked to each of the transmembrane domain and one of the one or more intracellular signaling domains.

In some aspects, the chimeric inhibitory receptor further comprises an intracellular spacer region positioned between the transmembrane domain and one of the one or more intracellular signaling domains and physically linked to each of the transmembrane domain and one of the one or more intracellular signaling domains.

In some aspects, the intracellular spacer region modulates sensitivity of the chimeric inhibitory receptor relative to an otherwise identical chimeric inhibitory receptor lacking the intracellular spacer region.

In some aspects, the intracellular spacer region increases sensitivity of the chimeric inhibitory receptor relative to an otherwise identical chimeric inhibitory receptor lacking the intracellular spacer region.

In some aspects, the intracellular spacer region reduces sensitivity of the chimeric inhibitory receptor relative to an otherwise identical chimeric inhibitory receptor lacking the intracellular spacer region.

In some aspects, the intracellular spacer region modulates potency of the chimeric inhibitory receptor relative to an otherwise identical chimeric inhibitory receptor lacking the intracellular spacer region.

In some aspects, the intracellular spacer region increases potency of the chimeric inhibitory receptor relative to an otherwise identical chimeric inhibitory receptor lacking the intracellular spacer region.

In some aspects, the intracellular spacer region reduces potency of the chimeric inhibitory receptor relative to an otherwise identical chimeric inhibitory receptor lacking the intracellular spacer region.

In some aspects, the intracellular spacer region modulates basal prevention, attenuation, or inhibition of activation of the tumor-targeting chimeric receptor when expressed on an immunomodulatory cell relative to an otherwise identical chimeric inhibitory receptor lacking the intracellular spacer region.

In some aspects, the intracellular spacer region reduces basal prevention, attenuation, or inhibition relative to an otherwise identical chimeric inhibitory receptor lacking the intracellular spacer region.

In some aspects, the intracellular spacer region increases basal prevention, attenuation, or inhibition relative to an otherwise identical chimeric inhibitory receptor lacking the intracellular spacer region.

In some aspects, the inhibitory chimeric receptor further comprises an enzymatic inhibitory domain.

In some aspects, the enzymatic inhibitory domain is capable of preventing, attenuating, or inhibiting activation of a tumor-targeting chimeric receptor when expressed on an immunomodulatory cell relative to an otherwise identical chimeric inhibitory receptor lacking the enzymatic inhibitory domain.

In some aspects, the enzymatic inhibitory domain comprises an enzyme catalytic domain.

In some aspects, the enzyme catalytic domain is derived from an enzyme selected from the group consisting of: CSK, SHP-1, PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c-CBL, CBL-b, PTPN22, LAR, PTPH1, SHIP-1, and RasGAP.

In some aspects, the enzymatic inhibitory domain comprises one or more modifications that modulate basal prevention, attenuation, or inhibition.

In some aspects, the one or more modifications reduce basal prevention, attenuation, or inhibition relative to an otherwise identical enzymatic inhibitory domain lacking the one or more modifications.

In some aspects, the one or more modifications increase basal prevention, attenuation, or inhibition relative to an otherwise identical enzymatic inhibitory domain lacking the one or more modifications.

In some aspects, the tumor-targeting chimeric receptor is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR).

In some aspects, the immunomodulatory cell is selected from the group consisting of: a T cell, a CD8+ T cell, a CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a viral-specific T cell, a Natural Killer T (NKT) cell, a Natural Killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a macrophage, a monocyte, a dendritic cell, an ESC-derived cell, and an iPSC-derived cell.

In some aspects, the immunomodulatory cell is a Natural Killer (NK) cell.

Also provided herein are compositions comprising the chimeric inhibitory receptor as described herein and a pharmaceutically acceptable carrier.

Also provided herein are engineered nucleic acids encoding the chimeric inhibitory receptor as described herein.

Also provided herein are expression vectors comprising the engineered nucleic acid as described herein.

Also provided herein are composition comprising the engineered nucleic acid as described herein or the expression vector as described herein, and a pharmaceutically acceptable carrier

Also provided herein are isolated immunomodulatory cells comprising the chimeric inhibitory receptor as described herein.

In some aspects, the cell further comprises a tumor-targeting chimeric receptor expressed on the surface of the cell.

In some aspects, upon binding of the protein to the chimeric inhibitory receptor, the chimeric inhibitory receptor prevents, attenuates, or inhibits activation of the tumor-targeting chimeric receptor relative to an otherwise identical cell lacking a chimeric inhibitory receptor.

Also provided herein are isolated immunomodulatory cells comprising a chimeric inhibitory receptor, wherein the chimeric inhibitory receptor comprises: an extracellular protein binding domain, a transmembrane domain, wherein the transmembrane domain is operably linked to the extracellular protein binding domain, and one or more intracellular signaling domains, wherein the one or more intracellular signaling domains is operably linked to the transmembrane domain, and wherein the one or more intracellular signaling domain are each derived from a protein selected from the group consisting of: SLAP1, SLAP2, Dok-1, Dok-2, LAIR1, GRB-2, CD200R, SIRPα, HAVR, GITR, PD-L1, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, CD94, KLRG-1, CEACAM1, LIR2, LIR3, LIR5, SIGLEC-2, and SIGLEC-10; and wherein upon binding of the protein to the chimeric inhibitory receptor, the chimeric inhibitory receptor prevents, attenuates, or inhibits activation of a tumor-targeting chimeric receptor expressed on the surface of the cell.

In some aspects, the cell further comprises a tumor-targeting chimeric receptor expressed on the surface of the cell.

Also provided herein are isolated cells comprising: a chimeric inhibitory receptor, wherein and the chimeric inhibitory receptor comprises: an extracellular protein binding domain, a transmembrane domain, wherein the transmembrane domain is operably linked to the extracellular protein binding domain, and one or more intracellular signaling domains, wherein the one or more intracellular signaling domains are operably linked to the transmembrane domain, and wherein the one or more intracellular signaling domain are each derived from a protein selected from the group consisting of: SLAP1, SLAP2, Dok-1, Dok-2, LAIR1, GRB-2, CD200R, SIRPα, HAVR, GITR, PD-L1, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, CD94, KLRG-1, CEACAM1, LIR2, LIR3, LIR5, SIGLEC-2, and SIGLEC-10; and a tumor-targeting chimeric receptor expressed on the surface of the cell, wherein upon binding of the protein to the chimeric inhibitory receptor, the chimeric inhibitory receptor prevents, attenuates, or inhibits activation of the tumor-targeting chimeric receptor.

In some aspects, the chimeric inhibitory receptor is recombinantly expressed.

In some aspects, the chimeric inhibitory receptor is expressed from a vector or a selected locus from the genome of the cell.

In some aspects, the tumor-targeting chimeric receptor is a chimeric antigen receptor (CAR) or an engineered T cell receptor.

In some aspects, prior to binding of the protein to the chimeric inhibitory receptor, the tumor-targeting chimeric receptor is capable of activating the cell.

In some aspects, upon binding of the protein to the chimeric inhibitory receptor, the chimeric inhibitory receptor suppresses cytokine production from the activated cell.

In some aspects, upon binding of the protein to the chimeric inhibitory receptor, the chimeric inhibitory receptor suppresses a cell-mediated immune response to a target cell, wherein the immune response is induced by activation of the immunomodulatory cell.

In some aspects, the transmembrane domain is physically linked to the extracellular protein binding domain.

In some aspects, the intracellular signaling domain is physically linked to the transmembrane domain.

In some aspects, the target cell is a tumor cell.

In some aspects, the cell is selected from the group consisting of: a T cell, a CD8+ T cell, a CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a viral-specific T cell, a Natural Killer T (NKT) cell, a Natural Killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a macrophage, a monocyte, a dendritic cell, an ESC-derived cell, and an iPSC-derived cell.

In some aspects, the immunomodulatory cell is a Natural Killer (NK) cell.

In some aspects, the cell is autologous.

In some aspects, the cell is allogeneic.

Also provided herein are compositions comprising the isolated cell as described herein and a pharmaceutically acceptable carrier.

Also provided herein are methods of preventing, attenuating, or inhibiting a cell-mediated immune response induced by a tumor-targeting chimeric receptor expressed of the surface of an immunomodulatory cell, comprising: engineering the immunomodulatory cell to express the chimeric inhibitory receptor of any one of claims 1-75 on the surface of the immunomodulatory cell, wherein upon binding of a cognate antigen to the chimeric inhibitory receptor, the intracellular signaling domain prevents, attenuates, or inhibits activation of the tumor-targeting chimeric receptor.

Also provided herein are methods of preventing, attenuating, or inhibiting activation of a tumor-targeting chimeric receptor expressed on the surface of an immunomodulatory cell, comprising: contacting the isolated cell as described herein or the composition as described herein with a cognate antigen of the chimeric inhibitory receptor under conditions suitable for the chimeric inhibitory receptor to bind the cognate antigen, wherein upon binding of the antigen to the chimeric inhibitory receptor, the intracellular signaling domain prevents, attenuates, or inhibits activation of the tumor-targeting chimeric receptor.

In some aspects, the tumor-targeting chimeric receptor is a chimeric antigen receptor (CAR) or an engineered T cell receptor.

In some aspects, the CAR binds one or more antigens expressed on the surface of a tumor cell.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:

FIG. 1A shows an exemplary diagram of a T cell co-expressing an anti-CD19-SLAP iCAR and an anti-CD20-CD28/CD3ζ aCAR contacting a target cell expressing CD19 and CD20. FIG. 1B shows negative control cells with no expression of either CAR construct.

FIG. 1C shows anti-CD20-CD28/CD3ζ aCAR expression in transduced T cells. FIG. 1D shows anti-CD20-CD28/CD3 aCAR and anti-CD19-SLAP iCAR expression in transduced T cells.

FIG. 2A shows TNF-α production by T cells is reduced by co-expression of an anti-CD20 aCAR and an anti-CD19 iCAR as compared to an anti-CD20 aCAR alone. FIG. 2B shows IFN-γ production by T cells is reduced by co-expression of an anti-CD20 aCAR and an anti-CD19 iCAR as compared to an anti-CD20 aCAR alone. FIG. 2C shows IL-2 production by T cells is reduced by co-expression of an anti-CD20 aCAR and an anti-CD19 iCAR as compared to an anti-CD20 aCAR alone.

FIG. 3 shows expression profiles of an anti-FLT3 aCAR and various iCAR formats with an anti-EMCN binding domain, including co-expression, following transduction of NK cells as assessed by flow cytometry. Between 1 and 3 biological replicates per condition (indicated as separate points).

FIG. 4 shows NK cell mediated killing (top panels) and cytokine secretion (bottom panel). Shown are for the various NK cells engineered to co-express an anti-FLT3 aCAR and the indicated anti-EMCN iCARs. “Separate”=each type of SEM cell presented separately (top left panel). “Mixed”=both types of SEM cells mixed together in the same culture (top right panel). Between 1 and 3 biological replicates per condition (indicated as separate points). 3 technical replicates per measurement, X and Y SEM plotted where relevant. KLRG1 is not shown where its iCAR protection is negative.

DETAILED DESCRIPTION
Definitions

Terms used in the claims and specification are defined as set forth below unless otherwise specified.

The term “inhibitory chimeric receptor” or “inhibitory chimeric antigen receptor” or “chimeric inhibitory receptor” as used herein refers to a polypeptide or a set of polypeptides, which when expressed in an immune effector cell, provides the cell with specificity for a target cell, and with inhibitory intracellular signal generation. Inhibitory chimeric receptors typically include an extracellular protein binding domain (e.g., antibody fragment as an antigen-binding domain), a spacer domain, a transmembrane domain, and one or more intracellular signaling/co-signaling domains. An inhibitory chimeric receptor may also be called an “iCAR.”

The term “tumor targeting chimeric receptor” refers to activating chimeric receptors, tumor-targeting chimeric antigen receptors (CARs), or engineered T cell receptors. A tumor targeting chimeric receptor may also be called an “aCAR.”

The term “chimeric antigen receptor” or alternatively a “CAR” as used herein refers to a polypeptide or a set of polypeptides, which when expressed in an immune effector cell, provides the cell with specificity for a target cell, and with intracellular signal generation. CARs typically include an extracellular protein binding domain (e.g., antibody fragment as an antigen-binding domain), a spacer domain, a transmembrane domain, and one or more intracellular signaling/co-signaling domains. In some embodiments, a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as “an intracellular signaling domain”) comprising a functional signaling domain derived from a inhibitory molecule or a stimulatory molecule and/or costimulatory molecule. In some aspects, the set of polypeptides that comprise the inhibitory chimeric receptor or tumor targeting chimeric receptor are contiguous with each other. In some embodiments, the inhibitory chimeric receptor or tumor targeting chimeric receptor further comprises a spacer domain between the extracellular antigen binding domain and the transmembrane domain. In some embodiments, the set of polypeptides include recruitment domains, such as dimerization or multimerization domains, that can couple the polypeptides to one another. In some embodiments, an inhibitory chimeric receptor comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from an inhibitory molecule or a stimulatory molecule. In one aspect, an inhibitory chimeric receptor comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional inhibitory domain derived from an inhibitory molecule. In one aspect, a tumor targeting chimeric receptor comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule.

The term, “intracellular signaling domain” as used herein, refers to a functional domain of the inhibitory chimeric receptor or the tumor targeting chimeric receptor located inside the cell. In some embodiments, the intracellular signaling domain is an inhibitory signaling domain. Following binding of the molecular binding domain to an protein, for example, an inhibitory signaling domain represses receptor signaling while an activation signaling domain transmits a signal (e.g., proliferative/survival signal) to the cell.

The term, “transmembrane domain” as used herein, refers to a domain that spans a cellular membrane. In some embodiments, a transmembrane domain comprises a hydrophobic alpha helix.

The term, “extracellular protein binding domain” or “extracellular antigen binding domain” as used herein, refers to a molecular binding domain which is typically an ectodomain of a cell receptor or the antigen binding domains of an antibody and is located outside the cell, exposed to the extracellular space. An extracellular antigen binding domain can include any molecule (e.g., protein or peptide) capable of binding to another protein or peptide. In some embodiments, an extracellular protein or antigen binding domain comprises an antibody, an antigen-binding fragment thereof, F(ab), F(ab′), a single chain variable fragment (scFv), or a single-domain antibody (sdAb). In some embodiments, an extracellular protein or antigen binding domain binds to a cell-surface ligand (e.g., an antigen, such as a cancer antigen or a protein expressed on the surface of a cell).

The term “tumor” refers to tumor cells and the associated tumor microenvironment (TME). In some embodiments, tumor refers to a tumor cell or tumor mass. In some embodiments, tumor refers to the tumor microenvironment.

The term “not expressed” refers to expression that is at least 2-fold lower than the level of expression in non-tumor cells that would result in activation of the tumor-targeting chimeric antigen receptor. In some embodiments, the expression is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold or more lower than the level of expression in non-tumor cells that would result in activation of the tumor-targeting chimeric antigen receptor.

The term “ameliorating” refers to any therapeutically beneficial result in the treatment of a disease state, e.g., a cancer disease state, including prophylaxis, lessening in the severity or progression, remission, or cure thereof.

The term “in situ” refers to processes that occur in a living cell growing separate from a living organism, e.g., growing in tissue culture.

The term “in vivo” refers to processes that occur in a living organism.

The term “mammal” as used herein includes both humans and non-humans and include but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

The term percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/).

The term “sufficient amount” means an amount sufficient to produce a desired effect, e.g., an amount sufficient to modulate protein aggregation in a cell.

The term “therapeutically effective amount” is an amount that is effective to ameliorate a symptom of a disease. A therapeutically effective amount can be a “prophylactically effective amount” as prophylaxis can be considered therapy.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

Chimeric Inhibitory Receptors

In one aspect, provided herein are chimeric inhibitory receptors comprising (i) an extracellular protein binding domain; (ii) a transmembrane domain, wherein the transmembrane domain is operably linked to the extracellular protein binding domain; and (iii) one or more intracellular signaling domains, wherein the one or more intracellular signaling domains are operably linked to the transmembrane domain, and wherein at least one of the one or more intracellular signaling domains is capable of preventing, attenuating, or inhibiting activation of a tumor-targeting chimeric receptor expressed on an immunomodulatory cell.

Generally, an inhibitory or tumor targeting chimeric receptor is designed for a T cell, or NK cell, and is a chimera of an intracellular signaling domain and an antigen-recognizing domain (e.g., a single chain fragment (scFv) of an antibody) (Enblad et al., Human Gene Therapy. 2015; 26(8):498-505). A T cell that expresses a chimeric antigen receptor (CAR) is known in the art as a CAR T cell. An activating or tumor targeting CAR generally induces T cell signaling pathways upon binding to its cognate ligand via an intracellular signaling domain that results in activation of the T cell and an immune response. Activation CAR, activating CAR, and tumor-targeting CAR are interchangeable terms.

An inhibitory chimeric receptor, generally, is an artificial immune cell receptor engineered to recognize and bind to proteins expressed by cells. Inhibitory chimeric receptors generally recognize proteins that are not expressed on tumor cells, while activating or tumor targeting chimeric receptors (e.g., aCARs) generally recognize proteins that are expressed on tumor cells. Chimeric receptors in general typically include an antibody fragment as an extracellular protein binding domain, a spacer or hinge domains, a hydrophobic alpha helix transmembrane domain, and one or more intracellular signaling/co-signaling domains.

An inhibitory chimeric receptor generally follows the structure of activating CARs (aCARs) but uses an inhibitory domain for the intracellular signaling domain, instead of an activation signaling domain derived from a T-cell receptor (TCR). The intracellular signaling/co-signaling domain are inhibitory domains that reduce or inhibit signaling by other receptor proteins in the same cell. An inhibitory chimeric receptor cell can contain an antigen-specific inhibitory receptor, for example, to block nonspecific immunoactivation, which may result from extra-tumor target expression. In some embodiments, an inhibitory chimeric receptor blocks T cell responses in T cells activated by either their endogenous T cell receptor or an activating or tumor-targeting CAR. For example, an immunomodulatory cell can express both an inhibitory chimeric receptor that recognizes a non-tumor protein target and a tumor-targeting chimeric receptor that recognizes a tumor protein. When such an immunomodulatory cell contacts a tumor cell, only the tumor-targeting receptor recognizes and binds its cognate ligand and is activated, resulting in induction of cell signaling pathways and immune cell activation. In contrast, when the immunomodulatory cell contacts a non-tumor target, the inhibitory chimeric receptor binds to its cognate ligand and represses or inhibits any signaling induced by the activation of the tumor-targeting chimeric receptor. Thus, the immunomodulatory cell can be constructed so that immune signaling only occurs when the cell contacts tumor cells.

In some embodiments, the protein bound by the inhibitory chimeric receptor is not expressed on the target tumor. In some embodiments, the expression is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold or more lower than the level of expression in non-tumor cells that would result in activation of the tumor-targeting chimeric antigen receptor.

In some embodiments, the protein bound by the inhibitory chimeric receptor is expressed on a non-tumor cell.

In some embodiments, the protein bound by the inhibitory chimeric receptor is expressed on a non-tumor cell derived from a tissue selected from the group consisting of brain, neuronal tissue, endocrine, endothelial, bone, bone marrow, immune system, muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, urinary bladder, male reproductive organs, female reproductive organs, adipose, soft tissue, and skin.

Intracellular Signaling Domains

The inhibitory chimeric receptors of the present disclosure comprise intracellular signaling domains that are capable of preventing, attenuating, or inhibiting activation of a tumor-targeting chimeric receptor expressed on an immunomodulatory cell. In some embodiments, the chimeric inhibitory receptor comprises one or more intracellular signaling domains.

In some embodiments, the intracellular signaling domain comprises one or more modifications. In some embodiments, the one or more modifications modulate sensitivity of the chimeric inhibitory receptor relative to the otherwise identical, unmodified receptor. In some embodiments, the one or more modifications increase sensitivity of the chimeric inhibitory receptor relative to the otherwise identical, unmodified receptor. In some embodiments, the one or more modifications reduce sensitivity of the chimeric inhibitory receptor relative to the otherwise identical, unmodified receptor. In some embodiments, the one or more modifications modulate potency of the chimeric inhibitory receptor relative to the otherwise identical, unmodified receptor. In some embodiments, the one or more modifications increase potency of the chimeric inhibitory receptor relative to the otherwise identical, unmodified receptor. In some embodiments, the one or more modifications reduce potency of the chimeric inhibitory receptor relative to the otherwise identical, unmodified receptor.

In some embodiments, the one or more modifications modulate basal prevention, attenuation, or inhibition of activation of the tumor-targeting chimeric receptor expressed on an immunomodulatory cell relative to the otherwise identical, unmodified receptor. In some embodiments, the one or more modifications reduce basal prevention, attenuation, or inhibition relative to the otherwise identical, unmodified receptor. In some embodiments, the one or more modifications increase basal prevention, attenuation, or inhibition relative to the otherwise identical, unmodified receptor.

Inhibitory Domains

In some embodiments, the inhibitory intracellular signaling domain is derived from a protein selected from the group consisting of: SLAP1, SLAP2, LAIR1, GRB-2, Dok-1, Dok-2, CD200R, SIRPalpha (SIRPα), HAVR, GITR, PD-L1, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, CD94, KLRG-1, CEACAM1, LIR2, LIR3, LIR5, SIGLEC-2, and SIGLEC-10. In some embodiments, the inhibitory chimeric receptor described herein comprises an inhibitory intracellular signaling domain. In some embodiments, the inhibitory intracellular signaling domain is a SLAP1 domain. In some embodiments, the SLAP1 domain comprises amino acid residues 8-276 of the full length SLAP1 protein. In some embodiments, the SLAP1 domain comprises amino acid residues 8-247 of the full length SLAP1 protein. In some embodiments, the SLAP1 domain comprises amino acid residues 8-261 of the full length SLAP1 protein. In some embodiments, the inhibitory intracellular signaling domain is a SLAP2 domain. In some embodiments, the inhibitory intracellular signaling domain is a Dok-2 domain. In some embodiments, the inhibitory intracellular signaling domain is a Dok-1 domain. In some embodiments, the inhibitory intracellular signaling domain is a GRB2 domain. In some embodiments, the inhibitory intracellular signaling domain is a CD200R domain. In some embodiments, the inhibitory intracellular signaling domain is a SIRPα domain.

Src-like adaptor proteins 1 and 2 (SLAP1 and SLAP2) are adaptor proteins involved in intracellular signaling pathways and are expressed in lymphocytes. Both SLAP1 and SLAP2 contain common SH2 and SH3 domains. SH2 domains allow proteins to bind to phosphorylated tyrosine epitopes. SLAP1 and SLAP2 function as negative regulators of T cell receptor (TCR) signaling, likely by associating with the E3 ubiquitin ligase c-Cbl, which promotes the ubiquitination and degradation of the TCR-chain, resulting in decreased TCR signaling.

Docking protein 2 (Dok-2) is part of a negative signaling complex in T cells. Docking protein 1 (Dok-1) is part of the negative regulation of the insulin receptor signaling pathway. Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein involved in signal transduction and contains one SH2 domain and two SH3 domains. Signal-regulator protein alpha (SIRPα) is an inhibitory receptor that contains four immunoreceptor tyrosine-based inhibition motifs (ITIMs). Cell surface transmembrane glycoprotein CD200 receptor 1 (CD200R) is involved in signaling pathways that regulate the expression of pro-inflammatory molecules and associates with Dok-1 and Dok-2.

Exemplary inhibitory intracellular signaling domain amino acid sequences are shown in Table 1. Exemplary inhibitory intracellular signaling domain nucleic acid sequences are shown in Table 2.

TABLE 1

Exemplary inhibitory intracellular signaling domain amino acid sequences

SEQ ID

Amino Acid Sequence
NO:
Description

Inhibitory Intracellular Signaling Domains

MEAIAKYDFKATADDELSFKRGDILKVLNEECDQNWYKAELNGK
1
GRB2 intracellular

DGFIPKNYIEMKPHPWFFGKIPRAKAEEMLSKQRHDGAFLIRESES

signaling domain

APGDFSLSVKFGNDVQHFKVLRDGAGKYFLWVVKFNSLNELVDY

HRSTSVSRNQQIFLRDIEQVPQQPTYVQALFDFDPQEDGELGFRRG

DFIHVMDNSDPNWWKGACHGQTGMFPRNYVTPVNRNV

MDGAVMEGPLFLQSQRFGTKRWRKTWAVLYPASPHGVARLEFFD
2
Dok-1 intracellular

HKGSSSGGGRGSSRRLDCKVIRLAECVSVAPVTVETPPEPGATAFR

signaling domain

LDTAQRSHLLAADAPSSAAWVQTLCRNAFPKGSWTLAPTDNPPKL

SALEMLENSLYSPTWEGSQFWVTVQRTEAAERCGLHGSYVLRVE

AERLTLLTVGAQSQILEPLLSWPYTLLRRYGRDKVMFSFEAGRRCP

SGPGTFTFQTAQGNDIFQAVETAIHRQKAQGKAGQGHDVLRADSH

EGEVAEGKLPSPPGPQELLDSPPALYAEPLDSLRIAPCPSQDSLYSD

PLDSTSAQAGEGVQRKKPLYWDLYEHAQQQLLKAKLTDPKEDPIY

DEPEGLAPVPPQGLYDLPREPKDAWWCQARVKEEGYELPYNPAT

DDYAVPPPRSTKPLLAPKPQGPAFPEPGTATGSGIKSHNSALYSQV

QKSGASGSWDCGLSRVGTDKTGVKSEGST

MGDGAVKQGFLYLQQQQTFGKKWRRFGASLYGGSDCALARLEL
3
Dok-2 intracellular

QEGPEKPRRCEAARKVIRLSDCLRVAEAGGEASSPRDTSAFFLETK

signaling domain

ERLYLLAAPAAERGDWVQAICLLAFPGQRKELSGPEGKQSRPCME

ENELYSSAVTVGPHKEFAVTMRPTEASERCHLRGSYTLRAGESAL

ELWGGPEPGTQLYDWPYRFLRRFGRDKVTFSFEAGRRCVSGEGNF

EFETRQGNEIFLALEEAISAQKNAAPATPQPQPATIPASLPRPDSPYS

RPHDSLPPPSPTTPVPAPRPRGQEGEYAVPFDAVARSLGKNFRGILA

VPPQLLADPLYDSIEETLPPRPDHIYDEPEGVAALSLYDSPQEPRGE

AWRRQATADRDPAGLQHVQPAGQDFSASGWQPGTEYDNVVLKK

GPK

PAPAERPLPNPEGLDSDFLAVLSDYPSPDISPPIFRRGEKLRVISDEG
4
SLAP1_aa8-aa276

GWWKAISLSTGRESYIPGICVARVYHGWLFEGLGRDKAEELLQLP

intracellular signaling

DTKVGSFMIRESETKKGFYSLSVRHRQVKHYRIFRLPNNWYYISPR

domain

LTFQCLEDLVNHYSEVADGLCCVLTTPCLTQSTAAPAVRASSSPVT

LRQKTVDWRRVSRLQEDPEGTENPLGVDESLFSYGLRESIASYLSL

TSEDNTSFDRKKKSISLMYGGSKRKSSFFSSPPYFED

PAPAERPLPNPEGLDSDFLAVLSDYPSPDISPPIFRRGEKLRVISDEG
5
SLAP1_aa8-aa247

GWWKAISLSTGRESYIPGICVARVYHGWLFEGLGRDKAEELLQLP

intracellular signaling

DTKVGSFMIRESETKKGFYSLSVRHRQVKHYRIFRLPNNWYYISPR

domain

LTFQCLEDLVNHYSEVADGLCCVLTTPCLTQSTAAPAVRASSSPVT

LRQKTVDWRRVSRLQEDPEGTENPLGVDESLFSYGLRESIASYLSL

TSEDNTSF

RKSLPSPSLSSSVQGQGPVTMEAERSKATAVALGSFPAGGPAELSL
6
SLAP2_aa8-aa261

RLGEPLTIVSEDGDWWTVLSEVSGREYNIPSVHVAKVSHGWLYEG

intracellular signaling

LSREKAEELLLLPGNPGGAFLIRESQTRRGSYSLSVRLSRPASWDRI

domain

RHYRIHCLDNGWLYISPRLTFPSLQALVDHYSELADDICCLLKEPC

VLQRAGPLPGKDIPLPVTVQRTPLNWKELDSSLLFSEAATGEESLLS

EGLRESLSFYISLNDEAVSLDDA

KVNGCRKYKLNKTESTPVVEEDEMQPYASYTEKNNPLYDTTNKV
7
CD200R intracellular

KASEALQSEVDTDLHTL

signaling domain

RIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKP
8
SIRPα intracellular

APQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQP

signaling domain

APKPEPSFSEYASVQVPRK

HRWCSNKKNAAVMDQESAGNRTANSEDSDEQDPQEVTYTQLNH
60
KIR2DL1 intracellular

CVFTQRKITRPSQRPKTPPTDIIVYTELPNAESRSKVVSCP

signaling domain

MTDSVIYSMLELPTATQAQNDYGPQQKSSSSRPSCSCLGSG
61
KLRG1 intracellular

signaling domain

HRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPE
62
LAIR1 intracellular

KDRETDTSALAAGSSQEVTYAQLDHWALTQRTARAVSPQSTKPM

signaling domain

AESITYAAVARH

LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAAD
53
LIR2 intracellular

AQEENLYAAVKDTQPEDGVEMDTRAAASEAPQDVTYAQLHSLTL

signaling domain

RRKATEPPPSQEREPPAEPSIYATLAIH

RRQRHSKHRTSDQRKTDFQRPAGAAETEPKDRGLLRRSSPAADVQ
64
LIR3 intracellular

EENLYAAVKDTQSEDRVELDSQSPHDEDPQAVTYAPVKHSSPRRE

signaling domain

MASPPSSLSGEFLDTKDRQVEEDRQMDTEAAASEASQDVTYAQLH

SLTLRRKATEPPPSQEGEPPAEPSIYATLAIH

QHWRQGKHRTLAQRQADFQRPPGAAEPEPKDGGLQRRSSPAADV
65
LIR5 intracellular

QGENFCAAVKNTQPEDGVEMDTRQSPHDEDPQAVTYAKVKHSRP

signaling domain

RREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYA

QLHSFTLRQKATEPPPSQEGASPAEPSVYATLAIH

KLQRRWKRTQSQQGLQENSSGQSFFVRNKKVRRAPLSEGPHSLGC
66
SIGLEC-2 intracellular

YNPMMEDGISYTTLRFPEMNIPRTGDAESSEMQRPPPDCDDTVTYS

signaling domain

ALHKRQVGDYENVIPDFPEDEGIHYSELIQFGVGERPQAQENVDYV

ILKH

KILPKRRTQTETPRPRFSRHSTILDYINVVPTAGPLAQKRNQKATPN
67
SIGLEC-10 intracellular

SPRTPLPPGAPSPESKKNQKKQYQLPSFPEPKSSTQAPESQESQEEL

signaling domain

HYATLNFPGVRPRPEARMPKGTQADYAEVKFQ

TABLE 2

Exemplary inhibitory intracellular signaling domain nucleic acid sequences

SEQ ID

Nucleic Acid Sequence
NO:
Description

Inhibitory Intracellular Signaling Domains

ATGGAAGCCATTGCCAAATACGACTTCAAGGCCACCGCCGACG
9
GRB2 intracellular

ACGAGCTGAGCTTCAAGAGAGGCGACATCCTGAAGGTGCTGAA

signaling domain

CGAGGAATGCGACCAGAACTGGTACAAGGCCGAGCTGAACGGC

AAGGACGGCTTCATCCCCAAGAACTACATCGAGATGAAGCCCC

ATCCATGGTTCTTCGGCAAGATCCCCAGAGCCAAGGCCGAAGA

GATGCTGAGCAAGCAGAGACACGACGGCGCCTTTCTGATCCGG

GAATCTGAATCTGCCCCTGGCGACTTCAGCCTGAGCGTGAAGTT

CGGCAACGACGTGCAGCACTTCAAGGTCCTGAGAGATGGCGCC

GGAAAGTACTTCCTGTGGGTCGTGAAGTTTAACAGCCTGAACG

AGCTGGTGGACTACCACAGATCCACCAGCGTGTCCCGGAACCA

GCAGATCTTCCTGCGGGACATCGAACAGGTGCCACAGCAGCCA

ACATACGTGCAGGCCCTGTTCGACTTCGACCCTCAAGAGGATGG

CGAGCTGGGCTTTAGACGGGGCGATTTCATCCACGTGATGGAC

AACAGCGACCCCAACTGGTGGAAGGGCGCTTGTCATGGACAGA

CCGGCATGTTCCCCAGAAACTACGTGACCCCTGTGAACCGGAA

CGTG

ATGGATGGCGCCGTGATGGAAGGCCCTCTGTTTCTCCAGAGCCA
10
Dok-1 intracellular

GAGATTCGGCACCAAGCGGTGGCGGAAAACATGGGCCGTTCTG

signaling domain

TACCCTGCCTCTCCTCATGGCGTGGCCCGGCTGGAATTTTTCGA

TCACAAGGGCTCTAGCAGCGGCGGAGGCAGAGGATCTAGTAGA

CGGCTGGACTGCAAAGTGATCCGGCTGGCCGAGTGTGTGTCTGT

GGCTCCTGTGACCGTGGAAACCCCTCCTGAACCTGGCGCCACAG

CCTTCAGACTGGATACAGCCCAGAGAAGCCATCTGCTGGCCGC

CGATGCTCCTTCTTCTGCTGCTTGGGTGCAGACCCTGTGCCGGA

ACGCTTTTCCTAAAGGCAGCTGGACACTGGCCCCTACCGACAAT

CCTCCTAAGCTGAGCGCCCTGGAAATGCTGGAAAACAGCCTGT

ACAGCCCCACCTGGGAGGGCTCTCAGTTTTGGGTCACCGTGCAG

AGAACAGAGGCCGCCGAAAGATGTGGCCTGCACGGCTCTTATG

TGCTGAGAGTGGAAGCCGAGAGACTGACCCTGCTGACAGTGGG

AGCCCAGTCTCAGATCCTGGAACCTCTGCTGAGCTGGCCCTACA

CACTGCTGAGAAGATACGGCCGGGACAAAGTGATGTTCAGCTT

CGAGGCCGGCAGAAGATGTCCTTCTGGCCCTGGCACATTCACAT

TTCAGACAGCCCAGGGCAACGACATCTTCCAGGCTGTGGAAAC

CGCCATCCACAGACAGAAGGCCCAGGGAAAAGCCGGCCAGGG

ACACGATGTTCTGAGAGCCGATTCTCACGAGGGCGAAGTGGCC

GAGGGAAAGCTTCCTTCTCCACCTGGACCTCAAGAGCTGCTGGA

TAGCCCTCCTGCTCTGTATGCCGAGCCTCTGGACAGCCTGAGAA

TCGCCCCTTGTCCAAGCCAGGACTCTCTGTACAGCGATCCCCTG

GATAGCACATCTGCCCAAGCTGGCGAAGGCGTGCAGAGGAAGA

AGCCCCTGTACTGGGATCTGTACGAGCACGCTCAGCAGCAACT

GCTGAAGGCCAAGCTGACAGACCCCAAAGAGGACCCCATCTAC

GACGAGCCTGAAGGACTTGCTCCAGTGCCACCTCAGGGCCTGT

ACGATCTGCCTAGAGAGCCTAAAGACGCCTGGTGGTGTCAGGC

CAGAGTGAAAGAGGAAGGCTACGAGCTGCCTTACAACCCCGCC

ACCGATGATTATGCCGTGCCTCCTCCAAGAAGCACCAAACCACT

GCTGGCCCCAAAGCCTCAGGGACCTGCTTTTCCTGAGCCTGGAA

CAGCCACAGGCAGCGGCATCAAGAGCCACAATAGCGCCCTGTA

TAGCCAGGTGCAGAAAAGCGGCGCCAGCGGCTCTTGGGATTGT

GGACTTAGCAGAGTGGGCACCGACAAGACCGGCGTGAAGTCTG

AGGGAAGCACA

ATGGGAGATGGCGCCGTGAAGCAGGGCTTTCTGTATCTCCAGC
11
Dok-2 intracellular

AGCAGCAGACCTTCGGCAAGAAGTGGCGGAGATTTGGCGCCTC

signaling domain

TCTGTACGGCGGCTCTGATTGTGCTCTGGCCCGACTGGAACTGC

AAGAGGGACCTGAGAAGCCCAGAAGATGCGAGGCCGCCAGAA

AAGTGATCCGGCTGAGCGATTGTCTGAGAGTGGCTGAAGCAGG

CGGCGAAGCCAGCTCTCCTAGAGATACCAGCGCATTCTTCCTGG

AAACAAAAGAGCGGCTGTACCTGCTGGCCGCTCCTGCTGCTGA

AAGAGGCGATTGGGTCCAAGCCATCTGCCTGCTGGCTTTTCCCG

GCCAGAGAAAAGAGCTGTCTGGCCCTGAGGGCAAGCAGAGCAG

GCCTTGCATGGAAGAGAACGAGCTGTACTCCAGCGCCGTGACA

GTGGGCCCTCACAAAGAATTTGCCGTGACCATGAGGCCCACCG

AGGCCAGCGAAAGATGTCACCTGAGAGGCAGCTACACCCTGAG

AGCCGGCGAATCTGCTCTGGAACTTTGGGGAGGACCTGAGCCT

GGCACACAGCTGTACGACTGGCCCTACAGATTCCTGCGGAGATT

CGGCCGGGACAAAGTGACCTTCAGCTTTGAGGCTGGCCGCAGA

TGTGTGTCCGGCGAGGGCAATTTCGAGTTCGAGACAAGACAGG

GCAACGAGATCTTCCTGGCTCTGGAAGAGGCCATCAGCGCCCA

GAAAAATGCTGCCCCTGCTACACCTCAGCCTCAGCCTGCTACAA

TCCCTGCCTCTCTGCCCAGACCTGACAGCCCTTATAGCAGACCC

CACGACTCTCTGCCTCCACCTTCTCCAACAACACCCGTGCCTGC

TCCTAGACCTAGAGGACAAGAGGGCGAGTACGCCGTGCCTTTT

GATGCCGTGGCTAGAAGCCTGGGCAAGAACTTCAGAGGCATCC

TGGCTGTGCCTCCACAGCTGCTGGCTGACCCTCTGTACGACAGC

ATCGAGGAAACCCTGCCTCCAAGACCTGACCACATCTACGACG

AGCCTGAAGGCGTGGCAGCCCTGTCTCTGTATGACTCCCCTCAA

GAGCCTAGAGGCGAAGCCTGGCGTAGACAGGCTACCGCCGATA

GAGATCCTGCCGGACTGCAACATGTGCAGCCAGCCGGCCAGGA

TTTTTCTGCCTCTGGATGGCAGCCAGGCACCGAGTACGATAACG

TGGTGCTGAAGAAGGGCCCCAAG

CCAGCCCCAGCGGAGCGACCGCTGCCAAACCCTGAAGGGCTCG
12
SLAP1_aa8-aa276

ACAGTGACTTTTTGGCTGTCCTCTCCGACTATCCTAGTCCCGATA

intracellular signaling

TCAGCCCCCCGATATTCAGGCGCGGTGAAAAACTCCGAGTCATC

domain

AGCGATGAGGGGGGTTGGTGGAAAGCCATCAGCCTGAGTACCG

GACGAGAGTCATACATTCCTGGAATATGTGTAGCGAGGGTGTA

CCACGGTTGGCTGTTCGAGGGTCTGGGAAGAGATAAGGCCGAG

GAACTTCTCCAACTCCCAGATACAAAAGTCGGTAGTTTTATGAT

TCGGGAAAGTGAAACTAAAAAGGGGTTCTATAGCCTCTCAGTT

CGGCATAGGCAGGTCAAGCATTATCGGATATTTCGCTTGCCTAA

CAACTGGTACTACATAAGTCCCCGACTCACATTCCAATGCCTGG

AGGACCTCGTGAATCACTATTCAGAAGTTGCAGATGGCCTCTGC

TGCGTACTCACGACACCCTGCCTTACCCAGAGTACAGCGGCCCC

GGCTGTTCGGGCATCTTCCAGCCCAGTAACACTCAGGCAAAAA

ACTGTGGATTGGCGCAGAGTCTCACGCCTTCAGGAAGATCCTGA

GGGTACGGAAAATCCGCTCGGTGTGGACGAGTCACTGTTCTCCT

ATGGCTTGAGGGAATCTATAGCGTCTTACCTTTCCTTGACGTCT

GAAGATAATACGTCTTTCGATCGCAAAAAAAAATCAATATCTCT

TATGTATGGCGGAAGCAAAAGGAAATCATCATTCTTTTCCTCTC

CACCATATTTCGAAGAT

CCAGCCCCAGCGGAGCGACCGCTGCCAAACCCTGAAGGGCTCG
13
SLAP1_aa8-aa247

ACAGTGACTTTTTGGCTGTCCTCTCCGACTATCCTAGTCCCGATA

intracellular signaling

TCAGCCCCCCGATATTCAGGCGCGGTGAAAAACTCCGAGTCATC

domain

AGCGATGAGGGGGGTTGGTGGAAAGCCATCAGCCTGAGTACCG

GACGAGAGTCATACATTCCTGGAATATGTGTAGCGAGGGTGTA

CCACGGTTGGCTGTTCGAGGGTCTGGGAAGAGATAAGGCCGAG

GAACTTCTCCAACTCCCAGATACAAAAGTCGGTAGTTTTATGAT

TCGGGAAAGTGAAACTAAAAAGGGGTTCTATAGCCTCTCAGTT

CGGCATAGGCAGGTCAAGCATTATCGGATATTTCGCTTGCCTAA

CAACTGGTACTACATAAGTCCCCGACTCACATTCCAATGCCTGG

AGGACCTCGTGAATCACTATTCAGAAGTTGCAGATGGCCTCTGC

TGCGTACTCACGACACCCTGCCTTACCCAGAGTACAGCGGCCCC

GGCTGTTCGGGCATCTTCCAGCCCAGTAACACTCAGGCAAAAA

ACTGTGGATTGGCGCAGAGTCTCACGCCTTCAGGAAGATCCTGA

GGGTACGGAAAATCCGCTCGGTGTGGACGAGTCACTGTTCTCCT

ATGGCTTGAGGGAATCTATAGCGTCTTACCTTTCCTTGACGTCT

GAAGATAATACGTCTTTC

AGAAAATCCCTCCCCAGTCCAAGCCTGTCTAGTAGCGTTCAGGG
14
SLAP2_aa8-aa261

TCAGGGCCCAGTTACTATGGAAGCGGAACGATCCAAGGCTACC

intracellular signaling

GCAGTTGCTCTGGGTTCATTCCCGGCTGGAGGGCCAGCGGAACT

domain

CTCCTTGCGCCTGGGTGAGCCACTTACCATCGTTTCTGAGGACG

GAGATTGGTGGACGGTTCTTTCTGAAGTATCTGGGAGAGAATAC

AACATACCGTCAGTTCATGTCGCAAAAGTTTCACACGGTTGGCT

TTACGAGGGACTCAGCAGGGAAAAAGCGGAGGAATTGTTGCTT

TTGCCCGGCAATCCTGGCGGAGCATTTTTGATAAGAGAGAGTCA

GACCCGGAGAGGCAGTTATTCCCTGTCAGTTAGACTCAGCAGA

CCTGCGAGTTGGGATAGGATTCGGCACTACAGGATTCACTGCCT

TGATAACGGCTGGCTGTATATATCTCCTCGCCTGACATTCCCTA

GCCTCCAAGCGTTGGTTGATCATTACTCTGAACTGGCAGACGAT

ATCTGCTGCCTGCTCAAGGAACCGTGCGTCCTCCAGAGGGCAG

GCCCACTTCCTGGCAAGGATATTCCACTTCCAGTCACGGTTCAA

AGAACCCCCCTCAATTGGAAGGAGCTGGATAGCTCTCTCCTCTT

TTCCGAGGCCGCTACAGGTGAAGAATCTCTGTTGTCTGAAGGAT

TGAGAGAGAGTCTCTCCTTCTACATCTCTCTGAATGATGAAGCA

GTGTCATTGGACGACGCA

AAAGTGAACGGCTGCCGGAAGTACAAGCTGAACAAGACCGAG
15
CD200R intracellular

AGCACCCCTGTGGTGGAAGAGGACGAGATGCAGCCTTACGCCA

signaling domain

GCTACACCGAGAAGAACAACCCTCTGTACGACACCACCAACAA

AGTGAAGGCCAGCGAGGCCCTCCAGAGCGAGGTTGACACAGAT

CTGCACACCCTG

CACCGGTGGTGCAGCAACAAGAAAAACGCCGCCGTGATGGACC
68
KIR2DL1 intracellular

AAGAGAGCGCCGGAAATAGAACCGCCAACAGCGAGGATAGCG

signaling domain

ACGAGCAGGACCCTCAAGAAGTGACCTACACACAGCTGAACCA

CTGCGTGTTCACCCAGCGGAAGATCACCAGACCTAGCCAGCGG

CCTAAGACACCACCTACCGACATCATCGTGTACACCGAGCTGCC

CAACGCCGAGAGCAGATCCAAGGTCGTGTCCTGTCCT

ATGACCGACAGCGTGATCTACAGCATGCTCGAGCTGCCTACAG
69
KLRG1 intracellular

CCACACAGGCCCAGAATGATTACGGCCCTCAGCAGAAGTCCAG

signaling domain

CTCCAGCAGACCTAGCTGTAGCTGTCTTGGCTCCGGC

CACCGGCAGAACCAGATCAAGCAGGGCCCTCCTAGAAGCAAGG
70
LAIR1 intracellular

ACGAGGAACAGAAGCCTCAGCAGAGGCCTGATCTGGCCGTGGA

signaling domain

CGTGCTGGAAAGAACAGCCGATAAGGCCACCGTGAACGGCCTG

CCTGAGAAGGACAGAGAGACAGACACATCTGCCCTGGCCGCTG

GCAGCTCCCAAGAAGTGACATACGCCCAGCTGGACCACTGGGC

CCTGACACAGAGAACTGCCAGAGCTGTGTCCCCTCAGAGCACC

AAACCTATGGCCGAGAGCATCACCTATGCCGCCGTGGCCAGACAT

CTGCGGCACAGAAGGCAGGGCAAGCACTGGACAAGCACCCAG
71
LIR2 intracellular

AGAAAGGCCGATTTTCAGCACCCTGCTGGCGCCGTTGGACCTGA

signaling domain

GCCTACAGATAGAGGACTGCAGTGGCGGTCTAGCCCTGCTGCC

GATGCTCAAGAGGAAAACCTGTACGCCGCCGTGAAGGACACCC

AACCTGAAGATGGCGTGGAAATGGACACCAGAGCCGCCGCATC

TGAAGCCCCTCAGGATGTGACATACGCCCAGCTGCATAGCCTG

ACACTGAGGCGGAAAGCCACAGAGCCTCCACCTAGCCAAGAGA

GAGAGCCTCCTGCCGAGCCTAGCATCTATGCCACACTGGCCATT

CAC

CGGAGACAGAGACACAGCAAGCACAGAACCAGCGACCAGAGA
72
LIR3 intracellular

AAGACCGACTTCCAGAGGCCTGCTGGCGCCGCTGAGACAGAGC

signaling domain

CTAAAGATAGAGGACTGCTGCGGAGAAGCAGCCCTGCTGCCGA

TGTGCAAGAGGAAAATCTGTACGCCGCCGTGAAGGACACCCAG

AGCGAGGATAGAGTGGAACTGGACAGCCAGTCTCCTCACGACG

AAGATCCTCAGGCCGTGACATACGCCCCTGTGAAGCACAGCAG

CCCTCGGAGAGAAATGGCCTCTCCACCTTCTAGCCTGAGCGGCG

AGTTCCTGGACACCAAGGACAGACAGGTGGAAGAGGACCGGCA

GATGGATACAGAAGCTGCCGCCTCTGAAGCCAGCCAGGATGTG

ACATATGCCCAGCTGCATAGCCTGACACTGCGGCGGAAAGCTA

CAGAGCCTCCTCCATCTCAAGAGGGCGAGCCTCCAGCCGAGCC

TAGCATCTATGCCACACTGGCCATTCAC

CAGCATTGGAGACAGGGAAAGCACAGAACACTGGCCCAGCGGC
73
LIR5 intracellular

AGGCCGATTTCCAAAGACCTCCAGGTGCCGCCGAACCTGAGCC

signaling domain

TAAAGATGGTGGCCTGCAGCGGAGATCTTCTCCTGCTGCCGATG

TGCAGGGCGAGAATTTTTGTGCCGCCGTGAAGAACACCCAGCC

TGAGGATGGCGTGGAAATGGACACCAGACAGAGCCCTCACGAC

GAGGATCCACAGGCCGTGACATACGCCAAAGTGAAGCACAGCC

GGCCTCGGAGAGAAATGGCTAGCCCTCCAAGTCCTCTGAGCGG

CGAGTTCCTGGACACCAAAGACAGACAGGCCGAAGAGGACCGG

CAGATGGATACAGAAGCTGCCGCCTCTGAAGCCCCTCAGGATG

TGACATATGCCCAGCTGCATAGCTTCACCCTGCGGCAGAAAGCC

ACAGAGCCTCCACCTTCTCAAGAGGGCGCTTCTCCAGCCGAGCC

ATCTGTGTATGCCACACTGGCCATTCAC

AAGCTGCAGCGGAGATGGAAGAGAACACAGAGCCAGCAGGGC
74
SIGLEC-2 intracellular

CTGCAAGAGAATAGCAGCGGCCAGAGCTTCTTCGTGCGGAACA

signaling domain

AGAAAGTGCGGAGAGCCCCTCTGTCTGAGGGACCTCATAGCCT

GGGCTGTTACAACCCCATGATGGAAGATGGCATCAGCTACACC

ACACTGCGGTTCCCCGAGATGAACATCCCTAGAACAGGCGACG

CCGAGAGCAGCGAAATGCAAAGACCTCCTCCTGACTGCGACGA

CACCGTGACATATAGCGCCCTGCACAAGAGACAAGTGGGCGAC

TACGAGAACGTGATCCCTGACTTCCCTGAGGACGAGGGCATCC

ACTACAGCGAGCTGATCCAGTTTGGCGTGGGCGAAAGACCCCA

GGCTCAAGAGAATGTGGACTACGTGATCCTGAAGCAC

AAGATCCTGCCTAAGCGGCGCACCCAGACCGAGACTCCTAGAC
75
SIGLEC-10 intracellular

CTAGATTCAGCCGGCACAGCACCATCCTGGACTACATCAACGTG

signaling domain

GTGCCTACCGCCGGACCACTGGCTCAGAAGAGAAACCAGAAGG

CCACACCTAACAGCCCCAGAACACCTCTTCCACCTGGCGCACCT

TCTCCAGAGAGCAAGAAGAACCAGAAGAAGCAGTACCAGCTGC

CTAGCTTCCCCGAGCCTAAGAGCAGTACACAGGCCCCTGAGAG

CCAAGAGTCCCAAGAGGAACTGCACTACGCCACACTGAACTTC

CCCGGCGTCAGACCTAGACCTGAGGCCAGAATGCCTAAGGGCA

CCCAGGCCGATTACGCCGAAGTGAAGTTTCAA

In some embodiments, one of the one or more intracellular signaling domains is derived from a protein selected from the group consisting of: SLAP1, SLAP2, Dok-1, Dok-2, LAIR1, GRB-2, CD200R, SIRPα, HAVR, GITR, PD-L1, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, CD94, KLRG-1, CEACAM1, LIR2, LIR3, LIR5, SIGLEC-2, and SIGLEC-10.

In some embodiments, the transmembrane domain is derived from the same protein as one of the one or more intracellular signaling domains. In some embodiments, the transmembrane domain is derived from a first protein and one of the one or more the intracellular signaling domains is derived from a second protein that is distinct from the first protein.

In some embodiments, one of the one or more intracellular signaling domains is derived from SLAP1.

(SEQ ID NO: 4)

PAPAERPLPNPEGLDSDFLAVLSDYPSPDISPPIFRRGEKLRVISDEG

GWWKAISLSTGRESYIPGICVARVYHGWLFEGLGRDKAEELLQLPDTK

VGSFMIRESETKKGFYSLSVRHRQVKHYRIFRLPNNWYYISPRLTFQC

LEDLVNHYSEVADGLCCVLTTPCLTQSTAAPAVRASSSPVTLRQKTVD

WRRVSRLQEDPEGTENPLGVDESLFSYGLRESIASYLSLTSEDNTSFD

RKKKSISLMYGGSKRKSSFFSSPPYFED,

or

(SEQ ID NO: 5)

PAPAERPLPNPEGLDSDFLAVLSDYPSPDISPPIFRRGEKLRVISDEG

GWWKAISLSTGRESYIPGICVARVYHGWLFEGLGRDKAEELLQLPDTK

VGSFMIRESETKKGFYSLSVRHRQVKHYRIFRLPNNWYYISPRLTFQC

LEDLVNHYSEVADGLCCVLTTPCLTQSTAAPAVRASSSPVTLRQKTVD

WRRVSRLQEDPEGTENPLGVDESLFSYGLRESIASYLSLTSEDNTSF.