TRISPECIFIC ANTI-CD38, ANTI-CD28, AND ANTI-CD3 BINDING PROTEINS AND
METHODS OF USE FOR TREATING VIRAL INFECTION
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of International Application No. PCT/US2018/055084, filed October 9, 2018; U.S. Provisional Application Serial No. 62/831,572, filed April 9, 2019; U.S. Provisional Application Serial No. 62/831,608, filed April 9, 2019; and EP Application No. 19306097.7, filed September 11, 2019; ail of which are incorporated herein by reference in their entirety.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (filename: 183952032140SEQLIST.TXT, date recorded: October 2, 2019, size: 144 KB).
FIELD
[0003] The disclosure relates to methods of using trispecific binding proteins comprising four polypeptide chains that form three antigen binding sites that specifically bind a CD38 polypeptide (e.g., human and/or cynomolgus monkey CD38 polypeptides), a CD28 polypeptide, and a CD3 polypeptide for expanding memory T cells (e.g., virus-specific memory T cells) and/or treating chronic viral infection.
BACKGROUND
[0004] As part of the human adaptive immunity, T cell immunity plays crucial role in controlling viral infection, eliminating infected ceils which results in clearance of viral infection. In chronic infectious diseases such as Herpes viral infection (HSV, CMV, EBV, etc.), HIV, and HBV, viruses establish their persistence in humans by various mechanisms including immune suppression, T cell exhaustion, and latency establishment. Nevertheless, viral infection generally induces viral antigen specific immunity including antigen specific

CDS T cells that can readily recognize infected cells for controlling or killing through cytokine release or cytotoxic T cell (CTL) mediated killing processes. [0005] Thus, viral antigen specific T cell activation and/or amplification in vivo and/or ex vivo may provide therapeutic strategies against chronic viral infections.
BRIEF SUMMARY
[0006] Provided herein are anti-CD38/CD28xCD3 trispecific antibodies that were developed and evaluated for their potential in activating T cells, and subsequent proliferation and/or amplification of antigen specific T cells. These trispecific Abs can effectively expand CD4 and CDS effector and memory populations, including antigen specific CDS T central memory and effector memory cells in vitro. Specifically, in vitro expansion of CMV, EBV, HIV-l, Influenza specific CDS central memory and effector memory cells were demonstrated. The anti-CD38/CD28xCD3 trispecific antibodies described herein exhibited novel properties by engaging CD3/CD28/CD38, providing signaling pathways to stimulate and expand T cells, which may offer an effective strategy treating chronic infectious diseases such as HSV, CMV, EBV, HIV-l, and HBV infections. [0007] To meet these and other needs, provided herein are binding proteins that bind a CD38 polypeptide (e.g., human and cynomolgus monkey CD38 polypeptides), a CD28 polypeptide, and a CD3 polypeptide.
[0008] In some embodiments, provided herein is a method for expanding virus-specific memory T cells, comprising contacting a virus-specific memory T cell with a binding protein, wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
VL2-LI-VLI-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula:
VHl-L3-VH2-L4-CHl-hinge-CH2-CH3 [II]
and a third polypeptide chain comprises a structure represented by the formula:
VH3-CHi-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VLS-CL [IV]
wherein:
VLI is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
Vi-n is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavv chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain,
CL is an immunoglobulin light chain constant domain;
CHI is an immunoglobulin CHI heavy chain constant domain;
CH2 is an immunoglobulin CH2 heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CHI and Cm domains; and
Lj, L?, L3 and L4 are amino acid linkers; wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and
wherein Vm and VLI form a first antigen binding site that binds a CD28 polypeptide, wherein VH2 and VL,2 form a second antigen binding site that binds a CD3 polypeptide, and wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide [0009] In some embodiments, provided herein is a binding protein that comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
VL2-LI-VLI-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula;
VHI -L3-VH2-L4-CHI -hinge-Cm-Cro [II] and a third polypeptide chain comprises a structure represented by the formula;
VH3-CHi-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula;
VL3-CL [IV]
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain,
VL3 is a third immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;

CHI is an immunoglobulin CHI heavy chain constant domain;
CH2 is an immunoglobulin Cm heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CHI and Cm domains; and
Li, L2, L3 and L4 are amino acid linkers, wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and
wherein VHI and VLI form a first antigen binding site that binds a CD28 polypeptide, wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, and wherein VHS and VL:< form a third antigen binding site that binds a CD38 polypeptide for use in expanding virus-specific memory T cells.
[0010] In some embodiments, the virus-specific memory T cell is contacted with the binding protein in vitro or ex vivo. In some embodiments, contacting the virus-specific memory T cell with the binding protein causes activation and/or proliferation of virus-specific memory T cells.
[0011] In some embodiments, provided herein is a method for expanding T cells, comprising contacting a T ceil with a binding protein in vitro or ex vivo, wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a staicture represented by the formula:
VL2-LI-VLI-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula;
VHI -L3-VH2-L4-CHI -hinge-Cm-Cro [II] and a third polypeptide chain comprises a structure represented by the formula;
VH3-CHi-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula;
VL3-CL [IV]
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain,
VL3 is a third immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;

CHI is an immunoglobulin CHI heavy chain constant domain;
CH2 is an immunoglobulin Cm heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CHI and Cm domains; and
Li, L2, L3 and L4 are amino acid linkers, wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and
wherein VHI and VLI form a first antigen binding site that binds a CD28 polypeptide, wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, and wherein VHS and VL:< form a third antigen binding site that binds a CD38 polypeptide. [0012] In some embodiments, provided herein is a binding protein that comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
VL2-LI-VLI-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula:
VHl-L3-VH2-L4-CHl-hinge-CH2-CH3 [II]
and a third polypeptide chain comprises a structure represented by the formula:
Vm-Cm-hinge-Cm-Cm [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VL3~CL [IV]
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain,
CL is an immunoglobulin light chain constant domain;
CHI is an immunoglobulin CHI heavy chain constant domain;
CH2 is an immunoglobulin CH?. heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CHI and Cm domains; and
Lj, L?, L3 and L4 are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over
light chain-heavy chain pair; and
wherein Vm and VLI form a first antigen binding site that binds a CD28 polypeptide,
wherein Vm and VL2 form a second antigen binding site that binds a CD3 polypeptide, and
wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide for
use in a method for expanding T cells.
[0013] In some embodiments, the T cell is a memory T cell or an effector T cell. In
some embodiments, the T cell expresses a chimeric antigen receptor (CAR) on its cell
surface or comprises a polynucleotide encoding a CAR.
[0014] In some embodiments, provided herein is a method for treating chronic viral
infection, comprising administering to an individual in need thereof an effective amount of
a binding protein, wherein the binding protein comprises four polypeptide chains that form
the three antigen binding sites, wherein a first polypeptide chain comprises a structure
represented by the formula;
VL2-LI-VLI-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula:
VHl-L3-VH2-L4-CHl-hinge-CH2-CH3 [II]
and a third polypeptide chain comprises a structure represented by the formula:
VH3-CHi-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VLS-CL [IV]
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL.3 is a third immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CHI is an immunoglobulin CHI heavy chain constant domain;
CH2 is an immunoglobulin Cm heavy chain constant domain;
C1-13 is an immunoglobulin Cm heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CHI and Cm domains; and
Li, L2, L3 and 1A are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and
wherein VHI and VLI form a first antigen binding site that binds a CD28 polypeptide, wherein Vm and VL2 form a second antigen binding site that binds a CD3 polypeptide, and wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide. [0015] In some embodiments, provided herein is a binding protein that comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
VL.2-LI-VLI-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula:
VHi-L3-VH2-L4-Cm-hinge-CH2-CH3 [II] and a third polypeptide chain comprises a structure represented by the formula:
VH3-CHi-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VLS-CL [IV]
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain,
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain,
CHI is an immunoglobulin CHI heavy chain constant domain;
CH2 is an immunoglobulin Cm heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CHI and Cm domains; and
Li, L2, L3 and 1U are amino acid linkers; wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and wherein VHI and VLI form a first antigen binding site that binds a CD28 polypeptide, wherein Vm and VL2 form a second antigen binding site that binds a CD3 polypeptide, and wherein Vm and VL3 form a third antigen binding site that binds a CDS 8 polypeptide for use in a method for treating chronic viral infection, wherein said method comprises administering to an individual in need thereof an effective amount

of the binding protein. In some embodiments, provided herein is a binding protein for use in a method for treating chronic viral infection, wherein said method comprises administering to an individual in need thereof an effective amount of the binding protein, wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain of the binding protein comprises a structure represented by the formula:
VL2-LI-VLI-L2-CL [I]
and a second polypeptide chain of the binding protein comprises a structure represented by the formula:
VHi-L3-VH2-L4-Cm-hinge-CH2-CH3 [II] and a third polypeptide chain of the binding protein comprises a structure represented by the formula:
Vm-Cm-hinge-Cm-Cm [III]
and a fourth polypeptide chain of the binding protein comprises a structure represented by the formula:
VL3~CL [IV]
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
Vi-n is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavv chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain,
CL is an immunoglobulin light chain constant domain;
CHI is an immunoglobulin CHI heavy chain constant domain;
CHI is an immunoglobulin CH?. heavy chain constant domain;
Ci-D is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CHI and CH2 domains; and
Li, L2, L3 and YA are amino acid linkers; wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and
wherein Vm and VLI form a first antigen binding site that binds a CD28 polypeptide, wherein Vm and VL2 form a second antigen binding site that binds a CDS polypeptide, and wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide.

[0016] In some embodiments, the individual is a human. In some embodiments, the binding protein is administered to the individual in pharmaceutical formulation comprising the binding protein and a pharmaceutic ally acceptable carrier. In some embodiments, administration of the binding protein results in activation and/or proliferation of virus-specific memory T cells in the individual.
[0017] In some embodiments that may be combined with any other embodiments described herein, the memory T cells are CD8+ or CD4+ memory T cells. In some embodiments, the memory T cells are central memory T cells (TCM) or effector memory T cells (TEM).
[0018] In some embodiments that may be combined with any other embodiments described herein, the CD28 polypeptide is a human CD28 polypeptide, wherein the CD3 polypeptide is a human CD3 polypeptide, and wherein the CD38 polypeptide is a human CD38 polypeptide.
[0019] In some embodiments that may be combined with any other embodiments described herein, the Vro domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the VLS domain comprises a CDR-Ll sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the Vro domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of lYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the VL3 domain comprises a CDR-Ll sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the VFB domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and the Vo domain comprises a CDR-Ll sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2

sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:46). In some embodiments, the VI-B domain comprises the amino acid sequence of QVQLQQSGAELVRSGASVKMSCKASGYTFTSFNMHWVECETPGQGLEWIGYIYPG NGGTNYNQKFKGKATLTADTSSSTAYMQISSLTSEDSAVYFCARTGGLRRAYFTY WGQGTLVTVS (SEQ ID NO:5), and the VL.3 domain comprises the amino acid sequence of
DIVLTQSPASLAVSLGQRATISCRASESVDSYGNGFMHWYQQKPGQPPKLLIYLAS NLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:6). In some embodiments, the Vro domain comprises the amino acid sequence of
QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPG QGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAV\TCARTGGLRRAYFTY WGQGTLVTVSS (SEQ ID NO: 13), and the VL3 domain comprises the amino acid sequence of
DIVLTQSPATLSLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASS RATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO: 14). In some embodiments, the Vro domain comprises the amino acid sequence of
QVQLVQSGAEVVKPGAS\^VSCKASGYTFTSFNMHWVKEAPGQRLEWIGYIYPG NGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTY WGQGTLVTVSS (SEQ ID NO: 17), and the VL3 domain comprises the amino acid sequence of
DI\T.TQSPATLSLSPGERATISCRASESVDSYGNGFMFfWYQQKPGQPPRLLIYLASS RATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO: 18). In some embodiments, the VH3 domain comprises the amino acid sequence of
QVQLVQSGAEWKSGASVKVSCKASGYTFTSFNMHWVKEAPGQGLEWIGYIYPG NGGTNYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFCARTGGLRRAYFTY WGQGTLVTVSS (SEQ ID NO:21), and the Vi.,3 domain comprises the amino acid sequence of
DIVLTQSPATLSLSPGERATISCRASESVDSYGNGFMHWYQQKPGQPPRLLIYLASS RAT GIPARESGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:l 8). In some embodiments, the VI-B domain comprises the amino acid

sequence of
QVQLVQSGAEV\7KPGAS\7KMSCKASGYTFTSFNIVIHW\^KEAPGQRLEWIGYIYPG NGGTNYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFCARTGGLRRAYFTY WGQGTLVTVSS (SEQ ID NO:23), and the VL3 domain comprises the amino acid sequence of
DIVLTQSPATLSLSPGERATISCRASESVDSYGNGFMHWYQQKPGQPPRLLIYLASS RAT GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO: 18). In some embodiments, the VH3 domain comprises the amino acid sequence of
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYD GSNKYTADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWG QGTLVTVSS (SEQ ID NO:9), and the VL3 domain comprises the amino acid sequence of AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEK (SEQ ID NO: 10).
[0020] In some embodiments that may be combined with any other embodiments described herein, the VHI domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO: 108), a CDR-H2 sequence comprising the amino acid sequence of lYPGNVNT (SEQ ID NO: 109), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO: 110), and the VLI domain comprises a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO: 111), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:l 12), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO: 113). In some embodiments, the VHI domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFSLSDYG (SEQ ID NO: 114), a CDR-H2 sequence comprising the amino acid sequence of IWAGGGT (SEQ ID NO: 115), and a CDR-H3 sequence comprising the amino acid sequence of ARDKGYSYYYSMDY (SEQ ID NO: 116), and the VLI domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVEYYVTSL (SEQ ID NO:l 17), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 118), and a CDR-L3 sequence comprising the amino acid sequence of QQSRKVPYT (SEQ ID NO:l 19), In some embodiments, the VHI domain comprises the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIH\V\?RQAPGQGLEWIGSIYPGN VNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDV

WGKGTTVTVSS (SEQ ID NO:49), and the VLI domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHT
GWSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO:50). In some embodiments, the Vi-n domain comprises the amino acid sequence of QVQLQESGPGLVKPSQTLSLTCTVSGFSLSDYGVHWVRQPPGKGLEWLGVIWAGG GTNYNPSLKSRKTISKDTSKNQVSLKLSSVTAADTAVYYCARDKGYSYYYSMDY VVGQGTTVTVS (SEQ ID NO:51), and the VLI domain comprises the amino acid sequence of
DIVLTQSPASLAVSPGQRATITCRASESVEYYVTSLMQWYQQKPGQPPKLLIFAASN VESGWARFSGSGSGTDFTLTINPVEA1WVANYYCQQSRKWYTTGQGTKLEIK (SEQ ID NO;52),
[0021] In some embodiments that may be combined with any other embodiments described herein, the Vi-12 domain comprises a CDR-HI sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 120), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 121), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 122), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHNNANTY (SEQ ID NO: 123), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 124), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 125), In some embodiments, the Vi-12 domain comprises a CDR-HI sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 126), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 127), and a CDR-H3 sequence comprising the amino acid sequence of GVYYALSPFDY (SEQ ID NO: 128), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHNNGNTY (SEQ ID NO: 129), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 130), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 131). In some embodiments, the Vi-12 domain comprises the amino acid sequence of QVQLVESGGGVVQPGRSLIILSCAASGFTFTKAWMHWVRQAPGKQLEVVVAQIKD KSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPF DYWGQGTLVTVSS (SEQ ID NO:53), and the VL2 domain comprises the amino acid sequence of DIVMTQTPLSLSVTPGQPASISCKSSQSLVHNNANTYLSWYLQKPGQSPQSLIYKVS

NRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK
(SEQ ID NO:54). In some embodiments, the Vm domain comprises the amino acid sequence of
QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHW\rRQAPGKGLEWVAQIKD KSNSYATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPF DYWGQGTLVTVSS (SEQ ID NO:84), and the VL2 domain comprises the amino acid sequence of
DIVMTQTPLSLSVTPGQPASISCKSSQSLVHNNGNTYLSWYLQKPGQSPQLLIYKVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGGGTKVEIK (SEQ ID NO:85).
[0022] In some embodiments that may be combined with any other embodiments described herein, at least one of Li, 1,2,1,3 or L4 is independently 0 amino acids in iength. In some embodiments, Li, L2, L3 and L4 each independently are zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPKAAP (SEQ ID NO: 58), and GGSGSSGSGG (SEQ ID NO: 59), In some embodiments, Li, L2, L3 and 1U each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO: 58), and GGSGSSGSGG (SEQ ID NO:59). In some embodiments, Li comprises the sequence GQPKAAP (SEQ ID NO: 58), L2 comprises the sequence TKGPS (SEQ ID NO:57), L3 comprises the sequence S, and L4 comprises the sequence RT. [0023] In some embodiments that may be combined with any other embodiments described herein, the hinge-Cm-Cm domains of the second and the third polypeptide chains are human IgG4 hinge-Cm-Cm domains, and wherein the hinge-Cm-Cm domains each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A and L235A. In some embodiments, the hinge-Cm-Cm domains of the second and the third polypeptide chains are human IgG4 hinge-Cm-Cm domains, and wherein the hinge-Cm-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to EU Index, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236, In some embodiments, the hinge-Cm-Cm domains of the second and the third polypeptide chains are human IgG4 hinge-Cm-Cm domains, and wherein the hinge-Cm-Cm domains each comprise amino acid substitutions at

positions corresponding to positions 228 and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the hinge-Cn2-Ci-i3 domains of the second and the third polypeptide chains are human IgGl hinge-Cm-Cra domains, and wherein the hinge-Cro-Cro domains each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgGl according to EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the hinge-Cm-Cro domains of the second and the third polypeptide chains are human IgGl hinge-Cm-Cm domains, and wherein the hinge-Cm-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgGl according to EU Index, wherein the amino acid substitutions are S298N, T299A, and Y300S. In some embodiments, the hinge-Cm-Cm domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the hinge-Cm-CtB domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W. In some embodiments, the hinge-Cm-Cro domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W; and wherein the hinge-Cm-G-B domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V.
[0024] In certain embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:60, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:62, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:63. In certain embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:64, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:65, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:63. In certain embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises the amino acid

sequence of SEQ ID NO:66, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:67, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:63. In certain embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:60, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:68, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:69. In certain embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:64, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:70, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:69. In certain embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:66, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:71, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:69.
[0025] In some embodiments that may be combined with any other embodiments described herein, the virus is a human immunodeficiency virus (HIV), influenza virus, cytomegalovirus (CMV), hepatitis B virus (HBV), human papillomavirus (HPV), Epstein-barr virus (EBV), human foamy virus (HFV), herpes simplex virus 1 (HSV-1), or herpes simplex virus 1 (HSV-2).
[0026] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the art. These and other embodiments of the invention are further described by the detailed description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 provides a schematic representation of a trispecific binding protein comprising four polypeptide chains that form three antigen binding sites that binds three target proteins: CD28, CD3, and CD38. A first pair of polypeptides possess dual variable domains having a cross-over orientation (VH1-VH2 and VL2-VL1) forming two antigen binding sites that recognize CD3 and CD28, and a second pair of polypeptides possess a single variable domain (VH3 and VL3) forming a single antigen binding site that

recognizes CD38. The trispecific binding protein shown in FIG. 1 uses an IgG4 constant region with a "knobs-into-holes" mutation, where the knob is on the second pair of polypeptides with a single variable domain.
[0028] FIG. 2 summarizes the binding affinities of indicated trispecific binding proteins against their cognate antigens (human CD3, CD28, and CD38) as measured by SPR.
[0029] FIG. 3 summarizes the binding affinity of the indicated anti-CD38xanti-CD28xanti-CD3 trispecific binding proteins for human CD38, as measured by SPR or flow cytometry (FACS).
[0030] FIGS. 4A-4D show the characterization of in vitro T cell subset expansion in response to CD38VHl/CD3midxCD28sup trispecific antibodies. Evaluation of T cell subset expansion was performed by coating wells with 350 ng/well of the CD38 trispecific Ab in the absence of exogenous cytokines. T cell populations were measured at indicated time points. A trispecific Ab having three mutated antigen binding domains was used as negative control. Flow cytometry was used to determine central (TCm) and effector memory (Tem) CD4 T ceils (FIG. 4A), T helper cells (Thl, Thl7, Th:2) (FIG. 4B), central (Tan) and effector memory (Tem) CD8 T cells (FIG. 4C), and cytomegalovirus (CMV) pp65-specific CDS cells (FIG. 4D) as described in Example 3. Analysis of CMV-specific pp65 effector ceils was performed by pentamer staining of peripheral blood mononuclear cells (PBMCs) from HLA-A2 CMV+ donors treated with the CD38 trispecific or the triple negative control antibodies,
[0031] FIGS. 5A-5B show the characterization of in vitro T cell subset expansion in PBMCs collected from CMV-infected Donor B in response to CD38vm/CD28sup x CD3mid trispecific antibody. T cell populations were measured at indicated time points. The triple mutant trispecific antibody was used as negative control. Flow cytometry was used to quantify CMV-specific memory CD8+ T cells (FIG. 5A), as well as CMV-specific central memory (TCm) and effector memory (Tem) CD8+ T cells (FIG. 5B). CD38vHi/CD28sup x CD3mid trispecific antibody activated T cells and promoted the proliferation of CMV-specific memory CD8+ T cells.
[0032] FIGS. 6A-6B show the characterization of in vitro T cell subset expansion in PBMCs collected from CMV-infected Donor C in response to CD38vm/CD28sup x CD3mid trispecific antibody. T cell populations were measured at indicated time points. The triple mutant trispecific antibody was used as negative control. Flow cytometry was used to quantify CMV-specific memory CD8+ T cells (FIG. 6A), as well as CMV-specific

central memory (Tcm) and effector memory (Tem) CD8+ T cells (FIG. 6B). CD38vHi/CD28sup x CD3mid trispecific antibody activated T cells and promoted the proliferation of CMV-specific memory CD8+ T cells.
[0033] FIGS. 7A-7B show the characterization of in vitro T cell subset expansion in PBMCs collected from Epstein-ban- virus (EBV)-infected Donor A in response to CD38vm/CD28sup x CD3mid trispecific antibody. T cell populations were measured at indicated time points. The triple mutant trispecific antibody was used as negative control. Flow cytometry' was used to quantify EBV-specific memory CD8+ T cells (FIG. 7A), as well as EBV-specific central memory (Tcm) and effector memory (Tem) CD8+ T cells (FIG. 7B). CD38vHi/CD28sup x CD3mid trispecific antibody activated T cells and promoted the proliferation of EBV-specific memory CD8+ T cells.
[0034] FIGS. 8A-8B show the characterization of in vitro T cell subset expansion in PBMCs collected from EBV-infected Donor B in response to CD38vHi/CD28sup x CD3mid trispecific antibody. T cell populations were measured at indicated time points. The triple mutant trispecific antibody was used as negative control. Flow cytometry' was used to quantify EBV-specific memory CD8+ T cells (FIG, 8A), as well as EBV-specific central memory (Tcm) and effector memory (Tem) CD8+ T cells (FIG. SB). CD38vm/CD28sup x CD3mid trispecific antibody activated T cells and promoted the proliferation of EBV-specific memory CD8+ T cells.
[0035] FIG. 9 shows flow cytometry profiles of PBMCs from the indicated human immunodeficiency virus (HlV)-positive donors (bottom panels and top right panel) assayed for HIV Gag-specific CD8+ T cells (A*02:01 - SLYNTVATL (HTV-1 gag pl7 76-84) Pentamer conjugated to PE, Prolmmune) at baseline (day 0; prior to incubation with trispecific antibodies). PBMCs from an HTV-negative donor were used as negative control (top left panel). The percentages of Gag-specific CD8+ T cell population are provided and shown as inset boxes. At baseline PBMCs from HIV-positive donors contain HIV Gag-specific CD8+ T cells. Donors A-C in FIG. 9 are the same as donors D-F shown in FIGS. 10A-12B.
[0036] FIGS. 10A-10B show the characterization of in vitro T cell subset expansion in
PBMCs collected from HIV-positive Donor D in response to CD38vm/CD28sup x CD3mid trispecific antibody. T cell populations were measured at indicated time points. The triple mutant trispecific antibody was used as negative control. Flow cytometry was used to quantify HIV-specific memory CD8+ T cells (FIG. 10A), as well as HIV-specific central memory (Tcm) and effector memory (Tem) CD8+ T cells (FIG. 10B). CD38vm/CD28sup x

CD3mid trispecific antibody activated T cells and promoted the proliferation of effector memory (Tem) CD8+ T cells.
[0037] FIGS. 11A-11.B show the characterization of in vitro T cell subset expansion in PBMCs collected from HIV-positive Donor E in response to CD38vHi/CD28sup x CD3mid trispecific antibody. T cell populations were measured at indicated time points. The triple mutant trispecific antibody was used as negative control. Flow cytometry' was used to quantify HIV-specific memory CD8+ T cells (FIG. 11 A), as well as FflV-specific central memory (Tcm) and effector memory (Tem) CD8+ T ceils (FIG. 11B). CD38vm/CD28sup x CD3mid trispecific antibody activated T cells and promoted the proliferation of effector memory (Tem) CD8+ T cells.
[0038] FIGS. 12A-12B show the characterization of in vitro T cell subset expansion in PBMCs collected from HIV-positive Donor F in response to CD38vm/CD28sup x CD3mid trispecific antibody. T cell populations were measured at indicated time points. The triple mutant trispecific antibody was used as negative control. Flow cytometry was used to quantify HIV-specific memory CD8+ T cells (FIG. 12A), as well as HIV-specific central memory (Tcm) and effector memory (Tem) CD8+ T cells (FIG. 12B). CD38vm/CD28sup x CD3mid trispecific antibody activated T cells and promoted the proliferation of effector memory (Tem) CD8+ T cells.
[0039] FIGS. 13A-13B show the characterization of in vitro T ceil subset expansion in PBMCs collected from influenza-infected Donor A in response to CD38vm/CD28sup x CD3mid trispecific antibody. T cell populations were measured at indicated time points. The triple mutant trispecific antibody was used as negative control. Flow cytometry was used to quantify influenza (Fiu)-specific memory CD8+ T cells (FIG. 13A), as well as Flu-specific central memory (Tcm) and effector memory (Tem) CD8+ T cells (FIG. 13B). CD38vHi/CD28sup x CD3mid trispecific antibody activated T cells and promoted the proliferation of Tem CD8+ T ceils (e.g., see days 7, 11) and Tcm CD8+ T cells (e.g., see day 7),
DETAILED DESCRIPTION
[0040] The disclosure provides trispecific binding proteins comprising four polypeptide chains that form three antigen binding sites that specifically bind a CD38 polypeptide (e.g., human and cynomolgus monkey CD38 polypeptides), a CD28 polypeptide, and a CDS

polypeptide, which may find use, e.g., in expanding memory T cells (e.g., virus-specific memory T cells) and/or treating chronic viral infection.
I. General Definitions
[0041] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and the like.
[0042] It is understood that aspects and embodiments of the present disclosure described herein include "comprising," "consisting," and "consisting essentially of aspects and embodiments,
[0043] The term "polynucleotide" as used herein refers to single-stranded or double-stranded nucleic acid polymers of at least 10 nucleotides in length. In certain embodiments, the nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Such modifications include base modifications such as bromuridine, ribose modifications such as arabinoside and 2',3'~dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroaniiothioate, phoshoraniladate and phosphoroamidate. The term "polynucleotide" specifically includes single-stranded and double-stranded forms of DNA. [0044] An "isolated polynucleotide" is a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which: (1) is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, (2) is linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.
[0045] An "isolated polypeptide" is one that: (1) is free of at least some other polypeptides with which it would normally be found, (2) is essentially free of other polypeptides from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or noncovalent interaction) with portions of a

polypeptide with which the "isolated polypeptide" is associated in nature, (6) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature. Such an isolated polypeptide can be encoded by genomic DNA, cDNA, mKNA or other RNA, of synthetic origin, or any combination thereof. Preferably, the isolated polypeptide is substantially free from polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise). [0046] Naturally occurring antibodies typically comprise a tetramer. Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one full-length "light" chain (typically having a molecular weight of about 25 kDa) and one full-length "heavy" chain (typically having a molecular weight of about 50-70 kDa). The terms "heavy chain" and "light chain" as used herein refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The ami no-terminal portion of each light and heavy chain typically includes a variable domain of about 100 to 110 or more amino acids that typically is responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant domain responsible for effector function. Thus, in a naturally occurring antibody, a full-length heavy chain immunoglobulin polypeptide includes a variable domain (VH) and three constant domains (CHI, CH?., and Cm), wherein the VH domain is at the amino-terminus of the polypeptide and the Cro domain is at the carboxyl-terminus, and a full-length light chain immunoglobulin polypeptide includes a variable domain (VL) and a constant domain (CL), wherein the VL domain is at the amino-terminus of the polypeptide and the CL domain is at the carboxyl-terminus.
[0047] Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgGl, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgMl and IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgAl and IgA2. Within full-length light and heavy chains, the variable and constant domains typically are joined by a "J" region of about 12 or more amino acids, with the heavv chain also including a "D" region of about 10 more amino acids. See, e.g.. FUNDAMENTAL IMMUNOLOGY (Paul, W., ed., Raven Press, 2nd ed., 1989), which is incorporated by reference in its entirety for all purposes. The variable regions of each light/heavy chain pair typically form an antigen binding site. The variable domains of

naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs, The CDRs from the two chains of each pair typically are aligned by the framework regions, which may enable binding to a specific epitope. From the ammo-terminus to the carboxyl-terminus, both light and heavy chain variable domains typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDRS, and FR4.
[0048] The term "CDR set" refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al, SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia and Lesk, 1987, J. Mol Biol 196: 901-17; Chothia et al, 1989, Nature 342: 877-83) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as LI, L2, and L3 or HI, H2, and H3 where the "L" and the "H" designates the light chain and the heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs, Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan, 1995, FASEBJ. 9: 133-39; MacCallum, 1996,,/. Mol Biol 262(5): 732-45; and Lefranc, 2003, Dev. Camp. Immunol 27: 55-11. Still other CDR boundary definitions may not strictly follow one of the herein systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use Kabat or Chothia defined CDRs. Identification of predicted CDRs using the amino acid sequence is well known in the field, such as in Martin, A.C. "Protein sequence and structure analysis of antibody variable domains," In Antibody Engineering, Vol. 2, Kontermann R., Dub el S., eds. Springer-Verlag, Berlin, p. 33-51 (2010). The amino acid sequence of the heavy and/or light chain variable domain may be also inspected to identify the sequences of the CDRs by other conventional

methods, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. The numbered sequences may be aligned by eye, or by employing an alignment program such as one of the CLUSTAL suite of programs, as described in Thompson, 1994, Nucleic Acids Res. 22: 4673-80. Molecular models are conventionally used to correctly delineate framework and CDR regions and thus correct the sequence-based assignments. [0049] In some embodiments, CDR/FR definition in an immunoglobulin light or heavy chain is to be determined based on IMGT definition (Lefranc et al. Dev. Comp. Immunol., 2003, 27(l):55-77; www.imgt.org).
[OOSOj The term "Fc" as used herein refers to a molecule comprising the sequence of a non-antigen-binding fragment resulting from digestion of an antibody or produced by other means, whether in monomelic or multimeric form, and can contain the hinge region. The original immunoglobulin source of the native Fc is preferably of human origin and can be any of the immunoglobulins. Fc molecules are made up of monomelic polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomelic subunits of native Fc molecules ranges from 1 to 4 depending on class {e.g., IgG, IgA, and IgE) or subclass {e.g., IgGl, IgG2, IgG3, IgAl, IgGA2, and IgG4). One example of a Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG. The term "native Fc" as used herein is generic to the monomelic, dimeric, and multimeric forms. [0051] A F(ab) fragment typically includes one light chain and the VH and CHI domains of one heavy chain, wherein the VH-CHI heavy chain portion of the F(ab) fragment cannot form a disulfide bond with another heavy chain polypeptide. As used herein, a F(ab) fragment can also include one light chain containing two variable domains separated by an amino acid linker and one heavy chain containing two variable domains separated by an amino acid linker and a CHI domain.
[0052] A F(ab') fragment typically includes one light chain and a portion of one heavy chain that contains more of the constant region (between the CHI and Cm domains), such that an interchain disulfide bond can be formed between two heavy chains to form a F(ab')2 molecule.
[0053] The term "binding protein" as used herein refers to a non-naturally occurring (or recombinant or engineered) molecule that specifically binds to at least one target antigen, e.g., a CD38 polypeptide of the present disclosure

[0054] A "recombinant" molecule is one that has been prepared, expressed, created, or isolated by recombinant means.
[0055] One embodiment of the disclosure provides binding proteins having biological and immunological specificity to between one and three target antigens. Another embodiment of the disclosure provides nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such binding proteins. Another embodiment of the disclosure provides expression vectors comprising nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such binding proteins. Yet another embodiment of the disclosure provides host cells that express such binding proteins (i.e., comprising nucleic acid molecules or vectors encoding polypeptide chains that form such binding proteins).
[0056] The term "swapability" as used herein refers to the interchangeability of variable domains within the binding protein format and with retention of folding and ultimate binding affinity. "Full swapability" refers to the ability to swap the order of both VHI and VH2 domains, and therefore the order of VLI and VL2 domains, in the polypeptide chain of formula I or the polypeptide chain of formula II (i.e., to reverse the order) while maintaining full functionality of the binding protein as evidenced by the retention of binding affinity. Furthermore, it should be noted that the designations VH and VL refer only to the domain's location on a particular protein chain in the final format. For example, VHI and VH2 could be derived from VLI and VL2 domains in parent antibodies and placed into the VHI and Vi-12 positions in the binding protein. Likewise, VLI and VL2 could be derived from VHI and Vm domains in parent antibodies and placed in the VHI and VH?. positions in the binding protein. Thus, the VH and VL designations refer to the present location and not the original location in a parent antibody. VH and VL domains are therefore "swappable." [0057] The term "antigen" or "target antigen" or "antigen target" as used herein refers to a molecule or a portion of a molecule that is capable of being bound by a binding protein, and additionally is capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen. A target antigen may have one or more epitopes. With respect to each target antigen recognized by a binding protein, the binding protein is capable of competing with an intact antibody that recognizes the target antigen. [0058] "CD38" is cluster of differentiation 38 polypeptide and is a glycoprotein found on the surface of many immune cells. In some embodiments, a binding protein of the present disclosure binds the extracellular domain of one or more CDS 8 polypeptide. Exemplar}' CD38 extracellular domain polypeptide sequences include, but are not limited

to, the extracellular domain of human CD38 (e.g., as represented by SEQ ID NO:l) and the extracellular domain of cynomolgus monkey CD38 (e.g., as represented by SEQ ID NO:30).
[0059] The term "T-cell engager" refers to binding proteins directed to a host's immune system, more specifically the T cells' cytotoxic activity as well as directed to a tumor target-protein.
[0060] The term "monospecific binding protein" refers to a binding protein that specifically binds to one antigen target.
[0061] The term "monovalent binding protein" refers to a binding protein that has one antigen binding site,
[0062] The term "bispecific binding protein" refers to a binding protein that specifically binds to two different antigen targets. In some embodiments, a bispecific binding protein binds to two different antigens. In some embodiments, a bispecific binding protein binds to two different epitopes on the same antigen,
[0063] The term "bivalent binding protein" refers to a binding protein that has two binding sites.
[0064] The term "trispecific binding protein" refers to a binding protein that specifically binds to three different antigen targets. In some embodiments, a trispecific binding protein binds to three different antigens. In some embodiments, a trispecific binding protein binds to one, two, or three different epitopes on the same antigen.
[0065] The term "trivalent binding protein" refers to a binding protein that has three binding sites. In particular embodiments the trivalent binding protein can bind to one antigen target. In other embodiments, the trivalent binding protein can bind to two antigen targets. In other embodiments, the trivalent binding protein can bind to three antigen targets,
[0066] An "isolated" binding protein is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the binding protein, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the binding protein will be purified: (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using

Coomassie blue or, preferably, silver stain. Isolated binding proteins include the binding protein in situ within recombinant ceils since at least one component of the binding protein's natural environment will not be present.
[0067] The terms "substantially pure" or "substantially purified" as used herein refer to a compound or species that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition). In some embodiments, a substantially purified fraction is a composition wherein the species comprises at least about 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition will comprise more than about 80%, 85%>, 90%, 95%, or 99% of all macromolar species present in the composition. In still other embodiments, the species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. [0068] The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specifically binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody or binding protein. In certain embodiments, a binding protein is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. In some embodiments, a binding protein is said to specifically bind an antigen when the equilibrium dissociation constant is < 10"8 M, more preferably when the equilibrium dissociation constant is < 10"9 M, and most preferably when the dissociation constant is < 10"10 M.
[0069] The dissociation constant (KD) of a binding protein can be determined, for example, by surface plasmon resonance. Generally, surface plasmon resonance analysis measures real-time binding interactions between ligand (a target antigen on a biosensor matrix) and analyte (a binding protein in solution) by surface plasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor; Piscataway, NT). Surface plasmon analysis can also be performed by immobilizing the analyte (binding protein on a biosensor matrix) and presenting the ligand (target antigen). The term "KD," as used herein refers to the dissociation constant of the interaction between a particular binding protein and a target antigen.

[0070] The term "binds to" as used herein in reference to a binding protein refers to the ability of a binding protein or an antigen-binding fragment thereof to bind to an antigen containing an epitope with an Kd of at least about 1 x 10"6 M, 1 x 10'7 M, 1 x 10"s M, 1 x 10'9 M, 1 x 10"10 M, 1 x 10"11 M, 1 x 10"12 M, or more, and/or to bind to an epitope with an affinity that is at least two-fold greater than its affinity for a nonspecific antigen. In some embodiments, a binding protein of the present disclosure binds to two or more antigens, e.g., a human and a cynomolgus monkey CD38 polypeptide,
[0071] In some embodiments, an antigen binding domain and/or binding protein of the present disclosure "cross reacts" with human and cynomolgus monkey CD38 polypeptides, e.g., CD38 extracellular domains, such as SEQ ID NO:l (human CD38 isoform A), SEQ ID NO: 105 (human CD38 isoform E) and SEQ ID NO:30 (cynomolgus monkey CD38). A binding protein binding to antigen 1 (Agl) is "cross-reactive" to antigen 2 (Ag2) when the EC.50S are in a similar range for both antigens. In the present application, a binding protein binding to Agl is cross-reactive to Ag2 when the ratio of affinity of Ag2 to affinity of Agl is equal or less than 10 (for instance 5, 2, 1 or 0.5), affinities being measured with the same method for both antigens.
[0072] A binding protein binding to Agl is "notsignificantlycross-reactive" to Ag2 when the affinities are very different for the two antigens. Affinity for Ag2 may not be measurable if the binding response is too low. In the present application, a binding protein binding to Agl is not significantly cross-reactive to Ag2, when the binding response of the binding protein to Ag2 is less than 5% of the binding response of the same binding protein to Agl in the same experimental setting and at the same antibody concentration. In practice, the binding protein concentration used can be the EC50 or the concentration required to reach the saturation plateau obtained with Agl. [0073] The term "linker" as used herein refers to one or more amino acid residues inserted between immunoglobulin domains to provide sufficient mobility for the domains of the light and heavy chains to fold into cross over dual variable region immunoglobulins. A linker is inserted at the transition between variable domains or between variable and constant domains, respectively, at the sequence level. The transition between domains can be identified because the approximate size of the immunoglobulin domains are well understood. The precise location of a domain transition can be determined by locating peptide stretches that do not form secondary structural elements such as beta-sheets or alpha-helices as demonstrated by experimental data or as can be assumed by techniques of

modeling or secondary structure prediction. The linkers described herein are referred to as Li, which is located on the light chain between the C-terminus of the VL2 and the N-terminus of the VLI domain; and L.2, which is located on the light chain between the C-terminus of the VLI and the N-terminus of the CL domain. The heavy chain linkers are known as L3, which is located between the C-terminus of the VHI and the N-terminus of the VH2 domain; and Li, which is located between the C-terminus of the VH?. and the N-terminus of the CHI domain.
[0074] The term "vector" as used herein refers to any molecule (e.g., nucleic acid, plasmid, or virus) that is used to transfer coding information to a host cell. The term "vector" includes a nucleic acid molecule that is capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA molecule into which additional DNA segments may be inserted. Another type of vector is a viral vector, wherein additional DNA segments may be inserted into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. The terms "plasmid" and "vector" may be used interchangeably herein, as a plasmid is the most commonly used form of vector. However, the disclosure is intended to include other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.
[0075] The phrase "recombinant host cell" (or "host cell") as used herein refers to a cell into which a recombinant expression vector has been introduced. A recombinant host cell or host cell is intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but such cells are still included within the scope of the term "host cell" as used herein. A wide variety of host cell expression systems can be used to express the binding proteins, including bacterial, yeast, baculoviral, and mammalian expression

systems (as well as phage display expression systems). An example of a suitable bacterial expression vector is pUC19. To express a binding protein recombinantly, a host ceil is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding the polypeptide chains of the binding protein such that the polypeptide chains are expressed in the host cell and, preferably, secreted into the medium in which the host cells are cultured, from which medium the binding protein can be recovered. [0076] The term "transformation" as used herein refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain a new DNA. For example, a cell is transformed where it is genetically modified from its native state. Following transformation, the transforming DNA may recombine with that of the cell by physically integrating into a chromosome of the ceil, or may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid, A cell is considered to have been stably transformed when the DNA is replicated with the division of the cell. The term "transfection" as used herein refers to the uptake of foreign or exogenous DNA by a ceil, and a ceil has been "transfected" when the exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are well known in the art. Such techniques can be used to introduce one or more exogenous DNA molecules into suitable host cells.
[0077] The term "naturally occurring" as used herein and applied to an object refers to the fact that the object can be found in nature and has not been manipulated by man. For example, a polynucleotide or polypeptide that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man is naturally-occurring. Similarly, "non-naturally occurring" as used herein refers to an object that is not found in nature or that has been structurally modified or synthesized by man.
[0078] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids; unnatural amino acids and analogs such as a-, a-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for the polypeptide chains of the binding proteins. Examples of unconventional amino acids include: 4-hydroxyproline, y-carboxyglutamate, e-N,N,N-trimethyllysine, s-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, G-N-methylarginine, and other similar amino acids and

imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl-terminal direction, in accordance with standard usage and convention. [0079] Naturally occurring residues may be divided into classes based on common side chain properties:
(1) hydrophobic: Met, Ala, Val, Leu, He, Phe, Trp, Tyr, Pro;
(2) polar hydrophilic: Arg, Asn, Asp, Gin, Glu, His, Lys, Ser, Thr ;
(3) aliphatic: Ala, Gly, He, Leu, Val, Pro;
(4) aliphatic hydrophobic: Ala, He, Leu, Val, Pro;
(5) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;
(6) acidic: Asp, Glu;
(7) basic: His, Lys, Arg;
(8) residues that influence chain orientation: Gly, Pro;
(9) aromatic: His, Trp, Tyr, Phe; and
(10) aromatic hydrophobic: Phe, Trp, Tyr.
[0080] Conservative amino acid substitutions may involve exchange of a member of
one of these classes with another member of the same class. Non-conservative substitutions
may involve the exchange of a member of one of these classes for a member from another
class,
[0081] A skilled artisan will be able to determine suitable variants of the polypeptide
chains of the binding proteins using well-known techniques. For example, one skilled in
the art may identify suitable areas of a polypeptide chain that may be changed without
destroying activity by targeting regions not believed to be important for activity.
Alternatively, one skilled in the art can identify residues and portions of the molecules that
are conserved among similar polypeptides. In addition, even areas that may be important
for biological activity or for structure may be subject to conservative amino acid
substitutions without destroying the biological activity or without adversely affecting the
polypeptide structure.
[0082] The term "patient" as used herein includes human and animal subjects (e.g.,
mammals, such as dogs, pigs, horses, cats, cows, etc.).
[0083] The terms "treatment" or "treat" as used herein refer to both therapeutic
treatment and prophylactic or preventative measures. Those in need of treatment include
those having a disorder as well as those prone to have the disorder or those in which the

disorder is to be prevented. In particular embodiments, binding proteins can be used to treat humans with chronic viral infection, or ameliorate chronic viral infection in a human subject,
[0084] The terms "pharmaceutical composition" or "therapeutic composition" as used herein refer to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.
[0085] The term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" as used herein refers to one or more formulation materials suitable for accomplishing or enhancing the deliver}' of a binding protein.
[0086] The terms "effective amount" and "therapeutically effective amount" when used in reference to a pharmaceutical composition comprising one or more binding proteins refer to an amount or dosage sufficient to produce a desired therapeutic result. More specifically, a therapeutically effective amount is an amount of a binding protein sufficient to inhibit, for some period of time, one or more of the clinically defined pathological processes associated with the condition being treated. The effective amount may vary depending on the specific binding protein that is being used, and also depends on a variety of factors and conditions related to the patient being treated and the severity of the disorder. For example, if the binding protein is to be administered in vivo, factors such as the age, weight, and health of the patient as well as dose response curves and toxicity data obtained in preclinical animal work would be among those factors considered. The determination of an effective amount or therapeutically effective amount of a given pharmaceutical composition is well within the ability of those skilled in the art.
[0087] One embodiment of the disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a binding protein,
Trispecific binding proteins
[0088] Certain aspects of the present disclosure relate to trispecific binding proteins (e.g., that bind CD38, CD28, and CD3 polypeptides). Any of the CDRs or variable domains of any of the antigen binding proteins described herein may find use in a trispecific binding protein of the present disclosure.
[0089] In some embodiments, the binding protein of the disclosure is a trispecific binding protein comprising four polypeptide chains that form three antigen binding sites

that bind one or more (e.g., three) different antigen targets or target proteins. In some embodiments, a first poiypeptide chain comprises a structure represented by the formula:
VL2-LI-VLI-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula;
VHl-L3-VH2-L4-CHl-hinge-CH2-CH3 [II]
and a third polypeptide chain comprises a structure represented by the formula:
VH3-CHi-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VL3-CL [IV]
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL.3 is a third immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CHI is an immunoglobulin CHI heavy chain constant domain;
C1-12 is an immunoglobulin CH2 heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CHI and Cm domains; and
Li, L2, L3 and 1A are amino acid linkers;
wherein the polypeptide of formula I and the polypeptide of formula II form a cross¬over light chain-heavy chain pair. In some embodiments, the first polypeptide chain and the second polypeptide chain have a cross-over orientation that forms two distinct antigen binding sites.
[0090] In some embodiments, VHI and VLI form a first antigen binding site that binds a CD28 polypeptide, wherein Vm and VL2 form a second antigen binding site that binds a CD3 polypeptide, and wherein VH3 and VLS form a third antigen binding site that binds a CD38 polypeptide. In some embodiments, the CD28 polypeptide is a human CD28 polypeptide. In some embodiments, the CD3 polypeptide is a human CDS polypeptide. In some embodiments, the CDS8 polypeptide is a human CDS8 polypeptide. In some

embodiments, the trispecific binding protein comprises one or more antigen binding sites described infra.
[0091] The binding proteins of the disclosure may be prepared using domains or sequences obtained or derived from any human or non-human antibody, including, for example, human, murine, or humanized antibodies. In some embodiments, a binding protein of the present disclosure is an antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric, humanized, or human antibody.
Anti-CD38 Binding Sites
[0092] Certain aspects of the present disclosure relate to binding proteins that comprise an antigen binding site that binds a CD38 polypeptide {e.g., human and cynomolgus monkey CD38 polypeptides).
[0093] In some embodiments, a binding protein or antigen-binding fragment thereof cross-reacts with human CD38 {e.g., a human CD38 isoform A and/or isoform E polypeptide) and cynomolgus monkey CD38. In some embodiments, a binding protein induces apoptosis of a CD38+ cell. In some embodiments, a binding protein recaiits a T cell to a CD38+ cell and optionally activates the T cell {e.g., though TCR stimulation and/or costimulation).
[0094] In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of lYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:3 7), a CDR-H2 sequence comprising the amino acid sequence of lYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino

acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); and/or an antibody light chain variable (VX) domain comprising a CDR-Ll sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG(SEQ ID NO: 132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NQ:36). In some embodiments, the binding proteins comprise 1, 2, 3, 4, 5, or 6 CDRs from an antibody VH and/or VL domain sequence of antiCD38 C2-CD38-1, antiCD3 8_C2-CD3 8- 1_VH1 - VL 1, antiCD3 8_C2-CD3 8- 1_VH3 - VL3, antiCD3 8_C2-CD38-1_VH5-VL3, antiCD38_C2-CD38-l_VH6-VL3, CD38i-n-iYi37o(may also be referred to herein as antiCD38_1370), antiCD38_C2-CD38-l_VHl-VLlxCD28supxCD3mid IgG4 FALA, antiCD38_C2-CD38-l_VHl-VLlxCD28supxCD3mid IgGlLALA P329A, antiCD38_C2-CD38-l_VHl-VLlxCD28supxCD3midIgGlNNSA, CD38HHYi37oxCD28supxCD3mid IgG4 FALA, CD38HHYi37oxCD28supxCD3mid IgGlLALA P329A, or CD38HHYi37oxCD28supxCD3mid IgGl NNSA, as shown in Table G, H, or I.
[0095] In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); and an antibody light chain variable (VL) domain comprising a CDR-Ll sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36).
[0096] In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); and/or an antibody light chain variable (VX) domain comprising a CDR-Ll sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino

acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-Hl sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In other embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-Hl sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36), In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-Hl sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ) ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36).
[0097] In some embodiments, the VH domain comprises the sequence, from N-terminus to C-terminus, FR1— CDR-Hl—FR2—CDR-H2—FR3—CDR-H3—FR4; where Fill comprises the sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO:86), QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO:87), or
QVQLVQSGAEVVKPGASVKMSCKAS (SEQ ID NO:88); where FR2 comprises the sequence MHWVKEAPGQRLEWIGY (SEQ ID NO:90) or MHWVKEAPGQGLEWIGY (SEQ ID NO:91); where FR3 comprises the sequence

NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO:93) or NYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO:94); and where FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO:96). In some embodiments, the VL domain comprises the sequence, from N-terminus to C-terminus, FR1—CDR-L1— FR2—CDR-L2—FR3—CDR-L3—FR4; where FR1 comprises the sequence DIVLTQSPATLSLSPGERATISCRAS (SEQ ID NO:97); where FR2 comprises the sequence MHWYQQKPGQPPRLLIY (SEQ ID NO:99); where FR3 comprises the sequence SRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYC (SEQ ID NO: 101); and where FR4 comprises the sequence FGGGTKLEIK (SEQ ID NO: 103). [0098] In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%o, at least 86%, at least 87%, at least 88%o, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%>, or 100% identical to the amino acid sequence of SEQ ID NO:5; and/or the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:6. In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17; and/or the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18, In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%>, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%>, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:21; and/or the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%*, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:l 8. In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%,

at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:23; and/or the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%>, at least 90%, at least 91%, at least 92%>, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%>, at least 879-4, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%>, at least 9794 at least 98%, at least 99%>, or 100% identical to the amino acid sequence of SEQ ID NO: 13, and/or the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%o, at least 88%, at least 89%, at least 90%o, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 9994 or 100% identical to the amino acid sequence of SEQ ID NO: 14. [0099] In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%>, or 100% identical to the amino acid sequence of SEQ ID NO:5; and the VL domain comprises an amino acid sequence that is at least 859-4, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:6. In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 8694 at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ) ID NO: 17; and the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%>, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%>, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%>, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:21; and the VL domain comprises an amino acid sequence that is at least 85%, at least 8694 at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%,

at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%o, at least 88%, at least 89%>, at least 90%, at least 91%, at least 92%>, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:23; and the VL domain comprises an amino acid sequence that is at least 859-4, at least 86%, at least 87%>, at least 889-4, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%>, at least 9894 at least 99%, or 1Q0%> identical to the amino acid sequence of SEQ ID NO: 1.8. In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%o, at least 88%, at least 89%, at least 90%o, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 9994 or 100% identical to the amino acid sequence of SEQ ID NO: 13; and the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 14.
[0100] In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:5; and the VL domain comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 17; and the VL domain comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:21; and the VL domain comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:23; and the VL domain comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 13; and the VL domain comprises the amino acid sequence of SEQ ID NO: 14.
[0101] In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:7 and/or an antibody light chain comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ

ID NO:22 and/or an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:24 and/or an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and/or an antibody light chain comprising the amino acid sequence of SEQ ID NO: 16.
[0102] In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:7 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ) ID NO: 19 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:22 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:24 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and an antibody light chain comprising the amino acid sequence of SEQ ID NO: 16.
[0103] In some embodiments, a binding site that binds CDS8 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:46). In some embodiments, a binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43); and an

Results [0253] The CD38vm/CD28sup x CD3mid trispecific antibody activated T cells and promoted the proliferation of influenza-specific memory CD8+ T cells following incubation for up to 11 days with PBMCs isolated from a known influenza-infected human donor FIGS.13A-13B (Influenza Donor A), As shown in FIG. 13A, the CD38WCD28sup x CD3mid trispecific antibody led to increases in influenza-specific memory CD8+ T cells (cells/ul) relative to the triple mutant control antibody. In addition, CD38vm/CD28sup x CD3mid trispecific antibody increased the percentage of influenza (Flu)-specific CD8+ Tem cells (e.g., see days 7 and 11) and TCm cells (e.g., see day 7) relative to the triple mutant control antibody (FIG. 13B).
[0254] Taken together, the data presented in Examples 1-7 demonstrate trispecific anti-CD38/CD3/CD28 antibodies stimulate potent anti-viral immunity against diverse viruses. Without wishing to be bound by theory, it is believed that CD38/CD3/CD28 trispecific antibodies can activate and promote the proliferation of T cells by engaging all three Hgands on T cells. In particular, it is believed that engagement of CD3/CD28 on T cells by CD38/CD3/CD28 trispecific antibodies initiates T cell activation, proliferation, and differentiation into memory T cells. Further, without wishing to be bound by theory, it is believed that engagement of CD28 provides an advantageous co-stimulatory signal which enhances the duration and magnitude of the immune response, and promotes T cell proliferation and survival.
[0255] While the disclosure includes various embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations that come within the scope of the disclosure. In addition, the section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described, [0256] Each embodiment herein described may be combined with any other embodiment or embodiments unless clearly indicated to the contrary. In particular, any feature or embodiment indicated as being preferred or advantageous may be combined with any other feature or features or embodiment or embodiments indicated as being preferred or advantageous, unless clearly indicated to the contrary'.
[0257] All references cited in this application are expressly incorporated by reference herein.

CLAIMS
What is claimed is:
1, A method for expanding virus-specific memory T cells, comprising contacting a
virus-specific memory T cell with a binding protein, wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
VL2-LI-VLI-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula:
VHl-L3-VH2-L4-CHl-hinge-CH2-CH3 [II]
and a third polypeptide chain comprises a structure represented by the formula:
Vro-Cm-hinge-Ci-E-Cro [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VLS-CL [IV]
wherein:
VLI is a first immunoglobulin light chain variable domain;
Vi.2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain,
VHI is a first immunoglobulin heavy chain variable domain;
V1-12 is a second immunoglobulin heavy chain variable domain,
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CHI is an immunoglobulin CHI heavy chain constant domain;
CH2 is an immunoglobulin Cm heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CHI and Cm domains; and
Li, L2, L3 and L4 are amino acid linkers, wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and
wherein VHI and VLI form a first antigen binding site that binds a CD28 polypeptide, wherein VH2 and V1.2 form a second antigen binding site that binds a CD3 polypeptide, and wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide.

2. The method of claim 1, wherein the vims-specific memory T cell is contacted with the binding protein in vitro or ex vivo.
3. The method of claim 1 or claim 2, wherein contacting the virus-specific memory T cell with the binding protein causes activation and/or proliferation of virus-specific memory T cells.
4. A method for expanding T cells, comprising contacting a T cell with a binding protein in vitro or ex vivo, wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a staicture represented by the formula:
VL2-LI-VLI-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula;
VHI -L,3- VH2-L4-CHI -hinge-Cm-Cro [II] and a third polypeptide chain comprises a structure represented by the formula;
VH3-CHi-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VL3-CL [IV]
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain,
VL3 is a third immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CHI is an immunoglobulin CHI heavy chain constant domain;
C1-12 is an immunoglobulin CH2 heavy chain constant domain;
CH3 is an immunoglobulin Cm heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CHI and Ci-12 domains, and
Li, L2, L3 and I_4 are amino acid linkers; wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and

wherein VHI and VLI form a first antigen binding site that binds a CD28 polypeptide, wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, and wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide.
5. The method of claim 4, wherein the T cell is a memory T cell or an effector T cell.
6. The method of claim 4 or claim 5, wherein the T cell expresses a chimeric antigen receptor (CAR) on its cell surface or comprises a polynucleotide encoding a CAR.
7. A method for treating chronic viral infection, comprising administering to an individual in need thereof an effective amount of a binding protein, wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:
VL2-LI-VLI-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula;
VHI -L3-VH2-L4-CHI -hinge-Cm-Cro [II] and a third polypeptide chain comprises a structure represented by the formula;
VH3-CHi-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VL3-CL [IV]
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain,
VL3 is a third immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CHI is an immunoglobulin CHI heavy chain constant domain;
C1-12 is an immunoglobulin CH2 heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CHI and Ci-12 domains, and
Li, L2, L3 and I_4 are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and
wherein VHI and VLI form a first antigen binding site that binds a CD28 polypeptide, wherein VH2 and VL.2 form a second antigen binding site that binds a CD3 polypeptide, and wherein VHS and VL3 form a third antigen binding site that binds a CD38 polypeptide.
8. The method of claim 7, wherein the individual is a human.
9. The method of claim 7 or claim 8, wherein the binding protein is administered to the individual in pharmaceutical formulation comprising the binding protein and a pharmaceutical!}' acceptable carrier.
10. The method of any one of claims 7-9, wherein administration of the binding protein results in activation and/or proliferation of virus-specific memory T cells in the individual.
11. The method of any one of claims 1-6 and 10, wherein the memory T cells are CD8+ or CD4+ memory T cells.
12. The method of any one of claims 1-6, 10, and 11, wherein the memory T cells are central memory T cells (TCM) or effector memory T cells (TEM).
13. The method of any one of claims 1-12, wherein the CD28 polypeptide is a human CD28 polypeptide, wherein the CDS polypeptide is a human CD3 polypeptide, and wherein the CD38 polypeptide is a human CD38 polypeptide.
14. The method of any one of claims 1-13, wherein:

(a) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NQ:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the VL3 domain comprises a CDR-LI sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 3 5), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36);
(b) the VI-B domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid

sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ It) NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36); or
(c) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and the VL3 domain comprises a CDR-Ll sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:46).
15. The method of claim 14, wherein:
(a) the VH3 domain comprises the amino acid sequence of QVQLQQSGAEL\11SGASVKMSCKASGYTFTSFNMHWVKETPGQGLEWIGYIYPGN GGTNYNQKFKGKATLTADTSSSTAYMQISSLTSEDSAVYFCARTGGLRRAYFTYWG QGTLVTVS (SEQ ID NO:5), and the VLS domain comprises the amino acid sequence of DIVLTQSPASLAVSLGQRATISCRASESVDSYGNGFMHWYQQKPGQPPKLLIYLASN LESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNKEDPWTFGGGTKLEIK (SEQIDNO:6);
(b) the VH3 domain comprises the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQ GGlTSfYTSTQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWG QGTLVTVSS (SEQ ID NO: 13), and the VLS domain comprises the amino acid sequence of DrVLTQSPATLSLSPGERAHSCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSR ATGIPARF SGSGSGTDFTLTISPLEPEDF AVY YCQQNKEDPWTFGGGTKLEIK (SEQ ID NO: 14);
(c) the VH3 domain comprises the amino acid sequence of QVQLVQSGAEVVKPGAS\^VSCKASGYTFTSFNMHWVKEAPGQRLEWIGYTYPGN GGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWG

QGTLVTVSS (SEQ ID NO: 17), and the VL3 domain comprises the amino acid sequence of DIVLTQSPATLSLSPGERATISCRASESVDSYGNGFMHWYQQKPGQPPRLLIYLASSR AT GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO: 18);
(d) the VH3 domain comprises the amino acid sequence of
QVQLVQSGAEVVKSGASVKVSCKASGYTFTSFNMHWVKEAPGQGLEWIGYIYPGN
GGTNYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFCARTGGLRRAYFTYWG QGTLVTVSS (SEQ ID NO:21), and the VL3 domain comprises the amino acid sequence of DIVLTQSPATLSLSPGERATISCRASESVDSYGNGFMHWYQQKPGQPPRLLIYLASSR
AT GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO: 18);
(e) the Vi-B domain comprises the amino acid sequence of QVQLVQSGAEV\7KPGAS\7KAISCKASGYTFTSFNlVlFrvV\^KEAPGQRLEWIGYIYPGN GGTNYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFCARTGGLRRAYFTYWG QGTLVTVSS (SEQ ID NO:23), and the VL3 domain comprises the amino acid sequence of DIVLTQSPATLSLSPGERATISCRASESVDSYGNGFMHWYQQKPGQPPRLLIYLASSR AT GIPARF SGSGSGTDFTLTISPLEPEDF AVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO: 18); or
(f) the VH3 domain comprises the amino acid sequence of QVQLVESGGGVVQPGRSLIILSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDG SNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQG TLVTVSS (SEQ ID NO:9), and the VL3 domain comprises the amino acid sequence of AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY'QQKPGKAPKLLIYAASSLQSG VPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIK (SEQ ID NO: 10).
16. The method of any one of claims 1-15, wherein:
(a) the VHI domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO: 108), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO: 109), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO: 110), and the VLI domain comprises a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:l 11), a

CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO: 112), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO: 113); or
(b) the Vi-n domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFSLSDYG (SEQ ID NO: 114), a CDR-H2 sequence comprising the amino acid sequence of IWAGGGT (SEQ ID NO: 115), and a CDR-H3 sequence comprising the amino acid sequence of ARDKGYSYYYSMDY (SEQ ID NO: 116), and the VLI domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVEYYVTSL (SEQ ID NO: 117), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 118), and a CDR-L3 sequence comprising the amino acid sequence of QQSRKVPYT (SEQ ID NO: 119).
17, The method of claim 16, wherein:
(a) the Vi-n domain comprises the amino acid sequence of QVQLVQSGAEWKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNV NTNT^AQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWG KGTTVTVSS (SEQ ID NO:49), and the VLI domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTG VPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO:50); or
(b) the VHI domain comprises the amino acid sequence of QVQLQESGPGLVKPSQTLSLTCTVSGFSLSDYGVHWVRQPPGKGLEWLGVIWAGGG TNYNPSLKSRKTISKDTSKNQVSLKLSSVTAADTAVYYCARDKGYSYYYSMDYWG QGTTVTVS (SEQ ID NO;51), and the VLI domain comprises the amino acid sequence of DIVLTQSPASLAVSPGQRATITCRASESVEYYVTSLMQWYQQKPGQPPKLLIFAASNV ESGVPARFSGSGSGTDFTLTINPVEANDVANYYCQQSRKVPYTFGQGTKLEIK(SEQ ff)NO:52).
18. The method of any one of claims 1-17, wherein:
(a) the VHI domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 120), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 121), and a CDR-H3 sequence comprising the

amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 122), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHNNANTY (SEQ ID NO: 123), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 124), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 125); or
(b) the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 126), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 127), and a CDR-H3 sequence comprising the amino acid sequence of GVYYALSPFDY (SEQ ID NO: 128), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHNNGNTY (SEQ ID NO: 129), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ) ID NO: 130), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 131),
19. The method of claim 18, wherein:
(a) the VH2 domain comprises the amino acid sequence of
QVQLVESGGGVVQPGRSLIILSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKS
NSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDY
WGQGTLVTVSS (SEQ ID NO:53), and the VL2 domain comprises the amino acid sequence
of
DIVMTQTPLSLSVTPGQPASISCKSSQSLVHNNANTYLSWYLQKPGQSPQSLIYKVSN RFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:54); or
(b) the VH2 domain comprises the amino acid sequence of
QVQLWSGGGVVQPGRSLRLSCAASGFTFTKAWMmYVRQAPGKGLEWVAQIKDKS
NSYATYYADSVKGRFTISRDNSKTMTLYLQMNSLRAEDTAVYYCRGVYYALSPFDY
WGQGTLVTVSS (SEQ ID NO:84), and the VL2 domain comprises the amino acid sequence
of
DIVMTQTPLSLSVTPGQPASISCKSSQSLVHNNGNTYLSWYLQKPGQSPQLLIYKVSN RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQ YPFTFGGGTK VEIK (SEQ IDNQ:85).

20. The method of any one of claims 1.-19, wherein at least one of Li, L2, L:< or Ls is independently 0 amino acids in length.
21. The method of any one of claims 1-19, wherein (a) Li, L2, L3 and L4 each independently are zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPKAAP (SEQ ID NO: 58), and GGSGSSGSGG (SEQ ID NO: 59); or (b) Li, L2, L3 and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO: 58), and GGSGSSGSGG (SEQ ID NO:59).
22. The method of any one of claims 1-19, wherein Li comprises the sequence GQPKAAP (SEQ ID NO: 58), L2 comprises the sequence TKGPS (SEQ ID NO:57), U comprises the sequence S, and L4 comprises the sequence RT.
23. The method of any one of claims 1-22, wherein the hinge-Cm-Cm domains of the second and the third polypeptide chains are human IgG4 hinge-Cm-Cm domains, and wherein the hinge-Cm-Cm domains each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A and L235A.
24. The method of any one of claims 1-22, wherein the hinge-Cm-Cm domains of the second and the third polypeptide chains are human IgG4 hinge-Cm-Cm domains, and wherein the hinge-Cm-Cm domains each comprise amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to EU Index, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236.
25. The method of any one of claims 1-24, wherein the hinge-Cm-Cm domains of the second and the third polypeptide chains are human IgG4 hinge-Cm-Cm domains, and wherein the hinge-Cm-Cm domains each comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P and R409K.
26. The method of any one of claims 1-22, wherein the hinge-Cm-Cm domains of the second and the third polypeptide chains are human IgGl hinge-Cm-Cm domains, and

wherein the hinge-Cm-Cro domains each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgGl according to EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A.
27. The method of any one of claims 1-22, wherein the hinge-Ci-i2-C:n3 domains of the second and the third polypeptide chains are human IgGl hinge-Cm-CHs domains, and wherein the hinge-Cm-Cro domains each comprise amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgGl according to EU Index, wherein the amino acid substitutions are S298N, T299A, and Y300S.
28. The method of any one of claims 1-27, wherein the hinge-Cm-Cro domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the hinge-Ci-12-CHS domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W.
29. The method of any one of claims 1-27, wherein the hinge-Cm-CtB domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W; and wherein the hinge-Cm-Cro domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V.
30. The binding protein of claim 1, wherein:

(a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:60, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:62, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:63;
(b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:64, the third

polypeptide chain comprises the amino acid sequence of SEQ ID NO:65, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:63;
(c) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:66, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:67, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:63;
(d) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:60, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:68, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:69;
(e) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:6I, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:64, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:70, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:69; or
(f) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:66, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:71, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:69,
31, The method of any one of claims 1-30, wherein the virus is a human immunodeficiency virus (HIV), influenza virus, cytomegalovirus (CMV), hepatitis B virus (HBV), human papillomavirus (HPV), Epstein-barr virus (EBV), human foamy virus (HFV), herpes simplex virus 1 (HSV-1), or herpes simplex virus 1 (HSV-2).