RU2820351C2 - Trispecific binding proteins binding cd38, cd28 and cd3, as well as methods of use for treating viral infection - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is a method for propagation of virus-specific memory T-cells, involving the introduction of a virus-specific memory T-cell into contact with a binding protein, where the binding protein contains four polypeptide chains which form three antigen-binding sites. Described is a method for propagation of T-cells, involving the introduction of a T-cell into contact with a binding protein in vitro or ex vivo, where the binding protein contains four polypeptide chains which form three antigen-binding sites. Disclosed is a method of treating a chronic viral infection, comprising administering to a patient an effective amount of a binding protein, where the binding protein contains four polypeptide chains which form three antigen-binding sites.

EFFECT: invention extends the range of products for treating chronic viral infections.

31 cl, 13 dwg, 13 tbl, 7 ex

Description

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to International Application No. PCT/US2018/055084, filed October 9, 2018, US Provisional Patent Application Serial No. 62/831572, filed April 9, 2019, US Provisional Patent Application no. Serial No. 62/831608, filed April 9, 2019, and European Patent Application No. 19306097.7, filed September 11, 2019, all of which are incorporated herein by reference in their entirety.

REPRESENTATION OF A LIST OF SEQUENCES IN AN ASCII TEXT FILE

[0002] The contents of the following ASCII text file provided are incorporated herein by reference in their entirety: Sequence Listing Computer Readable Form (CRF) (File Name: 183952032140SEQLIST.TXT, Date Compiled: October 2, 2019, Size: 144 KB).

Field of technology to which the invention relates

[0003] The present invention relates to methods of using trispecific binding proteins containing four polypeptide chains that form three antigen binding sites that specifically bind a CD38 polypeptide ( eg , human and/or cynomolgus CD38 polypeptides), a CD28 polypeptide, and a CD3 polypeptide, to expansion of memory T cells ( eg , virus-specific memory T cells) and/or treatment of chronic viral infection.

PREREQUISITES FOR CREATION OF THE INVENTION

[0004] As part of human adaptive immunity, T cell immunity plays a critical role in the control of viral infection by eliminating infected cells, resulting in clearance of the viral infection. In chronic infectious diseases such as herpes virus infection (HSV, CMV, EBV, etc.), HIV and HBV, viruses maintain their persistence in humans through various mechanisms, including immune suppression, T cell depletion and establishment of latency. . However, viral infection typically induces viral antigen-specific immunity, including antigen-specific CD8 T cells, which can readily recognize infected cells for control or killing through cytokine release or cytotoxic T cell (CTL)-mediated killing processes.

[0005] Thus, activation and/or amplification of viral antigen-specific T cells in vivo and/or ex vivo may provide therapeutic strategies against chronic viral infections.

BRIEF DESCRIPTION OF THE INVENTION

[0006] Presented herein are anti-CD38/CD28xCD3 trispecific antibodies that have been developed and evaluated for their ability to activate T cells and subsequent proliferation and/or amplification of antigen-specific T cells. These trispecific Abs can effectively expand CD4 and CD8 effector and memory cell populations, including antigen-specific CD8 central memory T cells and memory effector cells in vitro . In particular, in vitro proliferation of CMV, EBV, HIV-1, and influenza virus-specific CD8 central memory cells and effector memory cells was demonstrated. The anti-CD38/CD28xCD3 trispecific antibodies described herein exhibit novel properties by engaging CD3/CD28/CD38 to provide a signal transduction pathway for T cell stimulation and expansion, which may provide an effective strategy for the treatment of chronic infectious diseases such as HSV infections, CMV, EBV, HIV-1 and HBV.

[0007] To address these and other needs, herein provided are binding proteins that bind CD38 polypeptide ( eg , human and cynomolgus CD38 polypeptides), CD28 polypeptide, and CD3 polypeptide.

[0008] In some embodiments, provided herein is a method of propagating virus-specific memory T cells, comprising contacting the virus-specific memory T cell with a binding protein, wherein the binding protein comprises four polypeptide chains that form three antigen-binding region where the first polypeptide chain contains the structure represented by the formula:

V _L2 -L ₁ -V _L1 -L ₂ -C _L [I],

and the second polypeptide chain contains a structure represented by the formula:

V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge-C _H2 -C _H3 [II];

and the third polypeptide chain contains the structure represented by the formula:

V _H3 -C _H1 -hinge-C _H2 -C _H3 [III];

and the fourth polypeptide chain contains the structure represented by the formula:

V _L3 -C _L [IV];

Where

V _L1 is the first variable domain of the immunoglobulin light chain;

V _L2 is the second variable domain of the immunoglobulin light chain;

V _L3 is the third variable domain of the immunoglobulin light chain;

V _H1 is the first variable domain of the immunoglobulin heavy chain;

V _H2 is the second variable domain of the immunoglobulin heavy chain;

V _H3 is the third variable domain of the immunoglobulin heavy chain;

C _L is an immunoglobulin light chain constant domain;

C _H1 is the immunoglobulin heavy chain C _H1 constant domain;

C _H2 is the immunoglobulin heavy chain C _H2 constant domain;

C _H3 is the immunoglobulin heavy chain C _H3 constant domain;

the hinge is the hinge region of an immunoglobulin connecting the C _H1 and C _H2 domains; And

L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers,

wherein the polypeptide of formula I and the polypeptide of formula II form a light_chain-heavy_chain crossover pair, and

where V _H1 and V _L1 form a first antigen binding site that binds the CD28 polypeptide, where V _H2 and V _L2 form a second antigen binding region that binds the CD3 polypeptide, and where V _H3 and V _L3 form a third antigen binding region that binds the CD38 polypeptide

[0009] In some embodiments, provided herein is a binding protein that contains four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain contains the structure represented by the formula:

V _L2 -L ₁ -V _L1 -L ₂ -C _L [I],

and the second polypeptide chain contains a structure represented by the formula:

V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge-C _H2 -C _H3 [II];

and the third polypeptide chain contains the structure represented by the formula:

V _H3 -C _H1 -hinge-C _H2 -C _H3 [III];

and the fourth polypeptide chain contains the structure represented by the formula:

V _L3 -C _L [IV];

Where

V _L1 is the first variable domain of the immunoglobulin light chain;

V _L2 is the second variable domain of the immunoglobulin light chain;

V _L3 is the third variable domain of the immunoglobulin light chain;

V _H1 is the first variable domain of the immunoglobulin heavy chain;

V _H2 is the second variable domain of the immunoglobulin heavy chain;

V _H3 is the third variable domain of the immunoglobulin heavy chain;

C _L is an immunoglobulin light chain constant domain;

C _H1 is the immunoglobulin heavy chain C _H1 constant domain;

C _H2 is the immunoglobulin heavy chain C _H2 constant domain;

C _H3 is the immunoglobulin heavy chain C _H3 constant domain;

the hinge is the hinge region of an immunoglobulin connecting the C _H1 and C _H2 domains; And

L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers,

wherein the polypeptide of formula I and the polypeptide of formula II form a light_chain-heavy_chain crossover pair, and

wherein V _H1 and V _L1 form a first antigen binding site that binds the CD28 polypeptide, where V _H2 and V _L2 form a second antigen binding region that binds the CD3 polypeptide, and where V _H3 and V _L3 form a third antigen binding region that binds the CD38 polypeptide, for applications in the propagation of virus-specific memory T cells.

[0010] In some embodiments, a virus-specific memory T cell is contacted with a binding protein in vitro or ex vivo . In some embodiments, contacting a virus-specific memory T cell with a binding protein causes activation and/or proliferation of virus-specific memory T cells.

[0011] In some embodiments, provided herein is a method of T cell expansion comprising contacting the T cell with a binding protein in vitro or ex vivo , wherein the binding protein comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain the chain contains the structure represented by the formula:

V _L2 -L ₁ -V _L1 -L ₂ -C _L [I],

and the second polypeptide chain contains a structure represented by the formula:

V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge-C _H2 -C _H3 [II];

and the third polypeptide chain contains the structure represented by the formula:

V _H3 -C _H1 -hinge-C _H2 -C _H3 [III];

and the fourth polypeptide chain contains the structure represented by the formula:

V _L3 -C _L [IV];

Where

V _L1 is the first variable domain of the immunoglobulin light chain;

V _L2 is the second variable domain of the immunoglobulin light chain;

V _L3 is the third variable domain of the immunoglobulin light chain;

V _H1 is the first variable domain of the immunoglobulin heavy chain;

V _H2 is the second variable domain of the immunoglobulin heavy chain;

V _H3 is the third variable domain of the immunoglobulin heavy chain;

C _L is an immunoglobulin light chain constant domain;

C _H1 is the immunoglobulin heavy chain C _H1 constant domain;

C _H2 is the immunoglobulin heavy chain C _H2 constant domain;

C _H3 is the immunoglobulin heavy chain C _H3 constant domain;

the hinge is the hinge region of an immunoglobulin connecting the C _H1 and C _H2 domains; And

L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers,

wherein the polypeptide of formula I and the polypeptide of formula II form a light_chain-heavy_chain crossover pair, and

wherein V _H1 and V _L1 form a first antigen binding site that binds the CD28 polypeptide, where V _H2 and V _L2 form a second antigen binding region that binds the CD3 polypeptide, and where V _H3 and V _L3 form a third antigen binding region that binds the CD38 polypeptide.

[0012] In some embodiments, provided herein is a binding protein that contains four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain contains the structure represented by the formula:

V _L2 -L ₁ -V _L1 -L ₂ -C _L [I],

and the second polypeptide chain contains a structure represented by the formula:

V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge-C _H2 -C _H3 [II];

and the third polypeptide chain contains the structure represented by the formula:

V _H3 -C _H1 -hinge-C _H2 -C _H3 [III];

and the fourth polypeptide chain contains the structure represented by the formula:

V _L3 -C _L [IV];

Where

V _L1 is the first variable domain of the immunoglobulin light chain;

V _L2 is the second variable domain of the immunoglobulin light chain;

V _L3 is the third variable domain of the immunoglobulin light chain;

V _H1 is the first variable domain of the immunoglobulin heavy chain;

V _H2 is the second variable domain of the immunoglobulin heavy chain;

V _H3 is the third variable domain of the immunoglobulin heavy chain;

C _L is an immunoglobulin light chain constant domain;

C _H1 is the immunoglobulin heavy chain C _H1 constant domain;

C _H2 is the immunoglobulin heavy chain C _H2 constant domain;

C _H3 is the immunoglobulin heavy chain C _H3 constant domain;

the hinge is the hinge region of an immunoglobulin connecting the C _H1 and C _H2 domains; And

L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers,

wherein the polypeptide of formula I and the polypeptide of formula II form a light_chain-heavy_chain crossover pair, and

wherein V _H1 and V _L1 form a first antigen binding site that binds the CD28 polypeptide, where V _H2 and V _L2 form a second antigen binding region that binds the CD3 polypeptide, and where V _H3 and V _L3 form a third antigen binding region that binds the CD38 polypeptide, for use in the method of propagation of 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 contains a polynucleotide encoding a CAR.

[0014] In some embodiments, provided herein is a method of treating a 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 three antigen binding sites, wherein the first polypeptide chain comprises the structure , represented by the formula:

V _L2 -L ₁ -V _L1 -L ₂ -C _L [I],

and the second polypeptide chain contains a structure represented by the formula:

V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge-C _H2 -C _H3 [II];

and the third polypeptide chain contains the structure represented by the formula:

V _H3 -C _H1 -hinge-C _H2 -C _H3 [III];

and the fourth polypeptide chain contains the structure represented by the formula:

V _L3 -C _L [IV];

Where

V _L1 is the first variable domain of the immunoglobulin light chain;

V _L2 is the second variable domain of the immunoglobulin light chain;

V _L3 is the third variable domain of the immunoglobulin light chain;

V _H1 is the first variable domain of the immunoglobulin heavy chain;

V _H2 is the second variable domain of the immunoglobulin heavy chain;

V _H3 is the third variable domain of the immunoglobulin heavy chain;

C _L is an immunoglobulin light chain constant domain;

C _H1 is the immunoglobulin heavy chain C _H1 constant domain;

C _H2 is the immunoglobulin heavy chain C _H2 constant domain;

C _H3 is the immunoglobulin heavy chain C _H3 constant domain;

the hinge is the hinge region of an immunoglobulin connecting the C _H1 and C _H2 domains; And

L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers,

wherein the polypeptide of formula I and the polypeptide of formula II form a light_chain-heavy_chain crossover pair, and

wherein V _H1 and V _L1 form a first antigen binding site that binds the CD28 polypeptide, where V _H2 and V _L2 form a second antigen binding region that binds the CD3 polypeptide, and where V _H3 and V _L3 form a third antigen binding region that binds the CD38 polypeptide.

[0015] In some embodiments, provided herein is a binding protein that contains four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain contains the structure represented by the formula:

V _L2 -L ₁ -V _L1 -L ₂ -C _L [I],

and the second polypeptide chain contains a structure represented by the formula:

V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge-C _H2 -C _H3 [II];

and the third polypeptide chain contains the structure represented by the formula:

V _H3 -C _H1 -hinge-C _H2 -C _H3 [III];

and the fourth polypeptide chain contains the structure represented by the formula:

V _L3 -C _L [IV];

Where

V _L1 is the first variable domain of the immunoglobulin light chain;

V _L2 is the second variable domain of the immunoglobulin light chain;

V _L3 is the third variable domain of the immunoglobulin light chain;

V _H1 is the first variable domain of the immunoglobulin heavy chain;

V _H2 is the second variable domain of the immunoglobulin heavy chain;

V _H3 is the third variable domain of the immunoglobulin heavy chain;

C _L is an immunoglobulin light chain constant domain;

C _H1 is the immunoglobulin heavy chain C _H1 constant domain;

C _H2 is the immunoglobulin heavy chain C _H2 constant domain;

C _H3 is the immunoglobulin heavy chain C _H3 constant domain;

the hinge is the hinge region of an immunoglobulin connecting the C _H1 and C _H2 domains; And

L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers,

wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light-chain-heavy-chain pair; and where V _H1 and V _L1 form a first antigen binding site that binds a CD28 polypeptide, where V _H2 and V _L2 form a second antigen binding region that binds a CD3 polypeptide, and where V _H3 and V _L3 form a third antigen binding region that binds a CD38 polypeptide, for use in a method of treating a chronic viral infection, wherein the method comprises administering to an individual in need thereof an effective amount of a binding protein. In some embodiments, provided herein is a binding protein for use in a method of treating a chronic viral infection, wherein the method comprises administering to an individual in need thereof an effective amount of a binding protein, wherein the binding protein comprises four polypeptide chains that form three antigen binding sites, wherein The first polypeptide chain of the binding protein contains the structure represented by the formula:

V _L2 -L ₁ -V _L1 -L ₂ -C _L [I],

and the second polypeptide chain of the binding protein contains the structure represented by the formula:

V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge-C _H2 -C _H3 [II],

and the third polypeptide chain of the binding protein contains the structure represented by the formula:

V _H3 -C _H1 -hinge-C _H2 -C _H3 [III],

and the fourth polypeptide chain of the binding protein contains the structure represented by the formula:

V _L3 -C _L [IV],

Where

V _L1 is the first variable domain of the immunoglobulin light chain;

V _L2 is the second variable domain of the immunoglobulin light chain;

V _L3 is the third variable domain of the immunoglobulin light chain;

V _H1 is the first variable domain of the immunoglobulin heavy chain;

V _H2 is the second variable domain of the immunoglobulin heavy chain;

V _H3 is the third variable domain of the immunoglobulin heavy chain;

C _L is an immunoglobulin light chain constant domain;

C _H1 is the immunoglobulin heavy chain C _H1 constant domain;

C _H2 is the immunoglobulin heavy chain C _H2 constant domain;

C _H3 is the immunoglobulin heavy chain C _H3 constant domain;

the hinge is the hinge region of an immunoglobulin connecting the C _H1 and C _H2 domains; And

L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers,

wherein the polypeptide of formula I and the polypeptide of formula II form a light_chain-heavy_chain crossover pair, and

wherein V _H1 and V _L1 form a first antigen binding site that binds the CD28 polypeptide, where V _H2 and V _L2 form a second antigen binding region that binds the CD3 polypeptide, and where V _H3 and V _L3 form a third antigen binding region that binds the CD38 polypeptide.

[0016] In some embodiments, the individual is a human. In some embodiments, the binding protein is administered to an individual in a pharmaceutical composition comprising the binding protein and a pharmaceutically acceptable carrier. In some embodiments, administration of the binding protein results in the activation and/or proliferation of virus-specific memory T cells in an individual.

[0017] In some embodiments, which may be a combination of 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, which may be a combination of 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, which may be a combination of any other embodiments described herein, the V _H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence IYPGNGGT (SEQ ID NO:32), and the CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33), and the _L3 domain V containing the CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34) , a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the _H3 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence sequence ARTGGLRRAYFTY (SEQ ID NO:33), and domain V _L3 contains a CDR-L1 sequence containing the amino acid sequence QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence GAS (SEQ ID NO:40), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the _H3 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARMFRGAFDY (SEQ ID NO:43), and the V _L3 domain contains a CDR-L1 sequence containing the amino acid sequence QGIRND (SEQ ID NO:44), a CDR-L2 sequence containing the amino acid sequence AAS (SEQ ID NO:45), and a CDR-L3 sequence containing the amino acid sequence LQDYIYYPT (SEQ ID NO:46). In some embodiments, the V _H3 domain comprises the amino acid sequence QVQLQQSGAELVRSGASVKMSCKASGYTFTSFNMHWVKETPGQGLEWIGYIYPGNGGTNYNQKFKGKATLTADTSSSTAYMQISSLTSEDSAVYFCARTGGLRRAYFTYWGQGTLVTVS (SEQ ID NO:5), and the V _L3 domain comprises the amino acid sequence DIVLTQSPASLAVSLGQRATI SCRASESVDSYGNGFMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:6). In some embodiments, the V _H3 domain contains the amino acid sequence Qvqlvqsgaevvkpgasvkvsckasgytftsyamhwvkeapgqrlewigyiypgqggtnynqkfqgratltadtsastaymelsslrsedtavyfcartgglrrayftywgqgtlvtvss (SEQ ID NO:13), and the V _L3 domain contains the amino acid sequence Divltqspatlsl spgeratiscrasqsvssygqgfmhwyqqkpgqpprlliygassratgiparfsgsgsgtdftltisplepedfavyycqqnkedpwtfgggtkleik (SEQ ID NO:14). In some embodiments, the V _H3 domain comprises the amino acid sequence QVQLVQSGAEVVKPGASVKVSCKASGYTFTSFNMHWVKEAPGQRLEWIGYIYPGNGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO:17), and the V _L3 domain comprises the amino acid sequence DIVLTQSPATLSLSPGERATISCR ASESVDSYGNGFMHWYQQKPGQPPRLLIYLASSRAT GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:18). In some embodiments, the V _H3 domain contains the amino acid sequence QVQLVQSGAEVVKSGASVKVSCKASGYTFTSFNMHWVKEAPGQGLEWIGYIYPGNGGTNYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO:21), and the V _L3 domain contains the amino acid sequence DIVLTQSPATLSLSPGERATISCRASE DSYGNGFMHWYQQKPGQPPRLLIYLASSRAT GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:18). In some embodiments, the V _H3 domain contains the amino acid sequence QVQLVQSGAEVVKPGASVKMSCKASGYTFTSFNMHWVKEAPGQRLEWIGYIYPGNGGTNYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO:23), and the V _L3 domain contains the amino acid sequence DIVLTQSPATLSLSPGERATISCRASE SVDSYGNGFMHWYQQKPGQPPRLLIYLASSRAT GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:18). In some embodiments, the V _H3 domain contains the amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSS (SEQ ID NO:9), and the V _L3 domain contains the amino acid sequence AIQMTQSPSSLSASVGDRVTITCRASQ GIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIK (SEQ ID NO:10).

[0020] In some embodiments, which may be a combination of any other embodiments described herein, the V _H1 domain comprises a CDR-H1 sequence comprising the amino acid sequence GYTFTSYY (SEQ ID NO:108), a CDR-H2 sequence comprising the amino acid sequence IYPGNVNT (SEQ ID NO:109), and the CDR-H3 sequence containing the amino acid sequence TRSHYGLDWNFDV (SEQ ID NO:110), and the _L1 domain V containing the CDR-L1 sequence containing the amino acid sequence QNIYVW (SEQ ID NO:111) , a CDR-L2 sequence containing the amino acid sequence KAS (SEQ ID NO:112), and a CDR-L3 sequence containing the amino acid sequence QQGQTYPY (SEQ ID NO:113). In some embodiments, the _H1 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GFSLSDYG (SEQ ID NO:114), a CDR-H2 sequence comprising the amino acid sequence IWAGGGT (SEQ ID NO:115), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARDKGYSYYYSMDY (SEQ ID NO:116), and the V _L1 domain contains a CDR-L1 sequence containing the amino acid sequence ESVEYYVTSL (SEQ ID NO:117), a CDR-L2 sequence containing the amino acid sequence AAS (SEQ ID NO:118), and a CDR-L3 sequence containing the amino acid sequence QQSRKVPYT (SEQ ID NO:119). In some embodiments, the V _H1 domain contains the amino acid sequence qvqlvqsgaevvkpgasvkvsckasgytftsyyihwvrqapgqglewigsiypgnvntnyaqkfqgratltvdtsistaymelsrlrsddtavyyctrshygldwnfdvwgkgttvtvss (SEQ ID NO:49), and the V _L1 domain contains the amino acid sequence diqmtq spsslsasvgdrvtitcqasqniyvwlnwyqqkpgkapklliykasnlhtgvpsrfsgsgsgtdftltisslqpediatyycqqgqtypytfgqgtkleik (SEQ ID NO:50). In some embodiments, the V _H1 domain contains the amino acid sequence qvqlqesgpglvkpsqtlsltctvsgfslsdygvhwvrqppgkglewlgviwaggtnynpslksrktiskdtsknqvslklssvtaadtavyycardkgysyyysmdywgqgttvtvs (SEQ ID NO:51), and the V _L1 domain contains the amino acid sequence divltq spaslavspgqratitcrasesveyyvtslmqwyqqkpgqppkllifaasnvesgvparfsgsgsgtdftltinpveandvanyycqqsrkvpytfgqgtkleik (SEQ ID NO:52).

[0021] In some embodiments, which may be a combination of any other embodiments described herein, the V _H2 domain comprises a CDR-H1 sequence containing the amino acid sequence gftftkaw (SEQ ID NO:120), a CDR-H2 sequence containing the amino acid sequence IKDKSNSYAT (SEQ ID NO:121), and the CDR-H3 sequence containing the amino acid sequence RGVYYALSPFDY (SEQ ID NO:122), and the _L2 domain V containing the CDR-L1 sequence containing the amino acid sequence QSLVHNNANTY (SEQ ID NO:123) , a CDR-L2 sequence containing the amino acid sequence KVS (SEQ ID NO:124), and a CDR-L3 sequence containing the amino acid sequence GQGTQYPFT (SEQ ID NO:125). In some embodiments, the _H2 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GFTFTKAW (SEQ ID NO:126), a CDR-H2 sequence comprising the amino acid sequence IKDKSNSYAT (SEQ ID NO:127), and a CDR-H3 sequence comprising the amino acid sequence the sequence GVYYALSPFDY (SEQ ID NO:128), and the V _L2 domain contains a CDR-L1 sequence containing the amino acid sequence QSLVHNNGNTY (SEQ ID NO:129), a CDR-L2 sequence containing the amino acid sequence KVS (SEQ ID NO:130), and a CDR-L3 sequence containing the amino acid sequence GQGTQYPFT (SEQ ID NO:131). In some embodiments, the V _H2 domain contains the amino acid sequence qvqlvesgggvvqpgrslrlscaasgftftkawmhwvrqapgkqlewvaqikdksnsyatyyadsvkgrftisrddskntlylqmnslraedtavyycrgvyyalspfdywgqgtlvtvss (SEQ ID NO:53), and the V _L2 domain contains the amino acid sequence divmtqt plslsvtpgqpasisckssqslvhnnantylswylqkpgqspqsliykvsnrfsgvpdrfsgsgsgtdftlkisrveaedvgvyycgqgtqypftfgsgtkveik (SEQ ID NO:54). In some embodiments, the V _H2 domain contains the amino acid sequence qvqlvesgggvvqpgrslrlscaasgftftkawmhwvrqapgkglewvaqikdksnsyatyyadsvkgrftisrdnskntlylqmnslraedtavyycrgvyyalspfdywgqgtlvtvss (SEQ ID NO:84), and the V _L2 domain contains the amino acid sequence divmtqtpl slsvtpgqpasisckssqslvhnngntylswylqkpgqspqlliykvsnrfsgvpdrfsgsgsgtdftlkisrveaedvgvyycgqgtqypftfgggtkveik (SEQ ID NO:85).

[0022] In some embodiments, which may be a combination of any other embodiments described herein, at least one of L ₁ , L ₂ , L ₃ or L ₄ independently has a length of 0 amino acids. In some embodiments, L ₁ , L ₂ , L ₃ , and L ₄ are each independently zero amino acids in length or contain 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, L ₁ , L ₂ , L ₃ , and L ₄ 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, L ₁ contains the sequence GQPKAAP (SEQ ID NO: 58), L ₂ contains the sequence TKGPS (SEQ ID NO: 57), L ₃ contains the sequence S, and L ₄ contains the sequence RT.

[0023] In some embodiments, which may be a combination of any other embodiments described herein, the hinge-C _H2 -C _H3 domains of the second and third polypeptide chains are human IgG4-C _H2 -C _H3 hinge domains, and when In this case, each of the hinge-C _H2 -C _H3 domains contains amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, where the amino acid substitutions are F234A and L235A. In some embodiments, the hinge-C _H2 -C _H3 domains of the second and third polypeptide chains are human hinge-IgG4-C _H2 -C _H3 domains, and wherein each of the hinge-C _H2 -C _H3 domains contains amino acid substitutions at positions corresponding to 233-236 human IgG4 according to the EU index, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at position 236 In some embodiments, the hinge-C _H2 -C _H3 domains of the second and third polypeptide chains are hinge-IgG4- domains human C _H2 -C _H3 , and wherein each of the hinge-C _H2 -C _H3 domains contains amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, where the amino acid substitutions are S228P and R409K. In some embodiments, the hinge-C _H2 -C _H3 domains of the second and third polypeptide chains are human hinge-IgG1-C _H2 -C _H3 domains, and wherein each of the hinge-C _H2 -C _H3 domains contains amino acid substitutions at positions corresponding to 234, 235 and 329 human IgG1 according to the EU index, where the amino acid substitutions are L234A, L235A and P329A. In some embodiments, the hinge-C _H2 -C _H3 domains of the second and third polypeptide chains are human hinge-IgG1-C _H2 -C _H3 domains, and wherein each of the hinge-C _H2 -C _H3 domains contains amino acid substitutions at positions corresponding to 298, 299 and 300 human IgG1 according to the EU index, where the amino acid substitutions are S298N, T299A and Y300S. In some embodiments, the hinge-C _H2 -C _H3 domain of the second polypeptide chain contains amino acid substitutions at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are Y349C, T366S, L368A and Y407V; and wherein the hinge-C _H2 -C _H3 domain of the third polypeptide chain contains amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are S354C and T366W. In some embodiments, the hinge-C _H2 -C _H3 domain of the second polypeptide chain contains amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are S354C and T366W; and wherein the hinge-C _H2 -C _H3 domain of the third polypeptide chain contains amino acid substitutions at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A and Y407V.

[0024] In certain embodiments, the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:60, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:62, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO:63. In certain embodiments, the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:64, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:65, and the fourth polypeptide chain contains the amino acid sequence under SEQ ID NO:63. In certain embodiments, the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:66, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:67, and the fourth polypeptide chain contains the amino acid sequence under SEQ ID NO:63. In certain embodiments, the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:60, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:68, and the fourth polypeptide chain contains the amino acid sequence under SEQ ID NO:69. In certain embodiments, the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:64, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:70, and the fourth polypeptide chain contains the amino acid sequence under SEQ ID NO:69. In certain embodiments, the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:66, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:71, and the fourth polypeptide chain contains the amino acid sequence under SEQ ID NO:69.

[0025] In some embodiments, which may be a combination of any other embodiments described herein, the virus is human immunodeficiency virus (HIV), influenza virus, cytomegalovirus (CMV), hepatitis B virus (HBV), human papillomavirus (HPV), Epstein-Barr virus (EBV), human spumavirus (HFV), herpes simplex virus 1 (HSV-1), or herpes simplex virus 2 (HSV-2).

[0026] It should be understood that one, more, or all of the features of the various embodiments described herein can be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to one skilled in the art. These and other embodiments of the present invention are further described in the following detailed description.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0027] In FIG. 1 is a schematic representation of a trispecific binding protein containing four polypeptide chains that form three antigen binding sites that bind three target proteins: CD28, CD3 and CD38. The first pair of polypeptides has double variable domains having a crossover orientation (VH1-VH2 and VL2-VL1), forming two antigen-binding regions that recognize CD3 and CD28, and the second pair of polypeptides has one variable domain (VH3 and VL3), forming one antigen-binding region region that recognizes CD38. In the trispecific binding protein shown in FIG. 1, an IgG4 constant region with a knob-to-trench mutation is used, where the knob is on the second pair of polypeptides with a single variable domain.

[0028] In FIG. 2 shows a summary of the binding affinities of these trispecific binding proteins to their cognate antigens (human CD3, CD28 and CD38) as measured by SPR.

[0029] In FIG. 3 summarizes the binding affinities of the indicated CD38x-CD28x-CD3 trispecific binding proteins for human CD38 as measured by SPR or flow cytometry (FACS).

[0030] In FIG. 4A-4D show the in vitro expansion pattern of a subset of T cells in response to CD38VH1/CD3midxCD28sup trispecific antibodies. T cell subset expansion was assessed by coating wells with 350 ng/well CD38 trispecific Ab in the absence of exogenous cytokines. T cell populations were measured at the indicated time points. A trispecific Ab having three mutant antigen binding domains was used as a negative control. Flow cytometry was used to determine central ( _Tcm ) and effector ( _Tem ) CD4 memory T cells (FIG. 4A), T helper cells (Th1, Th17, Th2) (FIG. 4B), central ( _Tcm ) and effector (T _em ) memory CD8 T cells (FIG. 4C) and cytomegalovirus (CMV) pp65-specific CD8 cells (FIG. 4D) as described in Example 3. Analysis of CMV pp65-specific effector cells was performed by staining pentamer of peripheral blood mononuclear cells (PBMCs) from HLA-A2 CMV+ donors treated with CD38 trispecific or triple-negative control.

[0031] In FIG. 5A-5B show the in vitro expansion pattern of a subset of T cells in PBMC collected from CMV-infected donor B in response to the CD38 _VH1 /CD28sup x CD3mid trispecific antibody. T cell populations were measured at the indicated time points. The triple mutant trispecific antibody was used as a negative control. Flow cytometry was used to quantify CMV-specific memory CD8+ T cells (FIG. 5A), as well as CMV-specific central memory T cells ( _Tcm ) and effector ( _Tem ) CD8+ memory T cells (FIG. 5A). 5B). The CD38 _VH1 /CD28sup x CD3mid trispecific antibody activated T cells and promoted proliferation of CMV-specific CD8+ memory T cells.

[0032] In FIG. 6A-6B show the in vitro expansion of a subset of T cells in PBMC collected from CMV-infected donor C in response to the CD38 _VH1 /CD28sup x CD3mid trispecific antibody. T cell populations were measured at the indicated time points. The triple mutant trispecific antibody was used as a negative control. Flow cytometry was used to quantify CMV-specific memory CD8+ T cells (FIG. 6A), as well as CMV-specific central memory T cells ( _Tcm ) and effector ( _Tem ) CD8+ memory T cells (FIG. 6A). 6B). The CD38 _VH1 /CD28sup x CD3mid trispecific antibody activated T cells and promoted proliferation of CMV-specific CD8+ memory T cells.

[0033] In FIG. 7A-7B show the in vitro expansion of a subset of T cells in PBMC collected from Epstein-Barr virus (EBV)-infected donor A in response to the CD38 _VH1 /CD28sup x CD3mid trispecific antibody. T cell populations were measured at the indicated time points. The triple mutant trispecific antibody was used as a negative control. Flow cytometry was used to quantify EBV-specific memory CD8+ T cells (FIG. 7A), as well as EBV-specific central memory T cells ( _Tcm ) and effector ( _Tem ) CD8+ memory T cells (FIG. 7B). The trispecific antibody CD38 _VH1 /CD28sup x CD3mid activated T cells and promoted the proliferation of EBV-specific CD8+ memory T cells.

[0034] In FIG. 8A-8B show the in vitro expansion of a subset of T cells in PBMCs collected from EBV-infected donor B in response to the CD38 _VH1 /CD28sup x CD3mid trispecific antibody. T cell populations were measured at the indicated time points. The triple mutant trispecific antibody was used as a negative control. Flow cytometry was used to quantify EBV-specific memory CD8+ T cells (FIG. 8A), as well as EBV-specific central memory T cells ( _Tcm ) and effector ( _Tem ) CD8+ memory T cells (FIG. 8A). 8B). The trispecific antibody CD38 _VH1 /CD28sup x CD3mid activated T cells and promoted the proliferation of EBV-specific CD8+ memory T cells.

[0035] In FIG. 9 shows flow cytometry profiles of PBMC from the indicated human immunodeficiency virus (HIV) positive donors (lower panels and upper right panel) analyzed for HIV Gag specific CD8+ T cells (A*02:01 - SLYNTVATL (HIV- 1 gag p17 76-84) PE-conjugated pentamer, ProImmune) at baseline (day 0; before incubation with trispecific antibodies). PBMC from an HIV-negative donor were used as a negative control (top left panel). Population percentages of Gag-specific CD8+ T cells are presented and shown within the box. At baseline, PBMC 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 FIG. 10A-12B.

[0036] In FIG. 10A-10B show the in vitro expansion pattern of T cell subsets in PBMCs collected from HIV-positive donor D in response to the CD38 _VH1 /CD28sup x CD3mid trispecific antibody. T cell populations were measured at the indicated time points. The triple mutant trispecific antibody was used as a negative control. Flow cytometry was used to quantify HIV-specific memory CD8+ T cells (FIG. 10A), as well as HIV-specific central memory T cells ( _Tcm ) and effector ( _Tem ) CD8+ memory T cells (FIG. 10B). The CD38 _VH1 /CD28sup x CD3mid trispecific antibody activated T cells and promoted the proliferation of effector ( _Tem ) CD8+ memory T cells.

[0037] In FIG. 11A-11B show the in vitro expansion pattern of T cell subsets in PBMCs collected from HIV-positive donor E in response to the CD38 _VH1 /CD28sup x CD3mid trispecific antibody. T cell populations were measured at the indicated time points. The triple mutant trispecific antibody was used as a negative control. Flow cytometry was used to quantify HIV-specific memory CD8+ T cells (FIG. 11A), as well as HIV-specific central memory T cells ( _Tcm ) and effector ( _Tem ) CD8+ memory T cells (FIG. 11B). The CD38 _VH1 /CD28sup x CD3mid trispecific antibody activated T cells and promoted the proliferation of effector ( _Tem ) CD8+ memory T cells.

[0038] In FIG. 12A-12B show the in vitro expansion pattern of T cell subsets in PBMCs collected from HIV-positive donor F in response to the CD38 _VH1 /CD28sup x CD3mid trispecific antibody. T cell populations were measured at the indicated time points. The triple mutant trispecific antibody was used as a negative control. Flow cytometry was used to quantify HIV-specific memory CD8+ T cells (FIG. 12A), as well as HIV-specific central memory T cells ( _Tcm ) and effector ( _Tem ) CD8+ memory T cells (FIG. 12B). The CD38 _VH1 /CD28sup x CD3mid trispecific antibody activated T cells and promoted the proliferation of effector ( _Tem ) CD8+ memory T cells.

[0039] In FIG. 13A-13B show the in vitro expansion pattern of T cell subsets in PBMC collected from influenza A virus-infected donor in response to the CD38 _VH1 /CD28sup x CD3mid trispecific antibody. T cell populations were measured at the indicated time points. The triple mutant trispecific antibody was used as a negative control. Flow cytometry was used to quantify influenza virus-specific (Flu) memory CD8+ T cells (FIG. 13A), as well as Flu-specific central memory T cells (T _cm ) and effector (T _em ) CD8+ T cells memory (FIG. 13B). The CD38 _VH1 /CD28sup x CD3mid trispecific antibody activated T cells and promoted proliferation of T _em CD8+ T cells ( eg , see days 7, 11) and T _cm CD8+ T cells ( eg , see day 7).

DETAILED DESCRIPTION

[0040] The present invention relates to trispecific binding proteins containing four polypeptide chains that form three antigen-binding sites that specifically bind CD38 polypeptide ( for example , human and cynomolgus CD38 polypeptides), CD28 polypeptide and CD3 polypeptide, which can be used, for example , in the expansion of memory T cells ( eg , virus-specific memory T cells) and/or in the treatment of chronic viral infection.

I. General definitions

[0041] As used in accordance with the present invention, the following terms, unless otherwise indicated, should be understood to have the following meanings. Unless the context otherwise requires, terms in the singular will include the plural forms, and terms in the plural will include the singular form. As used in this detailed description and the accompanying claims, the singular form in the singular includes a reference to the plural form unless the content expressly states otherwise. Thus, for example, reference to a “molecule” does not necessarily include a combination of two or more such molecules and the like.

[0042] It is understood that aspects and embodiments of the present invention described herein include “comprising,” “consisting of,” and/or “substantially consisting of” aspects and embodiments.

[0043] As used herein, the term “polynucleotide” refers to single-stranded or double-stranded nucleic acid polymers having a length of at least 10 nucleotides. In certain embodiments, the nucleotides constituting a polynucleotide may be ribonucleotides, or deoxyribonucleotides, or a modified form of any 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 phosphothioate, phosphodithioate, phosphoselenoate, phosphodiselenoate, phosphoanilotioate, phosphoaniladate and phosphoamidate. The term "polynucleotide" specifically refers to single-stranded and double-stranded forms of DNA.

[0044] An "isolated polynucleotide" is a polynucleotide, whether of genomic, cDNA or synthetic origin, or characterized by some combination thereof, that: (1) is not associated with all or part of a polynucleotide in which the isolated polynucleotide occurs naturally, (2 ) is associated with a polynucleotide to which it is not naturally associated, or (3) does not occur in nature as part of a larger sequence.

[0045] An "isolated polypeptide" is one that: (1) does not contain at least some of the other polypeptides with which it is commonly found, (2) does not contain substantially other polypeptides from the same source, e.g. from the same species, (3) is expressed by a cell from another species, (4) has been separated from at least approximately 50 percent of the polynucleotides, lipids, carbohydrates or other substances with which it is naturally associated, (5) is not associated ( by covalent or non-covalent interaction) to portions of the polypeptide to which the "isolated polypeptide" is naturally associated, (6) is operably linked (by covalent or non-covalent interaction) to a polypeptide to which it is not naturally associated, or (7) is not found in nature. Such an isolated polypeptide may be encoded by genomic DNA, cDNA, mRNA or other RNA of synthetic origin, or any combination thereof. Preferably, the isolated polypeptide is substantially free of polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).

[0046] Naturally occurring antibodies are typically a tetramer. Each such tetramer typically consists of two identical pairs of polypeptide chains, with each pair having one full-length "light" chain (typically having a molecular weight of approximately 25 kDa) and one full-length "heavy" chain (typically having a molecular weight of approximately 50-70 kDa) . As used herein, the terms “heavy chain” and “light chain” refer to any immunoglobulin polypeptide containing a variable domain sequence sufficient to confer specificity for a target antigen. The amino terminal portion of each light and heavy chain typically contains a variable domain of approximately 100-110 amino acids or more, which is typically responsible for antigen recognition. The carboxy-terminal part of each chain usually defines a constant domain responsible for effector function. Thus, in a naturally occurring antibody, the full-length immunoglobulin heavy chain polypeptide contains a variable domain ( _VH ) and three constant domains ( _CH1 , _CH2 and _CH3 ), where the _VH domain is at the amino terminus of the polypeptide and the C _H3 is at the carboxyl terminus, and the full-length immunoglobulin light chain polypeptide contains a variable domain (V _L ) and a constant domain ( _CL ), where the V _L domain is at the amino terminus of the polypeptide and the C _L 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 they define the antibody isotype IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. IgA is similarly divided into subclasses, including but not limited to IgA1 and IgA2. In full-length light and heavy chains, the variable and constant domains are typically joined by a "J" region of about 12 or more amino acids, with the heavy chain also containing a "D" region of about 10 or more amino acids. Cm., For example, 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 region. The variable domains of naturally occurring antibodies are typically characterized by the same overall structure of relatively conserved framework regions (FRs) connected by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs of the two strands of each pair are typically aligned using framework regions, which may allow binding to a specific epitope. From the amino terminus to the carboxyl terminus, the variable domains of both the light and heavy chains typically contain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains.

[0048] The term “set of CDRs” refers to a group of three CDRs that are contained in a single variable region capable of binding an antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD. (1987) and (1991)), not only provides a unique residue numbering system applicable to any antibody variable region, but also provides precise residue boundaries defining three CDR. These CDRs may be called CDRs according to Kabat. Chothia and colleagues (Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-17; Chothia et al., 1989, Nature 342: 877-83) found that certain subfragments within the CDRs according to Kabat adopt almost identical peptide backbone conformations, despite the presence of large differences at the amino acid sequence level. These subfragments were designated L1, L2 and L3 or H1, H2 and H3, where “L” and “H” denote the light chain and heavy chain regions, respectively. These areas may be called CDR according to Chothia, the boundaries of which coincide with the CDR according to Kabat. Other boundaries defining the CDR, which coincide with the CDR according to Kabat, have been described by Padlan, 1995. FASEB J. 9: 133-39; MacCallum, 1996 J. Mol. Biol. 262(5): 732-45; and Lefranc, 2003, Dev. Comp. Immunol. 27: 55-77. Still other definitions of CDR boundaries may not strictly correspond to one of the systems given in this document, but will nevertheless coincide with CDR according to Kabat, although they may be shortened or lengthened based on predictive or experimental conclusions about whether that specific residues or groups of residues, or even all CDRs, do not significantly affect antigen binding. The methods used herein may use CDRs defined in accordance with any of these systems, although in certain embodiments CDRs defined in accordance with Kabat or Chothia are used. Identification of predicted CDRs using amino acid sequence is well known in the art, for example, Martin, A.C. "Protein sequence and structure analysis of antibody variable domains" on Antibody Engineering, Vol. 2. Kontermann R., Dübel S., eds. Springer-Verlag, Berlin, p. 33-51 (2010). The amino acid sequence of the heavy and/or light chain variable domain can also be examined to identify CDR sequences using other conventional methods, for example, by comparison with known amino acid sequences of other variable regions of the heavy and light chains, identifying areas of hypervariability in the sequence. Numbered sequences can be aligned manually or by using an alignment program such as one from the CLUSTAL software package, as described in Thompson, 1994. Nucleic Acids Res. 22: 4673-80. Molecular models are traditionally used to correctly identify framework and CDR regions and thus correct sequence-based alignments.

[0049] In some embodiments, the determination of CDR/FR in an immunoglobulin light or heavy chain should be based on the IMGT determination (Lefranc et al. Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt .org).

[0050] As used herein, the term "Fc" refers to a molecule, whether in monomeric or multimeric form, containing a sequence that is not part of an antigen binding fragment, which is obtained by cleavage of an antibody or obtained by other means, and may contain a hinge region . The original immunoglobulin source of the native Fc is preferably human and can be any of the immunoglobulins. Fc molecules are composed of monomeric polypeptides that can be linked into dimeric or multimeric forms through covalent (i.e..disulfide bonds) and non-covalent bonds. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules varies from 1 to 4 depending on the class (for example, IgG, IgA and IgE) or subclass (e.g. IgG1, IgG2, IgG3, IgA1, IgGA2 and IgG4). One example of an Fc is a disulfide-linked dimer resulting from the cleavage of IgG by papain. The term "native Fc" as used herein is intended to cover monomeric, dimeric and multimeric forms.

[0051] The F(ab) fragment typically contains one light chain and the _VH and _CH1 domains of one heavy chain, wherein the _VH - _CH1 portion of the heavy chain of the F(ab) fragment cannot form a disulfide bond with another heavy chain polypeptide . As used herein, an F(ab) fragment may also comprise one light chain comprising 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 C _H1 domain.

[0052] The F(ab') fragment typically contains one light chain and part of one heavy chain, which contains most of the constant region (between the C _H1 and C _H2 domains), whereby an interchain disulfide bond can be formed between the two heavy chains with the formation of the F(ab') ₂ molecule.

[0053] As used herein, the term “binding protein” refers to a non-naturally occurring (or recombinant or engineered) molecule that specifically binds to at least one target antigen, e.g. CD38 polypeptide of the present invention.

[0054] A "recombinant" molecule is a molecule that has been produced, expressed, generated, or isolated by recombinant means.

[0055] In one embodiment of the present invention, binding proteins are provided that have biological and immunological specificity for one to three target antigens. In another embodiment, the present invention provides nucleic acid molecules containing nucleotide sequences encoding polypeptide chains that form such binding proteins. In another embodiment, the present invention provides expression vectors containing nucleic acid molecules containing nucleotide sequences encoding polypeptide chains that form such binding proteins. In yet another embodiment, the present invention provides host cells that express such binding proteins (i.e. containing nucleic acid molecules or vectors encoding the polypeptide chains that form such binding proteins).

[0056] As used herein, the term "exchangeability" refers to the ability to interchange variable domains within a binding protein format while maintaining fold and highest binding affinity. "Full Exchange Capacity" refers to the ability to change the order of domains as V_H1, and V_H2 and therefore the order of domains V_L1 and V_L2 in a polypeptide chain of formula I or a polypeptide chain of formula II (i.e. reverse order) while maintaining full functionality of the binding protein, as evidenced by retention of binding affinity. In addition, it should be noted that the notation V_H and V_L refer only to the position of the domain in a particular protein chain in the final format. For example, V_H1 and V_H2 can be derived from V domains_L1 and V_L2 original antibodies and introduced into positions corresponding to V_H1 and V_H2 binding protein. Same as V_L1 and V_L2 can be derived from V domains_H1 and V_H2 original antibodies and placed in positions corresponding to V_H1 and V_H2 binding protein. Thus, the notation V_H and V_L refer to the currently existing position and not to the original position in the original antibody. Therefore, domains V_H and V_L are "capable of exchange".

[0057] As used herein, the term "antigen" or "target antigen" or "target antigen" means a molecule or part of a molecule that can be bound by a binding protein and can further be used in an animal to produce antibodies capable of binding to an epitope thereof. antigen. The target antigen may have one or more epitopes. Given that each target antigen is recognized by a binding protein, the binding protein is able to compete with an intact antibody that recognizes the target antigen.

[0058] "CD38" refers to cluster of differentiation polypeptide 38 and is a glycoprotein found on the surface of many immune cells. In some embodiments, the binding protein of the present invention binds the extracellular domain of one or more CD38 polypeptides. Exemplary CD38 polypeptide extracellular domain sequences include, but are not limited to, human CD38 extracellular domain (e.g. presented under SEQ ID NO:1) and the extracellular domain of cynomolgus monkey CD38 (e.g. presented under SEQ ID NO:30).

[0059] The term "T cell recruiter" refers to binding proteins directed to the host immune system, more specifically to the cytotoxic activity of T cells, and also directed to a tumor protein target.

[0060] The term “monospecific binding protein” refers to a binding protein that specifically binds to one target antigen.

[0061] The term "monovalent binding protein" refers to a binding protein that has a single antigen binding site.

[0062] The term "bispecific binding protein" refers to a binding protein that specifically binds to two different target antigens. In some embodiments, the bispecific binding protein binds to two different antigens. In some embodiments, the 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 target antigens. In some embodiments, the trispecific binding protein binds to three different antigens. In some embodiments, the 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 specific embodiments, the trivalent binding protein may bind to a single target antigen. In other embodiments, the trivalent binding protein can bind to two target antigens. In other embodiments, the trivalent binding protein can bind to three target antigens.

[0066] An "isolated" binding protein is a binding protein that has been identified and separated and/or recovered from a component of its natural environment. Contaminants from its natural environment are substances that would be problematic in diagnostic or therapeutic applications of the binding protein, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the binding protein will be purified: (1) to greater than 95% by weight of the antibody, as determined by the Lowry method, and most preferably to greater than 99% by weight, (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spin-beaker sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver dye. Isolated binding proteins include the binding protein in situ in recombinant cells, since at least one component of the natural environment of the binding protein will not be present.

[0067] As used herein, the terms “substantially pure” or “substantially purified” refer to a compound or structure that is the predominant structure present (i.e. per mole it is more abundant than any other single structure in the composition). In some embodiments, the substantially purified fraction is a composition wherein the structure represents at least about 50% (on a mole basis) of all macromolecular structures present. In other embodiments, the substantially pure composition will contain more than about 80%, 85%, 90%, 95%, or 99% of all macromolecular structures present in the composition. In still other embodiments, the structure is purified to the desired homogeneity (contaminant structures cannot be detected in the composition using traditional detection methods), where the composition consists of essentially a single macromolecular structure.

[0068] The term "epitope" means any determinant, preferably a polypeptide determinant, capable of specifically binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include reactive surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments may have specific three-dimensional structure 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 considered to specifically bind an antigen if it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. In some embodiments, a binding protein is considered to specifically bind an antigen if the equilibrium dissociation constant is ≤ 10 ^-8 M, more preferably if the equilibrium dissociation constant is ≤ 10 ^-9 M, and most preferably if the dissociation constant is ≤ 10 ^-10 M.

[0069] The dissociation constant (K _D ) of the binding protein can be determined, for example, using surface plasmon resonance. In general, surface plasmon resonance assays measure binding interactions between a ligand (target antigen on a biosensor array) and an analyte (binding protein in solution) in real time using surface plasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor; Piscataway, New -Jersey). Surface plasmon resonance analysis can also be performed by immobilizing an analyte (binding protein on a biosensor array) and presenting a ligand (target antigen). As used herein, the term “K _D ” refers to the dissociation constant of the interaction between a particular binding protein and a target antigen.

[0070] As used herein, the term “binds to” in reference to a binding protein means the ability of a binding protein or an antigen binding fragment thereof to bind an antigen containing an epitope with a Kd of at least about 1×10 ^-6 M, 1×10 ^-7 M, 1×10 ^-8 M, 1×10 ^-9 M, 1×10 ^-10 M, 1×10 ^-11 M, 1×10 ^-12 M or more, and/or bind to an epitope with affinity, which is at least two times higher than its affinity for a nonspecific antigen. In some embodiments, the binding protein of the present invention binds to two or more antigens, for example, human and cynomolgus CD38 polypeptide.

[0071] In some embodiments, the antigen binding domain and/or binding protein of the present invention "cross-reacts" with human and cynomolgus CD38 polypeptides, for example, CD38 extracellular domains such as SEQ ID NO:1 (human CD38 isoform A), SEQ ID NO:105 (human CD38 isoform E) and SEQ ID NO:30 (cynomolgus CD38). Binding protein binding to antigen 1 (Ag1) is “cross-reactive” to antigen 2 (Ag2) if the EC50 values are in a similar range for both antigens. As used herein, binding of a binding protein to Ag1 is cross-reactive to Ag2 if the ratio of affinity for Ag2 to affinity for Ag1 is 10 or less (e.g., 5, 2, 1, or 0.5), with affinities measured using one and the same method in the case of both antigens.

[0072] The binding of a binding protein to Ag1 is not “significantly cross-reactive” to Ag2 if the affinities for the two antigens are very different. Affinity for Ag2 may not be measurable if the binding reaction is too weak. In this application, the binding of a binding protein to Ag1 is not significantly cross-reactive with Ag2 if the binding reaction of the binding protein to Ag2 is less than 5% of the binding reaction of the same binding protein to Ag1 under the same experimental conditions and under the same experimental conditions. antibody concentrations. In practice, the concentration of binding protein used may be the EC50 or the concentration required to achieve the saturation plateau observed with Ag1.

[0073] As used herein, the term “linker” refers to one or more amino acid residues inserted between immunoglobulin domains to provide mobility sufficient for the light and heavy chain domains to fold into immunoglobulins with two variable regions crossover. The linker is inserted into the transition region between variable domains or between variable and constant domains, respectively, at the sequence level. The transition region between the domains can be identified because the approximate size of the immunoglobulin domains is well known. The exact location of the transition region between domains can be determined by determining the position of peptide stretches that do not form secondary structure elements such as beta sheets or alpha helices, as demonstrated by experimental data or as predicted by modeling or secondary structure prediction techniques . The linkers described herein are designated L ₁ , which is located on the light chain between the C-terminus of the V _L2 domain and the N-terminus of the V _L1 domain; and L ₂ , which is located on the light chain between the C-terminus of the V _L1 domain and the N-terminus of the C _L domain. The heavy chain linkers are referred to as L ₃ , which is located between the C-terminus of the V _H1 domain and the N-terminus of the V _H2 domain; and L ₄ , which is located between the C-terminus of the V _H2 domain and the N-terminus of the C _H1 domain.

[0074] As used herein, the term “vector” refers to any molecule (e.g. nucleic acid, plasmid, or virus) used to carry encoded information into a host cell. The term "vector" refers to 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 can be inserted. Another type of vector is a viral vector, whereby additional DNA segments can be inserted into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal vectors for mammals). Other vectors (for example, non-episomal mammalian vectors) can be integrated into the host cell genome after introduction into the host cell, and thereby replicate along with the host genome. In addition, certain vectors are capable of controlling the expression of genes to which they are functionally linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”). Typically, expression vectors useful in recombinant DNA technologies are often in the form of plasmids. The terms "plasmid" and "vector" may be used interchangeably herein since plasmid is the most widely used form of vector. However, other forms of expression vectors are intended to be included in the present invention, such as viral vectors (eg, retroviruses, adenoviruses and replication-defective adeno-associated viruses) that perform equivalent functions.

[0075] As used herein, the phrase “recombinant host cell” (or “host cell”) refers to a cell into which a recombinant expression vector has been introduced. Recombinant host cell or host cell is intended to refer not only to the particular cell in question, but also to the progeny of such cell. Because certain modifications may occur in subsequent generations due to mutation or environmental influences, such progeny may not actually 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 binding proteins, including bacterial, yeast, baculovirus, and mammalian cell-based expression systems (as well as phage display-based expression systems). An example of a suitable bacterial expression vector is pUC19. For recombinant expression of a binding protein, a host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding polypeptide chains of the binding protein, whereby the polypeptide chains are expressed in the host cell and preferably secreted into the medium in which the host cells are cultured, in this case, the binding protein can be extracted from this medium.

[0076] As used herein, the term "transformation" refers to a change in the genetic characteristics of a cell, and the cell has been transformed if it has been modified to contain new DNA. For example, a cell is transformed if it is genetically modified from its native state. After transformation, the transforming DNA may undergo recombination with the cell's DNA by physical integration into the cell's chromosome, or may be temporarily retained as an episomal element without replication, or may replicate independently as a plasmid. A cell is said to be stably transformed if the DNA is replicated when the cell divides. As used herein, the term "transfection" refers to the uptake of foreign or exogenous DNA into a cell, and the cell has been "transfected" if the exogenous DNA is introduced into 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] As used herein, the term "naturally occurring" and applied to an object refers to the fact that the object can be found in nature and has not been manipulated by humans. For example, a polynucleotide or polypeptide present in an organism (including viruses), which can be isolated from a natural source and which has not been intentionally modified by humans, is naturally occurring. Likewise, as used herein, the term “non-naturally occurring” refers to an entity that is not found in nature or that has been structurally modified or synthesized by humans.

[0078] As used herein, the twenty standard amino acids and their abbreviations are in accordance with generally accepted practice. Stereoisomers (for example, d-amino acids) twenty standard amino acids; non-naturally occurring amino acids and analogs such as α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid and other non-standard amino acids may also be suitable components of the polypeptide chains of binding proteins. Examples of non-standard amino acids include 4-hydroxyproline, γ-carboxyglutamate, ε-N, N,N-trimethyllysine, ε-N-acetyl-lysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ- N-methylarginine and other similar amino acids and imino acids (for example, 4-hydroxyproline). In the polypeptide naming system used herein, the left direction represents the direction towards the amino terminus and the right direction represents the direction towards the carboxyl terminus, in accordance with standard practice and rules.

[0079] Naturally occurring residues can be divided into classes based on general side chain properties:

(1) hydrophobic: Met, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Pro;

(2) polar hydrophilic: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr;

(3) aliphatic: Ala, Gly, Ile, Leu, Val, Pro;

(4) aliphatic hydrophobic: Ala, Ile, Leu, Val, Pro;

(5) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(6) acidic: Asp, Glu;

(7) basic: His, Lys, Arg;

(8) residues that affect chain orientation: Gly, Pro;

(9) aromatic: His, Trp, Tyr, Phe; And

(10) aromatic hydrophobic: Phe, Trp, Tyr.

[0080] Conservative amino acid substitutions may involve exchanging a member of one of these classes for another member of the same class. Non-conservative substitutions may involve exchanging a member of one of these classes for a member of another class.

[0081] One skilled in the art will be able to determine suitable variants of the polypeptide chains of binding proteins using generally known techniques. For example, one skilled in the art can identify suitable regions in a polypeptide chain that can be altered without interfering with activity by targeting regions that are not expected to be important in terms of activity. Alternatively, one of ordinary skill in the art can identify residues and portions of molecules that are conserved among similar polypeptides. In addition, even regions that may be biologically or structurally important can be subject to conservative amino acid substitutions without impairing biological activity or adversely affecting the structure of the polypeptide.

[0082] As used herein, the term "patient" includes humans and animals (eg, mammals such as dogs, pigs, horses, cats, cows, etc.).

[0083] As used herein, the terms “treating” or “treating” refer to both therapeutic treatment and prophylactic or preventive measures. Those in need of treatment include those who have the disorder, those who are susceptible to the disorder, or those in whom the disorder needs to be prevented. In specific embodiments, the binding proteins can be used to treat people with chronic viral infection or to alleviate chronic viral infection in a human subject.

[0084] As used herein, the terms “pharmaceutical composition” or “therapeutic composition” mean a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[0085] As used herein, the term “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” refers to one or more substances of the composition suitable for achieving or improving the delivery of a binding protein.

[0086] The terms "effective amount" and "therapeutically effective amount" when used in relation to a pharmaceutical composition containing one or more binding proteins mean an amount or dose sufficient to obtain the desired therapeutic result. More specifically, a therapeutically effective amount is an amount of binding protein sufficient to inhibit, for a period of time, one or more clinically defined pathological processes associated with the condition being treated. The effective amount may vary depending on the particular binding protein to be used and also depends on a number 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 , then factors such as the age, body weight and health status of the patient, as well as dose-response curves and toxicity data obtained from a preclinical animal study, will be included. factors taken into account. Determination of the effective amount or therapeutically effective amount of the specified pharmaceutical composition is within the competence of specialists in the art.

[0087] In one embodiment, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a binding protein.

Trispecific binding proteins

[0088] Some aspects of the present invention relate to trispecific binding proteins ( eg , those 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 the preparation of the trispecific binding protein of the present invention.

[0089] In some embodiments, the binding protein of the present invention is a trispecific binding protein comprising four polypeptide chains that form three antigen binding sites that bind one or more ( eg, three) different target antigens or target proteins. In some embodiments, the first polypeptide chain comprises a structure represented by the formula:

V _L2 -L ₁ -V _L1 -L ₂ -C _L [I];

and the second polypeptide chain contains a structure represented by the formula:

V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge-C _H2 -C _H3 [II];

and the third polypeptide chain contains the structure represented by the formula:

V _H3 -C _H1 -hinge-C _H2 -C _H3 [III];

and the fourth polypeptide chain contains the structure represented by the formula:

V _L3 -C _L [IV];

Where

V _L1 is the first variable domain of the immunoglobulin light chain;

V _L2 is the second variable domain of the immunoglobulin light chain;

V _L3 is the third variable domain of the immunoglobulin light chain;

V _H1 is the first variable domain of the immunoglobulin heavy chain;

V _H2 is the second variable domain of the immunoglobulin heavy chain;

V _H3 is the third variable domain of the immunoglobulin heavy chain;

C _L is an immunoglobulin light chain constant domain;

C _H1 is the immunoglobulin heavy chain C _H1 constant domain;

C _H2 is the immunoglobulin heavy chain C _H2 constant domain;

C _H3 is the immunoglobulin heavy chain C _H3 constant domain;

the hinge is the hinge region of an immunoglobulin connecting the C _H1 and C _H2 domains; And

L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers,

wherein the polypeptide of formula I and the polypeptide of formula II form a light_chain-heavy_chain crossover pair. In some embodiments, the first polypeptide chain and the second polypeptide chain have a crossover orientation such that they form two different antigen binding sites.

[0090] In some embodiments, V _H1 and V _L1 form a first antigen binding site that binds a CD28 polypeptide, wherein V _H2 and V _L2 form a second antigen binding site that binds a CD3 polypeptide, and where V _H3 and V _L3 form a third antigen binding region, which binds the CD38 polypeptide. In some embodiments, the CD28 polypeptide is a human CD28 polypeptide. In some embodiments, the CD3 polypeptide is a human CD3 polypeptide. In some embodiments, the CD38 polypeptide is a human CD38 polypeptide. In some embodiments, the trispecific binding protein comprises one or more antigen binding sites described below.

[0091] The binding proteins of the present invention can be prepared using domains or sequences derived from or derived from any human or non-human antibody, including, for example, human, mouse, or humanized antibodies. In some embodiments, the binding protein of the present invention 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 invention relate to binding proteins that contain an antigen binding region that binds a CD38 polypeptide (e.g. human and cynomolgus CD38 polypeptides).

[0093] In some embodiments, the binding protein or antigen binding fragment thereof cross-reacts with human CD38 (eg, human CD38 isoform A and/or isoform E polypeptide) and cynomolgus CD38. In some embodiments, the binding protein induces apoptosis of a CD38+ cell. In some embodiments, the binding protein recruits a T cell to a CD38+ cell and optionally activates the T cell (e.g. via TCR stimulation and/or costimulation).

[0094] In some embodiments, the binding region that binds CD38 comprises a heavy chain variable domain (VH) of an antibody comprising a CDR-H1 sequence comprising the amino acid sequence GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37) , a CDR-H2 sequence containing the amino acid sequence IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33); and/or a light chain variable domain (VL) of an antibody containing a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the binding region that binds CD38 comprises a heavy chain variable domain (VH) of an antibody comprising a CDR-H1 sequence comprising the amino acid sequence GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR sequence -H2 containing the amino acid sequence IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and the sequence CDR-H3 containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33); and/or a light chain variable domain (VL) of an antibody containing a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the binding proteins comprise 1, 2, 3, 4, 5, or 6 CDRs from the VH and/or VL domain sequence antiCD38_C2-CD38-1, antiCD38_C2-CD38-1_VH1-VL1, antiCD38_C2-CD38-1_VH3-VL3, antiCD38_C2 -CD38-1_VH5-VL3, antiCD38_C2-CD38-1_VH6-VL3, CD38 _HHY1370 (may also be called antiCD38_1370 in this document), antiCD38_C2-CD38-1_VH1-VL1xCD28supxCD3mid IgG4 FALA, antiCD38_C2-CD38-1_VH1-VL1 xCD28supxCD3mid IgG1LALA P329A, antiCD38_C2-CD38 -1_VH1-VL1xCD28supxCD3mid IgG1 NNSA, CD38 _HHY1370 xCD28supxCD3mid IgG4 FALA, CD38 _HHY1370 xCD28supxCD3mid IgG1LALA P329A or CD38 _HHY1370 xCD28supxCD3mid IgG1 NNSA, presented in Tables G, H, or I.

[0095] In some embodiments, the binding region that binds CD38 comprises a heavy chain variable domain (VH) of an antibody comprising a CDR-H1 sequence comprising the amino acid sequence GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37) , a CDR-H2 sequence containing the amino acid sequence IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33); and an antibody light chain variable domain (VL) comprising a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO :35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36).

[0096] In some embodiments, the binding region that binds CD38 comprises a heavy chain variable domain (VH) of an antibody comprising a CDR-H1 sequence comprising the amino acid sequence GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33); and/or a light chain variable domain (VL) of an antibody comprising a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35), and a CDR sequence -L3, containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the binding region that binds CD38 comprises a heavy chain variable domain (VH) of an antibody comprising a CDR-H1 sequence comprising the amino acid sequence GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33); and an antibody light chain variable domain (VL) comprising a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35), and a CDR-L3 sequence , containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In other embodiments, the binding region that binds CD38 comprises a heavy chain variable domain (VH) of an antibody comprising a CDR-H1 sequence comprising the amino acid sequence GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33); and/or a light chain variable domain (VL) of an antibody comprising a CDR-L1 sequence containing the amino acid sequence QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence GAS (SEQ ID NO:40), and a CDR sequence -L3, containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the binding region that binds CD38 comprises a heavy chain variable domain (VH) of an antibody comprising a CDR-H1 sequence comprising the amino acid sequence GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33); and an antibody light chain variable domain (VL) comprising a CDR-L1 sequence containing the amino acid sequence QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence GAS (SEQ ID NO:40), and a CDR-L3 sequence , containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36).

[0097] In some embodiments, the VH domain comprises, in an N-terminal to C-terminal direction, the sequence FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; where FR1 contains the sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO:86), QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO:87) or QVQLVQSGAEVVKPGASVKMSCKAS (SEQ ID NO:88); where FR2 contains the sequence MHWVKEAPGQRLEWIGY (SEQ ID NO:90) or MHWVKEAPGQGLEWIGY (SEQ ID NO:91); where FR3 contains the sequence NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO:93) or NYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO:94); and where FR4 contains the sequence WGQGTLVTVSS (SEQ ID NO:96). In some embodiments, the VL domain comprises, in an N-terminal to C-terminal direction, the sequence FR1-CDR-L1-FR2-CDR-L2-FR3-CDR-L3-FR4; where FR1 contains the sequence DIVLTQSPATLSLSPGERATISCRAS (SEQ ID NO:97); where FR2 contains the sequence MHWYQQKPGQPPRLLIY (SEQ ID NO:99); where FR3 contains the sequence SRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYC (SEQ ID NO:101); and where FR4 contains the sequence FGGGTKLEIK (SEQ ID NO:103).

[0098] 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%, according to at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:5; and/or the VL domain contains 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 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 contains 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 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 contains 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 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 contains 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 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:13; and/or the VL domain contains 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.

[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%, according to at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:5; and the VL domain contains 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 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 contains 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 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 contains 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 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 contains 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 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:13; and the VL domain contains 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 contains the amino acid sequence of SEQ ID NO:5, and the VL domain contains the amino acid sequence of SEQ ID NO:6. In some embodiments, the VH domain contains the amino acid sequence of SEQ ID NO:17, and the VL domain contains the amino acid sequence of SEQ ID NO:18. In some embodiments, the VH domain contains the amino acid sequence of SEQ ID NO:21, and the VL domain contains the amino acid sequence of SEQ ID NO:18. In some embodiments, the VH domain contains the amino acid sequence of SEQ ID NO:23, and the VL domain contains the amino acid sequence of SEQ ID NO:18. In some embodiments, the VH domain contains the amino acid sequence of SEQ ID NO:13, and the VL domain contains the amino acid sequence of SEQ ID NO:14.

[0101] In some embodiments, the binding region 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, the binding protein of the present invention 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, the binding protein of the present invention 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, the binding protein of the present invention 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, the binding protein of the present invention 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, the binding protein of the present invention 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, the binding protein of the present invention 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, the binding protein of the present invention 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, the binding protein of the present invention 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, the binding protein of the present invention 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, the binding region that binds CD38 comprises a heavy chain variable domain (VH) of an antibody comprising a CDR-H1 sequence comprising the amino acid sequence GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence containing the amino acid sequence ARMFRGAFDY (SEQ ID NO:43); and/or a light chain variable domain (VL) of an antibody comprising a CDR-L1 sequence containing the amino acid sequence QGIRND (SEQ ID NO:44), a CDR-L2 sequence containing the amino acid sequence AAS (SEQ ID NO:45), and a CDR sequence -L3, containing the amino acid sequence LQDYIYYPT (SEQ ID NO:46). In some embodiments, the binding region that binds CD38 comprises a heavy chain variable domain (VH) of an antibody comprising a CDR-H1 sequence comprising the amino acid sequence GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence containing the amino acid sequence ARMFRGAFDY (SEQ ID NO:43); and an antibody light chain variable domain (VL) comprising a CDR-L1 sequence containing the amino acid sequence QGIRND (SEQ ID NO:44), a CDR-L2 sequence containing the amino acid sequence AAS (SEQ ID NO:45), and a CDR-L3 sequence , containing the amino acid sequence LQDYIYYPT (SEQ ID NO:46).

[0104] In some embodiments, the VH domain comprises, in an N-terminal to C-terminal direction, the sequence FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; where FR1 contains the sequence QVQLVESGGGVVQPGRSLRLSCAAS (SEQ ID NO:89); where FR2 contains the sequence MHWVRQAPGKGLEWVAV (SEQ ID NO:92); where FR3 contains the sequence YYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO:95); and where FR4 contains the sequence WGQGTLVTVSS (SEQ ID NO:96). In some embodiments, the VL domain comprises, in an N-terminal to C-terminal direction, the sequence FR1-CDR-L1-FR2-CDR-L2-FR3-CDR-L3-FR4; where FR1 contains the sequence AIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO:98); where FR2 contains the sequence GWYQQKPGKAPKLLIY (SEQ ID NO:100); where FR3 contains the sequence SLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYC (SEQ ID NO:102); and where FR4 contains the sequence WGQGTLVTVSS (SEQ ID NO:104).

[0105] 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%, according to at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:9; and/or the VL domain contains 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:10.

[0106] 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%, according to at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:9; and the VL domain contains 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:10. In some embodiments, the VH domain contains the amino acid sequence of SEQ ID NO:9, and the VL domain contains the amino acid sequence of SEQ ID NO:10.

[0107] In some embodiments, the binding protein of the present invention comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:11, or an antibody light chain comprising the amino acid sequence of SEQ ID NO:12. In some embodiments, the binding protein of the present invention comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:11 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:12.

Anti-CD28 binding sites

[0108] Some aspects of the present invention relate to binding proteins that contain an antigen binding region that binds a CD28 polypeptide ( eg , human CD28 polypeptide).

[0109] In some embodiments, the binding region that binds CD28 comprises a heavy chain variable domain (VH) of an antibody comprising a CDR-H1 sequence comprising the amino acid sequence GYTFTSYY (SEQ ID NO:108), a CDR-H2 sequence comprising the amino acid sequence IYPGNVNT (SEQ ID NO:109), and a CDR-H3 sequence containing the amino acid sequence TRSHYGLDWNFDV (SEQ ID NO:110), and/or an antibody light chain variable domain (VL) containing the CDR-L1 sequence containing the amino acid sequence QNIYVW ( SEQ ID NO:111), a CDR-L2 sequence containing the amino acid sequence KAS (SEQ ID NO:112), and a CDR-L3 sequence containing the amino acid sequence QQGQTYPY (SEQ ID NO:113). In some embodiments, the binding region that binds CD28 comprises a heavy chain variable domain (VH) of an antibody comprising a CDR-H1 sequence comprising the amino acid sequence GYTFTSYY (SEQ ID NO:108), a CDR-H2 sequence comprising the amino acid sequence IYPGNVNT (SEQ ID NO:109), and a CDR-H3 sequence containing the amino acid sequence TRSHYGLDWNFDV (SEQ ID NO:110), and an antibody light chain variable domain (VL) containing the CDR-L1 sequence containing the amino acid sequence QNIYVW (SEQ ID NO:111 ), a CDR-L2 sequence containing the amino acid sequence KAS (SEQ ID NO:112), and a CDR-L3 sequence containing the amino acid sequence QQGQTYPY (SEQ ID NO:113).

[0110] In some embodiments, the binding region that binds CD28 comprises a heavy chain variable domain (VH) of an antibody comprising a CDR-H1 sequence comprising the amino acid sequence GFSLSDYG (SEQ ID NO:114), a CDR-H2 sequence comprising the amino acid sequence IWAGGGT (SEQ ID NO:115), and a CDR-H3 sequence containing the amino acid sequence ARDKGYSYYYSMDY (SEQ ID NO:116), and/or an antibody light chain variable domain (VL) containing the CDR-L1 sequence containing the amino acid sequence ESVEYYVTSL ( SEQ ID NO:117), a CDR-L2 sequence containing the amino acid sequence AAS (SEQ ID NO:118), and a CDR-L3 sequence containing the amino acid sequence QQSRKVPYT (SEQ ID NO:119). In some embodiments, the binding region that binds CD28 comprises a heavy chain variable domain (VH) of an antibody comprising a CDR-H1 sequence comprising the amino acid sequence GFSLSDYG (SEQ ID NO:114), a CDR-H2 sequence comprising the amino acid sequence IWAGGGT (SEQ ID NO:115), and a CDR-H3 sequence containing the amino acid sequence ARDKGYSYYYSMDY (SEQ ID NO:116), and an antibody light chain variable domain (VL) containing the CDR-L1 sequence containing the amino acid sequence ESVEYYVTSL (SEQ ID NO:117 ), a CDR-L2 sequence containing the amino acid sequence AAS (SEQ ID NO:118), and a CDR-L3 sequence containing the amino acid sequence QQSRKVPYT (SEQ ID NO:119).

Anti-CD3 binding sites

[0111] Some aspects of the present invention relate to binding proteins that contain an antigen binding region that binds a CD3 polypeptide ( eg , human CD3 polypeptide).

[0112] In some embodiments, the binding region that binds CD3 comprises a heavy chain variable domain (VH) of an antibody comprising a CDR-H1 sequence comprising the amino acid sequence GFTFTKAW (SEQ ID NO:120), a CDR-H2 sequence comprising the amino acid sequence IKDKSNSYAT (SEQ ID NO:121), and a CDR-H3 sequence containing the amino acid sequence RGVYYALSPFDY (SEQ ID NO:122), and/or an antibody light chain variable domain (VL) containing the CDR-L1 sequence containing the amino acid sequence QSLVHNNANTY ( SEQ ID NO:123), a CDR-L2 sequence containing the amino acid sequence KVS (SEQ ID NO:124), and a CDR-L3 sequence containing the amino acid sequence GQGTQYPFT (SEQ ID NO:125). In some embodiments, the binding region that binds CD3 comprises a heavy chain variable domain (VH) of an antibody comprising a CDR-H1 sequence comprising the amino acid sequence GFTFTKAW (SEQ ID NO:120), a CDR-H2 sequence comprising the amino acid sequence IKDKSNSYAT (SEQ ID NO:121), and a CDR-H3 sequence containing the amino acid sequence RGVYYALSPFDY (SEQ ID NO:122), and an antibody light chain variable domain (VL) containing the CDR-L1 sequence containing the amino acid sequence QSLVHNNANTY (SEQ ID NO:123 ), a CDR-L2 sequence containing the amino acid sequence KVS (SEQ ID NO:124), and a CDR-L3 sequence containing the amino acid sequence GQGTQYPFT (SEQ ID NO:125).

[0113] In some embodiments, the binding region that binds CD3 comprises a heavy chain variable domain (VH) of an antibody comprising a CDR-H1 sequence comprising the amino acid sequence GFTFTKAW (SEQ ID NO:126), a CDR-H2 sequence comprising the amino acid sequence IKDKSNSYAT (SEQ ID NO:127), and a CDR-H3 sequence containing the amino acid sequence GVYYALSPFDY (SEQ ID NO:128), and/or an antibody light chain variable domain (VL) containing the CDR-L1 sequence containing the amino acid sequence QSLVHNNGNTY ( SEQ ID NO:129), a CDR-L2 sequence containing the amino acid sequence KVS (SEQ ID NO:130), and a CDR-L3 sequence containing the amino acid sequence GQGTQYPFT (SEQ ID NO:131).

[0114] In some embodiments of any of the trispecific binding proteins of the present invention, one antigen binding domain binds a CD3 polypeptide (e.g. human CD3), and one antigen binding domain binds the CD28 polypeptide (e.g. human CD28). In some embodiments, domain V_H1 contains three CDRs from SEQ ID NO:49 or 51, as shown in Table H, and domain V_L1 contains three CDRs from SEQ ID NO:50 or 52, as shown in Table H. In some embodiments, domain V_H2 contains three CDRs from SEQ ID NO:49 or 51, as shown in Table H, and domain V_L2 contains three CDRs from SEQ ID NO:50 or 52, as shown in Table H. In some embodiments, domain V_H1 contains three CDRs from SEQ ID NO:53 or 84, as shown in Table H, and domain V_L1 contains three CDRs from SEQ ID NO:54 or 85, as shown in Table H. In some embodiments, domain V_H2 contains three CDRs from SEQ ID NO:53 or 84, as shown in Table H, and domain V_L2 contains three CDRs from SEQ ID NO:54 or 85 presented in Table H.

[0115] In some embodiments, the V _H1 domain contains the amino acid sequence of SEQ ID NO:49, the V _L1 domain contains the amino acid sequence of SEQ ID NO:50, the V _H2 domain contains the amino acid sequence of SEQ ID NO:53, and the V _L2 domain contains the amino acid sequence of SEQ ID NO:54. In some embodiments, the V _H2 domain contains the amino acid sequence of SEQ ID NO:49, the V _L2 domain contains the amino acid sequence of SEQ ID NO:50, the V _H1 domain contains the amino acid sequence of SEQ ID NO:53, and the V _L1 domain contains the amino acid sequence under SEQ ID NO:54. In some embodiments, the V _H1 domain contains the amino acid sequence of SEQ ID NO:51, the V _L1 domain contains the amino acid sequence of SEQ ID NO:52, the V _H2 domain contains the amino acid sequence of SEQ ID NO:53, and the V _L2 domain contains the amino acid sequence under SEQ ID NO:54. In some embodiments, the V _H2 domain contains the amino acid sequence of SEQ ID NO:51, the V _L2 domain contains the amino acid sequence of SEQ ID NO:52, the V _H1 domain contains the amino acid sequence of SEQ ID NO:53, and the V _L1 domain contains the amino acid sequence under SEQ ID NO:54.

[0116] In some embodiments, the V _H1 domain contains the amino acid sequence of SEQ ID NO:49, the V _L1 domain contains the amino acid sequence of SEQ ID NO:50, the V _H2 domain contains the amino acid sequence of SEQ ID NO:53, the V _L2 domain contains the amino acid sequence of SEQ ID NO:54, the V _H3 domain contains the amino acid sequence of SEQ ID NO:13, and the V _L3 domain contains the amino acid sequence of SEQ ID NO:14. In some embodiments, the V _H1 domain contains the amino acid sequence of SEQ ID NO:49, the V _L1 domain contains the amino acid sequence of SEQ ID NO:50, the V _H2 domain contains the amino acid sequence of SEQ ID NO:53, the V _L2 domain contains the amino acid sequence of SEQ ID NO:54, the V _H3 domain contains the amino acid sequence of SEQ ID NO:9, and the V _L3 domain contains the amino acid sequence of SEQ ID NO:10.

[0117] In some embodiments, the first polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:60, the third polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:62, and the fourth polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:63. In some embodiments, the first polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 64, the third polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 65, and the fourth polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: :63. In certain embodiments, the first polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 66, the third polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 67, and the fourth polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: :63. In certain embodiments, the first polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 60, the third polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:68, and the fourth polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:69 . In certain embodiments, the first polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 64, the third polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:70, and the fourth polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:69 . In certain embodiments, the first polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 66, the third polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:71, and the fourth polypeptide chain contains a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:69 .

[0118] In certain embodiments, the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:60, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:62, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO:63. In certain embodiments, the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:64, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:65, and the fourth polypeptide chain contains the amino acid sequence under SEQ ID NO:63. In certain embodiments, the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:66, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:67, and the fourth polypeptide chain contains the amino acid sequence under SEQ ID NO:63. In certain embodiments, the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:60, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:68, and the fourth polypeptide chain contains the amino acid sequence under SEQ ID NO:69. In certain embodiments, the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:64, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:70, and the fourth polypeptide chain contains the amino acid sequence under SEQ ID NO:69. In certain embodiments, the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:66, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:71, and the fourth polypeptide chain contains the amino acid sequence under SEQ ID NO:69.

[0119] In some embodiments, the binding protein of the present invention contains 1, 2, 3, 4, 5, or 6 CDR sequences from the antibody sequence shown in Table G. In some embodiments, the binding protein of the present invention contains 1, 2, 3 , 4, 5, or 6 CDR sequences, a VH domain sequence, and/or a VL domain sequence from the antibody sequence shown in Table H. In some embodiments, the binding protein of the present invention contains 1, 2, 3, 4, 5, or 6 CDR sequences , a VH domain sequence and/or a VL domain sequence from the antibody sequence shown in Table I. In some embodiments, the binding protein of the present invention contains 1, 2, 3 or 4 polypeptide sequences shown in Table I.

Table G. CDR sequences of CD38 binding proteins

Ab CDR_H1 CDR_H2 CDR_H3 CDR_L1 CDR_L2 CDR_L3 antiCD38_C2-CD38-1 GYTFTSFN
(SEQ ID NO:31) IYPGNGGT
(SEQ ID NO:32) ARTGGLRRAYFTY
(SEQ ID NO:33) ESVDSYGNGF
(SEQ ID NO:34) LAS
(SEQ ID NO:35) QQNKEDPWT
(SEQ ID NO:36) antiCD38_C2-CD38-1_VH1-VL1 GYTFTSYA
(SEQ ID NO:37) IYPGQGGT
(SEQ ID NO:38) ARTGGLRRAYFTY
(SEQ ID NO:33) QSVSSYGQGF
(SEQ ID NO:39) G.A.S.
(SEQ ID NO:40) QQNKEDPWT
(SEQ ID NO:36) antiCD38_C2-CD38-1_VH3-VL3 GYTFTSFN
(SEQ ID NO:31) IYPGNGGT
(SEQ ID NO:32) ARTGGLRRAYFTY
(SEQ ID NO:33) ESVDSYGNGF
(SEQ ID NO:34) LAS
(SEQ ID NO:35) QQNKEDPWT
(SEQ ID NO:36) antiCD38_C2-CD38-1_VH5-VL3 GYTFTSFN
(SEQ ID NO:31) IYPGNGGT
(SEQ ID NO:32) ARTGGLRRAYFTY
(SEQ ID NO:33) ESVDSYGNGF
(SEQ ID NO:34) LAS
(SEQ ID NO:35) QQNKEDPWT
(SEQ ID NO:36) antiCD38_C2-CD38-1_VH6-VL3 GYTFTSFN
(SEQ ID NO:31) IYPGNGGT
(SEQ ID NO:32) ARTGGLRRAYFTY
(SEQ ID NO:33) ESVDSYGNGF
(SEQ ID NO:34) LAS
(SEQ ID NO:35) QQNKEDPWT
(SEQ ID NO:36) CD38 _HHY1370 GFTFSSYG
(SEQ ID NO:41) IWYDGSNK
(SEQ ID NO:42) ARMFRGAFDY
(SEQ ID NO:43) QGIRND
(SEQ ID NO:44) A.A.S.
(SEQ ID NO:45) LQDYIYYPT (SEQ ID NO:46)

[0146]

Table H. Sequences of variable domains of CD38 binding proteins and other binding proteins

Ab VH (protein) VL (protein) antiCD38_C2-CD38-1 QVQLQQSGAELVRSGASVKMSCKASGYTFTSFNMHWVKETPGQGLEWIGYIYPGNGGTNYNQKFKGKATLTADTSSSTAYMQISSLTSEDSAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO:5) DIVLTQSPASLAVSLGQRATISCRASESVDSYGNGFMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:6 antiCD38_C2-CD38-1_VH1-VL1 QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO:13) DIVLTQSPATLSLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRAT
GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:14) antiCD38_C2-CD38-1_VH3-VL3 QVQLVQSGAEVVKPGASVKVSCKASGYTFTSFNMHWVKEAPGQRLEWIGYIYPGNGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO:17) DIVLTQSPATLSLSPGERATISCRASESVDSYGNGFMHWYQQKPGQPPRLLIYLASSRAT
GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:18) antiCD38_C2-CD38-1_VH5-VL3 QVQLVQSGAEVVKSGASVKVSCKASGYTFTSFNMHWVKEAPGQGLEWIGYIYPGNGGTNYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO:21) DIVLTQSPATLSLSPGERATISCRASESVDSYGNGFMHWYQQKPGQPPRLLIYLASSRAT
GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:18) antiCD38_C2-CD38-1_VH6-VL3 QVQLVQSGAEVVKPGASVKMSCKASGYTFTSFNMHWVKEAPGQRLEWIGYIYPGNGGTNYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO:23) DIVLTQSPATLSLSPGERATISCRASESVDSYGNGFMHWYQQKPGQPPRLLIYLASSRAT
GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:18) CD38 _HHY1370 QVQLVESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSS (SEQ ID NO:9) AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIK (SEQ ID NO:10) antiCD38_SB19 Qvqlvqsgaevakpgtsvklsckasgytftdywmqwvkqrpgqglewigtiypgdgdtgyaqkfqgkatltadkssktvymhlsslasedsavyycargdyygsnsldywgqgtsvtvss (SEq ID NO:47) DIVMTQSHLSMSTSLGDPVSITCKASQDVSTVVAWYQQKPGQSPRRLIYSASYRYIGVPDRFTGSGAGTDFTFTISSVQAEDLAVYYCQQHYSPPYTFGGGTKLEIK (SEQ ID NO:48) Anti-CD28 _sup qvqlvqsgaevvkpgasvkvsckasgytftsyyihwvrqapgqglewigsiypgnvntnyaqkfqgratltvdtsistaymelsrlrsddtavyyctrshygldwnfdvwgkgttvtvss (SEQ ID NO:49) diqmtqspsslsasvgdrvtitcqasqniyvwlnwyqqkpgkapklliykasnlhtgvpsrfsgsgsgtdftltisslqpediatyycqqgqtypytfgqgtkleik (SEq ID NO:50) Anti-CD28 _cvn qvqlqesgpglvkpsqtlsltctvsgfslsdygvhwvrqppgkglewlgviwagggtnynpslksrktiskdtsknqvslklssvtaadtavyycardkgysyyysmdywgqgttvtvss (SEQ ID NO:51) divltqspaslavspgqratitcrasesveyyvtslmqwyqqkpgqppkllifaasnvesgvparfsgsgsgtdftltinpveandvanyycqqsrkvpytfgqgtkleik (SEQ ID NO:52) Anti-CD3 _mid qvqlvesgggvvqpgrslrlscaasgftftkawmhwvrqapgkqlewvaqikdksnsyatyyadsvkgrftisrddskntlylqmnslraedtavyycrgvyyalspfdywgqgtlvtvss (SEQ ID NO:53) divmtqtplslsvtpgqpasisckssqslvhnnantylswylqkpgqspqsliykvsnrfsgvpdrfsgsgsgtdftlkisrveaedvgvyycgqgtqypftfgsgtkveik (SEQ ID NO:54) Anti-CD3 _low qvqlvesgggvvqpgrslrlscaasgftftkawmhwvrqapgkglewvaqikdksnsyatyyadsvkgrftisrdnskntlylqmnslraedtavyycrgvyyalspfdywgqgtlvtvss (SEQ ID NO:84) divmtqtplslsvtpgqpasisckssqslvhnngntylswylqkpgqspqlliykvsnrfsgvpdrfsgsgsgtdftlkisrveaedvgvyycgqgtqypftfgggtkveik (SEq ID NO:85)

Note: In the amino acid sequences above, the CDR sequences are in bold and underlined.

Table I. Full-length binding protein sequences

antiCD38_C2-CD38-1_VH1-VL1xCD28supxCD3mid IgG4 FALA heavy chain 1 FALA CD28supxCD3mid IgG4(hollow)
(for example, the second polypeptide chain of the trispecific binding protein of the present invention) Qvqlvqsgaevvkpgasvkvsckasgytftsyyihwvrqapgqglewigsiypgnvntnyaqkfqgratltvdtsistaymelsrlrsddtavyyctrshygldwnfdvwgkgttvtvsssqvqlvesgggvvqpgrslrlscaasgftftkawmhwvrqapgkqlewvaqikd ksnsyatyyadsvkgrftisrddskntlylqmnslraedtavyycrgvyyalspfdywgqgtlvtvssrtastkgpsvfplapcsrs tsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtktytcnvdhkpsntkvdkrveskygpp cppcpapeaaggpsvflfppkpkdtlmisrtpevtcvvvdvsqedpevqfnwyvdgvevhnaktkpreeqfnstyrvvsvltvlhqdwlngkeykckvsnkglpssiektiskakgqprepqvctlppsqeemtknqvslscavkgfypsdiavewesngqpennykttppvldsdgs fflvskltvdksrwqegnvfscsvmhealhnhytqkslslslg SEQ ID NO: 60 light chain 1 CD28supxCD3mid
(for example, the first polypeptide chain of the trispecific binding protein of the present invention) DivmtqtplsvtpgqpasisssssslvnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnylqskspsliykvsnrfsgsgsgsgsgtlkisriveycgqgtqgtktkveikgqpkapdiqmtqsmtqstqstqstqstqstqstqstqstqstqstqstqstqstqstqstqstqstqstqstqstqmatqstqstqmatqstqmatqstqmatqStqmatqStQ n SSLSASSVGDRVTITCQAQSQSNIVLNWYVYQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTSSLQPEATYCQTYPGQGTKSRAPSVFIFIPSDEQLKSGTA SVVCLLNNNFYPRAKVKVDNALQSGNSXVTEQDSKDSTYSSTLTLSKACHKVYACEVTHSSPVTKSFNRGEC SEQ ID NO: 61 heavy chain 2 FALA antiCD38_C2-CD38-1_VH1-VL1 IgG4(overhang)
(for example, the third polypeptide chain of the trispecific binding protein of the present invention) Qvqlvqsgaevvkpgasvkvsckasgytftsyamhwvkeapgqrlewigyiypgqggtnynqkfqgratltadtsastaymelsslrsedtavyfcartgglrrayftywgqgtlvtvssastkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlq ssglyslssvvtvpssslgtktytcnvdhkpsntkvdkrveskygppcppcpapeaaggpsvflfppkpkdtlmisrtpevtcvvvdvsqedpevqfnwyvdgvevhnaktkpreeqfnstyrvvsvltvlhqdwlngkeykckvsnkglpssiektiskakgqprepqvy tlppcqeemtknqvslwclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqegnvfscsvmhealhnhytqkslslslg SEQ ID NO: 62 light chain 2 antiCD38_C2-CD38-1_VH1-VL1
(for example, the fourth polypeptide chain of the trispecific binding protein of the present invention) Divltqspatlslspgeratiscrasqsvssygqgfmhwyqqkpgqpprlliygassratgiparfsgsgsgtdftltisplepedfavyycqqnkedpwtfgggtkleikrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstys lsstltlskadyekhkvyacevthqglsspvtksfnrgec SEQ ID NO: 63 antiCD38_C2-CD38-1_VH1-VL1xCD28supxCD3mid IgG1LALA P329A heavy chain 1 LALA P329A CD28supxCD3mid IgG1(hollow)
(for example, the second polypeptide chain of the trispecific binding protein of the present invention) QVQLVQSGAEVKPGASVKVSKKAGYTSYIHWVRQAPGSIYPGSIYPGNVNYAQKKFKGRATIMELSRSRSRSRSDTAVYGLDVNFDVNFDVSSSASSSQVQVQVQVQVQVQVQVQVQVAKLVESG GGVVQpgrsLrlscaasgftkawvrqpgqpgqpgkkikkiksnsnsyatSvkrftiDSRDDLYLALRADADVYADADYADYALSPFDYALSPFDVTVTVTVTVTASTASTASTASTASTASTASTASTASTASTASTASTASTASTASTASTASTASTASTASTASTKGPSVFPPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGVHTFPAVHTFPAVTFPAVSSLSSVTVPSSLGTYICNHKPSNTKVDKVDKSCDKPPSVFLFPKPKDTLMISRT PEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKPREQYNSTYNSTYVLTVLHQDWLNGKEKKVSNKAPIEKGQPSRDELSCAVSCAVSCAVKGFYPSDIAVEVWESNGS Qpennykttppvldsdgskltvdksrwqqgnvfssvmhealhnhytqslslspg SEQ ID NO: 64 light chain 1 CD28supxCD3mid
(for example, the first polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO: 61 heavy chain 2 LALA P329A antiCD38_C2-CD38-1_VH1-VL1 IgG1(overhang)
(for example, the third polypeptide chain of the trispecific binding protein of the present invention) Qvqlvqsgaevvkpgasvkvsckasgytftsyamhwvkeapgqrlewigyiypgqggtnynqkfqgratltadtsastaymelsslrsedtavyfcartgglrrayftywgqgtlvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqss glyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapeaaggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvvsvltvlhqdwlngkeykckvsnkalaapiektiskakgqprepqvytl ppcrdeltknqvslwclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg SEQ ID NO: 65 light chain 2 antiCD38_C2-CD38-1_VH1-VL1
(for example, the fourth polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO: 63 antiCD38_C2-CD38-1_VH1-VL1xCD28supxCD3mid IgG1 NNSA heavy chain 1 NNSA CD28supxCD3mid IgG1(hollow)
(for example, the second polypeptide chain of the trispecific binding protein of the present invention) Qvqlvqsgaevvkpgasvkvsckasgytftsyyihwvrqapgqglewigsiypgnvntnyaqkfqgratltvdtsistaymelsrlrsddtavyyctrshygldwnfdvwgkgttvtvsssqvqlvesgggvvqpgrslrlscaasgftftkawmhwvrqapgkqlewvaqik dksnsyatyyadsvkgrftisrddskntlylqmnslraedtavyycrgvyyalspfdywgqgtlvtvssrtastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepks cdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynnasrvvsvltvlhq dwlngkeykckvsnkalpapiektiskakgqprepqvctlppsrdeltknqvslscavkgfypsdiavewesngqpennykttppvldsdgs fflvskltvdksrwqqgnvfscsvmhealhnhytqkslslspg SEQ ID NO: 66 light chain 1 CD28supxCD3mid
(for example, the first polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO: 61 heavy chain 2 NNSA antiCD38_C2-CD38-1_VH1-VL1 IgG1(overhang)
(for example, the third polypeptide chain of the trispecific binding protein of the present invention) qvqlvqsgaevvkpgasvkvsckasgytftsyamhwvkeapgqrlewigyiypgqggtnynqkfqgratltadtsastaymelsslrsedtavyfcartgglrrayftywgqgtlvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqs sglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcv vvdvshedpevkfnwyvdgvevhnaktkpreeqynnasrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvy tlppcrdeltknqvslwclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg SEQ ID NO: 67 light chain 2 CD38VH1
(for example, the fourth polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO: 63 CD38 _HHY1370 xCD28supxCD3mid IgG4 FALA heavy chain 1 FALA CD28supxCD3mid IgG4(hollow)
(for example, the second polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO: 60 light chain 1 CD28supxCD3mid
(for example, the first polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO: 61 heavy chain 2 FALA CD38 _HHY1370 IgG4(overhang)
(for example, the third polypeptide chain of the trispecific binding protein of the present invention) Qvqlvesgggvvqpgrslrlscaasgftfssygmhwvrqapgkglewvaviwydgsnkyyadsvkgrftisgdnskntlylqmnslraedtavyycarmfrgafdywgqgtlvtvssastkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqs sglyslssvvtvpssslgtktytcnvdhkpsntkvdkrveskygppcppcpapeaaggpsvflfppkpkdtlmisrtpevtcvvvdvsqedpevqfnwyvdgvevhnaktkpreeqfnstyrvvsvltvlhqdwlngkeykckvsnkglpssiektiskakgqprepqvy tlppcqeemtknqvslwclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqegnvfscsvmhealhnhytqkslslslg SEQ ID NO: 68 light chain 2 CD38 _HHY1370
(for example, the fourth polypeptide chain of the trispecific binding protein of the present invention) aiqmtqspsslsasvgdrvtitcrasqgirndlgwyqqkpgkapklliyaasslqsgvpsrfsgsgsgtdftltisglqpedsatyyclqdyiyyptfgqgtkveikrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskd styslsstltlskadyekhkvyacevthqglsspvtksfnrgec SEQ ID NO: 69 CD38 _HHY1370 xCD28supxCD3mid IgG1LALA P329A heavy chain 1 LALA P329A CD28supxCD3mid IgG1(hollow)
(for example, the second polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO: 64 light chain 1 CD28supxCD3mid
(for example, the first polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO: 61 heavy chain 2 LALA P329A CD38 _HHY1370 IgG1(overhang)
(for example, the third polypeptide chain of the trispecific binding protein of the present invention) qvqlvesgggvvqpgrslrlscaasgftfssygmhwvrqapgkglewvaviwydgsnkyyadsvkgrftisgdnskntlylqmnslraedtavyycarmfrgafdywgqgtlvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlq ssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapeaaggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalaapiektiskakgqprep qvytlppcrdeltknqvslwclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg SEQ ID NO: 70 light chain 2 CD38 _HHY1370
(for example, the fourth polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO: 69 CD38 _HHY1370 xCD28supxCD3mid IgG1 NNSA heavy chain 1 NNSA CD28supxCD3mid IgG1(hollow)
(for example, the second polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO: 66 light chain 1 CD28supxCD3mid
(for example, the first polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO: 61 heavy chain 2 NNSA CD38 _HHY1370 IgG1(overhang)
(for example, the third polypeptide chain of the trispecific binding protein of the present invention) Qvqlvesgggvvqpgrslrlscaasgftfssygmhwvrqapgkglewvaviwydgsnkyyadsvkgrftisgdnskntlylqmnslraedtavyycarmfrgafdywgqgtlvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqs sglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynnasrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprep qvytlppcrdeltknqvslwclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg SEQ ID NO: 71 light chain 2 CD38 _HHY1370
(for example, the fourth polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO: 69 monovalent antibody antiCD38_C2-CD38-1 heavy chain antiCD38_C2-CD38-1 QVQLQQSGAELVRSGASVKMSCKASGYTFTSFNMHWVKETPGQGLEWIGYIYPGNGGTNYNQKFKGKATLTADTSSSTAYMQISSLTSEDSAVYFCART GGLRRAYFTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 7 light chain antiCD38_C2-CD38-1 DIVLTQSPASLAVSLGQRATISCRASESVDSYGNGFMHWYQQKPGQPPKLLIYLASNLES
GVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNKEDPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 8 monovalent antibody antiCD38_C2-CD38-1_VH1-VL1 heavy chain antiCD38_C2-CD38-1_VH1-VL1 QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTG GLRRAYFTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 15 light chain antiCD38_C2-CD38-1_VH1-VL1 DIVLTQSPATLSLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRAT
GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIKRTVAAPSVF
IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 16 monovalent antibody antiCD38_C2-CD38-1_VH3-VL3 heavy chain antiCD38_C2-CD38-1_VH3-VL3 QVQLVQSGAEVVKPGASVKVSCKASGYTFTSFNMHWVKEAPGQRLEWIGYIYPGNGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTG GLRRAYFTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 19 light chain antiCD38_C2-CD38-1_VH3-VL3 DIVLTQSPATLSLSPGERATISCRASESVDSYGNGFMHWYQQKPGQPPRLLIYLASSRAT
GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIKRTVAAPSVF
IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 20 monovalent antibody antiCD38_C2-CD38-1_VH5-VL3 heavy chain antiCD38_C2-CD38-1_VH5-VL3 QVQLVQSGAEVVKSGASVKVSCKASGYTFTSFNMHWVKEAPGQGLEWIGYIYPGNGGTNYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFCARTGGL RRAYFTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 22 light chain antiCD38_C2-CD38-1_VH5-VL3 DIVLTQSPATLSLSPGERATISCRASESVDSYGNGFMHWYQQKPGQPPRLLIYLASSRAT
GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIKRTVAAPSVF
IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 20 monovalent antibody antiCD38_C2-CD38-1_VH6-VL3 heavy chain antiCD38_C2-CD38-1_VH6-VL3 QVQLVQSGAEVVKPGASVKMSCKASGYTFTSFNMHWVKEAPGQRLEWIGYIYPGNGGTNYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFCARTGG LRRAYFTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 24 light chain antiCD38_C2-CD38-1_VH6-VL3 DIVLTQSPATLSLSPGERATISCRASESVDSYGNGFMHWYQQKPGQPPRLLIYLASSRAT
GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIKRTVAAPSVF
IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 20 monovalent CD38 antibody _HHY1370 heavy chain CD38 _HHY1370 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 11 light chain CD38 _HHY1370 AIQMTQSPSSLSSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPS
RFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIKRTVAAPSVFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 12 monovalent antibody antiCD38_SB19 heavy chain antiCD38_SB19 QVQLVQSGAEVAKPGTSVKLSCKASGYTFTDYWMQWVKQRPGQGLEWIGTIYPGDGDTGYAQKFQGKATLTADKSSKTVYMHLSSLASEDSAVYYC ARGDYYGSNSLDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 107 light chain antiCD38_SB19 DIVMTQSHLSMSTSLGDPVSITCKASQDVSTVVAWYQQKPGQSPRRLIYSASYRYIGVPDRFTGSGAGTDFTFTISSVQAEDLAVYYCQQHYSPPYTFGGGTKLEIKRTVAAPSVFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 106

Table J. Full-length polynucleotide sequences of binding proteins

antiCD38_C2-CD38-1_VH1-VL1xCD28supxCD3mid IgG4 FALA heavy chain 1 FALA CD28supxCD3mid IgG4(hollow)
(for example, the sequence encoding the second polypeptide chain of the trispecific binding protein of the present invention) caggtgcagctggtgcagtctggcgccgaggtcgtgaaacctggcgcctctgtgaaggtgtcctgcaaggccagcggctacacctttaccagctactacatccactgggtgcgccaggcccctggacagggactggaatggatcggcagcatctaccccggcaacgtgaacaccaactacgcccagaagttccagggcagagccaccctg accgtggacaccagcatcagcaccgcctacatggaactgagccggctgagaagcgacgacaccgccgtgtactactgcacccggtcccactacggcctggattgga acttcgacgtgtggggcaagggcaccaccgtgacagtgtctagcagccaggtgcagctggtggaatctggcggcggagtggtgcagcctggcagaagcctgagact gagctgtgccgccagcggcttcaccttcaccaaggcctggatgcactgggtgcgccaggcccctggaaagcagctggaatgggtggcccagatcaaggacaagagcaacagctacgccacctactacgccgacagcgtgaagggccggttcaccatcagccgggacgacagcaagaacaccctgtacctgcagatgaacagcctgc gggccgaggacaccgccgtgtactactgtcggggcgtgtactatgccctgagccccttcgattactggggccagggaaccctcgtgaccgtgtctagtcggaccgccagcacaaagggcccatcggtgttccctctggccccttgcagcagaagcaccagcgaatctacagccgccctgggctgcctcgtgaaggactactttc ccgagcccgtgaccgtgtcctggaactctggcgctctgacaagcggcgtgcacacctttccagccgtgctccagagcagcggcctgtactctctgag cagcgtcgtgacagtgcccagcagcagcctgggcaccaagacctacacctgtaacgtggaccacaagcccagcaacaccaaggtggacaagcgggtgg aatctaagtacggccctccctgccctccttgcccagcccctgaagctgccggcggaccctccgtgttcctgttccccccaaagcccaaggacaccctgatgatcagccggacccccgaagtgacctgcgtggtggtggatgtgtcccaggaagatcccgaggtgcagttcaattggtacgtggacggcgtggaagt gcacaacgccaagaccaagcccagagaggaacagttcaacagcacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgcccagctccatcgagaaaaccatcagcaaggccaagggccagccccgcgagcctcaagtgtgtaccctgccccc tagccaggaagagatgaccaagaaccaggtgtccctgagctgtgccgtgaaaggcttctaccccagcgacattgccgtggaatggggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctcattcttcctggtgtccaagctgaccgtggacaagagccggtggcaggaaggcaacgtgtt cagctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtctctgtccctgggc SEQ ID NO:72 light chain 1 CD28supxCD3mid
(for example, the sequence encoding the first polypeptide chain of the trispecific binding protein of the present invention) gacatcgtgatgacccagacccccctgagcctgagcgtgacacctggacagcctgccagcatcagctgcaagagcagccagagcctggtgcacaacaacgccaacacctacctgagctggtatctgcagaagcccggccagagcccccagtccctgatctacaaggtgtccaacagattcagcggcgtgcccgacagattctccggca gcggctctggcaccgacttcaccctgaagatcagccggggtggaagccgaggacgtgggcgtgtactattgtggccagggcacccagtaccccttcacctttggcagcggcaccaaggtggaaatcaagggccagcccaaggccgcccccgacatccagatgacccagagccccagcagcctgtctgccagcgtgggcgacaga gtgaccatcacctgtcaggccagccagaacatctacgtgtggctgaactggtatcagcagaagcccggcaaggcccccaagctgctgatctacaaggccagcaacctgc acaccggcgtgcccagcagattttctggcagcggctccggcaccgacttcaccctgacaatcagctccctgcagcccgaggacattgccacctactactgccagcaggg ccagacctacccctacacctttggccagggcaccaagctggaaatcaagaccaagggccccagccgtacggtggccgctcccagcgtgttcatcttcccacctagcgacgagcagctgaagtccggcacagcctctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaagtgcagtggaaggtggacaacgccct gcagagcggcaacagccaggaaagcgtgaccgagcaggacagcaaggactccacctacagcctgagcagcaccctgacactgagcaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccaggcctgtctagccccgtgaccaagagcttcaaccggggcgagtgt SEQ ID NO:73 heavy chain 2 FALA antiCD38_C2-CD38-1_VH1-VL1 IgG4(overhang)
(for example, the sequence encoding the third polypeptide chain of the trispecific binding protein of the present invention) caggtgcagctggtgcagtctggcgccgaagtcgtgaaacctggcgcctccgtgaaggtgtcctgcaaggccagcggctacacctttaccagctacgccatgcactgggtcaaagaggcccctggccagagactggaatggatcggctacatctaccccggccaggcggcaccaactacaaccagaagttccagggcagagccaccctgacc gccgatacaagcgccagcaccgcctacatggaactgagcagcctgcggagcgaggataccgccgtgtacttctgtgccagaacaggcggcctgaggcgggcctactttacctattggggccagggcaccctcgtgaccgtgtctagcgctagcacaaagggccatcggtgttccctctggccccttgcagcagaagcaccagcga atctacagccgccctgggctgcctcgtgaaggactactttcccgagcccgtgaccgtgtcctggaactctggcgctctgacaagcggcgtgcacacctttccagccgtgctccagagcagcggcctgtactctctgagcagcgtcgtgacagtgcccagcagcagcctgggcaccaagacctacacctgtaacg tggaccacaagcccagcaacaccaaggtggacaagcgggtggaatctaagtacggccctccctgccctccttgcccagcccctgaagctgccggcggaccctccgtgttcctgttccccccaaagcccaaggacaccctgatgatcagccggacccccgaagtgacctgcgtggtggtggatgtgtcccaggaagatcccga ggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaacagcacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgcccagctccatcgagaaaaccatcagcaaggccaag ggccagccccgcgagcctcaagtgtataccctgcccccttgccaggaagagatgaccaagaaccaggtgtccctgtggtgtctcgtgaaaggcttctaccccagcg acattgccgtggaatgggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctcattcttcctgtactccaagctgacc gtggacaagagccggtggcaggaaggcaacgtgttcagctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtctctgtccctgggc SEQ ID NO:74 light chain 2 antiCD38_C2-CD38-1_VH1-VL1
(for example, encoding the fourth polypeptide chain of the trispecific binding protein of the present invention) gacatcgtgctgacacagagccctgccaccctgtctctgagccctggcgagagagccaccatcagctgtagagccagccagagcgtgtccagctacggccagggcttcatgcactggtatcagcagaagcccggccagccccccagactgctgatctatggcgccagcagcagagccacaggcatccccgccagattttctggctctggcagcggcaccgacttcaccctgacaatcagccccctggaacccgaggacttcgccgtgtactactgccagcagaacaaagaggacccctggaccttcggcggaggcaccaagctggaaatcaagcgtacggtggccgctcccagcgtgttcatcttcccacctagcgacgagcagctgaagtccggcacagcctctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaatgccctgcagagcggcaacagccaggaaagcgtgaccgagcaggacagcaaggactccacctacagcctgagcagcaccctgaccctgtccaaggccgattacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagagcttcaaccggggcgagtgc SEQ ID NO:75 antiCD38_C2-CD38-1_VH1-VL1xCD28supxCD3mid IgG1LALA P329A heavy chain 1 LALA P329A CD28supxCD3mid IgG1(hollow)
(for example, the sequence encoding the second polypeptide chain of the trispecific binding protein of the present invention) caggtgcagctggtgcagtctggcgccgaggtcgtgaaacctggcgcctctgtgaaggtgtcctgcaaggccagcggctacacctttaccagctactacatccactgggtgcgccaggcccctggacagggactggaatggatcggcagcatctaccccggcaacgtgaacaccaactacgcccagaagttccagggcagagccaccct gaccgtggacaccagcatcagcaccgcctacatggaactgagccggctgagaagcgacgacaccgccgtgtactactgcacccggtcccactacggcctggattggaacttcgacgtgtggggcaagggcaccaccgtgacagtgtctagcagccaggtgcagctggtggaatctggcggcggagtggtgcagcctggcagaagcctgag actgagctgtgccgccagcggcttcaccttcaccaaggcctggatgcactgggtgcgccaggcccctggaaagcagctggaatgggtggcccagatcaaggacaagagcaacagctacgccacctactacgccgacagcgtgaagggccggttcaccatcagccgggacgacagcaagaacaccctgtacctgcagatgaacagcctg cgggccgaggacaccgccgtgtactactgtcggggcgtgtactatgccctgagccccttcgattactggggccagggaaccctcgtgaccgtgtctagtcggaccgccagcacaaagggccccagcgtgttccctctggcccctagcagcaagagcacatctggcggaacagccgccctgggctgcctcgtgaaggactactttc ccgagcccgtgaccgtgtcctggaattctggcgccctgaccagcggcgtgcacacctttccagctgtgctgcagtccagcggcctgtacagcctgagcagcgtcgtgacagtgcccagcagctctctgggcacccagacctacatctgcaacgtgaaccacaagcccagcaacaccaaggtggacaagaaggt ggaacccaagagctgcgacaagacccacacctgtcccccttgtcctgcccccgaagccgccggaggcccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatcagccggacccccgaagtgacctgcgtggtggtggatgtgtcccacgaggaccctgaagtgaagttcaattggtacgtggacggcg tggaagtgcacaacgccaagaccaagccaagagaggaacagtacaacagcacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctggccgcccccatcgagaaaaccatcagcaaggccaagggccagccccgcgaaccccaggtgtgcacactgcccccaag cagggacgagctgaccaagaaccaggtgtccctgagctgtgccgtgaaaggcttctacccctccgatatcgccgtggaatggggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctcattcttcctggtgtccaagctgacagtggacaagtcccggtggcagcagggcaacgtg ttcagctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgagcctgagccccggc SEQ ID NO:76 light chain 1 CD28supxCD3mid
(for example, the sequence encoding the first polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO:73 heavy chain 2 LALA P329A antiCD38_C2-CD38-1_VH1-VL1 IgG1(overhang)
(for example, encoding the third polypeptide chain of the trispecific binding protein of the present invention) caggtgcagctggtgcagtctggcgccgaagtcgtgaaacctggcgcctccgtgaaggtgtcctgcaaggccagcggctacacctttaccagctacgccatgcactgggtcaaagaggcccctggccagagactggaatggatcggctacatctaccccggccaggcggcaccaactacaaccagaagttccagggcagagccaccctg accgccgatacaagcgccagcaccgcctacatggaactgagcagcctgcggagcgaggataccgccgtgtacttctgtgccagaacaggcggcctgaggcgggcc tactttacctattggggccagggcaccctcgtgaccgtgtctagcgctagcacaaagggccccagcgtgttccctctggcccctagcagcaagagcacatctggc ggaacagccgccctgggctgcctcgtgaaggactactttcccgagcccgtgaccgtgtcctggaattctggcgccctgaccagcggcgtgcacacctttccagctgtgctgcagtccagcggcctgtacagcctgagcagcgtcgtgacagtgcccagcagctctctgggcacccagacctacatctgcaac gtgaaccacaagcccagcaacaccaaggtggacaagaaggtggaacccaagagctgcgacaagacccacacctgtcccccttgtcctgcccccgaagccgccggaggcccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatcagccggacccccgaagtgacctgcgtggtggtggatgtgtcccac gaggaccctgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagccaagagaggaacagtacaacagcacctaccgggtggtgtccg tgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctggccgcccccatcgagaaaaccatcagcaaggccaaggg ccagccccgcgaaccccaggtgtacacactgcccccatgcagggacgagctgaccaagaaccaggtgtccctgtggtgtctggtgaaaggcttctacccctccgata tcgccgtggaatgggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctcattcttcctgtactccaagctgacagtgg acaagtcccggtggcagcagggcaacgtgttcagctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgagcctgagccccggc SEQ ID NO:77 light chain 2 antiCD38_C2-CD38-1_VH1-VL1
(for example, encoding the fourth polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO:75 antiCD38_C2-CD38-1_VH1-VL1xCD28supxCD3mid IgG1 NNSA heavy chain 1 NNSA CD28supxCD3mid IgG1(hollow)
(for example, the sequence encoding the second polypeptide chain of the trispecific binding protein of the present invention) caggtgcagctggtgcagtctggcgccgaggtcgtgaaacctggcgcctctgtgaaggtgtcctgcaaggccagcggctacacctttaccagctactacatccactgggtgcgccaggcccctggacagggactggaatggatcggcagcatctaccccggcaacgtgaacaccaactacgcccagaagttccagggcagagccaccct gaccgtggacaccagcatcagcaccgcctacatggaactgagccggctgagaagcgacgacaccgccgtgtactactgcacccggtcccactacggcctggattggaacttcgacgtgtggggcaagggcaccaccgtgacagtgtctagcagccaggtgcagctggtggaatctggcggcggagtggtgcagcctggcagaagcctgag actgagctgtgccgccagcggcttcaccttcaccaaggcctggatgcactgggtgcgccaggcccctggaaagcagctggaatgggtggcccagatcaaggacaagagcaacagctacgccacctactacgccgacagcgtgaagggccggttcaccatcagccgggacgacagcaagaacaccctgtacctgcagatgaacagcctg cgggccgaggacaccgccgtgtactactgtcggggcgtgtactatgccctgagccccttcgattactggggccagggaaccctcgtgaccgtgtctagtcggaccgccagcacaaagggccccagcgtgttccctctggcccctagcagcaagagcacatctggcggaacagccgccctgggctgcctcgtgaaggactactttc ccgagcccgtgaccgtgtcctggaattctggcgccctgaccagcggcgtgcacacctttccagctgtgctgcagtccagcggcctgtacagcctgagcagcgtcgtgacagtgcccagcagctctctgggcacccagacctacatctgcaacgtgaaccacaagcccagcaacaccaaggtggacaagaaggt ggaacccaagagctgcgacaagacccacacctgtcccccttgtcctgcccccgaactgctgggaggcccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatcagccggacccccgaagtgacctgcgtggtggtggatgtgtcccacgaggaccctgaagtgaagttcaattggtacgtggacggcgt ggaagtgcacaacgccaagaccaagccaagagaggaacagtacaacaatgcctcccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctgcccccatcgagaaaaccatcagcaaggccaagggccagccccgcgaaccccaggtgtgcacactgccc ccaagcagggacgagctgaccaagaaccaggtgtccctgagctgtgccgtgaaaggcttctacccctccgatatcgccgtggaatggggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctcattcttcctggtgtccaagctgacagtggacaagtcccggtggcagcagggcaacg tgttcagctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgagcctgagccccggc SEQ ID NO:78 light chain 1 CD28supxCD3mid
(for example, the sequence encoding the first polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO:73 heavy chain 2 NNSA antiCD38_C2-CD38-1_VH1-VL1 IgG1(overhang)
(for example, encoding the third polypeptide chain of the trispecific binding protein of the present invention) caggtgcagctggtgcagtctggcgccgaagtcgtgaaacctggcgcctccgtgaaggtgtcctgcaaggccagcggctacacctttaccagctacgccatgcactgggtcaaagaggcccctggccagagactggaatggatcggctacatctaccccggccaggcggcaccaactacaaccagaagttccagggcagagccaccctg accgccgatacaagcgccagcaccgcctacatggaactgagcagcctgcggagcgaggataccgccgtgtacttctgtgccagaacaggcggcctgaggcgggcct actttacctattggggccagggcaccctcgtgaccgtgtctagcgctagcacaaagggcccatcggtcttccccctggcaccctcctccaagagcacctctggggg cacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagccca gcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcct cttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggt atgttgacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacaatgcctcccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccaca ggtgtacaccctgcccccatgccgggatgagctgaccaagaatcaagtcagcctgtggtgcctggtaaaaggcttctatcccagcgacatcgccgtggagtggg agagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctactcaaaactcaccgtggacaagagcaggt ggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggt SEQ ID NO:79 light chain 2 antiCD38_C2-CD38-1_VH1-VL1
(for example, encoding the fourth polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO:75 CD38 _HHY1370 xCD28supxCD3mid IgG4 FALA heavy chain 1 FALA CD28supxCD3mid IgG4(hollow)
(for example, the sequence encoding the second polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO:72 light chain 1 CD28supxCD3mid
(for example, the sequence encoding the first polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO:73 heavy chain 2 FALA CD38 _HHY1370 IgG4(overhang)
(for example, encoding the third polypeptide chain of the trispecific binding protein of the present invention) caggtgcagctggtggaaagcggcggaggcgtggtgcagcctggcaggtctctgagactgagctgtgccgccagcggcttcaccttcagcagctacggaatgcactgggtgcgccaggcccctggcaaaggactggaatgggtggccgtgatttggtacgacggcagcaacaagtactacgccgacagcgtgaagggccggttcaccatcagcggcgacaacagcaagaacaccctgtacctgcagatgaacagcctgcgggccgaggacaccgccgtgtactactgcgccagaatgttcagaggcgccttcgactactggggccagggcacactcgtgaccgtgtctagtgcgtcgaccaagggcccatcggtgttccctctggccccttgcagcagaagcaccagcgaatctacagccgccctgggctgcctcgtgaaggactactttcccgagcccgtgaccgtgtcctggaactctggcgctctgacaagcggcgtgcacacctttccagccgtgctccagagcagcggcctgtactctctgagcagcgtcgtgacagtgcccagcagcagcctgggcaccaagacctacacctgtaacgtggaccacaagcccagcaacaccaaggtggacaagcgggtggaatctaagtacggccctccctgccctccttgcccagcccctgaagctgccggcggaccctccgtgttcctgttccccccaaagcccaaggacaccctgatgatcagccggacccccgaagtgacctgcgtggtggtggatgtgtcccaggaagatcccgaggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaacagcacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgcccagctccatcgagaaaaccatcagcaaggccaagggccagccccgcgagcctcaagtgtataccctgcccccttgccaggaagagatgaccaagaaccaggtgtccctgtggtgtctcgtgaaaggcttctaccccagcgacattgccgtggaatgggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctcattcttcctgtactccaagctgaccgtggacaagagccggtggcaggaaggcaacgtgttcagctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtctctgtccctgggc SEQ ID NO:80 light chain 2 CD38 _HHY1370
(for example, encoding the fourth polypeptide chain of the trispecific binding protein of the present invention) gccatccagatgacccagagccccagcagcctgtctgccagcgtgggcgacagagtgaccatcacctgtagagccagccagggcatccggaacgacctgggctggtatcagcagaagcctggcaaggcccccaagctgctgatctacgccgctagctctctgcagtccggcgtgcccagcagattttctggcagcggctccggcacc gacttcaccctgacaatctctggcctgcagcccgaggacagcgccacctactactgtctgcaagactacatctactaccccaccttcggccagggcaccaaggt ggaaatcaagcgtacggtggccgctcccagcgtgttcatcttcccacctagcgacgagcagctgaagtccggcacagcctctgtcgtgtgcctgctgaacaactt ctacccccgcgaggccaaagtgcagtggaaggtggacaacgccctgcagagcggcaacagccaggaaagcgtgaccgagcaggacagcaaggactccacctacag cctgagcagcaccctgacactgagcaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagagctt caaccggggcgagtgt SEQ ID NO:81 CD38 _HHY1370 xCD28supxCD3mid IgG1LALA P329A heavy chain 1 LALA P329A CD28supxCD3mid IgG1(hollow)
(for example, the sequence encoding the second polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO:76 light chain 1 CD28supxCD3mid
(for example, the sequence encoding the first polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO:73 heavy chain 2 LALA P329A CD38 _HHY1370 IgG1(overhang)
(for example, encoding the third polypeptide chain of the trispecific binding protein of the present invention) caggtgcagctggtggaaagcggcggaggcgtggtgcagcctggcaggtctctgagactgagctgtgccgccagcggcttcaccttcagcagctacggaatgcactgggtgcgccaggcccctggcaaaggactggaatgggtggccgtgatttggtacgacggcagcaacaagtactacgccgacagcgtgaagggccggttcaccatcagcggcgacaacagcaagaacaccctgtacctgcagatgaacagcctgcgggccgaggacaccgccgtgtactactgcgccagaatgttcagaggcgccttcgactactggggccagggcacactcgtgaccgtgtctagtgcgtcgaccaagggccccagcgtgttccctctggcccctagcagcaagagcacatctggcggaacagccgccctgggctgcctcgtgaaggactactttcccgagcccgtgaccgtgtcctggaattctggcgccctgaccagcggcgtgcacacctttccagctgtgctgcagtccagcggcctgtacagcctgagcagcgtcgtgacagtgcccagcagctctctgggcacccagacctacatctgcaacgtgaaccacaagcccagcaacaccaaggtggacaagaaggtggaacccaagagctgcgacaagacccacacctgtcccccttgtcctgcccccgaagccgccggaggcccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatcagccggacccccgaagtgacctgcgtggtggtggatgtgtcccacgaggaccctgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagccaagagaggaacagtacaacagcacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctggccgcccccatcgagaaaaccatcagcaaggccaagggccagccccgcgaaccccaggtgtacacactgcccccatgcagggacgagctgaccaagaaccaggtgtccctgtggtgtctggtgaaaggcttctacccctccgatatcgccgtggaatgggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagcagggcaacgtgttcagctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgagcctgagccccggc SEQ ID NO:82 light chain 2 CD38 _HHY1370
(for example, encoding the fourth polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO:81 CD38 _HHY1370 xCD28supxCD3mid IgG1 NNSA heavy chain 1 NNSA CD28supxCD3mid IgG1(hollow)
(for example, the sequence encoding the second polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO:78 light chain 1 CD28supxCD3mid
(for example, the sequence encoding the first polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO:73 heavy chain 2 NNSA CD38 _HHY1370 IgG1(overhang)
(for example, encoding the third polypeptide chain of the trispecific binding protein of the present invention) caggtgcagctggtggaaagcggcggaggcgtggtgcagcctggcaggtctctgagactgagctgtgccgccagcggcttcaccttcagcagctacggaatgcactgggtgcgccaggcccctggcaaaggactggaatgggtggccgtgatttggtacgacggcagcaacaagtactacgccgacagcgtgaag ggccggttcaccatcagcggcgacaacagcaagaacaccctgtacctgcagatgaacagcctgcgggccgaggacaccgccgtgtactactgcgccagaatgttc agaggcgccttcgactactggggccaggcacactcgtgaccgtgtctagtgcgtcgaccaagggcccatcggtcttccccctggcaccctcctccaagagcacc tctggggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacacct tcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatca caagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtca gtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagtt caactggtatgttgacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacaatgcctcccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagcccc gagaaccacaggtgtacaccctgcccccatgccgggatgagctgaccaagaatcaagtcagcctgtggtgcctggtaaaaggcttctatcccagcgacatcgccgtggagtggggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctactcaaaactcaccgtggaca agagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggt SEQ ID NO:83 light chain 2 CD38 _HHY1370
(for example, encoding the fourth polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO:81

CD38 polypeptides

[0147] In some embodiments, the binding protein of the present invention comprises an antigen binding region that binds the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus CD38 polypeptide. Exemplary assays for determining whether an antigen binding site binds an antigen are described herein and are known in the art. In some embodiments, binding is determined using an ELISA assay, for example as described below. In some embodiments, binding is determined using an SPR assay, for example as described below. In some embodiments, binding is determined by flow cytometry analysis using cells expressing CD38 polypeptide on their cell surface, for example as described below.

[0148] In some embodiments, the binding protein of the present invention binds a purified polypeptide or fragment thereof containing the amino acid sequence of SEQ ID NO: 1 and/or 30 (such as measured by ELISA or SPR). In some embodiments, the binding protein of the present invention binds to a polypeptide or contains the amino acid sequence of SEQ ID NO:1 and/or 30 when expressed on the surface of a cell (such as measured by flow cytometry).

[0149] In some embodiments, the binding protein of the present invention binds to CD38 isoform A polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:1). In some embodiments, the binding protein of the present invention binds to an isoform E CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:105 and not containing the complete amino acid sequence of SEQ ID NO:1, consisting of the amino acid sequence of SEQ ID NO:105, or consisting essentially of the amino acid sequence of SEQ ID NO:105). In some embodiments, the binding protein of the present invention binds to CD38 isoform A polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:1) and a CD38 isoform E polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:105, and not containing the complete amino acid sequence of SEQ ID NO:1, consisting of the amino acid sequence of SEQ ID NO:105, or consisting essentially of the amino acid sequence of SEQ ID NO:105). Without being limited to any theory, it is believed that binding to CD38 isoform E polypeptide may be preferential, e.g. by targeting the binding protein of the present invention to the cell(s) expressing the CD38 isoform E polypeptide.

Polypeptide sequence of the extracellular domain of human CD38 isoform A

RWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKV QTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEI (SEQ ID NO:1)

Polypeptide sequence of human CD38 isoform E

RWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRHFWECGSP (SEQ ID NO:105)

[0150] In some embodiments, the extracellular domain of a human CD38 polypeptide comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the extracellular domain of the cynomolgus CD38 polypeptide comprises the amino acid sequence of SEQ ID NO:30.

CD38 polypeptide sequence of cynomolgus macaque

RWRQQWSGSGTTSRFPETVLARCVKYTEVHPEMRHVDCQSVWDAFKGAFISKYPCNITEEDYQPLVKLGTQTVPCNKTLLWSRIKDLAHQFTQVQRDMFTLEDMLLGYLADDLTWCGEFNTFEINYQSCPDWRKDCSNNPVSVFWKTVSRRFAETACGVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPE KVQALEAWVIHGGREDSRDLCQDPTIKELESIISKRNIRFFCKNIYRPDKFLQCVKNPEDSSCLSGI (SEQ ID NO:30)

Linkers

[0151] In some embodiments, the length of the linkers L ₁ , L ₂ , L ₃ , and L ₄ ranges from zero amino acids (length=0) to about 100 amino acids or less, 100, 50, 40, 30, 20, or 15 amino acids or less. The length of linkers can also be 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid. All of the L ₁ , L ₂ , L ₃ and L ₄ in one binding protein may have the same amino acid sequence, or they may all have different amino acid sequences.

[0152] Examples of suitable linkers include a single glycine (Gly) residue; diglycine peptide (Gly-Gly); tripeptide (Gly-Gly-Gly); peptide of four glycine residues; peptide of five glycine residues; peptide of six glycine residues; a peptide of seven glycine residues and a peptide of eight glycine residues. Other combinations of amino acid residues can be used, such as peptide GGGGSGGGGS (SEQ ID NO: 55), peptide GGGGSGGGGSGGGGS (SEQ ID NO: 56), peptide TKGPS (SEQ ID NO: 57), peptide GQPKAAP (SEQ ID NO: 58) , as well as the peptide GGSGSSGSGG (SEQ ID NO:59). The above examples are not intended to limit the scope of the present invention in any way, and it has been shown that linkers containing randomly selected amino acids selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, glycine and proline are suitable for binding proteins. For further description of linker sequences, see, for example, in WO2012135345 and international application No. PCT/US2017/027488.

[0153] The composition and sequence of amino acid residues in the linker may vary depending on the type of secondary structure element that needs to be achieved by the linker. For example, glycine, serine and alanine are the most suitable linkers for maximum flexibility. Some combinations of glycine, proline, threonine and serine are useful if a stiffer, longer linker is needed. If desired, any amino acid residue can be considered as a linker in combination with other amino acid residues to construct longer peptide linkers depending on the desired properties.

[0154] In some embodiments, the length of at least one of L ₁ , L ₂ , L ₃ or L ₄ is independently 0 amino acids. In some embodiments, L ₁ , L ₂ , L ₃ , or L ₄ are each independently at least one amino acid long. In some embodiments, the length of L ₁ is at least twice the length of L ₃ . In some embodiments, the length of L ₂ is at least twice the length of L ₄ . In some embodiments, the length of L ₁ is at least twice the length of L ₃ and the length of L ₂ is at least twice the length of L ₄ . In some embodiments, the length of L ₁ is 3-12 amino acid residues, the length of L ₂ is 3-14 amino acid residues, the length of L ₃ is 1-8 amino acid residues, and the length of L ₄ is 1-3 amino acid residues. In some embodiments, the length of L ₁ is 5-10 amino acid residues, the length of L ₂ is 5-8 amino acid residues, the length of L ₃ is 1-5 amino acid residues, and the length of L ₄ is 1-2 amino acid residues. In some embodiments, the length of L ₁ is 7 amino acid residues, the length of L ₂ is 5 amino acid residues, the length of L ₃ is 1 amino acid residue, and the length of L ₄ is 2 amino acid residues. In some embodiments, the length of L ₁ is 10 amino acid residues, the length of L ₂ is 10 amino acid residues, the length of L ₃ is 0 amino acid residues, and the length of L ₄ is 0 amino acid residues. In some embodiments, L ₁ , L ₂ , L ₃ , and L ₄ each have an independently selected length of 0 to 15 amino acids ( e.g. , 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14 or 15 amino acids), where at least two linkers are 1-15 amino acids in length ( for example , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids). In some embodiments, L ₁ , L ₂ , L ₃ , and L ₄ are each 0 amino acids in length.

[0155] In some embodiments, L₁, L₂, L₃ and/or L₄ contain a sequence derived from a naturally occurring sequence at the junction between the variable domain of an antibody and the constant domain of an antibody (e.g. described in WO2012/135345). For example, in some embodiments, the linker comprises a sequence located in the transition region between the endogenous V domains_H and C_H1 or between endogenous V domains_L and C_L (For example, kappa or lambda chains). In some embodiments, the linker comprises a sequence located in the transition region between the endogenous V domains_H and C_H1 human or between endogenous V domains_L and C_L person (for example, human kappa or lambda chains).

[0156] In some embodiments, L ₁ , L ₂ , L ₃ , and L ₄ are each independently zero amino acids in length or contain 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, L ₁ , L ₂ , L ₃ , and L ₄ 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).

[0157] In some embodiments, L ₁ contains the sequence GQPKAAP (SEQ ID NO: 58), L ₂ contains the sequence TKGPS (SEQ ID NO: 57), L ₃ contains the sequence S, and L ₄ contains the sequence RT. In some embodiments, L ₁ contains the sequence GGGGSGGGGS (SEQ ID NO: 55), L ₂ contains the sequence GGGGSGGGGS (SEQ ID NO: 55), L ₃ is 0 amino acids long, and L ₄ is 0 amino acids long. In some embodiments, L ₁ contains the sequence GGSGSSGSGG (SEQ ID NO: 59), L ₂ contains the sequence GGSGSSGSGG (SEQ ID NO: 59), L ₃ is 0 amino acids long, and L ₄ is 0 amino acids long. In some embodiments, L ₁ contains the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), L ₂ is 0 amino acids long, L ₃ contains the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), and L ₄ is 0 amino acids long.

Fc regions and constant domains

[0158] In some embodiments, the binding protein of the present invention comprises a full-length antibody heavy chain or a polypeptide chain containing an Fc region. In some embodiments, the Fc region is a human Fc region, for example, Fc region of human IgG1, IgG2, IgG3 or IgG4. In some embodiments, the Fc region includes a hinge region, C_H1, C_H2, C_H3 and optionally C domains_H4. In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, the Fc region is a human IgG4 Fc region. In some embodiments, the Fc region contains one or more mutations described below.

[0159] In some embodiments, the binding protein of the present invention contains one or two Fc variants. The term "Fc variant" as used herein refers to a molecule or sequence that is modified from the native Fc but still contains a binding site for the FcRn recycling receptor (neonatal Fc receptor). Exemplary Fc variants and their interaction with the salvage receptor are known in the art. Thus, the term “Fc variant” may include a molecule or sequence resulting from the humanization of a native Fc of non-human origin. In addition, the native Fc contains regions that can be removed because they provide structural features or biological activity that are not required for the antibody-like binding proteins of the present invention. Thus, the term "Fc variant" refers to a molecule or sequence that lacks one or more native Fc regions or residues or in which one or more Fc regions or residues have been modified to affect or involve: (1) disulfide bond formation, (2) incompatibility with the selected host cell, (3) heterogeneity of N-termini when expressed in the selected host cell, (4) glycosylation, (5) interaction with the complement system, (6) binding to an Fc receptor other than the receptor recycling, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0160] In some embodiments, the Fc region contains one or more mutations that reduce or eliminate Fc receptor binding and/or Fc region effector function (e.g., antibody-dependent cellular phagocytosis mediated by Fc receptors (ADCP), complement-dependent cytotoxicity (CDC) and/or antibody-dependent cellular cytotoxicity (ADCC)).

[0161] In some embodiments, the Fc region is a human IgG1 Fc region comprising one or more amino acid substitutions at positions corresponding to human IgG1 positions 234, 235 and/or 329 according to the EU index. In some embodiments, the amino acid substitutions are L234A, L235A, and/or P329A. In some embodiments, the Fc region is a human IgG1 Fc region comprising amino acid substitutions at positions corresponding to human IgG1 positions 298, 299 and/or 300 according to the EU index. In some embodiments, the amino acid substitutions are S298N, T299A, and/or Y300S.

[0162] In some embodiments, the Fc region is a human IgG4 Fc region containing one or more mutations that reduce or eliminate FcγI and/or FcγII binding. In some embodiments, the Fc region is a human IgG4 Fc region containing one or more mutations that reduce or eliminate FcγI and/or FcγII binding but do not affect FcRn binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to human IgG4 positions 228 and/or 409 according to the EU index. In some embodiments, the amino acid substitutions are S228P and/or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to human IgG4 positions 234 and/or 235 according to the EU index. In some embodiments, the amino acid substitutions are F234A and/or L235A. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to human IgG4 EU index positions 228, 234, 235 and/or 409. In some embodiments, the amino acid substitutions are S228P, F234A, L235A, and/or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to human IgG4 positions 233-236 according to the EU index. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and a deletion at position 236. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid mutations at positions corresponding to positions 228, 233-236, and/or or 409 human IgG4 according to the EU index. In some embodiments, the amino acid mutations are S228P; E233P, F234V, L235A, and deletion at position 236; and/or R409K.

[0163] In some embodiments, the binding protein of the present invention contains one or more mutations to improve purification, for example, by modulating the affinity for the purification reagent. For example, it is known that heterodimeric binding proteins can be selectively purified from their homodimeric forms if one of the two Fc regions of the heterodimeric form contains a mutation(s) that reduces or eliminates binding to protein A, since the heterodimeric form will have intermediate affinity to a Protein A-based purifier over any homodimeric form and can be selectively eluted from Protein A, e.g. by using a different pH value (see e.g.Smith, E. J. et al. (2015) Sci. Rep.5:17943). In some embodiments, the mutation involves substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are H435R and Y436F. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to C_H1, while the first Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain, and wherein the third polypeptide chain additionally contains a second Fc region linked to C_H1, while the second Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain; and wherein only one of the first and second Fc regions contains amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are H435R and Y436F. In some embodiments, the binding protein of the present invention contains knob and valley mutations and one or more mutations to improve clearance. In some embodiments, the first and/or second Fc regions are human IgG1 Fc regions. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions.

[0164] In some embodiments, one or both Fc regions are human IgG4 Fc regions containing amino acid substitutions at positions corresponding to human IgG4 positions 233-236 according to the EU index. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and a deletion at position 236. In some embodiments, the Fc regions are human IgG4 Fc regions containing amino acid mutations at positions corresponding to positions 228, 233-236, and/or or 409 human IgG4 according to the EU index. In some embodiments, the amino acid mutations are S228P; E233P, F234V, L235A, and deletion at position 236; and/or R409K. In some embodiments, one or both Fc regions are human IgG1 Fc regions containing one or more amino acid substitutions at positions corresponding to human IgG1 positions 234, 235 and/or 329 according to the EU index. In some embodiments, the amino acid substitutions are L234A, L235A, and/or P329A. In some embodiments, the Fc regions are human IgG1 Fc regions containing amino acid substitutions at positions corresponding to human IgG1 positions 298, 299 and/or 300 according to the EU index. In some embodiments, the amino acid substitutions are S298N, T299A, and/or Y300S.

[0165] To increase the yield of certain binding proteins (e.g. bispecific or trispecific binding proteins) domains C_H3 can be modified using ridge-in-groove technology, which is described in detail with several examples, for example in International Publication No. WO 96/027011, Ridgway et al., 1996, Protein Eng. 9: 617-21; and Merchant et al., 1998, Nat. Biotechnol. 16: 677-81. In particular, the interacting surfaces of two domains C_H3 modified to increase heterodimerization of both heavy chains containing these two C domains_H3. Each of the two C domains_H3 (of two heavy chains) may represent a "bump" while the other represents a "valley". The introduction of a disulfide bridge further stabilizes heterodimers (Merchant et al., 1998; Atwell et al., 1997, J. Mol. Biol. 270: 26-35) and increases yield. In certain embodiments, the overhang is on the second pair of single variable domain polypeptides. In other embodiments, the protrusion is on the first pair of polypeptides characterized by cross orientation. In yet other embodiments, domains C_H3 do not contain ridge-in-groove.

[0166] In some embodiments, the binding protein of the present invention (e.g. trispecific binding protein) contains a knob mutation on the second polypeptide chain and a trench mutation on the third polypeptide chain. In some embodiments, the binding protein of the present invention comprises a knob mutation on a third polypeptide chain and a trench mutation on a second polypeptide chain. In some embodiments, the overhang mutation involves substitution(s) at positions corresponding to positions 354 and/or 366 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitutions are S354C, T366W, T366Y, S354C and T366W, or S354C and T366Y. In some embodiments, the overhang mutation involves substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitutions are S354C and T366W. In some embodiments, the hole mutation involves substitution(s) at positions corresponding to positions 407 and optionally 349, 366 and/or 368 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitutions are Y407V or Y407T and optionally Y349C, T366S and/or L368A. In some embodiments, the hole mutation involves substitutions at positions corresponding to human IgG1 or IgG4 EU index positions 349, 366, 368 and 407. In some embodiments, the amino acid substitutions are Y349C, T366S, L368A, and Y407V.

[0167] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region comprises an immunoglobulin hinge region and the CH2 and CH3 constant domains of an immunoglobulin heavy chain, wherein the first Fc region comprises an amino acid(s) ) substitution(s) at positions corresponding to provisions 366 and optionally 354 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are T366W or T366Y and optionally S354C; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises amino acid substitution(s) at positions corresponding to positions 407 and optionally 349, 366 and/or 368 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are Y407V or Y407T and optionally Y349C, T366S and/or L368A.

[0168] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region comprises an immunoglobulin hinge region and the CH2 and CH3 constant domains of an immunoglobulin heavy chain, wherein the first Fc region comprises an amino acid(s) ) substitution(s) at positions corresponding to provisions 407 and optionally 349, 366 and/or 368 of human IgG1 or IgG4 in accordance with the EU index, where the amino acid substitutions are Y407V or Y407T and optionally Y349C, T366S and/or L368A; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises amino acid substitution(s) at positions corresponding to positions 366 and optionally 354 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are T366W or T366Y and optionally S354C.

[0169] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region comprises an immunoglobulin hinge region and the CH2 and CH3 constant domains of an immunoglobulin heavy chain, wherein the first Fc region comprises an amino acid substitution at a position corresponding to position 366 of human IgG1 or IgG4 according to the EU index, where the amino acid substitution is T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises amino acid substitution(s) at positions corresponding to positions 366, 368 and/or 407 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are T366S, L368A and/or Y407V.

[0170] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region comprises an immunoglobulin hinge region and the CH2 and CH3 constant domains of an immunoglobulin heavy chain, wherein the first Fc region comprises an amino acid(s) ) substitution(s) at positions corresponding to positions 366, 368 and/or 407 of human IgG1 or IgG4 in accordance with the EU index, where the amino acid substitutions are T366S, L368A and/or Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises an amino acid substitution at a position corresponding to position 366 Human IgG1 or IgG4 according to the EU index, where the amino acid substitution is T366W.

[0171] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region comprises an immunoglobulin hinge region and the immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are S354C and T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 349 , 366, 368 and 407 human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are Y349C, T366S, L368A and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region contains amino acid substitutions at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are Y349C, T366S, L368A and Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are S354C and T366W. In some embodiments, the first and/or second Fc regions are human IgG1 Fc regions. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions.

[0172] In some embodiments, the second polypeptide chain further comprises a first CH1-linked Fc region, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first The Fc region contains amino acid substitutions at positions corresponding to positions 228, 354, 366 and 409 of human IgG4 according to the EU index, where the amino acid substitutions are S228P, S354C, T366W and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 constant domains of an immunoglobulin heavy chain, wherein the second Fc region comprises amino acids substitutions at positions corresponding to positions 228, 349, 366, 368, 407 and 409 of human IgG4 according to the EU index, where the amino acid substitutions are S228P, Y349C, T366S, L368A, Y407V and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region contains amino acid substitutions at positions corresponding to positions 228, 349, 366, 368, 407 and 409 of human IgG4 in accordance with the EU index, where the amino acid substitutions are S228P, Y349C, T366S, L368A, Y407V and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 constant domains of an immunoglobulin heavy chain, wherein the second Fc region comprises amino acids substitutions at positions corresponding to positions 228, 354, 366 and 409 of human IgG4 according to the EU index, where the amino acid substitutions are S228P, S354C, T366W and R409K.

[0173] In some embodiments, the second polypeptide chain further comprises a first CH1-linked Fc region, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first The Fc region contains amino acid substitutions at positions corresponding to positions 234, 235, 354 and 366 of human IgG4 according to the EU index, where the amino acid substitutions are F234A, L235A, S354C and T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 constant domains of an immunoglobulin heavy chain, wherein the second Fc region comprises amino acids substitutions at positions corresponding to positions 234, 235, 349, 366, 368 and 407 of human IgG4 according to the EU index, where the amino acid substitutions are F234A, L235A, Y349C, T366S, L368A and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region contains amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368 and 407 of human IgG4 in accordance with the EU index, where the amino acid substitutions are F234A, L235A, Y349C, T366S, L368A and Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 constant domains of an immunoglobulin heavy chain, wherein the second Fc region comprises amino acids substitutions at positions corresponding to positions 234, 235, 354 and 366 of human IgG4 according to the EU index, where the amino acid substitutions are F234A, L235A, S354C and T366W.

[0174] In some embodiments, the second polypeptide chain further comprises a first CH1-linked Fc region, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first The Fc region contains amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366 and 409 of human IgG4 according to the EU index, where the amino acid substitutions are S228P, F234A, L235A, S354C, T366W and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 constant domains of an immunoglobulin heavy chain, wherein the second Fc region comprises amino acids substitutions at positions corresponding to EU index human IgG4 positions 228, 234, 235, 349, 366, 368, 407 and 409, where the amino acid substitutions are S228P, F234A, L235A, Y349C, T366S, L368A, Y407V and R409K . In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region contains amino acid substitutions at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407 and 409 of the human IgG4 according to the EU index, where the amino acid substitutions are S228P, F234A, L235A, Y349C, T366S, L368A, Y407V and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 constant domains of an immunoglobulin heavy chain, wherein the second Fc region comprises amino acids substitutions at positions corresponding to EU index human IgG4 positions 228, 234, 235, 354, 366 and 409, where the amino acid substitutions are S228P, F234A, L235A, S354C, T366W and R409K.

[0175] In some embodiments, the binding protein of the present invention contains one or more mutations to increase serum half-life (see, e.g., Hinton, P.R. et al. (2006) J. Immunol. 176(1):346- 56). In some embodiments, the mutation involves substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are M428L and N434S. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first CH1-linked Fc region, wherein the first Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region contains an immunoglobulin hinge region and the immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first and/or second Fc regions contain amino acid substitutions at positions corresponding to positions 428 and 434 of IgG1 or human IgG4 according to the EU index, where the amino acid substitutions are M428L and N434S. In some embodiments, the binding protein of the present invention contains knob and valley mutations and one or more mutations to increase serum half-life. In some embodiments, the first and/or second Fc regions are human IgG1 Fc regions. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions.

[0176] In some embodiments, the binding protein of the present invention contains one or more mutations to improve stability, for example, hinge region and/or contact surface of the IgG4 dimer (see For example, Spiess, C. et al.(2013) J Biol. Chem.288:26583-26593). In some embodiments, the mutation involves substitutions at positions corresponding to human IgG4 EU index positions 228 and 409, where the amino acid substitutions are S228P and R409K. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to C_H1, while the first Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain, and wherein the third polypeptide chain additionally contains a second Fc region linked to C_H1, while the second Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain; wherein the first and second Fc regions are human IgG4 Fc regions; and wherein each of the first and second Fc regions contains amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, where the amino acid substitutions are S228P and R409K. In some embodiments, the binding protein of the present invention contains knob and trench mutations and one or more mutations to improve stability. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions.

[0177] In some embodiments, the binding protein of the present invention contains one or more mutations to improve purification, for example, by modulating the affinity for the purification reagent. For example, it is known that heterodimeric binding proteins can be selectively purified from their homodimeric forms if one of the two Fc regions of the heterodimeric form contains a mutation(s) that reduces or eliminates binding to protein A, since the heterodimeric form will have intermediate affinity to a Protein A-based purifier over any homodimeric form and can be selectively eluted from Protein A, e.g. by using a different pH value (see e.g.Smith, E. J. et al. (2015) Sci. Rep.5:17943). In some embodiments, the mutation involves substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are H435R and Y436F. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to C_H1, while the first Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain, and wherein the third polypeptide chain additionally contains a second Fc region linked to C_H1, while the second Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain; and wherein only one of the first and second Fc regions contains amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are H435R and Y436F. In some embodiments, the binding protein of the present invention contains knob and valley mutations and one or more mutations to improve clearance. In some embodiments, the first and/or second Fc regions are human IgG1 Fc regions. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions.

[0178] In some embodiments, the binding protein of the present invention contains one or more mutations to increase serum half-life (see, e.g., Hinton, P.R. et al. (2006) J. Immunol. 176(1):346- 56). In some embodiments, the mutation involves substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are M428L and N434S. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first CH1-linked Fc region, wherein the first Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region contains an immunoglobulin hinge region and the immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first and/or second Fc regions contain amino acid substitutions at positions corresponding to positions 428 and 434 of IgG1 or human IgG4 according to the EU index, where the amino acid substitutions are M428L and N434S. In some embodiments, the binding protein of the present invention contains knob and valley mutations and one or more mutations to increase serum half-life. In some embodiments, the first and/or second Fc regions are human IgG1 Fc regions. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions.

[0179] In some embodiments, the binding protein of the present invention contains one or more mutations to reduce effector function, for example, antibody-dependent cellular phagocytosis mediated by Fc receptors (ADCP), complement-dependent cytotoxicity (CDC) and/or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the second polypeptide chain further comprises a first Fc region linked to C_H1, while the first Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain; where the third polypeptide chain additionally contains a second Fc region linked to C_H1, while the second Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain; wherein the first and second Fc regions are human IgG1 Fc regions; and wherein each of the first and second Fc regions contains amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG1 according to the EU index, where the amino acid substitutions are L234A and L235A. In some embodiments, the Fc regions of the second and third polypeptide chains are Fc regions of human IgG1, and wherein each of the Fc regions contains amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG1 according to the EU index, wherein the amino acid substitutions the replacements are L234A and L235A. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to C_H1, while the first Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain; where the third polypeptide chain additionally contains a second Fc region linked to C_H1, while the second Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain; wherein the first and second Fc regions are human IgG1 Fc regions; and wherein each of the first and second Fc regions contains amino acid substitutions at positions corresponding to positions 234, 235 and 329 of human IgG1 according to the EU index, where the amino acid substitutions are L234A, L235A and P329A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG1 Fc regions, and wherein each of the Fc regions comprises amino acid substitutions at positions corresponding to human IgG1 positions 234, 235, and 329 according to the EU index, wherein the amino acid substitutions are L234A, L235A and P329A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG4 Fc regions, and each of the Fc regions comprises amino acid substitutions at positions corresponding to human IgG4 positions 234 and 235 according to the EU index, wherein the amino acid substitutions represent are F234A and L235A. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to C_H1, while the first Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain, and wherein the third polypeptide chain additionally contains a second Fc region linked to C_H1, while the second Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain; and wherein each of the first and second Fc regions contains amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, where the amino acid substitutions are F234A and L235A.

[0180] In some embodiments, the binding protein of the present invention contains knob and trench mutations and one or more mutations to reduce effector function. In some embodiments, the first and/or second Fc regions are human IgG1 Fc regions. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions. For further description of Fc mutations at position 329, see, for example, Shields, R.L. et al.(2001) J Biol. Chem.276:6591-6604 and WO1999051642.

[0181] In some embodiments, the types of mutations described above can be combined in any order or combination. For example, the binding protein of the present invention may contain two or more knob and valley mutations, one or more mutations to increase serum half-life, one or more mutations to improve IgG4 stability, one or more mutations to improve purifications and/or one or more mutations to reduce effector function described above.

Nucleic acids

[0182] Standard recombinant DNA technologies are used to construct polynucleotides that encode polypeptides that form binding proteins, insert these polynucleotides into recombinant expression vectors, and introduce such vectors into host cells. Cm., eg Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 3rd ed.). Enzymatic reactions and purification procedures can be carried out in accordance with manufacturers' specifications that are routinely performed in the art or as described herein. Unless specific definitions are provided, the terminology used in the context of analytical chemistry, synthetic organic chemistry and medicinal and pharmaceutical chemistry, as well as the laboratory procedures and techniques described herein, are well known and widely used in the art. Likewise, conventional techniques can be used for chemical synthesis options, chemical analysis options, pharmaceutical preparation, formulation, delivery and patient treatment.

[0183] Other aspects of the present invention relate to isolated nucleic acid molecules containing a nucleotide sequence encoding any of the binding proteins described herein. In some embodiments, the isolated nucleic acid molecules contain a sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 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 sequences under SEQ ID NO:60-83 and/or presented in table J.

[0184] Certain aspects of the present invention relate to sets of polynucleotides. In some embodiments, the one or more polynucleotides are a vector (for example, expression vector). The kits may find use, among other things, in the production of one or more of the binding proteins described herein, for example, trispecific binding protein of the present invention. In some embodiments, the kit contains one, two, three, or four polynucleotides shown in Table J (For example, antiCD38_C2-CD38-1_VH1-VL1xCD28supxCD3mid IgG4 FALA, antiCD38_C2-CD38-1_VH1-VL1xCD28supxCD3mid IgG1LALA P329A, antiCD38_C2-CD38-1_VH1-VL1xCD28supxCD3mid IgG1 NNSA, CD38_HHY1370xCD28supxCD3mid IgG4 FALA, CD38_HHY1370xCD28supxCD3mid IgG1LALA P329A or CD38_HHY1370CD28supxCD3mid IgG1 NNSA). In some embodiments, the set of polynucleotides comprises a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide containing the sequence of SEQ ID NO:72, a third polynucleotide containing the sequence of SEQ ID NO:74, and a fourth polynucleotide containing the sequence of SEQ ID NO:75. In some embodiments, the set of polynucleotides comprises a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide containing the sequence of SEQ ID NO:76, a third polynucleotide containing the sequence of SEQ ID NO:77, and a fourth polynucleotide containing the sequence of SEQ ID NO:75. In some embodiments, the set of polynucleotides comprises a first polynucleotide containing the sequence of SEQ ID NO:73, a second polynucleotide containing the sequence of SEQ ID NO:78, a third polynucleotide containing the sequence of SEQ ID NO:79, and a fourth polynucleotide containing the sequence of SEQ ID NO:75. In some embodiments, the set of polynucleotides comprises a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide containing the sequence of SEQ ID NO:72, a third polynucleotide containing the sequence of SEQ ID NO:80, and a fourth polynucleotide containing the sequence of SEQ ID NO:81. In some embodiments, the set of polynucleotides comprises a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide containing the sequence of SEQ ID NO:76, a third polynucleotide containing the sequence of SEQ ID NO:82, and a fourth polynucleotide containing the sequence of SEQ ID NO:81. In some embodiments, the set of polynucleotides comprises a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide containing the sequence of SEQ ID NO:78, a third polynucleotide containing the sequence of SEQ ID NO:83, and a fourth polynucleotide containing the sequence of SEQ ID NO:81.

[0185] In some embodiments, the isolated nucleic acid is operably linked to a heterologous promoter to drive transcription of the nucleic acid sequence encoding the binding protein. A promoter may refer to nucleic acid control sequences that direct transcription of the nucleic acid. A first nucleic acid sequence is operably linked to a second nucleic acid sequence, wherein the first nucleic acid sequence is operably linked to a second nucleic acid sequence. For example, a promoter is operably linked to a binding protein coding sequence if the promoter affects transcription or expression of the coding sequence. Examples of promoters may include, but are not limited to, promoters derived from the genomes of viruses (such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, simian virus 40 (SV40), etc.), heterologous eukaryotic promoters (such as actin gene promoter, immunoglobulin gene promoter, heat shock protein gene promoters, etc.), CAG promoter (Niwa et al., Gene 108(2 ):193-9, 1991), phosphoglycerate kinase (PGK) gene promoter, tetracycline inducible promoter (Masui et al., Nucleic Acids Res. 33:e43, 2005), lac system, trp system, tac system, trc- system, the main operator and promoter regions of lambda phage, the promoter of the 3-phosphoglycerate kinase gene, the promoters of the yeast acid phosphatase gene and the promoter of the yeast mating factor alpha gene. The polynucleotides encoding the proteins of the present invention may be under the control of a constitutive promoter, an inducible promoter, or any other suitable promoter described herein, or other suitable promoter that is readily recognized by one of ordinary skill in the art.

[0186] In some embodiments, the isolated nucleic acid is inserted into a vector. In some embodiments, the vector is an expression vector. Expression vectors may contain one or more regulatory sequences operably linked to the polynucleotide to be expressed. The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g. polyadenylation signals). Examples of suitable enhancers may include, but are not limited to, enhancer sequences from mammalian genes (such as globin, elastase, albumin, α-fetoprotein, insulin, etc.) and enhancer sequences from eukaryotic cell virus (such as the SV40 late origin enhancer (100-270 bp), early promoter/enhancer of cytomegalovirus, polyoma enhancer at the late origin of replication, enhancers of adenoviruses, etc.). Examples of suitable vectors may include, for example, plasmids, cosmids, episomes, transposons, and viral vectors (e.g. vectors based on adenovirus, vaccinia virus, Sindbis virus, measles virus, herpes virus, lentivirus, retrovirus, adeno-associated virus, etc.). Expression vectors can be used to transfect host cells, such as, for example, bacterial cells, yeast cells, insect cells and mammalian cells. Biologically functional viral DNA and plasmid DNA vectors capable of expression and replication in a host are known in the art and can be used to transfect any cell of interest.

[0187] Other aspects of the present invention relate to a vector system comprising one or more vectors encoding the first, second, third and fourth polypeptide chains of any of the binding proteins described herein. In some embodiments, the vector system comprises a first vector encoding a first polypeptide chain of the binding protein, a second vector encoding a second polypeptide chain of the binding protein, a third vector encoding a third polypeptide chain of the binding protein, and a fourth vector encoding a fourth polypeptide chain of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and second polypeptide chains of the binding protein and a second vector encoding the third and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and third polypeptide chains of the binding protein and a second vector encoding the second and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and fourth polypeptide chains of the binding protein, and a second vector encoding the second and third polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first, second, third, and fourth polypeptide chains of the binding protein. The one or more vectors of the vector system may be any of the vectors described herein. In some embodiments, the one or more vectors are expression vectors.

Isolated host cells

[0188] Other aspects of the present invention relate to an isolated host cell containing one or more isolated polynucleotides, sets of polynucleotides, vectors and/or vector systems described herein. In some embodiments, the host cell is a bacterial cell (e.g. cell E. coli). In some embodiments, the host cell is a yeast cell (e.g. cell S. cerevisiae). In some embodiments, the host cell is an insect cell. Examples of insect host cells may include, for example, cells Drosophila (For example, S2 cells), cells Trichoplusia ni (For example, High Five cells^™) and cells Spodoptera frugiperda (For example, Sf21 or Sf9 cells). In some embodiments, the host cell is a mammalian cell. Examples of mammalian host cells may include, for example, human embryonic kidney cells (e.g. 293 cells or 293 cells subcloned for growth in suspension culture), Expi293™ cells, CHO cells, neonatal hamster kidney cells (e.g. BHK, ATCC CCL 10), mouse Sertoli cells (e.g. TM4 cells), monkey kidney cells (e.g. CV1 ATCC CCL 70), African green monkey kidney cells (e.g. VERO-76, ATCC CRL-1587), human cervical carcinoma cells (e.g. HELA, ATCC CCL 2), canine kidney cells (e.g. MDCK, ATCC CCL 34), Buffalo rat liver cells (e.g. BRL 3A, ATCC CRL 1442), human lung cells (e.g. W138, ATCC CCL 75), human liver cells (e.g. Hep G2, HB 8065), mouse mammary tumor cells (e.g. MMT 060562, ATCC CCL51), TRI cells, MRC 5 cells, FS4 cells, human hepatoma cell line (e.g. Hep G2) and myeloma cells (eg NS0 and Sp2/0 cells).

Methods and uses of binding proteins

[0189] Certain aspects of the present invention relate to methods for expanding virus-specific memory T cells. In some embodiments, the methods comprise contacting a virus-specific memory T cell with a binding protein of the present invention, for example , a trispecific binding protein that comprises a first antigen binding site that binds a CD28 polypeptide, a second antigen binding site that binds a CD3 polypeptide, and a third antigen binding site that binds the CD38 polypeptide.

[0190] In some embodiments, a virus-specific memory T cell is contacted with a binding protein in vitro or ex vivo .

[0191] In some embodiments, contacting a virus-specific memory T cell with a binding protein causes activation and/or proliferation of virus-specific memory T cells.

[0192] Other aspects of the present invention relate to methods for expanding T cells. In some embodiments, the methods include contacting the T cell with a binding protein of the present invention, for example , a trispecific binding protein that contains a first antigen binding site that binds a CD28 polypeptide, a second antigen binding site that binds a CD3 polypeptide, and a third antigen binding site. which binds the CD38 polypeptide.

[0193] In some embodiments, the T cell is a memory T cell or an effector T cell.

[0194] In some embodiments, the T cell expresses a chimeric antigen receptor (CAR) on its cell surface or contains a polynucleotide encoding a CAR.

[0195] Other aspects of the present invention relate to methods of treating a chronic viral infection, for example , in an individual in need thereof. In some embodiments, the methods comprise administering to an individual in need thereof an effective amount of a binding protein of the present invention, for example , a trispecific binding protein that comprises a first antigen binding site that binds a CD28 polypeptide, a second antigen binding site that binds a CD3 polypeptide, and a third antigen binding site. site that binds the CD38 polypeptide.

[0196] In some embodiments, the individual is a human.

[0197] In some embodiments, the binding protein is administered to an individual in a pharmaceutical composition comprising the binding protein and a pharmaceutically acceptable carrier.

[0198] In some embodiments, administration of the binding protein results in the activation and/or proliferation of virus-specific memory T cells in an individual.

[0199] In any of the above methods, the memory T cells may be CD8+ or CD4+ memory T cells. In any of the above methods, the memory T cells may be central memory T cells (T _CM ) or effector memory T cells (T _EM ).

[0200] Any of the binding proteins described herein may find use in the methods of the present invention.

[0201] Binding proteins can be used in any known assay, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays to detect and quantify one or more target antigens. Binding proteins will bind one or more target antigens with an affinity that is appropriate for the assay used.

[0202] For diagnostic applications, in certain embodiments, binding proteins can be labeled with a detectable moiety. The detectable fragment can be any fragment that is capable of directly or indirectly generating a detectable signal. For example, the detected fragment may be a radioisotope such as ³ H, ¹⁴ C, ³² P, ³⁵ S, ¹²⁵ I, ⁹⁹ Tc, ¹¹¹ In or ⁶⁷ Ga; a fluorescent or chemiluminescent compound such as fluorescein isothiocyanate, rhodamine or luciferin; or an enzyme such as alkaline phosphatase, β-galactosidase or horseradish peroxidase.

[0203] Binding proteins are also useful for in vivo imaging. A binding protein labeled with a detectable fragment can be administered to an animal, preferably into the bloodstream, and the presence and position of the labeled antibody in the host analyzed. The binding protein can be labeled with any moiety that can be detected in the animal by nuclear magnetic resonance, x-ray, or other detection methods known in the art.

[0204] For clinical or research applications, in some embodiments, the binding proteins may be conjugated to a cytotoxic agent. A variety of cytotoxic agent-related antibodies have been used to target cytotoxic payloads to specific tumor cells (i.e. antibody-drug conjugates). Cytotoxic agents and linkers that provide conjugation of the agent to the antibody are known in the art; see for example Parslow, A.C. et al.(2016) Biomedicines 4:14 and Kalim, M. et al.(2017) Drug Des. Devel. Ther.11:2265-2276.

[0205] The present invention also provides a kit containing a binding protein and other reagents useful for detecting levels of target antigens in biological samples. Such reagents may include detection tag, serum blocking, positive and negative control samples, and detection reagents. In some embodiments, the kit contains a composition comprising any binding protein, polynucleotide, vector, vector system, and/or host cell described herein. In some embodiments, the kit includes a container and a label or package insert on or with the container. Suitable containers include, for example, bottles, vials, syringes, IV bags, etc. Containers can be made from a number of materials, such as glass or plastic. The container contains a composition that, alone or in combination with another composition, is effective for treating, preventing and/or diagnosing a condition and may have a sterile opening (for example, the container may be a bag of intravenous solution or a vial with a stopper that can be pierced with a hypodermic needle). In some embodiments, the label or package insert states that the composition is used to prevent, diagnose, and/or treat a preferred condition. Alternatively or additionally, the article or kit may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline Ringer's solution, and dextrose solution. It may further include other substances required from a commercial and consumer point of view, including other buffers, diluents, fillers, needles and syringes.

Therapeutic compositions based on binding proteins and their administration

[0206] Therapeutic or pharmaceutical compositions containing binding proteins are within the scope of the present invention. Such therapeutic or pharmaceutical compositions may contain a therapeutically effective amount of a binding protein or binding protein-drug conjugate in admixture with a pharmaceutically or physiologically acceptable formulation agent selected in accordance with the route of administration. These pharmaceutical compositions may find use in any of the methods and applications described herein ( eg , ex vivo, in vitro and/or in vivo ).

[0207] Suitable formulation agents are preferably non-toxic to patients receiving them at the doses and concentrations used.

[0208] The pharmaceutical composition may contain formulation agents designed to modify, maintain or maintain, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or permeation of the composition. Suitable formulation agents include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine), antimicrobial agents, antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen sulfite), buffers (such as borate, bicarbonate, Tris-HCl , citrates, phosphates or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin ), fillers, monosaccharides, disaccharides and other carbohydrates (such as glucose, mannose or dextrins), proteins (such as serum albumin, gelatin or immunoglobulins), colors, flavors and diluents, emulsifiers, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides , salt-forming counterions (such as sodium ion), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide), solvents (such as glycerin, propylene glycol or polyethylene glycol ), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitol esters; polysorbates such as polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxapol), stability enhancers (such as sucrose and sorbitol), tonicity enhancers (such as alkali metal halides - preferably sodium or potassium chloride - or mannitol, sorbitol), delivery media, diluents, excipients and/ or pharmaceutical adjuvants (see for example, Remington's Pharmaceutical Sciences (18th Ed., A.R. Gennaro, ed., Mack Publishing Company 1990) and its subsequent editions, which are incorporated herein by reference for all purposes).

[0209] The optimal pharmaceutical composition will be determined by one skilled in the art depending, for example, on the intended route of administration, delivery format, and dosage required. Such compositions may be influenced by the physical state, stability, in vivo release rate, and in vivo release rate of the binding protein.

[0210] The base medium or carrier in the pharmaceutical composition may be aqueous or non-aqueous in nature. For example, a suitable vehicle or vehicle for injection may be water, physiological saline, or artificial cerebrospinal fluid, possibly with the addition of other substances conventional for compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are additional exemplary media. Other exemplary pharmaceutical compositions contain a Tris buffer with a pH of approximately 7.0-8.5, or an acetate buffer with a pH of approximately 4.0-5.5, which may further include sorbitol or a suitable substitute. In one embodiment of the present invention, binding protein compositions can be prepared for storage in the form of a lyophilized mass or an aqueous solution by mixing the selected composition at the required degree of purity with optional formulation aids. In addition, the binding protein can be formulated in the form of a lyophilisate using suitable excipients such as sucrose.

[0211] The pharmaceutical compositions of the present invention may be intended for parenteral or subcutaneous delivery. Alternatively, the compositions may be intended for inhalation or for delivery through the digestive tract, for example, orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the person skilled in the art.

[0212] The components of the formulation are present in concentrations that are acceptable at the site of administration. For example, buffers are used to maintain the pH of the composition at physiological levels or at slightly lower pH levels, typically in the pH range of about 5 to about 8.

[0213] If parenteral administration is contemplated, the therapeutic compositions to be administered may be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired binding protein in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, with which the binding protein is formulated as a sterile isotonic solution with suitable preservatives. Another formulation may be formulated based on the desired molecule with a vehicle, such as injectable microspheres, biodegradable particles, polymeric compounds (such as polylactic acid or polyglycolic acid), granules or liposomes, which provides a controlled or sustained release of the product, which can then be delivered via depot injection. Hyaluronic acid can also be used and may have the effect of providing a long stay in the bloodstream. Other suitable means for administering the desired molecule include implantable drug delivery devices.

[0214] In one embodiment, the pharmaceutical composition may be formulated for inhalation. For example, the binding protein may be formulated as a dry powder for inhalation. Binding protein inhalation solutions can also be formulated with a propellant for aerosol delivery. In yet another embodiment, the solutions can be sprayed.

[0215] It is also contemplated that certain formulations may be administered orally. In one embodiment of the present invention, the binding proteins that are administered in this manner can be formulated with or without such carriers as are typically used in the formulation of solid dosage forms such as tablets and capsules. For example, the capsule may be designed to release the active portion of the formulation upon entry into the digestive tract, where bioavailability is maximized and disintegration prior to entry into the systemic circulation is minimized. Additional substances may be included to facilitate absorption of the binding protein. Diluents, flavoring agents, low-melting waxes, vegetable oils, lubricants, suspending agents, disintegrating agents and binders may also be used.

[0216] Another pharmaceutical composition may include an effective amount of binding proteins in admixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water or other suitable medium, solutions can be prepared in unit dose form. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate; or binders such as starch, gelatin or gum acacia; or lubricants such as magnesium stearate, stearic acid or talc.

[0217] Additional pharmaceutical compositions of the present invention will be apparent to those skilled in the art, including formulations comprising binding proteins in a sustained or controlled delivery form. Techniques for formulating a number of other delayed or controlled delivery agents, such as liposomal carriers, biodegradable microparticles or porous granules and depot injection forms, are also known to those skilled in the art. Additional examples of sustained release formulations include semi-permeable polymer matrices in the form of shaped articles, e.g. films or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, poly(2-hydroxyethyl methacrylate), ethylene vinyl acetate or poly-D(-)-3-hydroxybutyric acid. Sustained release compositions may also include liposomes, which can be prepared by any of several methods known in the art.

[0218] Pharmaceutical compositions to be used for in vivo administration generally must be sterile. This can be achieved by filtration through sterile filtration membranes. If the composition is lyophilized, then sterilization using this method can be carried out either before or after lyophilization and reconstitution. The composition for parenteral administration may be stored in lyophilized form or in solution form. In addition, compositions for parenteral administration are typically placed in a container having a sterile entry port, such as an intravenous solution bag or vial with a stopper that can be pierced with a hypodermic injection needle.

[0219] Once the pharmaceutical composition is formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations can be stored either in a ready-to-use form or in a form (eg, lyophilized) that requires reconstitution before administration.

[0220] The present invention also includes kits for preparing a single dose unit. Each of the kits may contain both a first container with a dried protein and a second container with an aqueous composition. Also included within the scope of the present invention are kits containing single and multi-chamber prefilled syringes (e.g. syringes with liquid and syringes with lyophilisate).

[0221] The effective amount of the binding protein pharmaceutical composition to be used therapeutically will depend, for example, on the therapeutic case and objectives. One skilled in the art will appreciate that suitable dosage levels for treatment will therefore vary depending in part on the molecule being delivered, the indication for which the binding protein will be used, the route of administration and metric characteristics (body weight, body surface area or organ size) and condition (age and general health) of the patient. Accordingly, the doctor can select the dose and modify the route of administration to obtain the optimal therapeutic effect.

[0222] The frequency of dosing will depend on the pharmacokinetic parameters of the binding protein in the formulation to be used. Typically, the physician will administer the composition until the dosage reaches a level at which the desired effect is achieved. Therefore, the composition can be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over a period of time, or as a continuous infusion via an implantable device or catheter. Further determination of the appropriate dosage is performed in a standard manner by those of ordinary skill in the art and is within the range of tasks that they typically perform. Suitable dosages can be determined using appropriate dose-response data.

[0223] The route of administration of the pharmaceutical composition corresponds to known methods, for example, orally; by injection via intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; through sustained release systems or through implantable devices. If necessary, the compositions can be administered as a bolus injection or continuously by infusion or via an implantable device.

[0224] The composition can also be administered topically by implantation of a membrane, sponge, or other suitable material into which the desired molecule has been absorbed or encapsulated. If an implantable device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be through diffusion, bolus administration with time-controlled release, or continuous administration.

EXAMPLES

[0225] The following examples illustrate specific embodiments of the present invention and various applications thereof. They are provided for explanatory purposes only and should not be construed as limiting the scope of the present invention in any way.

[0226] The following terminology may be used interchangeably in examples and graphics provided herein to refer to specific antigen binding domains or antibodies to CD38:

antiCD38_C2-CD38-1_VH1-VL1 or CD38 _VH1

antiCD38_1370 or CD38 _HHY1370

antiCD38_SB19 or isatuximab.

Example 1: Cross-reactivity and induction of apoptosis by anti-CD38 antibodies

[0227] Humanized variants of the anti-CD38 antibody were characterized for binding to human and cynomolgus CD38 polypeptides and inducing apoptosis.

Materials and methods

Binding affinity for soluble extracellular domains of CD38

[0228] The binding properties of CD38 mAbs were assessed using BIAcore 2000 (BIAcore Inc., Uppsala, NJ). Briefly, the CM5 BIAcore biosensor chip was fixed in this instrument and activated with 250 μL of 1:1 NHS/EDC at room temperature. Mouse anti-human IgG1 Fc antibody (GE Healthcare, no. BR-1008-39) (13.5 μg/ml in 0.05 M acetate buffer, pH 5) was immobilized on activated chips in flow cells 1. Immobilization was carried out at a flow rate of 5 µl/min. The chip was then blocked by injecting 55 μl ethanolamine-HCl, pH 8.5, followed by washing five times with 50 mM NaOH, 1 M NaCl. To measure the binding of anti-CD38 mAb to human CD38 or cynomolgus CD38, antibodies were used at a concentration of 2 μg/ml in BIAcore running buffer (HBS-EP). Antigens (human CD38-his-tag (ID2) or cynomolgus CD38-his-tag (ID3)) were administered at 3 to 1000 nM. After completion of the injection phase, dissociation was monitored in BIAcore running buffer at the same flow rate for 360 s. The surface was regenerated between injections using 30 μl of 50 mM NaOH-1 M NaCl. Individual sensorgrams were analyzed using BIAsimulation software.

Binding affinity for B cell progenitors expressing human CD38

[0229] The binding of anti-CD38 antibodies to CD38 expressed on the surface of recombinant mouse preB::300.19 cells was determined by flow cytometry. The recombinant cell line was described in J. Deckket et al. 2014 Clin. Cancer Res 20:4574–4583. Mouse preB::300.19 CD38-expressing cells were transferred at 40,000 cells/well into a 96-well High Bind plate (MSD L15XB-3) and 100 μl/well of anti-CD38 antibody was added for 45 minutes at 4°C and washed three times. using PBS 1% BSA. 100 μl/well of goat anti-human IgG conjugated to Alexa488 (Jackson ImmunoResearch; no. 109-545-098) was added over 45 min at 4°C and washed three times with PBS 1% BSA. Antibody binding was assessed after centrifugation and resuspension of cells by adding 200 μl/well of 1% BSA PBS and reading using a Guava® easyCyte™ 8HT flow cytometry system. Apparent KD and EC50 values were estimated using BIOST@T-BINDING and BIOST@T-SPEED programs, respectively.

Recombinant CD38 proteins

[0230] Various recombinant CD38 proteins were used, derived from isoform A, with different tags and point mutations (SEQ ID NO: 2, 3, 4 and 28), and a tagged version of CD38 isoform E (SEQ ID NO: 105), covering the extracellular CD38 domain R45-P203. Proteins were produced by transient expression in mammalian cells. Coding DNA sequences were cloned into mammalian expression plasmids under the influence of the CMV enhancer/promoter and SV40 polyA signals. HEK293 cells (Invitrogen; #K9000-10) were transiently transfected with expression plasmids using the FreeStyle™ MAX 293 expression system according to the manufacturer's instructions.

Apoptosis Induction Assay

[0231] Cells were incubated at 2 x 10 ⁵ cells/ml in complete medium (RPMI-1640, 10% FBS, 2 mM L-glutamine) with 1.5 μg/ml (10 nM) of the indicated antibodies for 20 hours at 37 °C with 5% CO2. Cells were stained with Annexin V-FITC according to the manufacturer's instructions (Life Technologies). Samples were analyzed by flow cytometry on a BD FACSAria™ flow cytometer using BD FACSDiva software to control data acquisition and analysis (both BD Biosciences).

ELISA assays

[0232] 96-well plates were coated with CD38 at 0.5 μg/well in PBS and antibodies were added to the plate at a concentration of 100 μl/well of antibodies. The plate was incubated at 37°C for 1 hour and washed five times with PBS containing 0.05% Tween-20 (PBS-T). Then, 100 μl of a 1:25,000 dilution of human anti-IgG antibody conjugated to horseradish peroxidase (Jackson Ref: 109-035-098) was added to each well. After incubation at 37°C for 1 h in the dark, the plates were washed with PBS-T five times. Antibody binding was visually monitored by adding TMB-H ₂ O ₂ buffer and read at 450 nm. EC50 values were estimated using BIOST@T-SPEED software.

results

[0233] The binding properties of selected anti-human CD38 antibodies to soluble human CD38 and cynomolgus CD38 were examined using ELISA and surface plasmon resonance (SPR) analysis using the BIAcore system (Pharmacia Biosensor; Piscataway, NJ). ELISA data were used to determine the EC50 binding of human and cynomolgus CD38 antibodies for humanized anti-CD38 antibodies antiCD38_C2-CD38-1_VH1-VL1, antiCD38_C2-CD38-1_VH3-VL3, antiCD38_C2-CD38-1_VH5-VL3, antiCD38_C2-CD38-1_VH6 -VL3 and human anti-CD38 antibody antiCD38_1370.

[0234] The binding of humanized variants of anti-CD38 antibodies or human anti-CD38 mAb to CD38 was also assessed using SPR assays. SPR assay data were used to determine the K _D and k _{off of} antibody binding to human and cynomolgus CD38 for humanized anti-CD38 antibodies antiCD38_C2-CD38-1_VH1-VL1, antiCD38_C2-CD38-1_VH3-VL3, antiCD38_C2-CD38-1_VH5-VL3, antiCD38_C2 -CD38-1_VH6-VL3 and human anti-CD38 antibody antiCD38_1370. The binding data summarized in Table K shows that all CD38 mAbs bind to CD38 with similar binding characteristics.

Table K. Binding affinity of CD38 mAb to the soluble extracellular domain of human CD38 and cynomolgus CD38 determined by surface plasmon resonance

hCD38-his (SEQ ID NO:2) cCD38-his (SEQ ID NO:4) Kd (s-1) KD(M) Kd (s-1) KD(M) antiCD38_C2-CD38-1 2.66E-04 3.36E-10 9.85E-05 3.90E-10 antiCD38_C2-CD38-1_VH1-VL1 3.90E-04 3.32E-10 7.84E-04 3.44E-09 antiCD38_C2-CD38-1_VH3-VL3 2.83E-04 4.83E-10 1.29E-04 7.10E-10 antiCD38_C2-CD38-1_VH5-VL3 5.29E-04 8.22E-10 2.01E-04 1.14E-09 antiCD38_C2-CD38-1_VH6-VL3 3.33E-04 3.12E-10 1.25E-04 5.63E-10 antiCD38_1370 2.03E-04 1.44E-09 1.90E-04 1.38E-09

[0235] The ability of humanized anti-CD38 antibody variants to bind to cells expressing CD38 was assessed using the FACS-based binding assay described above. FACS data were used to determine EC50 antibody binding to human and cynomolgus CD38 for humanized anti-CD38 antibodies antiCD38_C2-CD38-1_VH1-VL1, antiCD38_C2-CD38-1_VH3-VL3, antiCD38_C2-CD38-1_VH5-VL3, antiCD38_C2-CD38-1_VH6 -VL3 and human anti-CD38 antibody antiCD38_1370. The binding assay data shown in Table L shows that all humanized anti-CD38 antibody variants exhibited similar binding affinities to cell surface CD38.

Table L. Binding affinity of CD38 mAb to CD38-expressing murine preB::300.19 cells

Apparent KD at FACS (M) hCD38-expressing cells cCD38-expressing cells antiCD38_C2-CD38-1 2.80E-10 2.20E-10 antiCD38_C2-CD38-1_VH1-VL1 3.30E-10 7.50E-10 antiCD38_C2-CD38-1_VH3-VL3 7.80E-10 1.31E-09 antiCD38_C2-CD38-1_VH5-VL3 5.50E-10 1.15E-09 antiCD38_C2-CD38-1_VH6-VL3 6.80E-10 1.07E-09 antiCD38_1370 2.07E-09 1.14E-09

[0236] Binding assay data from the ELISA, SPR, and FACS assays above are summarized in Table L2, along with the sequence identity of the VH and VL domains to human V regions.

Table L2. Summary of ELISA, SPR and FACS assays characterizing the binding of these antibodies to human (Hu) and cynomolgus (Cyno) CD38 polypeptides.

INN
Nomenclature ELISA
EC50, nM SPR
KD, nM FACS
EC50, nM Identity with the human V region Hu Cyno Hu Cyno Hu Cyno Hu Cyno antiCD38_C2-CD38-1_VH1-VL1 -zumAb 0.11 0.10 0.33 3.44 0.33 0.76 84.7% 87.1% antiCD38_C2-CD38-1_VH3-VL3 -zumAb 0.16 0.15 0.48 0.71 0.78 1.31 83.7% 83.9% antiCD38_C2-CD38-1_VH5-VL3 -zumAb 0.16 0.17 0.82 1.14 0.55 1.15 80.6% 83.9% antiCD38_C2-CD38-1_VH6-VL3 -zumAb 0.14 0.14 0.31 0.56 0.68 1.06 81.6% 83.9% antiCD38_1370 -umAb 0.05 0.09 1.44 1.38 2.00 1.14 99.0% 95.8%

[0237] The ability of anti-CD38 antibodies to bind to both isoforms A and E of human CD38 was also examined. To assess binding to CD38 isoform A and isoform E, an enzyme-linked immunosorbent assay (ELISA) was performed using isoform A and isoform E proteins (prepared as described above) as capture antigen. 96-well plates were coated by adding each antibody isoform at a concentration of 0.5 μg/well in PBS and 100 μl/well. The plate was incubated at 37°C for 1 hour and washed five times with PBS containing 0.05% Tween-20 (PBS-T). Then, 100 μl of a 1:25,000 dilution of human anti-IgG antibody conjugated to horseradish peroxidase (Jackson Ref: 109-035-098) was added to each well. After incubation at 37°C for 1 h in the dark, the plates were washed with PBS-T five times. Antibody binding was visually monitored by adding TMB-H ₂ O ₂ buffer and read at 450 nm. EC50 values were estimated using BIOST@T-SPEED software.

[0238] The binding affinities of various antibodies to CD38 isoform A (SEQ ID NO:1) and isoform E (SEQ ID NO:105) were determined as shown in Table L3. Table M provides a comparison of binding properties for various anti-CD38 antibodies.

Table L3. Binding affinity of anti-CD38 antibodies to CD38 isoforms A and E based on EC50 according to ELISA analysis

Antibody CD38 isoform A, EC50 (nM) CD38 isoform E, EC50 (nM) antiCD38_C2-CD38-1 0.11 (CV 9%) 0.08 (CV 7%) antiCD38_C2-CD38-1_VH1-VL1 0.14 (CV 13%) 0.10 (CV 12%) antiCD38_1370 0.47 (CV 3.7%) 0.32 (CV 5%) antiCD38_SB19 0.10 (CV 7.1%) No binding

Table M. Binding characteristics of various anti-CD38 antibodies

Antibody to CD38 H11 (Santa Cruz) Daratumumab antiCD38_SB19 antiCD38_C2-CD38-1 antiCD38_1370 Binding to human CD38 isoform A + + + + + Binding to human CD38 isoform E + - - + + Binding to CD38 in cynomolgus monkey + - - + +

[0239] In conclusion, only antiCD38_C2-CD38-1 bound to both human and cynomolgus CD38 with subnanomolar affinity and bound to CD38 isoforms A and E.

Example 2: Preparation of Trispecific CD38 Binding Proteins

[0240] The binding properties of the antigen binding domains of the selected anti-CD38 antibodies described in Example 1 were then analyzed in the trispecific format shown in FIG. 1.

Materials and methods

Preparation and purification of trispecific binding proteins

[0241] Trispecific binding proteins were produced by transiently transfecting Expi293 cells with 4 expression plasmids using the ExpiFectamine™ 293 Transfection Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. Briefly, 25% (w/w) of each plasmid was diluted in Opti-MEM, mixed with prediluted ExpiFectamine reagent for 20-30 minutes at room temperature (RT) and added to Expi293 cells (2.5 x 10 ⁶ cells /ml). Transfection optimization to determine the best ratio of plasmids has often been used to obtain trispecific binding protein in good yield and purity.

[0242] 4-5 days after transfection, the culture supernatant of the transfected cells was collected and filtered through a 0.45 μm pore size filter block (Nalgene). Trispecific binding protein in the supernatant was purified using a 3-step procedure. In the first step, protein A affinity purification was used and bound Ab was eluted using “IgG elution buffer” (Thermo Fisher Scientific). In the second step, the product was dialyzed against PBS (pH 7.4) overnight with 2 changes of PBS buffer. Any precipitate was cleared by filtration through a 0.45 μm filter block (Nalgene) before the next step. In the third step, size exclusion chromatography (SEC) purification (Hiload 16/600 Superdex 200 pg or Hiload 26/600 Superdex 200 pg, GE Healthcare) was used to remove aggregates and other structures in the sample. Before pooling, fractions were analyzed by SDS-PAGE under reducing and non-reducing conditions to identify fractions containing monomeric trispecific binding protein. The purified antibody can be aliquoted and stored at -80°C long-term.

ELISA assays

[0243] The binding properties of the purified antibodies were analyzed using ELISA or SPR methods. For ELISA, the corresponding antigens for each binding site in the trispecific binding protein were used to coat a 96-well Immuno Plate (Nunc 439454, Thermo Fisher Scientific) overnight at 4°C using 2 μg/ml of each antigen in PBS (pH 7.4 ). The coated plate was blocked with 5% skim milk+2% BSA in PBS for one hour at RT, followed by three washes in PBS+0.25% Tween 20 (Aqua Max 400, Molecular Devices). Antibodies (trispecific and control Abs) were prepared in serial dilutions and added to ELISA plates (100 μl/well in duplicate), incubated at room temperature for an hour, followed by washing 5 times with PBS + 0.25% Tween 20.

[0244] After washing, HRP-conjugated anti-human Fab secondary antibody (1:5000, Cat# 109-035-097, Jackson ImmunoResearch Inc) was added to each well and incubated at RT for 30 minutes. After washing 5 times with PBS+0.25% Tween 20, 100 μl of TMB Microwell Peroxidase Substrate (KPL, Gaithersburg, MD, USA) was added to each well. The reaction was stopped by adding 50 μl of 1 MH ₂ SO ₄ and OD ₄₅₀ was measured using SpectraMax M5 (Molecular Devices) and analyzed using SoftMax Pro6.3 software (Molecular Devices). The final data were transferred to GraphPad Prism software (GraphPad Software, California, USA) and plotted as shown. EC50 was calculated using the same software.

[0245] To determine the binding of anti-CD38xCD28xCD3 trispecific antibodies or isotype control antibodies (human IgG4) to human CD3 (Cambridge Biologics LLC, cat. no. 03-01-0051), CD28 (Cambridge Biologics LLC, cat. no. 03-01 -0303) and CD38 (Cambridge Biologics LLC, cat. no. 03-01-0369) ELISA assay was used. Bound antibodies were detected using horseradish peroxidase (HRP)-conjugated anti-Fab secondary antibody (Jackson ImmunoResearch Inc., #109-035-097).

results

[0246] Using SPR, antigen binding domains that bind CD38 were examined in a trispecific format (binding CD38x-CD28x-CD3) for the ability to bind CD38 when other antigen binding domains are bound to their cognate ligands. To sequentially bind the three antigens to each trispecific Ab, a saturating concentration (>10 kDa) of each antigen was administered for 8 min, followed by dissociation for 5 min. Surface regeneration was performed by injecting 10 mM glycine-HCl pH 2.5 for 60 s at 30 μL/min. Data were fitted to a 1:1 kinetic binding model and analyzed using Biacore S200 Evaluation Software v 1.0. The equilibrium dissociation constant (K _D ) was calculated using the association rate constant (k _on ) and the dissociation rate constant (k _off ).

[0247] This SPR-based assay showed that the trispecific binding proteins were able to bind CD38 regardless of whether the CD3 and/or CD28 antigen binding domains were also bound to their cognate antigen. Kinetic parameters measured by SPR are presented in Table M2.

Table M2. Binding of trispecific binding proteins that bind CD38x-CD28x-CD3 to 1, 2 or 3 cognate antigens

State of the binding protein before CD38 binding k _a (M ^-1 s ^-1 ) k _d (s ^-1 ) _KD (M) No pre-binding 9.02E+05 1.42E-03 1.57E-09 Pre-binding to CD3 8.35E+05 1.24E-03 1.48E-09 Prebinding to CD28 7.39E+05 1.32E-03 1.79E-09 Pre-binding to CD3, then CD28 8.18E+05 1.23E-03 1.50E-09 Pre-binding to CD28, then CD3 8.37E+05 1.23E-03 1.47E-09

[0248] These results demonstrate that all three targets can simultaneously bind to trispecific binding proteins. Prebinding trispecific binding proteins to CD28, CD3, or both (in any order) did not alter binding kinetics or binding affinity to CD38.

[0249] Each antigen binding domain of the trispecific binding protein CD38 _SB19 xCD28 _sup xCD3 _mid was then assessed by SPR for the ability to bind the cognate antigen with and without two other antigen binding domains at saturation. Tables M3 and M4 show the results of these analyses.

Table M3. Binding of a target in the absence of other targets

Target k _a (M ^-1 s ^-1 ) k _d (s ^-1 ) _KD (M) CD38 8.04E+05 1.41E-03 1.75E-09 CD28 1.16E+05 3.14E-04 2.71E-09 CD3 2.90E+04 6.73E-04 2.32E-08

Table M4. Binding of a target to other targets upon saturation

Target k _a (M ^-1 s ^-1 ) k _d (s ^-1 ) _KD (M) CD38 5.93E+05 1.44E-03 2.42E-09 CD28 1.05E+05 3.96E-04 3.77E-09 CD3 1.27E+05 2.36E-03 1.86E-08

[0250] As shown in Tables M3 and M4, the presence of two targets loaded with pre-antigen binding did not affect the kinetics or binding affinity of the third target for CD38 or CD28. In the case of CD3 binding, prebound CD38 and/or CD28 resulted in faster kinetics (approximately 4-fold effect on k _on and k _off values).

[0251] CD38-binding antigen-binding domains were examined in a trispecific format with two CD28-binding antigen-binding domains (a superagonist, "sup" and a traditional agonist, "cvn") and two CD3-binding antigen-binding domains ("mid" and "low"). The variable domain sequences for these antigen binding domains are as follows: anti-CD28 _sup : SEQ ID NO:49 (VH) and SEQ ID NO:50 (VL); anti-CD28 _cvn : SEQ ID NO:51 (VH) and SEQ ID NO:52 (VL); anti-CD3 _mid : SEQ ID NO:53 (VH) and SEQ ID NO:54 (VL); anti-CD3 _low : SEQ ID NO:84 (VH) and SEQ ID NO:85 (VL). The results of SPR assays assessing the binding of trispecific binding proteins are shown in FIG. 2. The three CD38 binding domains had approximately the same binding affinities in the trispecific binding protein format as in the monospecific format. Both CD3 binding domains had approximately the same binding affinities in mono-, bi- and tri-specific formats. CD28 binding domains showed slightly lower (but still nanomolar) binding affinities in the bi- or trispecific format compared to the monospecific format. When the other two antigen binding domains were saturated, the CD38 _SB19 and CD28 _sup binding domains had similar binding affinities compared to when the other two antigen binding domains were not antigen bound. However, the CD3 _mid binding domain showed faster kinetics when the other two antigen binding domains were in a saturated state. These results demonstrate that the CD38, CD28 and CD3 binding domains are compatible for use in a trispecific binding protein format.

[0252] The CD38-binding antigen-binding domains, the preparation of which are described herein, were also compared to the existing CD38-binding antigen-binding domain, antiCD38_SB19 (see SEQ ID NO:47 for VH and SEQ ID NO:48 for VL sequences, respectively). Binding of the trispecific molecules to CD38 expressed on the surface of recombinant mouse preB::300.19 cells was determined by flow cytometry, and the corresponding monovalent anti-CD38 antibodies were analyzed in parallel. The recombinant cell line was described in J. Deckket et al. 2014 Clin. Cancer Res 20:4574–4583. Mouse preB::300.19 CD38-expressing cells were transferred at 40,000 cells/well into a 96-well High Bind plate (MSD L15XB-3), and 100 μl/well of trispecific molecules were added for 45 minutes at 4°C and washed three times with PBS 1% BSA. 100 μl/well of goat anti-human IgG conjugated to Alexa488 (Jackson ImmunoResearch; no. 109-545-098) was added over 45 min at 4°C and washed three times with PBS 1% BSA. Antibody binding was assessed after centrifugation and resuspension of cells by adding 200 μl/well of 1% BSA PBS and reading using a Guava® easyCyte™ 8HT flow cytometry system. Apparent KD and EC50 values were estimated using BIOST@T-BINDING and BIOST@T-SPEED programs, respectively.

[0253] Flow cytometry was used as described above to examine the binding of the antiCD38_SB19 antibody or trispecific binding protein to the CD38-binding antigen binding domain, antiCD38_SB19, to murine progenitor B cells expressing human or cynomolgus CD38 polypeptide on their cell surface . The trispecific binding protein CD38xCD28 _sup xCD3 _mid with the CD38-binding antigen-binding domain antiCD38_SB19 bound to cells expressing human CD38 with an apparent affinity that was 8-fold lower (EC50=4 nM) than the monospecific antibody antiCD38_SB19 (EC50=0.5 nM). Neither the monospecific antibody antiCD38_SB19 nor the trispecific antigen binding domain CD38 binding protein antiCD38_SB19 bound to cells expressing cynomolgus monkey CD38.

[0254] The binding domain of the humanized anti-CD38 antibody antiCD38_C2-CD38-1_VH1-VL1 was also tested in trispecific formats for binding to cells expressing human or cynomolgus CD38 polypeptides. Unlike Anticd38_SB19 TRISPECICIAL BETTERS CD38XCD28 _SUP XCD3 _MID and CD38XCD28 _CVN XCD3 _MID with an antigen-binding domain connecting CD38, ANTICD38_C2-CD38-1_VH1-VL1 C2-CD38-1_VH1-VL1 were able to bind polypeptides CD38 and humans, and the cynomolgus macaque. The trispecific binding protein CD38xCD28 _cvn xCD3 _mid with the CD38-binding antigen-binding domain antiCD38_C2-CD38-1_VH1-VL1 bound to cells expressing human CD38 with an apparent affinity that was 9-fold lower (EC50=4.4 nM) than the parent antibodies antiCD38_C2-CD38-1_VH1-VL1 (EC50=0.5 nM). Trispecific binding protein CD38xCD28 _cvn xCD3 _mid with CD38-binding antigen-binding domain antiCD38_C2-CD38-1_VH1-VL1 bound to cells expressing cynomolgus CD38 with an apparent affinity that was 7.5-fold lower (EC50=7.5 nM) than the parent antibody antiCD38_C2-CD38-1_VH1-VL1 (EC50=1 nM). The trispecific binding protein CD38xCD28 _sup xCD3 _mid with the CD38-binding antigen binding domain antiCD38_C2-CD38-1_VH1-VL1 bound to cells expressing human CD38 with an apparent affinity that was 2.5-fold lower (EC50=11 nM) than the trispecific binding protein CD38xCD28 _cvn xCD3 _mid with antigen binding domain binding CD38, antiCD38_C2-CD38-1_VH1-VL1 (EC50=4.4 nM).

[0255] The binding domain of the humanized anti-CD38 antibody antiCD38_1370 was also compared with the monospecific antibody antiCD38_1370 for binding to cells expressing human or cynomolgus CD38 polypeptides. Although the monospecific antibody antiCD38_1370 bound to cells expressing human (EC50=11.2 nM) or cynomolgus (EC50=6.6 nM) CD38 polypeptides in the nM range, trispecific binding protein CD38xCD28 _sup xCD3 _mid trispecific binding protein with an antigen binding domain, binding to CD38, antiCD38_1370 bound to cells expressing human or cynomolgus CD38 polypeptides in the absence of saturation.

[0256] In conclusion, the binding affinity of the trispecific binding protein CD38 _SB19 xCD28 _sup xCD3 _mid (CD38 binding domain, antiCD38_SB19) to human CD38 was found to be in the same range regardless of whether binding to recombinant human CD38 was examined. by SPR or with human CD38 expressed on the cell surface by flow cytometry (FIG. 3). Similarly, the affinity of the trispecific binding proteins CD38 _VH1 xCD28 _sup xCD3 _mid/low (CD38 binding domain, antiCD38_C2-CD38-1_VH1-VL1) and CD38 _VH1 xCD28 _cvn xCD3 _mid/low (CD38 binding domain, antiCD38_C2-CD38-1_VH1-VL1 ) for binding to human CD38 was also in the same range in both assays. For CD38 _HHY1370 xCD28 _sup xCD3 _mid (CD38 binding domain, antiCD38_1370), the _KD for human CD38 binding determined by SPR was 1 nM, whereas the exact EC50 value could not be assessed by flow cytometry (FIG. 3). A summary description of the apparent KD values (obtained by FACS analysis) for trispecific binding proteins with different CD38 binding domains is presented in Table M5.

Table M5. Summary of apparent KD values obtained by flow cytometry analysis

Apparent KD at FACS (M) hCD38-expressing cells cCD38-expressing cells Trispecific antibody with CD38 binding domain, antiCD38_C2-CD38-1_VH1-VL1 4.4 nM 7.5 nM Trispecific antibody with CD38 binding domain, antiCD38_1370 No saturation No saturation Trispecific antibody with CD38 binding domain, antiCD38_SB19 4 nM No binding Apparent KD at FACS (M) hCD38-expressing cells cCD38-expressing cells antiCD38_C2-CD38-1_VH1-VL1 0.5 nM 1 nM antiCD38_1370 11.2 nM 6.6 nM antiCD38_SB19 0.5 nM No binding

[0257] As expected, the ΔCD38xCD28 _sup xCD3 _mid trispecific binding protein, lacking a CD38 binding domain, did not bind to cells expressing human or cynomolgus CD38 polypeptides. This indicates that the binding observed in this assay was specific to the antigen binding domains of the anti-CD38 antibody.

Example 3: CD38/CD3xCD28 Ab Stimulates CD4 and CD8 Central Memory Cells, Th1 and Antigen-Specific Responses

[0258] To determine whether the CD38/CD3xCD28 trispecific Ab could enhance cellular immune function, the phenotype of in vitro expanded T cells was assessed.

Materials and methods

[0259] Peripheral blood mononuclear cells were isolated from healthy donor blood collected by Research Blood Components, LLC (Boston, MA). PBMCs were added to antibody-coated plates (350 ng/well) (5 x 10 ⁵ cells/ml) as described above and incubated at 37°C for 3 and 7 days. Cells were collected at specific time points and analyzed by flow cytometry for T cell subsets: naïve (CCR7+ CD45RO-), Tcm (CCR7+ CD45RO+), Tem (CCR7- CD45RO+), Treg (CD4+ Foxp3+ CD25hi). Cells were also treated with monensin (GolgiStop) (BD Biosciences, California) for at least 6 hours before staining for flow cytometry to determine the expression of intracellular cytokines: Th1 (CD4+ IFN-γ+), Th2 (CD4+ IL-4+) and Th17 (CD4+ IL-17+). CMV pp65-specific CD8+ T cells were detected using a fluorescent-conjugated pentamer, HLA-restricted to donor PBMCs (A*02:01/NLVPMVATV) (ProImmune, Oxford, UK). PBMCs were obtained from HemaCare (Van Nuys, CA) from donors with known CMV-positive populations and HLA types. PMBC from donors negative for the restrictive HLA type were used as negative controls. Staining was performed according to the manufacturer's protocol.

results

[0260] Human PBMCs from CMV-infected donors were incubated for 7 days with a trispecific Ab or trispecific antigen binding site mutant antibody as a negative control in the absence of cytokines. Analysis of CD4 subsets revealed the greatest proliferation in the pool of central memory cells, with a smaller increase in the number of effector memory cells (FIG. 4A). Analysis of CD4 subsets also revealed the greatest proliferation of Th1 cells (>6-fold) compared with Th2 or Th17 cells (FIG. 4B). In the CD8 subset, a >150-fold increase in the CD8 subset of central memory cells was observed by day 7, with a smaller increase in effector memory cells (FIG. 4C). Importantly, pre-existing antigen-specific CD8 responses to CMV targeting the pp65 epitope in HLA-A2 seropositive donors using tetramer staining (Gratama JW, van Esser JW, et al. Blood 98:1358-1364(2001)) increased >44-fold in the presence of CD38 trispecific protein antibody compared with the negative control (FIG. 4D).

[0261] Taken together, these data indicate that CD38 trispecific Ab stimulates Th1 function and protective memory CD8 T cell responses that can enhance antitumor and antiviral immunity in vivo .

Example 4 CD38/CD28xCD3 trispecific antibodies provide a CMV-specific immune response

[0262] Activation and/or proliferation of viral antigen-specific T cells may provide a therapeutic strategy against viral infections, such as those caused by cytomegalovirus (CMV). The ability of CD38/CD28xCD3 trispecific antibodies to ensure the activation and expansion of CMV-specific T cells was determined.

Materials and methods

ELISA assays

[0263] Binding affinities for each target antigen by CD38/CD28xCD3 T cell recruiters were measured using ELISA. Briefly, each antigen was used to coat a 96-well Immuno Plate (Thermo Fisher Scientific) overnight at 4°C using 200 ng/well in PBS (pH 7.4) of each antigen. The coated plate was blocked with 5% skim milk+2% BSA in PBS for one hour at RT, followed by three washes in PBS+0.25% Tween 20 (Aqua Max 400, Molecular Devices). Antibodies (trispecific and control Abs) were prepared in serial dilutions and added to ELISA plates (100 μl/well in duplicate), incubated at room temperature for an hour, followed by washing 5 times with PBS + 0.25% Tween 20. After washing, HRP-conjugated anti-human Fab secondary antibody (1:5000, cat. no. 109-035-097, Jackson ImmunoResearch Inc) was added to each well and incubated at room temperature for 30 minutes. After washing 5 times with PBS+0.25% Tween 20, 100 μl of TMB Microwell Peroxidase Substrate (KPL, Gaithersburg, MD, USA) was added to each well. The reaction was stopped by adding 50 µl of 1 M H2SO4, OD450 was measured using SpectraMax M5 (Molecular Devices) and analyzed using SoftMax Pro6.3 software (Molecular Devices). The final data were transferred to GraphPad Prism software (GraphPad Software, California, USA) and plotted. EC50 was calculated using the same software.

SPR assays

[0264] Human CD38-His antigens (Cambridge Biologics, Cambridge, MA) were used for the full kinetic analysis. Kinetic characterization of purified antibodies was performed using SPR technology on a BIACORE 3000 (GE Healthcare). A capture assay was used using capture with human IgG1-specific antibodies and the orientation of the antibodies of interest. A human antibody capture kit (GE Healthcare) was used to capture Fc protein constructs. A His-binding antibody capture kit (GE Healthcare) was used to capture His-tagged antigen. Capture antibody was immobilized via primary amine groups (11,000 RU) onto a high purity CM5 chip (GE Life Sciences) using standard procedures. The antibody to be analyzed was captured at a flow rate of 10 μL/min. with an adjusted RU value that would result in a maximum analyte binding signal of typically 30 RU. Binding kinetics were measured with trispecific antibodies. HBS-EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% P20 surfactant) was used as the assay buffer at a flow rate of 30 μL/min. The chip surfaces were regenerated using the regenerating solution from the appropriate capture kit. Kinetic parameters were analyzed and calculated using the BIA evaluation v4.1 software package using a flow cell without captured antibody as a reference and a Langmuir model for 1:1 binding with mass transfer.

Measuring T cell proliferation in vitro

[0265] T cells were isolated from human peripheral blood mononuclear cell (PBMC) donors by negative selection using the Pan T Cell Isolation Kit (Miltenyi Biotec GmbH, Germany) in a magnetic field. Antibodies were applied to 96-well cell culture plates by preparing antibodies in sterile PBS and dispensing 50 μL into each well (350 ng/well). The plates were then incubated at 37°C for at least 2 hours and then washed with sterile PBS. Unattached T cells were added to antibody-coated plates (5 x 10 ⁵ cells/ml) and incubated at 37°C for several days. On day 4, cells were subcultured with new cell culture medium onto plates freshly coated with antibodies. In some experiments with 7-day incubations, only fresh medium was added without replacing the plate freshly coated with antibodies. Cells were collected at specific time points and cell numbers were calculated using CountBright™ counting beads.

T cell proliferation assay and T cell subset determination in vitro

[0266] Peripheral blood mononuclear cells were isolated from healthy donor blood collected by Research Blood Components, LLC (Boston, MA). PBMCs were added to antibody-coated plates (350 ng/well) (5 x 10 ⁵ cells/ml) as described above and incubated at 37°C for 3 and 7 days. Cells were collected at specific time points and analyzed by flow cytometry for T cell subsets: naïve (CCR7+ CD45RO-), T _cm (CCR7+ CD45RO+), T _em (CCR7- CD45RO+), T _reg (CD4+ Foxp3+ CD25hi) . Cells were also treated with monensin (GolgiStop) (BD Biosciences, California) for at least 6 hours before staining for flow cytometry to determine the expression of intracellular cytokines: Th1 (CD4+ IFN-γ+), Th2 (CD4+ IL-4+) and Th17 (CD4+ IL-17+). CMV pp65-specific, EBV-specific BMLF, influenza A virus-specific MP, and HIV-1 Gag-specific CD8+ T cells were detected using a fluorescent-conjugated pentamer, restricting donor PBMCs by HLA/viral peptide (A *02:01/NLVPMVATV; SEQ ID NO:26), (A*02:01/GLCTLVAML; SEQ ID NO:27), (A*02:01/GILGFVFTL; SEQ ID NO:28) and (A*02 :01/SLYNTVATL; SEQ ID NO:25), respectively (ProImmune, Oxford, UK). PBMC were obtained from HemaCare (Van Nuys, CA) from donors with known CMV, EBV, or influenza A virus infection and from BioIVT (Westbury, NY) from donors known to be positive for HIV-1 and HLA types . PMBC from donors negative for the restrictive HLA type were used as negative controls. Staining was performed according to the manufacturer's protocol.

Quantification of CMV-specific T cells

[0267] As described above, PBMC were isolated from the blood of human donors confirmed to be infected with CMV and added to plates containing trispecific antibodies or control antibodies. The plates were incubated at 37°C. Cells were collected at the indicated time points and analyzed as described above.

results

[0268] The CD38 _VH1 /CD28sup x CD3mid trispecific antibody activated T cells and promoted proliferation of CMV-specific CD8+ memory T cells after incubation for 10 days with PBMCs isolated from CMV-infected human donor B (FIG. 5A-5B ) and CMV-infected human donor C (FIG. 6A-6B). As shown in FIG. 5A (CMV-infected donor C) and FIG. 6A (CMV-infected donor C), the trispecific CD38 _VH1 /CD28sup x CD3mid antibody resulted in increased numbers of CMV-specific CD8+ memory T cells (cells/μl) compared to the triple mutant control antibody. In addition, the CD38 _VH1 /CD28sup x CD3mid trispecific antibody increased the percentage of CMV-specific CD8+ effector memory T cells (T _em ) and central memory T cells (T _cm ) compared with the triple mutant control antibody (CMV-infected donor B, FIG. 5B; CMV-infected donor C, FIG.

[0269] Taken together, these data indicate that CD38/CD28xCD3 trispecific antibodies mediate the activation and expansion of CMV-specific T cells, such as CMV-specific CD8+ T cells, CMV-specific effector (T _em ) CD8+ Memory T cells and CMV-specific central (T _cm ) CD8+ memory T cells.

Example 5 CD38/CD28xCD3 trispecific antibodies provide an EBV-specific immune response

[0270] The ability of CD38/CD28xCD3 trispecific antibodies to promote the activation and expansion of Epstein-Barr virus (EBV)-specific T cells was further determined.

Materials and methods

Quantification of EBV-specific T cells

[0271] As described above, PBMC were isolated from the blood of human donors with known EBV infection and added to plates containing trispecific antibodies or control antibodies. The plates were incubated at 37°C. Cells were collected at the indicated time points and analyzed as described above.

results

[0272] The CD38 _VH1 /CD28sup x CD3mid trispecific antibody activated T cells and promoted proliferation of EBV-specific CD8+ memory T cells after incubation for 11 days with PBMC isolated from EBV-infected human donor A (FIG. 7A-7B ) and EBV-infected human donor B (FIG. 8A-8B). As shown in FIG. 7A (EBV-infected donor A) and FIG. 8A (EBV-infected donor B), the trispecific CD38 _VH1 /CD28sup x CD3mid antibody resulted in increased numbers of EBV-specific CD8+ memory T cells (cells/μl) compared to the triple mutant control antibody. In addition, the CD38 _VH1 /CD28sup x CD3mid trispecific antibody increased the percentage of EBV-specific CD8+ T _em cells and T _cm cells compared to the triple mutant control antibody (EBV-infected donor A, FIG. 7B; EBV-infected donor B, FIG. 8B, for example , see day 7).

[0273] Taken together, these data indicate that CD38/CD28xCD3 trispecific antibodies mediate the activation and expansion of EBV-specific T cells, such as EBV-specific CD8+ T cells, EBV-specific effector ( _Tem ) CD8+ Memory T cells and EBV-specific central (T _cm ) memory CD8+ T cells.

Example 6 CD38/CD28xCD3 trispecific antibodies provide an HIV-specific immune response

[0274] The ability of CD38/CD28xCD3 trispecific antibodies to promote the activation and expansion of human immunodeficiency virus (HIV)-specific T cells was further determined.

Materials and methods

Quantification of HIV-specific T cells

[0275] As described above, PBMC were isolated from the blood of human donors confirmed to be infected with HIV and added to plates containing trispecific antibodies or control antibodies. The plates were incubated at 37°C. Cells were collected at the indicated time points and analyzed as described above.

results

[0276] On day 0 (before incubation with trispecific antibodies), PBMCs from HIV-positive donors exhibited HIV Gag CD8 T cell-specific (A*02:01 - SLYNTVATL (HIV-1 gag p17 76-84) pentamer, PE-conjugated, ProImmune) (FIG. 9). For example, three HIV-positive PBMC donors had 0.86% (HIV-infected donor A), 0.95% (HIV-infected donor B), and 2.27% (HIV-infected donor C) Gag-specific CD8 T cells by compared with 0.33% in PBMC from an HIV-negative donor.

[0277] Incubation of PBMCs for 10 days with the CD38 _VH1 /CD28sup x CD3mid trispecific antibody activated T cells and allowed proliferation of HIV-specific T cells. As shown in FIG. 10A (HIV-infected donor D), FIG. 11A (HIV-infected donor E) and FIG. 12A (HIV-infected donor F), the trispecific CD38 _VH1 /CD28sup x CD3mid antibody resulted in increased numbers of HIV-specific CD8+ memory T cells (cells/μl) compared to the triple mutant control antibody. In addition, the CD38 _VH1 /CD28sup x CD3mid trispecific antibody increased the percentage of HIV-specific CD8+ effector (T _em ) memory cells ( e.g. , see days 7 and 10), and also, to a lesser extent, CD8+ central (T _cm ) memory cells by compared to a triple mutant control antibody (HIV-infected donor D, FIG. 10B, HIV-infected donor E, FIG. 11B, HIV-infected donor F, FIG. 12B).

Example 7 CD38/CD28xCD3 trispecific antibodies provide an influenza virus-specific immune response

[0278] The ability of CD38/CD28xCD3 trispecific antibodies to promote the activation and expansion of influenza virus-specific T cells was determined.

Materials and methods

Quantification of influenza virus-specific T cells

[0279] As described above, PBMC were isolated from the blood of human donors confirmed to be infected with influenza A virus and added to plates containing trispecific antibodies or control antibodies. The plates were incubated at 37°C. Cells were collected at the indicated time points and analyzed as described above.

results

[0280] The CD38 _VH1 /CD28sup x CD3mid trispecific antibody activated T cells and promoted the proliferation of influenza virus-specific CD8+ memory T cells after incubation for 11 days with PBMC isolated from a human donor with known influenza virus infection (FIG. 13A-13B, influenza virus-infected donor A). As shown in FIG. 13A, the trispecific CD38 _VH1 /CD28sup x CD3mid antibody resulted in increased numbers of influenza virus-specific memory CD8+ T cells (cells/μl) compared to the triple mutant control antibody. In addition, the CD38 _VH1 /CD28sup x CD3mid trispecific antibody increased the percentage of influenza virus (Flu) specific CD8+ T _em cells ( eg , see days 7 and 11) and T _cm cells ( eg , see day 7) by compared to the triple mutant control antibody (FIG. 13B).

[0281] Taken together, the data presented in Examples 1-7 demonstrate that CD38/CD3/CD28 trispecific antibodies stimulate potent antiviral immunity against a variety of viruses. Without being limited to any theory, it is believed that CD38/CD3/CD28 trispecific antibodies can activate and promote T cell proliferation by engaging all three ligands on T cells. In particular, engagement of CD3/CD28 on T cells by CD38/CD3/CD28 trispecific antibodies is believed to initiate T cell activation, proliferation, and differentiation into memory T cells. Furthermore, without being limited by any theory, it is believed that CD28 engagement provides a beneficial costimulatory signal that increases the duration and magnitude of the immune response and promotes T cell proliferation and survival.

[0282] Although the present invention includes various embodiments, it should be understood that variations and modifications will be apparent to those skilled in the art. Thus, the appended claims are intended to cover all such equivalent variations that fall within the scope of the present invention. In addition, the section titles used herein are provided for purposes of organization only and should not be construed as limiting in any way the subject matter of the present invention.

[0283] Each embodiment described herein may be combined with any other embodiment or embodiments unless expressly stated otherwise. In particular, any feature or embodiment as preferred or advantageous may be combined with another feature or features or embodiment or embodiments stated as preferred or advantageous unless expressly stated to the contrary.

[0284] All literature cited in this application is expressly incorporated by reference herein.

--->

LIST OF SEQUENCES

<110> Sanofi

<120> TRISPECIFIC BINDING PROTEINS BINDING CD38, CD28 AND CD3

AND ALSO METHODS OF USE FOR TREATING VIRAL INFECTION

<130> 18395-20321.40

<140> Not assigned yet

<141> At the same time

<150>EP 19306097.7

<151> 2019-09-11

<150> US 62/831,608

<151> 2019-04-09

<150> US 62/831,572

<151> 2019-04-09

<150> PCT/US2018/055084

<151> 2018-10-09

<160> 132

<170> FastSEQ for Windows version 4.0

<210> 1

<211> 256

<212> PROTEIN

<213> Homo sapiens

<400> 1

Arg Trp Arg Gln Gln Trp Ser Gly Pro Gly Thr Thr Lys Arg Phe Pro

1.5 10 15

Glu Thr Val Leu Ala Arg Cys Val Lys Tyr Thr Glu Ile His Pro Glu

20 25 30

Met Arg His Val Asp Cys Gln Ser Val Trp Asp Ala Phe Lys Gly Ala

35 40 45

Phe Ile Ser Lys His Pro Cys Asn Ile Thr Glu Glu Asp Tyr Gln Pro

50 55 60

Leu Met Lys Leu Gly Thr Gln Thr Val Pro Cys Asn Lys Ile Leu Leu

65 70 75 80

Trp Ser Arg Ile Lys Asp Leu Ala His Gln Phe Thr Gln Val Gln Arg

85 90 95

Asp Met Phe Thr Leu Glu Asp Thr Leu Leu Gly Tyr Leu Ala Asp Asp

100 105 110

Leu Thr Trp Cys Gly Glu Phe Asn Thr Ser Lys Ile Asn Tyr Gln Ser

115 120 125

Cys Pro Asp Trp Arg Lys Asp Cys Ser Asn Asn Pro Val Ser Val Phe

130 135 140

Trp Lys Thr Val Ser Arg Arg Phe Ala Glu Ala Ala Cys Asp Val Val

145 150 155 160

His Val Met Leu Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys Asn Ser

165 170 175

Thr Phe Gly Ser Val Glu Val His Asn Leu Gln Pro Glu Lys Val Gln

180 185 190

Thr Leu Glu Ala Trp Val Ile His Gly Gly Arg Glu Asp Ser Arg Asp

195 200 205

Leu Cys Gln Asp Pro Thr Ile Lys Glu Leu Glu Ser Ile Ile Ser Lys

210 215 220

Arg Asn Ile Gln Phe Ser Cys Lys Asn Ile Tyr Arg Pro Asp Lys Phe

225 230 235 240

Leu Gln Cys Val Lys Asn Pro Glu Asp Ser Ser Cys Thr Ser Glu Ile

245 250 255

<210> 2

<211> 265

<212> PROTEIN

<213> Homo sapiens

<400> 2

Arg Trp Arg Gln Gln Trp Ser Gly Pro Gly Thr Thr Lys Arg Phe Pro

1.5 10 15

Glu Thr Val Leu Ala Arg Cys Val Lys Tyr Thr Glu Ile His Pro Glu

20 25 30

Met Arg His Val Asp Cys Gln Ser Val Trp Asp Ala Phe Lys Gly Ala

35 40 45

Phe Ile Ser Lys His Pro Cys Asn Ile Thr Glu Glu Asp Tyr Gln Pro

50 55 60

Leu Met Lys Leu Gly Thr Gln Thr Val Pro Cys Asn Lys Ile Leu Leu

65 70 75 80

Trp Ser Arg Ile Lys Asp Leu Ala His Gln Phe Thr Gln Val Gln Arg

85 90 95

Asp Met Phe Thr Leu Glu Asp Thr Leu Leu Gly Tyr Leu Ala Asp Asp

100 105 110

Leu Thr Trp Cys Gly Glu Phe Asn Thr Ser Lys Ile Asn Tyr Gln Ser

115 120 125

Cys Pro Asp Trp Arg Lys Asp Cys Ser Asn Asn Pro Val Ser Val Phe

130 135 140

Trp Lys Thr Val Ser Arg Arg Phe Ala Glu Ala Ala Cys Asp Val Val

145 150 155 160

His Val Met Leu Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys Asn Ser

165 170 175

Thr Phe Gly Ser Val Glu Val His Asn Leu Gln Pro Glu Lys Val Gln

180 185 190

Thr Leu Glu Ala Trp Val Ile His Gly Gly Arg Glu Asp Ser Arg Asp

195 200 205

Leu Cys Gln Asp Pro Thr Ile Lys Glu Leu Glu Ser Ile Ile Ser Lys

210 215 220

Arg Asn Ile Gln Phe Ser Cys Lys Asn Ile Tyr Arg Pro Asp Lys Phe

225 230 235 240

Leu Gln Cys Val Lys Asn Pro Glu Asp Ser Ser Cys Thr Ser Glu Ile

245 250 255

Ser Ala Ser His His His His His

260 265

<210> 3

<400> 3

000

<210> 4

<211> 265

<212> PROTEIN

<213> Macaca fascicularis

<400> 4

Arg Trp Arg Gln Gln Trp Ser Gly Ser Gly Thr Thr Ser Arg Phe Pro

1.5 10 15

Glu Thr Val Leu Ala Arg Cys Val Lys Tyr Thr Glu Val His Pro Glu

20 25 30

Met Arg His Val Asp Cys Gln Ser Val Trp Asp Ala Phe Lys Gly Ala

35 40 45

Phe Ile Ser Lys Tyr Pro Cys Asn Ile Thr Glu Glu Asp Tyr Gln Pro

50 55 60

Leu Val Lys Leu Gly Thr Gln Thr Val Pro Cys Asn Lys Thr Leu Leu

65 70 75 80

Trp Ser Arg Ile Lys Asp Leu Ala His Gln Phe Thr Gln Val Gln Arg

85 90 95

Asp Met Phe Thr Leu Glu Asp Met Leu Leu Gly Tyr Leu Ala Asp Asp

100 105 110

Leu Thr Trp Cys Gly Glu Phe Asn Thr Phe Glu Ile Asn Tyr Gln Ser

115 120 125

Cys Pro Asp Trp Arg Lys Asp Cys Ser Asn Asn Pro Val Ser Val Phe

130 135 140

Trp Lys Thr Val Ser Arg Arg Phe Ala Glu Thr Ala Cys Gly Val Val

145 150 155 160

His Val Met Leu Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys Asn Ser

165 170 175

Thr Phe Gly Ser Val Glu Val His Asn Leu Gln Pro Glu Lys Val Gln

180 185 190

Ala Leu Glu Ala Trp Val Ile His Gly Gly Arg Glu Asp Ser Arg Asp

195 200 205

Leu Cys Gln Asp Pro Thr Ile Lys Glu Leu Glu Ser Ile Ile Ser Lys

210 215 220

Arg Asn Ile Arg Phe Phe Cys Lys Asn Ile Tyr Arg Pro Asp Lys Phe

225 230 235 240

Leu Gln Cys Val Lys Asn Pro Glu Asp Ser Ser Cys Leu Ser Gly Ile

245 250 255

Ser Ala Ser His His His His His

260 265

<210> 5

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 5

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Ala

1. 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Phe

20 25 30

Asn Met His Trp Val Lys Glu Thr Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Ile Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser

115

<210> 6

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 6

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1.5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr

20 25 30

Gly Asn Gly Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp

65 70 75 80

Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Lys

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 7

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 7

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Ala

1. 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Phe

20 25 30

Asn Met His Trp Val Lys Glu Thr Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Ile Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly

225 230 235 240

Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 8

<211> 218

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 8

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1. 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr

20 25 30

Gly Asn Gly Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp

65 70 75 80

Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Lys

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 9

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 9

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Phe Arg Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 10

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 10

Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1. 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro

65 70 75 80

Glu Asp Ser Ala Thr Tyr Tyr Cys Leu Gln Asp Tyr Ile Tyr Tyr Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 11

<211> 446

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 11

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Phe Arg Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly Pro Asp Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 12

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 12

Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1. 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro

65 70 75 80

Glu Asp Ser Ala Thr Tyr Tyr Cys Leu Gln Asp Tyr Ile Tyr Tyr Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 13

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 13

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1.5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ala Met His Trp Val Lys Glu Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Gln Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 14

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 14

Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1. 5 10 15

Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Gly Gln Gly Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Pro Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asn Lys

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 15

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 15

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1. 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ala Met His Trp Val Lys Glu Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Gln Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly

225 230 235 240

Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 16

<211> 218

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 16

Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1.5 10 15

Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Gly Gln Gly Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Pro Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asn Lys

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 17

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 17

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1. 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Phe

20 25 30

Asn Met His Trp Val Lys Glu Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 18

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 18

Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1. 5 10 15

Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr

20 25 30

Gly Asn Gly Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Ala Ser Ser Arg Ala Thr Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Pro Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asn Lys

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 19

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 19

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1.5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Phe

20 25 30

Asn Met His Trp Val Lys Glu Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly

225 230 235 240

Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 20

<211> 218

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 20

Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1.5 10 15

Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr

20 25 30

Gly Asn Gly Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Ala Ser Ser Arg Ala Thr Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Pro Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asn Lys

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 21

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 21

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Ser Gly Ala

1.5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Phe

20 25 30

Asn Met His Trp Val Lys Glu Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Ile Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 22

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 22

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Ser Gly Ala

1.5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Phe

20 25 30

Asn Met His Trp Val Lys Glu Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Ile Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly

225 230 235 240

Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 23

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 23

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1. 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Phe

20 25 30

Asn Met His Trp Val Lys Glu Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Ile Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 24

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 24

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1. 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Phe

20 25 30

Asn Met His Trp Val Lys Glu Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Ile Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly

225 230 235 240

Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 25

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 25

Ser Leu Tyr Asn Thr Val Ala Thr Leu

15

<210> 26

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 26

Asn Leu Val Pro Met Val Ala Thr Val

15

<210> 27

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 27

Gly Leu Cys Thr Leu Val Ala Met Leu

15

<210> 28

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 28

Gly Ile Leu Gly Phe Val Phe Thr Leu

15

<210> 29

<400> 29

000

<210> 30

<211> 256

<212> PROTEIN

<213> Macaca fascicularis

<400> 30

Arg Trp Arg Gln Gln Trp Ser Gly Ser Gly Thr Thr Ser Arg Phe Pro

1.5 10 15

Glu Thr Val Leu Ala Arg Cys Val Lys Tyr Thr Glu Val His Pro Glu

20 25 30

Met Arg His Val Asp Cys Gln Ser Val Trp Asp Ala Phe Lys Gly Ala

35 40 45

Phe Ile Ser Lys Tyr Pro Cys Asn Ile Thr Glu Glu Asp Tyr Gln Pro

50 55 60

Leu Val Lys Leu Gly Thr Gln Thr Val Pro Cys Asn Lys Thr Leu Leu

65 70 75 80

Trp Ser Arg Ile Lys Asp Leu Ala His Gln Phe Thr Gln Val Gln Arg

85 90 95

Asp Met Phe Thr Leu Glu Asp Met Leu Leu Gly Tyr Leu Ala Asp Asp

100 105 110

Leu Thr Trp Cys Gly Glu Phe Asn Thr Phe Glu Ile Asn Tyr Gln Ser

115 120 125

Cys Pro Asp Trp Arg Lys Asp Cys Ser Asn Asn Pro Val Ser Val Phe

130 135 140

Trp Lys Thr Val Ser Arg Arg Phe Ala Glu Thr Ala Cys Gly Val Val

145 150 155 160

His Val Met Leu Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys Asn Ser

165 170 175

Thr Phe Gly Ser Val Glu Val His Asn Leu Gln Pro Glu Lys Val Gln

180 185 190

Ala Leu Glu Ala Trp Val Ile His Gly Gly Arg Glu Asp Ser Arg Asp

195 200 205

Leu Cys Gln Asp Pro Thr Ile Lys Glu Leu Glu Ser Ile Ile Ser Lys

210 215 220

Arg Asn Ile Arg Phe Phe Cys Lys Asn Ile Tyr Arg Pro Asp Lys Phe

225 230 235 240

Leu Gln Cys Val Lys Asn Pro Glu Asp Ser Ser Cys Leu Ser Gly Ile

245 250 255

<210> 31

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 31

Gly Tyr Thr Phe Thr Ser Phe Asn

15

<210> 32

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 32

Ile Tyr Pro Gly Asn Gly Gly Thr

15

<210> 33

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 33

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr

1.5 10

<210> 34

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 34

Glu Ser Val Asp Ser Tyr Gly Asn Gly Phe

1.5 10

<210> 35

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 35

Leu Ala Ser

1

<210> 36

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 36

Gln Gln Asn Lys Glu Asp Pro Trp Thr

15

<210> 37

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 37

Gly Tyr Thr Phe Thr Ser Tyr Ala

15

<210> 38

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 38

Ile Tyr Pro Gly Gln Gly Gly Thr

15

<210> 39

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 39

Gln Ser Val Ser Ser Tyr Gly Gln Gly Phe

1.5 10

<210> 40

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 40

Gly Ala Ser

1

<210> 41

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 41

Gly Phe Thr Phe Ser Ser Tyr Gly

15

<210> 42

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 42

Ile Trp Tyr Asp Gly Ser Asn Lys

15

<210> 43

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 43

Ala Arg Met Phe Arg Gly Ala Phe Asp Tyr

1.5 10

<210> 44

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 44

Gln Gly Ile Arg Asn Asp

15

<210> 45

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 45

Ala Ala Ser

1

<210> 46

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 46

Leu Gln Asp Tyr Ile Tyr Tyr Pro Thr

15

<210> 47

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 47

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Ala Lys Pro Gly Thr

1.5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Thr Ile Tyr Pro Gly Asp Gly Asp Thr Gly Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Lys Thr Val Tyr

65 70 75 80

Met His Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Asp Tyr Tyr Gly Ser Asn Ser Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 48

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 48

Asp Ile Val Met Thr Gln Ser His Leu Ser Met Ser Thr Ser Leu Gly

1. 5 10 15

Asp Pro Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Arg Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ile Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ala Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Pro Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 49

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 49

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1.5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ser Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Lys

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 50

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 50

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1.5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asn Ile Tyr Val Trp

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Thr Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 51

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 51

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1. 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr

20 25 30

Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Lys Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser

115

<210> 52

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 52

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1.5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr

20 25 30

Val Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Phe Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asn Asp Val Ala Asn Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Lys Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 53

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 53

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Lys Ala

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gln Leu Glu Trp Val

35 40 45

Ala Gln Ile Lys Asp Lys Ser Asn Ser Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Arg Gly Val Tyr Tyr Ala Leu Ser Pro Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 54

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 54

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1. 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Val His Asn

20 25 30

Asn Ala Asn Thr Tyr Leu Ser Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Ser Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gly Gln Gly

85 90 95

Thr Gln Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 55

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 55

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1.5 10

<210> 56

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 56

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1.5 10 15

<210> 57

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 57

Thr Lys Gly Pro Ser

15

<210> 58

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 58

Gly Gln Pro Lys Ala Ala Pro

15

<210> 59

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 59

Gly Gly Ser Gly Ser Ser Gly Ser Gly Gly

1.5 10

<210> 60

<211> 570

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 60

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1. 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ser Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Lys

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ser Gln Val Gln Leu Val Glu Ser

115 120 125

Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala

130 135 140

Ala Ser Gly Phe Thr Phe Thr Lys Ala Trp Met His Trp Val Arg Gln

145 150 155 160

Ala Pro Gly Lys Gln Leu Glu Trp Val Ala Gln Ile Lys Asp Lys Ser

165 170 175

Asn Ser Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr

180 185 190

Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser

195 200 205

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Arg Gly Val Tyr Tyr

210 215 220

Ala Leu Ser Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser Arg Thr Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

245 250 255

Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu

260 265 270

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

275 280 285

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

290 295 300

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

305 310 315 320

Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr

325 330 335

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

340 345 350

Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro

355 360 365

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

370 375 380

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

385 390 395 400

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

405 410 415

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

420 425 430

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

435 440 445

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

450 455 460

Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Glu

465 470 475 480

Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe

485 490 495

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

500 505 510

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

515 520 525

Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

530 535 540

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

545 550 555 560

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

565 570

<210> 61

<211> 338

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 61

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1. 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Val His Asn

20 25 30

Asn Ala Asn Thr Tyr Leu Ser Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Ser Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gly Gln Gly

85 90 95

Thr Gln Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Val Glu Ile Lys

100 105 110

Gly Gln Pro Lys Ala Ala Pro Asp Ile Gln Met Thr Gln Ser Pro Ser

115 120 125

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala

130 135 140

Ser Gln Asn Ile Tyr Val Trp Leu Asn Trp Tyr Gln Gln Lys Pro Gly

145 150 155 160

Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser Asn Leu His Thr Gly

165 170 175

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

180 185 190

Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln

195 200 205

Gln Gly Gln Thr Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu

210 215 220

Ile Lys Thr Lys Gly Pro Ser Arg Thr Val Ala Ala Pro Ser Val Phe

225 230 235 240

Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val

245 250 255

Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp

260 265 270

Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr

275 280 285

Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

290 295 300

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

305 310 315 320

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly

325 330 335

Glu Cys

<210> 62

<211> 446

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 62

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1.5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ala Met His Trp Val Lys Glu Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Gln Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Cys Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440 445

<210> 63

<211> 218

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 63

Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1. 5 10 15

Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Gly Gln Gly Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Pro Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asn Lys

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 64

<211> 573

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 64

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1.5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ser Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Lys

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ser Gln Val Gln Leu Val Glu Ser

115 120 125

Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala

130 135 140

Ala Ser Gly Phe Thr Phe Thr Lys Ala Trp Met His Trp Val Arg Gln

145 150 155 160

Ala Pro Gly Lys Gln Leu Glu Trp Val Ala Gln Ile Lys Asp Lys Ser

165 170 175

Asn Ser Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr

180 185 190

Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser

195 200 205

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Arg Gly Val Tyr Tyr

210 215 220

Ala Leu Ser Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser Arg Thr Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

245 250 255

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

260 265 270

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

275 280 285

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

290 295 300

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

305 310 315 320

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

325 330 335

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

340 345 350

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe

355 360 365

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

370 375 380

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

385 390 395 400

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

405 410 415

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

420 425 430

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

435 440 445

Lys Val Ser Asn Lys Ala Leu Ala Ala Pro Ile Glu Lys Thr Ile Ser

450 455 460

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro

465 470 475 480

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val

485 490 495

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

500 505 510

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

515 520 525

Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

530 535 540

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

545 550 555 560

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

565 570

<210> 65

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 65

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1.5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ala Met His Trp Val Lys Glu Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Gln Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Ala Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 66

<211> 573

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 66

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1. 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ser Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Lys

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ser Gln Val Gln Leu Val Glu Ser

115 120 125

Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala

130 135 140

Ala Ser Gly Phe Thr Phe Thr Lys Ala Trp Met His Trp Val Arg Gln

145 150 155 160

Ala Pro Gly Lys Gln Leu Glu Trp Val Ala Gln Ile Lys Asp Lys Ser

165 170 175

Asn Ser Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr

180 185 190

Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser

195 200 205

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Arg Gly Val Tyr Tyr

210 215 220

Ala Leu Ser Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser Arg Thr Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

245 250 255

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

260 265 270

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

275 280 285

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

290 295 300

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

305 310 315 320

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

325 330 335

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

340 345 350

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

355 360 365

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

370 375 380

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

385 390 395 400

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

405 410 415

Lys Pro Arg Glu Glu Gln Tyr Asn Asn Ala Ser Arg Val Val Ser Val

420 425 430

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

435 440 445

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

450 455 460

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro

465 470 475 480

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val

485 490 495

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

500 505 510

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

515 520 525

Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

530 535 540

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

545 550 555 560

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

565 570

<210> 67

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 67

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1. 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ala Met His Trp Val Lys Glu Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Gln Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Asn Ala Ser Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 68

<211> 443

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 68

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Phe Arg Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210> 69

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 69

Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1.5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro

65 70 75 80

Glu Asp Ser Ala Thr Tyr Tyr Cys Leu Gln Asp Tyr Ile Tyr Tyr Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 70

<211> 446

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 70

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1. 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Phe Arg Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Ala Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 71

<211> 446

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 71

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1. 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Phe Arg Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Asn Ala Ser Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 72

<211> 1710

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 72

caggtgcagc tggtgcagtc tggcgccgag gtcgtgaaac ctggcgcctc tgtgaaggtg 60

tcctgcaagg ccagcggcta cacctttacc agctactaca tccactgggt gcgccaggcc 120

cctggacagg gactggaatg gatcggcagc atctaccccg gcaacgtgaa caccaactac 180

gcccagaagt tccagggcag agccaccctg accgtggaca ccagcatcag caccgcctac 240

atggaactga gccggctgag aagcgacgac accgccgtgt actactgcac ccggtcccac 300

tacggcctgg attggaactt cgacgtgtgg ggcaagggca ccaccgtgac agtgtctagc 360

agccaggtgc agctggtgga atctggcggc ggagtggtgc agcctggcag aagcctgaga 420

ctgagctgtg ccgccagcgg cttcaccttc accaaggcct ggatgcactg ggtgcgccag 480

gcccctggaa agcagctgga atgggtggcc cagatcaagg acaagagcaa cagctacgcc 540

acctactacg ccgacagcgt gaagggccgg ttcaccatca gccgggacga cagcaagaac 600

accctgtacc tgcagatgaa cagcctgcgg gccgaggaca ccgccgtgta ctactgtcgg 660

ggcgtgtact atgccctgag ccccttcgat tactggggcc agggaaccct cgtgaccgtg 720

tctagtcgga ccgccagcac aaagggccca tcggtgttcc ctctggcccc ttgcagcaga 780

agcaccagcg aatctacagc cgccctgggc tgcctcgtga aggactactt tcccgagccc 840

gtgaccgtgt cctggaactc tggcgctctg acaagcggcg tgcacacctt tccagccgtg 900

ctccagagca gcggcctgta ctctctgagc agcgtcgtga cagtgcccag cagcagcctg 960

ggcaccaaga cctacacctg taacgtggac cacaagccca gcaacaccaa ggtggacaag 1020

cgggtggaat ctaagtacgg ccctccctgc cctccttgcc cagcccctga agctgccggc 1080

ggaccctccg tgttcctgtt ccccccaaag cccaaggaca ccctgatgat cagccggacc 1140

cccgaagtga cctgcgtggt ggtggatgtg tcccaggaag atcccgaggt gcagttcaat 1200

tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcccagaga ggaacagttc 1260

aacagcacct accgggtggt gtccgtgctg accgtgctgc accaggactg gctgaacggc 1320

aaagagtaca agtgcaaggt gtccaacaag ggcctgccca gctccatcga gaaaaccatc 1380

agcaaggcca agggccagcc ccgcgagcct caagtgtgta ccctgccccc tagccaggaa 1440

gagatgacca agaaccaggt gtccctgagc tgtgccgtga aaggcttcta ccccagcgac 1500

attgccgtgg aatgggagag caacggccag cccgagaaca actacaagac caccccccct 1560

gtgctggaca gcgacggctc attcttcctg gtgtccaagc tgaccgtgga caagagccgg 1620

tggcaggaag gcaacgtgtt cagctgctcc gtgatgcacg aggccctgca caaccactac 1680

acccagaagt ccctgtctct gtccctgggc 1710

<210> 73

<211> 1014

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 73

gacatcgtga tgacccagac ccccctgagc ctgagcgtga cacctggaca gcctgccagc 60

atcagctgca agagcagcca gagcctggtg cacaacaacg ccaacaccta cctgagctgg 120

tatctgcaga agcccggcca gagcccccag tccctgatct acaaggtgtc caacagattc 180

agcggcgtgc ccgacagatt ctccggcagc ggctctggca ccgacttcac cctgaagatc 240

agccgggtgg aagccgagga cgtgggcgtg tactattgtg gccagggcac ccagtacccc 300

ttcacctttg gcagcggcac caaggtggaa atcaagggcc agcccaaggc cgcccccgac 360

atccagatga cccagagccc cagcagcctg tctgccagcg tgggcgacag agtgaccatc 420

acctgtcagg ccagccagaa catctacgtg tggctgaact ggtatcagca gaagcccggc 480

aaggccccca agctgctgat ctacaaggcc agcaacctgc acaccggcgt gcccagcaga 540

ttttctggca gcggctccgg caccgacttc accctgacaa tcagctccct gcagcccgag 600

gacattgcca cctactactg ccagcagggc cagacctacc cctacacctt tggccagggc 660

accaagctgg aaatcaagac caagggcccc agccgtacgg tggccgctcc cagcgtgttc 720

atcttcccac ctagcgacga gcagctgaag tccggcacag cctctgtcgt gtgcctgctg 780

aacaacttct acccccgcga ggccaaagtg cagtggaagg tggacaacgc cctgcagagc 840

ggcaacagcc aggaaagcgt gaccgagcag gacagcaagg actccaccta cagcctgagc 900

agcaccctga cactgagcaa ggccgactac gagaagcaca aggtgtacgc ctgcgaagtg 960

acccaccagg gcctgtctag ccccgtgacc aagagcttca accggggcga gtgt 1014

<210> 74

<211> 1338

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 74

caggtgcagc tggtgcagtc tggcgccgaa gtcgtgaaac ctggcgcctc cgtgaaggtg 60

tcctgcaagg ccagcggcta cacctttacc agctacgcca tgcactgggt caaagaggcc 120

cctggccaga gactggaatg gatcggctac atctaccccg gccagggcgg caccaactac 180

aaccagaagt tccagggcag agccaccctg accgccgata caagcgccag caccgcctac 240

atggaactga gcagcctgcg gagcgaggat accgccgtgt acttctgtgc cagaacaggc 300

ggcctgaggc gggcctactt tacctattgg ggccagggca ccctcgtgac cgtgtctagc 360

gctagcacaa agggcccatc ggtgttccct ctggcccctt gcagcagaag caccagcgaa 420

tctacagccg ccctgggctg cctcgtgaag gactactttc ccgagcccgt gaccgtgtcc 480

tggaactctg gcgctctgac aagcggcgtg cacacctttc cagccgtgct ccagagcagc 540

ggcctgtact ctctgagcag cgtcgtgaca gtgcccagca gcagcctggg caccaagacc 600

tacacctgta acgtggacca caagcccagc aacaccaagg tggacaagcg ggtggaatct 660

aagtacggcc ctccctgccc tccttgccca gcccctgaag ctgccggcgg accctccgtg 720

ttcctgttcc ccccaaagcc caaggacacc ctgatgatca gccggacccc cgaagtgacc 780

tgcgtggtgg tggatgtgtc cccaggaagat cccgaggtgc agttcaattg gtacgtggac 840

ggcgtggaag tgcacaacgc caagaccaag cccagagagg aacagttcaa cagcacctac 900

cgggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa agagtacaag 960

tgcaaggtgt ccaacaaggg cctgcccagc tccatcgaga aaaccatcag caaggccaag 1020

ggccagcccc gcgagcctca agtgtatacc ctgccccctt gccaggaaga gatgaccaag 1080

aaccaggtgt ccctgtggtg tctcgtgaaa ggcttctacc ccagcgacat tgccgtggaa 1140

tggggagagca acggccagcc cgagaacaac tacaagacca ccccccctgt gctggacagc 1200

gacggctcat tcttcctgta ctccaagctg accgtggaca agagccggtg gcaggaaggc 1260

aacgtgttca gctgctccgt gatgcacgag gccctgcaca accactacac ccagaagtcc 1320

ctgtctctgt ccctggggc 1338

<210> 75

<211> 654

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 75

gacatcgtgc tgacacagag ccctgccacc ctgtctctga gccctggcga gagagccacc 60

atcagctgta gagccagcca gagcgtgtcc agctacggcc agggcttcat gcactggtat 120

cagcagaagc ccggccagcc ccccagactg ctgatctatg gcgccagcag cagagccaca 180

ggcatccccg ccagattttc tggctctggc agcggcaccg acttcaccct gacaatcagc 240

cccctggaac ccgaggactt cgccgtgtac tactgccagc agaacaaaga ggacccctgg 300

accttcggcg gaggcaccaa gctggaaatc aagcgtacgg tggccgctcc cagcgtgttc 360

atcttcccac ctagcgacga gcagctgaag tccggcacag cctctgtcgt gtgcctgctg 420

aacaacttct acccccgcga ggccaaggtg cagtggaagg tggacaatgc cctgcagagc 480

ggcaacagcc aggaaagcgt gaccgagcag gacagcaagg actccaccta cagcctgagc 540

agcaccctga ccctgtccaa ggccgattac gagaagcaca aggtgtacgc ctgcgaagtg 600

acccaccagg gcctgtctag ccccgtgacc aagagcttca accggggcga gtgc 654

<210> 76

<211> 1719

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 76

caggtgcagc tggtgcagtc tggcgccgag gtcgtgaaac ctggcgcctc tgtgaaggtg 60

tcctgcaagg ccagcggcta cacctttacc agctactaca tccactgggt gcgccaggcc 120

cctggacagg gactggaatg gatcggcagc atctaccccg gcaacgtgaa caccaactac 180

gcccagaagt tccagggcag agccaccctg accgtggaca ccagcatcag caccgcctac 240

atggaactga gccggctgag aagcgacgac accgccgtgt actactgcac ccggtcccac 300

tacggcctgg attggaactt cgacgtgtgg ggcaagggca ccaccgtgac agtgtctagc 360

agccaggtgc agctggtgga atctggcggc ggagtggtgc agcctggcag aagcctgaga 420

ctgagctgtg ccgccagcgg cttcaccttc accaaggcct ggatgcactg ggtgcgccag 480

gcccctggaa agcagctgga atgggtggcc cagatcaagg acaagagcaa cagctacgcc 540

acctactacg ccgacagcgt gaagggccgg ttcaccatca gccgggacga cagcaagaac 600

accctgtacc tgcagatgaa cagcctgcgg gccgaggaca ccgccgtgta ctactgtcgg 660

ggcgtgtact atgccctgag ccccttcgat tactggggcc agggaaccct cgtgaccgtg 720

tctagtcgga ccgccagcac aaagggcccc agcgtgttcc ctctggcccc tagcagcaag 780

agcacatctg gcggaacagc cgccctgggc tgcctcgtga aggactactt tcccgagccc 840

gtgaccgtgt cctggaattc tggcgccctg accagcggcg tgcacacctt tccagctgtg 900

ctgcagtcca gcggcctgta cagcctgagc agcgtcgtga cagtgcccag cagctctctg 960

ggcacccaga cctacatctg caacgtgaac cacaagccca gcaacaccaa ggtggacaag 1020

aaggtggaac ccaagagctg cgacaagacc cacacctgtc ccccttgtcc tgcccccgaa 1080

gccgccggag gcccttccgt gttcctgttc cccccaaagc ccaaggacac cctgatgatc 1140

agccggaccc ccgaagtgac ctgcgtggtg gtggatgtgt cccacgagga ccctgaagtg 1200

aagttcaatt ggtacgtgga cggcgtggaa gtgcacaacg ccaagaccaa gccaagagag 1260

gaacagtaca acagcaccta ccgggtggtg tccgtgctga ccgtgctgca ccaggactgg 1320

ctgaacggca aagagtacaa gtgcaaggtg tccaacaagg ccctggccgc ccccatcgag 1380

aaaaccatca gcaaggccaa gggccagccc cgcgaacccc aggtgtgcac actgccccca 1440

agcagggacg agctgaccaa gaaccaggtg tccctgagct gtgccgtgaa aggcttctac 1500

ccctccgata tcgccgtgga atgggagagc aacggccagc ccgagaacaa ctacaagacc 1560

accccccctg tgctggacag cgacggctca ttcttcctgg tgtccaagct gacagtggac 1620

aagtcccggt ggcagcaggg caacgtgttc agctgctccg tgatgcacga ggccctgcac 1680

aaccactaca cccagaagtc cctgagcctg agccccggc 1719

<210> 77

<211> 1347

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 77

caggtgcagc tggtgcagtc tggcgccgaa gtcgtgaaac ctggcgcctc cgtgaaggtg 60

tcctgcaagg ccagcggcta cacctttacc agctacgcca tgcactgggt caaagaggcc 120

cctggccaga gactggaatg gatcggctac atctaccccg gccagggcgg caccaactac 180

aaccagaagt tccagggcag agccaccctg accgccgata caagcgccag caccgcctac 240

atggaactga gcagcctgcg gagcgaggat accgccgtgt acttctgtgc cagaacaggc 300

ggcctgaggc gggcctactt tacctattgg ggccagggca ccctcgtgac cgtgtctagc 360

gctagcacaa agggccccag cgtgttccct ctggccccta gcagcaagag cacatctggc 420

ggaacagccg ccctgggctg cctcgtgaag gactactttc ccgagcccgt gaccgtgtcc 480

tggaattctg gcgccctgac cagcggcgtg cacacctttc cagctgtgct gcagtccagc 540

ggcctgtaca gcctgagcag cgtcgtgaca gtgcccagca gctctctggg cacccagacc 600

tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaagaa ggtggaaccc 660

aagagctgcg acaagaccca cacctgtccc ccttgtcctg cccccgaagc cgccggaggc 720

ccttccgtgt tcctgttccc cccaaagccc aaggacaccc tgatgatcag ccggaccccc 780

gaagtgacct gcgtggtggt ggatgtgtcc cacgaggacc ctgaagtgaa gttcaattgg 840

tacgtggacg gcgtggaagt gcacaacgcc aagaccaagc caagagagga acagtacaac 900

agcacctacc gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaaa 960

gagtacaagt gcaaggtgtc caacaaggcc ctggccgccc ccatcgagaa aaccatcagc 1020

aaggccaagg gccagccccg cgaaccccag gtgtacacac tgcccccatg cagggacgag 1080

ctgaccaaga accaggtgtc cctgtggtgt ctggtgaaag gcttctaccc ctccgatatc 1140

gccgtggaat gggagagcaa cggccagccc gagaacaact acaagaccac cccccctgtg 1200

ctggacagcg acggctcatt cttcctgtac tccaagctga cagtggacaa gtcccggtgg 1260

cagcagggca acgtgttcag ctgctccgtg atgcacgagg ccctgcacaa ccactacacc 1320

cagaagtccc tgagcctgag ccccggc 1347

<210> 78

<211> 1719

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 78

caggtgcagc tggtgcagtc tggcgccgag gtcgtgaaac ctggcgcctc tgtgaaggtg 60

tcctgcaagg ccagcggcta cacctttacc agctactaca tccactgggt gcgccaggcc 120

cctggacagg gactggaatg gatcggcagc atctaccccg gcaacgtgaa caccaactac 180

gcccagaagt tccagggcag agccaccctg accgtggaca ccagcatcag caccgcctac 240

atggaactga gccggctgag aagcgacgac accgccgtgt actactgcac ccggtcccac 300

tacggcctgg attggaactt cgacgtgtgg ggcaagggca ccaccgtgac agtgtctagc 360

agccaggtgc agctggtgga atctggcggc ggagtggtgc agcctggcag aagcctgaga 420

ctgagctgtg ccgccagcgg cttcaccttc accaaggcct ggatgcactg ggtgcgccag 480

gcccctggaa agcagctgga atgggtggcc cagatcaagg acaagagcaa cagctacgcc 540

acctactacg ccgacagcgt gaagggccgg ttcaccatca gccgggacga cagcaagaac 600

accctgtacc tgcagatgaa cagcctgcgg gccgaggaca ccgccgtgta ctactgtcgg 660

ggcgtgtact atgccctgag ccccttcgat tactggggcc agggaaccct cgtgaccgtg 720

tctagtcgga ccgccagcac aaagggcccc agcgtgttcc ctctggcccc tagcagcaag 780

agcacatctg gcggaacagc cgccctgggc tgcctcgtga aggactactt tcccgagccc 840

gtgaccgtgt cctggaattc tggcgccctg accagcggcg tgcacacctt tccagctgtg 900

ctgcagtcca gcggcctgta cagcctgagc agcgtcgtga cagtgcccag cagctctctg 960

ggcacccaga cctacatctg caacgtgaac cacaagccca gcaacaccaa ggtggacaag 1020

aaggtggaac ccaagagctg cgacaagacc cacacctgtc ccccttgtcc tgcccccgaa 1080

ctgctgggag gcccttccgt gttcctgttc cccccaaagc ccaaggacac cctgatgatc 1140

agccggaccc ccgaagtgac ctgcgtggtg gtggatgtgt cccacgagga ccctgaagtg 1200

aagttcaatt ggtacgtgga cggcgtggaa gtgcacaacg ccaagaccaa gccaagagag 1260

gaacagtaca acaatgcctc ccgggtggtg tccgtgctga ccgtgctgca ccaggactgg 1320

ctgaacggca aagagtacaa gtgcaaggtg tccaacaagg ccctgcctgc ccccatcgag 1380

aaaaccatca gcaaggccaa gggccagccc cgcgaacccc aggtgtgcac actgccccca 1440

agcagggacg agctgaccaa gaaccaggtg tccctgagct gtgccgtgaa aggcttctac 1500

ccctccgata tcgccgtgga atgggagagc aacggccagc ccgagaacaa ctacaagacc 1560

accccccctg tgctggacag cgacggctca ttcttcctgg tgtccaagct gacagtggac 1620

aagtcccggt ggcagcaggg caacgtgttc agctgctccg tgatgcacga ggccctgcac 1680

aaccactaca cccagaagtc cctgagcctg agccccggc 1719

<210> 79

<211> 1347

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 79

caggtgcagc tggtgcagtc tggcgccgaa gtcgtgaaac ctggcgcctc cgtgaaggtg 60

tcctgcaagg ccagcggcta cacctttacc agctacgcca tgcactgggt caaagaggcc 120

cctggccaga gactggaatg gatcggctac atctaccccg gccagggcgg caccaactac 180

aaccagaagt tccagggcag agccaccctg accgccgata caagcgccag caccgcctac 240

atggaactga gcagcctgcg gagcgaggat accgccgtgt acttctgtgc cagaacaggc 300

ggcctgaggc gggcctactt tacctattgg ggccagggca ccctcgtgac cgtgtctagc 360

gctagcacaa agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840

tatgttgacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900

aatgcctccc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatg ccgggatgag 1080

ctgaccaaga atcaagtcag cctgtggtgc ctggtaaaag gcttctatcc cagcgacatc 1140

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200

ctggactccg acggctcctt cttcctctac tcaaaactca ccgtggacaa gagcaggtgg 1260

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320

cagaagagcc tctccctgtc tccggggt 1347

<210> 80

<211> 1329

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 80

caggtgcagc tggtggaaag cggcggaggc gtggtgcagc ctggcaggtc tctgagactg 60

agctgtgccg ccagcggctt caccttcagc agctacggaa tgcactgggt gcgccaggcc 120

cctggcaaag gactggaatg ggtggccgtg atttggtacg acggcagcaa caagtactac 180

gccgacagcg tgaagggccg gttcaccatc agcggcgaca acagcaagaa caccctgtac 240

ctgcagatga acagcctgcg ggccgaggac accgccgtgt actactgcgc cagaatgttc 300

agaggcgcct tcgactactg gggccagggc acactcgtga ccgtgtctag tgcgtcgacc 360

aagggcccat cggtgttccc tctggcccct tgcagcagaa gcaccagcga atctacagcc 420

gccctgggct gcctcgtgaa ggactacttt cccgagcccg tgaccgtgtc ctggaactct 480

ggcgctctga caagcggcgt gcacaccttt ccagccgtgc tccagagcag cggcctgtac 540

tctctgagca gcgtcgtgac agtgcccagc agcagcctgg gcaccaagac ctacacctgt 600

aacgtggacc acaagcccag caacaccaag gtggacaagc gggtggaatc taagtacggc 660

cctccctgcc ctccttgccc agcccctgaa gctgccggcg gaccctccgt gttcctgttc 720

cccccaaagc ccaaggacac cctgatgatc agccggaccc ccgaagtgac ctgcgtggtg 780

gtggatgtgt cccaggaaga tcccgaggtg cagttcaatt ggtacgtgga cggcgtggaa 840

gtgcacaacg ccaagaccaa gcccagagag gaacagttca acagcaccta ccgggtggtg 900

tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aagagtacaa gtgcaaggtg 960

tccaacaagg gcctgcccag ctccatcgag aaaaccatca gcaaggccaa gggccagccc 1020

cgcgagcctc aagtgtatac cctgccccct tgccaggaag agatgaccaa gaaccaggtg 1080

tccctgtggt gtctcgtgaa aggcttctac cccagcgaca ttgccgtgga atggggagagc 1140

aacggccagc ccgagaacaa ctacaagacc accccccctg tgctggacag cgacggctca 1200

ttcttcctgt actccaagct gaccgtggac aagagccggt ggcaggaagg caacgtgttc 1260

agctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgtctctg 1320

tccctgggc 1329

<210> 81

<211> 642

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 81

gccatccaga tgacccagag ccccagcagc ctgtctgcca gcgtgggcga cagagtgacc 60

atcacctgta gagccagcca gggcatccgg aacgacctgg gctggtatca gcagaagcct 120

ggcaaggccc ccaagctgct gatctacgcc gctagctctc tgcagtccgg cgtgcccagc 180

agattttctg gcagcggctc cggcaccgac ttcaccctga caatctctgg cctgcagccc 240

gaggacagcg ccacctacta ctgtctgcaa gactacatct actaccccac cttcggccag 300

ggcaccaagg tggaaatcaa gcgtacggtg gccgctccca gcgtgttcat cttcccacct 360

agcgacgagc agctgaagtc cggcacagcc tctgtcgtgt gcctgctgaa caacttctac 420

ccccgcgagg ccaaagtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480

gaaagcgtga ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgaca 540

ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600

ctgtctagcc ccgtgaccaa gagcttcaac cggggcgagt gt 642

<210> 82

<211> 1338

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 82

caggtgcagc tggtggaaag cggcggaggc gtggtgcagc ctggcaggtc tctgagactg 60

agctgtgccg ccagcggctt caccttcagc agctacggaa tgcactgggt gcgccaggcc 120

cctggcaaag gactggaatg ggtggccgtg atttggtacg acggcagcaa caagtactac 180

gccgacagcg tgaagggccg gttcaccatc agcggcgaca acagcaagaa caccctgtac 240

ctgcagatga acagcctgcg ggccgaggac accgccgtgt actactgcgc cagaatgttc 300

agaggcgcct tcgactactg gggccagggc acactcgtga ccgtgtctag tgcgtcgacc 360

aagggcccca gcgtgttccc tctggcccct agcagcaaga gcacatctgg cggaacagcc 420

gccctgggct gcctcgtgaa ggactacttt cccgagcccg tgaccgtgtc ctggaattct 480

ggcgccctga ccagcggcgt gcacaccttt ccagctgtgc tgcagtccag cggcctgtac 540

agcctgagca gcgtcgtgac agtgcccagc agctctctgg gcacccagac ctacatctgc 600

aacgtgaacc acaagcccag caacaccaag gtggacaaga aggtggaacc caagagctgc 660

gacaagaccc acacctgtcc cccttgtcct gcccccgaag ccgccggagg cccttccgtg 720

ttcctgttcc ccccaaagcc caaggacacc ctgatgatca gccggacccc cgaagtgacc 780

tgcgtggtgg tggatgtgtc ccacgaggac cctgaagtga agttcaattg gtacgtggac 840

ggcgtggaag tgcacaacgc caagaccaag ccaagagagg aacagtacaa cagcacctac 900

cgggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa agagtacaag 960

tgcaaggtgt ccaacaaggc cctggccgcc cccatcgaga aaaccatcag caaggccaag 1020

ggccagcccc gcgaacccca ggtgtacaca ctgcccccat gcagggacga gctgaccaag 1080

aaccaggtgt ccctgtggtg tctggtgaaa ggcttctacc cctccgatat cgccgtggaa 1140

tggggagagca acggccagcc cgagaacaac tacaagacca ccccccctgt gctggacagc 1200

gacggctcat tcttcctgta ctccaagctg acagtggaca agtcccggtg gcagcagggc 1260

aacgtgttca gctgctccgt gatgcacgag gccctgcaca accactacac ccagaagtcc 1320

ctgagcctga gccccggc 1338

<210> 83

<211> 1338

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 83

caggtgcagc tggtggaaag cggcggaggc gtggtgcagc ctggcaggtc tctgagactg 60

agctgtgccg ccagcggctt caccttcagc agctacggaa tgcactgggt gcgccaggcc 120

cctggcaaag gactggaatg ggtggccgtg atttggtacg acggcagcaa caagtactac 180

gccgacagcg tgaagggccg gttcaccatc agcggcgaca acagcaagaa caccctgtac 240

ctgcagatga acagcctgcg ggccgaggac accgccgtgt actactgcgc cagaatgttc 300

agaggcgcct tcgactactg gggccagggc acactcgtga ccgtgtctag tgcgtcgacc 360

aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 420

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 480

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 540

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 600

aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 660

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 720

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 780

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtatgttgac 840

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa caatgcctcc 900

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 960

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1020

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat gccgggatga gctgaccaag 1080

aatcaagtca gcctgtggtg cctggtaaaa ggcttctatc ccagcgacat cgccgtggag 1140

tggggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200

gacggctcct tcttcctcta ctcaaaactc accgtggaca agagcaggtg gcagcagggg 1260

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1320

ctctccctgt ctccggggt 1338

<210> 84

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 84

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1. 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Lys Ala

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Lys Asp Lys Ser Asn Ser Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Arg Gly Val Tyr Tyr Ala Leu Ser Pro Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 85

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 85

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1.5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Val His Asn

20 25 30

Asn Gly Asn Thr Tyr Leu Ser Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gly Gln Gly

85 90 95

Thr Gln Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 86

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 86

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1.5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser

20 25

<210> 87

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 87

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Ser Gly Ala

1. 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser

20 25

<210> 88

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 88

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1. 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser

20 25

<210> 89

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 89

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 90

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 90

Met His Trp Val Lys Glu Ala Pro Gly Gln Arg Leu Glu Trp Ile Gly

1.5 10 15

Tyr

<210> 91

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 91

Met His Trp Val Lys Glu Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly

1.5 10 15

Tyr

<210> 92

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 92

Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

1.5 10 15

Val

<210> 93

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 93

Asn Tyr Asn Gln Lys Phe Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr

1. 5 10 15

Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val Tyr Phe Cys

35

<210> 94

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 94

Asn Tyr Asn Gln Lys Phe Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr

1. 5 10 15

Ser Ala Ser Thr Ala Tyr Met Glu Ile Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val Tyr Phe Cys

35

<210> 95

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 95

Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn

1.5 10 15

Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 96

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 96

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1.5 10

<210> 97

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 97

Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1.5 10 15

Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser

20 25

<210> 98

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 98

Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1. 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

20 25

<210> 99

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 99

Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile

1. 5 10 15

Tyr

<210> 100

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 100

Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

1. 5 10 15

<210> 101

<211> 36

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 101

Ser Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

1. 5 10 15

Thr Asp Phe Thr Leu Thr Ile Ser Pro Leu Glu Pro Glu Asp Phe Ala

20 25 30

Val Tyr Tyr Cys

35

<210> 102

<211> 36

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 102

Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

1. 5 10 15

Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro Glu Asp Ser Ala

20 25 30

Thr Tyr Tyr Cys

35

<210> 103

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 103

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

1.5 10

<210> 104

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 104

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1.5 10

<210> 105

<211> 159

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 105

Arg Trp Arg Gln Gln Trp Ser Gly Pro Gly Thr Thr Lys Arg Phe Pro

1.5 10 15

Glu Thr Val Leu Ala Arg Cys Val Lys Tyr Thr Glu Ile His Pro Glu

20 25 30

Met Arg His Val Asp Cys Gln Ser Val Trp Asp Ala Phe Lys Gly Ala

35 40 45

Phe Ile Ser Lys His Pro Cys Asn Ile Thr Glu Glu Asp Tyr Gln Pro

50 55 60

Leu Met Lys Leu Gly Thr Gln Thr Val Pro Cys Asn Lys Ile Leu Leu

65 70 75 80

Trp Ser Arg Ile Lys Asp Leu Ala His Gln Phe Thr Gln Val Gln Arg

85 90 95

Asp Met Phe Thr Leu Glu Asp Thr Leu Leu Gly Tyr Leu Ala Asp Asp

100 105 110

Leu Thr Trp Cys Gly Glu Phe Asn Thr Ser Lys Ile Asn Tyr Gln Ser

115 120 125

Cys Pro Asp Trp Arg Lys Asp Cys Ser Asn Asn Pro Val Ser Val Phe

130 135 140

Trp Lys Thr Val Ser Arg Arg His Phe Trp Glu Cys Gly Ser Pro

145 150 155

<210> 106

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 106

Asp Ile Val Met Thr Gln Ser His Leu Ser Met Ser Thr Ser Leu Gly

1.5 10 15

Asp Pro Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Arg Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ile Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ala Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Pro Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 107

<211> 450

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 107

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Ala Lys Pro Gly Thr

1. 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Thr Ile Tyr Pro Gly Asp Gly Asp Thr Gly Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Lys Thr Val Tyr

65 70 75 80

Met His Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Asp Tyr Tyr Gly Ser Asn Ser Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 108

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 108

Gly Tyr Thr Phe Thr Ser Tyr Tyr

15

<210> 109

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 109

Ile Tyr Pro Gly Asn Val Asn Thr

15

<210> 110

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 110

Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val

1.5 10

<210> 111

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 111

Gln Asn Ile Tyr Val Trp

15

<210> 112

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 112

Lys Ala Ser

1

<210> 113

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 113

Gln Gln Gly Gln Thr Tyr Pro Tyr

15

<210> 114

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 114

Gly Phe Ser Leu Ser Asp Tyr Gly

15

<210> 115

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 115

Ile Trp Ala Gly Gly Gly Thr

15

<210> 116

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 116

Ala Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr

1.5 10

<210> 117

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 117

Glu Ser Val Glu Tyr Tyr Val Thr Ser Leu

1.5 10

<210> 118

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 118

Ala Ala Ser

1

<210> 119

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 119

Gln Gln Ser Arg Lys Val Pro Tyr Thr

15

<210> 120

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 120

Gly Phe Thr Phe Thr Lys Ala Trp

15

<210> 121

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 121

Ile Lys Asp Lys Ser Asn Ser Tyr Ala Thr

1.5 10

<210> 122

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 122

Arg Gly Val Tyr Tyr Ala Leu Ser Pro Phe Asp Tyr

1.5 10

<210> 123

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 123

Gln Ser Leu Val His Asn Asn Ala Asn Thr Tyr

1.5 10

<210> 124

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 124

Lys Val Ser

1

<210> 125

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 125

Gly Gln Gly Thr Gln Tyr Pro Phe Thr

15

<210> 126

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 126

Gly Phe Thr Phe Thr Lys Ala Trp

15

<210> 127

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 127

Ile Lys Asp Lys Ser Asn Ser Tyr Ala Thr

1.5 10

<210> 128

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 128

Gly Val Tyr Tyr Ala Leu Ser Pro Phe Asp Tyr

1.5 10

<210> 129

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 129

Gln Ser Leu Val His Asn Asn Gly Asn Thr Tyr

1.5 10

<210> 130

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 130

Lys Val Ser

1

<210> 131

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 131

Gly Gln Gly Thr Gln Tyr Pro Phe Thr

15

<210> 132

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 132

Gln Ser Val Ser Ser Tyr Gly Gln Gly

15

<---

Claims (120)

1. A method of propagating virus-specific memory T cells, comprising contacting the virus-specific memory T cell with a binding protein, where the binding protein contains four polypeptide chains that form three antigen-binding sites, where the first polypeptide chain contains a structure, represented by the formula: V _L2 -L ₁ -V _L1 -L ₂ -C _L [I], and the second polypeptide chain contains a structure represented by the formula: V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge-C _H2 -C _H3 [II], and the third polypeptide chain contains the structure represented by the formula: V _H3 -C _H1 -hinge-C _H2 -C _H3 [III], and the fourth polypeptide chain contains the structure represented by the formula: V _L3 -C _L [IV], Where V _L1 is the first variable domain of the immunoglobulin light chain; V _L2 is the second variable domain of the immunoglobulin light chain; V _L3 is the third variable domain of the immunoglobulin light chain; V _H1 is the first variable domain of the immunoglobulin heavy chain; V _H2 is the second variable domain of the immunoglobulin heavy chain; V _H3 is the third variable domain of the immunoglobulin heavy chain; C _L is an immunoglobulin light chain constant domain; C _H1 is the immunoglobulin heavy chain C _H1 constant domain; C _H2 is the immunoglobulin heavy chain C _H2 constant domain; C _H3 is the immunoglobulin heavy chain C _H3 constant domain; the hinge is the hinge region of an immunoglobulin connecting the C _H1 and C _H2 domains; And L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers, wherein the polypeptide of formula I and the polypeptide of formula II form a light_chain-heavy_chain crossover pair, and wherein V _H1 and V _L1 form a first antigen binding site that binds the CD28 polypeptide, where V _H2 and V _L2 form a second antigen binding region that binds the CD3 polypeptide, and where V _H3 and V _L3 form a third antigen binding region that binds the CD38 polypeptide. 2. The method of claim 1, wherein the virus-specific memory T cell is contacted with the binding protein in vitro or ex vivo . 3. The method according to claim 1 or 2, wherein introducing the virus-specific memory T cell into contact with the binding protein causes activation and/or proliferation of the virus-specific memory T cell. 4. A method of propagating T cells, comprising contacting the T cell with a binding protein in vitro or ex vivo , wherein the binding protein contains four polypeptide chains that form three antigen-binding sites, where the first polypeptide chain contains a structure represented by the formula: V _L2 -L ₁ -V _L1 -L ₂ -C _L [I], and the second polypeptide chain contains a structure represented by the formula: V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge-C _H2 -C _H3 [II], and the third polypeptide chain contains the structure represented by the formula: V _H3 -C _H1 -hinge-C _H2 -C _H3 [III], and the fourth polypeptide chain contains the structure represented by the formula: V _L3 -C _L [IV], Where V _L1 is the first variable domain of the immunoglobulin light chain; V _L2 is the second variable domain of the immunoglobulin light chain; V _L3 is the third variable domain of the immunoglobulin light chain; V _H1 is the first variable domain of the immunoglobulin heavy chain; V _H2 is the second variable domain of the immunoglobulin heavy chain; V _H3 is the third variable domain of the immunoglobulin heavy chain; C _L is an immunoglobulin light chain constant domain; C _H1 is the constant domain of immunoglobulin heavy chain C _H1 ; C _H2 is the immunoglobulin heavy chain C _H2 constant domain; C _H3 is the immunoglobulin heavy chain C _H3 constant domain; the hinge is the hinge region of an immunoglobulin connecting the C _H1 and C _H2 domains; And L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers, wherein the polypeptide of formula I and the polypeptide of formula II form a light_chain-heavy_chain crossover pair, and wherein V _H1 and V _L1 form a first antigen binding site that binds the CD28 polypeptide, where V _H2 and V _L2 form a second antigen binding region that binds the CD3 polypeptide, and where V _H3 and V _L3 form a third antigen binding region that binds the 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 5, wherein the T cell expresses a chimeric antigen receptor (CAR) on its cell surface or contains a polynucleotide encoding a CAR. 7. A method of treating a chronic viral infection, comprising administering to a patient an effective amount of a binding protein, where the binding protein contains four polypeptide chains that form three antigen-binding sites, where the first polypeptide chain contains a structure represented by the formula: V _L2 -L ₁ -V _L1 -L ₂ -C _L [I], and the second polypeptide chain contains a structure represented by the formula: V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge-C _H2 -C _H3 [II], and the third polypeptide chain contains the structure represented by the formula: V _H3 -C _H1 -hinge-C _H2 -C _H3 [III], and the fourth polypeptide chain contains the structure represented by the formula: V _L3 -C _L [IV], Where V _L1 is the first variable domain of the immunoglobulin light chain; V _L2 is the second variable domain of the immunoglobulin light chain; V _L3 is the third variable domain of the immunoglobulin light chain; V _H1 is the first variable domain of the immunoglobulin heavy chain; V _H2 is the second variable domain of the immunoglobulin heavy chain; V _H3 is the third variable domain of the immunoglobulin heavy chain; C _L is an immunoglobulin light chain constant domain; C _H1 is the constant domain of immunoglobulin heavy chain C _H1 ; C _H2 is the immunoglobulin heavy chain C _H2 constant domain; C _H3 is the immunoglobulin heavy chain C _H3 constant domain; the hinge is the hinge region of an immunoglobulin connecting the C _H1 and C _H2 domains; And L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers, wherein the polypeptide of formula I and the polypeptide of formula II form a light_chain-heavy_chain crossover pair, and wherein V _H1 and V _L1 form a first antigen binding site that binds the CD28 polypeptide, where V _H2 and V _L2 form a second antigen binding region that binds the CD3 polypeptide, and where V _H3 and V _L3 form a third antigen binding region that binds the CD38 polypeptide. 8. The method according to claim 7, where the patient is a human. 9. The method according to claim 7 or 8, wherein the binding protein is administered to the patient in a pharmaceutical composition containing the binding protein and a pharmaceutically acceptable carrier. 10. The method according to any one of claims 7-9, wherein the introduction of the binding protein leads to the activation and/or proliferation of virus-specific memory T cells in the patient. 11. The method according to any one of claims 1 to 6 and 10, wherein the memory T cells are CD8+ or CD4+ memory T cells. 12. The method according to any one of claims 1 to 6, 10 and 11, wherein the memory T cells are central memory T cells (T _CM ) or effector memory T cells (T _EM ). 13. The method according to any one of claims 1 to 12, wherein the CD28 polypeptide is a human CD28 polypeptide, where the CD3 polypeptide is a human CD3 polypeptide, and wherein the CD38 polypeptide is a human CD38 polypeptide. 14. Method according to any one of claims 1-13, where: (a) domain V _{of H3} contains a CDR-H1 sequence containing the amino acid sequence GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence containing the amino acid sequence IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33), and domain V _L3 contains a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35), and the sequence CDR-L3 containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36); (b) domain V _{of H3} contains a CDR-H1 sequence containing the amino acid sequence GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence containing the amino acid sequence IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33), and domain V _L3 contains a CDR-L1 sequence containing the amino acid sequence QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence GAS (SEQ ID NO:40), and the sequence CDR-L3 containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36); or (c) domain V _{of H3} contains a CDR-H1 sequence containing the amino acid sequence GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence containing the amino acid sequence IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence containing the amino acid sequence ARMFRGAFDY (SEQ ID NO:43), and domain V _L3 contains the sequence CDR-L1 containing the amino acid sequence QGIRND (SEQ ID NO:44), the sequence CDR-L2 containing the amino acid sequence AAS (SEQ ID NO:45), and the sequence CDR-L3 containing the amino acid sequence LQDYIYYPT (SEQ ID NO:46). 15. Method according to claim 14, where: (a) the V _H3 domain contains the amino acid sequence QVQLQQSGAELVRSGASVKMSCKASGYTFTSFNMHWVKETPGQGLEWIGYIYPGNGGTNYNQKFKGKATLTADTSSSTAYMQISSLTSEDSAVYFCARTGGLRRAYFTYWGQGTLVTVS (SEQ ID NO:5), and the V _L3 domain contains the amino acid sequence DIVLTQSPASLAVSLGQRATISCR ASESVDSYGNGFMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:6); (b) domain V _H3 contains the amino acid sequence Qvqlvqsgaevvkpgasvkvsckasgytftsyamhwvkeapgqrlewigyiypgqggtnynqkfqgratltadtsastaymelsslrsedtavyfcartgglrrayftywgqgtlvtvss (SEQ ID NO:13), and domain V _L3 contains the amino acid sequence Divltqspatlslspger atiscrasqsvssygqgfmhwyqqkpgqpprlliygassratgiparfsgsgsgtdftltisplepedfavyycqqnkedpwtfgggtkleik (SEQ ID NO:14); (c) the V _H3 domain contains the amino acid sequence QVQLVQSGAEVVKPGASVKVSCKASGYTFTSFNMHWVKEAPGQRLEWIGYIYPGNGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO:17), and the V _L3 domain contains the amino acid sequence DIVLTQSPATLSLSPGERATISCRASE DSYGNGFMHWYQQKPGQPPRLLIYLASSRAT GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:18); (d) the V _H3 domain contains the amino acid sequence QVQLVQSGAEVVKSGASVKVSCKASGYTFTSFNMHWVKEAPGQGLEWIGYIYPGNGGTNYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO:21), and the V _L3 domain contains the amino acid sequence DIVLTQSPATLSLSPGERATISCRASESVDSY GNGFMHWYQQKPGQPPRLLIYLASSRAT GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:18); (e) the V _H3 domain contains the amino acid sequence QVQLVQSGAEVVKPGASVKMSCKASGYTFTSFNMHWVKEAPGQRLEWIGYIYPGNGGTNYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO:23), and the V _L3 domain contains the amino acid sequence DIVLTQSPATLSLSPGERATISCRASESVDS YGNGFMHWYQQKPGQPPRLLIYLASSRAT GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:18); or (f) the V domain _{of H3} contains the amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSS (SEQ ID NO:9), and the V domain of _L3 contains the amino acid sequence AIQMTQSPSSLSASVGDRVTITCRASQGIRN DLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIK (SEQ ID NO:10). 16. Method according to any one of claims 1-15, where: (a) domain V _{of H1} contains a CDR-H1 sequence containing the amino acid sequence GYTFTSYY (SEQ ID NO:108), a CDR-H2 sequence containing the amino acid sequence IYPGNVNT (SEQ ID NO:109), and a CDR-H3 sequence containing the amino acid sequence TRSHYGLDWNFDV (SEQ ID NO:110), and the V _L1 domain contains a CDR-L1 sequence containing the amino acid sequence QNIYVW (SEQ ID NO:111), a CDR-L2 sequence containing the amino acid sequence KAS (SEQ ID NO:112), and the sequence CDR-L3 containing the amino acid sequence QQGQTYPY (SEQ ID NO:113); or ( b) domain V _{of H1} contains a CDR-H1 sequence containing the amino acid sequence GFSLSDYG (SEQ ID NO:114), a CDR-H2 sequence containing the amino acid sequence IWAGGGT (SEQ ID NO:115), and a CDR-H3 sequence containing the amino acid sequence ARDKGYSYYYSMDY (SEQ ID NO:116), and domain V _L1 contains a CDR-L1 sequence containing the amino acid sequence ESVEYYVTSL (SEQ ID NO:117), a CDR-L2 sequence containing the amino acid sequence AAS (SEQ ID NO:118), and the sequence CDR-L3 containing the amino acid sequence QQSRKVPYT (SEQ ID NO:119). 17. Method according to claim 16, where: (a) the V _H1 domain contains the amino acid sequence qvqlvqsgaevvkpgasvkvsckasgytftsyyihwvrqapgqglewigsiypgnvntnyaqkfqgratltvdtsistaymelsrlrsddtavyyctrshygldwnfdvwgkgttvtvss (SEQ ID NO:49), and the V _L1 domain contains the amino acid sequence diqmtqsps slsasvgdrvtitcqasqniyvwlnwyqqkpgkapklliykasnlhtgvpsrfsgsgsgtdftltisslqpediatyycqqgqtypytfgqgtkleik (SEQ ID NO:50); or (b) the V _H1 domain contains the amino acid sequence qvqlqesgpglvkpsqtlsltctvsgfslsdygvhwvrqppgkglewlgviwagggtnynpslksrktiskdtsknqvslklssvtaadtavyycardkgysyyysmdywgqgttvtvs (SEQ ID NO:51), and the V _L1 domain contains the amino acid sequence divltqspa slavspgqratitcrasesveyyvtslmqwyqqkpgqppkllifaasnvesgvparfsgsgsgtdftltinpveandvanyycqqsrkvpytfgqgtkleik (SEQ ID NO:52). 18. Method according to any one of claims 1-17, where: (a) the V domain _{of H2} contains a CDR-H1 sequence containing the amino acid sequence GFTFTKAW (SEQ ID NO:120), a CDR-H2 sequence containing the amino acid sequence IKDKSNSYAT (SEQ ID NO:121), and a CDR-H3 sequence containing the amino acid sequence RGVYYALSPFDY (SEQ ID NO:122), and the V _L2 domain contains a CDR-L1 sequence containing the amino acid sequence QSLVHNNANTY (SEQ ID NO:123), a CDR-L2 sequence containing the amino acid sequence KVS (SEQ ID NO:124), and the sequence CDR-L3 containing the amino acid sequence GQGTQYPFT (SEQ ID NO:125); or (b) the V domain _{of H2} contains a CDR-H1 sequence containing the amino acid sequence GFTFTKAW (SEQ ID NO:126), a CDR-H2 sequence containing the amino acid sequence IKDKSNSYAT (SEQ ID NO:127), and a CDR-H3 sequence containing the amino acid sequence GVYYALSPFDY (SEQ ID NO:128), and the V _L2 domain contains a CDR-L1 sequence containing the amino acid sequence QSLVHNNGNTY (SEQ ID NO:129), a CDR-L2 sequence containing the amino acid sequence KVS (SEQ ID NO:130), and the sequence CDR-L3 containing the amino acid sequence GQGTQYPFT (SEQ ID NO:131). 19. Method according to claim 18, where: (a) the V _H2 domain contains the amino acid sequence qvqlvesgggvvqpgrslrlscaasgftftkawmhwvrqapgkqlewvaqikdksnsyatyyadsvkgrftisrddskntlylqmnslraedtavyycrgvyyalspfdywgqgtlvtvss (SEQ ID NO:53), and the V _L2 domain contains the amino acid sequence divmtqtpls lsvtpgqpasisckssqslvhnnantylswylqkpgqspqsliykvsnrfsgvpdrfsgsgsgtdftlkisrveaedvgvyycgqgtqypftfgsgtkveik (SEQ ID NO:54); or (b) the V _H2 domain contains the amino acid sequence qvqlvesgggvvqpgrslrlscaasgftftkawmhwvrqapgkglewvaqikdksnsyatyyadsvkgrftisrdnskntlylqmnslraedtavyycrgvyyalspfdywgqgtlvtvss (SEQ ID NO:84), and the V _L2 domain contains the amino acid sequence divmtqtplsls vtpgqpasisckssqslvhnngntylswylqkpgqspqlliykvsnrfsgvpdrfsgsgsgtdftlkisrveaedvgvyycgqgtqypftfgggtkveik (SEQ ID NO:85). 20. The method according to any one of claims 1 to 19, wherein at least one of L ₁ , L ₂ , L ₃ or L ₄ independently has a length of 0 amino acids. 21. The method according to any one of claims 1 to 19, wherein (a) each of L ₁ , L ₂ , L ₃ and L ₄ independently has a length of zero amino acids, or contains 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) each of L ₁ , L ₂ , L ₃ and L ₄ independently contains 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 according to any one of claims 1 to 19, wherein L ₁ contains the sequence GQPKAAP (SEQ ID NO: 58), L ₂ contains the sequence TKGPS (SEQ ID NO: 57), L ₃ contains the sequence S, and L ₄ contains the sequence RT. 23. The method according to any one of claims 1 to 22, wherein the hinge-C _H2 -C _H3 domains of the second and third polypeptide chains are human hinge-IgG4-C _H2 -C _H3 domains, and wherein each of the hinge-C _H2 -C _H3 domains contains amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, where the amino acid substitutions are F234A and L235A. 24. The method according to any one of claims 1 to 22, wherein the hinge-C _H2 -C _H3 domains of the second and third polypeptide chains are human hinge-IgG4-C _H2 -C _H3 domains, and wherein each of the hinge-C _H2 -C _H3 domains contains amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to the EU index, where the amino acid substitutions are E233P, F234V, L235A, and a deletion at position 236. 25. The method according to any one of claims 1 to 24, wherein the hinge-C _H2 -C _H3 domains of the second and third polypeptide chains are human hinge-IgG4-C _H2 -C _H3 domains, and wherein each of the hinge-C _H2 -C _H3 domains contains amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, where the amino acid substitutions are S228P and R409K. 26. The method according to any one of claims 1 to 22, wherein the hinge-C _H2 -C _H3 domains of the second and third polypeptide chains are human hinge-IgG1-C _H2 -C _H3 domains, and wherein each of the hinge-C _H2 -C _H3 domains contains amino acid substitutions at positions corresponding to positions 234, 235 and 329 of human IgG1 according to the EU index, where the amino acid substitutions are L234A, L235A and P329A. 27. The method according to any one of claims 1 to 22, wherein the hinge-C _H2 -C _H3 domains of the second and third polypeptide chains are human hinge-IgG1-C _H2 -C _H3 domains, and wherein each of the hinge-C _H2 -C _H3 domains contains amino acid substitutions at positions corresponding to positions 298, 299 and 300 of human IgG1 according to the EU index, where the amino acid substitutions are S298N, T299A and Y300S. 28. The method according to any one of claims 1 to 27, wherein the hinge-C _H2 -C _H3 domain of the second polypeptide chain contains amino acid substitutions at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or IgG4 in accordance with the EU index, where the amino acid substitutions are Y349C, T366S, L368A and Y407V; and wherein the hinge-C _H2 -C _H3 domain of the third polypeptide chain contains amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are S354C and T366W. 29. The method according to any one of claims 1 to 27, wherein the hinge-C _H2 -C _H3 domain of the second polypeptide chain contains amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 in accordance with the EU index, where the amino acid substitutions represent are S354C and T366W; and wherein the hinge-C _H2 -C _H3 domain of the third polypeptide chain contains amino acid substitutions at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A and Y407V. thirty. The method according to claim 1, where: (a) the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:60, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:62, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO:63; (b) the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:64, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:65, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO:63; (c) the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:66, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:67, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO:63; (d) the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:60, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:68, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO:69; (e) the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:64, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:70, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO:69; or (f) the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:66, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:71, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO:69. 31. The method according to any one of claims 1 to 30, where the virus is human immunodeficiency virus (HIV), influenza virus, cytomegalovirus (CMV), hepatitis B virus (HBV), human papillomavirus (HPV), Epstein-Barr virus (EBV ), human spumavirus (HFV), herpes simplex virus 1 (HSV-1), or herpes simplex virus 2 (HSV-2).

Images