WO2018215937A1

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Info

Publication number: WO2018215937A1
Application number: PCT/IB2018/053624
Authority: WO
Inventors: Michael DIDONATO; Rodrigo Andreas HESS; Glen Spraggon
Original assignee: Novartis AG
Current assignee: Novartis AG
Priority date: 2017-05-24
Filing date: 2018-05-22
Publication date: 2018-11-29
Anticipated expiration: 2019-11-24

Abstract

The present invention provides antibody cytokine engrafted proteins that bind to and stimulate intracellular signaling through an interleukin receptor superfamily member (i.e., IL7Ra), and stimulate signaling of immune cells. Methods of treating cancer with antibody cytokine engrafted proteins are also included.

Description

INTERLEUKIN-7 ANTIBODY CYTOKINE ENGRAFTED PROTEINS AND METHODS OF USE IN THE TREATMENT OF CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of U.S. Provisional Application No.

62/510,547 filed May 24, 2017, the content of which is hereby incorporated by reference in its entirety.

FIELD

[002] The present invention relates to interleukin-7 antibody cytokine engrafted compositions that bind the interleukin-7 receptor (CD 127), and methods of cancer treatment.

SEQUENCE LISTING

[003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on March 30, 2018, is named PAT057625-WO-PCT_SL.txt and is 123,221 bytes in size.

BACKGROUND

[004] IL7 was first cloned in 1989 (Goodwin et al, PNAS USA 1989; 86(1):302-

306). IL7 is a single chain 25 kD glycoprotein with a typical hematopoietic type 1 cytokine (Lupton et al, J. Immunol. 1990; 144(9):3592-3601). IL7 mediates its action through binding to a heterodimer of an IL7 binding subunit (IL7Ra, CD 127) and the common gamma chain (IL2Ry), which is shared by the cytokine receptors for IL2, IL4, IL9, IL15 and IL21.

[005] The first in vivo studies of IL7 in mice demonstrated that it had a proliferative and survival effect on both B and T cells (Morrissey et al., J. Immunol. 1991 ;147(2):561-568 and Grabenstein et al, J. Exp. Med. 1993: 178(l);257-264). Elimination of IL7 or IL7Ra in knockout mice resulted in complete loss of both T and B cells (von Freeden-Jeffry et al., J. Exp. Med. 1995; 181(4):1519-1526 and Moore et al, J. Immunol. 1996; 157(6)2366-2373). In contrast, inherited mutations in IL7Ra in humans result in autosomal recessive severe combined immune deficiency, characterized by a complete absence of T cells, but not of B cells or NK cells (Puel et al, Nat. Genet. 1998; 20(4):394-397). The surviving B cells usually exhibit defects, such as low IgG production. Further supporting the effect of IL7 on T cells in human, hematopoietic stem cells taken from human fetal bone marrow commit to the B cell lineage regardless of the presence of IL7 (Prieyl et al., PNAS USA 1996;

93(19):10348-10353).

[006] In the clinic, it was observed that IL7 contributed to the stimulation of the tumor microenvironment for T-cell acute lymphoblastic leukemia (T-ALL), and that IL7 actually contributed to leukemia progression (Silva et al., Cancer Res. 2009; 71 :4780-4789). A phase 1/2A trial examined the outcome of thrice weekly subcutaneous human recombinant IL7 administration in patients with idiopathic CD4 lymphopenia (Sheikh et al., Blood 2016; 127(8):977-988). The IL7 therapy resulted in increase in circulating CD4 and CD8 T helper cells and resident CD3 T cells in bone marrow and gut mucosa. Injections were well tolerated with the exceptions of a hypersensitivity reaction in one patient and the development of systemic lupus erythematosus by another (Sheikh et al., supra). This demonstrates the further need to develop useful IL7 therapeutics for the treatment of cancer.

DESCRIPTION

[007] The present disclosure provides for an IL7 cytokine engrafted into the CDR sequences of an antibody having preferred therapeutic profiles over known IL7 molecules. In particular, the provided antibody cytokine engrafted protein compositions increase or maintain T cells. Additionally, provided compositions convey improved half-life, stability and produceability over recombinant human IL7 formulations. The present disclosure thus provides antibody cytokine engrafted proteins that bind to and promote signalling through IL7Ra. Provided herein are antibody cytokine engrafted proteins comprising (i) an immunoglobulin heavy chain sequence comprising a heavy chain variable region (VH) and (ii) an immunoglobulin light chain sequence comprising a light chain variable region (VL), and wherein an IL7 molecule is engrafted into a complementarity determining region (CDR) of the VH or the VL of the antibody.

[008] The antibody cytokine engrafted protein wherein the IL7 molecule is engrafted into a heavy chain CDR.

[009] The antibody cytokine engrafted protein, wherein heavy chain CDR is selected from complementarity determining region 1 (HCDR1), complementarity determining region 2 (HCDR2) or complementarity determining region 3 (HCDR3).

[0010] The antibody cytokine engrafted protein, wherein the IL7 molecule engrafted into the HCDR2. [0011] The antibody cytokine engrafted protein, wherein the IL7 molecule engrafted into the HCDR3.

[0012] The antibody cytokine engrafted protein, wherein the IL7 molecule engrafted into a light chain CDR.

[0013] The antibody cytokine engrafted protein, wherein the light chain CDR is selected from complementarity determining region 1 (LCDRl), complementarity determining region 2 (LCDR2) or complementarity determining region 3 (LCDR3).

[0014] The antibody cytokine engrafted protein, wherein the antibody cytokine engrafted protein stimulates CD 8+ cell proliferation greater than recombinant IL7.

[0015] The antibody cytokine engrafted protein, wherein the antibody cytokine engrafted protein stimulates CD 4+ cell proliferation greater than recombinant IL7.

[0016] The antibody cytokine engrafted protein, wherein the antibody cytokine engrafted protein has a longer half-life than recombinant IL7.

[0017] The antibody cytokine engrafted protein, wherein the IL7 molecule consists of

SEQ ID NO:2.

[0018] The antibody cytokine engrafted protein, further comprising an IgG class antibody heavy chain.

[0019] The antibody cytokine engrafted protein, wherein the IgG class heavy chain is selected from IgGl, IgG2, or IgG4.

[0020] The antibody cytokine engrafted protein, wherein the binding specificity of the

CDRs to the target protein is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%, by the engrafted IL7 molecule.

[0021] The antibody cytokine engrafted protein, wherein the binding specificity of the

CDRs to the target protein is retained by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,

95%, 98%, 99%, or 100%, in the presence of the engrafted IL7 molecule.

[0022] The antibody cytokine engrafted protein, wherein the binding specificity of the

CDRs is to a target distinct from the binding specificity of the IL7 molecule.

[0023] The antibody cytokine engrafted protein, wherein the binding specificity of the

CDRs is to a non-human antigen.

[0024] The antibody cytokine engrafted protein, wherein the non-human antigen is a virus.

[0025] The antibody cytokine engrafted protein, wherein the virus is respiratory syncytial virus (RSV). [0026] The antibody cytokine engrafted protein, wherein the RSV is selected from

RSV subgroup A or RSV subgroup B.

[0027] The antibody cytokine engrafted protein, wherein the antibody scaffold portion of the antibody cytokine engrafted protein is humanized or human.

[0028] An antibody cytokine engrafted protein comprising:

(i) a heavy chain variable region that comprises (a) a HCDRl of SEQ ID NO: 24, (b) a HCDR2 of SEQ ID NO: 25, (c) a HCDR3 of SEQ ID NO: 26 and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 33, (e) a LCDR2 of SEQ ID NO: 34, and (f) a LCDR3 of SEQ ID NO: 35;

(ii) a heavy chain variable region that comprises (a) a HCDRl of SEQ ID NO: 42, (b) a HCDR2 of SEQ ID NO: 43, (c) a HCDR3 of SEQ ID NO: 44; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 51, (e) a LCDR2 of SEQ ID NO:52, and (f) a LCDR3 of SEQ ID NO:53;

(iii) a heavy chain variable region that comprises (a) a HCDRl of SEQ ID NO: 6, (b) a HCDR2 of SEQ ID NO: 7, (c) a HCDR3 of SEQ ID NO: 8; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 15, (e) a LCDR2 of SEQ ID NO: 16, and (f) a LCDR3 of SEQ ID NO: 17;

(iv) a heavy chain variable region that comprises (a) a HCDRl of SEQ ID NO: 60, (b) a HCDR2 of SEQ ID NO: 61, (c) a HCDR3 of SEQ ID NO: 62; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 69, (e) a LCDR2 of SEQ ID NO:70, and (f) a LCDR3 of SEQ ID NO:71 ;

(v) a heavy chain variable region that comprises (a) a HCDRl of SEQ ID NO: 78, (b) a HCDR2 of SEQ ID NO:79, (c) a HCDR3 of SEQ ID NO:80; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 87, (e) a LCDR2 of SEQ ID NO: 88, and (f) a LCDR3 of SEQ ID NO: 89; and

(vi) a heavy chain variable region that comprises (a) a HCDRl of SEQ ID NO: 96, (b) a HCDR2 of SEQ ID NO: 97, (c) a HCDR3 of SEQ ID NO: 98; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 105, (e) a LCDR2 of SEQ ID NO: 106, and (f) a LCDR3 of SEQ ID NO: 107.

[0029] An antibody cytokine engrafted protein comprising:

(i) a heavy chain variable region (VH) that comprises SEQ ID NO: 27, and a light chain variable region (VL) that comprises SEQ ID NO: 36; (ii) a heavy chain variable region (VH) that comprises SEQ ID NO:45, and a light chain variable region (VL) that comprises SEQ ID NO:54;

(iii) a heavy chain variable region (VH) that comprises SEQ ID NO: 9, and a light chain variable region (VL) that comprises SEQ ID NO: 18;

(iv) a heavy chain variable region (VH) that comprises SEQ ID NO:63, and a light chain variable region (VL) that comprises SEQ ID NO:72;

(v) a heavy chain variable region (VH) that comprises SEQ ID NO:81, and a light chain variable region (VL) that comprises SEQ ID NO:90; or

(vi) a heavy chain variable region (VH) that comprises SEQ ID NO: 99, and a light chain variable region (VL) that comprises SEQ ID NO: 108.

[0030] The antibody cytokine engrafted protein, further comprising a modified Fc region corresponding with reduced effector function.

[0031] The antibody cytokine engrafted protein, wherein the modified Fc region comprises a mutation selected from one or more of D265A, P329A, P329G, N297A, L234A, and L235A.

[0032] The antibody cytokine engrafted protein, wherein the modified Fc region comprises a combination of mutations selected from one or more of D265A/P329A, D265A/N297A, L234/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[0033] An antibody cytokine engrafted protein comprising a HCDR1 of SEQ ID NO:

24, a HCDR2 of SEQ ID NO: 25, a HCDR3 of SEQ ID NO: 26, a LCDRl of SEQ ID NO: 33, a LCDR2 of SEQ ID NO: 34, a LCDR3 of SEQ ID NO: 35, a modified Fc region containing the mutation D265A/P329A, wherein the antibody cytokine engrafted protein induces the proliferation of CD8+ T cells.

[0034] An antibody cytokine engrafted protein comprising a HCDR1 of SEQ ID NO:

42, a HCDR2 of SEQ ID NO: 43, a HCDR3 of SEQ ID NO: 44, a LCDRl of SEQ ID NO: 51, a LCDR2 of SEQ ID NO: 52, a LCDR3 of SEQ ID NO: 53, a modified Fc region containing the mutation D265A/P329A, wherein the antibody cytokine engrafted protein induces the proliferation of CD8+ T cells.

[0035] An isolated nucleic acid encoding an antibody cytokine engrafted protein comprising: (i) a heavy chain variable region of SEQ ID NO: 28 and/or a light chain variable region of SEQ ID NO:37;

(ii) a heavy chain variable region of SEQ ID NO: 46 and/or a light chain variable region of SEQ ID NO:55;

(iii) a heavy chain variable region of SEQ ID NO: 10 and/or a light chain variable region of SEQ ID NO: 19;

(vi) a heavy chain variable region of SEQ ID NO: 64 and/or a light chain variable region of SEQ ID NO: 73;

(v) a heavy chain variable region of SEQ ID NO: 82 and/or a light chain variable region of SEQ ID NO: 91 ; or

(ii) a heavy chain variable region of SEQ ID NO: 100 and/or a light chain variable region of SEQ ID NO: 109.

[0036] A recombinant host cell suitable for the production of an antibody cytokine engrafted protein, comprising the nucleic acids encoding the heavy and light chain polypeptides of the antibody cytokine engrafted protein, and optionally, secretion signals.

[0037] The host cell which is a mammalian cell line.

[0038] The mammalian cell line, wherein the mammalian cell line is a CHO cell line.

[0039] A pharmaceutical composition comprising the antibody cytokine engrafted protein and a pharmaceutically acceptable carrier.

[0040] A method of treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of the antibody cytokine engrafted protein or the pharmaceutical composition.

[0041] The method of treating cancer, wherein the cancer is selected from the group consisting of: melanoma, lung cancer, colorectal cancer, prostate cancer, breast cancer, leukemia and lymphoma.

[0042] The method of treating cancer, wherein the antibody cytokine engrafted protein or the pharmaceutical composition is administered in combination with another therapeutic agent.

[0043] The method of treating cancer, wherein the therapeutic agent is an immune checkpoint inhibitor.

[0044] The method of treating cancer, wherein the immune checkpoint is selected from the group consisting of: PD-1, PD-Ll, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR. [0045] The method of treating cancer, wherein the immune checkpoint inhibitor is an anti-PD-Ll antibody.

[0046] The method of treating cancer, wherein the immune checkpoint inhibitor is an anti-TIM3 antibody.

[0047] A method of expanding CD8+ T cells in a patient in need thereof, comprising administering to the individual a therapeutically effective amount of the antibody cytokine engrafted protein or the pharmaceutical composition.

[0048] The method of expanding CD8+ T cells, further comprising administration of an immune checkpoint inhibitor.

[0049] The method of expanding CD8+ T cells, wherein the immune checkpoint is selected from the group consisting of: PD-1, PD-Ll, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIRl, CD160, 2B4 and TGFR.

[0050] The method of expanding CD8+ T cells, wherein the immune checkpoint inhibitor is an anti-PD-Ll antibody.

[0051] The method of expanding CD8+ T cells, wherein the immune checkpoint inhibitor is an anti-TIM3 antibody.

[0052] A method of reverting the exhaustion of CD8+ T cells in a patient in need thereof, comprising administering to the individual a therapeutically effective amount of the antibody cytokine engrafted protein or the pharmaceutical composition.

[0053] The method of reverting the exhaustion of CD8+ T cells, further comprising administration of an immune checkpoint inhibitor.

[0054] The method of reverting the exhaustion of CD8+ T cells, wherein the immune checkpoint is selected from the group consisting of: PD-1, PD-Ll, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIRl, CD160, 2B4 and TGFR.

[0055] The method of reverting the exhaustion of CD8+ T cells, wherein the immune checkpoint inhibitor is an anti-PD-Ll antibody.

[0056] The method of reverting the exhaustion of CD8+ T cells, wherein the immune checkpoint inhibitor is an anti-TIM3 antibody.

[0057] A method of treating a viral infection in a patient in need thereof, comprising administering to the individual a therapeutically effective amount of the antibody cytokine engrafted protein.

[0058] The method of treating a viral infection, further comprising administration of an immune checkpoint inhibitor. [0059] The method of treating a viral infection, wherein the immune checkpoint is selected from the group consisting of: PD-1, PD-Ll, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIRl, CD160, 2B4 and TGFR.

[0060] The method of treating a viral infection, wherein the immune checkpoint inhibitor is an anti-PD-Ll antibody.

[0061] The method of treating a viral infection, wherein the immune checkpoint inhibitor is an anti-TIM3 antibody.

[0062] Use of an antibody cytokine engrafted protein comprising:

(i) a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 24, (b) a HCDR2 of SEQ ID NO: 25, (c) a HCDR3 of SEQ ID NO: 26 and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 33, (e) a LCDR2 of SEQ ID NO: 34, and (f) a LCDR3 of SEQ ID NO: 35;

(ii) a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 42, (b) a HCDR2 of SEQ ID NO: 43, (c) a HCDR3 of SEQ ID NO: 44; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 51, (e) a LCDR2 of SEQ ID NO:52, and (f) a LCDR3 of SEQ ID NO:53;

(iii) a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 6, (b) a HCDR2 of SEQ ID NO: 7, (c) a HCDR3 of SEQ ID NO: 8; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 15, (e) a LCDR2 of SEQ ID NO: 16, and (f) a LCDR3 of SEQ ID NO: 17;

(iv) a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 60, (b) a HCDR2 of SEQ ID NO: 61, (c) a HCDR3 of SEQ ID NO: 62; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 69, (e) a LCDR2 of SEQ ID NO:70, and (f) a LCDR3 of SEQ ID NO:71 ;

(v) a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 78, (b) a HCDR2 of SEQ ID NO:79, (c) a HCDR3 of SEQ ID NO:80; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 87, (e) a LCDR2 of SEQ ID NO: 88, and (f) a LCDR3 of SEQ ID NO: 89; or

(vi) a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 96, (b) a HCDR2 of SEQ ID NO: 97, (c) a HCDR3 of SEQ ID NO: 98; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 105, (e) a LCDR2 of SEQ ID NO: 106, and (f) a LCDR3 of SEQ ID NO: 107, in the treatment of cancer. [0063] The use of the antibody cytokine engrafted protein in the treatment of cancer, wherein the antibody cytokine engrafted protein is administered in combination with another therapeutic agent.

[0064] The use of the antibody cytokine engrafted protein in the treatment of cancer, wherein the therapeutic agent is an immune checkpoint inhibitor.

[0065] The use of the antibody cytokine engrafted protein in the treatment of cancer, wherein the immune checkpoint is selected from the group consisting of: PD-1, PD-L1, PD- L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 and TGFR.

[0066] The use of the antibody cytokine engrafted protein in the treatment of cancer, wherein the immune checkpoint inhibitor is an anti-PD-Ll antibody.

[0067] The use of the antibody cytokine engrafted protein in the treatment of cancer, wherein the immune checkpoint inhibitor is an anti-TIM3 antibody.

[0068] In certain embodiments, the antibody cytokine engrafted protein comprises an

IgG class antibody Fc region. In particular embodiments, the immunoglobulin is selected from IgGl, IgG2, or IgG4 subclass Fc region. The antibody, antibody fragment, or antigen binding molecule optionally contains at least one modification that modulates (i.e., increases or decreases) binding of the antibody or antibody fragment to an Fc receptor. The immunoglobulin heavy chain can optionally comprise a modification conferring modified effector function. In particular embodiments the immunoglobulin heavy chain can comprise a mutation conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A/N297A, L234/L235A, P329A/L234A/L235A, and

P329G/L234A/L235A.

[0069] In some embodiments, the antibody cytokine engrafted protein also comprises variations in the IL7 portion of the molecule. The variations can be single amino acid changes, single amino acid deletions, multiple amino acid changes and multiple amino acid deletions. These changes in the IL7 cytokine portion of the molecule can increase or decrease the signaling of the antibody cytokine engrafted protein on the IL7 receptor.

[0070] Furthermore, the disclosure provides polynucleotides encoding at least a heavy chain and/or a light chain protein of an antibody cytokine engrafted protein as described herein. In another related aspect, host cells are provided that are suitable for the production of an antibody cytokine engrafted protein as described herein. In particular embodiments, host cells comprise nucleic acids encoding a light chain and/or heavy chain polypeptide of the antibody cytokine engrafted protein. In still another aspect, methods for producing antibody cytokine engrafted proteins are provided, comprising culturing provided host cells as described herein under conditions suitable for expression, formation, and secretion of the antibody cytokine engrafted protein and recovering the antibody cytokine engrafted protein from the culture. In a further aspect, the disclosure further provides kits comprising an antibody cytokine engrafted protein, as described herein.

[0071] In another related aspect, the disclosure further provides compositions comprising an antibody cytokine engrafted protein, as described herein, and a

pharmaceutically acceptable carrier. In some embodiments, the disclosure provides pharmaceutical compositions comprising an antibody cytokine engrafted protein for administering to an individual.

[0072] In another aspect, methods of treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an antibody cytokine engrafted protein, as described herein. In a further aspect, an antibody cytokine engrafted protein for use in treatment or prophylaxis of cancer in an individual is provided.

[0073] In some embodiments, the patient has a cell proliferation disorder or cancer, for example, melanoma, lung cancer, colorectal cancer, prostate cancer, breast cancer, leukemia and lymphoma.

DEFINITIONS

[0074] An "antibody" refers to a molecule of the immunoglobulin family comprising a tetrameric structural unit. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD), connected through a disulfide bond. Recognized immunoglobulin genes include the κ, λ, α, γ, δ, ε, and μ constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. Antibodies can be of any isotype/class (e.g., IgG, IgM, IgA, IgD, and IgE), or any subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, IgA2).

[0075] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used structurally and functionally. The N-terminus of each chain defines a variable (V) region or domain of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these regions of light and heavy chains respectively. The pairing of a VH and VL together forms a single antigen- binding site. In addition to V regions, both heavy chains and light chains contain a constant (C) region or domain. A secreted form of a immunoglobulin C region is made up of three C domains, CHI, CH2, CH3, optionally CH4 ( μ), and a hinge region. A membrane-bound form of an immunoglobulin C region also has membrane and intracellular domains. Each light chain has a VL at the N-terminus followed by a constant domain (C) at its other end. The constant domains of the light chain (CL) and the heavy chain (CHI, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention, the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino- terminus of the antibody. The N-terminus is a variable region and at the C-terminus is a constant region; the CH3 and CL domains actually comprise the carboxy-terminal domains of the heavy and light chain, respectively. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain. As used herein, an "antibody"

encompasses conventional antibody structures and variations of antibodies. Thus, within the scope of this concept are antibody cytokine engrafted proteins, full length antibodies, chimeric antibodies, humanized antibodies, human antibodies, and antibody fragments thereof.

[0076] Antibodies exist as intact immunoglobulin chains or as a number of well- characterized antibody fragments produced by digestion with various peptidases. The term "antibody fragment," as used herein, refers to one or more portions of an antibody that retains six CDRs. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab' which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into an Fab' monomer. The Fab' monomer is essentially a Fab with a portion of the hinge region (Paul, Fundamental Immunology 3d ed. (1993)). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. As used herein, an "antibody fragment" refers to one or more portions of an antibody, either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies, that retain binding specificity and functional activity. Examples of antibody fragments include Fv fragments, single chain antibodies (ScFv), Fab, Fab', Fd (Vh and CHI domains), dAb (Vh and an isolated CDR); and multimeric versions of these fragments (e.g. , F(ab')2,) with the same binding specificity. Antibody cytokine engrafted proteins can also comprise antibody fragments necessary to achieve the desired binding specificity and activity.

[0077] A "Fab" domain as used in the context comprises a heavy chain variable domain, a constant region CHI domain, a light chain variable domain, and a light chain constant region CL domain. The interaction of the domains is stabilized by a disulfide bond between the CHI and CL domains. In some embodiments, the heavy chain domains of the Fab are in the order, from N-terminus to C-terminus, VH-CH and the light chain domains of a Fab are in the order, from N-terminus to C-terminus, VL-CL. In some embodiments, the heavy chain domains of the Fab are in the order, from N-terminus to C-terminus, CH-VH and the light chain domains of the Fab are in the order CL-VL. Although the Fab fragment was historically identified by papain digestion of an intact immunoglobulin, in the context of this disclosure, a "Fab" is typically produced recombinantly by any method. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen- binding site.

[0078] "Complementarity-determining domains" or "complementary-determining regions" ("CDRs") interchangeably refer to the hypervariable regions of VL and VH. CDRS are the target protein-binding site of antibody chains that harbor specificity for such target protein. There are three CDRs (CDR 1-3, numbered sequentially from the N-terminus) in each human VL or VH, constituting about 15-20% of the variable domains. CDRs are structurally complementary to the epitope of the target protein and are thus directly responsible for the binding specificity. The remaining stretches of the VL or VH, the so-called framework regions (FR), exhibit less variation in amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4. W.H. Freeman & Co., New York, 2000).

[0079] Positions of CDRs and framework regions can be determined using various well known definitions in the art, e.g., Kabat, Chothia, and AbM (see, e.g., Kabat et al. 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, Johnson et al. , Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al. , Nature, 342:877-883 (1989); Chothia et al, J. Mol. Biol, 227:799-817 (1992); Al-Lazikani et al., J.Mol.Biol., 273:927-748 (1997)). Definitions of antigen combining sites are also described in the following: Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); and Lefranc, M.P., Nucleic Acids Res., 29:207-209 (2001); (ImMunoGenTics (IMGT) numbering) Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al, Dev. Comp. Immunol., 27, 55-77 (2003); MacCallum et al, J. Mol. Biol, 262:732-745 (1996); and Martin et al, Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); Martin et al, Methods Enzymol., 203: 121-153 (1991); and Rees et al, In Sternberg M.J.E. (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996).

[0080] Under Kabat, CDR amino acid residues in the VH are numbered 31-35

(HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the V_L are numbered 24-34 (LCDRl), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia, CDR amino acids in the V_H are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in V_L are numbered 26-32 (LCDRl), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102

(HCDR3) in human VH and amino acid residues 24-34 (LCDRl), 50-56 (LCDR2), and 89- 97 (LCDR3) in human VL.

[0081] An "antibody variable light chain" or an "antibody variable heavy chain" as used herein refers to a polypeptide comprising the VL or VH, respectively. The endogenous VL is encoded by the gene segments V (variable) and J (junctional), and the endogenous VH by V, D (diversity), and J. Each of VL or VH includes the CDRs as well as the framework regions (FR). The term "variable region" or "V-region" interchangeably refer to a heavy or light chain comprising FR 1 -CDR 1 -FR2-CDR2-FR3 -CDR3 -FR4. A V-region can be naturally occurring, recombinant or synthetic. In this application, antibody light chains and/or antibody heavy chains can be collectively referred to as "antibody chains." As provided and further described herein, an "antibody variable light chain" or an "antibody variable heavy chain" and/or a "variable region" and/or an "antibody chain" optionally comprises a cytokine polypeptide sequence incorporated into a CDR.

[0082] The C-terminal portion of an immunoglobulin heavy chain herein, comprising, e.g., CH2 and CH3 domains, is the "Fc" domain. An "Fc region" as used herein refers to the constant region of an antibody excluding the first constant region (CHI) immunoglobulin domain. Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM Fc may include the J chain. For IgG, Fc comprises immunoglobulin domains Cy2 and Cy3 and the hinge between Cyl and Cy. It is understood in the art that boundaries of the Fc region may vary, however, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, using the numbering is according to the EU index as in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). "Fc region" may refer to this region in isolation or this region in the context of an antibody or antibody fragment. "Fc region" includes naturally occurring allelic variants of the Fc region, e.g., in the CH2 and CH3 region, including, e.g., modifications that modulate effector function. Fc regions also include variants that don't result in alterations to biological function. For example, one or more amino acids are deleted from the N-terminus or C- terminus of the Fc region of an immunoglobulin without substantial loss of biological function. For example, in certain embodiments a C-terminal lysine is modified replaced or removed. In particular embodiments one or more C-terminal residues in the Fc region is altered or removed. In certain embodiments one or more C-terminal residues in the Fc (e.g., a terminal lysine) is deleted. In certain other embodiments one or more C-terminal residues in the Fc is substituted with an alternate amino acid (e.g., a terminal lysine is replaced). Such variants are selected according to general rules known in the art so as to have minimal effect on activity (see, e.g., Bowie, et al., Science 247:306-1310, 1990). The Fc domain is the portion of the immunoglobulin (Ig) recognized by cell receptors, such as the FcR, and to which the complement-activating protein, CI q, binds. The lower hinge region, which is encoded in the 5' portion of the CH2 exon, provides flexibility within the antibody for binding to FcR receptors.

[0083] A "chimeric antibody" is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g. , an enzyme, toxin, hormone, growth factor, and drug; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.

[0084] A "humanized" antibody is an antibody that retains the reactivity (e.g., binding specificity, activity) of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining non-human CDR regions and replacing remaining parts of an antibody with human counterparts. See, e.g. , Morrison et al. , Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984); Morrison and Oi, Adv. Immunol, 44:65-92 (1988); Verhoeyen et al, Science, 239:1534-1536 (1988); Padlan, Molec. Immun. , 28:489- 498 (1991); Padlan, Molec. Immun., 31(3):169-217 (1994).

[0085] A "human antibody" includes antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin.

Furthermore, if an antibody contains a constant region, the constant region also is derived from such human sequences, e.g. , human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik et al., J. Mol. Biol. 296:57-86, 2000). Human antibodies may include amino acid residues not encoded by human sequences {e.g. , mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo, or a conservative substitution to promote stability or manufacturing).

[0086] The term "corresponding human germline sequence" refers to a nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest determined amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other all other known variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences. A corresponding human germline sequence can also refer to the human variable region amino acid sequence or subsequence with the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other evaluated variable region amino acid sequences. A corresponding human germline sequence can be framework regions only, complementary determining regions only, framework and complementary determining regions, a variable segment (as defined above), or other combinations of sequences or sub-sequences that comprise a variable region.

Sequence identity can be determined using the methods described herein, for example, aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. The corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference variable region nucleic acid or amino acid sequence.

[0087] The term "valency" as used herein refers to the number of potential target binding sites in a polypeptide. Each target binding site specifically binds one target molecule or a specific site on a target molecule. When a polypeptide comprises more than one target binding site, each target binding site may specifically bind the same or different molecules (e.g., may bind to different molecules, e.g., different antigens, or different epitopes on the same molecule). A conventional antibody, for example, has two binding sites and is bivalent; "trivalent" and "tetravalent" refer to the presence of three binding sites and four binding sites, respectively, in an antibody molecule. The antibody cytokine engrafted proteins can be monovalent (i.e. , bind one target molecule), bivalent, or multivalent (i.e., bind more than one target molecule).

[0090] As used herein, the term "epitope" or "binding region" refers to a domain in the antigen protein that is responsible for the specific binding between the antibody CDRs and the antigen protein.

[0091] As used herein, the term "receptor-cytokine binding region" refers to a domain in the engrafted cytokine portion of the antibody cytokine engrafted protein that is responsible for the specific binding between the engrafted cytokine and its receptor (e.g. the IL7Ra). There is at least one such receptor-cytokine binding region present in each antibody cytokine engrafted protein, and each of the binding regions may be identical or different from the others.

[0092] The term "agonist" refers to an antibody capable of activating a receptor to induce a full or partial receptor-mediated response. For example, an agonist of the IL7Ra binds to the IL7Ra and induces IL7-mediated intracellular signaling, cell activation and/or proliferation of T cells. The antibody cytokine engrafted protein agonist stimulates signaling through the IL7Ra similarly in some respects to the native IL7 ligand. The binding of IL7 to IL7Ra induces Jakl and Jak3 activation which results in STAT5 phosphorylation. In some embodiments, an antibody cytokine engrafted protein agonist can be identified by its ability to bind IL7Ra and induce STAT5 phosphorylation, and/or the proliferation of T cells.

[0093] The term "interleukin 7" or "IL7" or "interleukin-7" or "IL-7"

interchangeably, refers to an alpha helical cytokine family member that functions in the proliferation and homeostasis of T cells. IL7 comprising residues of full length human IL7 is utilized in the context of the agonist antibody cytokine engrafted proteins. The human IL7 as disclosed herein has over its full length at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid SEQ ID NO:2, and retains preferential agonist activity of the antibody cytokine engrafted proteins as described herein and has been published as GenBank Accession No: NP_000871. SEQ ID NO: l is the human IL7 cDNA sequence. The human IL7 nucleic acid encoding for the IL7 protein as disclosed herein has over its full length at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: l, and was published under GenBank Accession No: NM_000880.

[0094] The term "antibody cytokine engrafted protein" or "antibody cytokine graft" or "engrafted" means that at least one cytokine is incorporated directly within a CDR of the antibody, interrupting the sequence of the CDR. The cytokine can be incorporated within HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3. The cytokine can be incorporated within HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 and incorporated toward the N-terminal sequence of the CDR or toward the C-terminal sequence of the CDR. The cytokine incorporated within a CDR can reduce the specific binding of the antibody portion to the original target protein or the antibody cytokine engrafted protein can retain its specific binding to its target protein.

[0095] The term "isolated," when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state. It can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames that flank the gene and encode a protein other than the gene of interest. The term "purified" denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.

[0096] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acids

(DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al, J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al, Mol. Cell. Probes 8:91-98 (1994)).

[0097] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

[0098] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ- carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e. , an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g. , homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups {e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0099] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[00100] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid.

Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles. The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[00101] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e. , gaps) as compared to the reference sequence (e.g. , a polypeptide), which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[00102] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same sequences. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over a specified region, or, when not specified, over the entire sequence of a reference sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. The disclosure provides polypeptides or polynucleotides that are substantially identical to the polypeptides or polynucleotides, respectively, exemplified herein (e.g. , the variable regions exemplified in any one of: SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 36, SEQ ID NO: 45, SEQ ID NO: 54, SEQ ID NO: 63, SEQ ID NO: 72, SEQ ID NO: 81, SEQ ID NO: 90, SEQ ID NO: 99, or SEQ ID NO: 108). The identity exists over a region that is at least about 15, 25 or 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length, or over the full length of the reference sequence. With respect to amino acid sequences, identity or substantial identity can exist over a region that is at least 5, 10, 15 or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75 or 100 amino acids in length, optionally at least about 150, 200 or 250 amino acids in length, or over the full length of the reference sequence. With respect to shorter amino acid sequences, e.g. , amino acid sequences of 20 or fewer amino acids, substantial identity exists when one or two amino acid residues are conservatively substituted, according to the conservative

substitutions defined herein.

[00103] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[00104] A "comparison window," as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) /. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[00105] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) /. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. , supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[00106] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g. , Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[00107] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[00108] The term "link," when used in the context of describing how the binding regions are connected within an antibody cytokine engrafted protein of this invention, encompasses all possible means for physically joining the regions. The multitude of binding regions are frequently joined by chemical bonds such as a covalent bond (e.g. , a peptide bond or a disulfide bond) or a non-covalent bond, which can be either a direct bond (i.e. , without a linker between two binding regions) or indirect bond (i.e. , with the aid of at least one linker molecule between two or more binding regions).

[00109] The terms "subject," "patient," and "individual" interchangeably refer to a mammal, for example, a human or a non-human primate mammal. The mammal can also be a laboratory mammal, e.g. , mouse, rat, rabbit, hamster. In some embodiments, the mammal can be an agricultural mammal (e.g., equine, ovine, bovine, porcine, camelid) or domestic mammal (e.g., canine, feline).

[00110] As used herein, the terms "treat," "treating," or "treatment" of any disease or disorder refer in one embodiment, to ameliorating the disease or disorder (i.e. , slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment, "treat," "treating," or "treatment" refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, "treat," "treating," or "treatment" refers to modulating the disease or disorder, either physically, (e.g. , stabilization of a discernible symptom), physiologically, (e.g. , stabilization of a physical parameter), or both. In yet another embodiment, "treat," "treating," or "treatment" refers to preventing or delaying the onset or development or progression of a disease or disorder. [00111] The term "therapeutically acceptable amount" or "therapeutically effective dose" interchangeably refer to an amount sufficient to effect the desired result (i.e. , reduction in tumor volume). In some embodiments, a therapeutically acceptable amount does not induce or cause undesirable side effects. A therapeutically acceptable amount can be determined by first administering a low dose, and then incrementally increasing that dose until the desired effect is achieved. A "prophylactically effective dosage," and a

"therapeutically effective dosage," of an IL7 antibody cytokine engrafted protein can prevent the onset of, or result in a decrease in severity of, respectively, disease symptoms, including symptoms associated with cancer and cancer treatment.

[00112] The term "co-administer" refers to the simultaneous presence of two (or more) active agents in an individual. Active agents that are co-administered can be concurrently or sequentially delivered.

[00113] As used herein, the phrase "consisting essentially of refers to the genera or species of active pharmaceutical agents included in a method or composition, as well as any inactive carrier or excipients for the intended purpose of the methods or compositions. In some embodiments, the phrase "consisting essentially of expressly excludes the inclusion of one or more additional active agents other than an IL7 antibody cytokine engrafted protein. In some embodiments, the phrase "consisting essentially of expressly excludes the inclusion of more additional active agents other than an IL7 antibody cytokine engrafted protein and a second co-administered agent.

[00114] The terms "a," "an," and "the" include plural referents, unless the context clearly indicates otherwise.

BRIEF DESCRIPTION OF THE DRAWINGS

[00115] Figures 1A and IB show the activity of IL7 antibody cytokine engrafted proteins on CD8 (Figure 1A) and CD4 (Figure IB) cells.

[00116] Figure 2A compares pSTAT5 activity of IL7 antibody cytokine engrafted proteins on CD4 T cells, CD8 T cells, B cells and NK cells. Figure 2B shows the effects of increasing concentrations of IgG.IL7.H2 and IgG.IL7.H3 on CD8 T cells as measured by pSTAT5. Figure 2C shows the effects of increasing concentrations of IgG.IL7.H2 and IgG.IL7.H3 on CD4 T cells as measured by pSTAT5. [00117] Figures 3A-3D show the pharmacodynamics of IL7 antibody cytokine engrafted proteins, demonstrating increased CD8 T cell proliferation.

[00118] Figures 4A and 4B demonstrate that IgG.IL7.H2 and IgG.IL7.H3 reduce tumor growth as a single agent. Figure 4C shows the increase in CD 8 T cells in the blood. Figure 4D shows the increase of CD 8 tumor infiltrating lymphocytes upon administration of IL7 antibody cytokine engrafted protein. Figure 4E shows the increase of CD4 tumor infiltrating lymphocytes (TILs) upon administration of IgG.IL7.H2 and IgG.IL7.H3.

[00119] Figure 5 is a graphical representation of the synergistic combination of IL7 antibody cytokine engrafted proteins with an anti-PD-Ll antibody.

[00120] Figure 6 is a structural diagram of IgG.IL7.H2 and IgG.IL7.H3, respectively.

[00121] Figure 7 is a graph of the binding of various IL7 antibody cytokine engrafted proteins to RSV.

[00122] Figure 8 is a Gyros assay showing that IL7 antibody cytokine engrafted proteins have a longer half-life than recombinant IL7.

[00123] Figure 9 is a FACS plot and graphs showing the expansion of CD8+ cells in the blood upon administration of IL7 antibody cytokine engrafted proteins as a single agent and upon administration of IL7 antibody cytokine engrafted proteins in combination with an anti-PD-Ll antibody.

[00124] Figure 10 shows that IL7 antibody cytokine engrafted proteins induces the reduction of Tim-3, either alone or in combination with anti-PD-Ll.

[00125] Figure 11 shows the increase in total numbers of naive, central memory and effector memory CD8+ T cells in the blood upon dosing with IL7 antibody cytokine engrafted proteins as a single agent or in combination with an anti-PD-Ll antibody.

[00126] Figure 12 demonstrates that administration of IL7 antibody cytokine engrafted proteins also induces an increase in CD8+ PD-1+ cells.

[00127] Figure 13 shows that administration of IL7 antibody cytokine engrafted proteins were able to reduce viral load as a single agent, or in combination with an anti-PD- Ll antibody.

[00128] Figure 14 demonstrates that the administration of IL7 antibody cytokine engrafted proteins resulted in the increase of IFN-gamma either as a single agent or in combination with an anti-PD-Ll antibody

Antibody Cytokine engrafted proteins targeting the IL7Ra [00129] Provided herein are antibody cytokine engrafted proteins comprising an IL7 molecule engrafted into the complementarity determining region (CDR) of an antibody. The antibody cytokine engrafted proteins of the present disclosure show suitable properties to be used in human patients, for example, they retain immunostimulatory activity similar to that of native or recombinant human IL7. Other activities and characteristics are also demonstrated throughout the specification. Thus, provided are antibody cytokine engrafted proteins having an improved therapeutic profile over previously known IL7 and modified IL7 therapeutic agents, and methods of use of the provided antibody cytokine engrafted proteins in cancer treatment.

[00130] Accordingly, the present disclosure provides antibody cytokine engrafted proteins that are agonists of the IL7Ra, with selective activity profiles. Provided antibody cytokine engrafted proteins comprise an immunoglobulin heavy chain sequence and an immunoglobulin light chain sequence. Each immunoglobulin heavy chain sequence comprises a heavy chain variable region (VH) and a heavy chain constant region (CH), wherein the heavy chain constant region consists of CHI, CH2, and CH3 constant regions. Each immunoglobulin light chain sequence comprises a light chain variable region (VL) and a light chain constant region (CL). In each antibody cytokine engrafted protein an IL7 molecule is incorporated into a complementarity determining region (CDR) of the VH or VL.

[00131] In some embodiments, the antibody cytokine engrafted protein comprises an

IL7 molecule incorporated into a heavy chain CDR. In certain embodiments the IL7 molecule is incorporated into heavy chain complementarity determining region 1 (HCDR1). In certain embodiments the IL7 molecule is incorporated into heavy chain complementarity determining region 2 (HCDR2). In certain embodiments the IL7 molecule is incorporated into heavy chain complementarity determining region 3 (HCDR3).

[00132] In some embodiments, the antibody cytokine engrafted protein comprises an

IL7 molecule incorporated into a light chain CDR. In certain embodiments the IL7 molecule is incorporated into light chain complementarity determining region 1 (LCDR1). In certain embodiments the IL7 molecule is incorporated into light chain complementarity determining region 2 (LCDR2). In certain embodiments the IL7 molecule is incorporated into light chain complementarity determining region 3 (LCDR3).

[00133] In some embodiments, the antibody cytokine engrafted comprises an IL7 sequence incorporated into a CDR, whereby the IL7 sequence is inserted into the CDR sequence. The insertion may be at or near the N-terminal region of the CDR, in the middle region of the CDR or at or near the C-terminal region of the CDR. In other embodiments, the antibody cytokine engrafted comprises an IL7 molecule incorporated into a CDR, whereby the IL7 sequence does not frameshift the CDR sequence. In other embodiments, the antibody cytokine engrafted comprises an IL7 molecule incorporated into a CDR, whereby the IL7 sequence replaces all or part of a CDR sequence. A replacement can be at or near the beginning of the CDR, in the middle region of the CDR or at or near the end of the CDR. A replacement can be as few as one or two amino acids of a CDR sequence, or the entire CDR sequence.

[00134] In some embodiments the IL7 molecule is engrafted directly into a CDR without a peptide linker, i.e., with no additional amino acids between the CDR sequence and the IL7 sequence.

[00135] In some embodiments antibody cytokine engrafted proteins comprise immunoglobulin heavy chains of an IgG class antibody heavy chain. In certain embodiments an IgG heavy chain is any one of an IgGl, an IgG2 or an IgG4 subclass.

[00136] In some embodiments antibody cytokine engrafted proteins comprise heavy and light chain immunoglobulin sequences selected from a known, clinically utilized immunoglobulin sequence. In certain embodiments antibody cytokine engrafted proteins comprise heavy and light chain immunoglobulin sequences which are humanized sequences. In other certain embodiments antibody cytokine engrafted proteins comprise heavy and light chain immunoglobulin sequences which are human sequences.

[00137] In some embodiments antibody cytokine engrafted proteins comprise heavy and light chain immunoglobulin sequences selected from germline immunoglobulin sequences.

[00138] In some embodiments antibody cytokine engrafted proteins comprise heavy and light chain immunoglobulin sequences having binding specificity of the immunoglobulin variable domains to a target distinct from the binding specificity of the IL7 molecule. In some embodiments the binding specificity of the immunoglobulin variable domain to its target is retained by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%, in the presence of the engrafted IL7 cytokine. In certain embodiments the retained binding specificity is to a non-human target. In other embodiments the binding specificity is to a human target having therapeutic utility in conjunction with IL7 therapy. In certain embodiments, targeting the binding specificity of the immunoglobulin conveys additional therapeutic benefit to the IL7 component. In certain embodiments the binding specificity of the immunoglobulin to its target conveys synergistic activity with IL7.

[00139] In still other embodiments, the binding specificity of the immunoglobulin is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%, by the engrafting of the IL7 molecule.

[00140] Provided antibody cytokine engrafted proteins comprise an IL7 molecule engrafted into a complementarity determining region (CDR) of the VH or VL. In some embodiments, the IL7 sequence has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the IL7 molecule comprises the sequence of SEQ ID NO: 2. In some embodiments, the IL7 molecule consists of SEQ ID NO: 2.

[00141] Provided antibody cytokine engrafted proteins comprise an IL7 molecule engrafted into a complementarity determining region (CDR) of the VH or VL. In some embodiments, the IL7 sequence has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the IL7 molecule comprises the sequence of SEQ ID NO: 2. In some embodiments, the IL7 molecule consists of SEQ ID NO: 2.

[00142] In some embodiments, the antibody cytokine engrafted protein confers pro immunomodulatory properties superior to human IL7, recombinant human IL7, or IL7 fused to an Fc. The IL7 antibody cytokine engrafted protein confers increased proliferation of T cells and homeostasis of mature T cells.

[00143] In some embodiments, the antibody cytokine engrafted proteins comprise a modified immunoglobulin IgG having a modified Fc conferring modified effector function. In certain embodiments the modified Fc region comprises a mutation selected from one or more of D265A, P329A, P329G, N297A, L234A, and L235A. In particular embodiments the immunoglobulin heavy chain may comprise a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A/N297A, L234/L235A, P329A/L234A/L235A, and

P329G/L234A/L235A.

[00144] In some embodiments, the antibody cytokine engrafted proteins comprise (i) a heavy chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO: 9 and (ii) a light chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO: 18. The immunoglobulin chain is an IgG class selected from IgGl, IgG2, or IgG4. In certain embodiments the immunoglobulin optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A/N297A, L234/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00145] In some embodiments, the antibody cytokine engrafted proteins comprise (i) a heavy chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO: 27 and (ii) a light chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO: 36. The immunoglobulin chain is an IgG class selected from IgGl, IgG2, or IgG4. In certain embodiments the immunoglobulin optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A,

D265A/N297A, L234/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00146] In some embodiments, the antibody cytokine engrafted proteins comprise (i) a heavy chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO: 45 and (ii) a light chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO: 54. The immunoglobulin chain is an IgG class selected from IgGl, IgG2, or IgG4. In certain embodiments the immunoglobulin optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A,

D265A/N297A, L234/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00147] In some embodiments, the antibody cytokine engrafted proteins comprise (i) a heavy chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO:63 and (ii) a light chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO:72. The immunoglobulin chain is an IgG class selected from IgGl, IgG2, or IgG4. In certain embodiments the immunoglobulin optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A,

D265A/N297A, L234/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00148] In some embodiments, the antibody cytokine engrafted proteins comprise (i) a heavy chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO: 81 and (ii) a light chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO: 90. The immunoglobulin chain is an IgG class selected from IgGl, IgG2, or IgG4. In certain embodiments the immunoglobulin optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A,

D265A/N297A, L234/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00149] In some embodiments, the antibody cytokine engrafted proteins comprise (i) a heavy chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO: 99 and (ii) a light chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO: 108. The immunoglobulin chain is an IgG class selected from IgGl, IgG2, or IgG4. In certain embodiments the immunoglobulin optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A,

D265A/N297A, L234/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

Engineered and Modified Antibody Cytokine Engrafted Proteins

[00150] In certain aspects, antibody cytokine engrafted constructs are generated by engrafting an IL7 sequence into a CDR region of an immunoglobulin scaffold. Both heavy and light chain immunoglobulin chains are produced to generate final antibody engrafted proteins. Antibody cytokine engrafted proteins confer preferred therapeutic activity on T cells, and the antibody cytokine engrafted proteins as compared with native or recombinant human IL7 (rhIL7) or IL7 fused to an Fc.

[00151] To engineer antibody cytokine engrafted proteins, an IL7 sequence (e.g., SEQ

ID NO: 2) is inserted into a CDR loop of an immunoglobulin chain scaffold protein.

Engrafted constructs can be prepared using any of a variety of known immunoglobulin sequences which have been utilized in clinical settings, known immunoglobulin sequences which are in current discovery and/or clinical development, human germline antibody sequences, as well as sequences of novel antibody immunoglobulin chains. Constructs are produced using standard molecular biology methodology utilizing recombinant DNA encoding relevant sequences. Sequences of an IL7 molecule engrafted in an exemplary scaffold, referred to as GFTX3b, are depicted in TABLE 2. Insertion points were selected to be the mid-point of the loop based on available structural or homology model data, however, insertion points can be adjusted toward one or another end of the CDR loop.

[00152] Thus the present disclosure provides antibodies or fragments thereof that specifically bind to IL7Ra comprising an IL7 protein recombinantly inserted into a heterologous antibody protein or polypeptide to generate antibody cytokine engrafted proteins. In particular, the disclosure provides antibody cytokine engrafted proteins comprising an antibody or antigen-binding fragment of an antibody described herein or any other relevant scaffold antibody polypeptide (e.g., a full antibody immunoglobulin protein, a Fab fragment, Fc fragment, Fv fragment, F(ab)2 fragment, a VH domain, a VH CDR, a VL domain, a VL CDR, etc.) and a heterologous cytokine protein, polypeptide, or peptide, e.g., an IL7 molecule. Methods for fusing or conjugating proteins, polypeptides, or peptides to an antibody or an antibody fragment are known in the art. See, e.g. , U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; European Patent Nos. EP 307,434 and EP 367,166; International Publication Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al, 1991, Proc. Natl. Acad. Sci. USA 88: 10535-10539; Zheng et al, 1995, J. Immunol. 154:5590-5600; and Vil et al , 1992, Proc. Natl. Acad. Sci. USA 89: 11337- 11341. Additionally, antibody cytokine engrafted proteins may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as "DNA shuffling"). DNA shuffling may be employed to prepare engrafted protein constructs and/or to alter the activities of antibodies or fragments thereof (e.g. , antibodies or fragments thereof with higher affinities and lower dissociation rates). See, generally, U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al, 1997 ', Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson, et al, 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308- 313. Antibodies or fragments thereof, or the encoded antibodies or fragments thereof, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. A polynucleotide encoding an antibody or fragment thereof that specifically binds to an antigen protein of interest may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous cytokine molecules, e.g., IL7, for preparation of antibody cytokine engrafted proteins as provided herein.

[00153] An antibody Fab contains six CDR loops, 3 in the light chain (CDRL1 ,

CDRL2, CDRL3) and 3 in the heavy chain (CDRH1 , CDRH2, CDRH3) which can serve as potential insertion sites for a cytokine protein. Structural and functional considerations are taken into account in order to determine which CDR loop(s) to insert the cytokine. As a CDR loop size and conformation vary greatly across different antibodies, the optimal CDR for insertion can be determined empirically for each particular antibody/protein combination. Additionally, since a cytokine protein will be inserted into a CDR loop, this can put additional constraints on the structure of the cytokine protein as discussed in Example 1.

[00154] CDRs of immunoglobulin chains are determined by well-known numbering systems known in the art, including those described herein. For example, CDRs have been identified and defined by (1) using the numbering system described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), NIH publication No. 91- 3242; and (2) Chothia, see Al-Lazikani et al., (1997) "Standard conformations for the canonical structures of immunoglobulins," J.Mol.Biol. 273:927-948. For identified CDR amino acid sequences less than 20 amino acids in length, one or two conservative amino acid residue substitutions can be tolerated while still retaining the desired specific binding and/or agonist activity.

[00155] An antibody cytokine engrafted protein further can be prepared using an antibody having one or more of the CDRs and/or VH and/or VL sequences shown herein (e.g. , TABLE 2) as starting material to engineer a modified antibody cytokine engrafted protein, which may have altered properties from the starting antibody engrafted protein. Alternatively any known antibody sequences may be utilized as a scaffold to engineer modified antibody cytokine engrafted protein. For example, any known, clinically utilized antibody may be utilized as a starting materials scaffold for preparation of antibody engrafted protein. Known antibodies and corresponding immunoglobulin sequences include, e.g., palivizumab, alirocumab, mepolizumab, necitumumab, nivolumab, dinutuximab, secukinumab, evolocumab, blinatumomab, pembrolizumab, ramucirumab, vedolizumab, siltuximab, obinutuzumab, trastuzumab, raxibacumab, pertuzumab, belimumab, ipilimumab, denosumab, tocilizumab, ofatumumab, canakinumab, golimumab, ustekinumab,

certolizumab, catumaxomab, eculizumab, ranibizumab, panitumumab, natalizumab, bevacizumab, cetuximab, efalizumab, omalizumab, tositumomab, ibritumomab tiuxetan, adalimumab, alemtuzumab, gemtuzumab, infliximab, basiliximab, daclizumab, rituximab, abciximab, muromonab, or modifications thereof. Known antibodies and immunoglobulin sequences also include germline antibody sequences. Framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database, as well as in Kabat, E. A., et al., 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al, 1992 J. fol. Biol. 227:776-798; and Cox, J. P. L. et al , 1994 Eur. J Immunol. 24:827-836. In still other examples, antibody and corresponding immunoglobulin sequences from other known entities which can be in early discovery and/or drug development can be similarly adapted as starting material to engineer a modified antibody cytokine engrafted protein.

[00156] A wide variety of antibody/immunoglobulin frameworks or scaffolds can be employed so long as the resulting polypeptide includes at least one binding region which accommodates incorporation of a cytokine (e.g., IL7). Such frameworks or scaffolds include the 5 main idiotypes of human immunoglobulins, or fragments thereof, and include immunoglobulins of other animal species, preferably having humanized and/or human aspects. Novel antibodies, frameworks, scaffolds and fragments continue to be discovered and developed by those skilled in the art.

[00157] Antibodies can be generated using methods that are known in the art. For preparation of monoclonal antibodies, any technique known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al , Immunology Today 4:72 (1983); Cole et al, Monoclonal Antibodies and Cancer Therapy, pp. 77-96. Alan R. Liss, Inc. 1985). Techniques for the production of single chain antibodies (U.S. Patent No. 4,946,778) can be adapted to produce antibodies for use in antibody cytokine engrafted proteins. Also, transgenic mice, or other organisms such as other mammals, may be used to express and identify primatized or humanized or human antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens for use in antibody cytokine engrafted proteins (see, e.g. ,

McCafferty et al. , supra; Marks et al. , Biotechnology, 10:779-783, (1992)).

[00158] Methods for primatizing or humanizing non-human antibodies are well known in the art. Generally, a primatized or humanized antibody has one or more amino acid residues introduced into it from a source which is non-primate or non-human. Such non- primate or non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers (see, e.g., Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239: 1534- 1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.

Accordingly, such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, primatized or humanized antibodies are typically primate or human antibodies in which some complementary determining region ("CDR") residues and possibly some framework ("FR") residues are substituted by residues from analogous sites in an originating species (e.g., rodent antibodies) to confer binding specificity.

[00159] Alternatively or additionally, an in vivo method for replacing a nonhuman antibody variable region with a human variable region in an antibody while maintaining the same or providing better binding characteristics relative to that of the nonhuman antibody may be utilized to convert non-human antibodies into engineered human antibodies. See, e.g., U.S. Patent Publication No. 20050008625, U.S. Patent Publication No. 2005/0255552. Alternatively, human V segment libraries can be generated by sequential cassette replacement in which only part of the reference antibody V segment is initially replaced by a library of human sequences; and identified human "cassettes" supporting binding in the context of residual reference antibody amino acid sequences are then recombined in a second library screen to generate completely human V segments (see, U.S. Patent Publication No.

2006/0134098).

[00160] Various antibodies or antigen-binding fragments for use in preparation of antibody cytokine engrafted proteins can be produced by enzymatic or chemical modification of the intact antibodies, or synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv), or identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554, 1990). For example, minibodies can be generated using methods described in the art, e.g., Vaughan and Sollazzo, Comb. Chem. High Throughput Screen 4:417-30 2001. Bispecific antibodies can be produced by a variety of methods including engrafted of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al, J. Immunol. 148, 1547-1553 (1992). Single chain antibodies can be identified using phage display libraries or ribosome display libraries, gene shuffled libraries. Such libraries can be constructed from synthetic, semisynthetic or native and immunocompetent sources. Selected immunoglobulin sequences may thus be utilized in preparation of antibody cytokine engrafted protein constructs as provided herein.

[00161] Antibodies, antigen-binding molecules or antibody cytokine engrafted molecules of use in the present disclosure further include bispecific antibodies. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Other antigen-binding fragments or antibody portions include bivalent scFv (diabody), bispecific scFv antibodies where the antibody molecule recognizes two different epitopes, single binding domains (dAbs), and minibodies. Selected immunoglobulin sequences may thus be utilized in preparation of antibody cytokine engrafted protein constructs as provided herein.

[00162] Antigen-binding fragments of antibodies e.g., a Fab fragment, scFv, can be used as building blocks to construct antibody cytokine engrafted proteins, and may optionally include multivalent formats. In some embodiments, such multivalent molecules comprise a constant region of an antibody (e.g., Fc).

[00163] Antibody cytokine engrafted proteins can be engineered by modifying one or more residues within one or both variable regions (i.e., VH and/or VL) of an antibody, for example, within one or more CDR regions, and such adapted VH and/or VL region sequences are utilized for engrafting a cytokine or for preparation of cytokine engrafting. Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a specific antibody by constructing expression vectors that include CDR sequences from a specific antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et ah , 1998 Nature 332:323-327; Jones, P. et ah, 1986 Nature 321 :522-525; Queen, C. et ah, 1989 Proc. Natl. Acad., U.S.A. 86: 10029-10033; U.S. Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530,101 ; 5,585,089; 5,693,762 and 6,180,370 to Queen et ah). In certain instances it is beneficial to mutate residues within the framework regions to maintain or enhance the antigen binding ability of the antibody (see e.g., U.S. Patent Nos. 5,530,101 ; 5,585,089; 5,693,762 and 6,180,370 to Queen et al).

[00164] In some aspects mutation of amino acid residues within the VH and/or VL

CDRl, CDR2, and/or CDR3 regions to thereby improve one or more binding properties {e.g. , affinity) of the antibody of interest, known as "affinity maturation," may be beneficial, e.g., to optimize antigen binding of an antibody in conjunction with the context of the cytokine engrafted protein. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and the effect on antibody binding, or other functional property of interest, can be evaluated in in vitro or in vivo assays as described herein and/or alternative or additional assays known in the art. Conservative modifications can be introduced. The mutations may be amino acid substitutions, additions or deletions. Moreover, typically no more than one, two, three, four or five residues within a CDR region are altered.

[00165] Engineered antibodies or antibody fragments include those in which modifications have been made to framework residues within VH and/or VL, e.g. to improve the properties of the antibody. In some embodiments such framework modifications are made to decrease immunogenicity of the antibody. For example, one approach is to change one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline

configuration, the somatic mutations can be "backmutated" to the germline sequence by, for example, site-directed mutagenesis. Additional framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043 by Carr et al.

[00166] Constant regions of the antibodies or antibody fragments utilized for preparation of the antibody cytokine engrafted protein can be any type or subtype, as appropriate, and can be selected to be from the species of the subject to be treated by the present methods (e.g. , human, non-human primate or other mammal, for example, agricultural mammal (e.g. , equine, ovine, bovine, porcine, camelid), domestic mammal (e.g. , canine, feline) or rodent (e.g., rat, mouse, hamster, rabbit). In some embodiments antibodies utilized in antibody cytokine engrafted proteins are engineered to generate humanized or Humaneered® antibodies. In some embodiments antibodies utilized in antibody cytokine engrafted proteins are human antibodies. In some embodiments, antibody constant region isotype is IgG, for example, IgGl, IgG2, IgG3, IgG4. In certain embodiments the constant region isotype is IgGi. In some embodiments, antibody cytokine engrafted proteins comprise an IgG. In some embodiments, antibody cytokine engrafted proteins comprise an IgGl Fc. In some embodiments, antibody cytokine engrafted proteins comprise an IgG2 Fc.

[00167] In addition or alternative to modifications made within framework or CDR regions, antibodies or antibody fragments utilized in preparation of antibody cytokine engrafted proteins may be engineered to include modifications within an Fc region, typically to alter one or more functional properties of the antibody, such as, e.g., serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody, antibody fragment thereof, or antibody cytokine engrafted protein can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody cytokine engrafted protein.

[00168] In one embodiment, a hinge region of CHI is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. For example, by the approach is described further in U.S. Patent No. 5,677,425 by Bodmer et al. wherein the number of cysteine residues in the hinge region of CHI is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody cytokine engrafted protein. In another embodiment, an Fc hinge region of an antibody is mutated to alter the biological half-life of the antibody cytokine engrafted protein. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc -hinge fragment such that the antibody cytokine engrafted protein has impaired Staphylococcyl protein A (SpA) binding relative to native Fc -hinge domain SpA binding. This approach is described in further detail in U.S. Patent No.

6,165,745 by Ward ei al. [00169] The present disclosure provides for antibody cytokine engrafted proteins that specifically bind to the IL7Ra which have an extended half-life in vivo. In another embodiment, an antibody cytokine engrafted protein is modified to increase its biological half-life. Various approaches are possible. Antibody cytokine engrafted proteins having an increased half-life in vivo can also be generated introducing one or more amino acid modifications (i.e. , substitutions, insertions or deletions) into an IgG constant domain, or FcRn binding fragment thereof (preferably a Fc or hinge Fc domain fragment). For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375 to Ward. See, e.g. , International Publication No. WO 98/23289; International Publication No. WO 97/34631 ; and U.S. Patent No. 6,277,375.

Alternatively, to increase the biological half-life, the antibody cytokine engrafted protein is altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos.

5,869,046 and 6,121,022 by Presta et al. In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody cytokine engrafted protein. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody cytokine engrafted protein has an altered affinity for an effector ligand but retains antigen- binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor (FcR) or the CI component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[00170] In another embodiment, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody cytokine engrafted protein has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent Nos. 6,194,551 by Idusogie et al.

[00171] Antibody cytokine engrafted proteins containing such mutations mediate reduced or no antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In some embodiments, amino acid residues L234 and L235 of the IgGl constant region are substituted to Ala234 and Ala235. In some embodiments, amino acid residue N267 of the IgGl constant region is substituted to Ala267. [00172] In another embodiment, one or more amino acid residues are altered to thereby alter the ability of the antibody cytokine engrafted protein to fix complement. This approach is described further in PCT Publication WO 94/29351 by Bodmer et al.

[00173] In yet another embodiment, an Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody cytokine engrafted protein for an Fey receptor by modifying one or more amino acids. This approach is described further in PCT Publication WO 00/42072 by Presta. Moreover, binding sites on human IgGl for FcyRl, FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R.L. et al, 2001 J. Biol. Chen. 276:6591-6604).

[00174] In still another embodiment, glycosylation of an antibody cytokine engrafted protein is modified. For example, an aglycoslated antibody cytokine engrafted protein can be made {i.e., the antibody cytokine engrafted protein lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for "antigen." Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[00176] In some embodiments, one or more domains, or regions, of an antibody cytokine engrafted protein are connected via a linker, for example, a peptide linker, such as those that are well known in the art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ., et al. (1994) Structure 2: 1121-1123). A peptide linker may vary in length, e.g., a linker can be 1-100 amino acids in length, typically a linker is from five to 50 amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.

[00177] In some embodiments IL7 is engrafted into the CDR sequence optionally with one or more peptide linker sequences. In certain embodiments one or more peptide linkers is independently selected from a (Gly_n-Ser)_m sequence (SEQ ID NO: 111), a (Gly_n-Ala)_m sequence (SEQ ID NO: 112), or any combination of a (Gly_n-Ser)m/(Gly_n-Ala)m sequence (SEQ ID NOS 111-112), wherein each n is independently an integer from 1 to 5 and each m is independently an integer from 0 to 10. Examples of linkers include, but are not limited to, glycine-based linkers or gly/ser linkers G/S such as (G_mS)_n wherein n is a positive integer equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 and m is an integer equal to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 113). In certain embodiments one or more linkers include G₄S (SEQ ID NO: 114) repeats, e.g., the Gly-Ser linker (G₄S)_n wherein n is a positive integer equal to or greater than 1 (SEQ ID NO: 114). For example, n=l, n=2, n=3. n=4, n=5 and n=6, n=7, n=8, n=9 and n=10. In some embodiments, Ser can be replaced with Ala e.g., linkers G/A such as (GmA)n wherein n is a positive integer equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 and m is an integer equal to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 115). In certain embodiments one or more linkers include G₄A (SEQ ID NO: 116) repeats, (G₄A)_n wherein n is a positive integer equal to or greater than 1 (SEQ ID NO: 116). For example, n=l, n=2, n=3. n=4, n=5 and n=6, n=7, n=8, n=9 and n=10. In some embodiments, the linker includes multiple repeats of linkers. In other embodiments, a linker includes combinations and multiples of G₄S (SEQ ID NO: 114) and G₄A (SEQ ID NO: 116).

[00178] Other examples of linkers include those based on flexible linker sequences that occur naturally in antibodies to minimize immunogenicity arising from linkers and junctions. For example, there is a natural flexible linkage between the variable domain and a CHI constant domain in antibody molecular structure. This natural linkage comprises approximately 10-12 amino acid residues, contributed by 4-6 residues from C-terminus of V domain and 4-6 residues from the N-terminus of the CHI domain. Antibody cytokine engrafted proteins can, e.g., employ linkers incorporating terminal 5-6 amino acid residues, or 11-12 amino acid residues, of CHI as a linker. The N-terminal residues of the CHI domain, particularly the first 5-6 amino acid residues, adopt a loop conformation without strong secondary structure, and, therefore, can act as a flexible linker. The N-terminal residues of the CHI domain are a natural extension of the variable domains, as they are part of the Ig sequences, and, therefore, minimize to a large extent any immunogenicity potentially arising from the linkers and junctions. In some embodiments a linker sequence includes a modified peptide sequence based on a hinge sequence.

[00179] Moreover, the antibody cytokine engrafted proteins can include marker sequences, such as a peptide to facilitate purification of antibody cytokine engrafted proteins. In preferred embodiments, a marker amino acid sequence is a hexa-histidine (SEQ ID NO: 117) peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., Chatsworth, CA), among others, many of which are commercially available. As described in Gentz et al. , 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for instance, hexa-histidine (SEQ ID NO: 117) provides for convenient purification of the engrafted protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin ("HA") tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767), and the "flag" tag.

[00180] Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Assays for Antibody Cytokine Engrafted Protein activity [00181] Assays for identifying antibody cytokine engrafted proteins are known in the art and described herein. Agonist antibody cytokine engrafted proteins bind to the IL7Ra and promote, induce, stimulate intracellular signalling resulting in T cell proliferation as well as other immunostimulatory effects.

[00182] Binding of the antibody cytokine engrafted proteins to the IL7Ra can be determined using any method known in the art. For example, binding to the IL7Ra can be determined using known techniques, including without limitation ELISA, Western blots, surface plasmon resonance (SPR) (e.g., BIAcore), and flow cytometry.

[00183] Intracellular signalling through the IL7Ra can be measured using any method known in the art. For example, activation of the IL7Ra by IL7 promotes STAT5 activation and signalling. Methods for measuring STAT5 activation are standard in the art (e.g., phosphorylation status of STAT5 protein, reporter gene assays, downstream signalling assays, etc.). Activation through the IL7Ra expands T cells, so the absolute numbers of T cells can be assayed for. In addition, either CD8+ or CD4+ T cells can be assayed for independently. Methods for measuring proliferation of cells are standard in the art (e.g. ,³H- thymidine incorporation assays, CFSE labelling). Methods for measuring cytokine production are well known in the art (e.g. , ELISA assays, ELISpot assays). In performing in vitro assays, test cells or culture supernatant from test cells contacted with antibody cytokine engrafted proteins can be compared to control cells or culture supernatants from control cells that have not been contacted with an antibody cytokine engrafted protein and/or those that have been contacted with recombinant human IL7 or an IL7-Fc fusion molecule.

Polynucleotides Encoding Antibody Cytokine Engrafted Proteins

[00185] In another aspect, isolated nucleic acids encoding heavy and light chain proteins of the antibody cytokine engrafted proteins are provided. Antibody cytokine engrafted proteins can be produced by any means known in the art, including but not limited to, recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers. Recombinant expression can be from any appropriate host cells known in the art, for example, mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[00186] Provided herein are polynucleotides that encode the variable regions exemplified in any one of SEQ ID NO: 10, SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ ID NO:82, SEQ ID NO:91, SEQ ID NO: 100, and SEQ ID NO: 109.

[00187] In some embodiments, the polynucleotide encoding the heavy chain variable regions comprises a sequence having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 28, SEQ ID NO: 46, SEQ ID NO: 64, SEQ ID NO: 82, and SEQ ID NO: 100. In some embodiments, the polynucleotide encoding the light chain variable regions comprises a sequence having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 73, SEQ ID NO: 91 and SEQ ID NO: 109.

[00188] Polynucleotides can encode only the variable region sequence of an antibody cytokine engrafted protein. They can also encode both a variable region and a constant region of the antibody cytokine engrafted protein. Some of the polynucleotide sequences encode a polypeptide that comprises variable regions of both the heavy chain and the light chain of one of the antibody cytokine engrafted proteins. Some other polynucleotides encode two polypeptide segments that respectively are substantially identical to the variable regions of the heavy chain and the light chain of one of the antibody cytokine engrafted proteins.

[00189] In certain embodiments polynucleotides or nucleic acids comprise DNA. In other embodiments polynucleotides or nucleic acids comprise RNA, which may be single stranded or double stranded.

[00190] In some embodiments a recombinant host cell comprising the nucleic acids encoding one or more immunoglobulin protein chain of an antibody cytokine engrafted protein, and optionally, secretion signals is provided. In certain embodiments a recombinant host cell comprises a vector encoding one immunoglobulin protein chain and secretion signals. In other certain embodiments a recombinant host cell comprises one or more vectors encoding two immunoglobulin protein chains of the antibody cytokine engrafted protein and secretion signals. In some embodiments a recombinant host cell comprises a single vector encoding two immunoglobulin protein chains of the antibody cytokine engrafted protein and secretion signals. In some embodiments a recombinant host cell comprises two vectors, one encoding a heavy chain immunoglobulin protein chain, and another encoding a light chain immunoglobulin protein chain of the antibody cytokine engrafted protein, with each including secretion signals. A recombinant host cell may be a prokaryotic or eukaryotic cell. In some embodiments a host cell is a eukaryotic cell line. In some embodiments a host cell is a mammalian cell line.

[00191] Additionally provided are methods for producing the antibody cytokine engrafted proteins. In some embodiments the method comprises the steps of (i) culturing a host cell comprising one or more vectors encoding immunoglobulin protein chains of an antibody cytokine engrafted protein under conditions suitable for expression, formation, and secretion of the antibody cytokine engrafted protein and (ii) recovering the antibody cytokine engrafted protein.

[00192] The polynucleotide sequences can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence (e.g., sequences as described herein) encoding a polypeptide chain of an antibody cytokine engrafted protein. Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90, 1979; the

phosphodiester method of Brown et al., Meth. Enzymol. 68: 109, 1979; the

diethylphosphoramidite method of Beaucage et al., Terra. Lett., 22: 1859, 1981 ; and the solid support method of U.S. Patent No. 4,458,066. Introducing mutations to a polynucleotide sequence by PCR can be performed as described in, e.g., PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al, PCR Methods and Applications 1 : 17, 1991.

[00193] Also provided in the disclosure are expression vectors and host cells for producing the antibody cytokine engrafted proteins described above. Various expression vectors can be employed to express polynucleotides encoding the immunoglobulin polypeptide chains, or fragments, of the antibody cytokine engrafted proteins. Both viral- based and nonviral expression vectors can be used to produce the immunoglobulin proteins in a mammalian host cell. Nonviral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g. , Harrington et al., Nat. Genet. 15:345, 1997). For example, nonviral vectors useful for expression of the antibody cytokine engrafted protein polynucleotides and polypeptides in mammalian (e.g. , human) cells include pThioHis A, B & C, pcDNA3.1/His, pEBVHis A, B & C (Invitrogen, San Diego, CA), MPSV vectors, and numerous other vectors known in the art for expressing other proteins. Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brent et al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al, Cell 68: 143, 1992.

[00194] The choice of expression vector depends on the intended host cells in which the vector is to be expressed. Typically, the expression vectors contain a promoter and other regulatory sequences (e.g., enhancers) that are operably linked to the polynucleotides encoding an immunoglobulin protein of the antibody cytokine engrafted protein. In some embodiments, an inducible promoter is employed to prevent expression of inserted sequences except under inducing conditions. Inducible promoters include, e.g., arabinose, lacZ, metallothionein promoter or a heat shock promoter. Cultures of transformed organisms can be expanded under noninducing conditions without biasing the population for coding sequences whose expression products are better tolerated by the host cells. In addition to promoters, other regulatory elements may also be required or desired for efficient expression of an immunoglobulin chain or fragment of the antibody cytokine engrafted proteins. These elements typically include an ATG initiation codon and adjacent ribosome binding site or other sequences. In addition, the efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (see, e.g. , Scharf et al., Results Probl. Cell Differ. 20: 125, 1994; and Bittner et al, Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or CMV enhancer may be used to increase expression in mammalian host cells.

[00195] Expression vectors may also provide a secretion signal sequence position to form an antibody cytokine engrafted protein that exported out of the cell and into the culture medium. In certain aspects, the inserted immunoglobulin sequences of the antibody cytokine engrafted proteins are linked to a signal sequences before inclusion in the vector. Vectors to be used to receive sequences encoding immunoglobulin light and heavy chain variable domains sometimes also encode constant regions or parts thereof. Such vectors allow expression of the variable regions as engrafted proteins with the constant regions thereby leading to production of intact antibody cytokine engrafted proteins or fragments thereof. Typically, such constant regions are human.

[00196] Host cells for harboring and expressing the antibody cytokine engrafted protein chains can be either prokaryotic or eukaryotic. E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present disclosure. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, one can also make expression vectors, which typically contain expression control sequences compatible with the host cell {e.g. , an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation. Other microbes, such as yeast, can also be employed to express antibody cytokine engrafted protein polypeptides. Insect cells in combination with baculovirus vectors can also be used.

[00197] In some preferred embodiments, mammalian host cells are used to express and produce the antibody cytokine engrafted protein polypeptides. For example, they can be either a mammalian cell line containing an exogenous expression vector. These include any normal mortal or normal or abnormal immortal animal or human cell. For example, a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed, including the CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B-cells and hybridomas. The use of mammalian tissue cell culture to express polypeptides is discussed generally in, e.g., Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y., 1987. Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g. , Queen et al., Immunol. Rev. 89:49-68, 1986), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell type-specific, stage-specific, and/or modulatable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPSV promoter, the tetracycline- inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[00198] Methods for introducing expression vectors containing the polynucleotide sequences of interest vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts (see generally Sambrook et al., supra). Other methods include, e.g., electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic methods, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, engrafted to the herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), agent-enhanced uptake of DNA, and ex vivo transduction. For long-term, high-yield production of recombinant proteins, stable expression will often be desired. For example, cell lines which stably express antibody cytokine engrafted protein immunoglobulin chains can be prepared using expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth of cells which successfully express the introduced sequences in selective media. Resistant, stably transfected cells can be proliferated using tissue culture techniques appropriate to the cell type. Compositions Comprising Antibody Cytokine Engrafted Proteins

[00199] Provided are pharmaceutical compositions comprising an antibody cytokine engrafted protein formulated together with a pharmaceutically acceptable carrier. Optionally, pharmaceutical compositions additionally contain other therapeutic agents that are suitable for treating or preventing a given disorder. Pharmaceutically acceptable carriers enhance or stabilize the composition, or facilitate preparation of the composition. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[00200] A pharmaceutical composition of the present disclosure can be administered by a variety of methods known in the art. Route and/or mode of administration vary depending upon the desired results. It is preferred that administration be by parenteral administration (e.g., selected from any of intravenous, intramuscular, intraperitoneal, intrathecal, intraarterial, or subcutaneous), or administered proximal to the site of the target. A pharmaceutically acceptable carrier is suitable for administration by any one or more of intravenous, intramuscular, intraperitoneal, intrathecal, intraarterial, subcutaneous, intranasal, inhalational, spinal or epidermal administration (e.g., by injection). Depending on the route of administration, active compound, e.g., antibody cytokine engrafted protein, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. In some embodiments the pharmaceutical composition is formulated for intravenous administration. In some embodiments the pharmaceutical composition is formulation for subcutaneous administration.

[00201] An antibody cytokine engrafted protein, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[00202] In some embodiments, a pharmaceutical composition is sterile and fluid.

Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin. In certain embodiments compositions can be prepared for storage in a lyophilized form using appropriate excipients (e.g., 20mM Histidine at pH 5.5, 220 mM sucrose and 0.04% polysorbate 20).

[00203] Pharmaceutical compositions can be prepared in accordance with methods well known and routinely practiced in the art. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions. Applicable methods for formulating an antibody cytokine engrafted protein and determining appropriate dosing and scheduling can be found, for example, in Remington: The Science and Practice of Pharmacy, 21^st Ed., University of the Sciences in Philadelphia, Eds., Lippincott Williams & Wilkins (2005); and in Martindale: The Complete Drug Reference, Sweetman, 2005, London: Pharmaceutical Press., and in Martindale, Martindale: The Extra Pharmacopoeia, 31st Edition., 1996, Amer Pharmaceutical Assn, and Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Pharmaceutical compositions are preferably manufactured under GMP conditions. Typically, a therapeutically effective dose or efficacious dose of an antibody cytokine engrafted protein is employed in the

pharmaceutical compositions. An antibody cytokine engrafted protein is formulated into pharmaceutically acceptable dosage form by conventional methods known to those of skill in the art. Dosage regimens are adjusted to provide the desired response (e.g., a therapeutic response). In determining a therapeutically or prophylactically effective dose, a low dose can be administered and then incrementally increased until a desired response is achieved with minimal or no undesired side effects. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[00204] Actual dosage levels of active ingredients in the pharmaceutical compositions can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular compositions employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors.

Articles of Manufacture/Kits

[00205] In some aspects an antibody cytokine engrafted protein is provided in an article of manufacture (i.e., a kit). A provided antibody cytokine engrafted protein is generally in a vial or a container. Thus, an article of manufacture comprises a container and a label or package insert, on or associated with the container. Suitable containers include, for example, a bottle, vial, syringe, solution bag, etc. As appropriate, the antibody cytokine engrafted protein can be in liquid or dried (e.g. , lyophilized) form. The container holds a composition which, by itself or combined with another composition, is effective for preparing a composition for treating, preventing and/or ameliorating cancer. The label or package insert indicates the composition is used for treating, preventing and/or ameliorating cancer. Articles of manufacture (kits) comprising an antibody cytokine engrafted protein, as described herein, optionally contain one or more additional agent. In some embodiments, an article of manufacture (kit) contains antibody cytokine engrafted protein and a

pharmaceutically acceptable diluent. In some embodiments an antibody cytokine engrafted protein is provided in an article of manufacture (kit) with one or more additional active agent in the same formulation (e.g. , as mixtures). In some embodiments an antibody cytokine engrafted protein is provided in an article of manufacture (kit) with a second or third agent in separate formulations (e.g. , in separate containers). In certain embodiments an article of manufacture (kit) contains aliquots of the antibody cytokine engrafted protein wherein the aliquot provides for one or more doses. In some embodiments aliquots for multiple administrations are provided, wherein doses are uniform or varied. In particular

embodiments varied dosing regimens are escalating or decreasing, as appropriate. In some embodiments dosages of an antibody cytokine engrafted protein and a second agent are independently uniform or independently varying. In certain embodiments, an article of manufacture (kit) comprises an additional agent such as an anti-cancer agent or immune checkpoint molecule. Selection of one or more additional agent will depend on the dosage, delivery, and disease condition to be treated.

Methods of Treatment and Use of Compositions for Treatment of Cancer

Conditions Subject to Treatment or Prevention

[00206] Antibody cytokine engrafted proteins find use in treatment, amelioration or prophylaxis of cancer. In one aspect, the disclosure provides methods of treatment of cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an antibody cytokine engrafted protein, as described herein. In some embodiment an antibody cytokine engrafted protein is provided for use as a therapeutic agent in the treatment or prophylaxis of cancer in an individual. In a further aspect, the disclosure provides a composition comprising such an antibody cytokine engrafted protein for use in treating or ameliorating cancer in an individual in need thereof.

[00207] Conditions subject to treatment include various cancer indications. For therapeutic purposes, an individual was diagnosed with cancer. For preventative or prophylactic purposes, an individual may be in remission from cancer or may anticipate future onset. In some embodiments, the patient has cancer, is suspected of having cancer, or is in remission from cancer. Cancers subject to treatment with an antibody cytokine engrafted protein derive benefit from activation of IL7Ra signalling, as described herein. Cancer indications subject to treatment include without limitation: melanoma, lung cancer, colorectal cancer, prostate cancer, breast cancer, leukemia and lymphoma.

Administration of Antibody Cytokine Engrafted Proteins

[00208] A physician or veterinarian can start doses of an antibody cytokine engrafted protein employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, effective doses of the compositions vary depending upon many different factors, including the specific disease or condition to be treated, means of administration, target site, physiological state of the patient, whether a patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Treatment dosages typically require titration to optimize safety and efficacy. For administration with an antibody cytokine engrafted protein, dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg. Dosing can be daily, weekly, bi-weekly, monthly, or more or less often, as needed or desired. An exemplary treatment regime entails administration once weekly, once per every two weeks or once a month or once every 3 to 6 months.

[00209] The antibody cytokine engrafted protein can be administered in single or divided doses. An antibody cytokine engrafted protein is usually administered on multiple occasions. Intervals between single dosages can be weekly, bi-weekly, monthly or yearly, as needed or desired. Intervals can also be irregular as indicated by measuring blood levels of antibody cytokine engrafted protein in the patient. In some methods, dosage is adjusted to achieve a plasma antibody cytokine engrafted protein concentration of 1-1000 μg/ml and in some methods 25-300 μg/ml. Alternatively, antibody cytokine engrafted protein can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody cytokine engrafted protein in the patient. In general, antibody engrafted proteins show longer half-life than that of recombinant cytokines (e.g. rhIL7). Dosage and frequency of administration can vary depending on whether treatment is prophylactic or therapeutic. In general for prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. In general for therapeutic applications, a relatively high dosage in relatively short intervals is sometimes required until progression of the cancer is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, a patient may be administered a prophylactic regime. Co-Administration with a Second Agent

[00210] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration encompasses co-administration in multiple, or in separate containers (e.g. , capsules, powders, and liquids) for each active ingredient.

Powders and/or liquids may be reconstituted or diluted to a desired dose prior to

administration. In addition, such administration also encompasses use of each type of therapeutic agent in a sequential manner, either at approximately the same time or at different times. In either case, the treatment regimen will provide beneficial effects of the drug combination in treating the conditions or disorders described herein. [00211] The combination therapy can provide "synergy" and prove "synergistic", i.e., the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately. A synergistic effect can be attained when the active ingredients are: (1) co-formulated and administered or delivered

simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect can be attained when the compounds are administered or delivered sequentially, e.g., by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e., serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.

[00212] In one aspect, the present disclosure provides a method of treating cancer by administering to a subject in need thereof an antibody cytokine engrafted protein in combination with one or more tyrosine kinase inhibitors, including but not limited to, EGFR inhibitors, Her2 inhibitors, Her3 inhibitors, IGFR inhibitors, and Met inhibitors.

[00213] For example, tyrosine kinase inhibitors include but are not limited to, Erlotinib hydrochloride (Tarceva®); Linifanib (N-[4-(3-amino-lH-indazol-4-yl)phenyl]-N'-(2-fluoro- 5-methylphenyl)urea, also known as ABT 869, available from Genentech); Sunitinib malate (Sutent®); Bosutinib (4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4- methylpiperazin-l-yl)propoxy]quinoline-3-carbonitrile, also known as SKI-606, and described in US Patent No. 6,780,996); Dasatinib (Sprycel®); Pazopanib (Votrient®);

Sorafenib (Nexavar®); Zactima (ZD6474); nilotinib (Tasigna®); Regorafenib (Stivarga®) and Imatinib or Imatinib mesylate (Gilvec® and Gleevec®).

[00214] Epidermal growth factor receptor (EGFR) inhibitors include but are not limited to, Erlotinib hydrochloride (Tarceva®), Gefitnib (Iressa®); N-[4-[(3-Chloro-4- fluorophenyl)amino]-7-[[(3"S")-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]- 4(dimethylamino)-2-butenamide, Tovok®); Vandetanib (Caprelsa®); Lapatinib (Tykerb®); (3R,4R)-4- Amino- 1 -((4-((3-methoxyphenyl)amino)pyrrolo[2, 1 -f] [ 1 ,2,4] triazin-5- yl)methyl)piperidin-3-ol (BMS690514); Canertinib dihydrochloride (CI-1033); 6-[4-[(4- Ethyl- 1 -piperaziny l)me thy 1] phenyl] -N- [( 1 R)- 1 -phenylethyl] - 7H-Pyrrolo[2,3-d]pyrimidin-4- amine (AEE788, CAS 497839-62-0); Mubritinib (TAK165); Pelitinib (EKB569); Afatinib (BIBW2992); Neratinib (HKI-272); N-[4-[[l-[(3-Fluorophenyl)methyl]-lH-indazol-5- yl]amino]-5-memylpyrrolo[2,l-f][l,2,4]triazin-6-yl]-carbarnic acid, (3S)-3- morpholinylmethyl ester (BMS599626); N-(3,4-Dichloro-2-fluorophenyl)-6-methoxy-7- [[(3aa,5 ,6aa)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]- 4-quinazolinamine (XL647, CAS 781613-23-8); and 4-[4-[[(lR)- l-Phenylethyl]amino]-7H-pyrrolo[2,3- d]pyrimidin-6-yl]-phenol (PKI166, CAS 187724-61-4).

[00215] EGFR antibodies include but are not limited to, Cetuximab (Erbitux®);

Panitumumab (Vectibix®); Matuzumab (EMD-72000); Nimotuzumab (hR3);

Zalutumumab; TheraCIM h-R3; MDX0447 (CAS 339151-96-1); and ch806 (mAb-806, CAS 946414-09-1).

[00216] Human Epidermal Growth Factor Receptor 2 (HER2 receptor) (also known as

Neu, ErbB-2, CD340, or pi 85) inhibitors include but are not limited to, Trastuzumab (Herceptin®); Pertuzumab (Omnitarg®); Neratinib (HKI-272, (2E)-N-[4-[[3-chloro-4- [(pyridin-2-yl)methoxy]phenyl]amino]-3-cyano-7-ethoxyquinolin-6-yl]-4- (dimethylamino)but-2-enamide, and described PCT Publication No. WO 05/028443);

Lapatinib or Lapatinib ditosylate (Tykerb®); (3R,4R)-4-amino-l-((4-((3- methoxyphenyl)amino)pyrrolo[2, 1-f] [1 ,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514); (2E)-N-[4-[(3-Chloro-4-fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-6- quinazolinyl]-4-(dimethylamino)-2-butenamide (BIBW-2992, CAS 850140-72-6); N-[4-[[l- [(3-Fluorophenyl)methyl]-lH-indazol-5-yl]amino]-5-methylpyrrolo[2, l-f] [l ,2,4]triazin-6-yl]- carbamic acid, (3S)-3-morpholinylmethyl ester (BMS 599626, CAS 714971-09-2);

Canertinib dihydrochloride (PD183805 or CI-1033); and N-(3,4-Dichloro-2-fluorophenyl)-6- methoxy-7-[[(3aa,5 ,6aa)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]- 4- quinazolinamine (XL647, CAS 781613-23-8).

[00217] HER3 inhibitors include but are not limited to, LJM716, MM-121 , AMG-888,

RG7116, REGN-1400, AV-203, MP-RM-1 , MM-111, and MEHD-7945A.

[00218] MET inhibitors include but are not limited to, Cabozantinib (XL 184, CAS

849217-68-1); Foretinib (GSK1363089, formerly XL880, CAS 849217-64-7); Tivantinib (ARQ197, CAS 1000873-98-2); l-(2-Hydroxy-2-methylpropyl)-N-(5-(7-methoxyquinolin-4- yloxy)pyridin-2-yl)-5-methyl-3-oxo-2-phenyl-2,3-dihydro-lH-pyrazole-4-carboxamide (AMG 458); Cryzotinib (Xalkori®, PF-02341066); (3Z)-5-(2,3-Dihydro-lH-indol- l- ylsulfonyl)-3 -( { 3 ,5-dimethyl-4- [(4-methylpiperazin- 1 -yl)carbonyl] - 1 H-pyrrol-2- yl}methylene)-l ,3-dihydro-2H-indol-2-one (SU11271); (3Z)-N-(3-Chlorophenyl)-3-({3,5- dimethyl-4- [(4-methylpiperazin- 1 -yl)carbonyl] - lH-pyrrol-2-yl } methylene)-N-methyl-2- oxoindoline-5-sulfonamide (SU11274); (3Z)-N-(3-Chlorophenyl)-3- { [3,5-dimethyl-4-(3- morpholin-4-ylpropyl)-lH-pyrrol-2-yl]methylene}-N-methyl-2-oxoindoline-5-sulfonamide (SU11606); 6-[Difluoro[6-(l-methyl-lH-pyrazol-4-yl)-l ,2,4-triazolo[4,3-b]pyridazin-3- yl] methyl] -quinoline (JNJ38877605, CAS 943540-75-8); 2-[4-[l-(Quinolin-6-ylmethyl)-lH- [l,2,3]triazolo[4,5-b]pyrazin-6-yl]-lH-pyrazol-l-yl]ethanol (PF04217903, CAS 956905-27- 4); N-((2R)-l ,4-Dioxan-2-ylmethyl)-N-methyl-N'-[3-(l-methyl- lH-pyrazol-4-yl)-5-oxo-5H- benzo[4,5]cyclohepta[l ,2-b]pyridin-7-yl]sulfamide (MK2461 , CAS 917879-39-1); 6-[[6-(l- Methyl-lH-pyrazol-4-yl)- l,2,4-triazolo[4,3-¾pyridazin-3-yl]thio]-quinoline (SGX523, CAS 1022150-57-7); and (3Z)-5-[[(2,6-Dichlorophenyl)methyl]sulfonyl]-3-[[3,5-dimethyl-4- [ [(2R)-2-( 1 -pyrrolidinylmethyl)- 1 -pyrrolidinyl]carbonyl] - lH-pyrrol-2-yl] methylene] -1,3- dihydro-2H-indol-2-one (ΡΗΑ665752, CAS 477575-56-7).

[00219] IGF1R inhibitors include but are not limited to, BMS-754807, XL-228, OSI-

906, GSK0904529A, A-928605, AXL1717, KW-2450, MK0646, AMG479, IMCA12, MEDI-573, and BI836845. See e.g., Yee, JNCI, 104; 975 (2012) for review.

[00220] In another aspect, the present disclosure provides a method of treating cancer by administering to a subject in need thereof an antibody cytokine engrafted protein in combination with one or more FGF downstream signaling pathway inhibitors, including but not limited to, MEK inhibitors, Braf inhibitors, PI3K/Akt inhibitors, SHP2 inhibitors, and also mTor inhibitors.

[00221] For example, mitogen-activated protein kinase (MEK) inhibitors include but are not limited to, XL-518 (also known as GDC-0973, Cas No. 1029872-29-4, available from ACC Corp.); 2-[(2-Chloro-4-iodophenyl)amino]-N-(cyclopropylmethoxy)-3,4-difluoro- benzamide (also known as CI-1040 or PD184352 and described in PCT Publication No. WO2000035436); N-[(2R)-2,3-Dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4- iodophenyl)amino]- benzamide (also known as PD0325901 and described in PCT Publication No. WO2002006213); 2,3-Bis[amino[(2-aminophenyl)thio]methylene]-butanedinitrile (also known as U0126 and described in US Patent No. 2,779,780); N-[3,4-Difluoro-2-[(2-fluoro- 4-iodophenyl)amino]-6-methoxyphenyl]-l-[(2R)-2,3-dihydroxypropyl]- cyclopropanesulfonamide (also known as RDEA119 or BAY869766 and described in PCT Publication No. WO2007014011); (3S,4R,5Z,8S,9S, l lE)-14-(Ethylamino)-8,9, 16- trihydroxy-3,4-dimethyl-3,4,9, 19-tetrahydro-lH-2-benzoxacyclotetradecine-l ,7(8H)-dione] (also known as E6201 and described in PCT Publication No. WO2003076424); 2'-Amino-3'- methoxyflavone (also known as PD98059 available from Biaffin GmbH & Co., KG, Germany); Vemurafenib (PLX-4032, CAS 918504-65-1); (R)-3-(2,3-Dihydroxypropyl)-6- fluoro-5-(2-fluoro-4-iodophenylamino)-8-methylpyrido[2 -d]pyrimidine-4,7(3H,8H)-dione

(TAK-733, CAS 1035555-63-5); Pimasertib (AS-703026, CAS 1204531-26-9); and

Trametinib dimethyl sulfoxide (GSK-1120212, CAS 1204531-25-80).

[00222] Phosphoinositide 3 -kinase (PI3K) inhibitors include but are not limited to, 4-

[2-(lH-Indazol-4-yl)-6-[[4-(memylsulfonyl)piperazin-l-yl]methyl]thieno[3,2-d]pyrimidin-4- yl]morpholine (also known as GDC 0941 and described in PCT Publication Nos. WO

09/036082 and WO 09/055730); 2-Methyl-2-[4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3- dihydroirnidazo[4,5-c]quinolin-l-yl]phenyl]propionitrile (also known as BEZ 235 or NVP-

BEZ 235, and described in PCT Publication No. WO 06/122806); 4-(trifluoromethyl)-5-(2,6- dimorpholinopyrimidin-4-yl)pyridin-2-amine (also known as B KM 120 or NVP-BKM120, and described in PCT Publication No. WO2007/084786); Tozasertib (VX680 or MK-0457,

CAS 639089-54-6); (5Z)-5-[[4-(4-Pyridinyl)-6-quinolinyl]methylene]-2,4-thiazolidinedione

(GSK1059615, CAS 958852-01-2); (lE,4S,4aR,5R,6aS,9aR)-5-(Acetyloxy)-l-[(di-2- propenylamino)methylene]-4,4a,5,6,6a,8,9,9a-octahydro-l l-hydroxy-4-(methoxymethyl)-

4a,6a-dimethyl-cyclopenta[5,6]naphtho[l,2-c]pyran-2,7,10(lH)-trione (PX866, CAS 502632-

66-8); and 8-Phenyl-2-(moφholin-4-yl)-chromen-4-one (LY294002, CAS 154447-36-6).

[00223] mTor inhibitors include but are not limited to, Temsirolimus (Torisel®);

Ridaforolimus (formally known as deferolimus, (lR,2R,45)-4-[(2R)-2

[(1R,9S,12S, 15R,16£,18R,19R,21R, 23S,24£,26£,28Z,30S,32S,35R)-l, 18-dihydroxy-19,30- dimethoxy-15, 17,21, 23, 29,35-hexamethyl-2,3,10,14,20-pentaoxo-l l,36-dioxa-4- azatricycloPOJ- LO⁴'⁹] hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2- methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669, and described in PCT Publication No. WO 03/064383); Everolimus (Afinitor® or RAD001);

Rapamycin (AY22989, Sirolimus®); Simapimod (CAS 164301-51-3); (5-{2,4-Bis[(35)-3- methylmo holin-4-yl]pyrido[2,3-ίi]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055);

2-Amino-8-[ira«5'-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl- pyrido[2,3- ]pyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4); and Λ^-[1,4-άίοχο-4-

[[4-(4-oxo-8-phenyl-4H-l-benzopyran-2-yl)mo holinium-4-yl]methoxy]butyl]-L- arginylglycyl-L-a-aspartylL-serine- (SEQ ID NO: 118), inner salt (SF1126, CAS 936487-67-

1)·

[00224] In yet another aspect, the present disclosure provides a method of treating cancer by administering to a subject in need thereof an antibody cytokine engrafted protein in combination with one or more pro-apoptotics, including but not limited to, IAP inhibitors, Bcl2 inhibitors, MCL1 inhibitors, Trail agents, Chk inhibitors.

[00225] For examples, IAP inhibitors include but are not limited to, NVP-LCL161,

GDC-0917, AEG-35156, AT406, and TL32711. Other examples of IAP inhibitors include but are not limited to those disclosed in WO04/005284, WO 04/007529, WO05/097791, WO 05/069894, WO 05/069888, WO 05/094818, US2006/0014700, US2006/0025347, WO 06/069063, WO 06/010118, WO 06/017295, and WO08/134679.

[00226] BCL-2 inhibitors include but are not limited to, 4-[4-[[2-(4-Chlorophenyl)-

5 , 5 -dimethyl- 1 -cyclohexen- 1 -yl] methyl] - 1 -piperazinyl] -N- [ [4- [ [( 1 R)-3 -(4-morpholinyl)- 1 - [(phenylthio)methyl]propyl] amino] -3- [(trifluoromethyl)sulfonyl]phenyl] sulfonyl] benzamide (also known as ABT-263 and described in PCT Publication No. WO 09/155386);

Tetrocarcin A; Antimycin; Gossypol ((-)BL-193); Obatoclax; Ethyl-2-amino-6- cyclopentyl-4-(l-cyano-2-ethoxy-2-oxoethyl)-4Hchromone-3-carboxylate (HA14 - 1); Oblimersen (G3139, Genasense®); Bak BH3 peptide; (-)-Gossypol acetic acid (AT-101); 4- [4- [(4'-Chloro[ 1 , 1 '-biphenyl] -2-yl)methyl] - 1 -piperazinyl] -N- [[4- [[( lR)-3-(dimethylamino)-

1- [(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]-benzamide (ABT-737, CAS 852808-04-9); and Navitoclax (ABT-263, CAS 923564-51-6).

[00227] Proapoptotic receptor agonists (PAR As) including DR4 (TRAILR1) and DR5

(TRAILR2), including but are not limited to, Dulanermin (AMG-951, RhApo2L/TRAIL); Mapatumumab (HRS-ETR1, CAS 658052-09-6); Lexatumumab (HGS-ETR2, CAS 845816-

02- 6); Apomab (Apomab®); Conatumumab (AMG655, CAS 896731-82-1); and

Tigatuzumab (CS1008, CAS 946415-34-5, available from Daiichi Sankyo).

[00228] Checkpoint Kinase (CHK) inhibitors include but are not limited to, 7- Hydroxystaurosporine (UCN-01); 6-Bromo-3-(l-methyl-lH-pyrazol-4-yl)-5-(3R)-3- piperidinyl-pyrazolo[l,5-fl]pyrimidin-7-amine (SCH900776, CAS 891494-63-6); 5-(3- Fluorophenyl)-3-ureidothiophene-2-carboxylic acid N-[(S)-piperidin-3-yl]amide (AZD7762, CAS 860352-01-8); 4-[((3S)-l-Azabicyclo[2.2.2]oct-3-yl)amino]-3-(lH-benzimidazol-2-yl)- 6-chloroquinolin-2(lH)-one (CHIR 124, CAS 405168-58-3); 7-Aminodactinomycin (7- AAD), Isogranulatimide, debromohymenialdisine; N-[5-Bromo-4-methyl-2-[(2S)-2- morpholinylmethoxy]-phenyl]-N'-(5-methyl-2-pyrazinyl)urea (LY2603618, CAS 911222-45- 2); Sulforaphane (CAS 4478-93-7, 4-Methylsulfinylbutyl isothiocyanate); 9, 10,11,12- Tetrahydro- 9,12-epoxy-lH-diindolo[l,2,3- g:3',2',r-¾]pyrrolo[3,4-i^'][l,6]benzodiazocine- l,3(2H)-dione (SB-218078, CAS 135897-06-2); and TAT-S216A (Sha et al, Mol. Cancer. Ther 2007; 6(1):147-153), and CBP501.

[00229] In one aspect, the present disclosure provides a method of treating cancer by administering to a subject in need thereof an antibody cytokine engrafted protein in combination with one or more FGFR inhibitors. For example, FGFR inhibitors include but are not limited to, Brivanib alaninate (BMS-582664, (5)-((R)-l-(4-(4-Fluoro-2-methyl-lH- indol-5-yloxy)-5-methylpyrrolo[2,l- |[l,2,4]triazin-6-yloxy)propan-2-yl)2- aminopropanoate); Vargatef (BIBF1120, CAS 928326-83-4); Dovitinib dilactic acid (TKI258, CAS 852433-84-2); 3-(2,6-Dichloro-3,5-dimethoxy-phenyl)-l-{6-[4-(4-ethyl- piperazin-l-yl)-phenylamino]-pyrimidin-4-yl}-l -methyl-urea (BGJ398, CAS 872511-34-7); Danusertib (PHA-739358); and (PD173074, CAS 219580-11-7). In a specific aspect, the present disclosure provides a method of treating cancer by administering to a subject in need thereof an antibody drug conjugate in combination with an FGFR2 inhibitor, such as 3-(2,6- dichloro-3,5-dimethoxyphenyl)- 1 -(6((4-(4-ethylpiperazin- 1 -yl)phenyl)amino)pyrimidin-4- yl)-l-methylurea (also known as BGJ-398); or 4-amino-5-fluoro-3-(5-(4-methylpiperazinl- yl)-lH-benzo[(f]imidazole-2-yl)quinolin-2(lH)-one (also known as dovitinib or TKI-258). AZD4547 (Gavine et al, 2012, Cancer Research 72, 2045-56, N-[5-[2-(3,5- Dimethoxyphenyl)ethyl]-2H-pyrazol-3-yl]-4-(3R,5S)-diemthylpiperazin-l-yl)benzamide), Ponatinib (AP24534; Gozgit et al, 2012, Mol Cancer Ther., 11; 690-99; 3-[2-(imidazo[l,2- b]pyridazin-3-yl)ethynyl]-4-methyl-N- { 4- [(4-methylpiperazin- 1 - yl)methyl] -3- (trifluoromethyl)phenyl}benzamide, CAS 943319-70-8).

[00230] The antibody cytokine engrafted proteins can also be administered in combination with another cytokine, or antibody cytokine engrafted protein. In some embodiments, the cytokine is IL15, IL15-Fc, IL15 linked to a sushi domain of IL15 receptor or IL15 complexed with soluble IL15Ra.

[00231] The antibody cytokine engrafted proteins can also be administered in combination with an immune checkpoint inhibitor. In one embodiment, the antibody cytokine engrafted proteins can be administered in combination with an inhibitor of an immune checkpoint molecule chosen from one or more of PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR. In one embodiment, the immune checkpoint inhibitor is an anti-PD-1 antibody, wherein the anti-PD-1 antibody is selected from Nivolumab, Pembrolizumab or Pidilizumab. In some embodiments, the anti-PD-1 antibody is Nivolumab. Alternative names for Nivolumab include MDX- 1106, MDX- 1106-04, ONO-4538, or BMS-936558. In some embodiments, the anti-PD- 1 antibody is Nivolumab (CAS Registry Number: 946414-94-4). Nivolumab is a fully human IgG4 monoclonal antibody which specifically blocks PDl. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PDl are disclosed in US 8,008,449 and WO2006/121168.

[00232] In some embodiments, the anti-PD- 1 antibody is Pembrolizumab.

Pembrolizumab (also referred to as Lambrolizumab, MK-3475, MK03475, SCH-900475 or KEYTRUDA^®; Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1.

Pembrolizumab and other humanized anti-PD- 1 antibodies are disclosed in Hamid, O. et al. (2013) New England Journal of Medicine 369 (2): 134-44, US 8,354,509 and

WO2009/114335.

[00233] In some embodiments, the anti-PD- 1 antibody is Pidilizumab. Pidilizumab

(CT-011 ; Cure Tech) is a humanized IgGlk monoclonal antibody that binds to PDl.

Pidilizumab and other humanized anti-PD- 1 monoclonal antibodies are disclosed in

WO2009/101611.

[00234] Other anti-PDl antibodies include AMP 514 (Amplimmune) and, e.g., anti-

PD 1 antibodies disclosed in US 8,609,089, US 2010/028330, and/or US 2012/0114649 and US2016/0108123.

[00235] In some embodiments, the antibody cytokine engrafted proteins can be administered with the anti-Tim3 antibody disclosed in US2015/0218274. In other embodiments, the antibody cytokine engrafted proteins can be administered with the anti-PD- Ll antibody disclosed in US2016/0108123, Durvalumab® (MEDI4736), Atezolizumab® (MPDL3280A) or Avelumab®, or the anti-PD-Ll antibody disclosed in WO2016/061142.

Methods of Treatment and Use of Compositions for Treatment of Viral Infection

[00236] For infections resulting from viral causes, the antibody cytokine engrafted proteins can be combined by application simultaneous with, prior to or subsequent to application of standard therapies for treating viral infections.

[00237] Some examples of pathogenic viruses causing infections treatable by antibody cytokine engrafted proteins include HIV, hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, poliovirus, rabies virus, BK virus, JC virus and arboviral encephalitis virus.

[00238] In one embodiment, the infection is an influenza infection. Influenza infection can result in fever, cough, myalgia, headache and malaise, which often occur in seasonal epidemics. Influenza is also associated with a number of postinfectious disorders, such as encephalitis, myopericarditis, Goodpasture's syndrome, and Reye's syndrome. Influenza infection also suppresses normal pulmonary antibacterial defenses, such that patients recovering from influenza have an increased risk of developing bacterial pneumonia.

Influenza viral surface proteins show marked antigenic variation, resulting from mutation and recombination. Thus, cytolytic T lymphocytes are the host's primary vehicle for the elimination of virus after infection. Influenza is classified into three primary types: A, B and C. Influenza A is unique in that it infects both humans and many other animals (e.g., pigs, horses, birds and seals) and is the principal cause of pandemic influenza. Also, when a cell is infected by two different influenza A strains, the segmented RNA genomes of two parental virus types mix during replication to create a hybrid replicant, resulting in new epidemic strains. Influenza B does not replicate in animals and thus has less genetic variation and influenza C has only a single serotype.

[00239] Most conventional therapies are palliatives of the symptoms resulting from infection, while the host's immune response actually clears the disease. However, certain strains (e.g., influenza A) can cause more serious illness and death. Influenza A may be treated both clinically and prophylactically by the administration of the cyclic amines inhibitors amantadine and rimantadine, which inhibit viral replication. However, the clinical utility of these drugs is limited due to the relatively high incidence of adverse reactions, their narrow anti-viral spectrum (influenza A only), and the propensity of the virus to become resistant. The administration of serum IgG antibody to the major influenza surface proteins, hemagglutinin and neuraminidase can prevent pulmonary infection, whereas mucosal IgA is required to prevent infection of the upper respiratory tract and trachea. The most effective current treatment for influenza is vaccination with the administration of virus inactivated with formalin or β-propiolactone. The antibody cytokine engrafted proteins can be used as a single agent or in combination with anti-PD-Ll antibodies or the standard of care treatment in order to reduce influenza virus infection.

[00240] In another embodiment, the infection is a hepatitis infection, e.g., a Hepatitis B or C infection. Hepatitis B virus (HBV) is the most infectious known bloodborne pathogen. It is a major cause of acute and chronic hepatitis and hepatic carcinoma, as well as life-long, chronic infection. Following infection, the virus replicates in hepatocytes, which also then shed the surface antigen HBsAg. The detection of excessive levels of HBsAg in serum is used a standard method for diagnosing a hepatitis B infection. An acute infection may resolve or it can develop into a chronic persistent infection. Current treatments for chronic HBV include a-interferon, which increases the expression of class I human leukocyte antigen (HLA) on the surface of hepatocytes, thereby facilitating their recognition by cytotoxic T lymphocytes. Additionally, the nucleoside analogs ganciclovir, famciclovir and lamivudine have also shown some efficacy in the treatment of HBV infection in clinical trials. Additional treatments for HBV include pegylated a-interferon, adenfovir, entecavir and telbivudine. While passive immunity can be conferred through parental administration of anti-HBsAg serum antibodies, vaccination with inactivated or recombinant HBsAg also confers resistance to infection. The antibody cytokine engrafted proteins can be used as a single agent or in combination with anti-PD-Ll antibodies to treat HBV.

[00241] Hepatitis C virus (HCV) infection may lead to a chronic form of hepatitis, resulting in cirrhosis. While symptoms are similar to infections resulting from Hepatitis B, in distinct contrast to HBV, infected hosts can be asymptomatic for 10-20 years. The antibody cytokine engrafted proteins can be administered as a monotherapy, or combined with anti- PD-Ll antibodies for HCV infection. Alternatively, the antibody cytokine engrafted protein can be administered with one or more of Sovaldi (sofosbuvir) Olysio (simeprevir), plus ribavirin or pegylated interferon. Although regimens that include Incivek (telaprevir) or Victrelis (boceprevir) plus ribavirin and pegylated interferon are also approved, they are associated with increased side effects and longer duration of treatment and are therefore not considered preferred regimens. The antibody cytokine engrafted proteins can be used as a single agent or in combination with anti-PD-Ll antibodies to treat HCV.

[00242] In another embodiment, the infection is HIV. HIV attacks CD4+ cells, including T-lymphocytes, monocyte-macrophages, follicular dendritic cells and Langerhan's cells, and CD4+ helper/inducer cells are depleted. As a result, the host acquires a severe defect in cell-mediated immunity. Infection with HIV results in AIDS in at least 50% of individuals, and is transmitted via sexual contact, administration of infected blood or blood products, artificial insemination with infected semen, exposure to blood-containing needles or syringes and transmission from an infected mother to infant during childbirth. [00243] A host infected with HIV may be asymptomatic, or may develop an acute illness that resembling mononucleosis— fever, headache, sore throat, malaise and rash.

Symptoms can progress to progressive immune dysfunction, including persistent fever, night sweats, weight loss, unexplained diarrhea, eczema, psoriasis, seborrheic dermatitis, herpes zoster, oral candidiasis and oral hairy leukoplakia. Opportunistic infections by a host of parasites are common in patients whose infections develop into AIDS.

[00244] Treatments for HIV include antiviral therapies including nucleoside analogs, zidovudine (AST) either alone or in combination with didanosine or zalcitabine, dideoxyinosine, dideoxycytidine, lamidvudine, stavudine; reverse transcriptive inhibitors such as delavirdine, nevirapine, loviride, and proteinase inhibitors such as saquinavir, ritonavir, indinavir and nelfinavir. The antibody cytokine engrafted proteins can be used as a single agent or in combination with anti-PD-Ll antibodies or the standard of care treatments for HIV.

[00245] In another embodiment, the infection is a Cytomegalovirus (CMV). CMV infection is often associated with persistent, latent and recurrent infection. CMV infects and remains latent in monocytes and granulocyte-monocyte progenitor cells. The clinical symptoms of CMV include mononucleosis-like symptoms (i.e., fever, swollen glands, malaise), and a tendency to develop allergic skin rashes to antibiotics. The virus is spread by direct contact. The virus is shed in the urine, saliva, semen and to a lesser extent in other body fluids. Transmission can also occur from an infected mother to her fetus or newborn and by blood transfusion and organ transplants. CMV infection results in general impairment of cellular immunity, characterized by impaired blastogenic responses to nonspecific mitogens and specific CMV antigens, diminished cytotoxic ability and elevation of CD8 lymphocyte number of CD4+ lymphocytes.

[00246] Treatments of CMV infection include the anti-virals ganciclovir, foscarnet and cidovir, but these drugs are typically only prescribed in immunocompromised patients. The antibody cytokine engrafted proteins can be used as a single agent or in combination with anti-PD-Ll antibodies or the standard of care treatments in order to reduce CMV infection.

[00247] In another embodiment, the infection is Epstein-Barr virus (EBV). EBV can establish persistent and latent infections and primarily attacks B cells. Infection with EBV results in the clinical condition of infectious mononucleosis, which includes fever, sore throat, often with exudate, generalized lymphadenopathy and splenomegaly. Hepatitis is also present, which can develop into jaundice. [00248] While typical treatments for EBV infections are palliative of symptoms, EBV is associated with the development of certain cancers such as Burkitt's lymphoma and nasopharyngeal cancer. Thus, clearance of viral infection before these complications result would be of great benefit. The antibody cytokine engrafted proteins can be used as a single agent or in combination with anti-PD-Ll antibodies to treat EBV infections.

[00249] In another embodiment, the infection is Herpes simplex virus (HSV). HSV is transmitted by direct contact with an infected host. A direct infection may be asymptomatic, but typically result in blisters containing infectious particles. The disease manifests as cycles of active periods of disease, in which lesions appear and disappear as the viral latently infect the nerve ganglion for subsequent outbreaks. Lesions may be on the face, genitals, eyes and/or hands. In some case, an infection can also cause encephalitis.

[00250] Treatments for herpes infections are directed primarily to resolving the symptomatic outbreaks, and include systemic antiviral medicines such as: acyclovir (e.g., Zovirax®), valaciclovir, famciclovir, penciclovir, and topical medications such as docosanol (Abreva®), tromantadine and zilactin. The antibody cytokine engrafted proteins can be used as a single agent or in combination with anti-PD-Ll antibodies or the conventional treatments in order to treat herpes infections.

[00251] In another embodiment, the infection is Human T-lymphotrophic virus

(HTLV-1, HTLV-2). HTLV is transmitted via sexual contact, breast feeding or exposure to contaminated blood. The virus activates a subset of TH cells called Thi cells, resulting in their overproliferation and overproduction of Thi related cytokines (e.g., IFN-γ and TNF-a). This in turn results in a suppression of Th2 lymphocytes and reduction of Th2 cytokine production (e.g., IL-4, IL-5, IL-10 and IL-13), causing a reduction in the ability of an infected host to mount an adequate immune response to invading organisms requiring a Th2- dependent response for clearance (e.g., parasitic infections, production of mucosal and humoral antibodies).

[00252] HTLV infections cause lead to opportunistic infections resulting in bronchiectasis, dermatitis and superinfections with Staphylococcus spp. and Strongyloides spp. resulting in death from polymicrobial sepsis. HTLV infection can also lead directly to adult T-cell leukemia/lymphoma and progressive demyelinating upper motor neuron disease known as HAM/TSP. The antibody cytokine engrafted proteins can be used as a single agent or in combination with anti-PD-Ll antibodies to treat HTLV infections. [00253] In another embodiment, the infection is Human papilloma virus (HPV). HPV primarily affects keratinocytes and occurs in two forms: cutaneous and genital. Transmission is believed to occur through direct contact and/or sexual activity. Both cutaneous and genital HPV infection, can result in warts and latent infections and sometimes recurring infections, which are controlled by host immunity which controls the symptoms and blocks the appearance of warts, but leaves the host capable of transmitting the infection to others.

Infection with HPV can also lead to certain cancers, such as cervical, anal, vulvar, penile and oropharynial cancer. There are no known cures for HPV infection, but current treatment is topical application of Imiquimod, which stimulates the immune system to attack the affected area. The antibody cytokine engrafted proteins can be used as a single agent or in combination with anti-PD-Ll antibodies to treat HPV infections.

EXAMPLES

Example 1: Creation of IL7 antibody cytokine engrafted proteins

[00254] Antibody cytokine engrafted proteins were generated by engineering an IL7 sequence into CDR regions of various immunoglobulin scaffolds, then both heavy and light chain immunoglobulin chains were used to generate final antibody cytokine proteins.

Antibody cytokine engrafted proteins confer the therapeutic properties of IL7, and have additional beneficial effects, such as increased half-life.

[00255] To create antibody cytokine engrafted proteins, the mature form of IL7 (SEQ

ID NO:2) was inserted into various CDR regions of an immunoglobulin chain scaffold. Antibody cytokine engrafted proteins were prepared using a variety of known

immunoglobulin sequences which have been utilized in clinical settings as well as germline antibody sequences. Sequences of IL7 in an exemplary scaffold, referred to as GFTX3b, are depicted in TABLE 2. Insertion points were selected to be the mid-point of the CDR loop based on available structural and/or homology model data. Antibody cytokine engrafted proteins were produced using standard molecular biology methodology utilizing recombinant DNA encoding the relevant sequences.

[00256] The selection of which CDR was chosen for cytokine engraftment was on the parameters of: the required biology, biophysical properties and a favorable development profile. Modeling software was only partially useful in predicting which CDR and which location within the CDR will provide the desired parameters, so therefore all six possible antibody cytokine grafts were made and then evaluated in biological assays. If the required biological activity is achieved, then the biophysical properties such as structural resolution as to how the antibody cytokine engrafted molecule interacts with the respective cytokine receptor are resolved.

[00257] For the antibody IL7 engrafted molecules, the structure of the antibody candidate considered for cytokine engrafting was initially solved. From this structure, it was noted that the paratope of the antibody was at the extreme N-terminus of the antibody "arm" and that a cytokine engrafted into this location would present the cytokine to its respective receptor. Because of the engrafting technology, each antibody IL7 engrafted protein is constrained by a CDR loop of different length, sequence and structural environments. As such, IL7 was engrafted into all six CDRs, corresponding to LCDR-1, LCDR-2, LCDR-3 and HCDR-1, HCDR-2 and HCDR-3.

[00258] Insertion of IL7 into HCDR-2 and HCDR-3 were chosen because these locations had the best combination of both biophysical and biological properties. For the selection of the insertion point, the structural center of the CDR loop was chosen as this would provide the most space on either side (of linear size 3.8 A x the number of residues) and without being bound by any one theory, this provided a stable molecule by allowing the IL7 to more readily fold independently. As the structure of the grafting scaffold GFTX3b was already known, so the structural center of each CDR was also known. This usually coincides with the center of the CDR loop sequence as defined using the Chothia numbering format.

[00259] In summary, the insertion point in each CDR was chosen on a structural basis, and which CDR graft was best for the IL7 cytokine was based on desired biology and biophysical properties. The nature of the cytokine receptor, the cytokine/receptor interactions and the mechanism of signaling also played a role and this was investigated by comparing each individual antibody cytokine molecule for their respective properties.

TABLE 1

SEQ ID IL7 DNA ACATCCGCGGCAACGCCTCCTTGGTGTCGTCCGCTTCC

AATAACCCAGCTTGCGTCCTGCACACTTGTGGCTTCCG NO: 1

TGCACACATTAACAACTCATGGTTCTAGCTCCCAGTCG CCAAGCGTTGCCAAGGCGTTGAGAGATCATCTGGGAAG TCTTTTACCCAGAATTGCTTTGATTCAGGCCAGCTGGT TTTTCCTGCGGTGATTCGGAAATTCGCGAATTCCTCTG GTCCTCATCCAGGTGCGCGGGAAGCAGGTGCCCAGGAG AGAGGGGATAATGAAGATTCCATGCTGATGATCCCAAA GATTGAACCTGCAGACCAAGCGCAAAGTAGAAACTGAA AGTACACTGCTGGCGGATCCTACGGAAGTTATGGAAAA GGCAAAGCGCAGAGCCACGCCGTAGTGTGTGCCGCCCC

CCTTGGGATGGATGAAACTGCAGTCGCGGCGTGGGTAA GAGGAACCAGCTGCAGAGATCACCCTGCCCAACACAGA CTCGGCAACTCCGCGGAAGACCAGGGTCCTGGGAGTGA CTATGGGCGGTGAGAGCTTGCTCCTGCTCCAGTTGCGG TCATCATGACTACGCCCGCCTCCCGCAGACCATGTTCC ATGTTTCTTTTAGGTATATCTTTGGACTTCCTCCCCTG ATCCTTGTTCTGTTGCCAGTAGCATCATCTGATTGTGA TATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTC TAATGGTCAGCATCGATCAATTATTGGACAGCATGAAA GAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTT TTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTA TGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTT CTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTT ATTAAAAGTTTCAGAAGGCACAACAATACTGTTGAACT GCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTG GGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAA ATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTT TCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGG AATAAAATTTTGATGGGCACTAAAGAACACTGAAAAAT ATGGAGTGGCAATATAGAAACACGAACTTTAGCTGCAT CCTCCAAGAATCTATCTGCTTATGCAGTTTTTCAGAGT GGAATGCTTCCTAGAAGTTACTGAATGCACCATGGTCA AAACGGATTAGGGCATTTGAGAAATGCATATTGTATTA CTAGAAGATGAATACAAACAATGGAAACTGAATGCTCC AGTCAACAAACTATTTCTTATATATGTGAACATTTATC AATCAGTATAATTCTGTACTGATTTTTGTAAGACAATC CATGTAAGGTATCAGTTGCAATAATACTTCTCAAACCT GTTTAAATATTTCAAGACATTAAATCTATGAAGTATAT AATGGTTTCAAAGATTCAAAATTGACATTGCTTTACTG TCAAAATAATTTTATGGCTCACTATGAATCTATTATAC TGTATTAAGAGTGAAAATTGTCTTCTTCTGTGCTGGAG ATGTTTTAGAGTTAACAATGATATATGGATAATGCCGG TGAGAATAAGAGAGTCATAAACCTTAAGTAAGCAACAG CATAACAAGGTCCAAGATACCTAAAAGAGATTTCAAGA GATTTAATTAATCATGAATGTGTAACACAGTGCCTTCA ATAAATGGTATAGCAAATGTTTTGACATGAAAAAAGGA CAATTTCAAAAAAATAAAATAAAATAAAAATAAATTCA CCTAGTCTAAGGATGCTAAACCTTAGTACTGAGTTACA TTGTCATTTATATAGATTATAACTTGTCTAAATAAGTT TGCAATTTGGGAGATATATTTTTAAGATAATAATATAT GTTTACCTTTTAATTAATGAAATATCTGTATTTAATTT TGACACTATATCTGTATATAAAATATTTTCATACAGCA TTACAAATTGCTTACTTTGGAATACATTTCTCCTTTGA TAAAATAAATGAGCTATGTATTAAAAAAAAAAAAAAA

SEQ ID Mature IL7 DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNE

FNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFD NO: 2 Protein

LHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLE ENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH

TABLE 2

IgG.IL7.Hl (DAPA-IL7-CDRH1) SEQ ID NO: 3 HCDR1 CDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICD (Rabat ) ANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQ

VKGRKPAALGEAQPTKSLEE KSLKEQKKLNDLCFLKRLLQEIKTCW

NKILMGTKEHSTSGMSVG

SEQ ID NO: 4 HCDR2 DIWWDDRRDYNPSLRS

(Rabat )

SEQ ID NO: 5 HCDR3 SMITNWYFDV

(Rabat )

SEQ ID NO: 6 HCDR1 GFSLDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEF FFK (Chothia) RHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILL

NCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQE

IKTCWNKILMGTKEHSTSGM

SEQ ID HCDR2 WWDDR

NO: 7 (Chothia)

SEQ ID NO: 8 HCDR3 SMITNWYFDV

(Chothia)

SEQ ID NO: 9 VH QVTLRESGPALVRPTQTLTLTCTFSGFSLDCDIEGKDGKQYESVLMV

SIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQ

FLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKS

LEENKSLKEQKKL DLCFLKRLLQEIKTCWNKILMGTKEHSTSGMSV

GWIRQPPGRALEWLADIWWDDRRDYNPSLRSRLTISRDTSRNQWLR VTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSS

SEQ ID NO:10 VH DNA CAGGTGACCCTCCGCGAGAGCGGACCCGCCCTCGTGAAGCCAACCCA

GACCCTGACCCTCACTTGCACCTTCTCTGGATTCTCCCTGGATTGTG ATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTC AGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTG CCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTA ATAAGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAA TTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAA AGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTA AAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGT TTGGAAGAAAATAAATCTTTAAAGGAACAGAAAAAACTGAATGACTT GTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAATA AAATTTTGATGGGCACTAAAGAACACTCTACCTCAGGGATGTCAGTC GGATGGATCAGACAGCCACCAGGGAAGGCACTTGAGTGGCTGGCCGA CATTTGGTGGGACGACAAAAAGGACTACAATCCCTCACTGAAGAGCA GGCTGACTATCTCCAAGGACACTTCCAAGAACCAGGTGGTGCTTAAA

GTCACCAACATGGACCCTGCTGATACTGCCACCTATTACTGTGCACG CAGCATGATCACTAACTGGTACTTTGACGTGTGGGGTGCTGGCACCA CCGTGACTGTCAGCTCA

SEQ ID NO:ll Heavy QVTLRESGPALVRPTQTLTLTCTFSGFSLDCDlEGKDGKQYESVLMV

Chain SIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQ

FLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKS

LEENKSLKEQKKL DLCFLKRLLQEIKTCWNKILMGTKEHSTSGMSV

GWIRQPPGRALEWLADIWWDDRRDYNPSLRSRLTISRDTSRNQWLR VTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSSASTRGPSVFP LAPSSRSTSGGTAALGCLVRDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSWTVPSSSLGTQTYICNVNHRPSNTRVDRRVEPRSCD RTHTCPPCPAPELLGGPSVFLFPPRPRDTLMISRTPEVTCVWAVSH EDPEVRFNWYVDGVEVHNARTRPREEQYNSTYRWSVLTVLHQDWLN GREYRCRVSNRALAAPIERTISRARGQPREPQVYTLPPSREEMTRNQ VSLTCLVRGFYPSDIAVEWESNGQPENNYRTTPPVLDSDGSFFLYSR LTVDRSRWQQGNVFSCSVMHEALHNHYTQRSLSLSPGR

SEQ ID LCDR1 RAQLSVGYMH

NO: 12 (Rabat)

SEQ ID NO: 13 LCDR2 DTSRLAS

(Rabat )

SEQ ID NO:14 LCDR3 FQGSGYPFT

(Rabat )

SEQ ID NO: 15 LCDR1 QLSVGY

(Chothia)

SEQ ID NO: 16 LCDR2 DTS

(Chothia)

SEQ ID NO:17 LCDR3 GSGYPF

(Chothia)

SEQ ID NO:18 VL DIQMTQSPSTLSASVGDRVT ITCRAQLSVGYMHWYQQRPGRAPRLLI

YDTSRLASGVPSRFSGSGSGTEFTLTI SSLQPDDFATYYCFQGSGYP FTFGGGTRLEIR

SEQ ID NO:19 VL DNA GACATTCAGATGACCCAGTCACCCAGCACTCTGAGCGCTTCCGTGGG

AGACAGAGTCACCATTACTTGCAAGGCCCAGCTCTCTGTGGGATATA TGCATTGGTACCAGCAGAAACCGGGAAAAGCACCTAAGCTGCTGATC TACGACACCTCAAAACTGGCCTCAGGCGTGCCAAGCCGGTTCAGCGG GTCCGGATCTGGTACTGAGTTTACCCTCACCATCTCATCTCTCCAGC

CAGACGATTTCGCTACCTATTACTGCTTCCAAGGATCCGGGTACCCA TTCACTTTTGGCGGAGGGACTAAGCTGGAAATCAAG

SEQ ID NO:20 Light DIQMTQSPSTLSASVGDRVT ITCRAQLSVGYMHWYQQRPGRAPRLLI

Chain YDTSRLASGVPSRFSGSGSGTEFTLTI SSLQPDDFATYYCFQGSGYP

FTFGGGTRLEIRRTVAAPSVFIFPPSDEQLRSGTASWCLLNNFYPR EARVQWRVDNALQSGNSQESVTEQDSRDSTYSLSSTLTLSRADYERH RVYACEVTHQGLSSPVTRSFNRGEC

IgG . IL7. H2 (DAPA-IL7-CDRH2 )

SEQ ID NO:21 HCDR1 TSGMSVG

(Rabat )

SEQ ID NO: 22 HCDR2 DIWWDDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFF (Rabat ) KRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTIL

LNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQ

EIKTCWNKILMGTKEHDRRDYNPSLRS

SEQ ID NO: 23 HCDR3 SMITNWYFDV

(Rabat )

SEQ ID NO:24 HCDR1 GFSLSTSGM

(Chothia)

SEQ ID NO: 25 HCDR2 WWDDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKR (Chothia) HICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLN

CTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEI

KTCWNKILMGTKEHDR

SEQ ID NO: 26 HCDR3 SMITNWYFDV

(Chothia)

SEQ ID NO:27 VH QVTLRESGPALVRPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGRAL

EWLADIWWDDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNE

FNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEG

TTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLK

RLLQEIKTCWNKILMGTKEHDRRDYNPSLRSRLTISRDTSRNQWLR

VTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSS

SEQ ID NO:28 VH DNA CAGGTGACCCTCCGCGAGAGCGGACCCGCCCTCGTGAAGCCAACCCA

GACCCTGACCCTCACTTGCACCTTCTCTGGATTCTCCCTGTCTACCT CAGGGATGTCAGTCGGATGGATCAGACAGCCACCAGGGAAGGCACTT GAGTGGCTGGCCGACATTTGGTGGGACGATTGTGATATTGAAGGTAA AGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAAT TATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAA

TTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTAT GTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGA ATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGC ACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACC AGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATA AATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTTTCCTAAAG AGACTATTACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGG CACTAAAGAACACGACAAAAAGGACTACAATCCCTCACTGAAGAGCA GGCTGACTATCTCCAAGGACACTTCCAAGAACCAGGTGGTGCTTAAA GTCACCAACATGGACCCTGCTGATACTGCCACCTATTACTGTGCACG CAGCATGATCACTAACTGGTACTTTGACGTGTGGGGTGCTGGCACCA CCGTGACTGTCAGCTCA

SEQ ID NO:29 Heavy QVTLRESGPALVRPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGRAL

Chain EWLADIWWDDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNE

FNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEG

TTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLK

RLLQEIKTCWNKILMGTKEHDRRDYNPSLRSRLTISRDTSRNQWLR

VTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSSASTRGPSVFP LAPSSRSTSGGTAALGCLVRDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSWTVPSSSLGTQTYICNVNHRPSNTRVDRRVEPRSCD RTHTCPPCPAPELLGGPSVFLFPPRPRDTLMISRTPEVTCVWAVSH EDPEVRFNWYVDGVEVHNARTRPREEQYNSTYRWSVLTVLHQDWLN GREYRCRVSNRALAAPIERTISRARGQPREPQVYTLPPSREEMTRNQ VSLTCLVRGFYPSDIAVEWESNGQPENNYRTTPPVLDSDGSFFLYSR LTVDRSRWQQGNVFSCSVMHEALHNHYTQRSLSLSPGR

SEQ ID NO:30 LCDR1 RAQLSVGYMH

(Rabat )

SEQ ID N0:31 LCDR2 DTSRLAS

(Rabat )

SEQ ID NO: 32 LCDR3 FQGSGYPFT

(Rabat )

SEQ ID NO: 33 LCDR1 QLSVGY

(Chothia)

SEQ ID NO:34 LCDR2 DTS

(Chothia) SEQ ID NO: 35 LCDR3 GSGYPF

(Chothia)

SEQ ID NO: 36 VL DIQMTQSPSTLSASVGDRVT ITCRAQLSVGYMHWYQQRPGRAPRLLI

YDTSRLASGVPSRFSGSGSGTEFTLTI SSLQPDDFATYYCFQGSGYP FTFGGGTRLEIR

SEQ ID NO:37 VL DNA GACATTCAGATGACCCAGTCACCCAGCACTCTGAGCGCTTCCGTGGG

AGACAGAGTCACCATTACTTGCAAGGCCCAGCTCTCTGTGGGATATA TGCATTGGTACCAGCAGAAACCGGGAAAAGCACCTAAGCTGCTGATC TACGACACCTCAAAACTGGCCTCAGGCGTGCCAAGCCGGTTCAGCGG GTCCGGATCTGGTACTGAGTTTACCCTCACCATCTCATCTCTCCAGC CAGACGATTTCGCTACCTATTACTGCTTCCAAGGATCCGGGTACCCA TTCACTTTTGGCGGAGGGACTAAGCTGGAAATCAAG

SEQ ID NO:38 Light DIQMTQSPSTLSASVGDRVT ITCRAQLSVGYMHWYQQRPGRAPRLLI

Chain YDTSRLASGVPSRFSGSGSGTEFTLTI SSLQPDDFATYYCFQGSGYP

IgG.IL7.H3 (DAPA-IL7-CDRH3)

SEQ ID NO:39 HCDR1 TSGMSVG

(Rabat )

SEQ ID NO: 40 HCDR2 DIWWDDRRDYNPSLRS

(Rabat )

SEQ ID NO: 41 HCDR3 SMITDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEF FFK (Rabat ) RHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILL

NCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQE

IKTCWNKILMGTKEHNWYFDV

SEQ ID NO: 42 HCDR1 GFSLSTSGM

(Chothia)

SEQ ID NO: 43 HCDR2 WWDDR

(Chothia)

SEQ ID NO: 44 HCDR3 SMITDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEF FFK (Chothia) RHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILL

NCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQE

IKTCWNKILMGTKEHNWYFDV

SEQ ID NO: 45 VH QVTLRESGPALVRPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGRAL

EWLADIWWDDRRDYNPSLRSRLTI SRDTSRNQWLRVTNMDPADTAT YYCARSMITDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNE

FNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEG

TTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLK

RLLQEIKTCWNKILMGTKEHNWYFDVWGAGTTVTVSS

SEQ ID NO: 46 VH DNA CAGGTGACCCTCCGCGAGAGCGGACCCGCCCTCGTGAAGCCAACCCA

GACCCTGACCCTCACTTGCACCTTCTCTGGATTCTCCCTGTCTACCT CAGGGATGTCAGTCGGATGGATCAGACAGCCACCAGGGAAGGCACTT GAGTGGCTGGCCGACATTTGGTGGGACGACAAAAAGGACTACAATCC CTCACTGAAGAGCAGGCTGACTATCTCCAAGGACACTTCCAAGAACC AGGTGGTGCTTAAAGTCACCAACATGGACCCTGCTGATACTGCCACC TATTACTGTGCACGCAGCATGATCACTGATTGTGATATTGAAGGTAA AGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAAT TATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAA TTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTAT GTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGA ATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGC ACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACC AGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATA AATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTTTCCTAAAG AGACTATTACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGG CACTAAAGAACACAACTGGTACTTTGACGTGTGGGGTGCTGGCACCA CCGTGACTGTCAGCTCA

SEQ ID NO: 47 Heavy QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKAL

Chain EWLADIWWDDKKDYNPSLKSRLTI SKDTSKNQWLKVTNMDPADTAT

YYCARSMITDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNE FNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEG TTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLK RLLQEIKTCWNKILMGTKEHNWYFDVWGAGTTVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWAVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN GKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 48 LCDR1 KAQLSVGYMH

(Rabat ) SEQ ID NO: 49 LCDR2 DTSRLAS

(Rabat )

SEQ ID NO: 50 LCDR3 FQGSGYPFT

(Rabat )

SEQ ID NO: 51 LCDR1 QLSVGY

(Chothia)

SEQ ID NO: 52 LCDR2 DTS

(Chothia)

SEQ ID NO: 53 LCDR3 GSGYPF

(Chothia)

SEQ ID NO: 54 VL DIQMTQSPSTLSASVGDRVT ITCRAQLSVGYMHWYQQRPGRAPRLLI

YDTSRLASGVPSRFSGSGSGTEFTLTI SSLQPDDFATYYCFQGSGYP FTFGGGTRLEIR

SEQ ID NO: 55 VL DNA GACATTCAGATGACCCAGTCACCCAGCACTCTGAGCGCTTCCGTGGG

SEQ ID NO: 56 Light DIQMTQSPSTLSASVGDRVT ITCRAQLSVGYMHWYQQRPGRAPRLLI

Chain YDTSRLASGVPSRFSGSGSGTEFTLTI SSLQPDDFATYYCFQGSGYP

IgG.IL7.Ll (DAPA-IL7-CDRL1)

SEQ ID NO: 57 HCDR1 TSGMSVG

(Rabat )

SEQ ID NO: 58 HCDR2 DIWWDDRRDYNPSLR

(Rabat )

SEQ ID NO: 59 HCDR3 SMITNWYFDV

(Rabat )

SEQ ID NO:60 HCDR1 GFSLSTSGM

(Chothia)

SEQ ID NO:61 HCDR2 WWDDR

(Chothia) SEQ ID NO: 62 HCDR3 SMITNWYFDV

(Chothia)

SEQ ID NO: 63 VH QVTLRESGPALVRPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGRAL

EWLADIWWDDRRDYNPSLRSRLTISRDTSANQWLRVTNMDPADTAT YYCARSMITNWYFDVWGAGTTVTVSS

SEQ ID NO:64 VH DNA CAGGTGACCCTCCGCGAGAGCGGACCCGCCCTCGTGAAGCCAACCCA

GACCCTGACCCTCACTTGCACCTTCTCTGGATTCTCCCTGTCTACCT CAGGGATGTCAGTCGGATGGATCAGACAGCCACCAGGGAAGGCACTT GAGTGGCTGGCCGACATTTGGTGGGACGACAAAAAGGACTACAATCC CTCACTGAAGAGCAGGCTGACTATCTCCAAGGACACTTCCAAGAACC AGGTGGTGCTTAAAGTCACCAACATGGACCCTGCTGATACTGCCACC TATTACTGTGCACGCAGCATGATCACTAACTGGTACTTTGACGTGTG GGGTGCTGGCACCACCGTGACTGTCAGCTCA

SEQ ID NO: 65 Heavy QVTLRESGPALVRPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGRAL

Chain EWLADIWWDDRRDYNPSLRSRLTISRDTSANQWLRVTNMDPADTAT

YYCARSMITNWYFDVWGAGTTVTVSSASTRGPSVFPLAPSSRSTSGG TAALGCLVRDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHRPSNTRVDRRVEPRSCDRTHTCPPCPAP ELLGGPSVFLFPPRPRDTLMISRTPEVTCVWAVSHEDPEVRFNWYV DGVEVHNARTRPREEQYNSTYRWSVLTVLHQDWLNGREYRCRVSNR ALAAPIERTISRARGQPREPQVYTLPPSREEMTRNQVSLTCLVRGFY PSDIAVEWESNGQPENNYRTTPPVLDSDGSFFLYSRLTVDRSRWQQG NVFSCSVMHEALHNHYTQRSLSLSPGR

SEQ ID NO: 66 LCDR1 RAQLSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFF (Rabat ) KRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTIL

LNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQ

EIKTCWNKILMGTKEHVGYMH

SEQ ID NO:67 LCDR2 DTSRLAS

(Rabat )

SEQ ID NO:68 LCDR3 FQGSGYPFT

(Rabat )

SEQ ID NO:69 LCDR1 QLSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKR (Chothia) HICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLN

CTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEI

KTCWNKILMGTKEHVGY SEQ ID NO: 70 LCDR2 DTS

(Chothia)

SEQ ID NO: 71 LCDR3 GSGYPF

(Chothia)

SEQ ID NO: 72 VL DIQMTQSPSTLSASVGDRVTITCKAQLSDCDIEGKDGKQYESVLMVS

IDQLLDSMKEIGSNCLNNEF FFKRHICDANKEGMFLFRAARKLRQF

LKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSL

EENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHVGYMHWYQ

QKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFA TYYCFQGSGYPFTFGGGTKLEIK

SEQ ID NO: 73 VL DNA GACATTCAGATGACCCAGTCACCCAGCACTCTGAGCGCTTCCGTGGG

AGACAGAGTCACCATTACTTGCAAGGCCCAGCTCTCTGATTGTGATA TTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGC ATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCT GAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATA AGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTT CTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGT TTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAG GAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTG GAAGAAAATAAATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTG TTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAATAAAA TTTTGATGGGCACTAAAGAACACGTGGGATATATGCATTGGTACCAG CAGAAACCGGGAAAAGCACCTAAGCTGCTGATCTACGACACCTCAAA ACTGGCCTCAGGCGTGCCAAGCCGGTTCAGCGGGTCCGGATCTGGTA CTGAGTTTACCCTCACCATCTCATCTCTCCAGCCAGACGATTTCGCT ACCTATTACTGCTTCCAAGGATCCGGGTACCCATTCACTTTTGGCGG AGGGACTAAGCTGGAAATCAAG

SEQ ID NO: 74 Light DIQMTQSPSTLSASVGDRVTITCKAQLSDCDIEGKDGKQYESVLMVS

Chain IDQLLDSMKEIGSNCLNNEF FFKRHICDANKEGMFLFRAARKLRQF

LKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSL

EENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHVGYMHWYQ

QKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFA TYYCFQGSGYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS WCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

IgG . IL7. L2 (DAPA-IL7-CDRL2 ) SEQ ID NO: 75 HCDR1 TSGMSVG

(Rabat )

SEQ ID NO: 76 HCDR2 DIWWDDRRDYNPSLR

(Rabat )

SEQ ID NO: 77 HCDR3 SMITNWYFDV

(Rabat )

SEQ ID NO: 78 HCDR1 GFSLSTSGM

(Chothia)

SEQ ID NO: 79 HCDR2 WWDDR

(Chothia)

SEQ ID NO:80 HCDR3 SMITNWYFDV

(Chothia)

SEQ ID NO:81 VH QVTLRESGPALVRPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGRAL

EWLADIWWDDRRDYNPSLRSRLTISRDTSANQWLRVTNMDPADTAT YYCARSMITNWYFDVWGAGTTVTVSS

SEQ ID NO: 82 VH DNA CAGGTGACCCTCCGCGAGAGCGGACCCGCCCTCGTGAAGCCAACCCA

SEQ ID NO: 83 Heavy QVTLRESGPALVRPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGRAL

Chain EWLADIWWDDRRDYNPSLRSRLTISRDTSANQWLRVTNMDPADTAT

SEQ ID NO:84 LCDR1 RAQLSVGYMH

(Rabat ) SEQ ID NO: 85 LCDR2 DTDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRH (Rabat ) ICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNC

TGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIK

TCWNKILMGTKEHSRLAS

SEQ ID NO: 86 LCDR3 FQGSGYPFT

(Rabat )

SEQ ID NO:87 LCDR1 QLSVGY

(Chothia)

SEQ ID NO:88 LCDR2 DTDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRH (Chothia) ICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNC

TGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIK

TCWNKILMGTKEHS

SEQ ID NO:89 LCDR3 GSGYPF

(Chothia)

SEQ ID NO:90 VL DIQMTQSPSTLSASVGDRVT ITCRAQLSVGYMHWYQQRPGRAPRLLI

YDTDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKR

HICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLN

CTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEI

KTCWNKILMGTKEHSRLASGVPSRFSGSGSGTEFTLTISSLQPDDFA

TYYCFQGSGYPFTFGGGTRLEIR

SEQ ID NO:91 VL DNA GACATTCAGATGACCCAGTCACCCAGCACTCTGAGCGCTTCCGTGGG

AGACAGAGTCACCATTACTTGCAAGGCCCAGCTCTCTGTGGGATATA TGCATTGGTACCAGCAGAAACCGGGAAAAGCACCTAAGCTGCTGATC TACGACACCGATTGTGATATTGAAGGTAAAGATGGCAAACAATATGA GAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAG AAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGA CATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGC TCGCAAGTTGAGGCAATTTCTTAAAATGAATAGCACTGGTGATTTTG ATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATACTGTTGAAC TGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGC CCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGA AAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATA AAAACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACACTCAAA ACTGGCCTCAGGCGTGCCAAGCCGGTTCAGCGGGTCCGGATCTGGTA CTGAGTTTACCCTCACCATCTCATCTCTCCAGCCAGACGATTTCGCT ACCTATTACTGCTTCCAAGGATCCGGGTACCCATTCACTTTTGGCGG

AGGGACTAAGCTGGAAATCAAG

SEQ ID NO: 92 Light DIQMTQSPSTLSASVGDRVT ITCRAQLSVGYMHWYQQRPGRAPRLLI

Chain YDTDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKR

HICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLN

CTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEI

KTCWNKILMGTKEHSRLASGVPSRFSGSGSGTEFTLTISSLQPDDFA

TYYCFQGSGYPFTFGGGTRLEIRRTVAAPSVFIFPPSDEQLRSGTAS WCLLNNFYPREARVQWRVDNALQSGNSQESVTEQDSRDSTYSLSST LTLSRADYERHRVYACEVTHQGLSSPVTRSFNRGEC

IgG.IL7.L3 (DAPA-IL7-CDRL3)

SEQ ID NO: 93 HCDR1 TSGMSVG

(Rabat )

SEQ ID NO:94 HCDR2 DIWWDDRRDYNPSLR

(Rabat )

SEQ ID NO: 95 HCDR3 SMITNWYFDV

(Rabat )

SEQ ID NO: 96 HCDR1 GFSLSTSGM

(Chothia)

SEQ ID NO:97 HCDR2 WWDDR

(Chothia)

SEQ ID NO:98 HCDR3 SMITNWYFDV

(Chothia)

SEQ ID NO:99 VH QVTLRESGPALVRPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGRAL

EWLADIWWDDRRDYNPSLRSRLTISRDTSANQWLRVTNMDPADTAT YYCARSMITNWYFDVWGAGTTVTVSS

SEQ ID NO:100 VH DNA CAGGTGACCCTCCGCGAGAGCGGACCCGCCCTCGTGAAGCCAACCCA

SEQ ID NO:101 Heavy QVTLRESGPALVRPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGRAL

Chain EWLADIWWDDRRDYNPSLRSRLTISRDTSANQWLRVTNMDPADTAT YYCARSMITNWYFDVWGAGTTVTVSSASTRGPSVFPLAPSSRSTSGG

TAALGCLVRDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHRPSNTRVDRRVEPRSCDRTHTCPPCPAP ELLGGPSVFLFPPRPRDTLMISRTPEVTCVWAVSHEDPEVRFNWYV DGVEVHNARTRPREEQYNSTYRWSVLTVLHQDWLNGREYRCRVSNR ALAAPIERTISRARGQPREPQVYTLPPSREEMTRNQVSLTCLVRGFY PSDIAVEWESNGQPENNYRTTPPVLDSDGSFFLYSRLTVDRSRWQQG NVFSCSVMHEALHNHYTQRSLSLSPGR

SEQ ID NO:102 LCDR1 RAQLSVGYMH

(Rabat )

SEQ ID NO:103 LCDR2 DTSRLAS

(Rabat )

SEQ ID NO:104 LCDR3 FQGSGDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFF (Rabat ) KRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTIL

LNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQ

EIKTCWNKILMGTKEHYPFT

SEQ ID NO:105 LCDR1 QLSVGY

(Chothia)

SEQ ID NO:106 LCDR2 DTS

(Chothia)

SEQ ID NO:107 LCDR3 GSGDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKR (Chothia) HICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLN

CTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEI

KTCWNKILMGTKEHYPF

SEQ ID NO:108 VL DIQMTQSPSTLSASVGDRVT ITCRAQLSVGYMHWYQQRPGRAPRLLI

YDTSRLASGVPSRFSGSGSGTEFTLTI SSLQPDDFATYYCFQGSGDC DIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDA

NKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQV

KGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN

KILMGTKEHYPFTFGGGTRLEIR

SEQ ID NO:109 VL DNA GACATTCAGATGACCCAGTCACCCAGCACTCTGAGCGCTTCCGTGGG

AGACAGAGTCACCATTACTTGCAAGGCCCAGCTCTCTGTGGGATATA TGCATTGGTACCAGCAGAAACCGGGAAAAGCACCTAAGCTGCTGATC TACGACACCTCAAAACTGGCCTCAGGCGTGCCAAGCCGGTTCAGCGG GTCCGGATCTGGTACTGAGTTTACCCTCACCATCTCATCTCTCCAGC CAGACGATTTCGCTACCTATTACTGCTTCCAAGGATCCGGGGATTGT GATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGT

CAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATT GCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCT AATAAGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCA ATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAA AAGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTT AAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAG TTTGGAAGAAAATAAATCTTTAAAGGAACAGAAAAAACTGAATGACT TGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAAT AAAATTTTGATGGGCACTAAAGAACACTACCCATTCACTTTTGGCGG AGGGACTAAGCTGGAAATCAAG

SEQ ID NO:110 Light DIQMTQSPSTLSASVGDRVT ITCKAQLSVGYMHWYQQKPGKAPKLLI

Chain YDTSKLASGVPSRFSGSGSGTEFTLTI SSLQPDDFATYYCFQGSGDC

DIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDA

NKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQV

KGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN

KILMGTKEHYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS

WCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Example 2: In vitro activity ofIL7 antibody cytokine engrafted proteins in mouse splenocytes

[00260] Cells were isolated from mouse spleens and single cell suspensions were added to each well. Each IL7 cytokine engrafted protein, recombinant human IL7, or IL7 Fc molecule was added to the wells, and incubated for 30 minutes at 37°C. After 20 minutes, cells were fixed with Cytofix buffer (BD#554655), washed and stained with surface markers. After 30 minutes at room temperature, samples were washed and re-suspended cell pellets were permeabilized with -20°C Perm Buffer III (BD#558050), washed and stained with pSTAT5 Ab (Becton Dickinson #612567). Cells were acquired on LSR Fortessa® and data analyzed with Flow Jo® software. Data was graphed with Prism software.

[00261] IL7 cytokine engrafted proteins were assessed for stimulation on the IL7Ra on mouse splenocytes. All of the IL7 antibody cytokine engrafted proteins displayed increased activation of the IL7Ra pathway on both CD 8 (Figure 1 A) and CD4 (Figure IB) T cells when compared to equimolar amounts of recombinant human IL7 (rec hIL7), as well as human IL- 7 combined to an Fc portion. Thus, IL7 antibody cytokine engrafted proteins were more effective at CD 8 and CD4 activation than the native IL7 cytokine.

Example 3: In vitro activity ofIL7 antibody cytokine engrafted proteins in human PBMCs

[00262] PBMC cells were placed in serum- free test media, and IL7 antibody cytokine engrafted protein or recombinant human IL7 was added to the cells and incubated for 20 minutes at 37°C. After 20 minutes, cells were fixed with 1.6% formaldehyde, washed and stained with surface markers. After 30 minutes at room temperature, samples were washed and re-suspended cell pellets were permeabilized with -20°C methanol, washed and stained with pSTAT5 Ab (BD Biosciences #612567) and DNA intercalators. Cells were run on Cytof and data analyzed with Flow Jo® software.

[00263] All the molecules tested, independent of its format, induced activation of the

IL7Ra pathway on both CD8 and CD4 T cells, but not B cells or NK cells, when compared to wild-type scaffold or unstimulated cells (Figure 2A). In addition, both CD8 (Figure 2B) and CD4 (Figure 2C) T cells were strongly activated, by either recombinant hIL7, IgG.IL7.H2 and IgG.IL7.H3, and independent of the concentration used. Thus, IL7 antibody cytokine engrafted proteins strongly stimulate both CD 8 and CD4 human T cells, without stimulating B cells or NK cells.

Example 4: In vivo activity of IL7 antibody cytokine engrafted proteins in C57BL6 mice

[00264] B6 female mice were administered IL7, IL7-Fc and IL7 cytokine engrafted proteins once a day for 4 days at different concentrations. One day after last treatment (day 5), spleens were processed to obtain a single cell suspension and washed in RPMI (10% FBS). Red blood cells were lysed with Red Blood Cell Lysis Buffer (Sigma #R7757) and cells counted for cell number and viability. FACS staining was performed under standard protocols using FACS buffer (lxPBS + 0.5% BSA + 0.05% sodium azide). Cells were stained with surface antibodies: Rat anti-mouse CD8-BUV737 (BD Biosciences #564297), Rat anti-mouse CD19-PeCF594/TR (BD Biosciences #562291), Rat anti-mouse CD3-PerCP (Biolegend #100218), Rat anti-mouse CD127-e450 (ebioscience #48-1273-82), Rat anti- mouse CD4-BV510 (BD Biosciences #563106), Rat anti-mouse CD44-BV711 (BD

Biosciences #563971), Rat anti-mouse CD62L-APC-Cy7 (BD Biosciences #560514), and subsequently fixed/permeabilized and stained for both Rat anti-mouse Ki-67-e660

(ebioscience #50-5698-82) and FoxP3 according to the Anti-Mouse/Rat FoxP3 FITC Staining Set (ebioscience #71-5775-40). Cells were analyzed on the BD LSR Fortessa® or BD FACS LSR II, and data analyzed with Flow Jo® software. Data was graphed with Prism software.

[00265] From the six different IL7 cytokine engrafted proteins tested, IgG.IL7.H2 and

IgG.IL7.H3 consistently increased CD8 Ki67+ T cells (Figure 3A-B), as well as the frequency of effector memory (CD44hi_gh CD62Li_0W) T cells (Figure 3C-D) after daily IP administration for 4 consecutive days. IgG.IL7.H2 and IgG.IL7.H3 consistently increased CD4+ T cells as well (data not shown). Of note, the molar amount of IL7 antibody cytokine engrafted proteins was 5 times lower than the amount used for Fc fusion IL7 to achieve the same relative expansion of CD8+ and CD4+ T cells. All of the IL7 antibody cytokine engrafted proteins were well tolerated by the mice, and no ill effects were seen.

Example 5: In vivo activity of IL7 antibody cytokine engrafted proteins in a CT26 syngeneic mouse tumor model

[00266] CT26 (ATCC #CRL-2638) cells are an aggressive, undifferentiated human colorectal cancer line and frequently used to test anti-cancer activity of molecules in syngeneic mouse models. CT26 cells were grown in sterile conditions in a 37°C incubator with 5% CO². The cells were cultured in RPMI 1640 media supplemented with 10% FBS. Cells were passaged every 3-4 days. For the day of injection, cells were harvested at passage 11 and re-suspended in HBSS at a concentration of 2.5x 10⁶/ml. Cells were Radii tested for mycoplasma and murine viruses. For each mouse, 0.25 x 10⁶ cells were implanted with subcutaneously injection into the right flank using a 28g needle (100 μΐ injection volume). After implantation, animals were calipered and weighed 3 times per week once tumors were palpable. Caliper measurements were calculated using (LxWxW)/2. Mice were fed with normal diet and housed in a SPF animal facility in accordance with the Guide for Care and Use of Laboratory Animals and regulations of the Institutional Animal Care and Use Committee.

[00267] When tumors reached about 100 mm³, mice were administered 20-100 μg of

IL7- flag, IL7-Fc-fusion, IgG.IL7.H2 and IgG.IL7.H3 intraperitoneally twice per week for a total of 4 doses. Tumors were measured twice a week. Average tumor volumes were plotted using Prism 5® (GraphPad) software. An endpoint for efficacy studies was achieved when tumor size reached a volume of 1000 mm³. Following injection, mice were also closely monitored for signs of clinical deterioration. The mice were monitored for any signs of morbidity, including respiratory distress, hunched posture, decreased activity, hind leg paralysis, tachypnea as a sign for pleural effusions, weight loss approaching 20% or 15% plus other signs, such as their ability to carry on normal activities (feeding, mobility).

[00268] One day after last treatment (day 13), spleens and tumors were collected.

Spleens were processed to obtain a single cell suspension and washed in RPMI (10% FBS). Red blood cells were lysed with Red Blood Cell Lysis Buffer (Sigma #R7757) and cells counted for cell number and viability. FACS staining was performed under standard protocols using FACS buffer (lxPBS + 0.5% BSA + 0.05% sodium azide). Cells were stained with the following surface antibodies: Rat anti-mouse CD8-BUV737 (BD

Biosciences #564297), Rat anti-mouse CD19-PeCF594/TR (BD Biosciences #562291), Rat anti-mouse CD3-PerCP (Biolegend #100218), Rat anti-mouse CD127-e450 (ebioscience #48- 1273-82), Rat anti-mouse CD4-BV510 (BD Biosciences #563106), Rat anti-mouse CD44- BV711 (BD Biosciences #563971), Rat anti-mouse CD62L-APC-Cy7 (BD Biosciences #560514), and subsequently fixed/permeabilized and stained for both Rat anti-mouse Ki-67- e660 (ebioscience #50-5698-82) and FoxP3 according to the Anti-Mouse/Rat FoxP3 FITC Staining Set (ebioscience #71-5775-40). Cells were analyzed on the BD LSR Fortessa® or BD FACS LSR II, and data analyzed with Flow Jo® software. Tumors were fixed in formalin and paraffin embedded for Immunohistochemistry staining against mouse CD8 (eBioscience #14-0808-82) and mouse CD4 (Abeam #abl 83685). The numbers of positive cells were quantified using Matlab software (MathWorks), Data was graphed with Prism software.

[00269] IgG.IL7.H2 and IgG.IL7.H3 were tested in vivo for their efficacy against

CT26 tumors. Administration of IgG.IL7.H2 or IgG.IL7.H3 significantly decreased tumor growth when compared to an IL7-flag protein or an Fc-fusion-IL7 at equimolar doses (Figure 4A). The same trend was observed at lower doses (Figure 4B). Also, the frequency of effector memory (CD44high/CD62Li_0W) CD8+ T cells was significantly increased in mice treated with IgG.IL7.H2 and IgG.IL7.H3 when compared to control groups (Figure 4C), one day after the last of 4 doses. In addition, the frequencies of both CD8+ and CD4+ Tumor Infiltrating Lymphocytes (TILs) were increased at high doses of IgG.IL7.H2 and IgG.IL7.H3 when compared to control groups (Figure 4D and Figure 4E, respectively). Therefore, IgG.IL7.H2 and IgG.IL7.H3 showed enhanced IL7 activity, reduced tumor volume as a single agent, and increased the number of TILs when compared to recombinant IL7.

Example 6: activity ofIL7 cytokine engrafted proteins in an Ex vivo model of exhaustion [00270] B6 female mice were intravenously (iv) infected with 2xl0⁶ PFU of

Lymphocytic Choriomeningitis Virus (LCMV) clone 13. Three weeks after infection, spleens were collected and processed to obtain a single cell suspension. After B cell depletion using the EasySep™ StemCell B cell depletion kit (Stemcell, Cambridge MA) cells were added to wells in RPMI (10% FBS) with a cocktail of three MHC-I and one MHC-II LCMV-specific peptides, with (Media + anti-PD-Ll) or without (Media) anti-PD-Ll antibody. INF gamma was measured after 24 hours by ELISA and data was graphed with Prism software.

[00271] IgG.IL7.H2 increased IFN-gamma production in synergy with anti-PD-Ll antibody. While the addition of recombinant human IL7 did not result in any further increase in IFN-gamma production respect to anti-PD-Ll treatment (DMSO), addition of IgG.IL7.H2 resulted in a significant increase of IFN-gamma (Figure 5). Thus, IL7 antibody cytokine engrafted proteins were able to revert the exhaustion phenotype of CD8+ T cells in an ex vivo model.

Example 7: binding of antibody cytokine engrafted proteins

[00272] Antibody cytokine engrafted proteins were prepared using a variety of known immunoglobulin sequences which have been utilized in clinical settings as well as germline antibody sequences. One of the antibodies used has RSV as its target antigen. To determine if engrafting IL7 into the CDRs of this antibody reduced binding to RSV, an ELISA assay was run on RSV proteins either in PBS or a carbonate buffer. As shown in Figure 7, this appears to be influenced by which CDR was chosen for IL7 engrafting. For example, IL7 engrafted into heavy chain CDR1 (CDR-H1) has RSV binding similar to the ungrafted (unmodified) original antibody. In contrast, engrafting IL7 into heavy chain CDR2 (CDR-H2) and into CDR-H3 reduces binding to RSV. IL7 engrafted into light chain CDR3 (CDR-L3) has almost no RSV binding. As expected, IL2 engrafted into a GFTX antibody scaffold which targets IgE produces no binding. This demonstrates that antibody cytokine engrafted proteins can retain binding to the original target of the antibody scaffold, or this binding can be reduced.

Example 8: In vivo pharmacokinetics of IL7 antibody cytokine engrafted proteins in CD1 mice

[00273] CD1 female mice were administered a single dose of equimolar amounts of

IL7, IL7-Fc and IL7 cytokine engrafted proteins, and IL7 protein was measured by Gyros assay in serum samples at different time points (Figure 8). Data was analysed and graphed with Prism software.

[00274] From the six different IL7 proteins tested, IgG.IL7.H2 showed the best exposure when compared to the other formats (Figure 8). Of note, the amount of recombinant human IL7 was below the Limit of Quantification (LOQ) after just 6 hours, while the same was true for the IL-Fc fusion after 24 hours. All of the IL7 antibody cytokine engrafted proteins were measurable up to 72 hours.

Example 9: activity of IgG.IL7.H2 cytokine engrafted protein in an In vivo model of T cell exhaustion

[00275] B6 female mice were intravenously (iv) infected with 2xl0⁶ PFU of

Lymphocytic Choriomeningitis Virus (LCMV) clone 13. Three weeks after infection, mice were administered with 20C^g of an Isotype control antibody alone, lOC^g of IgG.IL7.H2 alone, 20C^g of anti-PD-Ll alone, or co-dose with lOC^g of IgG.IL7.H2 plus 20C^g of anti- PD-L1 twice a week for 2 weeks. Three days after last dose (day 35), blood, spleen cells and liver were analysed.

[00276] Spleen and blood were processed to obtain a single cell suspension and washed in RPMI (10% FBS). Red blood cells were lysed with Red Blood Cell Lysis Buffer (Sigma #R7757) and cells counted for cell number and viability. FACS staining was performed under standard protocols using FACS buffer (lxPBS + 0.5% BSA + 0.05% sodium azide). Cells were stained with the following surface antibodies and tetramer molecules: Rat anti-mouse CD8-PerCP (BD Biosciences #553036), Rat anti-mouse CD19- APC-Cy7 (BD Biosciences #560143), Rat anti-mouse KLRG1-BV421 (BD Biosciences #560733), Rat anti-mouse CD127-PE-Cy7 (BD Biosciences #560733), Rat anti-mouse CD4- BUV395 (BD Biosciences #563790), Rat anti-mouse CD44-BUV737 (BD Biosciences #564392), Rat anti-mouse CD62L-FITC (Tonbo #35-0621-U100), Rat anti-mouse CD366- APC (Biolegend #119706), Rat anti-mouse CD279-BV605 (Biolegend #135219), T-Select H-2Db LCMV gp33 (C9M) Tetramer-PE (MBL #TS-M512-1) and T-Select H-2Db LCMV gp276-286 Tetramer-BV421 (MBL #TB-5009-4). Cells were analyzed on the BD LSR Fortessa® or BD FACS LSR II, and data analyzed with Flow Jo® software. Data was graphed with Prism® software.

[00277] An increase in virus-specific CD8+ T cells was observed in the blood upon dosing with IgG.IL7.H2 antibody cytokine engrafted protein, independent of the presence of anti-PD-Ll antibody (Figure 9). An increase in total numbers of naive, central memory and effector memory CD8+ T cells was also observed in the blood upon dosing with IgG.IL7.H2 cytokine engrafted protein, but not with anti-PD-Ll alone (Figure 11). Analysis of spleen cells also showed that IgG.IL7.H2 antibody cytokine engrafted protein induces the reduction of another checkpoint molecule Tim-3, either alone or in combination with anti-PD-Ll (Figure 10). In addition, dosing with IgG.IL7.H2 cytokine engrafted protein also induces an increase in CD8+ cells with an intermediate expression of PD-1, known to be the best responder upon PD-1 blocking therapies (Figure 12).

[00278] Addition of the IgG.IL7.H2 cytokine engrafted protein, in combination with anti-PD-Ll, resulted in a significant increase of IFN-gamma (Figure 14). Taken together these data indicate that the IgG.IL7.H2 antibody cytokine engrafted protein reverted the exhaustion phenotype of CD8+ T cells in an in vivo model. Analysis of viral RNA in the liver indicates that administration of IgG.IL7.H2 was able to reduce viral load as a single agent (Figure 13). The combination of IgG.IL7.H2 and an anti-PD-Ll antibody further reduced viral load (Figure 13).

[00279] It is understood that the examples and embodiments described herein are for illustrative purposes and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, sequence accession numbers, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS:

1. An antibody cytokine engrafted protein comprising:

(a) a heavy chain variable region (VH), comprising Complementarity

Determining Regions (CDR) HCDR1, HCDR2, HCDR3; and

(b) a light chain variable region (VL), comprising LCDR1, LCDR2, LCDR3; and

(c) an Interleukin 7 (IL7) molecule engrafted into a CDR of the VH or the VL.

2. The antibody cytokine engrafted protein of claim 1, wherein the IL7 molecule is engrafted into a heavy chain CDR.

3. The antibody cytokine engrafted protein of claim 2, wherein heavy chain CDR is selected from complementarity determining region 1 (HCDR1), complementarity determining region 2 (HCDR2) or complementarity determining region 3 (HCDR3).

4. The antibody cytokine engrafted protein of claim 3, wherein the IL7 molecule engrafted into the HCDR2.

5. The antibody cytokine engrafted protein of claim 3, wherein the IL7 molecule engrafted into the HCDR3.

6. The antibody cytokine engrafted protein of claim 1, wherein the IL7 molecule engrafted into a light chain CDR.

7. The antibody cytokine engrafted protein of claim 6, wherein the light chain CDR is selected from complementarity determining region 1 (LCDR1), complementarity determining region 2 (LCDR2) or complementarity determining region 3 (LCDR3).

8. The antibody cytokine engrafted protein of any one of claims 1-7, wherein the antibody cytokine engrafted protein stimulates CD 8+ cell proliferation greater than recombinant IL7.

9. The antibody cytokine engrafted protein of any one of claims 1-8, wherein the antibody cytokine engrafted protein stimulates CD 4+ cell proliferation greater than recombinant IL7.

10. The antibody cytokine engrafted protein of any one of claims 1-9, wherein the antibody cytokine engrafted protein has a longer half-life than recombinant IL7.

11. The antibody cytokine engrafted protein of any one of claims 1-10, wherein the IL7 molecule consists of SEQ ID NO:2.

12. The antibody cytokine engrafted protein of any one of claims 1-11, further comprising an IgG class antibody heavy chain.

13. The antibody cytokine engrafted protein of claim 12, wherein the IgG class heavy chain is selected from IgGl, IgG2, or IgG4.

14. The antibody cytokine engrafted protein of any one of claims 1-12, wherein the binding specificity of the CDRs to the target protein is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%, by the engrafted IL7 molecule.

15. The antibody cytokine engrafted protein of any one of claims 1-12, wherein the binding specificity of the CDRs to the target protein is retained by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%, in the presence of the engrafted IL7 molecule.

The antibody cytokine engrafted protein of any one of claims 1-15, wherein the binding specificity of the CDRs is to a target distinct from the binding specificity of the IL7 molecule.

17. The antibody cytokine engrafted protein of any one of claims 1-16, wherein the binding specificity of the CDRs is to a non-human antigen.

The antibody cytokine engrafted protein of claim 17, wherein the non-human antigen is a virus.

The antibody cytokine engrafted protein of claim 18, wherein the

respiratory syncytial virus (RSV).

The antibody cytokine engrafted protein of claim 19, wherein the RSV^' selected from RSV subgroup A or RSV subgroup B.

21. The antibody cytokine engrafted protein of any one of claims 1-20, wherein the antibody scaffold portion of the antibody cytokine engrafted protein is humanized or human.

An antibody cytokine engrafted protein comprising:

(iii) a heavy chain variable region that comprises (a) a HCDRl of SEQ ID NO: 6, (b) a HCDR2 of SEQ ID NO: 7, (c) a HCDR3 of SEQ ID NO: 8; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 15, (e) a LCDR2 of SEQ ID NO: 16, and (f) a LCDR3 of SEQ ID NO: 17; (iv) a heavy chain variable region that comprises (a) a HCDRl of SEQ ID NO: 60, (b) a HCDR2 of SEQ ID NO: 61, (c) a HCDR3 of SEQ ID NO: 62; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 69, (e) a LCDR2 of SEQ ID NO:70, and (f) a LCDR3 of SEQ ID NO:71 ;

(v) a heavy chain variable region that comprises (a) a HCDRl of SEQ ID NO: 78, (b) a HCDR2 of SEQ ID NO:79, (c) a HCDR3 of SEQ ID NO:80; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 87, (e) a LCDR2 of SEQ ID NO: 88, and (f) a LCDR3 of SEQ ID NO: 89; or

An antibody cytokine engrafted protein comprising:

(i) a heavy chain variable region (VH) that comprises SEQ ID NO: 27, and a light chain variable region (VL) that comprises SEQ ID NO: 36;

(ii) a heavy chain variable region (VH) that comprises SEQ ID NO:45, and a light chain variable region (VL) that comprises SEQ ID NO:54;

24. The antibody cytokine engrafted protein of any one of claims 1-23, further comprising a modified Fc region corresponding with reduced effector function.

25. The antibody cytokine engrafted protein of claim 24, wherein the modified Fc region comprises a mutation selected from one or more of D265A, P329A, P329G, N297A, L234A, and L235A.

26. The antibody cytokine engrafted protein of claim 25, wherein the modified Fc region comprises a combination of mutations selected from one or more of D265A/P329A, D265A/N297A, L234/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

27. An antibody cytokine engrafted protein comprising a HCDR1 of SEQ ID NO:

24, a HCDR2 of SEQ ID NO: 25, a HCDR3 of SEQ ID NO: 26, a LCDR1 of SEQ ID NO: 33, a LCDR2 of SEQ ID NO: 34, a LCDR3 of SEQ ID NO: 35, a modified Fc region containing the mutation D265A/P329A, and wherein the antibody cytokine engrafted protein induces the proliferation of CD8+ T cells.

28. An antibody cytokine engrafted protein comprising a HCDR1 of SEQ ID NO:

42, a HCDR2 of SEQ ID NO: 43, a HCDR3 of SEQ ID NO: 44, a LCDR1 of SEQ ID NO: 51, a LCDR2 of SEQ ID NO: 52, a LCDR3 of SEQ ID NO: 53, a modified Fc region containing the mutation D265A/P329A, and wherein the antibody cytokine engrafted protein induces the proliferation of CD8+ T cells.

29. An isolated nucleic acid encoding an antibody cytokine engrafted protein comprising:

(i) a heavy chain variable region of SEQ ID NO: 28 and/or a light chain variable region of SEQ ID NO:37;

(vi) a heavy chain variable region of SEQ ID NO: 64 and/or a light chain variable region of SEQ ID NO: 73; (v) a heavy chain variable region of SEQ ID NO: 82 and/or a light chain variable region of SEQ ID NO: 91 ; or

30. A recombinant host cell suitable for the production of an antibody cytokine engrafted protein, comprising the nucleic acid of claim 29 encoding the heavy and light chain polypeptides of the antibody cytokine engrafted protein, and optionally, secretion signals.

31. The recombinant host cell of claim 30, which is a mammalian cell line.

32. The recombinant host cell of claim 31, wherein the mammalian cell line is a CHO cell line.

33. A pharmaceutical composition comprising the antibody cytokine engrafted protein of any one of claims 1 to 28 and a pharmaceutically acceptable carrier.

34. A method of treating cancer in an individual in need thereof, comprising

administering to the individual a therapeutically effective amount of the antibody cytokine engrafted protein of any one of claims 1 to 28 or the pharmaceutical composition of claim 33.

35. The method of claim 34, wherein the cancer is selected from the group

consisting of: melanoma, lung cancer, colorectal cancer, prostate cancer, breast cancer, leukemia and lymphoma.

36. The method of claim 34 or 35, wherein the antibody cytokine engrafted

protein or the pharmaceutical composition is administered in combination with another therapeutic agent.

37. The method of claim 36, wherein the therapeutic agent is an immune

checkpoint inhibitor.

38. The method of claim 37, wherein the immune checkpoint is selected from the group consisting of: PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3,

CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR.

39. The method of claim 38, wherein the immune checkpoint inhibitor is an anti- PD-L1 antibody.

40. The method of claim 38, wherein the immune checkpoint inhibitor is an anti- TIM3 antibody.

41. A method of expanding CD8+ T cells in a patient in need thereof, comprising administering to the individual a therapeutically effective amount of the antibody cytokine engrafted protein of any one of claims 1 to 28 or the pharmaceutical composition of claim 33.

42. The method of claim 41, further comprising administration of an immune checkpoint inhibitor.

43. The method of claim 42, wherein the immune checkpoint is selected from the group consisting of: PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3,

CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR.

44. The method of claim 43, wherein the immune checkpoint inhibitor is an anti- PD-L1 antibody.

45. The method of claim 43, wherein the immune checkpoint inhibitor is an anti- TIM3 antibody.

46. A method of reverting the exhaustion of CD8+ T cells in a patient in need thereof, comprising administering to the individual a therapeutically effective amount of the antibody cytokine engrafted protein of any one of claims 1 to 28 or the pharmaceutical composition of claim 33.

47. The method of claim 46, further comprising administration of an immune checkpoint inhibitor.

48. The method of claim 47, wherein the immune checkpoint is selected from the group consisting of: PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIRl, CD160, 2B4 and TGFR.

49. The method of claim 48, wherein the immune checkpoint inhibitor is an anti- PD-L1 antibody.

50. The method of claim 48, wherein the immune checkpoint inhibitor is an anti- TIM3 antibody.

51. A method of treating a viral infection in a patient in need thereof, comprising administering to the individual a therapeutically effective amount of the antibody cytokine engrafted protein of any one of claims 1 to 28 or the pharmaceutical composition of claim 33.

52. The method of claim 51, further comprising administration of an immune checkpoint inhibitor.

53. The method of claim 52, wherein the immune checkpoint is selected from the group consisting of: PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIRl, CD160, 2B4 and TGFR.

54. The method of claim 53, wherein the immune checkpoint inhibitor is an anti- PD-L1 antibody.

55. The method of claim 53, wherein the immune checkpoint inhibitor is an anti- TIM3 antibody.

56. A use of an antibody cytokine engrafted protein comprising:

(i) a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 24, (b) a HCDR2 of SEQ ID NO: 25, (c) a HCDR3 of SEQ ID NO: 26 and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 33, (e) a LCDR2 of SEQ ID NO: 34, and (f) a LCDR3 of SEQ ID NO: 35; (ii) a heavy chain variable region that comprises (a) a HCDRl of SEQ ID NO: 42, (b) a HCDR2 of SEQ ID NO: 43, (c) a HCDR3 of SEQ ID NO: 44; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 51, (e) a LCDR2 of SEQ ID NO:52, and (f) a LCDR3 of SEQ ID NO:53;

(vi) a heavy chain variable region that comprises (a) a HCDRl of SEQ ID NO: 96, (b) a HCDR2 of SEQ ID NO: 97, (c) a HCDR3 of SEQ ID NO: 98; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 105, (e) a LCDR2 of SEQ ID NO: 106, and (f) a LCDR3 of SEQ ID NO: 107, in the treatment of cancer.

57. The use of claim 56, wherein the antibody cytokine engrafted protein is

administered in combination with another therapeutic agent.

58. The use of claim 57, wherein the therapeutic agent is an immune checkpoint inhibitor.

59. The use of claim 58, wherein the immune checkpoint is selected from the group consisting of: PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3,

CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR. The use of claim 59, wherein the immune checkpoint inhibitor is an anti-PD- Ll antibody.

The use of claim 59, wherein the immune checkpoint inhibitor is an anti-TIM3 antibody.