Humanized chimeric bovine antibodies and methods of use

Abstract

Chimeric antibodies containing ultralong CDR3, for example based on bovine antibody sequences or humanized sequences thereof, and related antibodies are provided, in which a portion of the CDR3 of the heavy chain is replaced by a heterologous sequence, for example, a sequence of interleukin (IL)-15 or IL-2. Molecules of the present disclosure include chimeric IL-15 modified antibody molecules that are further linked or complexed with an extracellular portion of IL15Rα, such as the IL15Rα sushi domain. The present disclosure also provides methods of making and using the chimeric antibodies. TIFF2024517759000027.tif98156

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 181,223, filed April 28, 2021, the contents of which are incorporated by reference in their entirety for all purposes.

[0002] INCORPORATION BY REFERENCE TO SEQUENCE LISTING This application is filed with an electronic sequence listing, which is provided as a file named 166262000340SeqList.txt, created on April 23, 2022, and is 152 kilobytes in size. The information in the electronic format of the sequence listing is incorporated by reference in its entirety.

[0003] Field The present disclosure relates to chimeric antibodies containing ultralong CDR3, such as based on bovine antibody sequences or humanized sequences thereof, in which a portion of the CDR3 of the heavy chain is replaced by a heterologous sequence, such as an interleukin (IL)-15 or IL-2 sequence, and related antibodies. Among the molecules of the present disclosure are chimeric IL-15 modified antibody molecules that are further linked or complexed with an extracellular portion of IL15Rα, such as the IL15Rα sushi domain. The present disclosure also provides methods of making and using the chimeric antibodies. [Background technology]

[0004] background Antibodies are natural proteins that vertebrate immune systems form in response to foreign substances (antigens), primarily to protect against infection. Antibodies contain complementarity determining regions (CDRs) that mediate binding to target antigens. Some bovine antibodies have unusually long variable heavy (VH) CDR3 sequences compared to other vertebrates. These long CDR3s, which can be up to 70 amino acids long, can form unique domains that protrude from the antibody surface, thereby enabling unique antibody platforms.

[0005] Interleukin (IL)-15 and IL-2 are cytokines that stimulate the proliferation and cytotoxicity of cytotoxic T lymphocytes and natural killer (NK) cells, and are therefore immunotherapeutic candidates for the treatment of cancer.However, such cytokines can be difficult to express as stable soluble proteins, and often have short half-lives in vitro and in vivo.There is still a need for improved cytokine therapeutics, such as IL-2 or IL-15 therapeutics, particularly for use in the treatment of cancer. Summary of the Invention

[0006] overview Provided herein in some aspects is a chimeric modified antibody comprising a heavy chain comprising: (a) a modified variable heavy (VH) region of a bovine antibody or antigen-binding fragment, or a humanized sequence thereof, wherein the modified VH region comprises a modified ultralong CDR3, and at least a portion of the ultralong CDR3 of the bovine antibody or antigen-binding fragment, or a humanized sequence thereof, is replaced by a cytokine sequence, or a biologically active portion thereof; and (b) a modified human IgG heavy chain constant region having reduced effector activity compared to a wild-type human IgG heavy chain constant region.

[0007] In some of the optional embodiments, the cytokine sequence or biologically active portion thereof replaces the knob region of the ultralong CDR3 region of a bovine antibody or antigen-binding fragment or humanized sequence thereof.

[0008] In some of the embodiments, the cytokine sequence or biologically active portion thereof is between the ascending stalk strand and the descending stalk strand of the modified ultralong CDR3, and the ascending stalk strand of the modified ultralong CDR3 is a variant compared to the ascending stalk strand of the ultralong CDR3 of the bovine antibody or antigen-binding fragment or humanized sequence thereof. In some of the embodiments, the cytokine sequence or biologically active portion thereof is linked to the ascending stalk strand and / or the descending stalk strand of the modified ultralong CDR3 via a flexible linker, optionally a GGS or GSG linker. In some of the embodiments, the cytokine sequence or biologically active portion thereof is linked to the ascending stalk strand and the descending stalk strand of the modified ultralong CDR3 via a flexible linker. In some of the embodiments, the flexible linker is a GGS linker. In some of the embodiments, the linker is a GSG linker. In some of the optional embodiments, the cytokine sequence or biologically active portion thereof is linked to the ascending stalk strand of the modified ultralong CDR3 via a GGS linker and to the descending stalk strand of the modified ultralong CDR3 via a GSG linker.

[0009] In some of the optional embodiments, the ascending stalk strand comprises the sequence CX2TVX5QETKKYQT, where X2 and X5 are any amino acid.

[0010] Provided herein in some aspects is a chimeric modified antibody comprising a heavy chain comprising a modified variable heavy (VH) region of a bovine antibody or antigen-binding fragment or a humanized sequence thereof, wherein the modified VH region comprises a modified ultralong CDR3 in which at least a portion of the ultralong CDR3 region of the bovine antibody or antigen-binding fragment or a humanized sequence thereof is replaced with a heterologous sequence, the heterologous sequence is between the ascending stalk strand and the descending stalk strand of the modified ultralong CDR3, and the ascending stalk strand of the modified ultralong CDR3 comprises the sequence CX2TVX5QETKKYQT, where X2 and X5 are any amino acids.

[0011] In some of the embodiments, X2 is Ser, Thr, Gly, Asn, Ala, or Pro, and X5 is His, Gln, Arg, Lys, Gly, Thr, Tyr, Phe, Trp, Met, Ile, Val, or Leu. In some of the embodiments, X2 is Ser, Ala, or Thr, and X5 is His or Tyr.

[0012] In some of the embodiments, the ascending stalk strand of the modified very long CDR3 comprises a sequence as set forth in any of SEQ ID NOs: 183-185. In some of the embodiments, the sequence of the ascending stalk strand of the modified very long CDR3 is set forth in any of SEQ ID NOs: 183-185.

[0013] In some of the embodiments, the ascending stalk strand of the modified ultralong CDR3 comprises the sequence shown in SEQ ID NO: 185. In some of the embodiments, the sequence of the ascending stalk strand of the modified ultralong CDR3 is shown in SEQ ID NO: 185.

[0014] In some of the embodiments, the heterologous sequence replaces the knob region of the ultralong CDR3 region of a bovine antibody or antigen-binding fragment or a humanized sequence thereof. In some of the embodiments, the heterologous sequence is linked to the ascending and / or descending stalk strands of the modified ultralong CDR3 via a flexible linker, optionally a GGS or GSG linker. In some of the embodiments, the flexible linker is a GGS linker. In some of the embodiments, the linker is a GSG linker. In some of the embodiments, the heterologous sequence is linked to the ascending and descending stalk strands of the modified ultralong CDR3 via a flexible linker. In some of the embodiments, the flexible linker is a GGS linker. In some of the embodiments, the linker is a GSG linker. In some of the optional embodiments, the heterologous sequence is linked to the ascending stalk strand of the modified very long CDR3 via a GGS linker and to the descending stalk strand of the modified very long CDR3 via a GSG linker.

[0015] In some of the optional embodiments, the heterologous sequence comprises a cytokine sequence or a biologically active portion thereof.

[0016] In some of the embodiments, the heavy chain further comprises a human IgG heavy chain constant region. In some of the embodiments, the human IgG heavy chain constant region is a modified human IgG heavy chain constant region having reduced effector activity compared to a wild-type human IgG heavy chain constant region.

[0017] In some of the optional embodiments, the human IgG is human IgG1.

[0018] In some of the embodiments, the modified human IgG heavy chain constant region is modified to reduce FcR binding. In some of the embodiments, the reduced effector activity comprises reduced antibody-dependent cell-mediated cytotoxicity (ADCC).

[0019] In some of any of the embodiments, the modified human IgG heavy chain constant region is altered at one or more of positions Glu233 (E233), Leu234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Asn297 (N297), Ser298 (S298), Asn325 (N325), Ala327 (A327) and Pro329 (P329). In some of any of the embodiments, the modified human IgG heavy chain constant region comprises one or more mutations selected from Leu234Ala (L234A), Leu235Ala (L235A), Leu235Glu (L235E), Asp265Asn (D265N), Asp265Ala (D265A), Asp270Asn (D270N), Ser298Asn (S298N), Asn325Glu (N325E), Ala327Ser (A327S), Pro329Ala (P329A), and Pro239Gly (P329G).

[0020] In some of any of the embodiments, the modified human IgG heavy chain constant region is altered at two or more of positions Glu233 (E233), Leu234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Asn297 (N297), Ser298 (S298), Asn325 (N325), Ala327 (A327), and Pro329 (P329). In some of the optional embodiments, the modified human IgG heavy chain constant region comprises Leu234Ala and Leu235Ala (L234A / L235A) mutations; Leu234Val and Leu235Ala (L234V / L235A) mutations; Leu234Ala, Leu235Ala and Asn297Ala (L234A / L235A / N297A) mutations; Leu234Ala, Leu235Ala and Pro239Ala (L234A / L235A / P329A) mutations; Asp265Ala and Pro329Ala (D329A) mutations; 265A / P329A) mutation; Asp265Ala and Pro329Gly (D265A / P329G) mutation; Leu234Ala, Leu235Ala and Asp265Ala (L234A / L235A / D265A) mutation; Leu234Ala, Leu235Ala and Pro329Gly (L234A / L235A / P329G) mutation; or Leu234Ala, Leu235Ala, Asp265Ala and Pro329Gly (L234A / L235A / D265A / P329G) mutation.

[0021] In some of the optional embodiments, the modified human IgG heavy chain constant region comprises Leu234Ala and Leu235Ala (L234A / L235A) mutations.

[0022] In some of the embodiments, the modified human IgG heavy chain constant region comprises the sequence shown in SEQ ID NO: 187 or SEQ ID NO: 188. In some of the embodiments, the modified human IgG heavy chain constant region comprises the sequence shown in SEQ ID NO: 187. In some of the embodiments, the modified human IgG heavy chain constant region comprises the sequence shown in SEQ ID NO: 188.

[0023] In some of the embodiments, the cytokine sequence or biologically active portion thereof comprises an interleukin-15 (IL-15) cytokine sequence or biologically active portion thereof. In some of the embodiments, the cytokine sequence or biologically active portion thereof comprises a sequence of amino acids that exhibits at least or at least about 85%, at least or at least about 90%, at least or at least about 90%, at least or at least about 92%, at least or at least about 92%, at least or at least about 95%, at least or at least about 96%, at least or at least about 96%, at least or at least about 97%, at least or at least about 97%, at least or at least about 98%, or at least or at least about 99% sequence identity to SEQ ID NO:1. In some of the embodiments, the cytokine sequence or biologically active portion thereof comprises a sequence set forth in SEQ ID NO:1.

[0024] In some of the embodiments, the cytokine sequence or biologically active portion thereof comprises an interleukin-12 (IL-2) cytokine sequence or biologically active portion thereof. In some of the embodiments, the cytokine sequence or biologically active portion thereof comprises a sequence of amino acids that exhibits at least or at least about 85%, at least or at least about 90%, at least or at least about 90%, at least or at least about 92%, at least or at least about 92%, at least or at least about 95%, at least or at least about 95%, at least or at least about 96%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, or at least or at least about 99% sequence identity to SEQ ID NO:165. In some of the embodiments, the cytokine sequence or biologically active portion thereof comprises a sequence of amino acids set forth in SEQ ID NO:165.

[0025] In some of any of the embodiments, the bovine antibody or antigen-binding fragment is the bovine antibody BLV1H12 or an antigen-binding fragment thereof.

[0026] In some optional embodiments, the descending stalk strand comprises the sequence shown in SEQ ID NO:10.

[0027] In some of the optional embodiments, the ascending stalk strand comprises the sequence set forth in SEQ ID NO:183, the cytokine sequence or biologically active portion thereof comprises the sequence of amino acids set forth in SEQ ID NO:1, and the descending stalk strand comprises the sequence set forth in SEQ ID NO:10; the ascending stalk strand comprises the sequence set forth in SEQ ID NO:184, the cytokine sequence or biologically active portion thereof comprises the sequence of amino acids set forth in SEQ ID NO:1, and the descending stalk strand comprises the sequence set forth in SEQ ID NO:10; or the ascending stalk strand comprises the sequence set forth in SEQ ID NO:185, the cytokine sequence or biologically active portion thereof comprises the sequence of amino acids set forth in SEQ ID NO:1, and the descending stalk strand comprises the sequence set forth in SEQ ID NO:10.

[0028] In some optional embodiments, the ascending stalk strand comprises the sequence set forth in SEQ ID NO:183, the cytokine sequence or biologically active portion thereof comprises the sequence of amino acids set forth in SEQ ID NO:1, and the descending stalk strand comprises the sequence set forth in SEQ ID NO:10.

[0029] In some optional embodiments, the ascending stalk strand comprises the sequence set forth in SEQ ID NO:184, the cytokine sequence or biologically active portion thereof comprises the sequence of amino acids set forth in SEQ ID NO:1, and the descending stalk strand comprises the sequence set forth in SEQ ID NO:10.

[0030] In some optional embodiments, the ascending stalk strand comprises the sequence set forth in SEQ ID NO:185, the cytokine sequence or biologically active portion thereof comprises the sequence of amino acids set forth in SEQ ID NO:1, and the descending stalk strand comprises the sequence set forth in SEQ ID NO:10.

[0031] In some of the embodiments, the modified ultralong CDR3 comprises a sequence set forth in any of SEQ ID NOs: 206-208. In some of the embodiments, the modified ultralong CDR3 comprises a sequence set forth in SEQ ID NO: 208.

[0032] In some of the optional embodiments, the modified VH region is a mutant of the VH region of BLV1H12.

[0033] In some of the embodiments, the heavy chain comprises the formula V1-X-V2-C, where the V1 region of the heavy chain comprises the sequence set forth in SEQ ID NO:182; the X region comprises a modified ultralong CDR3; the V2 region comprises the sequence set forth in SEQ ID NO:11; and the C region comprises a human IgG heavy chain constant region. In some of the embodiments, the human IgG heavy chain constant region is any of those described herein. In some of the embodiments, the human IgG heavy chain constant region is a modified human IgG heavy chain constant region having reduced effector activity compared to a wild-type human IgG heavy chain constant region. In some of the embodiments, the modified human IgG heavy chain constant region is any of those described herein.

[0034] In some of the optional embodiments, the heavy chain comprises the formula V1-X-V2-C, where the V1 region of the heavy chain comprises the sequence set forth in SEQ ID NO:182; the X region comprises a modified ultralong CDR3; the V2 region comprises the sequence set forth in SEQ ID NO:11; and the C region comprises a modified human IgG heavy chain constant region.

[0035] In some of the embodiments, the modified VH region comprises a sequence set forth in any of SEQ ID NOs:200-202. In some of the embodiments, the modified VH region comprises a sequence set forth in SEQ ID NO:202.

[0036] In some of the embodiments, the heavy chain comprises a sequence set forth in any of SEQ ID NOs: 189-191. In some of the embodiments, the heavy chain comprises a sequence set forth in SEQ ID NO:191.

[0037] In some of the optional embodiments, the modified VH region is a variant of the humanized sequence of the VH region of BLV1H12.

[0038] In some of the embodiments, the heavy chain comprises the formula V1-X-V2-C, where the V1 region of the heavy chain comprises a sequence set forth in SEQ ID NO:197 or a sequence exhibiting at least 65% sequence identity to SEQ ID NO:197; the X region comprises a modified ultralong CDR3; the V2 region comprises a sequence set forth in SEQ ID NO:11; and the C region comprises a human IgG heavy chain constant region. In some of the embodiments, the human IgG heavy chain constant region is any described herein. In some of the embodiments, the human IgG heavy chain constant region is a modified human IgG heavy chain constant region having reduced effector activity compared to a wild-type human IgG heavy chain constant region. In some of the embodiments, the modified human IgG heavy chain constant region is any described herein. In some of the embodiments, the V1 region has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO:197.

[0039] In some of the optional embodiments, the heavy chain comprises the formula V1-X-V2-C, where the V1 region of the heavy chain comprises the sequence set forth in SEQ ID NO:197, or a sequence exhibiting at least 65% sequence identity to SEQ ID NO:197; the X region comprises a modified ultralong CDR3; the V2 region comprises the sequence set forth in SEQ ID NO:11; and the C region comprises a modified human IgG heavy chain constant region. In some embodiments, the V1 region has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO:197.

[0040] In some of the embodiments, the heavy chain comprises the formula V1-X-V2-C, where the V1 region of the heavy chain comprises the sequence set forth in SEQ ID NO:197; the X region comprises a modified ultralong CDR3; the V2 region comprises the sequence set forth in SEQ ID NO:11; and the C region comprises a human IgG heavy chain constant region. In some of the embodiments, the human IgG heavy chain constant region is any of those described herein. In some of the embodiments, the human IgG heavy chain constant region is a modified human IgG heavy chain constant region having reduced effector activity compared to a wild-type human IgG heavy chain constant region. In some of the embodiments, the modified human IgG heavy chain constant region is any of those described herein.

[0041] In some of the optional embodiments, the heavy chain comprises the formula V1-X-V2-C, where the V1 region of the heavy chain comprises the sequence set forth in SEQ ID NO:197; the X region comprises a modified ultralong CDR3; the V2 region comprises the sequence set forth in SEQ ID NO:11; and the C region comprises a modified human IgG heavy chain constant region.

[0042] In some of the embodiments, the modified VH region comprises a sequence set forth in any of SEQ ID NOs:203-205. In some of the embodiments, the modified VH region comprises a sequence set forth in SEQ ID NO:205.

[0043] In some of the embodiments, the heavy chain comprises a sequence set forth in any of SEQ ID NOs: 192-194. In some of the embodiments, the heavy chain comprises a sequence set forth in SEQ ID NO: 205.

[0044] In some of the embodiments, the chimeric modified antibody further comprises a light chain. In some of the embodiments, the chimeric modified antibody comprises a humanized light chain. In some of the embodiments, the humanized light chain comprises a sequence set forth in SEQ ID NO:181, or a sequence that exhibits at least 85% sequence identity to SEQ ID NO:181. In some of the embodiments, the humanized light chain comprises a sequence set forth in SEQ ID NO:181. In some of the embodiments, the humanized light chain has at least at least 90%, at least 95%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO:181.

[0045] In some of the optional embodiments, the antibody is a full-length or intact antibody.

[0046] In some of any of the embodiments herein, the antibody is an antigen-binding fragment. In further embodiments, the antigen-binding fragment is a Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragment. In some embodiments, the antibody is a Fab.

[0047] In some of the embodiments, the chimeric engineered antibody is complexed with an extracellular domain of IL15Rα comprising an IL15Rα sushi domain. In some of the embodiments, the extracellular domain of IL15Rα comprising an IL15Rα sushi domain is non-covalently associated with an IL-15 sequence. In some of the embodiments, the extracellular domain of IL15Rα comprising an IL15Rα sushi domain is linked to a light chain of the chimeric engineered antibody, optionally linked via a peptide linker. In some of the embodiments, the extracellular domain of IL15Rα comprising an IL15Rα sushi domain is linked to a light chain of the chimeric engineered antibody via a peptide linker. In some of the embodiments, the extracellular domain of IL15Rα comprising the IL15Rα sushi domain comprises the sequence shown in SEQ ID NO:2.

[0048] Provided herein in some aspects is a polynucleotide encoding the chimeric engineered antibody of any of the aspects.

[0049] Provided herein in some aspects is a polynucleotide encoding the heavy chain or variable region thereof of the chimeric modified antibody of any of the aspects.

[0050] Provided herein in some aspects is a polynucleotide encoding the light chain or variable region thereof of the chimeric modified antibody of any of the aspects.

[0051] Provided herein, in some aspects, is an expression vector comprising a polynucleotide of any of the aspects.

[0052] Provided herein in some aspects is a host cell comprising the polynucleotide or expression vector of any aspect.

[0053] In some of the embodiments, the host cell further comprises a polynucleotide or vector encoding an extracellular domain of IL15Rα comprising an IL15Rα sushi domain, In some of the embodiments, the extracellular domain of IL15Rα comprising the IL15Rα sushi domain comprises the sequence shown in SEQ ID NO:2.

[0054] Provided herein in some aspects are methods for producing a chimeric modified antibody comprising culturing a host cell of any aspect under conditions for expression of the chimeric modified antibody by the host cell, and optionally further comprising recovering or purifying the chimeric modified antibody. In some aspects, the conditions are for expression by the host cell of the chimeric modified antibody, the heavy chain or variable region thereof of the chimeric modified antibody, or the light chain or variable region thereof of the chimeric modified antibody.

[0055] In some of the optional aspects, the method further comprises recovering or purifying the chimeric modified antibody, the heavy chain or variable region thereof, or the light chain or variable region thereof.

[0056] In some optional embodiments, the method further comprises recovering or purifying the chimeric modified antibody.

[0057] Provided herein in some aspects is a chimeric engineered antibody produced by the method of any of the aspects.

[0058] Provided herein in some aspects is a chimeric engineered antibody comprising the heavy chain or a variable region thereof, or the light chain or a variable region thereof, of a chimeric engineered antibody produced by the method of any aspect.

[0059] Provided herein in some aspects is a pharmaceutical composition comprising a chimeric engineered antibody of any aspect.

[0060] Provided herein, in some aspects, is a method of stimulating immune cells comprising contacting a population of immune cells with a chimeric engineered antibody of any aspect, thereby stimulating cells of the population of immune cells.

[0061] Provided herein, in some aspects, is a method of expanding immune cells comprising contacting a population of immune cells with a chimeric engineered antibody of any aspect, thereby promoting cell proliferation of the population of immune cells.

[0062] In some of the embodiments, the population of immune cells comprises cells expressing IL2 / 15Rβ and / or IL2 / 15Rβ γc receptor subunits. In some of the embodiments, the population of immune cells comprises cells expressing IL2 / 15Rβ and IL2 / 15Rβ γc receptor subunits.

[0063] In some of the embodiments, the population of immune cells comprises T cells or natural killer (NK) cells. In some of the embodiments, the population of immune cells comprises T cells. In some of the embodiments, the population of immune cells comprises NK cells.

[0064] In some of the embodiments, the method is performed ex vivo or in vitro. In some of the embodiments, the method is performed in vivo upon administration of the chimeric engineered antibody to a subject.

[0065] Provided herein, in some aspects, is a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a chimeric engineered antibody of any aspect.

[0066] Provided herein, in some aspects, is a method of treating cancer in a subject, comprising administering to the subject a pharmaceutical composition of any of the aspects.

[0067] In some of the embodiments, the method further comprises administering an anti-tumor agent to the subject. In some of the embodiments, the anti-tumor agent comprises a monoclonal antibody. In some of the embodiments, the anti-tumor agent comprises a checkpoint inhibitor. In some of the embodiments, the anti-tumor agent comprises a cell therapy, optionally comprising T cell therapy or NK cell therapy. In some of the embodiments, the cell therapy is T cell therapy. In some of the embodiments, the cell therapy is NK cell therapy. In some of the embodiments, the cell therapy comprises cells expressing a chimeric antigen receptor (CAR).

[0068] In some of the embodiments, the anti-tumor agent is an agent for treating cancer. In some of the embodiments, the anti-tumor agent is directed against an antigen associated with cancer.

[0069] In some of the optional embodiments, the cell therapy comprises cells expressing IL2 / 15Rβ and the IL2 / 15Rβ γc receptor subunit.

[0070] Provided herein, in some aspects, is the use of any of the provided chimeric engineered antibodies in the manufacture of a medicament for treating cancer in a subject.

[0071] Provided herein, in some aspects, is the use of a pharmaceutical composition comprising any of the provided chimeric engineered antibodies in a method of treating cancer in a subject.

[0072] In some of the optional embodiments, an anti-tumor agent is administered to the subject in combination with a pharmaceutical composition.

[0073] Provided herein, in some aspects, is the use of a pharmaceutical composition comprising an anti-tumor agent and any of the provided chimeric engineered antibodies in a method of treating cancer in a subject.

[0074] Provided herein in some aspects is the use of an anti-tumor agent in a method of treating cancer in a subject, wherein the anti-tumor agent is administered in combination with a pharmaceutical composition comprising any of the provided chimeric engineered antibodies.

[0075] In some optional embodiments, the method includes administering a pharmaceutical composition to a subject.

[0076] In some optional embodiments, the method includes administering an anti-tumor agent to the subject.

[0077] In some optional embodiments, the pharmaceutical composition and the anti-tumor agent are administered separately to the subject.

[0078] In some of the optional aspects, the method is any of those described herein.

[0079] Provided herein in some aspects is the use of any of the provided chimeric engineered antibodies in combination with an anti-tumor agent in the manufacture of a medicament for treating cancer in a subject.

[0080] Provided herein in some aspects is the use of an anti-tumor agent in the manufacture of a medicament for treating cancer in a subject, wherein the anti-tumor agent is administered in combination with a pharmaceutical composition comprising any of the provided chimeric engineered antibodies.

[0081] In some of the embodiments, the anti-tumor agent is an agent for treating cancer. In some of the embodiments, the anti-tumor agent is directed against an antigen associated with cancer.

[0082] In some of the embodiments, the anti-tumor agent comprises a monoclonal antibody. In some of the embodiments, the anti-tumor agent comprises a checkpoint inhibitor. In some of the embodiments, the anti-tumor agent comprises a cell therapy. In some of the embodiments, the cell therapy comprises cells expressing IL2 / 15Rβ and IL2 / 15Rβ γc receptor subunits. In some of the embodiments, the cell therapy is T cell therapy. In some of the embodiments, the cell therapy is NK cell therapy. In some of the embodiments, the cell therapy comprises cells expressing a chimeric antigen receptor (CAR).

[0083] Provided herein, in some aspects, is a pharmaceutical composition comprising any of the provided chimeric engineered antibodies for use in a method of treating cancer in a subject.

[0084] In some of the optional embodiments, an anti-tumor agent is administered to the subject in combination with a pharmaceutical composition.

[0085] Provided herein, in some aspects, is a combination therapy comprising a pharmaceutical composition comprising any of the provided chimeric engineered antibodies and an anti-tumor agent for use in a method of treating cancer in a subject.

[0086] Provided herein, in some aspects, are anti-tumor agents for use in methods of treating cancer in a subject, wherein the anti-tumor agent is administered in combination with any of the provided chimeric engineered antibodies.

[0087] In some optional embodiments, the method includes administering a pharmaceutical composition to a subject.

[0088] In some optional embodiments, the method includes administering an anti-tumor agent to the subject.

[0089] In some optional embodiments, the pharmaceutical composition and the anti-tumor agent are administered separately to the subject.

[0090] In some of the optional aspects, the method is any of those described herein.

[0091] In some of the embodiments, the anti-tumor agent is an agent for treating cancer. In some of the embodiments, the anti-tumor agent is directed against an antigen associated with cancer.

[0092] In some of the embodiments, the anti-tumor agent comprises a monoclonal antibody. In some of the embodiments, the anti-tumor agent comprises a checkpoint inhibitor. In some of the embodiments, the anti-tumor agent comprises a cell therapy. In some of the embodiments, the cell therapy comprises cells expressing IL2 / 15Rβ and IL2 / 15Rβ γc receptor subunits. In some of the embodiments, the cell therapy is T cell therapy. In some of the embodiments, the cell therapy is NK cell therapy. In some of the embodiments, the cell therapy comprises cells expressing a chimeric antigen receptor (CAR). [Brief description of the drawings]

[0093] [Figure 1A] Figures 1A and 1B show schematics of the fusion antibody constructs that were generated: Figure 1A shows the crystal structure of BLV1H12 (left) showing how two β-strand stalks protrude from the bovine VH immunoglobulin domain and terminate in an unusual three disulfide-linked knob domain, and the crystal structure of the BLV1H12-IL-15 Rαsushi (B15_Rαsushi) mutant (right) in which the knob region is replaced with an IL-15 cytokine sequence and the construct further contains an IL15Rα sushi domain. [Figure 1B] Figures 1A and 1B show schematic diagrams of the fusion antibody constructs that were generated. Figure 1B shows the different fusion antibody constructs BLV1H12-IL-15 (B15), BLV1H12-IL-15 Rasushi (B15_Rasushi) and BLV1H12-IL-15 GS-Rasushi (B15_GS_Rasushi). [Diagram 2]Activation of IL2 / 15Rβ and γc receptor subunits and STAT5 signaling by chimeric BLV1H12-IL-15 (B15) fusion antibodies is shown through induction and secretion of the STAT5-inducible alkaline phosphatase (SEAP) reporter gene in HEK-Blue IL2 reporter cells. [Figure 3A] Figures 3A-3C show the results of a rodent study in which rats were administered both chimeric B15 fusion antibodies with and without the IL15Rα sushi domain, and show the rat weight (Figure 3A), natural killer (NK) cell percentage (Figure 3B), and CD8 T cell percentage (Figure 3C) following administration of the B15 fusion antibodies. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 4A] Figures 4A-4D show the results of a non-human primate study in which monkeys were administered both a humanized chimeric B15 fusion antibody with and without the IL15Rα sushi domain. Figures 4A-4D show monkey body weights (Figure 4A), mature T cell and cytotoxic T cell counts (Figure 4B), NK cell and helper T cell counts (Figure 4C), and B cell and monocyte counts (Figure 4D) before and after administration of the humanized B15 fusion antibody. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0094] Detailed Description Provided herein are chimeric fusion antibodies in which a heterologous sequence, e.g., a cytokine sequence, or biologically active portion thereof, such as an IL-15 or IL-2 sequence, replaces a portion of the ultralong CDR3 region of the heavy chain of a bovine (cow) antibody or a humanized sequence thereof. In some embodiments, the ultralong CDR3 region contains the ascending stalk region, knob region, and descending stalk region, e.g., present in a bovine antibody, with all or a portion of the knob region being replaced by the cytokine sequence. In some embodiments, the cytokine sequence is the cytokine sequence of IL-2 or a biologically active portion thereof, e.g., having the sequence shown in SEQ ID NO:165. In some embodiments, the cytokine sequence is the cytokine sequence of IL-15 or a biologically active portion thereof, e.g., having the sequence shown in SEQ ID NO:1. In some embodiments, a further portion of the ultralong CDR3 region, e.g., the ascending stalk strand, is modified relative to the ascending stalk strand of a bovine antibody or a humanized sequence thereof. In some embodiments, the heavy chain constant region of the provided chimeric antibodies is modified, e.g., mutated, to reduce, e.g., reduce, compared to a wild-type heavy chain constant region, effector activity of the provided chimeric antibodies. Also provided herein are mutant chimeric IL-15 modified antibodies, including such antibodies linked or complexed to an extracellular portion of IL15Rα, e.g., the IL15Rα sushi domain (e.g., as shown in SEQ ID NO:2).

[0095] IL-15 and IL-2 are pleiotropic cytokines that play important roles in both innate and adaptive immunity. Originally, IL-15, like IL-2, was described as a T cell growth factor. For example, IL-15 is involved in the generation of multiple lymphocyte subsets, including natural killer (NK) cells, NK-T cells, and memory CD8 T cells. IL-15 is also chemotactic for T cells and acts on neutrophils to induce morphological cell shape changes and stimulate IL-8 production. Both cytokines belong to the four α-helical bundle family, and their membrane receptors share two subunits (IL-2R / IL-15R β and γ chains) that are responsible for signal transduction. IL-15 functions through the trimeric IL-15R complex, which is composed of a high-affinity binding α chain (IL-15Rα) and common IL-2R β and γ chains. The IL-2Rβ / γ complex is an intermediate affinity receptor for both cytokines that is expressed by most NK cells and can be activated in vitro by nanomolar concentrations of IL-2 or IL-15 (Wei et al. J Immunol. 2001, 167(1)277-282; Mortier et al. J Biol Chem. 2006, 281(3):1612-1619).

[0096] The IL-15Rα and IL-2Rα subunits form a subfamily of cytokine receptors that contain an extracellular portion at their N-terminus, a so-called "sushi" structural domain (one in IL-15Rα and two in IL-2Rα), which is also found in complement or adhesion molecules. The IL-15Rα Sushi domain is a common motif in protein-protein interactions. Sushi domains are also known as short consensus repeats or type 1 glycoprotein motifs. They have been identified in several protein-binding molecules, including the complement components C1r, C1s, factor H and C2m, as well as the non-immunological molecules factor XIII and β2-glycoprotein. A typical Sushi domain has approximately 60 amino acid residues and contains four cysteines. The first cysteine ​​forms a disulfide bond with the third cysteine, and the second cysteine ​​forms a disulfide bridge with the fourth cysteine. The two disulfide bonds are essential to maintain the tertiary structure of the protein (Kato et al. Biochemistry. 1991,30:11687; Bottenus et al. Biochemistry 1990,29:11195; Ranganathan et al. Pac. Symp. Biocomput. 2000,00:155). The high affinity receptor α (IL15Rα) is involved in augmenting IL-15-mediated trans-signaling to the receptor β and γ subunits (IL2 / 15Rβ and γc).

[0097] IL-2 can stimulate the proliferation, activation, and possibly cytotoxicity of cytotoxic T lymphocytes and natural killer (NK) cells. IL-15 can stimulate the proliferation, activation, and possibly cytotoxicity of cytotoxic T lymphocytes and natural killer (NK) cells. These activities make IL-2 and IL-15 desirable for therapeutic use, but IL-2 and IL-15 are difficult to express as stable soluble proteins and have short half-lives in vitro and in vivo.

[0098] The embodiments provided address these issues. Among the embodiments provided are chimeric antibodies in which an IL-2 or IL-15 cytokine sequence or a biologically active portion thereof replaces all or part of the knob region of a bovine antibody or a humanized variant thereof. The provided antibodies containing an IL-15 cytokine sequence or a biologically active portion thereof may be further linked or complexed with an extracellular portion of IL15Rα, such as the IL15Rα sushi domain, to further mediate IL15 activity. It is found herein that the provided chimeric antibodies, including chimeric IL-15 antibodies (e.g., B15) and variants thereof complexed or linked with the extracellular portion of IL15Rα, can be expressed and purified like a typical human antibody, and exhibit efficient binding and activity to IL2 / 15Rβ and γc subunits. In particular, the provided antibodies function similarly to soluble IL-15 in in vitro signaling assays, but can be easily produced in mammalian cells with improved stability. Furthermore, it is found herein that the antibodies provided exhibit biological activity in vivo, including inducing the proliferation of immune cells, such as NK cells and T cells.

[0099] Furthermore, in some embodiments, the provided antibodies have reduced effector activity, e.g., are modified or mutated to have reduced effector activity. In some embodiments, the provided antibodies are modified to reduce FcR binding and / or to reduce mediation or promotion of antibody-dependent cell-mediated cytotoxicity (ADCC). In some aspects, the methods of using or the use of the provided antibodies provide certain advantages, e.g., the ability to reduce or avoid ADCC against cells to which the provided antibodies bind. For example, in the case of the provided antibodies that include an IL-15 sequence or a biologically active portion thereof, the reduced effector activity of the provided antibodies can reduce or prevent ADCC against cells, e.g., immune cells, expressing IL2 / 15Rβ and / or IL2 / 15Rβ γc receptor subunits to which the provided antibodies can bind.

[0100] Such antibodies may be useful for treating or preventing various diseases, disorders or conditions, including inflammatory diseases, disorders or conditions, autoimmune diseases, disorders or conditions, metabolic diseases, disorders or conditions, neoplastic diseases, disorders or conditions, and cancer. In some aspects, methods and uses of the provided antibodies are provided for treating diseases or conditions, such as cancer. These methods may further include administering a combination agent, such as an antitumor agent, in combination with the provided antibody. The combination agent may promote or mediate ADCC against cells, such as tumor cells. In some aspects, the provided antibodies have reduced effector function and do not interfere with the ADCC-related effects of the combination agent. Thus, in some aspects, the provided antibodies have the additional advantage of being able to be used in combination therapy without affecting the ability of the combination agent to induce or promote ADCC against tumor cells.

[0101] The present disclosure also provides methods and materials for the preparation of the provided chimeric antibodies, including chimeric IL-15 modified antibodies and chimeric IL-2 modified antibodies.

[0102] All publications, including patent documents, scientific articles, and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. To the extent that a definition set forth herein is contrary to or inconsistent with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein takes precedence over the definition incorporated herein by reference.

[0103] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0104] I. Definition Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or usages used herein are intended to have the same meaning as commonly understood by those of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

[0105] As used herein, the articles "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0106] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, when a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, and any other stated or intervening value within that stated range, is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. When a stated range includes one or both of the limits, ranges excluding one or both of those included limits are also encompassed within the claimed subject matter. This applies regardless of the breadth of the range.

[0107] As used herein, the term "about" will be understood by those of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein, "about" when referring to a measurable value, such as an amount, duration, etc., is meant to encompass a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, even more preferably ±0.1% from the specified value, such variations being suitable for carrying out the disclosed methods.

[0108] "Ultralong CDR3" or "ultralong CDR3 sequence" as used interchangeably herein includes CDR3 or CDR3 sequence that is not derived from a human antibody sequence. Ultralong CDR3 can be 35 amino acids or more, for example 40 amino acids or more, 45 amino acids or more, 50 amino acids or more, 55 amino acids or more, or 60 amino acids or more. Typically, ultralong CDR3 is a heavy chain CDR3 (CDR-H3 or CDRH3). Ultralong CDR3H3 shows the characteristics of CDRH3 of ruminant (e.g., bovine) sequence. The structure of ultralong CDR3 includes a "stalk" composed of an ascending strand and a descending strand (e.g., each about 12 amino acids long) and a disulfide-rich "knob" located on the stalk. The unique "stalk and knob" structure of ultralong CDR3 results in two antiparallel β-strands (the ascending stalk strand and the descending stalk strand) supporting a disulfide-bonded knob that protrudes from the antibody surface to form a mini antigen-binding domain. In some embodiments, ultralong CDR3 antibodies include, in order, an ascending stalk region, a knob region and a descending stalk region. The length of ultralong CDR3 can include non-antibody sequences, for example cytokine sequences such as IL-15.

[0109] A modified ultralong CDR3 refers to an ultralong CDR3, at least a portion of which comprises a non-antibody sequence, for example a cytokine sequence such as IL-15. In some cases, at least a portion of the knob of the ultralong CDR3 is replaced with or comprises a non-antibody sequence.

[0110] "Substantially similar" or "substantially the same" refers to a sufficiently high degree of similarity between two numerical values ​​(generally one associated with an antibody disclosed herein and the other associated with a reference / comparator antibody) such that one of skill in the art would consider the difference between the two values ​​to have little or no biological and / or statistical significance within the context of the biological property measured by said values ​​(e.g., Kd values). The difference between said two values ​​is preferably less than about 50%, preferably less than about 40%, preferably less than about 30%, preferably less than about 20%, preferably less than about 10% as a function of the value of the reference / comparator antibody.

[0111] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, whereas high affinity antibodies generally bind antigens quickly and tend to remain bound longer. Various methods of measuring binding affinity are known in the art, any of which can be used for the purposes of this disclosure.

[0112] "Percent (%) amino acid sequence identity" with respect to a peptide or polypeptide sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps as necessary, without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways that are within the skill of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MegAlign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0113] "Polypeptide," "peptide," "protein," and "protein fragment" may be used interchangeably to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and to non-naturally occurring amino acid polymers.

[0114] "Amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to natural amino acids. Natural amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as natural amino acids, such as an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs can have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as natural amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function similarly to a natural amino acid.

[0115] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. "Amino acid variants" refers to amino acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids that code for the same or essentially identical amino acid sequences, or, if the nucleic acid does not code for an amino acid sequence, essentially the same or related (e.g., naturally adjacent) sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for most proteins. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Thus, at all positions where alanine is specified by a codon, the codon can be changed to another of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid mutations are "silent mutations," which are a type of conservatively modified mutation. All nucleic acid sequences herein that code for a polypeptide also describe silent mutations of the nucleic acid. Those skilled in the art will recognize that in certain circumstances, each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to obtain a functionally identical molecule. Thus, silent mutations of nucleic acids encoding polypeptides are included in the described sequences for expression products, but not for actual probe sequences. For amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, or protein sequences that change, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants", including cases where the change results in the replacement 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, the polymorphic variants, interspecies homologs, and alleles disclosed herein.Typically, conservative substitutions include: 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)).

[0116] "Humanized" or "human engineered" forms of non-human (e.g., bovine) antibodies are chimeric antibodies that contain amino acids represented in human immunoglobulin sequences, including, for example, those in which minimal sequence is derived from a non-human immunoglobulin. For example, a humanized or human engineered antibody can be a non-human (e.g., bovine) antibody in which some residues have been substituted with residues from analogous sites in human antibodies (see, e.g., U.S. Pat. No. 5,766,886). The humanized antibody can optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986), Riechmann et al., Nature 332:323-329 (1988), and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998), Harris, Biochem. Soc. Transactions 23:1035-1038 (1995), Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

[0117] "Variable domain" with respect to an antibody refers to a specific Ig domain of the heavy or light chain of an antibody that contains a sequence of amino acids that differ between different antibodies. Each light chain and each heavy chain has one variable region domain (VL and VH). The variable domain provides antigen specificity and is therefore responsible for antigen recognition. Each variable region contains the CDRs, which are part of the antigen binding site domain and the framework region (FR).

[0118] "Constant region domain" refers to a domain of an antibody heavy or light chain that contains a sequence of amino acids that is relatively more conserved among antibodies than the variable region domain. Each light chain has a single light chain constant region (CL) domain, and each heavy chain contains one or more heavy chain constant region (CH) domains, including CH1, CH2, CH3, and optionally CH4. Full-length IgA, IgD, and IgG isotypes contain CH1, CH2 CH3, and hinge regions, while IgE and IgM contain CH1, CH2 CH3, and CH4. The CH1 and CL domains extend the Fab arm of the antibody molecule, thus contributing to the interaction with antigens and the rotation of the antibody arm. The antibody constant region can perform effector functions, such as, but not limited to, clearance of antigens, pathogens, and toxins to which the antibody specifically binds, through interactions with various cells, biomolecules, and tissues.

[0119] An antibody containing a very long CDR3 is an antibody containing a variable heavy (VH) chain with a very long CDR3. The antibody may further comprise a pairing of a VH chain and a variable light (VL) chain. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region. Thus, the term antibody includes full-length antibodies and parts thereof, including antibody fragments, which contain a heavy chain or a part thereof and / or a light chain or a part thereof. An antibody may contain two heavy chains (which may be designated H and H') and two light chains (which may be designated L and L'), each L chain being linked to a H chain by a covalent disulfide bond, and the two H chains being linked to each other by disulfide bonds. The terms "full-length antibody" or "intact antibody" are used interchangeably to refer to an antibody in substantially intact form, as opposed to an antibody fragment. A full-length antibody is typically an antibody that has two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region.

[0120] The term "antibody" is used herein in the broadest sense and includes fragments antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, heavy chain variable (V) fragments capable of specific binding, H ) regions, and single chain variable fragments (scFv), as well as polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments.

[0121] "Antibody fragments" include portions of an intact antibody, the antigen-binding and / or variable regions of the intact antibody. Antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fd' fragments; single-chain antibody molecules including single-chain Fvs (scFv) or single-chain Fabs (scFab); antigen-binding fragments of any of the above and multispecific antibodies from antibody fragments.

[0122] A "Fab fragment" is an antibody fragment resulting from digestion of a full-length immunoglobulin with papain, or a fragment having the same structure produced synthetically, for example by recombinant methods. A Fab fragment is a fragment of an antibody that contains only a light chain (V L and C L ) and the variable domain of the heavy chain (V H ) and one constant region domain of the heavy chain (C H 1) and another strand containing

[0123] An "scFv fragment" is a fragment of variable light chains (VLCs) covalently connected by a polypeptide linker in any order. L ) and variable heavy chain (V H (Gly-Ser) with several Glu or Lys residues dispersed throughout to enhance solubility. n It is a residue.

[0124] A chimeric antibody refers to an antibody containing a modified ultralong CDR3 in which at least a portion of the knob of the CDR3 of the heavy chain is replaced with or includes a non-antibody sequence, e.g., a cytokine sequence such as IL-15.

[0125] The term "corresponding to" with respect to a protein position refers to a nucleotide or amino acid position identified when aligned with a disclosed sequence based on a structural sequence alignment or using a standard alignment algorithm such as the GAP algorithm, e.g., a statement that the nucleotide or amino acid position "corresponds to" a nucleotide or amino acid position in the disclosed sequence as set forth in the sequence listing. For example, corresponding residues of similar sequences (e.g., fragments or species variants) can be determined by alignment to a reference sequence by structural alignment methods. By aligning the sequences, one of skill in the art can identify corresponding residues, e.g., using conserved identical amino acid residues as a guide.

[0126] The term "effective amount" or "therapeutically effective amount" as used herein means an amount of a pharmaceutical composition sufficient to significantly and positively modify the symptoms and / or condition being treated (e.g., provide a positive clinical response). The effective amount of active ingredient for use in a pharmaceutical composition will vary depending on the particular condition being treated, the severity of the condition, the duration of treatment, the nature of any concurrent therapy, the particular active ingredient used, the particular pharma- ceutically acceptable excipients and / or carriers utilized, and similar factors associated with the knowledge and expertise of the attending physician.

[0127] As used herein, the term "pharmaceutical acceptable" refers to a material, such as a carrier or diluent, that does not abrogate the biological activity or properties of the compound and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0128] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0129] As used herein, the term "pharmaceutical composition" refers to a mixture of at least one compound of the present invention with other chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering the compound exist in the art, including, but not limited to, intravenous, oral, aerosol, parenteral, ocular, pulmonary, and topical administration.

[0130] As used herein, "disease or disorder" refers to a pathological condition in an organism resulting from a cause or condition, including but not limited to an infection, an acquired condition, or a genetic condition, and characterized by an identifiable symptom.

[0131] As used herein, the terms "treat," "treating," or "treatment" refer to ameliorating a disease or disorder, e.g., slowing or halting or reducing the onset of a disease or disorder, e.g., the underlying cause of the disorder, or at least one of its clinical symptoms.

[0132] As used herein, the term "subject" refers to an animal, including a mammal, such as a human. The terms subject and patient can be used interchangeably.

[0133] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes cases where said event or circumstance occurs and cases where it does not occur. For example, an optionally substituted group means that the group is unsubstituted or substituted.

[0134] II. Chimeric Antibodies Provided herein are chimeric antibodies in which a heterologous sequence, e.g., a cytokine sequence, e.g., an IL-2 sequence or a biologically active portion thereof, or an IL-15 sequence or a biologically active portion thereof, replaces a portion of the ultralong CDR3 region of the heavy chain of a bovine (cow) antibody or a humanized sequence thereof. In some embodiments, the IL-15 sequence may comprise a full-length IL-15 (e.g., human IL-15) sequence (e.g., the sequence shown in SEQ ID NO:1) or a biologically active portion of IL-15. IL-15 is a potent immunostimulatory cytokine and an essential survival factor for T cells and natural killer cells. Preclinical studies comparing IL2 and IL15 have shown that IL-15 is less toxic than IL-2. The IL-15 sequence may also be modified to increase its binding affinity to the IL-15 receptor. For example, asparagine can be replaced by aspartic acid at position 72 of ILLS (SEQ ID NO. 2 of US Patent Publication No. 20140134128, the contents of which are incorporated by reference in their entirety). Any portion of IL-15 that retains one or more functions of full-length or mature IL-15 can be useful in the present invention. Such functions include promoting NK cell survival, regulating NK cells, and supporting T cell activation and proliferation, as well as NK cell development from hematopoietic stem cells.

[0135] In some embodiments, further portions of the ultralong CDR3 region, such as the ascending stalk strand, are modified relative to the ascending stalk strand of a bovine antibody or its humanized sequence. In some embodiments, the heavy chain constant region of the provided chimeric antibody is modified, e.g., mutated, e.g., reduced, as compared to the wild-type heavy chain constant region, to reduce the effector activity of the provided chimeric antibody. The provided chimeric antibodies also include such antibodies linked or complexed to the extracellular portion of IL15Rα, e.g., the IL15Rα sushi domain (e.g., as shown in SEQ ID NO:2). In some embodiments, the IL-15 cytokine is formatted with the alpha subunit of the IL15 receptor (IL15Ra), or a portion thereof, which binds to and activates the membrane-bound IL15 beta / gamma receptor. A unique feature of IL-I5-mediated activation is the mechanism of trans-presentation, where IL-15 is presented either on the same cell or on a different cell as a complex with the alpha subunit of the IL15 receptor (IL15Ra), which binds to and activates the membrane-bound IL15 beta / gamma receptor. The IL15 / IL15Ra complex is more effective at activating IL-15 signaling than IL-15 itself. In some embodiments, full-length IL-15Ra or a portion of IL15Ra can be complexed or fused (e.g., linked) to the IL-15 cytokine sequence or a biologically active portion thereof. Any portion of IL-15 and IL-15Ra that retains one or more functions of full-length or mature IL15 or IL15Ra, respectively, can be useful in the embodiments provided. Such functions include promoting NK cell survival, regulating NK cells, and activating and expanding T cells, as well as supporting NK cell development from hematopoietic stem cells. IL15 receptor alpha contains an extracellular domain called the sushi domain, which contains most of the structural elements necessary for binding to IL15. Thus, in some embodiments, the portion of IL15Ra is or comprises the IL15Ra sushi domain.A portion of IL15Ra useful in the embodiments provided can include amino acids 31-205 or 31-95 of human IL15Ra (Uniprot ID: Q1326.1).

[0136] The antibodies provided exhibit the characteristics of bovine or cow antibodies with unique heavy chain variable region sequences containing ultralong CDR3 sequences up to 70 or more amino acids in length. CDR3 sequences identified in cattle include BLV1H12 (see SEQ ID NO:25), BLV5B8 (see SEQ ID NO:30), BLV5D3 (see SEQ ID NO:31), and BLV8C1 1 (see SEQ ID NO:32) (see, e.g., Saini, et al. (1999) Eur. Immunol. 29:2420-2426, and Saini and Kaushik (2002) Scand. J. Immunol. 55:140-148), BF4E9 (see SEQ ID NO:33) and BF1 H1 (see SEQ ID NO:34) (see, e.g., Saini and Kaushik (2002) Scand. J. Immunol. 55:140-148), and F18 (see SEQ ID NO:35) (see, e.g., Berens, et al. (see, e.g., et al. (1997) Int. Immunol. 9:189-199). Exemplary antibody variable region sequences comprising ultralong CDR3 sequences identified in cattle include BLV1H12. In some embodiments, the BLV1H12 ultralong CDR3 sequence is encoded by SEQ ID NO:25. Exemplary bovine antibodies include the bovine antibody BLVH12 (e.g., heavy chain variable region set forth in SEQ ID NO:26, and light chain variable region set forth in SEQ ID NO:27), and the bovine antibody BLV5B8 (e.g., heavy chain variable region set forth in SEQ ID NO:28, and light chain variable region set forth in SEQ ID NO:29).

[0137] In cow antibodies, ultralong CDR3 sequences form a structure in which a "stalk" is composed of two 12-residue antiparallel β-strands (ascending and descending strands) and a 39-residue disulfide-rich "knob" on the stalk, forming a subdomain with an unusual architecture that differs significantly from standard antibody paratopes. The long antiparallel β-ribbon serves as a bridge connecting the knob domain with the main backbone of the antibody. The unique "stalk and knob" structure of ultralong CDR3 results in two antiparallel β-strands (ascending and descending stalk strands) that support a disulfide-bonded knob that protrudes from the antibody surface to form a mini antigen-binding domain. In some embodiments, ultralong CDR3 antibodies include, in order, an ascending stalk region, a knob region, and a descending stalk region.

[0138] The unique "stalk" and knob structural features are conserved across different bovine or cow ultralong CDR3 sequences. The ascending strand of the stalk contains primarily hydrophobic side chains and a relatively conserved "T(T / S)VHQ" motif and its variants at the base that initiates the ascending strand. This conserved T(T / S)VHQ motif and its variants are typically found after the first cysteine ​​residue in the variable region sequence of the various bovine or cow sequences. The conserved T(T / S)VHQ motif is connected by a variable number of residues to a motif (CPDG in BLV1H12) that forms a β-turn at the base of each knob. The stalk can be of variable length, and the descending strands of the stalk contain alternating aromatics that form a ladder through stacking interactions that may contribute to the stability of the long solvent-exposed two-stranded β-ribbon (Wang et al. Cell. 2013,153(6):1379-1393).

[0139] In some embodiments, the chimeric antibodies provided herein are based on an antibody scaffold that may be derived from or based on a bovine antibody sequence or a humanized sequence thereof, but contain a heterologous sequence, such as a cytokine sequence, e.g., an IL-2 sequence or a biologically active portion thereof, or an IL-15 sequence or a biologically active portion thereof, inserted into or replacing a portion of the knob domain of the ultralong CDR3 of the heavy chain of the bovine antibody sequence or a humanized sequence thereof. In some embodiments, the ultralong CDR3 sequence of the heavy chain of the chimeric antibodies provided herein contains a stalk component that contains an ascending strand and a descending strand that are joined together by a region that contains a heterologous sequence. In some embodiments, the heterologous sequence replaces a portion of a bovine antibody or a humanized sequence thereof, e.g., replaces the knob region of a bovine antibody or a humanized sequence thereof.

[0140] In some embodiments, the heterologous sequence is a non-antibody sequence. In some embodiments, the heterologous sequence is a signaling molecule sequence. In some embodiments, the heterologous sequence is a hormone sequence. In some embodiments, the heterologous sequence is a neurotransmitter sequence. In some embodiments, the heterologous sequence is a growth factor. In some embodiments, the heterologous sequence is a cytokine sequence. In some embodiments, the heterologous sequence is a chemokine sequence. In some embodiments, the heterologous sequence is an interferon sequence. In some embodiments, the heterologous sequence is an interleukin sequence. In some embodiments, the heterologous sequence is a lymphokine sequence. In some embodiments, the heterologous sequence is a tumor necrosis factor sequence.

[0141] In some embodiments, the heterologous sequence is a cytokine sequence. Thus, the chimeric antibodies provided include chimeric cytokine modified antibodies in which a cytokine sequence replaces all or part of the knob region of a bovine antibody or a humanized sequence thereof. In some embodiments, the cytokine sequence is an IL-2 sequence or a biologically active portion thereof. In some embodiments, the cytokine sequence is an IL-15 sequence or a biologically active portion thereof.

[0142] In some embodiments, the heterologous sequence is inserted into the knob region of the CDR3 sequence of the antibody, optionally including removing a portion of the CDR3 (e.g., one or more amino acids of the CDR3) or the entire CDR3 sequence (e.g., all or substantially all of the amino acids of the CDR3). In some embodiments, the heterologous sequence can be inserted into the knob domain of an ultralong CDR3. In some embodiments, the heterologous sequence is contained between the ascending and descending stalk strands.

[0143] In some embodiments, the IL-15 sequence or biologically active portion thereof is inserted into the knob region of the CDR3 sequence of an antibody, optionally including removing a portion of the CDR3 (e.g., one or more amino acids of the CDR3) or the entire CDR3 sequence (e.g., all or substantially all of the amino acids of the CDR3). In some embodiments, the IL-15 or biologically active portion thereof may be inserted into the knob domain of an ultralong CDR3 (FIGS. 1A and 1B). In some embodiments, the IL-15 or biologically active portion thereof is contained between the ascending and descending stalk strands.

[0144] In some embodiments, the IL-2 sequence or biologically active portion thereof is inserted into the knob region of the CDR3 sequence of an antibody, optionally including removing a portion of the CDR3 (e.g., one or more amino acids of the CDR3) or the entire CDR3 sequence (e.g., all or substantially all of the amino acids of the CDR3). In some embodiments, the IL-2 or biologically active portion thereof may be inserted into the knob domain of an ultralong CDR3. In some embodiments, the IL-2 or biologically active portion thereof is contained between the ascending and descending stalk strands.

[0145] In some embodiments, the ultralong CDR3 can be 35 amino acids or more in length (e.g., 40 or more, 45 or more, 50 or more, 55 or more, 60 or more).

[0146] Any of the embodiments provided herein can contain any of the features described in PCT / US2013 / 020910, PCT / US2014 / 047315, or PCT / US2013 / 020903, all of which are incorporated by reference in their entireties.

[0147] Exemplary features of the antibody, including heavy and light chains, are described in the following subsections. In some of any of the embodiments herein, the antibody is a full-length antibody or an intact antibody. In some of any of the embodiments herein, the antibody is an antigen-binding fragment thereof. In further embodiments, the antigen-binding fragment thereof is a Fab, Fab'-SH, Fv, scFv or (Fab')2 fragment. In some embodiments, the antibody is a Fab.

[0148] A. Heavy Chain Region In provided embodiments, the heavy chain of the provided chimeric antibody is based on or derived from a framework sequence having an ultralong CDR3 in which a heterologous sequence, e.g., a cytokine sequence, e.g., IL-2 or a biologically active portion thereof, or IL-15 or a biologically active portion thereof, is inserted into or replaces at least a portion of the ultralong CDR3 sequence. The antibody framework can be derived from bovine sequences, such as VH-VL, human germline sequences, or modified human germline sequences.

[0149] In some embodiments, the heavy chain of the provided chimeric antibody is based on or derived from a bovine or cow framework sequence in which a heterologous sequence, such as a cytokine sequence, for example an IL-2 sequence or a biologically active portion thereof, or an IL-15 sequence or a biologically active portion thereof, can be inserted into or replace at least a portion of the ultralong CDR3 sequence of the bovine or cow sequence. The antibody can comprise at least a portion of a BLV1H12 antibody containing a heterologous sequence, such as an ultralong CDR3 fusion containing a cytokine sequence. Alternatively or additionally, the provided chimeric antibody comprises at least a portion of a BLV5D3, BLV8C11, BF1H1, BLV5B8 and / or F18 antibody containing a heterologous sequence, such as an ultralong CDR3 fusion containing a cytokine sequence. In some embodiments, a heterologous sequence, e.g., a cytokine sequence, e.g., an IL-15 sequence, or a biologically active portion thereof, may be inserted into or replace at least a portion of the ultralong CDR3 of the sequence shown in SEQ ID NO:26 or SEQ ID NO:28.

[0150] In some embodiments, the heavy chain of the provided chimeric antibody is based on or derived from a humanized heavy chain framework sequence that is humanized relative to bovine or cow sequences. In some embodiments, the heavy chain of the provided chimeric antibody is based on or derived from a human heavy chain framework sequence that exhibits sequence or structural similarity to bovine or cow sequences. In some cases, humanization can include engineering an ultralong CDR3 sequence derived from a bovine ultralong CDR3, such as any of those described above, into a human framework. The human framework can be of germline origin or can be derived from a non-germline (e.g., mutated or affinity matured) sequence. Genetic engineering techniques well known to those skilled in the art, including those disclosed herein, can be used to generate hybrid DNA sequences containing human frameworks and non-human ultralong CDR3s. Unlike human antibodies that may be encoded by V-region genes from one of seven families, bovine antibodies that produce ultralong CDR3 sequences appear to utilize a single V-region family that may be considered most homologous to the human VH4 family. In particular, in embodiments where a cattle-derived very long CDR3 sequence is humanized to produce an antibody comprising a very long CDR3, a human V-region sequence from the VH4 family can be genetically fused to a bovine-derived very long CDR3 sequence. Exemplary VH4 germline gene sequences in a human antibody locus include VH4-39, VH4-59*03, VH4-34*02, and VH4-34*09 human heavy chain germline sequences. In some embodiments, the human heavy chain germline sequence is a sequence set forth in any one of SEQ ID NOs:68-71. In some embodiments, the human heavy chain germline sequence is a sequence encoded by a sequence set forth in any one of SEQ ID NOs:169-172.

[0151] In some embodiments, a heterologous sequence, such as a cytokine sequence, such as an IL-2 sequence or a biologically active portion thereof, or an IL-15 sequence or a biologically active portion thereof, may be inserted into or replace at least a portion of the ultralong CDR3 of a human germline sequence comprising the sequence shown in SEQ ID NOs:68-71.

[0152] In some embodiments, the chimeric antibody provided comprises a fusion of a human VH4 framework sequence with an ultralong CDR3 from bovine, with at least a portion of the knob replaced with a heterologous sequence, such as an IL-15 or IL-2 sequence or a biologically active portion thereof. In some aspects, such a fusion can be generated by the following steps: First, a second cysteine ​​in the V-region gene sequence is identified along with a nucleotide sequence encoding the second cysteine. Generally, the second cysteine ​​marks the boundary between the framework and the CDR3 two residues upstream (N-terminus) of the CDR3. Second, a second cysteine ​​in the bovine-derived V-region sequence is identified, which also marks two residues upstream (N-terminus) of the CDR3. Third, the genetic material encoding the human V-region is combined with the genetic sequence encoding the ultralong CDR3. In this way, a genetic fusion can be performed in which the ultralong CDR3 sequence is placed in frame with the human V-region sequence. Preferably, the humanized antibody with ultralong CDR3 has an amino acid composition as close to human as possible. Optionally, the J region sequence can be mutated from a bovine-derived sequence to a human sequence. Optionally, the humanized heavy chain can also be paired with a human light chain.

[0153] In some embodiments, the modified VH region of the provided chimeric antibodies is a variant of the VH region of a bovine antibody, e.g., BLV1H12. In some embodiments, the modified VH region of the provided chimeric antibodies is a variant of the humanized sequence of the VH region of a bovine antibody, e.g., BLV1H12.

[0154] In some embodiments, the provided chimeric antibody or binding fragment thereof comprises a heavy chain variable region comprising a sequence of the formula V1-X-V2, where the V1 region of the heavy chain comprises a portion of the heavy chain sequence containing three framework regions (e.g., FR-1, FR-2 and FR-3) separating the two CDR regions (CDR1 and CDR2), the X region comprises a modified ultralong CDR3 sequence that may comprise a heterologous sequence, e.g., an IL-2 sequence or a biologically active portion thereof or an IL-15 sequence or a biologically active portion thereof, and the V2 region comprises a portion of the heavy chain comprising FR-4.

[0155] In some embodiments, the V1 region comprises the formula FR1-CDR1-FR2-CDR2-FR3. In some embodiments, the V1 region comprises an amino acid sequence selected from the group consisting of: (i) a bovine heavy chain region comprising the amino acids of SEQ ID NO:26 (encoded by the nucleotides of SEQ ID NO:5), or (i) a humanized heavy chain region comprising a human germline variable region comprising SEQ ID NOs:12-19. In some embodiments, the V1 region comprises the sequence set forth in SEQ ID NO:182 or SEQ ID NO:197.

[0156] In some embodiments, the modified VH region of the provided chimeric antibody is a variant of the VH region of a bovine antibody, e.g., BLV1H12. In some embodiments, the V1 region comprises the sequence shown in SEQ ID NO:182.

[0157] In some embodiments, the modified VH region of the provided chimeric antibody is a variant of the humanized sequence of the VH region of a bovine antibody, e.g., BLV1H12. In some embodiments, the V1 region has the amino acid sequence shown in SEQ ID NO:197, or SEQ ID NO: The present invention includes sequences that exhibit at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least about 99% sequence identity to the amino acid sequence set forth in NO:197. In some embodiments, the V1 region comprises the sequence set forth in SEQ ID NO:197.

[0158] In some embodiments, the X region comprises a heterologous sequence, e.g., a modified ultralong CDR3 sequence that may include an IL-15 sequence, or a biologically active portion thereof (e.g., a human IL-15 sequence, or a biologically active portion thereof). In some embodiments, the IL-15 sequence comprises an amino acid sequence set forth in SEQ ID NO:1, or a sequence of amino acids that exhibits at least or at least about 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the IL-15 sequence comprises an amino acid sequence found in SEQ ID NO:1.

[0159] In some embodiments, the IL-15 sequences exhibit activity in stimulating proliferation, activation or cytotoxicity of cytotoxic T lymphocytes and natural killer (NK) cells, such as in in vitro assays or in vivo. In some embodiments, the IL-15 sequences exhibit binding to IL2 / 15Rβ and / or γc subunits, such as in in vitro binding assays. In some embodiments, the activity or binding is similar to or retained compared to recombinant IL-15 monomer.

[0160] In some embodiments, the heterologous sequence, e.g., an IL-15 sequence or a biologically active portion thereof, is inserted into or replaces a portion of the knob of the ultralong CDR3 between the ascending and descending stalk regions. The heterologous sequence, e.g., an IL-15 sequence, may be located between the stalk regions, and the heterologous sequence, e.g., an IL-15 sequence, is directly or indirectly linked to each of the stalk regions. In some embodiments, the link to one or both of the stalk sequences is indirect via a linker. The linker can comprise an amino acid sequence of (GGGGS), where n=1-5. Alternatively, the linker comprises an amino acid sequence of (GSG), GGGSGGGGS, or GGGGSGGGS. In some cases, the linker has the sequence GGS (SEQ ID NO:151) or GSG (SEQ ID NO:186).

[0161] In some embodiments, the X region comprises a heterologous sequence, e.g., a modified ultralong CDR3 sequence that may include an IL-2 sequence or a biologically active portion thereof (e.g., a human IL-2 sequence or a biologically active portion thereof). In some embodiments, the IL-2 sequence comprises an amino acid sequence set forth in SEQ ID NO:165, or a sequence of amino acids that exhibits at least or at least about 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:165. In some embodiments, the IL-2 sequence comprises an amino acid sequence found in SEQ ID NO:165.

[0162] In some embodiments, the IL-2 sequences exhibit activity in stimulating proliferation, activation or cytotoxicity of cytotoxic T lymphocytes and natural killer (NK) cells, such as in in vitro assays or in vivo. In some embodiments, the IL-2 sequences exhibit binding to IL2 / 15Rβ and / or γc subunits, such as in in vitro binding assays. In some embodiments, the activity or binding is similar to or retained compared to recombinant IL-2 monomer.

[0163] In some embodiments, the heterologous sequence, e.g., an IL-2 sequence or a biologically active portion thereof, is inserted into or replaces a portion of the knob of the ultralong CDR3 between the ascending and descending stalk regions. The heterologous sequence, e.g., an IL-2 sequence, may be located between the stalk regions, and the heterologous sequence, e.g., an IL-2 sequence, is directly or indirectly linked to each of the stalk regions. In some embodiments, the link to one or both of the stalk sequences is indirect via a linker. The linker can comprise an amino acid sequence of (GGGGS), where n=1-5. Alternatively, the linker comprises an amino acid sequence of (GSG), GGGSGGGGS, or GGGGSGGGS. In some cases, the linker has the sequence GGS (SEQ ID NO:151) or GSG (SEQ ID NO:186).

[0164] The ultralong CDR3 may comprise at least a portion of the knob domain of the CDR3, at least a portion of the stalk domain of the CDR3, or a combination thereof. The portion of the knob domain of the CDR3 may comprise one or more conserved motifs derived from the knob domain of the ultralong CDR3. The stalk domain of the CDR3 may comprise one or more conserved motifs derived from the stalk domain of the ultralong CDR3.

[0165] In aspects of each or any of the above or below embodiments, the ultralong CDR3 is 35 amino acids or more, 40 amino acids or more, 45 amino acids or more, 50 amino acids or more, 55 amino acids or more, or 60 amino acids or more in length. In some embodiments of each or any of the above or below embodiments, the ultralong CDR3 is 35 amino acids or more in length.

[0166] In some embodiments, the X region of the provided chimeric antibody comprises an ascending stalk strand and a descending stalk strand. In some embodiments, the heterologous sequence, e.g., a cytokine sequence, e.g., an IL-15 sequence, of the provided chimeric antibody is between the ascending stalk strand and the descending stalk strand. In some embodiments, the provided chimeric antibody comprises an ascending stalk strand and a descending stalk strand of a bovine antibody or a humanized sequence thereof, e.g., an ascending stalk strand and a descending stalk strand of a BLV1H12 or a humanized sequence thereof. In some embodiments, one or both of the ascending stalk strand and the descending stalk strand are variants of the ascending or descending stalk strand of a bovine antibody or a humanized sequence thereof. In some embodiments, the ascending stalk strand of the provided chimeric antibody is a variant of the ascending stalk strand of a bovine antibody or a humanized sequence thereof.

[0167] In some embodiments, the X region of the provided chimeric antibody comprises the motif X 1 X 2 X 3 X 4 X 5 -[Heterogeneous sequence]-(X a X b In some embodiments, the ultralong CDR3 is 45 amino acids long or longer. In some embodiments, the one or more additional amino acids include the X 1 X 2 X 3 X 4 X 5 Between the motif and the heterologous sequence and / or (X a Xb ) z motif and the heterologous sequence.

[0168] In some embodiments, X 1 X 2 X 3 X 4 X 5 The motif is all or part of the ascending stalk strand. In some embodiments, the X on the ascending stalk strand 1 X 2 X 3 X 4 X 5 The motif comprises a sequence selected from TTVHQ (SEQ ID NO:36), TSVHQ (SEQ ID NO:37) or any one of SEQ ID NOs:38-67. In some embodiments, the ascending stalk strand comprises a sequence selected from SEQ ID NOs:72-75 or SEQ ID NO:158. In some embodiments, the ultralong CDR3 comprises an ascending stalk region encoded by SEQ ID NOs:9, SEQ ID NOs:81-121 or SEQ ID NO:157. In some embodiments, the motif comprises a sequence selected from CX 1 X 2 X 3 X 4 X 5 For example, in some cases, the ascending stalk region encoded by any of SEQ ID NOs:36-67, 72-75, or SEQ ID NO:158 may further contain an N-terminal Cys residue. Such an exemplary ascending stalk region is set forth in SEQ ID NO:159. In some embodiments, the ascending stalk strand further comprises the sequence ETKKYQT. In some embodiments, the ascending stalk strand further comprises the sequence ETKKYQS.

[0169] In some embodiments, the ascending stalk strand comprises the sequence CX2TVX5QETKKYQT. In some embodiments, X2 and X5 are any amino acid. In some embodiments, X2 is Ser, Thr, Gly, Asn, Ala, or Pro. In some embodiments, X5 is His, Gln, Arg, Lys, Gly, Thr, Tyr, Phe, Trp, Met, Ile, Val, or Leu. In some embodiments, X2 is Ser, Thr, Gly, Asn, Ala, or Pro, and X5 is His, Gln, Arg, Lys, Gly, Thr, Tyr, Phe, Trp, Met, Ile, Val, or Leu. In some embodiments, X2 is Ser, Ala, or Thr. In some embodiments, X5 is His or Tyr. In some embodiments, X2 is Ser, Ala, or Thr, and X5 is His or Tyr. In some embodiments, X2 is Ser and X5 is His. In some embodiments, X2 is Ala and X5 is His. In some embodiments, X2 is Thr and X5 is Tyr. In some embodiments, the ascending stalk region of the provided chimeric antibodies comprises a sequence set forth in any of SEQ ID NOs: 183-185. In some embodiments, the ascending stalk region of the provided chimeric antibodies comprises a sequence set forth in SEQ ID NO: 183. In some embodiments, the ascending stalk region of the provided chimeric antibodies comprises a sequence set forth in SEQ ID NO: 184. In some embodiments, the ascending stalk region of the provided chimeric antibodies comprises a sequence set forth in SEQ ID NO: 185.

[0170] In some embodiments, (X a X b ) z motif is part of the descending stalk strand, where X a is any amino acid residue, and X bis an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and z is 1-4. In some embodiments, the descending stalk strand comprises alternating aromatics having the formula YXYXYX, where X is any amino acid. In some embodiments, the descending stalk strand comprises a sequence contained in SEQ ID NO:76-80 or SEQ ID NO:161. In some embodiments, the ultralong CDR3 comprises a descending stalk region encoded by SEQ ID NO:122-149 or SEQ ID NO:160. In some embodiments, the descending stalk region of the provided chimeric antibodies comprises a sequence shown in SEQ ID NO:10.

[0171] In some embodiments, the chimeric antibody provided comprises a modified ultralong CDR3.

[0172] In some embodiments, the modified ultralong CDR3 comprises, in order, an ascending stalk region having an amino acid sequence encoded by SEQ ID NO:9, an IL15 cytokine sequence as represented by SEQ ID NO:1, and a descending stalk region having an amino acid sequence encoded by SEQ ID NO:10. In some embodiments, the ultralong CDR3 comprises, in order, an ascending stalk region having an amino acid sequence encoded by SEQ ID NO:157, an IL15 cytokine sequence as represented by SEQ ID NO:1, and a descending stalk region having an amino acid sequence encoded by SEQ ID NO:160.

[0173] In some embodiments, the modified ultralong CDR3 comprises, in order, an ascending stalk region having an amino acid sequence encoded by SEQ ID NO:9, an IL2 cytokine sequence as represented by SEQ ID NO:165, and a descending stalk region having an amino acid sequence encoded by SEQ ID NO:10. In some embodiments, the ultralong CDR3 comprises, in order, an ascending stalk region having an amino acid sequence encoded by SEQ ID NO:157, an IL2 cytokine sequence as represented by SEQ ID NO:165, and a descending stalk region having an amino acid sequence encoded by SEQ ID NO:160.

[0174] In some embodiments, the modified ultralong CDR3 comprises, in order, an ascending stalk strand having the amino acid sequence set forth in SEQ ID NO: 183, an IL-15 cytokine sequence set forth in SEQ ID NO: 1, and a descending stalk strand having the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the modified ultralong CDR3 comprises the sequence set forth in SEQ ID NO: 206.

[0175] In some embodiments, the modified ultralong CDR3 comprises, in order, an ascending stalk strand having the amino acid sequence set forth in SEQ ID NO: 184, an IL-15 cytokine sequence set forth in SEQ ID NO: 1, and a descending stalk strand having the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the modified ultralong CDR3 comprises the sequence set forth in SEQ ID NO: 207.

[0176] In some embodiments, the modified ultralong CDR3 comprises, in order, an ascending stalk strand having the amino acid sequence set forth in SEQ ID NO: 185, an IL-15 cytokine sequence set forth in SEQ ID NO: 1, and a descending stalk strand having the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the modified ultralong CDR3 comprises the sequence set forth in SEQ ID NO: 208.

[0177] In some embodiments, the modified ultralong CDR3 comprises, in order, an ascending stalk strand having the amino acid sequence set forth in SEQ ID NO: 159, an IL-15 cytokine sequence set forth in SEQ ID NO: 1, and a descending stalk strand having the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the modified ultralong CDR3 comprises the sequence set forth in SEQ ID NO: 209.

[0178] In some embodiments, the V2 region of the heavy chain comprises TIFF2024517759000002.tif24142. In some embodiments, the V2 region of the heavy chain comprises the sequence set forth in SEQ ID NO:11.

[0179] In some embodiments, the modified VH region of the provided chimeric antibodies is a variant of the VH region of a bovine antibody, such as BLV1H12.

[0180] In some embodiments, the heavy chain comprises the formula V1-X-V2-C, where the V1 region of the heavy chain comprises the sequence set forth in SEQ ID NO:182; the X region comprises a modified ultralong CDR3 sequence; the V2 region comprises the sequence set forth in SEQ ID NO:11; and the C region comprises an immunoglobulin constant region, e.g., a modified IgG (e.g., IgG1) constant region as described. In some embodiments, X comprises a sequence set forth in any of SEQ ID NOs:206-208. In some embodiments, the modified VH region comprises a sequence set forth in SEQ ID NO:200 or a sequence exhibiting at least or at least about 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:200. In some embodiments, the modified VH region comprises a sequence set forth in SEQ ID NO:200.In some embodiments, the modified VH region comprises a sequence set forth in SEQ ID NO:201 or a sequence exhibiting at least or at least about 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:201. In some embodiments, the modified VH region comprises a sequence set forth in SEQ ID NO:201. In some embodiments, the modified VH region comprises a sequence set forth in SEQ ID NO:202, or a sequence exhibiting at least or at least about 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:202. In some embodiments, the modified VH region comprises a sequence set forth in SEQ ID NO:202.

[0181] In some embodiments, the modified VH region of the provided chimeric antibodies is a variant of the humanized sequence of the VH region of a bovine antibody, e.g., BLV1H12. In some embodiments, the heavy chain comprises the formula V1-X-V2-C, where the X region comprises a modified ultralong CDR3 sequence; the V2 region comprises the sequence set forth in SEQ ID NO:11; and the C region comprises an immunoglobulin constant region, e.g., a modified IgG (e.g., IgG1) constant region as described. In some embodiments, the V1 region of the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:197, or the amino acid sequence set forth in SEQ ID NO:206. The present invention includes sequences that exhibit at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least about 99% sequence identity to the amino acid sequence set forth in NO:197. In some embodiments, the V1 region comprises a sequence set forth in SEQ ID NO: 197. In some embodiments, X comprises a sequence set forth in any of SEQ ID NOs: 206-208.In some embodiments, the modified VH region comprises a sequence set forth in SEQ ID NO:203, or a sequence exhibiting at least or at least about 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:203. In some embodiments, the modified VH region comprises a sequence set forth in SEQ ID NO:203. In some embodiments, the modified VH region comprises a sequence set forth in SEQ ID NO:204, or a sequence exhibiting at least or at least about 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:204. In some embodiments, the modified VH region comprises a sequence set forth in SEQ ID NO:204.In some embodiments, the modified VH region comprises a sequence set forth in SEQ ID NO:205, or a sequence exhibiting at least or at least about 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:205. In some embodiments, the modified VH region comprises a sequence set forth in SEQ ID NO:205.

[0182] In certain embodiments, the chimeric IL-15 modified antibody or antigen-binding fragment provided herein comprises a variable heavy chain sequence encoded by the sequence of nucleotides set forth in SEQ ID NO:7, or a variable heavy chain sequence encoded by the sequence of nucleotides set forth in SEQ ID NO: The modified antibody or antigen-binding fragment of the present invention comprises a sequence of nucleotides that exhibits at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:7, and comprises a modified ultralong CDR3 that contains an IL-15 sequence. In some embodiments, the chimeric IL-15 modified antibody or antigen-binding fragment provided herein comprises a variable heavy chain sequence encoded by a sequence of nucleotides set forth in SEQ ID NO:7. In some embodiments, the chimeric IL-15 modified antibody or antigen-binding fragment provided herein consists of, or consists essentially of, the variable heavy chain sequence encoded by the sequence of nucleotides set forth in SEQ ID NO:7.

[0183] In some embodiments, the heavy chain comprises a variable heavy chain as described above that is linked to a human constant region. In some embodiments, the human constant region comprises a CH1-CH2-CH3 constant domain. In some embodiments, the human constant region is that of human IgG1 (e.g., having the sequence shown in SEQ ID NO:196, or a naturally occurring variant thereof, such as a K97R, D239E, or L241M mutation).

[0184] In some embodiments, the human IgG is a human IgG1 (eg, having the sequence shown in SEQ ID NO:196, or a naturally occurring variant thereof, such as a K97R, D239E, or L241M mutation).

[0185] In some embodiments, the heavy chain constant region is mutated or modified, i.e., modified constant region. In some embodiments, the heavy chain constant region is a modified human IgG heavy chain constant region. In some cases, the mutation comprises one or more amino acid substitutions to reduce the effector activity of the heavy chain constant region. In some embodiments, the heavy chain constant region is modified to reduce the effector activity of the antibody. In some embodiments, the modified human IgG heavy chain constant region has reduced effector activity. In some embodiments, the effector activity is reduced compared to the wild-type human IgG heavy chain constant region. In some of any of the described embodiments, the modified human IgG heavy chain constant region is modified compared to the constant region of wild-type human IgG1. In some embodiments, the modified human IgG heavy chain constant region is modified by one or more amino acid substitutions compared to SEQ ID NO:196 (or a naturally occurring variant thereof, such as a variant having a K97R, D239E, or L241M mutation) and exhibits at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO:196 or a naturally occurring variant thereof, and has a sequence that contains one or more amino acid substitutions, e.g., to reduce an effector activity of the heavy chain constant region.

[0186] Various examples of mutations to heavy chain constant regions to alter, e.g., reduce, effector function are known, including any of those described below. In some embodiments, references to amino acid substitutions in heavy chain constant regions are according to EU numbering by Kabat (also referred to as Kabat numbering), unless otherwise noted with reference to a particular SEQ ID NO:. EU numbering is known and follows the latest IMGT Scientific Chart (IMGT®, International ImMunoGeneTics Information System®, http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (created: May 17, 2001, last updated: January 10, 2013) and the EU index, as reported in Kabat, EA et al. Sequences of Proteins of Immunological interest. 5th ed. US Department of Health and Human Services, NIH publication No. 91-3242 (1991).

[0187] In some embodiments, modified heavy chain constant regions exhibiting reduced effector functions may be desirable candidates for applications in which binding of the chimeric antibody to cell surface targets, e.g., binding of IL-15 sequences to IL-15 receptor subunits, is desired, but certain effector functions, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC), are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the provided chimeric antibody lacks FcγR binding (and thus likely lacks ADCC activity). In some embodiments, the provided chimeric antibody lacks FcγR binding and retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII and FcγRIII. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (Cell Technology, Inc. Mountain View, Calif.); and CytoTox 96™ Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively or additionally, the ADCC activity of the molecule of interest may be evaluated in vivo, for example in an animal model, such as the model disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be carried out to confirm that the multispecific polypeptide construct or its truncated components cannot bind C1q, and therefore lack CDC activity.See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0188] In some embodiments, the heavy chain constant region is modified to alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement dependent cytotoxicity (CDC), e.g., amino acid modifications are made to the heavy chain constant region as described in Natsume et al., 2008 Cancer Res, 68(10):3863-72; Idusogie et al., 2001 J Immunol, 166(4):2571-5; Moore et al., 2010 mAbs, 2(2):181-189; Lazar et al., 2006 PNAS, 103(11):4005-4010, Shields et al., 2001 JBC, 276(9):6591-6604; Stavenhagen et al., 2007 Cancer Res, 67(18):8882-8890; Stavenhagen et al., 2008 Advan. Enzyme Regul., 48:152-164; Alegre et al, 1992 J Immunol, 148:3461-3468; Reviewed in Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11.

[0189] In some embodiments, the heavy chain constant region has been altered at one or more of the following positions to reduce Fc receptor binding: Leu234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325), Ala327 (A327), or Pro329 (P329). For example, Leu 234Ala (L234A), Leu235Ala (L235A), Leu235Glu (L235E), Asp265Asn (D265N), Asp265Ala (D265A), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Pro329Ala (P329A) or Pro239Gly (P329G), Asn325Glu (N325E) or Ala327Ser (A327S). In some embodiments, modifications within the heavy chain constant region reduce binding to Fc receptor-gamma receptors but have minimal effect on binding to neonatal Fc receptor (FcRn).

[0190] In some embodiments, the heavy chain constant region is modified at amino acid Asn297 (Kabat numbering), e.g., Asn297Ala (N297A) or Asn297Asp (N297D), to prevent glycosylation of the chimeric antibody. In some embodiments, the heavy chain constant region is modified at amino acid Leu235 (Kabat numbering), e.g., Leu235Glu (L235E) or Leu235Ala (L235A), to alter Fc receptor interactions. In some embodiments, the heavy chain constant region of the chimeric antibody is modified at amino acid Leu234 (Kabat numbering), e.g., Leu234Ala (L234A), to alter Fc receptor interactions. In some embodiments, the heavy chain constant region of the chimeric antibody is modified at amino acid Leu234 (Kabat numbering), e.g., Leu235Glu (L235E), to alter Fc receptor interactions. In some embodiments, the heavy chain constant region of the chimeric antibody is altered at both amino acids 234 and 235, e.g., Leu234Ala and Leu235Ala (L234A / L235A) or Leu234Val and Leu235Ala (L234V / L235A). In some embodiments, the modified heavy chain constant region comprises Leu234Ala and Leu235Ala (L234A / L235A) mutations. In some embodiments, the heavy chain constant region of the chimeric antibody is altered at amino acids 234, 235, and 297, e.g., Leu234Ala, Leu235Ala, Asn297Ala (L234A / L235A / N297A). In some embodiments, the heavy chain constant region of the chimeric antibody is altered at amino acids 234, 235, and 329, e.g., Leu234Ala, Leu235Ala, Pro239Ala (L234A / L235A / P329A). In some embodiments, the heavy chain constant region of the chimeric antibody is altered at amino acid Asp265 (Kabat numbering), e.g., Asp265Ala (D265A), to alter Fc receptor interactions.In some embodiments, the heavy chain constant region of the chimeric antibody is modified at amino acid Pro329 (Kabat numbering), e.g., Pro329Ala (P329A) or Pro329Gly (P329G), to alter Fc receptor interactions. In some embodiments, the heavy chain constant region of the chimeric antibody is modified at both amino acids 265 and 329, e.g., Asp265Ala and Pro329Ala (D265A / P329A) or Asp265Ala and Pro329Gly (D265A / P329G). In some embodiments, the heavy chain constant region of the chimeric antibody is modified at amino acids 234, 235, and 265, e.g., Leu234Ala, Leu235Ala, Asp265Ala (L234A / L235A / D265A). In some embodiments, the heavy chain constant region of the chimeric antibody is modified at 234, 235, and 329 amino acids, such as Leu234Ala, Leu235Ala, Pro329Gly (L234A / L235A / P329G). In some embodiments, the heavy chain constant region of the chimeric antibody is modified at 234, 235, 265, and 329 amino acids, such as Leu234Ala, Leu235Ala, Asp265Ala, Pro329Gly (L234A / L235A / D265A / P329G). In some embodiments, the heavy chain constant region of the chimeric antibody is modified at Gly235 to reduce Fc receptor binding. For example, in this case, Gly235 is deleted from the heavy chain constant region of the chimeric antibody. In some embodiments, the heavy chain constant region of the chimeric antibody is modified at amino acid Gly236, e.g., Gly236Ala (G236A), to enhance interaction with CD32A. In some embodiments, the heavy chain constant region of the chimeric antibody lacks Lys447 (Kabat et al 1991 EU Index of Sequences of Proteins of Immunological Interest).

[0191] In some embodiments, the heavy chain constant region of the chimeric antibody lacks amino acids at one or more of the following positions: Glu233 (E233), Leu234 (L234), or Leu235 (L235) to reduce Fc receptor binding. In some embodiments, the heavy chain constant region of the chimeric antibody lacks amino acids at one or more of the following positions: Glu233 (E233), Leu234 (L234), or Leu235 (L235), and is modified at one or more of Asp265 (D265), Asn297 (N297), or Pro329 (P329) to reduce Fc receptor binding. In some embodiments, the heavy chain constant region of the chimeric antibody comprises three amino acid deletions in the lower hinge corresponding to IgG1 E233, L234, and L235. In some aspects, such heavy chain constant regions do not engage FcγR and are therefore referred to as "effector silent" or "effector null."

[0192] In some embodiments, the modified heavy chain constant region comprises Leu234Ala and Leu235Ala (L234A / L235A) mutations. In some embodiments, the modified heavy chain constant region comprises a sequence set forth in SEQ ID NO: 187, or a sequence with reduced effector activity that exhibits at least or at least about 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 187. In some embodiments, the modified VH region comprises a sequence set forth in SEQ ID NO: 187. In some embodiments, the modified heavy chain constant region comprises a sequence set forth in SEQ ID NO:188, or a sequence with reduced effector activity that exhibits at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99% sequence identity to the sequence set forth in SEQ ID NO:188.In some embodiments, the modified VH region comprises the sequence shown in SEQ ID NO:188.

[0193] B. Light Chain Region In some embodiments, the provided chimeric antibody or antigen-binding fragment further comprises a light chain variable region. In some embodiments, the chimeric antibody variable heavy chain region or heavy chain is based on bovine sequences and is paired with a variable light region or light chain of a bovine antibody. In some embodiments, the chimeric antibody variable heavy chain region or heavy chain is based on humanized sequences and is paired with a variable light region or light chain of a bovine antibody. In some embodiments, the chimeric antibody variable heavy chain region or heavy chain is based on humanized sequences and is paired with a humanized variable light region or light chain of a bovine antibody. In some embodiments, the light chain is a lambda light chain.

[0194] In some embodiments, the variable light region is a variable light region of a bovine antibody, such as the variable light region of BLVH12, BLV5D3, BLV8C11, BF1H1, BLV5B8, and / or F18. In some embodiments, the light chain variable region may comprise a sequence based on or derived from the polypeptide sequence of SEQ ID NO:27 or 29. In some embodiments, the light chain polypeptide sequence is encoded by a DNA sequence based on or derived from the DNA sequence of SEQ ID NO:8. In some embodiments, the light chain polypeptide sequence is encoded by a DNA sequence based on or derived from the DNA sequence of SEQ ID NO:168.

[0195] In some embodiments, the light chain comprises the variable light region of a bovine antibody bound to a human lambda light chain constant region (e.g., as shown in SEQ ID NO: 155). In some embodiments, a portion of the BLV1H12 light chain variable region (e.g., as shown in SEQ ID NO: 8 or SEQ ID NO: 168) is bound to a human lambda light chain constant region.

[0196] In some embodiments, the light chain is a humanized light chain or a human light chain. In some embodiments, the present disclosure provides for pairing of a humanized heavy chain comprising an ultralong CDR3 with a human light chain. In some embodiments, the light chain is homologous to a bovine light chain known to pair with a bovine ultralong CDR3 heavy chain. Some human VL sequences, including VL1-47, VL1-40, VL1-51, and VL2-18, which are homologous to lambda regions from Bos Taurus, can be used to pair with the above sequences. In some embodiments, the light chain variable region is a sequence shown in any one of SEQ ID NOs: 156 or 173-176. In some embodiments, the light chain variable sequence is a sequence encoded by a sequence shown in any one of SEQ ID NOs: 177-180. In some embodiments, the light chain variable region comprises the variable region of the VL1-51 germline sequence shown in SEQ ID NO:156.

[0197] In some embodiments, the light chain variable region is a human germline light chain sequence, such as any of those described above, containing one or more amino acid modifications. Such modifications may include substitutions of certain amino acid residues in the human light chain with residues at corresponding positions in the bovine light chain sequence. The modified light chain may improve the yield of the antibody comprising the ultralong CDR3 and / or increase its binding specificity. In some embodiments, the modifications include one or more of the amino acid substitutions S2A, T5N, P8S, A12G, A13S, and P14L according to the Kabat numbering. In some embodiments, the modifications include the amino acid substitutions S2A, T5N, P8S, A12G, A13S, and P14L according to the Kabat numbering. In some embodiments, the modifications are in CDR1 and include the amino acid substitutions I29V and N32G. In some embodiments, the modifications are in CDR2 and include the substitution of DNN for GDT. In some embodiments, the modifications are in CDR2 and include the substitution DNNKRP for GDTSRA. In some embodiments, the modifications include any combination of the foregoing. For example, provided modifications of human germline light chain sequences include amino acid substitutions S2A, T5N, P8S, A12G, A13S, and P14L based on Kabat numbering, and a substitution of DNN with GDT in CDR2.

[0198] In some embodiments, the light chain comprises a humanized variable light chain as described above that is linked to a human lambda light chain constant region (e.g., as shown in SEQ ID NO:155). In some embodiments, a portion of a light chain variable region, such as a modified human germline light chain, is linked to a human lambda light chain constant region.

[0199] In some embodiments, the light chain of the provided chimeric antibody is a humanized light chain. In some embodiments, the light chain comprises an amino acid sequence set forth in SEQ ID NO:181, or a sequence of amino acids exhibiting at least or at least about 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:181. In some embodiments, the light chain comprises the sequence set forth in SEQ ID NO: 181. In some embodiments, the sequence of the light chain is set forth in SEQ ID NO: 181.

[0200] C. IL-15Rα SUSHI domain In some embodiments, chimeric cytokine antibodies containing the IL-15 sequences or biologically active portions thereof provided herein may be further linked or complexed to all or a portion of the IL-15 high affinity receptor alpha (IL15Rα) receptor subunit, e.g., a portion containing the extracellular domain of IL15Rα. In some embodiments, all or a portion of IL15Rα is linked or complexed to the provided chimeric antibodies to increase trans-signaling to the receptor beta and gamma subunits (IL2 / 15Rβ and γc) receptor subunits.

[0201] In some embodiments, the chimeric antibodies provided are linked or complexed to a portion of the extracellular domain of IL15Rα. In some embodiments, the chimeric antibodies provided are linked or complexed to an IL15Rα sushi domain. In some embodiments, the IL15Rα sushi domain comprises the sequence shown in SEQ ID NO:2.

[0202] In some embodiments, provided herein is a chimeric IL-15 modified antibody or antigen-binding fragment, the heavy chain or variable sequence thereof comprising an IL-15 sequence or a biologically active portion thereof (e.g., the IL-15 sequence is disposed between the ascending and descending stalks of the ultralong CDR3) linked or complexed with an extracellular domain of IL15Rα, e.g., the IL15Rα sushi domain, and replacing all or a portion of the knob region of the ultralong CDR3 of a bovine antibody or humanized sequence thereof. In some embodiments, the chimeric IL-15 modified antibody or antigen-binding fragment is complexed with the IL15Rα sushi domain as shown in SEQ ID NO:2.

[0203] In some embodiments, a chimeric antibody can be generated by co-expressing all or a portion of an IL15Rα extracellular domain, e.g., an IL15Rα sushi domain, e.g., as set forth in SEQ ID NO:2, with the heavy and light chain regions of a chimeric antibody in a host cell. In some embodiments, an IL15Rα sushi domain, e.g., as set forth in SEQ ID NO:2, is co-expressed in a host cell with the heavy and light chain regions of a chimeric antibody.

[0204] In some embodiments, the IL-15 cytokine sequence is linked to all or a portion of the IL15Rα extracellular domain, e.g., the IL15Rα sushi domain, e.g., as shown in SEQ ID NO: 2. In some embodiments, the IL-15 sequence and the IL15Rα sushi domain sequence are disposed between the ascending and descending stalks of the ultralong CDR3.

[0205] In some embodiments, the heavy chain or variable sequence of the chimeric antibody is linked to an extracellular domain of IL15Rα, e.g., the IL15Rα sushi domain. In some embodiments, the light chain or variable sequence of the chimeric antibody is linked to an extracellular domain of IL15Rα, e.g., the IL15Rα sushi domain.

[0206] In some embodiments, provided herein is a chimeric IL-15 modified antibody or antigen-binding fragment that contains a heavy chain or variable sequence thereof in which an IL-15 sequence replaces all or part of the knob of an ultralong CDR3 (e.g., disposed between the ascending and descending stalks of the ultralong CDR3) and a light chain or variable sequence thereof linked to an extracellular domain of IL15Rα, e.g., the IL15Rα sushi domain. In some embodiments, the chimeric IL-15 modified antibody or antigen-binding fragment is linked to the IL15Rα sushi domain depicted in SEQ ID NO:2. In some embodiments, the light chain comprises a sequence encoded by SEQ ID NO:168 or is a variable sequence thereof. In some embodiments, the light chain comprises a sequence depicted in SEQ ID NO:181 or is a variable sequence thereof. The linkage between the extracellular domain of IL15Rα (e.g., IL15Rα sushi domain, e.g., as set forth in SEQ ID NO:2) and its light chain or variable sequence is via a peptide linker. In some embodiments, the linker is a flexible linker, such as a glycine linker or a glycine-serine (GS) linker. In some embodiments, the peptide linker is a GS linker. Exemplary GS linkers include, but are not limited to, any of the sequences set forth in SEQ ID NOs:150-154, or the sequences encoded by the nucleotide sequences set forth in SEQ ID NOs:163 or SEQ ID NO:164. In some embodiments, the linker is GS.

[0207] In some embodiments, a chimeric IL-15 modified antibody or antigen-binding fragment provided herein contains a heavy chain or variable sequence thereof in which an IL-15 sequence replaces all or part of the knob of an ultralong CDR3 (e.g., is located between the ascending and descending stalks of the ultralong CDR3), and a light chain or variable sequence thereof comprising a sequence of amino acids encoded by SEQ ID NO:3.

[0208] D. Vectors, Host Cells, and Recombinant Methods The provided chimeric antibodies or antigen-binding fragments can be produced according to any suitable method, including, for example, the use of polynucleotides encoding the antibodies or fragments thereof, or the heavy or light chains thereof. As an example, the polynucleotides can be inserted into replicable vectors that are used for the ultimate expression of the provided chimeric antibodies or antigen-binding fragments, for example, by a host cell into which the vector is introduced. Such polynucleotides, vectors, e.g., expression vectors, and host cells are also provided herein, including any of those described herein.

[0209] For recombinant production of the antibody or fragment thereof disclosed herein, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of DNA) or expression. The DNA encoding the antibody is easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody). In an exemplary embodiment, an antibody comprising an ultralong CDR3, a variable region comprising an ultralong CDR3, or a nucleic acid encoding an ultralong CDR3 is isolated and inserted into a replicable vector for further cloning (amplification of DNA) or expression. Many vectors are available. The choice of vector depends in part on the host cell to be used. In general, preferred host cells are of prokaryotic or eukaryotic (generally mammalian) origin. It will be understood that the constant region of any isotype, including IgG, IgM, IgA, IgD, and IgE constant regions, can be used for this purpose, and such constant regions can be obtained from any human or animal species.

[0210] Expression vectors containing regulatory elements derived from eukaryotic viruses are typically used in eukaryotic expression vectors, such as SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector that allows expression of proteins under the direction of the CMV promoter, the SV40 early promoter, the SV40 late promoter, the metallothionein promoter, the murine mammary tumor virus promoter, the Rous sarcoma virus promoter, the polyhedrin promoter, or other promoters that have been shown to be effective for expression in eukaryotic cells.

[0211] Some expression systems have markers that provide gene amplification, such as thymidine kinase and dihydrofolate reductase. Alternatively, high-yield expression systems without gene amplification are also suitable, for example, using baculovirus vectors in insect cells with nucleic acid sequences encoding partially human ultralong CDR3 antibody chains under the direction of the polyhedrin promoter or other strong baculovirus promoters.

[0212] Polynucleotide sequences encoding the polypeptide components of the antibodies disclosed herein can be obtained using standard recombinant techniques. In some embodiments, the polynucleotides can be synthesized using a nucleotide synthesizer or PCR techniques. Once obtained, the sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. For the purposes of this disclosure, many vectors available and known in the art can be used. The selection of an appropriate vector depends primarily on the size of the nucleic acid to be inserted into the vector and the specific host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification or expression of heterologous polynucleotide, or both) and its compatibility with the specific host cell in which it resides. Vector components generally include, but are not limited to, an origin of replication, a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, a heterologous nucleic acid insert, and a transcription termination sequence. In addition, a V region containing an ultralong CDR3 can be optionally fused to a C region to produce an antibody containing a constant region.

[0213] Generally, plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used in connection with these hosts. The vectors usually have a replication site, as well as marking sequences that can provide phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from E. coli species. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance, thus providing an easy means for identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophages may also contain, or be modified to contain, promoters that can be used by the microorganism for expression of endogenous proteins. Examples of pBR322 derivatives used for the expression of certain antibodies have been described (see, for example, U.S. Pat. No. 5,648,237).

[0214] Additionally, phage vectors containing replicon and control sequences compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, bacteriophages such as λGEM™-11 can be utilized to generate recombinant vectors that can be used to transform susceptible host cells such as E. coli LE392.

[0215] The expression vector disclosed herein may contain two or more promoter-cistron pairs, each encoding a polypeptide component. A promoter is a non-translated regulatory sequence located upstream (5') of a cistron that modulates its expression. Prokaryotic promoters are typically divided into two classes: inducible and constitutive. An inducible promoter is a promoter that initiates an increase in the transcription level of the cistron under its control in response to a change in culture condition, such as the presence or absence of a nutrient or a change in temperature.

[0216] A large number of promoters that are recognized by various potential host cells are well known. The selected promoter can be functionally linked to the cistron DNA encoding the light or heavy chain by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector disclosed herein. Both the native promoter sequence and many heterologous promoters can be used to direct the amplification and / or expression of the target gene. In some embodiments, heterologous promoters are utilized because they generally allow greater transcription and higher yield of expressed target genes compared to the native target polypeptide promoter.

[0217] Suitable promoters for use in prokaryotic hosts include ara B promoter, PhoA promoter, β-galactamase and lactose promoter systems, tryptophan (trp) promoter systems, and hybrid promoters such as tac or trc promoters. However, other promoters that are functional in bacteria (such as other known bacterial or phage promoters) are also suitable. Their nucleotide sequences are published, allowing those skilled in the art to operably ligate them to the cistrons encoding the target light and heavy chains using linkers or adapters (e.g., Siebenlist et al. (1980) Cell 20:269) and provide any necessary restriction sites.

[0218] Suitable bacterial promoters are well known in the art and are fully described in the scientific literature in Sambrook and Russell, supra, and Ausubel et al., supra. Bacterial expression systems for expressing antibody chains of recombinant catalytic polypeptides are available, for example, in E. coli, Bacillus species, and Salmonella (Palva et al., Gene, 22:229-235 (1983); Mosbach et al., Nature, 302:543-545 (1983)).

[0219] In one aspect disclosed herein, each cistron in the recombinant vector contains a secretion signal sequence component that directs translocation of the expressed polypeptide across the membrane. In general, the signal sequence may be a component of the vector or may be part of the target polypeptide DNA inserted into the vector. The signal sequence must be recognized and processed (e.g., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native signal sequence of the heterologous polypeptide, the signal sequence is replaced with a prokaryotic signal sequence selected from, for example, PelB, OmpA, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leader, LamB, PhoE, and MBP. In one embodiment disclosed herein, the signal sequence used in both cistrons of the expression system is a STII signal sequence or a variant thereof.

[0220] In another aspect, the production of immunoglobulins according to the present disclosure can occur in the cytoplasm of the host cell, and therefore does not require the presence of a secretion signal sequence in each cistron. In that regard, immunoglobulin light and heavy chains are expressed, folded, and assembled to form functional immunoglobulins in the cytoplasm. Certain host strains (e.g., E. coli trxB strains) provide cytoplasmic conditions that favor disulfide bond formation, thereby allowing proper folding and assembly of expressed protein subunits (see, e.g., Proba and Pluckthun Gene, 159:203 (1995)).

[0221] Suitable host cells for cloning or expressing the vector encoding the antibody include prokaryotic or eukaryotic cells as described herein.In one embodiment, the host cell is a eukaryotic cell, such as Chinese Hamster Ovary (CHO) cell, Human Embryonic Kidney (HEK) cell or lymphoid cell (e.g., YO, NSO, Sp20 cell).For example, the antibody can be produced in bacteria, especially when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes expression of antibody fragments in E. coli). After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns. See Gemgross, Nat. Biotech. 22:1409-1414(2004), and Li et al., Nat. Biotech. 24:210-215(2006). Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculoviral strains have been identified that can be used in conjunction with the transfection of insect cells, particularly Spodoptera frugiperda cells. These examples are illustrative and not limiting.Methods for constructing derivatives of any of the above bacteria with defined genotypes are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). In general, the appropriate bacterium should be selected taking into account the replicability of the replicon in the bacterial cell. For example, when the replicon is provided using well-known plasmids such as pBR322, pBR325, pACYC177 or pKN410, E. coli, Serratia or Salmonella species can be suitably used as hosts. Typically, the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors can be desirably incorporated into the cell culture.

[0222] Plant cell cultures can also be used as hosts.See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants).Vertebrate cells can also be used as hosts.For example, mammalian cell lines adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 lines transformed with SV40 (COS-7), human embryonic kidney lines (e.g., 293 or 293 cells described in Graham et al., Gen VlI'0l. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK, buffalo rat hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor cells (MMT 060562), e.g., Mather et al., Annals NI'. Acad. Sci. 383:44-68(1982), TR1 cells, MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR'CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)), and myeloma cell lines such as YO, NSO, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, N.), pp. 255-268(2003).

[0223] In one such embodiment, the host cell contains (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody.

[0224] Depending on the host cell used, transformation is performed using standard techniques appropriate for such cells. The calcium treatment using calcium chloride is generally used for bacterial cells that contain substantial cell wall barriers. Another method for transformation uses polyethylene glycol / DMSO. Yet another technique that can be used is electroporation.

[0225] The expressed polypeptides of the present disclosure are secreted into the periplasm of the host cell and are either recovered therefrom or transported into the medium. Protein recovery from the periplasm typically involves disrupting the microorganism, generally by means such as osmotic shock, sonication or lysis. Once the cells are disrupted, the cell debris or whole cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein transported into the medium can be isolated therein. For further purification of the produced protein, the cells can be removed from the culture and the culture supernatant can be filtered and concentrated. The expressed polypeptides can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay.

[0226] Antibody production can be carried out in large quantities by fermentation processes. A variety of large-scale fed-batch fermentation procedures are available for the production of recombinant proteins. Large-scale fermentations have a capacity of at least 1000 liters, preferably about 1,000 to 100,000 liters. These fermenters use stirring impellers to distribute oxygen and nutrients, especially glucose (the preferred carbon / energy source). Small-scale fermentation generally refers to fermentation in fermenters that are no larger than about 100 liters in volume, and can range from about 1 liter to about 100 liters.

[0227] In the fermentation process, induction of protein expression is typically initiated after the cells have grown under appropriate conditions to a desired density, for example an OD550 of about 180-220, at which stage the cells are in early stationary phase. As known in the art and described above, various inducers may be used according to the vector construct used. The cells may be allowed to grow for a shorter period before induction. The cells are usually induced for about 12-50 hours, although longer or shorter induction times may be used.

[0228] Various fermentation conditions can be modified to improve the production yield and quality of the polypeptides disclosed herein. For example, to improve the proper assembly and folding of secreted antibody polypeptides, additional vectors that overexpress chaperone proteins such as Dsb proteins (DsbA, DsbB, DsbC, DsbD, and / or DsbG) or FkpA (peptidyl prolyl cis,trans isomerase with chaperone activity) can be used to co-transform the host prokaryotic cells. Chaperone proteins have been demonstrated to promote proper folding and solubility of heterologous proteins produced in bacterial host cells (see, e.g., Chen et al. (1999) J Bio Chem 274:19601-19605; U.S. Pat. No. 6,083,715; U.S. Pat. No. 6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol. Microbiol. 39:199-210).

[0229] To minimize the proteolysis of expressed heterologous proteins (especially those that are proteolytically sensitive), certain host strains that are deficient in proteolytic enzymes can be used in the present disclosure.For example, host cell strains can be modified to introduce genetic mutations into genes that code for known bacterial proteases, such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof.Several E. coli protease-deficient strains are available (see, for example, Joly et al. (1998), supra; U.S. Patent No. 5,264,365; U.S. Patent No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996)).

[0230] E. coli strains that are deficient in proteolytic enzymes and transformed with plasmids that overexpress one or more chaperone proteins can be used as host cells in the expression systems disclosed herein.

[0231] Standard protein purification methods known in the art can be used. The following procedures are examples of suitable purification procedures: fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or cation exchange resins, such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration, such as using Sephadex G-75.

[0232] In one aspect, protein A immobilized on a solid phase is used for the immunoaffinity purification of the full-length antibody products disclosed herein. Protein A is a 41 kD cell wall protein from Staphylococcus aureas that binds with high affinity to the Fc region of antibodies (see, for example, Lindmark et al (1983) J. Immunol. Meth. 62:1-13). The solid phase on which protein A is immobilized is preferably a column that includes a glass or silica surface, more preferably a controlled pore glass column or a silicic acid column. In some applications, the column is coated with a reagent such as glycerin to prevent non-specific adhesion of contaminants.

[0233] As the first step of purification, the preparation from the cell culture as described above is applied to a protein A immobilized solid phase, and the antibody of interest is specifically bound to protein A. The solid phase is then washed to remove contaminants that are non-specifically bound to the solid phase. Finally, the antibody of interest is recovered from the solid phase by elution.

[0234] III. Pharmaceutical Compositions The antibody or antigen-binding fragment comprising the ultralong CDR3, nucleic acid, or vector disclosed herein can be formulated into a composition, particularly a pharmaceutical composition. Such a composition with an antibody comprising an ultralong CDR3 comprises a therapeutically effective or prophylactically effective amount of an antibody comprising the ultralong CDR3, antibody fragment, nucleic acid, or vector disclosed herein, mixed with a suitable carrier, such as a pharma-ceutically acceptable agent. Typically, the antibody comprising the ultralong CDR3, antibody fragment, nucleic acid, or vector disclosed herein is sufficiently purified for administration before being formulated into a pharmaceutical composition. Such pharmaceutical compositions are provided herein and include any of those described herein.

[0235] Pharmaceutically acceptable agents for use in the pharmaceutical compositions of the present invention include carriers, excipients, diluents, antioxidants, preservatives, colorants, flavorings and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, isotonicity agents, co-solvents, wetting agents, complexing agents, buffering agents, antibacterial agents, and surface active agents.

[0236] Neutral buffered saline or saline mixed with serum albumin is an exemplary suitable carrier. Pharmaceutical compositions can include antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as Tween, Pluronics, or polyethylene glycol (PEG). Also, by way of example, suitable tonicity enhancers include alkali metal halides (preferably sodium chloride or potassium chloride), mannitol, sorbitol, etc. Suitable preservatives include benzalkonium chloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, and the like. Hydrogen peroxide may also be used as a preservative. Suitable co-solvents include glycerin, propylene glycol, and PEG. Suitable complexing agents include caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxy-propyl-beta-cyclodextrin. Suitable surfactants or wetting agents include sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapal, and the like. The buffer may be a conventional buffer such as acetate, borate, citrate, phosphate, bicarbonate, or Tris-HCl. The acetate buffer may be about pH 4-5.5, and the Tris buffer may be about pH 7-8.5. Additional pharmaceutical agents are described in Remington's Pharmaceutical Sciences, 18th Edition, AR Gennaro, ed., Mack Publishing Company, 1990.

[0237] The composition may be in liquid form or lyophilized or freeze-dried form and may include one or more cryoprotectants, excipients, surfactants, high molecular weight structural additives and / or bulking agents (see, e.g., U.S. Pat. Nos. 6,685,940, 6,566,329, and 6,372,716). In one embodiment, a cryoprotectant is included that is a non-reducing sugar, such as sucrose, lactose, or trehalose. The amount of cryoprotectant typically included is such that the resulting formulation upon reconstitution is isotonic, although hypertonic or slightly hypotonic formulations may also be appropriate. Additionally, the amount of cryoprotectant should be sufficient to prevent unacceptable amounts of protein degradation and / or aggregation upon lyophilization. Exemplary cryoprotectant concentrations relative to sugar (e.g., sucrose, lactose, trehalose) in the pre-lyophilized formulation are from about 10 mM to about 400 mM. In another embodiment, surfactants, such as non-ionic surfactants and ionic surfactants, such as polysorbates (e.g., polysorbate 20, polysorbate 80); poloxamers (e.g., poloxamer 188); poly(ethylene glycol) phenyl ethers (e.g., Triton); sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl- sarcosine; linoleyl, myristyl, or cetyl betaine; lauroamidopropyl, cocamidopropyl, linoleamidopropyl, myristamidopropyl, palmidopropyl, or isostearamidopropyl betaine (e.g., lauroamidopropyl); myristamidopropyl, palmidopropyl, or isostearamidopropyl dimethylamine; sodium methyl cocoyl or disodium methyl ofeyl taurate; and the MONAQUAT™ series (Mona Industries, Inc., Paterson, NJ), polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, etc.).An exemplary amount of surfactant that may be present in the pre-lyophilized formulation is about 0.001-0.5%. High molecular weight structural additives (e.g., bulking agents, binders) include, for example, gum arabic, albumin, alginic acid, calcium phosphate (dibasic), cellulose, carboxymethylcellulose, sodium carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, dextran, dextrin, dextrates, sucrose, tylose, pregelatinized starch, calcium sulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose, disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfite, polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose, compressed sugar, magnesium aluminum silicate, maltodextrin, polyethylene oxide, polymethacrylate, povidone, sodium alginate, tragacanth microcrystalline cellulose, starch, and zein. Exemplary concentrations of the high molecular weight structural additive are 0.1% to 10% by weight. In other embodiments, bulking agents (e.g., mannitol, glycine) may be included.

[0238] The composition may be suitable for parenteral administration.Exemplary compositions are suitable for injection or infusion into animals by any route available to those skilled in the art, such as intraarticular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, or intralesional.Parenteral formulations are typically sterile, pyrogen-free, isotonic aqueous solutions, optionally containing pharmaceutically acceptable preservatives.

[0239] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, inert gases, and the like. See generally Remington's Pharmaceutical Science, 16th Ed., Mack Eds., 1980.

[0240] The pharmaceutical compositions described herein can be formulated for controlled or sustained delivery in a manner that provides local concentration (e.g., bolus, depot effect) and / or increased stability or half-life of the product in a particular local environment. The compositions can include microparticle preparations of polymeric compounds such as polylactic acid, polyglycolic acid, and the like, as well as preparations of antibodies comprising ultralong CDR3, antibody fragments, nucleic acids or vectors disclosed herein, with means such as biodegradable matrices, injectable microspheres, microcapsule particles, microcapsules, biodegradable particle beads, liposomes, and implantable delivery devices that provide controlled or sustained release of active agents and can then be delivered as depot injections. The techniques for formulating such sustained or controlled delivery means are known, and various polymers have been developed and used for controlled release and delivery of drugs. Such polymers are typically biodegradable and biocompatible. Polymer hydrogels, including those formed by the complexation of mirror-image polymer or polypeptide segments, and hydrogels with temperature or pH-sensitive properties may be desirable to provide a drug depot effect due to the mild, aqueous conditions involved in the entrapment of bioactive protein agents (e.g., antibodies with ultralong CDR3s). See, for example, the description of controlled release porous polymeric microparticles for the delivery of pharmaceutical compositions in WO 93 / 15722.

[0241] Suitable materials for this purpose include polylactide (see, e.g., U.S. Pat. No. 3,773,919), polymers of poly-(a-hydroxycarboxylic acids), such as poly-D-(-)-3-hydroxybutyric acid (EP 133,988A), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981), and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Other biodegradable polymers include poly(lactones), poly(acetals), poly(orthoesters), and poly(orthocarbonates). Sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art (see, for example, Eppstein et al., Proc. Natl. Acad. Sci. USA, 82:3688-92 (1985)). The carrier itself or its degradation products should be non-toxic in the target tissue and should not further exacerbate the condition. This can be determined by routine screening in animal models of the target disorder, or in normal animals if such models are not available.

[0242] Microencapsulation of recombinant proteins for sustained release has been successfully performed with human growth hormone (rhGH), interferon- (rhIFN-), interleukin-2 and MN rgp120. Johnson et al., Nat. Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993); Hora et al., Bio / Technology. 8:755-758 (1990); Cleland, "Design and Production of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere Systems", in Vaccine Design: The Subunit and Adjuvant Approach, Powell and Newman, eds, (Plenum Press: New York, 1995), pp. 439-462; WO 97 / 03692, WO 96 / 40072, WO 96 / 07399; and U.S. Pat. No. 5,654,010. Sustained release formulations of these proteins have been developed using poly-lactic-coglycolic acid (PLGA) polymers due to their biocompatibility and wide range of biodegradable properties. The degradation products of PLGA, lactic acid and glycolic acid can be rapidly cleared in the human body. Furthermore, the degradability of this polymer may depend on its molecular weight and composition. Lewis, "Controlled release of bioactive agents from lactide / glycolide polymer", in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pp. 1-41.Further examples of sustained release compositions include, for example, EP 58,481A, U.S. Pat. No. 3,887,699, EP 158,277A, Canadian Patent No. 1176565, U. Sidman et al., Biopolymers 22,547

[1983] , R. Langer et al., Chem. Tech. 12,98

[1982] , Sinha et al., J. Control. Release 90,261

[2003] , Zhu et al., Nat. Biotechnol. 18,24

[2000] , and Dai et al., Colloids Surf B Biointerfaces 41,117

[2005] .

[0243] Bioadhesive polymers are also contemplated for use in or with the compositions of the present disclosure. Bioadhesives are synthetic and natural materials that can adhere to biological substrates for extended periods of time. For example, Carbopol and Polycarbophil are both synthetic cross-linked derivatives of poly(acrylic acid). Bioadhesive delivery systems based on natural substances include, for example, hyaluronic acid, also known as hyaluronan. Hyaluronic acid is a natural mucopolysaccharide that consists of residues of D-glucuronic acid and N-acetyl-D-glucosamine. Hyaluronic acid is found in the extracellular tissue matrix of vertebrates, including connective tissue, synovial fluid, and the vitreous and aqueous humor of the eye. Esterified derivatives of hyaluronic acid have been used to produce microspheres for use in delivery that are biocompatible and biodegradable (see, e.g., Cortivo et al., Biomaterials (1991) 12:727-730; EP 517,565; WO 96 / 29998; Illum et al., J. Controlled Rel. (1994) 29:133-141). Exemplary hyaluronic acid-containing compositions of the present disclosure include hyaluronic acid ester polymers in an amount of about 0.1% to about 40% (w / w) of an antibody containing an ultralong CDR3 relative to the hyaluronic acid polymer.

[0244] Both biodegradable and non-biodegradable polymer matrices can be used to deliver the compositions of the present disclosure, and such polymer matrices can include natural or synthetic polymers. Biodegradable matrices are preferred. The period over which release occurs is based on the choice of polymer. Typically, release over a period ranging from a few hours to 3-12 months is most desirable. Exemplary synthetic polymers that can be used to form the biodegradable delivery system include polymers of lactic and glycolic acid, polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidones, polyglycolides, polysiloxanes, polyanhydrides, polyurethanes and their copolymers, poly(butyric acid), ... acid), poly(valeric acid), alkyl cellulose, hydroxyalkyl cellulose, cellulose ethers, cellulose esters, nitrocellulose, polymers of acrylic and methacrylic acid esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, sodium cellulose sulfate, poly(methyl methacrylate), poly(ethyl methacrylate), poly Poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohol), polyvinyl acetate, polyvinyl chloride, polystyrene, and polyvinylpyrrolidone.Exemplary natural polymers include alginate and other polysaccharides, including dextran and cellulose, collagen, its chemical derivatives (substitution, addition, hydroxylation, oxidation, and other modifications of chemical groups, such as alkyl, alkylene, and the like, routinely performed by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamins and hydrophobic proteins, copolymers and mixtures thereof. Generally, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, surface or bulk erosion. The polymer is optionally in the form of a hydrogel (e.g., WO 04 / 009664, WO 05 / 087201, Sawhney, et al., Macromolecules, 1993, 26, 581-587), which can absorb up to about 90% of its weight in water and is further optionally crosslinked with multivalent ions or other polymers.

[0245] Delivery systems also include non-polymeric systems that are lipids, including sterols such as cholesterol, cholesterol esters, and fatty acids, or neutral fats such as mono-, di-, and triglycerides; hydrogel release systems; silastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants, etc. Specific examples include, but are not limited to, (a) erosion systems in which the product is contained in the form of a matrix, such as those described in U.S. Patents 4,452,775, 4,675,189, and 5,736,152, and (b) diffusion systems in which the product permeates through a polymer at a controlled rate, such as those described in U.S. Patents 3,854,480, 5,133,974, and 5,407,686. Liposomes containing the products can be prepared by known methods, such as (German Patent No. 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030-4034 (1980); European Patent No. 52,322; European Patent No. 36,676; European Patent No. 88,046; European Patent No. 143,949; European Patent No. 142,641; JP 83-118008; U.S. Patent Nos. 4,485,045 and 4,544,545; and European Patent No. 102,324).

[0246] Alternatively or additionally, the composition may be administered locally by implantation into the affected area of ​​a membrane, sponge or other suitable material in which the antibody comprising the ultralong CDR3, antibody fragment, nucleic acid or vector disclosed herein is absorbed or encapsulated. When an implantable device is used, the device may be implanted into any suitable tissue or organ, and delivery of the antibody comprising the ultralong CDR3 antibody fragment, nucleic acid or vector disclosed herein may be via bolus or continuous administration, or directly through the device via a catheter using continuous infusion.

[0247] Pharmaceutical compositions comprising antibodies comprising ultralong CDR3, antibody fragments, nucleic acids or vectors disclosed herein may be formulated, for example, as dry powders for inhalation. Inhalation solutions may also be formulated into liquefied propellants for aerosol delivery. In yet another formulation, the solution may be nebulized. Additional pharmaceutical compositions for pulmonary administration include those described, for example, in WO 94 / 20069, which discloses pulmonary delivery of chemically modified proteins. For pulmonary delivery, the particle size should be suitable for delivery to the distal lung. For example, the particle size may be between 1 μm and 5 μm. However, larger particles may be used, for example, when each particle is fairly porous.

[0248] Certain formulations containing the antibody comprising the ultralong CDR3, antibody fragment, nucleic acid or vector disclosed herein may be administered orally.The formulations administered in this manner may be formulated with or without carriers that are normally used in the preparation of solid dosage forms such as tablets and capsules.For example, capsules can be designed to release the active portion of the formulation at the point in the gastrointestinal tract where bioavailability is at its highest and pre-systemic degradation is at its lowest.Additional agents can be included to promote the absorption of selective binding agents.Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be used.

[0249] Another preparation may contain an effective amount of the antibody comprising the ultralong CDR3, antibody fragment, nucleic acid or vector disclosed herein in a mixture with non-toxic excipients suitable for tablet manufacture.By dissolving tablets in sterile water or other suitable vehicle, the solution can be prepared in unit dose form.Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binders such as starch, gelatin, or gum arabic; or lubricants such as magnesium stearate, stearic acid, or talc.

[0250] Suitable and / or preferred pharmaceutical formulations can be determined based on the intended route of administration, delivery format, and desired dosage, taking into consideration the present disclosure and general knowledge of formulation technology.Regardless of the mode of administration, effective dosage can be calculated according to patient's weight, body surface area, or organ size.Further refinement of calculations to determine suitable dosage for treatment, including each of the formulations described herein, is routine in the art and falls within the scope of routine work in the art.Suitable dosage can be confirmed by using suitable dose-response data.

[0251] In some embodiments, the antibody comprising an ultralong CDR3 or a fragment thereof comprises a modified Fc region, in which the native Fc region is modified to increase the half-life of the antibody or fragment in a biological environment, for example, the half-life as measured by serum half-life or in vitro assay. Methods for altering the original form of the Fc region of IgG are also described in U.S. Patent No. 6,998,253.

[0252] In certain embodiments, it may be desirable to modify the antibody or fragment to increase serum half-life, for example, by adding a molecule such as PEG or other water-soluble polymers, including polysaccharide polymers, to the antibody fragment to increase half-life. This can also be achieved, for example, by incorporating a salvage receptor binding epitope into the antibody fragment (e.g., by mutating the appropriate region in the antibody fragment, or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at the end or in the middle, for example, by DNA or peptide synthesis) (see WO 96 / 32478). Salvage receptor binding epitope refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

[0253] The salvage receptor binding epitope may comprise a region in which any one or more amino acid residues from one or two loops of the Fc domain are transferred to a similar position of the antibody fragment. Even more preferably, three or more residues from one or two loops of the Fc domain are transferred. Even more preferably, the epitope is taken from the CH2 domain of the Fc region (e.g., of an IgG) and transferred to the CH1, CH3, or VH region of the antibody, or more than one such region. Alternatively, the epitope is taken from the CH2 domain of the Fc region and transferred to the CL or VL region of the antibody fragment, or both. See also WO 97 / 34631 and WO 96 / 32478, which describe Fc variants and their interactions with salvage receptors.

[0254] IV. How to use Provided herein are methods and uses of compositions containing chimeric cytokine (e.g., IL-15) modified antibodies or antigen-binding fragments, for example in connection with the modulation of immune cells such as T cells and NK cells, and in methods for treating diseases or conditions.

[0255] In some aspects, provided herein are methods of stimulating cells, e.g., immune cells, using chimeric antibodies. In some aspects, provided herein are methods of expanding cells, e.g., immune cells, using chimeric antibodies.

[0256] In some embodiments, a population of cells, e.g., immune cells, is contacted with a chimeric antibody, thereby stimulating the cells, e.g., cells of the population of immune cells. In some embodiments, a population of cells, e.g., immune cells, is contacted with a chimeric antibody, thereby promoting proliferation of the cells, e.g., cells of the population of immune cells.

[0257] In some embodiments, the population of cells, e.g., immune cells, comprises cells expressing an IL-15 receptor subunit, such as an IL2 / 15Rβ and / or an IL2 / 15Rβ γc receptor subunit. In some embodiments, the population of cells, e.g., immune cells, comprises T cells. In some embodiments, the population of cells, e.g., immune cells, comprises natural killer (NK) cells.

[0258] In some aspects, the methods provided are performed ex vivo or in vitro. In some aspects, the methods provided are performed in vivo. In some aspects, the methods provided are performed upon administration of a chimeric antibody to a subject, e.g., a subject having a disease or condition.

[0259] Methods and uses of chimeric antibodies or compositions containing cytokine (e.g., IL-15) modified antibodies or antigen-binding fragments to treat diseases or conditions are also provided herein. In certain embodiments, the disease or condition is treatable with a cytokine and the chimeric antibody comprises a cytokine sequence. In some embodiments, the disease or condition is treatable with IL-2 or IL-15 alone or in combination with another agent. In some embodiments, the chimeric antibody comprises an IL-2 or IL-25 sequence or a biologically active portion thereof. In some embodiments, the provided chimeric antibodies or antigen-binding fragments are particularly suitable for use as immunotherapy. In certain aspects, the provided chimeric antibodies or antigen-binding fragments, or compositions thereof, are used in some oncology applications, such as cancer, by promoting the activation and / or proliferation of T cells or the expansion of NK cells. In certain aspects, the provided chimeric antibodies or antigen-binding fragments, or compositions thereof, are modified to have reduced effector activity and to avoid the induction of certain effects, such as ADCC, for example, ADCC against cells targeted for stimulation by the chimeric antibody, while still promoting the activation and / or proliferation of T cells. In some embodiments, the chimeric antibody, when the chimeric antibody comprises IL-15 sequences or a biologically active portion thereof, does not induce ADCC against a cell to which it binds, e.g., an immune cell, e.g., a cell expressing IL2 / 15Rβ and / or IL2 / 15Rβ γc receptor subunit. In some embodiments, the provided chimeric antibodies or antigen-binding fragments are used to treat cancer in a subject in need of treatment.

[0260] Such methods and uses include therapeutic methods and uses that involve administering the molecule to a subject having a disease, condition, or disorder, such as cancer, to treat the disease or disorder. Uses include the use of the composition in such methods and treatments, as well as the use of such compositions in the preparation of a medicament for carrying out such therapeutic methods. In some embodiments, the methods and uses thereby treat a disease or condition or disorder, such as a tumor or cancer, in a subject.

[0261] In some embodiments, the cancer is a blood cancer, such as lymphoma, leukemia, or myeloma. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the cancer is a head and neck, breast, liver, colon, ovarian, prostate, pancreatic, brain, cervix, bone, skin, lung, or blood cancer. In some embodiments, the cancer may include a malignant tumor characterized by abnormal or uncontrolled cell growth. Other features that may be associated with cancer include metastasis, interference with the normal function of neighboring cells, release of abnormal levels of cytokines or other secretory products, and suppression or exacerbation of inflammatory or immunological responses, infiltration of surrounding or distant tissues or organs, such as lymph nodes, etc. Metastatic disease may refer to cancer cells that have left the original tumor site and traveled to other parts of the body, for example, via the bloodstream or lymphatic system.

[0262] In some embodiments, the methods provided result in an improvement and / or treatment of a disease or condition, such as cancer. In some aspects, the methods provided result in one or more improvements in the disease, such as a reduction in the number of neoplastic cells, an increase in neoplastic cell death, an inhibition of neoplastic cell survival, an inhibition (i.e., a slowing or halting to some extent) of tumor growth, an increase in patient survival, and / or some relief from one or more symptoms associated with the disease or condition.

[0263] In the aspect of the method provided, response can be evaluated or determined using disease or condition specific criteria.In some embodiments, tumor response can be evaluated for changes in tumor morphology (i.e., overall tumor burden, tumor size) using screening techniques such as magnetic resonance imaging (MRI) scan, X-ray image, computed tomography (CT) scan, bone scan image, endoscopy, and tumor biopsy sampling, including bone marrow aspirate (BMA) and circulating tumor cell count.

[0264] The provided method includes administering a therapeutically effective amount of the composition provided herein to a subject in need thereof, for example, a cancer subject. The therapeutically effective amount can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the drug to induce a desired response in the individual. The therapeutically effective amount is also an amount in which any toxic or harmful effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. In some cases, the therapeutically effective amount of a tumor or cancer therapy can also be measured by its ability to stabilize the progression of the disease. The ability of the provided antibody or antigen-binding fragment to inhibit cancer can be evaluated in an animal model system that predicts efficacy in human tumors.

[0265] Alternatively, this property of the composition can be evaluated by testing the ability of the antibody or antigen-binding fragment to inhibit cell growth or induce apoptosis by in vitro assays known to those skilled in the art. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise ameliorate symptoms in a subject. Those skilled in the art will be able to determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.

[0266] In some embodiments, the provided antibodies or antigen-binding fragments can be administered in a single dose or in several doses as needed to obtain the desired response, in some embodiments, the effective amount depends on the source applied, the subject being treated, the severity and type of the condition being treated, and the mode of administration.

[0267] The dosage regimen is adjusted to provide the optimum desired response (e.g., therapeutic response). 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. Parenteral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to a physically discrete unit suitable as a unitary dosage for the subject to be treated. Each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0268] In some embodiments, the therapeutically effective amount is between or about 0.1 mg / kg and 100 mg / kg, or any value between any of the foregoing.

[0269] In some embodiments, the provided methods and uses can be performed in combination with another therapy, such as another therapy for treating a disease or condition. In some embodiments, the disease or condition is a tumor or cancer, and the other therapy is an antitumor agent or antitumor therapy (also referred to herein as an anticancer agent or anticancer therapy). In particular, the provided methods can be used in conjunction with cancer immunotherapy. Cancer immunotherapy aims to eradicate cancer cells by restoring the tumor-killing function of tumor-reactive immune cells, such as T cells or NK cells. Cancer immunotherapy strategies, including checkpoint blockade, adoptive cell transfer (ACT) and cancer vaccines, which can increase antitumor immune effector cells, have produced remarkable results in some tumors. In some embodiments, the antitumor or anticancer therapy is an antibody therapeutic, such as a monoclonal antibody.

[0270] In some aspects, anti-tumor agents suitable for use in the methods and uses provided include those that promote ADCC against tumor cells.For example, in some aspects, chimeric antibodies are modified to reduce effector function, so that they do not interfere with or compete with the ability of anti-tumor agents to promote ADCC against tumor cells, for example, through the engagement of anti-tumor agents with immune cells, for example, through FcR binding.In some embodiments, for example, when the anti-tumor agent comprises a cell therapy, the chimeric antibody does not bind to the FcR expressed by the cell therapy, or has reduced binding to FcR, and the chimeric antibody does not induce ADCC of the anti-tumor agent against the cells to which the chimeric antibody binds.Anti-tumor agents that do not promote ADCC are also suitable for use in the methods and uses provided herein.

[0271] A problem with certain cancer immunotherapy approaches is that host antitumor immunity can hinder the efficacy of cancer immunotherapy. For example, in some cases, the formation of an immunosuppressive tumor microenvironment can affect the ability of natural tumor-reactive immune cells or adoptively transferred immune cells to successfully eradicate cancer cells. In particular, in solid tumors, the therapeutic efficacy of immunotherapy regimens remains unsatisfactory due to the lack of effective antitumor responses in the immunosuppressive tumor microenvironment. Tumor cells often induce immune tolerance or immune suppression, and such tolerance is acquired so that even truly foreign tumor antigens become tolerated. Such tolerance is similarly active and dominant, as cancer vaccines and adoptive transfer of preactivated immune effector cells (e.g., T cells) are subject to suppression by inhibitors within the tumor microenvironment (TME).

[0272] In some embodiments, the chimeric cytokine (e.g., IL-15) modified antibody or antigen-binding fragment is administered in combination with a checkpoint blockade agent (also called immune checkpoint inhibitor). Immune checkpoint inhibitors are molecules that completely or partially reduce, inhibit, interfere with or modulate one or more checkpoint proteins. Checkpoint proteins control the activation or function of T cells. These proteins are responsible for co-stimulatory or inhibitory interactions of T cell responses. Immune checkpoint proteins control and maintain self-tolerance as well as the duration and magnitude of physiological immune responses.

[0273] Immune checkpoint inhibitors include any agent that statistically significantly blocks or inhibits an inhibitory pathway of the immune system. Such inhibitors may include small molecule inhibitors that bind to and block or inhibit immune checkpoint receptor ligands, or may include antibodies or antigen-binding fragments thereof. Exemplary immune checkpoint molecules that can be targeted for blockade or inhibition include, but are not limited to, PD1 (CD279), PDL1 (CD274, B7-H1), PDL2 (CD273, B7-DC), CTLA-4, LAG3 (CD223), TIM3, 4-1BB (CD137), 4-1BBL (CD137L), GITR (TNFRSF18, AITR), CD40, Ox40 (CD134, TNFRSF4), CXCR2, tumor associated antigens (TAA), B7-H3, B7-H4, BTLA, HVEM, GAL9, B7H3, B7H4, VISTA, KIR, 2B4 (belonging to the CD2 molecule family and expressed on all NK, gamma delta and memory CD8+ (alpha beta) T cells), CD160 (also called BY55), and CGEN-15049. Immune checkpoint inhibitors include antibodies or antigen-binding fragments thereof, or other binding proteins that bind to and block or inhibit the activity of one or more of PD1, PDL1, PDL2, CTLA-4, LAG3, TIM3, 4-1BB, 4-1BBL, GITR, CD40, Ox40, CXCR2, TAA, B7-H3, B7-H4, BTLA, HVEM, GAL9, B7H3, B7H4, VISTA, KIR, 2B4, CD160, and CGEN-15049. Exemplary immune checkpoint inhibitors include tremelimumab (CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonal antibody (anti-B7-H1; MEDI4736), MK-3475 (PD-1 blocker), nivolumab (anti-PD1 antibody), CT-011 (anti-PD1 antibody), BY55 monoclonal antibody, AMP224 (anti-PDL1 antibody), BMS-936559 (anti-PDL1 antibody), MPLDL3280A (anti-PDL1 antibody), MSB0010718C (anti-PDL1 antibody), and Yervoy / ipilimumab (anti-CTLA-4 checkpoint inhibitor).

[0274] In some embodiments, the immune checkpoint inhibitor specifically binds to a molecule selected from among CD25, PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L, OX40, OX40L, CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28, and VISTA. In some embodiments, the immune checkpoint inhibitor is an antibody or antigen-binding fragment, a small molecule, or a polypeptide. In some embodiments, the immune checkpoint inhibitor is selected from among nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab, IMP31, BMS-986016, urelumab, TRX518, dacetuzumab, lucatumumab, SEQ-CD40, CP-870, CP-893, MED16469, MEDI4736, MOXR0916, AMP-224, and MSB001078C, or an antigen-binding fragment thereof. In some embodiments, the immune checkpoint inhibitor can be an anti-PD-1 or anti-PD-L1 antibody. Antibodies targeting PD-1 or PD-L1 include, but are not limited to, nivolumab, pembrolizumab, or atezolizumab.

[0275] In some embodiments, the anti-tumor agent comprises a monoclonal antibody. In some embodiments, the monoclonal antibody is any of those described in Zahavi et al. (2020), Antibodies (Basel) 9(3):34. In some embodiments, the monoclonal antibody is atezolizumab, avelumab, bevacizumab, cemiplimab, cetuximab, daratumumab, dinutuximab, durvalumab, elotuzumab, ipilimumab, isatuximab, mogamulizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, olaratumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, rituximab, or trastuzumab.

[0276] In some embodiments, the chimeric cytokine (e.g., IL-15) modified antibody or antigen-binding fragment is administered in combination with a cell therapy, such as an adoptive cell therapy. In some embodiments, administration of the provided chimeric cytokine (e.g., IL-15) modified antibody or antigen-binding fragment in combination with such an adoptive cell therapy can be used to stimulate T cells, such as in a TCR / CAR combination, to increase the expansion of NK cells, including engineered NK cells (e.g., CAR-engineered NK cells), in the manipulation or control of TILs. In some embodiments, the adoptive cell therapy can be an autologous cell therapy or an allogeneic cell therapy. In some embodiments, the immune cells for adoptive cell therapy in the provided combinations can be dendritic cells, T cells, such as CD8+T cells and CD4+T cells, natural killer (NK) cells, NK T cells, cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes (TILs), lymphokine-activated killer (LAK) cells, memory T cells, regulatory T cells (Tregs), helper T cells, cytokine-induced killer (CIK) cells, and any combination thereof. In other embodiments, the immune stimulatory cells for adoptive cell therapy can be generated from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). In some embodiments, autologous or allogeneic immune cells are used in adoptive cell therapy.

[0277] In some embodiments, administration of chimeric cytokine (e.g., IL-15) modified antibodies or antigen-binding fragments, whether as a single agent or a combination agent, results in the proliferation of immune cells. In some embodiments, administration of chimeric cytokine modified antibodies or antigen-binding fragments results in a 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.2-fold, 2.4-fold, 2.6-fold, 2.8-fold, 3-fold, 4-fold, or 5-fold increase in the number of immune cells. In some embodiments, the immune cells that proliferate are T cells. In some embodiments, the immune cells that proliferate are NK cells. In some embodiments, the immune cells that proliferate are T cells and NK cells.

[0278] In some embodiments, the immune cells that are expanded are immune cells that are administered as part of cell therapy, including any of the cell therapies described herein, such as cell therapy administered in combination with chimeric cytokine-modified antibodies or antigen-binding fragments.In some embodiments, the cell therapy is T cell therapy.In some embodiments, the cell therapy is NK cell therapy.

[0279] In some embodiments, the chimeric cytokine (e.g., IL-15) modified antibody or antigen-binding fragment is administered in combination with other agents effective in the treatment of cancer, infectious diseases and other immune deficiency disorders, such as anti-cancer agents.Anti-cancer agents can be any agent that can adversely affect a subject's cancer, for example, by killing cancer cells, inducing apoptosis of cancer cells, slowing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to tumors or cancer cells, promoting an immune response against cancer cells or tumors, preventing or inhibiting the progression of cancer, or extending the lifespan of subjects with cancer.

[0280] In some embodiments, the anti-cancer agent or therapy can be a chemotherapeutic agent, or radiation therapy, an immunotherapeutic agent, surgery, or any other therapeutic agent that improves the therapeutic efficacy of the treatment in combination with the chimeric cytokine (e.g., IL-15) modified antibody or antigen-binding fragment.

[0281] In one aspect, the chimeric cytokine (e.g., IL-15) modified antibody or antigen-binding fragment may be used in combination with an aminopyrimidine derivative, such as a Burkitt tyrosine receptor kinase (BTK) inhibitor, for example, using the methods taught in WO2016164580, the contents of which are incorporated herein by reference in their entirety.

[0282] In some embodiments, a chimeric cytokine (eg, IL-15) modified antibody or antigen-binding fragment may be used in combination with an antibody specific for some target molecule on the surface of a tumor cell.

[0283] Exemplary anti-cancer drugs include, but are not limited to, acivicin; aclarubicin; acodazole hydrochloride; acronine; adzelesin; aldesleukin; altretamine; ambomycin; amethanthrone acetate; amsacrine; anastrozole; anthramycin; asparaginase; asperin, sulindac, curcumin, alkylating agents such as nitrogen mustards, e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan, and chlorambucil; nitrosoureas, e.g., carmustine (BCU), lomustine (CCNU), semustine (methyl-CC U); thylenimines / methylmelamines such as triethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); alkylsulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites such as folic acid analogues such as methotrexate and trimetrexate, pyrrolidine analogues such as 5-Fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2'-difluorodeoxycytidine, purine analogues such as 6-mercaptopurine, 6-thioguanine, azathioprine, 2,'-deoxycoformycin (pentostatin), erythrohyckoxynonyladenine (EHNA), ffudarabine phosphate phosphate), and 2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products such as mitotic inhibitors, such as paclitaxei, vinca alkaloids, such as vinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine, and estramustine phosphate; epipodophyllotoxins, such as etoposide and teniposide;Antibiotics such as actimomycin D, daunomycin (ruhidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycin, plicamycin (mithramycin), mitomycin C and actinomycin; enzymes such as -[.-asparaginase, cytokines such as interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, TNF-beta and GM-CSF, anti-angiogenic factors such as angiostatin and endostatin, inhibitors of FGF or VEGF such as angiogenic factors soluble forms of receptors such as soluble VGF / VEGF receptors, platinum coordination complexes such as cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted ureas such as hydroxyurea, methylhydrazme derivatives such as N-methylhydrazme (MIFf) and procarbazine, adrenal cortical suppressants such as mitotane (ο,ρ'-DDD) and aminoglutethimide; hormones and antagonists such as corticosteroid antagonists. Anti-estrogens, such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestins, such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogens, such as diethylstilbestrol and ethinyl estradiol equivalents; anti-estrogens, such as tamoxifen; androgens, such as testosterone propionate and fiuoxymesterone / equivalents; anti-androgens, such as flutamide, gonadotropes, pin-releasing hormone analogues and leuprolide; non-steroidal antiandrogens, such as flutamide; kinase inhibitors, histone deacetylase inhibitors, methylation inhibitors, proteasome inhibitors, monoclonal antibodies, oxidants, antioxidants, telomerase inhibitors, BH3 mimetics, ubiquitin ligase inhibitors, stat inhibitors and receptor tyrosine kinase inhibitors, such as imatinib mesylate (marketed as Gleevac or Giivac) and erlotinib (an EGF receptor inhibitor), currently marketed as Tarveca;Antiviral drugs, such as oseltamivir phosphate; amphotericin B and palivizumab; Sdi 1 mimetics; Semusiine; Senescence derived inhibitor 1; Sparfosic acid; Spicamycin D; Spiromustine; Spienopentin; Spongestatin 1; Squaiamine; Stipiamide; Stromelysin inhibitors; Sulfinosine; Superactive vasoactive intestinal peptide antagonists; Veraresol; Veramine; Verdine; Verteporfin; Vinorelbine; Vmxaltme; Vitaxin; Vorozole; Zanoteron; Zeniplatin; Zilascorb; and Zinostatin stimalamer; ΡΟ β small molecule inhibitors, GSK2636771; pan-PI3 inhibitors (BKM120); BRAF inhibitors. Beniurafenib (Zeiboraf) and Dabrafenib (Tafmiar); or any analogs or derivatives and variants of the above.

[0284] In some embodiments, the anti-cancer agent is an antibody or antigen-binding antibody fragment thereof, examples of which include, but are not limited to, daclizumab (Zenapax), bevacizumab (Avastin®), basiliximab, ipilimumab, nivolumab, pembrolizumab, MPDL3280A, pidilizumab (CT-011), MK-3475, BMS-936559, MPDL3280A (atezolizumab), tremelimumab, IMP321, BMS-986016, LAG525, urelumab, PF-05082566, TRX518, MK-4166, dacetuzumab (SGN- 40), lucatumumab (HCD122), SEA-CD40, CP-870, CP-893, MEDI6469, MEDI6383, MOXR0916, AMP-224, MSB0010718C (avelumab), MEDI4736, PDR001, rHIgM12B7, urocuplumab, BKT140, valilumab (CDX-1127), ARGX-110, MGA271, lirilumab (BMS-986015, IPH2101), IPH2201, ARGX-115, emactuzumab, CC-90002 and MNRP1685A or antibody-binding fragments thereof.

[0285] Other agents may be used in combination with the chimeric cytokine (e.g., IL-15) modified antibody or antigen-binding fragment, including, but not limited to, agents that affect the upregulation of cell surface receptors and their ligands, such as Fas / Fas ligand, DR4 or DR5 / TRAIL and GAP junctions, cytostatic and differentiating agents, inhibitors of cell adhesion, such as focal adhesion kinase (FAK) inhibitors and lovastatin, or agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers, such as the antibody C225.

[0286] In some embodiments, the chimeric cytokine (e.g., IL-15) modified antibody or antigen-binding fragment is administered in combination with a cancer vaccine. In some embodiments, the cancer vaccine may include peptides and / or proteins derived from tumor-associated antigens (TAA). Such strategies may be utilized to induce an immune response in a subject, which may in some instances be a cytotoxic T lymphocyte (CTL) response. The peptides used in the cancer vaccine may also be modified to match the mutation profile of the subject. For example, EGFR-derived peptides with mutations matching those found in subjects in need of therapy have been successfully used in lung cancer patients (Li F et al. (2016) Oncoimmunology. Oct 7;5(12):el 238539, the contents of which are incorporated herein by reference in their entirety). In one embodiment, the cancer vaccine includes superagonist-modified peptide ligands (APLs) derived from TAAs. These are mutant peptide ligands that deviate from the natural peptide sequence by one or more amino acids that activate specific CTL clones more effectively than the natural epitope. These modifications may allow the peptide to better bind to restricted class I MHC molecules or to interact more favorably with the TCR of a given tumor-specific CTL subset. APLs can be selected using the methods taught in U.S. Patent Application Publication No. 20160317633, the entire contents of which are incorporated herein by reference.

[0287] In some embodiments, the combination may involve simultaneous or separate administration of the chimeric cytokine (e.g., IL-15) modified antibody or antigen-binding fragment and the other agent. Alternatively, the chimeric cytokine (e.g., IL-15) modified antibody or antigen-binding fragment may precede or follow the other agent / therapy by intervals ranging from minutes, days, weeks to months.

[0288] V. Illustrative Embodiments Among the aspects provided are: 1. (a) a modified variable heavy (VH) region of a bovine antibody or antigen-binding fragment, or a humanized sequence thereof, wherein the modified VH region comprises a modified ultralong CDR3, and at least a portion of the ultralong CDR3 of the bovine antibody or antigen-binding fragment, or a humanized sequence thereof, is replaced by a cytokine sequence, or a biologically active portion thereof; (b) a modified human IgG heavy chain constant region having reduced effector activity compared to a wild-type human IgG heavy chain constant region; A chimeric engineered antibody comprising a heavy chain comprising: 2. 2. The chimeric engineered antibody of embodiment 1, wherein said cytokine sequence, or a biologically active portion thereof, replaces the knob region of said ultralong CDR3 region of said bovine antibody or antigen-binding fragment, or humanized sequence thereof. 3. 3. The chimeric engineered antibody of embodiment 1 or embodiment 2, wherein said cytokine sequence, or a biologically active portion thereof, is between the ascending and descending stalk strands of said engineered very long CDR3, and said ascending stalk strand of said engineered very long CDR3 is mutant compared to the ascending stalk strand of said very long CDR3 of said bovine antibody or antigen-binding fragment, or humanized sequence thereof. 4. 4. The chimeric engineered antibody of embodiment 3, wherein said cytokine sequence, or a biologically active portion thereof, is linked to said ascending stalk strand and / or said descending stalk strand of said engineered ultralong CDR3 via a flexible linker, optionally a GGS or GSG linker. 5. The chimeric engineered antibody of embodiment 3 or embodiment 4, wherein said ascending stalk strand comprises the sequence CX2TVX5QETKKYQT, wherein X2 and X5 are any amino acid. 6. 1. A chimeric modified antibody comprising a heavy chain comprising a modified variable heavy (VH) region of a bovine antibody or antigen-binding fragment or a humanized sequence thereof, the modified VH region comprises a modified ultralong CDR3 in which at least a portion of the ultralong CDR3 region of the bovine antibody or antigen-binding fragment or humanized sequence thereof is replaced with a heterologous sequence, the heterologous sequence being between the ascending stalk strand and the descending stalk strand of the modified ultralong CDR3, the ascending stalk strand of the modified ultralong CDR3 comprising the sequence CX2TVX5QETKKYQT, where X2 and X5 are any amino acid; Chimeric engineered antibodies. 7. 7. The chimeric modified antibody of embodiment 5 or embodiment 6, wherein X2 is Ser, Thr, Gly, Asn, Ala, or Pro, and X5 is His, Gln, Arg, Lys, Gly, Thr, Tyr, Phe, Trp, Met, Ile, Val, or Leu. 8. The chimeric modified antibody of any of aspects 5 to 7, wherein X2 is Ser, Ala, or Thr, and X5 is His or Tyr. 9. The chimeric modified antibody of any of aspects 3 to 8, wherein the ascending stalk strand of the modified very long CDR3 comprises a sequence as shown in any of SEQ ID NOs: 183 to 185. 10. The chimeric modified antibody of any of aspects 3 to 8, wherein the sequence of the ascending stalk strand of the modified ultralong CDR3 is set forth in any of SEQ ID NOs: 183 to 185. 11. 11. The chimeric modified antibody of any of aspects 6 to 10, wherein said heterologous sequence replaces the knob region of said very long CDR3 region of said bovine antibody or antigen-binding fragment or humanized sequence thereof. 12. 12. The chimeric engineered antibody of any of aspects 6 to 11, wherein said heterologous sequence is linked to said ascending stalk strand and / or said descending stalk strand of said engineered ultralong CDR3 via a flexible linker, optionally a GGS or GSG linker. 13. 13. The chimeric modified antibody of any of aspects 6 to 12, wherein said heterologous sequence comprises a cytokine sequence or a biologically active portion thereof. 14. 14. The chimeric modified antibody of any of aspects 6 to 13, wherein the heavy chain further comprises a human IgG heavy chain constant region. 15. 15. The chimeric modified antibody of embodiment 14, wherein said human IgG heavy chain constant region is a modified human IgG heavy chain constant region having reduced effector activity compared to a wild-type human IgG heavy chain constant region. 16. The chimeric modified antibody of any of aspects 1 to 5 and 7 to 15, wherein the human IgG is human IgG1. 17. The modified chimeric antibody of any of aspects 1 to 5 and 7 to 16, wherein the modified human IgG heavy chain constant region has been modified to reduce FcR binding. 18. The chimeric modified antibody of any of embodiments 1 to 5 and 7 to 17, wherein said reduced effector activity comprises reduced antibody-dependent cell-mediated cytotoxicity (ADCC). 19. 19. The chimeric modified antibody of any of embodiments 1-5 and 7-18, wherein said modified human IgG heavy chain constant region is altered at one or more of positions Glu233 (E233), Leu234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Asn297 (N297), Ser298 (S298), Asn325 (N325), Ala327 (A327), and Pro329 (P329). 20. 20. The chimeric modified antibody of any of embodiments 1-5 and 7-19, wherein said modified human IgG heavy chain constant region comprises one or more mutations selected from Leu234Ala (L234A), Leu235Ala (L235A), Leu235Glu (L235E), Asp265Asn (D265N), Asp265Ala (D265A), Asp270Asn (D270N), Ser298Asn (S298N), Asn325Glu (N325E), Ala327Ser (A327S), Pro329Ala (P329A), and Pro239Gly (P329G). twenty one. 21. The chimeric modified antibody of any of embodiments 1-5 and 7-20, wherein said modified human IgG heavy chain constant region has been altered at two or more of positions Glu233 (E233), Leu234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Asn297 (N297), Ser298 (S298), Asn325 (N325), Ala327 (A327), and Pro329 (P329). twenty two. the modified human IgG heavy chain constant region comprises Leu234Ala and Leu235Ala (L234A / L235A) mutations; Leu234Val and Leu235Ala (L234V / L235A) mutations; Leu234Ala, Leu235Ala, and Asn297Ala (L234A / L235A / N297A) mutations; Leu234Ala, Leu235Ala, and Pro239Ala (L234A / L235A / P329A) mutations; Asp265Ala and Pro329Ala (D265A / P329A) mutations; 22. The chimeric modified antibody of any of aspects 1-5 and 7-21, comprising: 5Ala and Pro329Gly (D265A / P329G) mutations; Leu234Ala, Leu235Ala, and Asp265Ala (L234A / L235A / D265A) mutations; Leu234Ala, Leu235Ala, and Pro329Gly (L234A / L235A / P329G) mutations; or Leu234Ala, Leu235Ala, Asp265Ala, and Pro329Gly (L234A / L235A / D265A / P329G) mutations. twenty three. 23. The chimeric modified antibody of any of aspects 1 to 5 and 7 to 22, wherein the modified human IgG heavy chain constant region comprises Leu234Ala and Leu235Ala (L234A / L235A) mutations. twenty four. The chimeric modified antibody of embodiments 1 to 5 and 7 to 23, wherein said modified human IgG heavy chain constant region comprises the sequence shown in SEQ ID NO:187 or SEQ ID NO:188. twenty five. 25. The chimeric modified antibody of any of embodiments 1-5 and 7-24, wherein said cytokine sequence or biologically active portion thereof comprises an interleukin-15 (IL-15) cytokine sequence or a biologically active portion thereof. 26. 26. The chimeric engineered antibody of any of embodiments 1-5 and 7-25, wherein said cytokine sequence or biologically active portion thereof comprises a sequence of amino acids exhibiting at least 85%, or at least about 85%, at least 90%, or at least about 90%, at least 92%, or at least about 92%, at least 95%, or at least about 95%, at least 96%, or at least about 96%, at least 97%, or at least about 97%, at least 98%, or at least about 98%, or at least 99% or at least about 99% sequence identity to SEQ ID NO:1. 27. 27. The chimeric engineered antibody of any of embodiments 1 to 5 and 7 to 26, wherein said cytokine sequence or a biologically active portion thereof comprises the sequence shown in SEQ ID NO:1. 28. 25. The chimeric modified antibody of any of embodiments 1-5 and 7-24, wherein said cytokine sequence or biologically active portion thereof comprises an interleukin-12 (IL-2) cytokine sequence or a biologically active portion thereof. 29. 30. The chimeric engineered antibody of any of embodiments 1-5, 7-24, and 28, wherein said cytokine sequence or biologically active portion thereof comprises a sequence of amino acids exhibiting at least 85%, or at least about 85%, at least 90%, or at least about 90%, at least 92%, or at least about 92%, at least 95%, or at least about 95%, at least 96%, or at least about 96%, at least 97%, or at least about 97%, at least 98%, or at least about 98%, or at least 99% or at least about 99% sequence identity to SEQ ID NO:165. 30. 30. The chimeric engineered antibody of any of embodiments 1-5, 7-24, 28, and 29, wherein said cytokine sequence or biologically active portion thereof comprises the sequence of amino acids set forth in SEQ ID NO:165. 31. 31. The chimeric modified antibody of any of aspects 1 to 30, wherein the bovine antibody or antigen-binding fragment is the bovine antibody BLV1H12 or an antigen-binding fragment thereof. 32. 32. The chimeric modified antibody of any of aspects 3 to 31, wherein the descending stalk strand comprises the sequence shown in SEQ ID NO:10. 33. the ascending stalk strand comprises the sequence set forth in SEQ ID NO:183, the cytokine sequence or biologically active portion thereof comprises the sequence of amino acids set forth in SEQ ID NO:1, and the descending stalk strand comprises the sequence set forth in SEQ ID NO:10; the ascending stalk strand comprises the sequence set forth in SEQ ID NO:184, the cytokine sequence or biologically active portion thereof comprises the sequence of amino acids set forth in SEQ ID NO:1, and the descending stalk strand comprises the sequence set forth in SEQ ID NO:10; or the ascending stalk strand comprises the sequence set forth in SEQ ID NO:185, the cytokine sequence or biologically active portion thereof comprises the sequence of amino acids set forth in SEQ ID NO:1, and the descending stalk strand comprises the sequence set forth in SEQ ID NO:10. A chimeric modified antibody according to any one of embodiments 3 to 5, 7 to 27, 31, and 32. 34. 34. The chimeric modified antibody of any of aspects 1 to 27 and 31 to 33, wherein the modified ultralong CDR3 comprises a sequence as shown in any of SEQ ID NOs: 206 to 208. 35. 35. The chimeric modified antibody of any of aspects 1 to 34, wherein the modified VH region is a mutant of the VH region of BLV1H12. 36. 36. The chimeric modified antibody of any of aspects 1-35, wherein said heavy chain comprises the formula V1-X-V2-C, where the V1 region of said heavy chain comprises the sequence set forth in SEQ ID NO:182; the X region comprises said modified ultralong CDR3; the V2 region comprises the sequence set forth in SEQ ID NO:11; and the C region comprises said modified human IgG heavy chain constant region. 37. 37. The chimeric modified antibody of any of aspects 1 to 27 and 31 to 36, wherein the modified VH region comprises a sequence shown in any of SEQ ID NOs: 200 to 202. 38. 38. The chimeric modified antibody of any of aspects 1 to 27 and 31 to 37, wherein the heavy chain comprises a sequence as set forth in any of SEQ ID NOs: 189 to 191. 39. 35. The chimeric modified antibody of any of aspects 1 to 34, wherein the modified VH region is a mutant of a humanized sequence of the VH region of BLV1H12. 40. 40. The chimeric modified antibody of any of embodiments 1-34 and 39, wherein said heavy chain comprises the formula V1-X-V2-C, where the V1 region of said heavy chain comprises the sequence set forth in SEQ ID NO:197, or a sequence that exhibits at least 65% sequence identity to SEQ ID NO:197; the X region comprises said modified ultralong CDR3; the V2 region comprises the sequence set forth in SEQ ID NO:11; and the C region comprises said modified human IgG heavy chain constant region. 41. 41. The chimeric modified antibody of any of embodiments 1-34, 39, and 40, wherein said heavy chain comprises the formula V1-X-V2-C, where the V1 region of said heavy chain comprises the sequence set forth in SEQ ID NO:197; the X region comprises said modified ultralong CDR3; the V2 region comprises the sequence set forth in SEQ ID NO:11; and the C region comprises said modified human IgG heavy chain constant region. 42. The chimeric modified antibody of any of aspects 1 to 27, 31 to 34, and 39 to 41, wherein the modified VH region comprises a sequence shown in any of SEQ ID NOs: 203 to 205. 43. 43. The chimeric modified antibody of any of aspects 1 to 27, 31 to 34, and 39 to 42, wherein the heavy chain comprises a sequence as set forth in any of SEQ ID NOs: 192 to 194. 44. 44. The chimeric modified antibody of any of embodiments 1 to 43, further comprising a light chain. 45. 45. The chimeric modified antibody of any of embodiments 1 to 44, comprising a humanized light chain. 46. 46. ​​The chimeric engineered antibody of embodiment 45, wherein said humanized light chain comprises a sequence as shown in SEQ ID NO:181, or a sequence exhibiting at least 85% sequence identity to SEQ ID NO:181. 47. 46. ​​The chimeric modified antibody of embodiment 45, wherein said humanized light chain comprises the sequence as set forth in SEQ ID NO:181. 48. The chimeric modified antibody of any of embodiments 1 to 27 and 31 to 47, wherein said chimeric modified antibody is complexed with an extracellular domain of IL15Rα comprising the IL15Rα sushi domain. 49. 49. The chimeric engineered antibody of embodiment 48, wherein said extracellular domain of IL15Rα comprising an IL15Rα sushi domain is non-covalently associated with said IL-15 sequence. 50. 49. The chimeric engineered antibody of embodiment 48, wherein said extracellular domain of IL15Rα comprising the IL15Rα sushi domain is linked to said light chain of said chimeric engineered antibody, optionally via a peptide linker. 51. 51. The chimeric modified antibody of any of aspects 48 to 50, wherein the extracellular domain of IL15Rα comprising the IL15Rα sushi domain comprises the sequence shown in SEQ ID NO:2. 52. A polynucleotide encoding the chimeric modified antibody of any of embodiments 1 to 51. 53. A polynucleotide encoding the heavy chain or the variable region thereof of the chimeric modified antibody of any of embodiments 1 to 51. 54. A polynucleotide encoding the light chain or the variable region thereof of the chimeric modified antibody of any of embodiments 1 to 51. 55. An expression vector comprising the polynucleotide of any one of embodiments 52 to 54. 56. A host cell comprising the polynucleotide of any of embodiments 52 to 54 or the expression vector of embodiment 55. 57. 57. The host cell of embodiment 56, further comprising a polynucleotide or vector encoding the extracellular domain of IL15Rα comprising the IL15Rα sushi domain. 58. 58. The host cell of embodiment 57, wherein said extracellular domain of IL15Rα comprising the IL15Rα sushi domain comprises the sequence shown in SEQ ID NO:2. 59. A method for producing a chimeric modified antibody, comprising culturing a host cell of any of aspects 56 to 58 under conditions for expression of said chimeric modified antibody by said host cell, and optionally further comprising recovering or purifying said chimeric modified antibody. 60. A chimeric engineered antibody produced by the method of embodiment 59. 61. A pharmaceutical composition comprising the chimeric modified antibody of any of embodiments 1 to 51 and 60. 62. A method for stimulating immune cells, comprising the step of contacting a population of immune cells with a chimeric modified antibody of any of aspects 1 to 51 and 60, thereby stimulating cells of the population of immune cells. 63. A method for expanding immune cells, comprising the step of contacting a population of immune cells with a chimeric modified antibody of any of aspects 1 to 51 and 60, thereby promoting cell proliferation of the population of immune cells. 64. The method of embodiment 62 or embodiment 63, wherein said population of immune cells comprises cells expressing IL2 / 15Rβ and / or IL2 / 15Rβ γc receptor subunit. 65. The method of any of embodiments 62-64, wherein the population of immune cells comprises T cells or natural killer (NK) cells. 66. The method of any of embodiments 62 to 65, which is carried out ex vivo or in vitro. 67. 66. The method of any of embodiments 62 to 65, which is performed in vivo upon administration of said chimeric modified antibody to a subject. 68. A method for treating cancer in a subject, comprising the step of administering to the subject a therapeutically effective amount of the chimeric modified antibody of any of aspects 1 to 51 and 60. 69. A method of treating cancer in a subject, comprising administering to the subject the pharmaceutical composition of embodiment 61. 70. The method of any of aspects 67-69, further comprising the step of administering to the subject an anti-tumor agent. 71. The method of embodiment 70, wherein the anti-tumor agent comprises a monoclonal antibody. 72. The method of embodiment 70 or embodiment 71, wherein the anti-tumor agent comprises a checkpoint inhibitor. 73. The method of any of aspects 70-72, wherein the anti-tumor agent comprises a cell therapy, optionally a T cell therapy or a NK cell therapy. 74. The method of embodiment 73, wherein said cell therapy comprises cells expressing a chimeric antigen receptor (CAR). EXAMPLES

[0289] VI. Working Examples The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0290] Example 1 Generation of Chimeric Interleukin (IL)-15 Fusion Antibodies An exemplary chimeric BLV1H12-IL-15 (B15) fusion antibody containing IL-15 sequences was generated by modifying the ultralong CDR3 of the bovine antibody BLV1H12 or its humanized variants.

[0291] The heavy chain of BLV1H12 comprises a sequence having the formula V1-X-V2-C, where the V1 region of the heavy chain comprises a portion of the heavy chain sequence containing three framework regions (e.g., FR-1, FR-2, and FR-3) that separate the two CDR regions (CDR1 and CDR2), the X region comprises the ultralong CDR3 sequence including the knob region between the ascending and descending stalk strands, the V2 region comprises a portion of the heavy chain including FR-4, and the C region is the constant heavy chain region. The VH region of the B15 antibody was engineered by replacing the knob region of the ultralong CDR3 (SEQ ID NO:195) with an N-terminal GGS linker (SEQ ID NO:151), an IL-15 sequence (SEQ ID NO:1), and a C-terminal GSG linker (SEQ ID NO:186). The VH region was further engineered by modifying the ascending stalk strand of the ultralong CDR3 (unmodified sequence shown in SEQ ID NO:9). In addition to the engineered VH region, the heavy chain of the B15 antibody also contained an unmodified or modified heavy chain constant region of human IgG1 (unmodified sequence shown in SEQ ID NO:196), where the modified constant region contained Leu234Ala and Leu235Ala mutations (LALA double mutation).

[0292] Table E1 includes sequence identifiers (SEQ ID NOs) for the amino acid sequences of the B15 heavy chain. The heavy chains of B15 variants 1-3 and 7 were generated based on the BLV1H12 VH region, and the heavy chains of B15 variants 4-6 and 8 were generated based on humanized variants of the BLV1H12 VH region. The light chains of B15 variants 1-6 and 8 contained the humanized light chain sequence shown in SEQ ID NO:181, while the light chain of B15 variant 7 contained the bovine light chain sequence encoded by SEQ ID NO:168.

[0293] TABLE E1. SEQ ID NO. for exemplary B15 heavy chains TIFF2024517759000003.tif96150

[0294] To produce B15 antibody, the signal sequence and B15 heavy chain coding sequence were chemically synthesized with a 5'EcoRI site and cloned into pUC57 vector by GenScript, Inc. using EcoRI and NheI restriction enzymes. The 3' terminal NheI site was already present in the synthesized sequence. The expression vector encoding the heavy chain was then co-transfected into Freestyle HEK 293 cells (ThermoScientific) in parallel with the pFUSE expression vector encoding the humanized or bovine light chain. The cells were grown at 37°C, 8% CO2, and the secreted B15 antibody was harvested 96 hours after transfection. The B15 antibody was purified by CaptureSelect CH1-XL affinity matrix (ThermoScientific), then concentrated and buffer exchanged into phosphate buffered saline (PBS) using Amicon Ultra-4 centrifugal filters (MW cutoff = 10,000 kDa, Millipore Sigma). The recovered B15 antibody was quantified using Nanodrop based on molecular weight and extinction coefficient.

[0295] The left panel of Figure 1A shows the crystal structure of BLV1H12, and the left panel of Figure 1B shows a schematic of the generated B15 antibody.

[0296] Example 2 Chimeric IL-15 Fusion Antibody-Induced Receptor Activation and Signaling Activation of IL2 / 15Rβ and γc receptor subunits and STAT5 signaling by chimeric B15 fusion antibodies generated as described in Example 1 was examined using HEK-Blue IL2 reporter cells (InvivoGen) and analyzed through induction and secretion of the STAT5-inducible alkaline phosphatase (SEAP) reporter gene.

[0297] HEK-Blue IL2 reporter cells were placed in suspension by gently rinsing the cells twice with pre-warmed phosphate-buffered saline (PBS), detaching the cells in the presence of PBS using a cell scraper, and resuspending the cells in fresh pre-warmed test medium (DMEM with high glucose and 10% heat-inactivated FBS) to approximately 280,000 cells per mL. B15 variants 1-3, 7, and 8 were serially diluted 4-fold in PBS from 64 nM to 0.25 nM (based on the molar concentration of the Fab fragments) and 20 μL of each cytokine dilution was added to a 96-well tissue culture treated plate, with triplicates per dilution. 50,000 cells were then added to each well and cultured at 37 °C, 5% CO2 for 20 h. Twenty microliters of cell culture supernatant from each well containing secreted SEAP was mixed with 180 μL of Quanti-Blue substrate solution for 30 min at 37°C, and the color change (corresponding to the amount of secreted SEAP) was measured at 590 nm using a Molecular Devices plate reader.

[0298] As shown in Figure 2, B15 mutants 1-3, 7, and 8 induced STAT5 signaling in a dose-dependent manner. Table E2 shows the calculated EC50 values ​​for the B15 antibodies tested. These results demonstrate that the generated constructs are capable of inducing IL-15-mediated signaling activity.

[0299] Table E2: EC50 values ​​for B15 antibody binding to IL2 / 15R β and γc subunits TIFF2024517759000004.tif19151N / A: EC50 could not be determined from binding curve

[0300] Example 3 Generation of chimeric IL-15 fusion antibodies with IL15Rα sushi domain An exemplary chimeric B15 fusion antibody was generated that contains the extracellular sushi domain of IL15Rα (SEQ ID NO:2). The B15 Rαsushi antibody was generated by co-expressing the IL15Rα sushi domain with an exemplary B15 antibody (B15 variants 3 and 6 described in Example 1) to produce the B15Rαsushi antibody. Expression vectors encoding these sequences as well as a signal sequence were co-transfected into freestyle HEK 293 cells and then grown at 37° C., 8% CO2. The expressed B15 Rαsushi antibody was secreted and harvested 96 hours after transfection. The B15 Rαsushi antibody was purified by CaptureSelect CH1-XL affinity matrix (ThermoScientific), then concentrated and buffer exchanged into phosphate buffered saline (PBS) using an Amicon Ultra-4 centrifugal filter (MW cutoff=10,000 kDa, Millipore Sigma). Recovered antibody was quantified using Nanodrop based on molecular weight and extinction coefficient.

[0301] The right panel of FIG. 1A and the center and right panels of FIG. 1B show a schematic diagram of the B15 Rαsushi antibody that was generated.

[0302] Example 4 Receptor activation and signaling by chimeric IL-15 Rαsushi fusion antibodies Activation of IL2 / 15Rβ and γc receptor subunits and STAT5 signaling by the chimeric B15 Rαsushi fusion antibody generated as described in Example 3 is examined using HEK-Blue IL2 reporter cells (InvivoGen) and analyzed through induction and secretion of the STAT5-inducible alkaline phosphatase (SEAP) reporter gene.

[0303] HEK-Blue IL2 reporter cells are prepared as described in Example 2 and co-cultured with 4-fold serially diluted (64 nM to 0.25 nM based on Fab concentration) B15 Rαsushi antibody for 20 h at 37° C. and 5% CO2. 20 μL of cell culture supernatant from each well containing secreted SEAP is mixed with 180 μL of Quanti-Blue substrate solution for 30 min at 37° C. and the color change (corresponding to the amount of secreted SEAP) is measured at 590 nm using a Tecan plate reader.

[0304] Example 5 Expansion of NK-92 cells induced by chimeric IL-15 fusion antibodies The activity of chimeric B15 fusion antibodies generated as described in Example 1 and chimeric B15 Rasushi fusion antibodies generated as described in Example 3 is evaluated for their ability to expand NK-92 natural killer (NK) cells. NK-92 cells express IL2Rα, IL15Rα, IL2 / 15Rβ and γc subunits and their growth and proliferation are dependent on exogenous addition of IL2 or IL15 to bind and activate their receptors.

[0305] NK-92 cells are maintained in growth medium supplemented with 200 U / mL IL-2. Prior to the expansion assay, NK-92 cells are washed twice with growth medium without IL-2 to remove residual cell-bound IL-2 and seeded at 10,000 cells per well in tissue culture-treated 96-well plates. These cells are incubated with two-fold serial dilutions (1.33 nM to 0.005 nM) of IL-2 monomer, IL-15 monomer, B15 antibody, or B15 Rαsushi antibody for 48 hours at 37°C and 5% CO2. Incubation with half-molar concentrations of B15 or B15 Rαsushi antibody is compared to IL-2 and IL-15 monomer. Final NK92 cell numbers per well are assessed by reduction of the tetrazolium dye MTT to its insoluble formazan by the presence of metabolically active oxidoreductase enzymes (MTT assay kit, Promega).

[0306] Example 6 Evaluation of in vitro activity of chimeric IL-15 fusion antibodies in human PBMCs The activity of chimeric B15 fusion antibodies generated as described in Example 1 and chimeric B15 Rasushi fusion antibodies generated as described in Example 3 is evaluated for their ability to stimulate NK cells and T cells in human peripheral blood mononuclear cells (PBMCs) in vitro. Both NK cells and T cells express IL15Rα and IL2 / 15Rβ and γc subunits and their growth and proliferation are dependent on endogenous or exogenous IL15 to bind and activate the receptor.

[0307] Wash human PBMCs twice with PBS, count using a hemocytometer, and resuspend in RPMI1640 medium containing 10% FBS. Seed 100,000 cells in 100 μL per well in a tissue culture treated 96-well flat bottom or U-bottom (to promote cell contact) plate. Serially dilute B15 and B15 Rαsushi antibodies 5-fold from 500 nM to 0.032 nM in the same medium and add 100 μL of each dilution to the corresponding cells to achieve a final concentration of 250 nM to 0.016 nM. Also set up a control without the addition of fusion antibody. Incubate these cells for 96 h at 37 °C, 5% CO2. After treatment, PBMCs are stained with anti-CD3-FITC (SK7), anti-CD4-PE (OKT4), anti-CD8a-eFluor 450 (SK1), and anti-CD56-APC (AF12-7H3) to gate on the following cell types: CD3+CD4+ T cells, CD3+CD8+ T cells, and NK cells (CD3-CD56+). Intracellular Ki67, as a cell proliferation marker, is stained using anti-Ki67-PE-Cy7 (20Raj1) and Foxp3 / Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer's protocol. Stained samples are then analyzed using a Novocyte Advanteon Flow Cytometer (Agilent, Santa Clara, CA).

[0308] Example 7 In vivo activity of chimeric IL-15 fusion antibodies in rodents To examine the in vivo effects of chimeric B15 fusion antibodies on rodent NK and T cells, 18 female 7-9 week old Fischer 344 rats were randomly divided into six groups of three rats each based on day 0 body weight. On days 1 and 4, rats were administered either saline vehicle (vehicle, group 1), a "no knob" control antibody (NK-CTRL, heavy chain shown in SEQ ID NO:210, group 2), engineered IL-15 in the CDR H3 of a bovine VH scaffold (heavy chain of B15 variant 3 shown in SEQ ID NO:191, group 3), engineered IL-15 in the CDR H3 of a bovine scaffold complexed with the IL-15Rα sushi domain (B15 variant 3 with Rα, group 4), engineered IL-15 in the CDR H3 of a humanized scaffold (heavy chain of B15 variant 6 shown in SEQ ID NO:194, group 5), or engineered IL-15 in the CDR H3 of a humanized scaffold complexed with the IL-15Rα sushi domain (B15 variant 6 with Rα, group 6). The sequence of the IL-15Rα sushi domain is shown in SEQ ID NO:2. The fusion antibodies of groups 3-4 and groups 5-6 contained light chains derived from bovine V-lambda (encoded by the sequence shown in SEQ ID NO:168) or humanized V-lambda (SEQ ID NO:181), respectively. Group 2, a "no knob" negative control without engineered IL-15, contained bovine VH and VL regions. The constant region of each antibody was derived from human IgG1 (SEQ ID NO:188) with the LALA mutation. Each dose was 0.1 mg / kg intraperitoneally on days 1 and 4 in a volume of approximately 3 mL. Vehicle was administered in a volume of 3 mL. The rat groups for this study are shown in Table E3.

[0309] Table E3: Rodent studies of chimeric B15 fusion antibodies TIFF2024517759000005.tif57133 Vehicle = saline, NK-Ctrl = no knob negative control, ip = intraperitoneal

[0310] Body weight was monitored daily, blood was collected sublingually at different time points after the second fusion antibody administration, and a final blood collection by cardiac puncture was performed on day 5. Blood (up to 250 μL) was processed by adding 10x volume of room temperature ammonium chloride potassium (ACK) buffer to lyse red blood cells for 5 min, followed by 10x volume of cold PBS to stop the lysis reaction. Samples were centrifuged at 400 × g for 5 min and washed with PBS. T and NK cells were stained with fluorescent antibodies targeting rat CD4 (FITC-labeled, clone W3 / 25, BioLegend) and CD8 (PE-labeled, clone OX-8, BioLegend) for T cells and CD161 (APC-labeled, clone 3.3.3, BioLegend) for NK cells, and live cells were identified by staining with Live / Dead Aqua (Life Technologies).

[0311] The results showed that none of the engineered fusion antibodies significantly affected the rats' body weight (Table E4 and Figure 3A). 15 min after the last dose, a significant increase in NK cells was observed in groups 3 to 6, and the NK cell numbers were significantly higher in the groups that received the fusion antibody with the IL-15Rα chain (groups 4 and 6) (Figure 3B). 24 h after the last dose, an increase in CD8 T cells was observed for groups 3 to 6, and this increase was comparable between the groups that received and did not receive the IL-15Rα chain (Figure 3C). The results were comparable for the bovine and humanized fusion antibodies.

[0312] Table E4. Body weight changes and mortality events following administration of chimeric B15 fusion antibody TIFF2024517759000006.tif51134 Vehicle = Saline, Study Duration = 5 days, Mean BW Nadir = Lowest group mean body weight as % change from Day 1

[0313] Thus, engineered chimeric B15 fusion antibodies have biological activity in rodents in vivo, and B15 fusion antibodies containing the IL-15Rα sushi domain have enhanced activity in NK cells.

[0314] Example 8 In vivo activity of chimeric B15 fusion antibodies in non-human primates To examine the in vivo effects of B15 fusion antibodies on NK and T cells in non-human primates, nine male naive cynomolgus monkeys were randomized into three groups of three monkeys each based on body weight on day 0 (2.7-4.7 kg). On day 1, monkeys were administered a single dose of either engineered IL-15 in the CDR H3 of a humanized scaffold (heavy chain of B15 variant 6, SEQ ID NO:194, group 1), engineered IL-15 in the CDR H3 of a humanized scaffold complexed with the IL-15Rα sushi domain (group 2), or a "no knob" humanized control antibody (SEQ ID NO:211, group 3). The sequence of the IL-15Rα sushi domain is shown in SEQ ID NO:2. Fusion antibodies for all groups contained light chains derived from humanized V-lambda (SEQ ID NO:181). The constant region of each antibody was derived from human IgG1 (SEQ ID NO:188) with the "LALA mutation." Doses were administered intravenously at 0.1 mg / kg on day 1 in volumes ranging from 1.4 to 2.4 mL depending on body weight. Monkey groups for this study are shown in Table E5.

[0315] Table E5. Non-human primate studies of chimeric B15 fusion antibodies TIFF2024517759000007.tif50128NK-Ctrl=no knob negative control, iv=intravenous

[0316] Body weight was measured the day before dosing and on the last day of the experiment. Blood samples (0.5 mL) were collected from the femoral vein at different time points (days -3, 1, 2, 3, 4, 5, 6, 7, 10, 15, and 22) before and after fusion antibody dosing. Samples were collected for evaluation of leukocyte phenotype by flow cytometry. Samples were enrolled and processed on the day they were collected. Absolute cell counts and cell percentage values ​​were calculated for each phenotype for each sample. Absolute counts were determined using a dual platform method. In this dual approach, the cell percentage values ​​obtained by flow cytometry were used in conjunction with absolute leukocyte differential cell counts (i.e., lymphocytes or monocytes) determined by a hematology analyzer, respectively, to obtain the absolute number of each cell type per μL of whole blood for each individual sample. In addition to the determination of absolute leukocyte counts, the panel of tests contained monoclonal antibodies identifying the cell types in Table E6. Aliquots of whole blood specimens were stained with a predefined volume of pre-tested and titrated monoclonal antibodies specific for each phenotypic marker. After staining, red blood cells were lysed in each tube. Prepared samples were analyzed with BD FACSDiva v8.0.2. For pharmacodynamic analysis, values ​​of treated monkeys were compared to pre-treatment values. Fold changes (x) in peripheral blood leukocyte counts were determined by comparing the mean or individual values ​​of the treatment group with the mean or individual values ​​of the respective pre-treatment (day -3) group.

[0317] Table E6: Leukocyte phenotyping panel TIFF2024517759000008.tif33144

[0318] Results showed that none of the engineered antibodies significantly affected monkey body weight (Figure 4A). On day 2, there was a minimal to moderate decrease in absolute counts of all lymphocyte subtypes assessed by immunophenotyping (i.e., mature T cells [as low as 0.21x], helper T cells [as low as 0.24x], cytotoxic T cells [as low as 0.17x], NK cells [as low as 0.09x], and B cells [as low as 0.23x]), which was resolved by day 3 (Figure 4B-D). In animals receiving the fusion antibody (Groups 1 and 2), there was an increase in the absolute counts of all lymphocyte subtypes evaluated from days 4 or 5 to 6 or 7 (i.e., mature T cells [up to 1.67x], helper T cells [up to 1.48x], cytotoxic T cells [up to 2.01x], NK cells [up to 2.58x], and B cells [up to 1.83x]), which resolved by days 7 or 10, whereas there was no increase in lymphocyte counts in Group 3 (control) monkeys (Figures 4B-D). The increase in lymphocyte numbers in Group 2 (humanized B15 with IL-15Rα sushi domain) monkeys occurred one day later than in Group 1 (humanized B15 without IL-15Rα sushi domain) monkeys, and also resolved one day later (Figures 4B-D). The fusion antibody had no significant effect on monocytes from treated monkeys compared to controls (FIG. 4D).

[0319] Thus, B15 fusion antibodies both with and without the IL-15Rα sushi domain enhanced NK and T cell numbers in non-human primates.

[0320] The present invention is not intended to be limited in scope to the specific disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the descriptions and teachings herein. Such variations can be made without departing from the true scope and spirit of the present disclosure and are intended to fall within the scope of the present disclosure.

[0321] array TIFF2024517759000009.tif202152TIFF2024517759000010.tif215152TIFF2024517759000011.tif207152TIFF2024517759000012.tif217152TIFF2024517759000013.tif209152TIFF2024517759000014.tif218152TIFF2024517759000015.tif209152TIFF2024517759000016.tif209152TIFF2024517759000017.tif209152TIFF2024517759000018.tif214152TIFF2024517759000019.tif216152TIFF2024517759000020.tif216152TIFF2024517759000021.tif216152TIFF2024517759000022.tif215152TIFF2024517759000023.tif206152TIFF2024517759000024.tif211152TIFF2024517759000025.tif216152TIFF2024517759000026.tif90152

Claims

1. (a) a modified variable heavy (VH) region of a bovine antibody or antigen-binding fragment, or a humanized sequence thereof, wherein the modified VH region comprises a modified ultralong CDR3, and at least a portion of the ultralong CDR3 of the bovine antibody or antigen-binding fragment, or a humanized sequence thereof, is replaced by a cytokine sequence, or a biologically active portion thereof; (b) a modified human IgG heavy chain constant region having reduced effector activity compared to a wild-type human IgG heavy chain constant region; A chimeric engineered antibody comprising a heavy chain comprising:

2. 2. The chimeric engineered antibody of claim 1, wherein the cytokine sequence or a biologically active portion thereof replaces the knob region of the ultralong CDR3 region of the bovine antibody or antigen-binding fragment or humanized sequence thereof.

3. The chimeric modified antibody of claim 1, wherein the cytokine sequence or a biologically active portion thereof is located between the ascending stalk strand and the descending stalk strand of the modified ultralong CDR3, and the ascending stalk strand of the modified ultralong CDR3 is mutant compared to the ascending stalk strand of the ultralong CDR3 of the bovine antibody or antigen-binding fragment or humanized sequence thereof.

4. The chimeric modified antibody of claim 3, wherein the cytokine sequence or a biologically active portion thereof is linked to the ascending stalk strand and / or the descending stalk strand of the modified ultralong CDR3 via a flexible linker, optionally a GGS or GSG linker.

5. the ascending stalk strand has the sequence CX 2 TVX 5 Includes QETKKYQT, X 2 and X 5 The chimeric modified antibody of claim 3, wherein is any amino acid.

6. 1. A chimeric modified antibody comprising a heavy chain comprising a modified variable heavy (VH) region of a bovine antibody or antigen-binding fragment or a humanized sequence thereof, The modified VH region comprises a modified ultralong CDR3 in which at least a portion of the ultralong CDR3 region of the bovine antibody or antigen-binding fragment or humanized sequence thereof is replaced with a heterologous sequence, the heterologous sequence being between the ascending stalk strand and the descending stalk strand of the modified ultralong CDR3, and the ascending stalk strand of the modified ultralong CDR3 is selected from the group consisting of the sequence CX 2 TVX 5 Includes QETKKYQT, X 2 and X 5 is any amino acid, Chimeric engineered antibodies.

7. X 2 is Ser, Thr, Gly, Asn, Ala, or Pro, and X 5 The chimeric modified antibody of claim 6, wherein X is His, Gln, Arg, Lys, Gly, Thr, Tyr, Phe, Trp, Met, Ile, Val, or Leu, and optionally X2 is Ser, Ala, or Thr, and X5 is His or Tyr, and optionally the ascending stalk strand of the modified ultralong CDR3 comprises a sequence of amino acids set forth in any of SEQ ID NOs: 183-185.

8. X2 is Ser, Thr, Gly, Asn, Ala, or Pro, and X5 is His, Gln, Arg, Lys, Gly, Thr, Tyr, Phe, Trp, Met, Ile, Val, or Leu, and optionally X 2 is Ser, Ala, or Thr, and X 5 is His or Tyr, and optionally, the ascending stalk strand of the modified ultralong CDR3 comprises a sequence of amino acids set forth in any of SEQ ID NOs: 183-185.

9. the heterologous sequence replaces the knob region of the ultralong CDR3 region of the bovine antibody or antigen-binding fragment or humanized sequence thereof; and / or The heterologous sequence is linked to the ascending and / or descending stalk strands of the modified ultralong CDR3 via a flexible linker, optionally a GGS or GSG linker; The chimeric modified antibody of claim 6.

10. The chimeric modified antibody of claim 6, wherein the heterologous sequence comprises a cytokine sequence or a biologically active portion thereof.

11. 7. The chimeric modified antibody of claim 6, wherein the heavy chain further comprises a human IgG heavy chain constant region, optionally wherein the human IgG heavy chain constant region is a modified human IgG heavy chain constant region having reduced effector activity compared to a wild-type human IgG heavy chain constant region.

12. The chimeric modified antibody of claim 11, wherein the modified human IgG heavy chain constant region is modified to reduce FcR binding.

13. The chimeric modified antibody of claim 11, wherein the reduced effector activity comprises reduced antibody-dependent cell-mediated cytotoxicity (ADCC).

14. the modified human IgG heavy chain constant region is altered at one or two or more of the following positions: Glu233 (E233), Leu234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Asn297 (N297), Ser298 (S298), Asn325 (N325), Ala327 (A327), and Pro329 (P329); and / or comprising one or more mutations selected from Leu234Ala (L234A), Leu235Ala (L235A), Leu235Glu (L235E), Asp265Asn (D265N), Asp265Ala (D265A), Asp270Asn (D270N), Ser298Asn (S298N), Asn325Glu (N325E), Ala327Ser (A327S), Pro329Ala (P329A), and Pro239Gly (P329G); Leu234Ala and Leu235Ala (L234A / L235A) mutations; Leu234Val and Leu235Ala (L234V / L235A) mutations; Leu234Ala, Leu235Ala, and Asn297Ala (L234A / L235A / N297A) mutations; Leu234Ala, Leu235Ala, and Pro239Ala (L234A / L235A / P329A) mutations; Asp265Ala and Pro329Ala (D265A / P329A) mutations; A Leu234Ala, Leu235Ala, and Asp265Ala (L234A / L235A / D265A) mutations; Leu234Ala, Leu235Ala, and Pro329Gly (L234A / L235A / P329G) mutations; or Leu234Ala, Leu235Ala, Asp265Ala, and Pro329Gly (L234A / L235A / D265A / P329G) mutations, The chimeric modified antibody of claim 11.

15. 15. The chimeric modified antibody of claim 14, wherein said modified human IgG heavy chain constant region comprises Leu234Ala and Leu235Ala (L234A / L235A) mutations, and optionally said modified human IgG heavy chain constant region comprises the sequence depicted in SEQ ID NO:187 or SEQ ID NO:

188.

16. The chimeric modified antibody of claim 1, wherein the modified human IgG heavy chain constant region is modified to reduce FcR binding.

17. The chimeric modified antibody described in claim 1, wherein the reduced effector activity includes reduced antibody-dependent cell-mediated cytotoxicity (ADCC).

18. The modified human IgG heavy chain constant region, altered at one or two or more of the following positions: Glu233 (E233), Leu234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Asn297 (N297), Ser298 (S298), Asn325 (N325), Ala327 (A327), and Pro329 (P329); and / or comprising one or more mutations selected from Leu234Ala (L234A), Leu235Ala (L235A), Leu235Glu (L235E), Asp265Asn (D265N), Asp265Ala (D265A), Asp270Asn (D270N), Ser298Asn (S298N), Asn325Glu (N325E), Ala327Ser (A327S), Pro329Ala (P329A), and Pro239Gly (P329G); Leu234Ala and Leu235Ala (L234A / L235A) mutations; Leu234Val and Leu235Ala (L234V / L235A) mutations; Leu234Ala, Leu235Ala, and Asn297Ala (L234A / L235A / N297A) mutations; Leu234Ala, Leu235Ala, and Pro239Ala (L234A / L235A / P329A) mutations; Asp265Ala and Pro329Ala (D265A / P329A) mutations; A Leu234Ala, Leu235Ala, and Asp265Ala (L234A / L235A / D265A) mutations; Leu234Ala, Leu235Ala, and Pro329Gly (L234A / L235A / P329G) mutations; or Leu234Ala, Leu235Ala, Asp265Ala, and Pro329Gly (L234A / L235A / D265A / P329G) mutations, The chimeric modified antibody of claim 1.

19. The chimeric modified antibody of claim 18, wherein the modified human IgG heavy chain constant region comprises Leu234Ala and Leu235Ala (L234A / L235A) mutations, and optionally the modified human IgG heavy chain constant region comprises the sequence shown in SEQ ID NO:187 or SEQ ID NO:

188.

20. The chimeric modified antibody of claim 1, wherein the cytokine sequence or a biologically active portion thereof comprises an interleukin-15 (IL-15) cytokine sequence or an interleukin-2 (IL-2) cytokine sequence or a biologically active portion thereof.

21. The chimeric modified antibody of claim 10, wherein the cytokine sequence or a biologically active portion thereof comprises an interleukin-15 (IL-15) cytokine sequence or an interleukin-2 (IL-2) cytokine sequence or a biologically active portion thereof.

22. The cytokine sequence or a biologically active portion thereof, A sequence of amino acids that exhibits at least 85% or at least about 85% sequence identity to the sequence of amino acids set forth in SEQ ID NO: 165; The amino acid sequence shown in SEQ ID NO: 165, a sequence of amino acids that exhibits at least 85% or at least about 85% sequence identity to the sequence of amino acids set forth in SEQ ID NO:1, or The amino acid sequence shown in SEQ ID NO:1 The chimeric modified antibody of claim 1, comprising:

23. The cytokine sequence or biologically active portion thereof, A sequence of amino acids that exhibits at least 85% or at least about 85% sequence identity to the sequence of amino acids set forth in SEQ ID NO: 165; The amino acid sequence shown in SEQ ID NO: 165, a sequence of amino acids that exhibits at least 85% or at least about 85% sequence identity to the sequence of amino acids set forth in SEQ ID NO:1, or The amino acid sequence shown in SEQ ID NO:1 The chimeric modified antibody of claim 10, comprising:

24. 2. The chimeric modified antibody of claim 1, wherein the bovine antibody or antigen-binding fragment is the bovine antibody BLV1H12 or an antigen-binding fragment thereof.

25. The chimeric modified antibody of claim 6, wherein the bovine antibody or antigen-binding fragment is the bovine antibody BLV1H12 or an antigen-binding fragment thereof.

26. the ascending stalk strand comprises the sequence set forth in SEQ ID NO:183, the cytokine sequence or biologically active portion thereof comprises the sequence of amino acids set forth in SEQ ID NO:1, and the descending stalk strand comprises the sequence set forth in SEQ ID NO:10; the ascending stalk strand comprises the sequence set forth in SEQ ID NO:184, the cytokine sequence or biologically active portion thereof comprises the sequence of amino acids set forth in SEQ ID NO:1, and the descending stalk strand comprises the sequence set forth in SEQ ID NO:10; or the ascending stalk strand comprises the sequence set forth in SEQ ID NO:185, the cytokine sequence or biologically active portion thereof comprises the sequence of amino acids set forth in SEQ ID NO:1, and the descending stalk strand comprises the sequence set forth in SEQ ID NO:

10. The chimeric modified antibody of claim 3.

27. The method of claim 27, wherein the ascending stalk strand comprises the sequence set forth in SEQ ID NO:183, the cytokine sequence or a biologically active portion thereof comprises the sequence of amino acids set forth in SEQ ID NO:1, and the descending stalk strand comprises the sequence set forth in SEQ ID NO:10; the ascending stalk strand comprises the sequence set forth in SEQ ID NO:184, the cytokine sequence or biologically active portion thereof comprises the sequence of amino acids set forth in SEQ ID NO:1, and the descending stalk strand comprises the sequence set forth in SEQ ID NO:10; or the ascending stalk strand comprises the sequence set forth in SEQ ID NO:185, the cytokine sequence or biologically active portion thereof comprises the sequence of amino acids set forth in SEQ ID NO:1, and the descending stalk strand comprises the sequence set forth in SEQ ID NO:

10. The chimeric modified antibody of claim 10.

28. The chimeric modified antibody of claim 1, wherein the modified ultralong CDR3 comprises a sequence as set forth in any of SEQ ID NOs: 206 to 208.

29. The chimeric modified antibody described in claim 6, wherein the modified ultralong CDR3 comprises an array shown in any of SEQ ID NO:206 to 208.

30. The heavy chain comprises the formula V1-X-V2-C, wherein the V1 region of the heavy chain comprises the sequence set forth in SEQ ID NO:182; the X region comprises the modified ultralong CDR3; the V2 region comprises the sequence set forth in SEQ ID NO:11; and the C region comprises the modified human IgG heavy chain constant region; or the V1 region of the heavy chain comprises the sequence set forth in SEQ ID NO:197, or a sequence exhibiting at least 65% sequence identity to SEQ ID NO:197; the X region comprises the modified ultralong CDR3; the V2 region comprises the sequence set forth in SEQ ID NO:11; and the C region comprises the modified human IgG heavy chain constant region. The chimeric modified antibody of claim 1.

31. The heavy chain comprising the formula V1-X-V2-C, wherein: the V1 region of the heavy chain comprises the sequence set forth in SEQ ID NO:182; the X region comprises the modified ultralong CDR3; the V2 region comprises the sequence set forth in SEQ ID NO:11; and the C region comprises the modified human IgG heavy chain constant region; or the V1 region of the heavy chain comprises the sequence set forth in SEQ ID NO:197, or a sequence exhibiting at least 65% sequence identity to SEQ ID NO:197; the X region comprises the modified ultralong CDR3; the V2 region comprises the sequence set forth in SEQ ID NO:11; and the C region comprises the modified human IgG heavy chain constant region. The chimeric modified antibody of claim 11.

32. the modified VH region comprises a sequence as set forth in any of SEQ ID NOs:200-205; and / or the heavy chain comprises a sequence as set forth in any of SEQ ID NOs: 189-194; The chimeric modified antibody of claim 1.

33. The modified VH region comprises a sequence as set forth in any of SEQ ID NOs:200-205; and / or the heavy chain comprises a sequence as set forth in any of SEQ ID NOs: 189-194; The chimeric modified antibody of claim 11.

34. The chimeric modified antibody of claim 1, wherein the modified VH region is a mutant of the humanized sequence of the VH region of BLV1H12.

35. The chimeric modified antibody of claim 6, wherein the modified VH region is a mutant of the humanized sequence of the VH region of BLV1H12.

36. The chimeric modified antibody of claim 1, further comprising a light chain, optionally comprising a humanized light chain comprising the sequence shown in SEQ ID NO:181 or a sequence exhibiting at least 85% sequence identity to the sequence shown in SEQ ID NO:

181.

37. A chimeric modified antibody as described in claim 6, further comprising a light chain, optionally comprising a humanized light chain comprising the sequence shown in SEQ ID NO:181 or a sequence exhibiting at least 85% sequence identity to the sequence shown in SEQ ID NO:

181.

38. The chimeric modified antibody of claim 1, wherein the chimeric modified antibody is complexed with an extracellular domain of IL15Rα that includes the IL15Rα sushi domain.

39. The chimeric modified antibody of claim 6, wherein the chimeric modified antibody is complexed with an extracellular domain of IL15Rα comprising the IL15Rα sushi domain.

40. A polynucleotide encoding the heavy chain, light chain or variable region thereof of the chimeric modified antibody of any one of claims 1 to 39, or encoding the chimeric modified antibody of any one of claims 1 to 39.

41. 41. An expression vector comprising the polynucleotide of claim 40.

42. 41. A host cell comprising the polynucleotide of claim 40 or an expression vector comprising the polynucleotide of claim 40.

43. A method for producing a chimeric modified antibody, comprising culturing a host cell described in claim 42 under conditions for expressing the chimeric modified antibody, the heavy chain or a variable region thereof of the chimeric modified antibody, or the light chain or a variable region thereof of the chimeric modified antibody by the host cell.

44. 44. A chimeric engineered antibody produced by the method of claim 43 or comprising the heavy chain or the variable region thereof or the light chain or the variable region thereof produced by the method of claim 43.

45. A pharmaceutical composition comprising a chimeric modified antibody according to any one of claims 1 to 39.

46. 40. A method of stimulating or promoting the proliferation of immune cells, comprising contacting a population of immune cells with a chimeric modified antibody according to any one of claims 1 to 39, thereby stimulating cells of said population of immune cells.

47. 40. Use of a chimeric modified antibody according to any one of claims 1 to 39 in the manufacture of a medicament for treating cancer in a subject.

48. Use of a combination of a chimeric modified antibody according to any one of claims 1 to 39 and an antitumor agent in the manufacture of a medicament for treating cancer in a subject.

49. A pharmaceutical composition comprising the chimeric modified antibody of any one of claims 1 to 39 for use in a method for treating cancer in a subject.

50. A pharmaceutical composition for treating cancer in a subject, comprising a chimeric modified antibody described in any one of claims 1 to 39, said pharmaceutical composition being used in combination with an anti-tumor agent.

51. 51. The pharmaceutical composition of claim 50, wherein the anti-tumor agent comprises a monoclonal antibody, a checkpoint inhibitor, and / or a cell therapy, optionally wherein the anti-tumor agent comprises a cell therapy comprising cells expressing IL2 / 15Rβ and IL2 / 15Rβ γc receptor subunits, and / or wherein the cell therapy is a T cell therapy or a NK cell therapy.

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