Chimeric cytokine modified antibodies and methods of use thereof

Abstract

To provide improved cytokine therapeutics, such as IL-2 or IL-15 therapeutics.SOLUTION: Provided are chimeric cytokine modified antibodies containing an ultralong CDR3, such as based on a bovine antibody sequence or a humanized sequence thereof, in which a part of the CDR3 of the heavy chain is replaced by an interleukin (IL-15) or IL-2, and related antibodies. Among provided antibodies are chimeric IL-15 cytokine modified antibody molecules that are further linked or complexed with an extracellular portion of the IL15Rα, such as the IL15Rα sushi domain.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 925,740, filed October 24, 2019, entitled "CHIMERIC CYTOKINE MODIFIED ANTIBODIES AND METHODS OF USE THEREOF," the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] Sequence Listing This application is filed with an electronic Sequence Listing, which is provided as a file entitled 165772000140SeqList.txt, created on October 22, 2020, and is 79,015 bytes 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 cytokine-engineered antibodies containing ultralong CDR3s, such as those based on bovine antibody sequences or humanized sequences thereof, in which a portion of the CDR3 of the heavy chain is replaced by interleukin (IL-15) or IL-2, and related antibodies. Molecules of the present disclosure include chimeric IL-15 cytokine-engineered antibody molecules 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 cytokine-engineered antibodies. [Background technology]

[0004] background Antibodies are natural proteins formed by the immune system of vertebrates in response to foreign substances (antigens), primarily for defense against infection. Antibodies contain complementarity-determining regions (CDRs) that mediate binding to target antigens. Some bovine antibodies have unusually long VH CDR3 sequences compared to other vertebrates, which can be up to 70 amino acids long. The long CDR3 can form a unique domain that protrudes 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 cancer treatment. However, such cytokines can be difficult to express as stable soluble proteins and often have short half-lives in vitro and in vivo. There remains a need for improved cytokine therapeutics, such as IL-2 or IL-15 therapeutics, particularly for use in cancer treatment. Summary of the Invention

[0006] overview Provided herein are chimeric cytokine-modified antibodies or antigen-binding fragments comprising a modified ultralong CDR3 comprising an interleukin-15 (IL-15) cytokine sequence, or a biologically active portion thereof, replacing at least a portion of the ultralong CDR3 region of the heavy chain of a bovine antibody or antigen-binding fragment, or a humanized sequence thereof.

[0007] In some embodiments, the IL-15 cytokine sequence is human IL-15. In some embodiments, the IL-15 cytokine sequence comprises a sequence of amino acids that exhibits 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. In some embodiments, the IL-15 cytokine sequence comprises the sequence of amino acids set forth in SEQ ID NO:1. In some embodiments, the IL-15 cytokine sequence consists of the sequence of amino acids set forth in SEQ ID NO:1.

[0008] Provided herein are chimeric cytokine-modified antibodies or antigen-binding fragments comprising a modified ultralong CDR3 comprising an interleukin-2 (IL-2) cytokine sequence, or a biologically active portion thereof, replacing at least a portion of the ultralong CDR3 region of the heavy chain of a bovine antibody or antigen-binding fragment, or a humanized sequence thereof.

[0009] In some embodiments, the IL-2 cytokine sequence is human IL-2. In some embodiments, the IL-2 cytokine sequence comprises a sequence of amino acids that exhibits 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. In some embodiments, the IL-2 cytokine sequence comprises the sequence of amino acids set forth in SEQ ID NO:165. In some embodiments, the IL-2 cytokine sequence consists of the sequence of amino acids set forth in SEQ ID NO:165.

[0010] In some of the optional embodiments, the cytokine sequence replaces at least a portion of the ultralong CDR3 region of the heavy chain of a bovine antibody or antigen-binding fragment, hi some embodiments, the bovine antibody or antigen-binding fragment is the bovine antibody BLV1H12 or an antigen-binding fragment thereof.

[0011] In some embodiments, the bovine antibody or antigen-binding fragment comprises a variable heavy chain amino acid sequence encoded by the sequence set forth in SEQ ID NO: 5 and a variable light chain amino acid sequence encoded by the sequence set forth in SEQ ID NO: 8. In some embodiments, the bovine antibody or antigen-binding fragment comprises a variable heavy chain amino acid sequence encoded by the sequence set forth in SEQ ID NO: 167 and a variable light chain amino acid sequence encoded by the sequence set forth in SEQ ID NO: 168.

[0012] In some embodiments, the bovine antibody or antigen-binding fragment comprises a variable heavy chain set forth in SEQ ID NO:26 and a variable light chain set forth in SEQ ID NO:27.

[0013] In some of the optional embodiments, the cytokine sequence replaces at least a portion of the ultralong CDR3 region of the heavy chain of a humanized bovine antibody or antigen-binding fragment thereof. In some embodiments, the humanized bovine antibody or antigen-binding fragment thereof comprises a heavy chain or portion thereof that is or is derived from a human heavy chain germline sequence and a light chain or portion thereof that is or is derived from a human light chain germline sequence. In some embodiments, the human heavy chain germline sequence is a VH4-39, VH4-59*03, VH4-34*02, or VH4-34*09 germline sequence, or a sequence set forth in any one of SEQ ID NOs:68-71.

[0014] In some of any of the embodiments, the human light chain germline sequence is a VL1-51 germline sequence or a sequence based on the VL1-51 germline sequence comprising one or more mutations, optionally wherein the VL1-51 germline sequence is set forth in SEQ ID NO: 156. In some embodiments, the one or more mutations are selected from the following: one or more of the amino acid substitutions S2A, T5N, P8S, A12G, A13S, and P14L according to the Kabat numbering system; the amino acid substitutions S2A, T5N, P8S, A12G, A13S, and P14L according to the Kabat numbering system; a mutation in CDR1 comprising the amino acid substitutions I29V and N32G; a mutation in CDR2 comprising the substitution DNN for GDT; a mutation in CDR2 comprising the substitution DNNKRP for GDTSRA; or a combination of any of the foregoing.

[0015] In some of the embodiments, the antibody provided is an antigen-binding fragment comprising a variable heavy chain and a variable light chain. In some embodiments, the antibody comprises a variable heavy chain linked to a heavy chain constant domain (CH1-CH2-CH3) and a variable light chain linked to a light chain constant domain (CL1). In some embodiments, the heavy chain constant domain is derived from human IgG1. In some embodiments, the light chain constant domain is a lambda light chain region.

[0016] In some embodiments, at least a portion of the ultralong CDR3 region comprises a knob region, and the cytokine sequence is located between the ascending and descending stalk domains of the modified ultralong CDR3. In some embodiments, the cytokine sequence is linked to the ascending and / or descending stalk domains via a flexible linker, optionally a GGS or GSG linker. In some embodiments, the ascending stalk domain comprises the sequence set forth in SEQ ID NO: 158 or SEQ ID NO: 159. In some embodiments, the descending stalk domain comprises the sequence set forth in SEQ ID NO: 161.

[0017] In some embodiments, the antibodies or antigen-binding fragments provided comprise a variable heavy chain sequence encoded by a sequence of nucleotides set forth in SEQ ID NO:7, or a sequence of nucleotides that exhibits 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%, at least 99%, or at least about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:7, and contain a modified ultralong CDR3 containing an IL-15 sequence. In some embodiments, the antibodies or antigen-binding fragments provided comprise a variable heavy chain sequence encoded by a sequence of nucleotides set forth in SEQ ID NO:7. In some embodiments, the antibodies or antigen-binding fragments provided consist of a variable heavy chain sequence encoded by a sequence of nucleotides set forth in SEQ ID NO:7.

[0018] In some embodiments, the antibody or antigen-binding fragment is complexed with an extracellular domain of IL15Rα comprising an IL15Rα sushi domain. In some embodiments, the extracellular domain of IL15Rα comprising the IL15Rα sushi domain is non-covalently associated with the IL-15 sequence. In some embodiments, the extracellular domain of IL15Rα comprising the IL15Rα sushi domain is linked to the variable light chain. In some embodiments, the extracellular domain of IL15Rα comprising the IL15Rα sushi domain is linked to the variable light chain via a peptide linker. In some embodiments, the peptide linker is a glycine linker or a glycine-serine linker, and optionally the linker is GS.

[0019] In some of the embodiments, the extracellular domain of IL15Rα comprising the IL15Rα sushi domain comprises the sequence shown in SEQ ID NO: 2. In some of the embodiments, the extracellular domain of IL15Rα comprising the IL15Rα sushi domain consists of the sequence shown in SEQ ID NO: 2.

[0020] In some embodiments, the variable light chain comprises the sequence of amino acids encoded by SEQ ID NO:3.

[0021] Provided herein is a polynucleotide encoding the chimeric cytokine-modified antibody or antigen-binding fragment according to any of the previous aspects.

[0022] Provided herein is a polynucleotide encoding the heavy chain or variable region thereof of the chimeric cytokine-modified antibody or antigen-binding fragment according to any of the previous aspects.

[0023] Provided herein is a polynucleotide encoding the light chain, or variable region thereof, of the chimeric cytokine-modified antibody or antigen-binding fragment according to any of the previous aspects.

[0024] Provided herein is an expression vector comprising a polynucleotide according to any of the preceding aspects.

[0025] Provided herein is a host cell comprising the polynucleotide or expression vector of any of the preceding aspects. In some of any of the aspects, the host cell further comprises a polynucleotide or vector that expresses the extracellular domain of IL15Rα comprising the IL15Rα sushi domain. In some of any of the aspects, the extracellular domain of IL15Rα comprising the IL15Rα sushi domain comprises the sequence set forth in SEQ ID NO:2.

[0026] Provided herein is a method of producing a chimeric cytokine modified antibody or antigen-binding fragment, comprising culturing a host cell according to any of the preceding aspects under conditions for expression of said antibody or antigen-binding fragment by said cell, and optionally further comprising recovering said antibody or antigen-binding fragment by purification.

[0027] Provided herein is a chimeric cytokine-modified antibody or antigen-binding fragment produced by the method of any of the preceding aspects.

[0028] Provided herein is a pharmaceutical composition comprising a chimeric cytokine-modified antibody or antigen-binding fragment according to any of the preceding aspects.

[0029] Provided herein is a method of treating cancer in a subject, comprising administering a therapeutically effective amount of a chimeric cytokine-modified antibody or antigen-binding fragment of any of the previous aspects.

[0030] Provided herein is a method of treating cancer in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of any of the previous aspects. [Brief explanation of the drawings]

[0031] [Figure 1] Figures 1A and 1B show schematic diagrams of the generated constructs. Figure 1A shows the crystal structure of BLV1H12 (left), which shows 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 B15_IL15Rα sushi variant of BLV1H12 (right), in which the knob region is replaced with an IL-15 cytokine domain and additionally contains an IL15Rα sushi domain. Figure 1B shows the different fusion antibody constructs: BLV1H12-IL-15 (B15), BLV1H12-IL-15-Rα sushi (B15_Rα sushi), and BLV1H12-IL-15-GS-Rα sushi (B15_GS_Rα sushi). [Figure 2]Figure 1 shows the expression of purified B15 fusion antibody constructs expressed from HEK 293 cells BLV1H12-IL-15 (B15), BLV1H12-IL-15-Rαsushi (B15_Rαsushi), and BLV1H12-IL-15-GS-Rαsushi (B15_GS_Rαsushi) and analyzed by SDS-PAGE gel electrophoresis. [Figure 3] Figures 3A and 3B show the ability of the chimeric BLV1H12-IL-15 (B15) fusion antibody to bind to the IL2 / 15Rβ receptor, as determined by ELISA assay. Figure 3A shows the ability of the B15 antibody to bind to the IL2Rα or IL15Rα receptor subunit. Figure 3B shows the ability of the B15 antibody to bind to the IL2 / 15Rβ receptor subunit in the presence or absence of the IL15Rα subunit. [Figure 4] Activation of IL2 / 15Rβ and γc receptors and STAT5 signaling by chimeric B15 molecules is demonstrated through induction and secretion of the STAT5-inducible alkaline phosphatase (SEAP) reporter gene in HEK-Blue IL2 reporter cells. [Figure 5] We demonstrate activation of IL2 / 15Rβ and γc receptors and STAT5 signaling by alternative chimeric B15 molecules associated with the IL15Rα sushi domain through induction and secretion of the STAT5-inducible alkaline phosphatase (SEAP) reporter gene in HEK-Blue IL2 reporter cells. [Figure 6] The ability of chimeric B15 molecules to expand NK-92 natural killer cells is shown. NK-92 cells were incubated with 2-fold serial dilutions (1.33 nM to 0.005 nM) of IL2 or IL15 monomer (R&D Systems), or chimeric B15, chimeric mutant B15_Rαsushi, or chimeric B15 mutant B15_GS_Rαsushi antibodies, and analyzed by MTT assay. [Figure 7]FIG. 1 shows the ability of chimeric B15 antibodies compared to chimeric B15 mutant B15-Rαsushi or B15-GS-Rαsushi antibodies to expand NK-92 natural killer cells as demonstrated by MTT assay. [Figure 8] Figures 8A and 8B show schematics of the generated constructs. Figure 8A shows the crystal structure of BLV1H12 (left), which shows 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 a chimeric BLV1H12-IL-2(B2) fusion antibody (right), which was generated by replacing the IL-15 region of the chimeric B15 antibody with IL-2. Figure 8B shows a diagram of the BLV1H12-IL-2(B2) fusion antibody, which contains the IL-2 sequence in the knob domain. [Figure 9] FIG. 1 shows the expression of purified fusion antibody construct BLV1H12-IL-2(B2) expressed from HEK 293 cells and analyzed by SDS-PAGE gel electrophoresis. [Figure 10] 1 shows the ability of chimeric BLV1H12-IL-2(B2) fusion antibodies to bind to IL2Rα and IL15Rα as demonstrated by enzyme-linked immunosorbent assay (ELISA). [Figure 11] Activation of IL2 / 15Rβ and γc receptors and STAT5 signaling by chimeric B2 molecules is demonstrated through induction and secretion of the STAT5-inducible alkaline phosphatase (SEAP) reporter gene in HEK-Blue IL2 reporter cells. [Figure 12] The ability of chimeric B2 molecules to expand NK-92 natural killer cells is shown. NK-92 cells were incubated with either IL2 monomer (R&D Systems) or chimeric B2 antibody at two-fold serial dilutions (1.33 nM to 0.005 nM) and analyzed by MTT assay. [Figure 13]We demonstrate the ability of chimeric B15 molecules to stimulate NK cells and T cells in human PBMCs in vitro. PBMCs were incubated with B15 and B15_Rα sushi at final concentrations ranging from 250 nM to 0.016 nM. Afterwards, PBMCs were stained with anti-CD3-FITC (SK7), anti-CD4-PE (OKT4), anti-CD8a-eFluor 450 (SK1), and anti-CD56-APC (AF12-7H3), followed by analysis using a Novocyte Advanteon Flow Cytometer (Agilent, Santa Clara, CA). DETAILED DESCRIPTION OF THE INVENTION

[0032] Detailed Description Provided herein are chimeric cytokine-engineered antibody fusion molecules in which an IL-15 or IL-2 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 ultralong CDR3 region contains the ascending stalk region, knob region, and descending stalk region present in a bovine antibody, with all or a portion of the knob region replaced by the cytokine sequence. In some embodiments, the cytokine sequence is IL-2 or a biologically active portion thereof, e.g., IL-2 has the sequence set forth in SEQ ID NO:165. In some embodiments, the cytokine sequence is IL-15 or a biologically active portion thereof, e.g., IL-15 has the sequence set forth in SEQ ID NO:1. Also provided herein are variant chimeric IL-15 modified antibodies, including such antibodies linked or conjugated to the extracellular portion of IL15Rα, such as the IL15Rα sushi domain (eg, as shown in SEQ ID NO:2).

[0033] IL-15 and IL-2 are pleiotropic cytokines that play important roles in both innate and adaptive immunity. IL-15, like IL-2, was originally 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-alpha helix bundle family, and their membrane receptors share two signaling subunits (IL-2R / IL-15R beta and gamma chains). IL-15 functions via the trimeric IL-15R complex, which consists of a high-affinity binding alpha chain (IL-15Rα) and the common IL-2R beta and gamma 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).

[0034] The IL-15Rα and IL-2Rα subunits form a subfamily of cytokine receptors containing so-called "sushi" structural domains (one in IL-15Rα and two in IL-2Rα) at ​​their N-terminus, an extracellular domain also found in complement and 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 contains approximately 60 aa residues and 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. Two disulfide bonds are essential for maintaining 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 increasing IL15-mediated trans-signaling to the receptor β and γ subunits (IL2 / 15Rβ and γc).

[0035] In some embodiments, IL-2 stimulates the proliferation, activation, and sometimes cytotoxicity of cytotoxic T lymphocytes and natural killer (NK) cells. In some embodiments, IL-15 stimulates the proliferation, activation, and sometimes cytotoxicity of cytotoxic T lymphocytes and natural killer (NK) cells. IL-15 may be a better drug candidate than IL-2 because it does not cause vascular leak syndrome and does not stimulate regulatory T 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.

[0036] The provided embodiments address these issues. Among the provided embodiments 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 its humanized variant. The provided antibodies containing an IL-15 cytokine sequence, or a biologically active portion thereof, can be further linked or complexed with an extracellular portion of IL15Rα, such as the IL15Rα sushi domain, to further mediate IL15 activity. It is herein found that the provided chimeric molecules, including chimeric IL2 molecules (e.g., B2) or chimeric IL15 molecules (e.g., B15) and variants thereof complexed or linked with the extracellular portion of IL15Rα, can be expressed and purified similarly to typical human antibodies and exhibit efficient binding and activity to the IL2 / 15Rβ and γc subunits. In particular, the provided molecules function similarly to the respective IL-2 or IL15 soluble monomeric cytokines in in vitro signaling assays, but can be easily produced in mammalian cells and have increased stability.

[0037] Such antibodies may be useful in the treatment or prevention of a variety of 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.

[0038] The present disclosure also provides methods and materials for the preparation of the provided chimeric cytokine modified antibodies, including chimeric IL-15 modified antibodies and chimeric IL-2 modified antibodies.

[0039] 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 were individually incorporated by reference. To the extent that a definition set forth herein contradicts or is 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.

[0040] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0041] 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 one 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.

[0042] 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.

[0043] Throughout this disclosure, various aspects of the claimed subject matter are presented in 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. Accordingly, 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 independently be 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 either or both of those included limits are also encompassed within the claimed subject matter. This applies regardless of the width of the range.

[0044] As used herein, the term "about" will be understood by those of ordinary skill in the art and will vary to some extent depending 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%, and even more preferably ±0.1% from the specified value, such variations being appropriate for practicing the disclosed methods.

[0045] As used interchangeably herein, "ultralong CDR3" or "ultralong CDR3 sequence" includes CDR3 or CDR3 sequences that are not derived from human antibody sequences. Ultralong CDR3s 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, the ultralong CDR3 is a heavy chain CDR3 (CDR-H3 or CDRH3). Ultralong CDR3H3 exhibits the CDRH3 characteristics of ruminant (e.g., bovine) sequences. The length of the ultralong CDR3 can include non-antibody sequences, such as cytokine sequences, for example, IL-15.

[0046] "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.

[0047] "Binding affinity" generally refers to the strength of the sum total 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, which 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 expressed 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 for measuring binding affinity are known in the art, any of which can be used for the purposes of this disclosure.

[0048] "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 specific 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 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 appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared.

[0049] "Polypeptide," "peptide," "protein," and "protein fragment" can 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 corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0050] "Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as amino acids 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 naturally occurring amino acids, such as an alpha carbon bonded 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 naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that differs from the general chemical structure of an amino acid but function similarly to a naturally occurring amino acid.

[0051] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. "Amino acid variant" refers to an amino acid sequence. With respect to a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids encoding identical or essentially identical amino acid sequences, or, if the nucleic acid does not encode an amino acid sequence, to essentially identical or related (e.g., naturally adjacent) sequences. Due to the degeneracy of the genetic code, many functionally identical nucleic acids encode most proteins. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be altered to another of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," a type of conservatively modified variation. All nucleic acid sequences herein that encode a polypeptide also describe silent variations 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 altered to obtain a functionally identical molecule. Therefore, silent mutations of nucleic acids encoding polypeptides are included in the sequences described for expression products, but not in the actual probe sequences.With respect to 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.Typical 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)).

[0052] "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, e.g., minimal sequence derived from 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).

[0053] "Variable domain" in reference to an antibody refers to a specific Ig domain of an antibody's heavy or light chain that contains a sequence of amino acids that differs 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 an antigen-binding site domain and CDRs, which are part of the framework region (FR).

[0054] The term "constant region domain" refers to the domain of an antibody heavy or light chain that contains a sequence of amino acids that is 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 a hinge region, while IgE and IgM contain CH1, CH2, CH3, and CH4. The CH1 and CL domains extend the Fab arm of the antibody molecule and thus contribute to the interaction with antigens and 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 specifically bound by the antibody, through interactions with various cells, biomolecules, and tissues.

[0055] 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., by stating that the nucleotide or amino acid position "corresponds to" a nucleotide or amino acid position in a disclosed sequence as set forth in a sequence listing. For example, corresponding residues of similar sequences (e.g., fragments or species variants) can be determined by alignment to a reference sequence using structural alignment methods. By aligning the sequences, one skilled in the art can identify corresponding residues, e.g., using conserved and identical amino acid residues as a guide.

[0056] The term "effective amount" or "therapeutically effective amount," as used herein, refers to that amount of a pharmaceutical composition sufficient to significantly and positively alter 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 pharmaceutically acceptable excipients and / or carriers utilized, and similar factors associated with the knowledge and expertise of the attending physician.

[0057] As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not abrogate the biological activity or properties of the compound and that 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.

[0058] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells, which may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0059] 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. Pharmaceutical compositions facilitate administration of the compound to an organism. Multiple techniques for administering compounds exist in the art, including, but not limited to, intravenous, oral, aerosol, parenteral, ocular, pulmonary, and topical administration.

[0060] 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 identifiable symptoms.

[0061] As used herein, the terms "treat," "treating," or "treatment" refer to ameliorating a disease or disorder, e.g., delaying or arresting 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.

[0062] 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.

[0063] 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.

[0064] II. Chimeric Cytokine-Engineered Antibodies Provided herein are chimeric modified antibodies in which 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, replaces a portion of the ultralong CDR3 region of the heavy chain of a bovine (cow) antibody or a humanized sequence thereof. The provided chimeric modified IL-15 antibodies also include such antibodies linked or conjugated to the extracellular portion of IL15Rα, e.g., the IL15Rα sushi domain (e.g., as shown in SEQ ID NO:2).

[0065] 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 amino acids in length or longer. CDR3 sequences identified in cattle include BLV1 H12 (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., BLV1H12, BLV1H13, BLV5B8, BLV5B9, BLV5B10, BLV5B11, BLV5B12, BLV5B13, BLV5B14, BLV5B15, BLV5B16, BLV5B17, BLV5B18, BLV5B19, BLV5B20, BLV5B21, BLV5B22, BLV5B23, BLV5B24, BLV5B25, BLV5B26, BLV5B27, BLV5B28, BLV5B29, BLV5B30, BLV5B31, BLV5B32, BLV5B33, BLV5B34, BLV5B35, BLV5B36, BLV5B37, BLV5B38, BLV5B39, BLV5B40, BLV5B41, BLV5B42, BLV5B43, BLV5B44, BLV5B45, BLV5B46, BLV5B47, BLV5B48, BLV5B49, BLV5B51, BLV5B52, BLV5B53, BLV5B54, BLV5B55, BLV5B56, BLV5B57, BLV5B58, BLV5B60, BLV5B61, BLV5B62, BLV5B63, BLV5B64, BLV5B65, BLV5B66, BLV5B67, BLV5B68, BLV5B69, BLV5B70, BLV5B71, BLV5B72, BLV5B73, BLV5B74, BLV5B75, BLV5B76, BLV5B77, BLV5B78, BLV5B79, B

[0066] In bovine antibodies, ultralong CDR3 sequences form a structure consisting of a "stalk" composed of two 12-residue antiparallel β-strands (an ascending strand and a descending strand) and a 39-residue disulfide-rich "knob" located on the stalk, forming a subdomain with an unusual architecture significantly different from that of a standard antibody paratope. The long antiparallel β-ribbon functions as a bridge connecting the knob domain to the main backbone of the antibody. The unique "stalk and knob" structure of ultralong CDR3s results in two antiparallel β-strands, i.e., the ascending and descending stalk strands, supporting the disulfide-bonded knob that protrudes from the antibody surface to form a mini-antigen-binding domain. In some embodiments, ultralong CDR3 antibodies comprise, in order, an ascending stalk region, a knob region, and a descending stalk region.

[0067] 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 its base, which 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 sequences of various bovine or cow sequences. The conserved T(T / S)VHQ motif is connected by a variable number of residues to a β-turn-forming motif (CPDG in BLV1H12) at the base of each knob. The stalk can be of variable length, and the descending strands of the stalk contain alternating aromatic groups that form ladders through stacking interactions that may contribute to the stability of long, solvent-exposed, two-stranded β-ribbons (Wang et al. Cell. 2013,153(6):1379-1393).

[0068] The ultralong CDR3 sequences of the heavy chains of the chimeric antibodies provided herein contain a stalk component containing an ascending strand and a descending strand connected to each other by a knob domain containing 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. In some embodiments, the provided antibodies comprise cytokine (e.g., IL-2 or IL-15)-modified ultralong CDR3 fusions in which the antibody sequence is based on or derived from bovine or cow sequences or humanized sequences thereof, having an ultralong CDR3 in the heavy chain, but the ultralong CDR3 has been modified to contain a non-antibody cytokine sequence compared to the ultralong CDR3 derived from the antibody sequence. In some embodiments, the non-antibody sequence is IL-2 or a biologically active portion thereof, and the IL-2 or a biologically active portion thereof can be inserted into the ultralong CDR3 portion. In some embodiments, the non-antibody sequence is IL-15 or a biologically active portion thereof, and the IL-15 or a biologically active portion thereof can be inserted into a portion of the ultralong CDR3. For example, an antibody scaffold can be derived from or based on a bovine antibody sequence or a humanized version thereof, but includes 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, inserted into or substituting a portion of the knob domain of the ultralong CDR3 of the heavy chain of the bovine antibody sequence or a humanized version thereof.

[0069] In some embodiments, the IL-15 sequence, or a biologically active portion thereof, is inserted into the knob region of the CDR3 sequence of an antibody, optionally including removal of 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 sequence, or a 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 sequence, or a biologically active portion thereof, is contained between the ascending and descending stalk strands.

[0070] In some embodiments, the IL-2 sequence, or a biologically active portion thereof, is inserted into the knob region of the CDR3 sequence of an antibody, optionally including removal of 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 sequence, or a biologically active portion thereof, may be inserted into the knob domain of an ultralong CDR3 (Figures 8A and 8B). In some embodiments, the IL-2 sequence, or a biologically active portion thereof, is contained between the ascending and descending stalk strands.

[0071] 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).

[0072] 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 entirety.

[0073] A. Heavy Chain Region In provided embodiments, the heavy chain of the provided chimeric cytokine modified antibodies is based on or derived from a framework sequence having an ultralong CDR3 in which 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.

[0074] In some embodiments, the heavy chain of the provided chimeric cytokine-engineered antibody is based on or derived from a bovine or cow framework sequence in which a cytokine sequence, such as IL-2 or a biologically active portion thereof, or IL-15 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 an ultralong CDR3 fusion containing a cytokine sequence. Alternatively or additionally, the antibody can comprise at least a portion of a BLV5D3, BLV8C11, BF1H1, BLV5B8, and / or F18 antibody containing an ultralong CDR3 fusion containing a cytokine sequence. In some embodiments, IL-15 or a biologically active portion thereof can be inserted into or replace at least a portion of the ultralong CDR3 of the sequence set forth in SEQ ID NO:26 or SEQ ID NO:28.

[0075] In some embodiments, the heavy chain of the provided chimeric IL-15 modified antibodies is based on or derived from a humanized heavy chain framework sequence that is humanized compared to a bovine or cow sequence. In some embodiments, the heavy chain of the provided chimeric cytokine modified antibodies is based on or derived from a human heavy chain framework sequence that exhibits sequence or structural similarity to a bovine or cow sequence. In some cases, humanization can involve 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 a human framework and a non-human ultralong CDR3. Unlike human antibodies, which can 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 cattle-derived ultralong CDR3 sequences are humanized to produce antibodies comprising ultralong CDR3s, a human V-region sequence from the VH4 family can be genetically fused to the bovine-derived ultralong CDR3 sequence. Exemplary VH4 germline gene sequences in human antibody loci include, but are not limited to, the VH4-39, VH4-59*03, VH4-34*02, or 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 the sequence encoded by the sequence set forth in any one of SEQ ID NOs:169-172.

[0076] In some embodiments, a cytokine sequence, such as IL-2 or a biologically active portion thereof, or IL-15 or a biologically active portion thereof, may be inserted into or replace at least a portion of an ultralong CDR3 of a human germline sequence comprising the sequence set forth in SEQ ID NOs:68-71.

[0077] In some embodiments, the provided antibody comprises a fusion of a human VH4 framework sequence with a bovine-derived ultralong CDR3, with at least a portion of the knob replaced with IL-15 or IL-2, or a biologically active portion thereof. In some aspects, such a fusion can be produced 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-terminal) of the CDR3. Second, a second cysteine ​​in the bovine-derived V-region sequence, which also marks two residues upstream (N-terminal) of the CDR3, is identified. Third, genetic material encoding a human V-region is combined with a genetic sequence encoding the ultralong CDR3. In this way, a genetic fusion can be performed in which the ultralong CDR3 sequence is positioned in frame with the human V-region sequence. Preferably, a humanized antibody comprising an 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, and optionally, the humanized heavy chain can be paired with a human light chain.

[0078] In some embodiments, the antibody or binding fragment thereof comprises a heavy chain variable region comprising a sequence of the formula V1-X-V2, wherein 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 two CDR regions (CDR1 and CDR3), X comprises an ultralong CDR3 sequence that may comprise an IL-2 sequence or a biologically active portion thereof or an IL-15 sequence or a biologically active portion thereof, and V2 comprises a portion of the heavy chain that comprises FR-4.

[0079] 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.

[0080] In some embodiments, X comprises an ultralong CDR3 sequence that can 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 the 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%, or at least about 86%, at least 87%, or at least about 87%, at least 88%, or at least about 88%, at least 89%, or at least about 89%, at least 90%, or at least about 90%, at least 91%, or at least about 91%, at least 92%, or at least about 92%, at least 93%, or at least about 93%, at least 94%, or at least about 94%, 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 the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the IL-15 sequence comprises the amino acid sequence found in SEQ ID NO: 1.

[0081] In some embodiments, the IL-15 sequences exhibit activity in stimulating the 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 the IL2 / 15Rβ and / or γc subunit, such as in an in vitro binding assay. In some embodiments, the activity or binding is similar to or retained compared to recombinant IL-15 monomer.

[0082] In some embodiments, the IL-15 sequence or biologically active portion is inserted into the knob of the ultralong CDR3 between the ascending and descending stalk regions. The IL-15 sequence may be located between the stalk regions, and the IL-15 sequence is directly or indirectly linked to each stalk region. In some embodiments, the link to one or both of the stalk sequences is indirect via a linker. The linker can comprise the amino acid sequence (GGGGS), where n=1-5. Alternatively, the linker comprises the amino acid sequence (GSG), GGGSGGGGS, or GGGGSGGGS. In some cases, the linker has the sequence GGS or GSG.

[0083] In some embodiments, X comprises an ultralong CDR3 sequence that can 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 the 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%, or at least about 86%, at least 87%, or at least about 87%, at least 88%, or at least about 88%, at least 89%, or at least about 89%, at least 90%, or at least about 90%, at least 91%, or at least about 91%, at least 92%, or at least about 92%, at least 93%, or at least about 93%, at least 94%, or at least about 94%, 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%, 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 the amino acid sequence found in SEQ ID NO: 165.

[0084] In some embodiments, the IL-2 sequences exhibit activity in stimulating the 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 the IL2 / 15Rβ and / or γc subunit, such as in an in vitro binding assay. In some embodiments, the activity or binding is similar to or retained compared to recombinant IL-2 monomer.

[0085] In some embodiments, the IL-2 sequence or biologically active portion is inserted into the knob of the ultralong CDR3 between the ascending and descending stalk regions. The IL-2 sequence may be located between the stalk regions, and the IL-2 sequence is directly or indirectly linked to each stalk region. In some embodiments, the link to one or both stalk sequences is indirect via a linker. The linker can comprise the amino acid sequence (GGGGS), where n=1-5. Alternatively, the linker comprises the amino acid sequence (GSG), GGGSGGGGS, or GGGGSGGGS. In some cases, the linker has the sequence GGS or GSG.

[0086] 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.

[0087] In each or any aspect of the above or below embodiments, the ultralong CDR3 is 35 or more amino acids in length, 40 or more amino acids in length, 45 or more amino acids in length, 50 or more amino acids in length, 55 or more amino acids in length, or 60 or more amino acids in length. In some embodiments of each or any of the above or below embodiments, the ultralong CDR3 is 35 or more amino acids in length.

[0088] In some embodiments, the X portion of the heavy chain comprising the ultralong CDR3 comprises the motif X 1 X 2 X 3 X 4 X 5 -[cytokine sequence]-(X a X b )z motif. In some embodiments, the ultralong CDR3 is 45 amino acids in length or longer. In some embodiments, the one or more additional amino acids are selected from the group consisting of X 1 X 2 X 3 X4 X 5 Between the motif and the cytokine sequence and / or (X a X b )z motif and the cytokine sequence.

[0089] 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 NO: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. An exemplary such ascending stalk region is shown in SEQ ID NO:159.

[0090] 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 aromatic residues having the formula YXYXYX, where X is any amino acid. In some embodiments, the descending stalk strand comprises a sequence contained in SEQ ID NOs:76-80 or SEQ ID NO:161. In some embodiments, the ultralong CDR3 comprises a descending stalk region encoded by SEQ ID NOs:10, 122-149, or 160.

[0091] In some embodiments, the ultralong CDR3 comprises, in order, an ascending stalk region having the amino acid sequence encoded by SEQ ID NO:9, an IL15 cytokine sequence represented by SEQ ID NO:1, and a descending stalk region having the amino acid sequence encoded by SEQ ID NO:10. In some embodiments, the ultralong CDR3 comprises, in order, an ascending stalk region having the amino acid sequence encoded by SEQ ID NO:157, an IL15 cytokine sequence represented by SEQ ID NO:1, and a descending stalk region having the amino acid sequence encoded by SEQ ID NO:160.

[0092] In some embodiments, the ultralong CDR3 comprises, in order, an ascending stalk region having the amino acid sequence encoded by SEQ ID NO:9, an IL2 cytokine sequence represented by SEQ ID NO:165, and a descending stalk region having the amino acid sequence encoded by SEQ ID NO:10. In some embodiments, the ultralong CDR3 comprises, in order, an ascending stalk region having the amino acid sequence encoded by SEQ ID NO:157, an IL2 cytokine sequence represented by SEQ ID NO:165, and a descending stalk region having the amino acid sequence encoded by SEQ ID NO:160.

[0093] In some embodiments, the V2 region of the heavy chain comprises TIFF2025169343000002.tif26145.

[0094] In certain embodiments, the chimeric IL-15 modified antibodies or antigen-binding fragments provided herein contain a variable heavy chain sequence encoded by the sequence of nucleotides set forth in SEQ ID NO:7, or a sequence of nucleotides exhibiting at least 85%, or at least about 85%, at least 86%, or at least about 86%, at least 87%, or at least about 87%, at least 88%, or at least about 88%, at least 89%, or at least about 89%, at least 90%, or at least about 90%, at least 91%, or at least about 91%, at least 92%, or at least about 92%, at least 93%, or at least about 93%, at least 94%, or at least about 94%, 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%, at least 99%, or at least about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:7, and contain a modified ultralong CDR3 containing an IL-15 sequence. In some embodiments, the chimeric IL-15 modified antibodies or antigen-binding fragments provided herein comprise a variable heavy chain sequence encoded by the sequence of nucleotides set forth in SEQ ID NO: 7. In some embodiments, the chimeric IL-15 modified antibodies or antigen-binding fragments provided herein consist of, or consist essentially of, a variable heavy chain sequence encoded by the sequence of nucleotides set forth in SEQ ID NO: 7.

[0095] In some embodiments, the heavy chain comprises a variable heavy chain as described above linked to a human constant region. In some embodiments, the human constant region comprises CH1-CH2-CH3 constant domains. In some embodiments, the human constant region is that of human IgG1.

[0096] B. Light Chain Region In some embodiments, the antibody or antigen-binding fragment further comprises a light chain variable region. In some embodiments, the chimeric cytokine-engineered antibody variable chain is based on bovine sequences and is paired with the variable light chain of a bovine antibody. In another embodiment, the present disclosure provides pairing of a humanized ultralong CDR3 heavy chain with a bovine light chain. In a specific embodiment, the light chain is a lambda light chain.

[0097] In some embodiments, the variable light chain is the variable light chain of a bovine antibody, e.g., the variable light chain 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.

[0098] In some embodiments, the light chain comprises the variable light chain of a bovine antibody linked 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 linked to a human lambda light chain constant region.

[0099] In some embodiments, the light chain is a humanized light chain or a human light chain. In some embodiments, the present disclosure provides 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. Several human VL sequences, including VL1-47, VL1-40, VL1-51, and VL2-18, which are homologous to lambda regions derived from Bos Taurus, can be used for pairing with the above sequences. In some embodiments, the light chain variable region is a sequence set forth 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 set forth 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.

[0100] 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 substitution of specific 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 and / or increase the binding specificity of antibodies comprising ultralong CDR3. 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 system. In some embodiments, the modifications include the amino acid substitutions S2A, T5N, P8S, A12G, A13S, and P14L according to the Kabat numbering system. 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 with GDT. In some embodiments, the modifications are n 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 for GDT in CDR2.

[0101] In some embodiments, the light chain comprises a humanized variable light chain as described above joined 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 joined to a human lambda light chain constant region.

[0102] C. IL-15Rα sushi domain In some embodiments, the chimeric interleukin-15 antibody molecules provided herein may be further linked or complexed with all or a portion of the IL-15 high-affinity receptor alpha (IL15Rα), e.g., a portion containing the extracellular domain of IL15Rα, e.g., the IL15Rα sushi domain. In some embodiments, the IL-15 cytokine sequence is linked to all or a portion of the IL-15 high-affinity receptor alpha (IL15Rα). In some embodiments, the IL15Rα is expressed to increase transsignaling to the receptor beta and gamma subunits (IL2 / 15Rβ and γc). The IL-15 high-affinity receptor comprises the IL15Rα sushi domain. In some embodiments, the IL15Rα sushi domain comprises the sequence set forth in SEQ ID NO:2.

[0103] In some embodiments, provided herein are chimeric IL-15 engineered antibodies or antigen-binding fragments, wherein the heavy chain or variable sequence thereof comprises an IL-15 sequence that replaces all or part of the knob of an ultralong CDR3 (e.g., inserted into the knob region between the ascending and descending stalks), complexed with an extracellular domain of IL15Rα, such as an IL15Rα sushi domain. In some embodiments, the chimeric IL-15 engineered antibody or antigen-binding fragment is complexed with the IL15Rα sushi domain set forth in SEQ ID NO:15. Such antibody molecules can be produced by co-expressing an IL15Rα extracellular domain, such as a sushi domain such as that set forth in SEQ ID NO:2, with heavy and light chain regions in a host cell.

[0104] In some embodiments, provided herein are chimeric IL-15 engineered antibodies or antigen-binding fragments containing 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., inserted into the knob region between the ascending and descending stalks), and a light chain or variable sequence thereof linked to an extracellular domain of IL15Ra, e.g., the IL15Ra sushi domain. In some embodiments, the chimeric IL-15 engineered antibody or antigen-binding fragment is linked to the IL15Ra sushi domain shown in SEQ ID NO:2. The link between the extracellular domains of IL15Ra (e.g., the IL15Ra sushi domain, e.g., that shown in SEQ ID NO:2) 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 NO: 163 or SEQ ID NO: 164. In some embodiments, the linker is GS.

[0105] In some embodiments, the chimeric IL-15 modified antibodies or antigen-binding fragments provided herein contain a heavy chain or variable sequence thereof in which the IL-15 sequence replaces all or part of the knob of an ultralong CDR3 (e.g., is inserted into the knob region between the ascending and descending stalks), and a light chain or variable sequence thereof comprising the sequence of amino acids encoded by SEQ ID NO:3.

[0106] D. Vectors, Host Cells and Recombinant Methods For recombinant production of the antibodies or fragments thereof disclosed herein, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (DNA amplification) or expression. The DNA encoding the antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding 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 (DNA amplification) or expression. Many vectors are available. The choice of vector depends in part on the host cell to be used. Generally, preferred host cells are of prokaryotic or eukaryotic (generally mammalian) origin. It will be understood that constant regions of any isotype, including IgG, IgM, IgA, IgD, and IgE constant regions, can be used for this purpose, and that such constant regions can be obtained from any human or animal species.

[0107] Expression vectors containing regulatory elements derived from eukaryotic viruses are typically used in eukaryotic expression vectors, such as SV40 vectors, papilloma virus vectors, and Epstein-Barr virus-derived vectors. Other exemplary eukaryotic vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector that allows protein expression under the direction of a CMV promoter, an SV40 early promoter, an SV40 late promoter, a metallothionein promoter, a murine mammary tumor virus promoter, a Rous sarcoma virus promoter, a polyhedrin promoter, or other promoters shown to be effective for expression in eukaryotic cells.

[0108] 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 promoter.

[0109] Polynucleotide sequences encoding the polypeptide components of the antibodies disclosed herein can be obtained using standard recombinant techniques. In some embodiments, polynucleotides can be synthesized using a nucleotide synthesizer or PCR technology. Once obtained, the polypeptide-encoding sequence is 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 the 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, the heterologous nucleic acid insert, and a transcription termination sequence. Furthermore, a V region containing an ultralong CDR3 can be optionally fused to a C region to produce an antibody containing a constant region.

[0110] Plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are generally used in connection with these hosts. The vector usually contains a replication site and marking sequences capable of providing phenotypic selection in transformed cells. For example, Escherichia coli (E. coli) is typically transformed using pBR322, a plasmid derived from the 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 to express specific antibodies have been described (see, e.g., U.S. Pat. No. 5,648,237).

[0111] 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, bacteriophage 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.

[0112] The expression vectors 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 classified 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 conditions, such as the presence or absence of nutrients or a change in temperature.

[0113] Numerous promoters recognized by various potential host cells are well known. The selected promoter can be operably 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 for greater transcription and higher yields of the expressed target gene compared to the native target polypeptide promoter.

[0114] Promoters suitable for use in prokaryotic hosts include the ara B promoter, the PhoA promoter, the β-galactamase and lactose promoter systems, the tryptophan (trp) promoter system, and hybrid promoters such as the tac or trc promoter. However, other promoters functional in bacteria (such as other known bacterial or phage promoters) are similarly suitable. Their nucleotide sequences have been 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.

[0115] Suitable bacterial promoters are well known in the art and are fully described in the scientific literature by Sambrook and Russell, supra, and Ausubel et al., supra. Bacterial expression systems for expressing antibody chains of recombinant catalytic polypeptides are available in, for example, E. coli, Bacillus species, and Salmonella (Palva et al., Gene, 22:229-235 (1983); Mosbach et al., Nature, 302:543-545 (1983)).

[0116] In one aspect disclosed herein, each cistron in the recombinant vector contains a secretory signal sequence component that directs translocation of the expressed polypeptide across a membrane. Generally, 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 one that is recognized and processed (e.g., cleaved by a signal peptidase) by the host cell. In the case of prokaryotic host cells that do not recognize and process the signal sequence native to the heterologous polypeptide, the signal sequence is replaced with a prokaryotic signal sequence selected from, for example, PelB, OmpA, alkaline phosphatase, penicillinase, Ipp, or the 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 the STII signal sequence or a variant thereof.

[0117] In another aspect, 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 secretory signal sequence within each cistron. In this 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 the expressed protein subunits (see, e.g., Proba and Pluckthun Gene, 159:203 (1995)).

[0118] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell, a Human Embryonic Kidney (HEK) cell, or a lymphoid cell (e.g., YO, NSO, Sp20 cell). For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., 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, describing 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 connection 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). Generally, the appropriate bacterium must be selected taking into account the replicability of the replicon within the bacterial cell. For example, when the replicon is supplied using a well-known plasmid such as pBR322, pBR325, pACYC177, or pKN410, Escherichia coli, Serratia, or Salmonella species can be suitably used as the host. Typically, the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors can be desirably incorporated into the cell culture.

[0119] 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 SV40 (COS-7) transformed monkey kidney CV1 lines, human embryonic kidney lines (e.g., 293 or 293 cells described in Graham et al., Gen VlI'01. 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 (MMT 060562), e.g., Mather et al., Annals of NI'. Acad. Sci. 383:44-68 (1982). 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 specific 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).

[0120] 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.

[0121] Depending on the host cell used, transformation is carried out using standard techniques appropriate for such cells. Calcium treatment using calcium chloride is commonly 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.

[0122] The expressed polypeptides of the present disclosure are secreted into the periplasm of the host cell and recovered therefrom, or transported into the culture medium. Protein recovery from the periplasm typically involves disrupting the microorganisms, generally by means such as osmotic shock, sonication, or lysis. Once the cells are disrupted, 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, proteins transported into the culture 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 polypeptide can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay.

[0123] Antibody production can be carried out in large quantities via fermentation. Various large-scale fed-batch fermentation procedures are available for the production of recombinant proteins. Large-scale fermentations have a capacity of at least 1,000 liters, preferably between about 1,000 and 100,000 liters. These fermentors use agitation impellers to distribute oxygen and nutrients, especially glucose (the preferred carbon / energy source). Small-scale fermentation generally refers to fermentation in fermentors with a volume of about 100 liters or less, which can range from about 1 liter to about 100 liters.

[0124] In fermentation processes, induction of protein expression typically begins after cells have grown under appropriate conditions to a desired density, e.g., an OD550 of approximately 180-220, at which point the cells are in early stationary phase. Various inducers, known in the art and described above, may be used depending on the vector construct used. Cells may be grown for a shorter period before induction. Cells are typically induced for approximately 12-50 hours, although longer or shorter induction times may be used.

[0125] Various fermentation conditions can be modified to improve the production yield and quality of the polypeptides disclosed herein. For example, to improve proper assembly and folding of secreted antibody polypeptides, host prokaryotic cells can be co-transformed with additional vectors overexpressing chaperone proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD, and / or DsbG) or FkpA (a peptidyl prolyl cis,trans isomerase with chaperone activity). 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).

[0126] To minimize proteolysis of expressed heterologous proteins (especially those that are proteolytically sensitive), certain host strains 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 encoding 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, e.g., 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)).

[0127] 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.

[0128] 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 using, for example, Sephadex G-75.

[0129] In one aspect, solid-phase-immobilized protein A is used for immunoaffinity purification of full-length antibody products disclosed herein. Protein A is a 41 kD cell wall protein from Staphylococcus aureus that binds with high affinity to the Fc region of antibodies (see, e.g., Lindmark et al. (1983) J. Immunol. Meth. 62:1-13). The solid phase to which protein A is immobilized is preferably a column containing 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 nonspecific adhesion of contaminants.

[0130] 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 nonspecifically bound to the solid phase. Finally, the antibody of interest is recovered from the solid phase by elution.

[0131] III. Pharmaceutical Compositions Antibodies or antigen-binding fragments comprising the ultralong CDR3, nucleic acid or vector disclosed herein can be formulated into compositions, particularly pharmaceutical compositions. Such compositions comprising antibodies comprising ultralong CDR3s comprise a therapeutically 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 pharmaceutically acceptable agent. Typically, antibodies comprising the ultralong CDR3, antibody fragment, nucleic acid or vector disclosed herein are sufficiently purified for administration before being formulated into pharmaceutical compositions.

[0132] 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 surfactants.

[0133] 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 nonionic surfactants such as Tween, Pluronics, or polyethylene glycol (PEG). Also, for example, suitable isotonicity 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, etc. Hydrogen peroxide may also be used as a preservative. Suitable cosolvents include glycerin, propylene glycol, and PEG. Suitable complexing agents include caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin. Suitable surfactants or wetting agents include sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapal, etc. The buffer may be a conventional buffer such as acetate, borate, citrate, phosphate, bicarbonate, or Tris-HCl. Acetate buffers may have a pH of about 4-5.5, and Tris buffers may have a pH of about 7-8.5. Additional pharmaceutical agents are described in Remington's Pharmaceutical Sciences, 18th Edition, AR Gennaro, ed., Mack Publishing Company, 1990.

[0134] The composition may be in liquid or lyophilized or freeze-dried form and may contain one or more cryoprotectants, excipients, surfactants, high molecular weight structural additives, and / or bulking agents (see, e.g., U.S. Patent 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 is isotonic upon reconstitution, although hypertonic or slightly hypotonic formulations may also be appropriate. Furthermore, 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 about 10 mM to about 400 mM. In another embodiment, surfactants, such as nonionic 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- Included are 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%. Examples of high molecular weight structural additives (e.g., fillers, binders) include gum arabic, albumin, alginic acid, dibasic calcium phosphate, 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 metabisulfite, polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose, compressible sugar, magnesium aluminum silicate, maltodextrin, polyethylene oxide, polymethacrylate, povidone, sodium alginate, microcrystalline cellulose tragacanth, starch, and zein. An exemplary concentration of the high molecular weight structural additive is 0.1% to 10% by weight. In other embodiments, bulking agents (eg, mannitol, glycine) may be included.

[0135] The composition can 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 route.Parenteral formulations are typically sterile, pyrogen-free, isotonic aqueous solutions, optionally containing pharmaceutically acceptable preservatives.

[0136] 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 solution, 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, such as antibacterial agents, antioxidants, chelating agents, inert gases, and the like, may also be present. See generally Remington's Pharmaceutical Science, 16th Ed., Mack Eds., 1980.

[0137] The pharmaceutical compositions described herein can be formulated for controlled or sustained delivery in a manner that provides local product concentration (e.g., bolus, depot effect) and / or increased stability or half-life in a specific local environment. The compositions can include microparticle preparations of polymeric compounds such as polylactic acid, polyglycolic acid, and the like, as well as formulations of antibodies containing 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. 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 conjugation of enantiomeric polymer or polypeptide segments, and hydrogels with temperature- or pH-sensitive properties may be desirable for providing a drug depot effect due to the mild, aqueous conditions involved in entrapment of bioactive protein agents (e.g., antibodies containing ultralong CDR3s). See, e.g., the description of controlled-release porous polymeric microparticles for delivery of pharmaceutical compositions in WO 93 / 15722.

[0138] Suitable materials for this purpose include polylactide (see, e.g., U.S. Pat. No. 3,773,919), polymers of poly-(α-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 aggravate the condition. This can be determined by routine screening in animal models of the target disorder, or in normal animals if such models are unavailable.

[0139] 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 polylactic-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 polymers," 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] .

[0140] 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 long 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 composed 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 prepare biocompatible and biodegradable microspheres for use in delivery (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 comprising an ultralong CDR3 relative to the hyaluronic acid polymer.

[0141] 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 biodegradable delivery systems include polymers of lactic and glycolic acid, polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyanhydrides, polyurethanes and their copolymers, poly(butyric acid), and the like. 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, cellulose sulfate sodium salt, 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 and alkylene, as 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 by enzymatic hydrolysis or exposure to water in vivo, either by surface or bulk erosion. The polymers are optionally in the form of hydrogels (e.g., WO 04 / 009664; WO 05 / 087201; Sawhney, et al., Macromolecules, 1993, 26, 581-587), which can absorb up to about 90% of their weight in water and are optionally crosslinked with multivalent ions or other polymers.

[0142] Delivery systems also include non-polymeric systems that are lipids, including sterols such as cholesterol, cholesterol esters, and fatty acids, or neutral lipids 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 a matrix, such as those described in U.S. Patent Nos. 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. Patent Nos. 3,854,480, 5,133,974, and 5,407,686. Liposomes containing the product 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).

[0143] Alternatively or additionally, the composition may be administered locally by implanting a membrane, sponge, or other suitable material into the affected area into which an antibody comprising an 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 an ultralong CDR3 antibody fragment, nucleic acid, or vector disclosed herein may be via a bolus, or via continuous administration, or directly through the device via a catheter using continuous infusion.

[0144] Pharmaceutical compositions containing antibodies comprising the ultralong CDR3, antibody fragments, nucleic acids, or vectors disclosed herein can be formulated for inhalation, for example, as dry powders. Inhalation solutions may also be formulated in liquefied propellants for aerosol delivery. In yet another formulation, the solution may be nebulized. Additional pharmaceutical compositions for pulmonary administration include those described in International Publication No. 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 1 μm to 5 μm. However, larger particles may be used, for example, if each particle is fairly porous.

[0145] Certain formulations containing antibodies comprising ultralong CDR3, antibody fragments, nucleic acids or vectors disclosed herein may be orally administered.The formulations administered in this manner may be formulated with or without carriers that are commonly used in the preparation of solid dosage forms such as tablets and capsules.For example, capsules can be designed to release the active part of the formulation at the time in the gastrointestinal tract when bioavailability is at its maximum and pre-systemic degradation is at its minimum.Additional agents can be included to promote the absorption of selective binding agents.Diluents, flavoring agents, low-melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents and binders can also be used.

[0146] Another preparation can comprise 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 sodium bicarbonate, lactose, or calcium phosphate; or binders such as starch, gelatin, or gum arabic; or lubricants such as magnesium stearate, stearic acid, or talc.

[0147] Suitable and / or preferred pharmaceutical formulations can be determined based on the intended route of administration, delivery form and desired dosage, taking into account the present disclosure and general knowledge of formulation technology.Regardless of the mode of administration, effective dosage can be calculated according to the patient's body weight, body surface area or organ size.Further refinement of the calculation for determining the 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.Appropriate dosage can be confirmed by using appropriate dose-response data.

[0148] In some embodiments, the antibody comprising an ultralong CDR3 or a fragment thereof comprises a modified Fc region, in which the native Fc region has been modified to increase the half-life of the antibody or fragment in a biological environment, e.g., serum half-life or half-life as measured by an in vitro assay. Methods for altering the original form of the Fc region of an IgG are also described in U.S. Patent No. 6,998,253.

[0149] In certain embodiments, it may be desirable to modify the antibody or fragment to increase its 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 its half-life. This can also be achieved, for example, by incorporating a salvage receptor binding epitope into the antibody fragment (e.g., by mutating an appropriate region in the antibody fragment, or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at its end or in the middle, for example, by DNA or peptide synthesis) (see WO 96 / 32478). A 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.

[0150] 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 in 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, or more than one such region, of the antibody. Alternatively, the epitope is taken from the CH2 domain of the Fc region and transferred to the CL or VL region, or both, of the antibody fragment. See also WO 97 / 34631 and WO 96 / 32478, which describe Fc variants and their interactions with salvage receptors.

[0151] IV. Treatment Methods and Uses Provided herein are methods for and uses of compositions containing chimeric cytokine-modified antibodies or antigen-binding fragments to treat diseases or conditions. In certain embodiments, the disease or condition is treatable with a cytokine present in the chimeric molecule. For example, the disease or condition is treatable with IL-2 or IL-15. In some embodiments, the provided chimeric cytokine-modified antibodies or antigen-binding fragments are particularly suitable for use as immunotherapies. In certain aspects, the provided chimeric cytokine-modified antibodies or antigen-binding fragments, or compositions thereof, are used in several oncological applications, such as cancer, by promoting T cell activation and / or proliferation. In some embodiments, the provided chimeric cytokine-modified antibodies or antigen-binding fragments are used to treat cancer in a subject in need thereof.

[0152] Such methods and uses include, for example, therapeutic methods and uses that involve administering a 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 aspects, the methods and uses thereby treat a disease or condition or disorder, such as a tumor or cancer, in a subject.

[0153] In some embodiments, the cancer is a cancer of the head and neck, breast, liver, colon, ovary, prostate, pancreas, brain, cervix, bone, skin, lung, or blood. In some embodiments, cancer can include malignant tumors characterized by abnormal or uncontrolled cell growth. Other characteristics that can 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 can refer to cancer cells that have left the original tumor site and migrated to other parts of the body, for example, via the bloodstream or lymphatic system.

[0154] In some embodiments, the provided methods result in an improvement and / or treatment of a disease or condition, such as cancer. In some aspects, the provided methods 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.

[0155] In aspects of the provided methods, response can be assessed or determined using criteria specific to the disease or condition. In some embodiments, screening techniques such as magnetic resonance imaging (MRI) scan, X-ray imaging, computed tomography (CT) scan, bone scan imaging, endoscopy, and tumor biopsy sampling, including bone marrow aspirate (BMA) and circulating tumor cell counting, can be used to assess tumor response for changes in tumor morphology (i.e., overall tumor burden, tumor size).

[0156] The provided method includes administering a therapeutically effective amount of a composition provided herein to a subject in need thereof, such as a cancer subject. The therapeutically effective amount can vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the pharmaceutical to induce a desired response in the individual. A 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 disease progression. 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.

[0157] Alternatively, this property of the composition can be evaluated by examining 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 improve 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.

[0158] 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, hi 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.

[0159] 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 can 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.

[0160] 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.

[0161] V. Illustrative Embodiments Among the aspects provided are: 1. A modified ultralong CDR3 comprising an interleukin-15 (IL-15) cytokine sequence or a biologically active portion thereof replacing at least a portion of the ultralong CDR3 region of the heavy chain of a bovine antibody or antigen-binding fragment or a humanized sequence thereof. A chimeric cytokine-modified antibody or antigen-binding fragment comprising: 2. The chimeric cytokine engineered antibody or antigen-binding fragment of embodiment 1, wherein said IL-15 cytokine sequence is human IL-15. 3. The chimeric cytokine engineered antibody or antigen-binding fragment of embodiment 1 or embodiment 2, wherein said IL-15 cytokine sequence 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. 4. The chimeric cytokine engineered antibody or antigen-binding fragment of any of aspects 1-3, wherein said IL-15 cytokine sequence comprises the sequence of amino acids set forth in SEQ ID NO:1. 5. A modified ultralong CDR3 comprising an interleukin-2 (IL-2) cytokine sequence or a biologically active portion thereof replacing at least a portion of the ultralong CDR3 region of the heavy chain of a bovine antibody or antigen-binding fragment or a humanized sequence thereof. A chimeric cytokine-modified antibody or antigen-binding fragment comprising: 6. The chimeric cytokine modified antibody or antigen-binding fragment of embodiment 5, wherein said IL-2 cytokine sequence is human IL-2. 7. The chimeric cytokine engineered antibody or antigen-binding fragment of embodiment 5 or embodiment 6, wherein said IL-2 cytokine sequence 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. 8. The chimeric cytokine engineered antibody or antigen-binding fragment of any of aspects 5-7, wherein said IL-2 cytokine sequence comprises the sequence of amino acids set forth in SEQ ID NO:165. 9. The chimeric cytokine-modified antibody or antigen-binding fragment of any of aspects 1-8, wherein said cytokine sequence replaces at least a portion of the ultralong CDR3 region of the heavy chain of a bovine antibody or antigen-binding fragment. 10. The chimeric cytokine modified antibody or antigen-binding fragment of embodiment 9, wherein said bovine antibody or antigen-binding fragment is the bovine antibody BLV1H12 or an antigen-binding fragment thereof. 11. The chimeric cytokine-modified antibody or antigen-binding fragment of embodiment 9 or embodiment 10, wherein said bovine antibody or antigen-binding fragment comprises a variable heavy chain amino acid sequence encoded by the sequence set forth in SEQ ID NO:5 and a variable light chain amino acid sequence encoded by the sequence set forth in SEQ ID NO:8. 12. The chimeric cytokine-modified antibody or antigen-binding fragment of embodiment 9 or embodiment 10, wherein said bovine antibody or antigen-binding fragment comprises a variable heavy chain amino acid sequence encoded by the sequence set forth in SEQ ID NO:167 and a variable light chain amino acid sequence encoded by the sequence set forth in SEQ ID NO:168. 13. The chimeric cytokine-modified antibody or antigen-binding fragment of embodiment 9 or embodiment 10, wherein said bovine antibody or antigen-binding fragment comprises a variable heavy chain as set forth in SEQ ID NO:26 and a variable light chain as set forth in SEQ ID NO:27. 14. The chimeric cytokine-modified antibody or antigen-binding fragment of any of aspects 1-8, wherein said cytokine sequence replaces at least a portion of the ultralong CDR3 region of the heavy chain of a humanized bovine antibody or antigen-binding fragment thereof. 15. The humanized bovine antibody or antigen-binding fragment thereof, a heavy chain or portion thereof that is or is derived from a human heavy chain germline sequence; a light chain or portion thereof that is or is derived from a human light chain germline sequence; 15. The chimeric cytokine modified antibody or antigen-binding fragment of embodiment 14, comprising: 16. The chimeric cytokine engineered antibody or antigen-binding fragment of embodiment 15, wherein said human heavy chain germline sequence is a VH4-39, VH4-59*03, VH4-34*02, or VH4-34*09 germline sequence, or a sequence set forth in any one of SEQ ID NOs:68-71. 17. The chimeric cytokine modified antibody or antigen-binding fragment of embodiment 15 or embodiment 16, wherein said human light chain germline sequence is a VL1-51 germline sequence or a sequence based on said VL1-51 germline sequence comprising one or more mutations, and optionally said VL1-51 germline sequence is set forth in SEQ ID NO:156. 18. The one or more mutations are selected from the following: one or more of the amino acid substitutions S2A, T5N, P8S, A12G, A13S, and P14L based on Kabat numbering; amino acid substitutions S2A, T5N, P8S, A12G, A13S, and P14L based on Kabat numbering; mutations in CDR1, including amino acid substitutions I29V and N32G; mutations in CDR2, including substitutions of DNN with GDT; a mutation in CDR2, including a substitution of DNNKRP with GDTSRA; or Any combination of the above 18. The chimeric cytokine modified antibody or antigen-binding fragment of embodiment 17, selected from: 19. The chimeric cytokine-modified antibody or antigen-binding fragment of any of aspects 1-18, wherein said antibody is an antigen-binding fragment comprising a variable heavy chain and a variable light chain. 20. The chimeric cytokine-modified antibody or antigen-binding fragment of any of aspects 1-19, wherein said antibody comprises a variable heavy chain attached to a heavy chain constant domain (CH1-CH2-CH3) and a variable light chain attached to a light chain constant domain (CL1). 21. The chimeric cytokine-modified antibody or antigen-binding fragment of embodiment 20, wherein said heavy chain constant domain is derived from human IgG1. 22. The chimeric cytokine-modified antibody or antigen-binding fragment of embodiment 20 or embodiment 21, wherein said light chain constant domain is a lambda light chain region. 23. The chimeric cytokine-engineered antibody or antigen-binding fragment of any of aspects 1-22, wherein at least a portion of the ultralong CDR3 region comprises a knob region, and wherein the cytokine sequence is located between the ascending and descending stalk domains of the engineered ultralong CDR3. 24. The chimeric cytokine engineered antibody or antigen-binding fragment of embodiment 23, wherein the cytokine sequence is linked to the ascending stalk domain and / or the descending stalk domain via a flexible linker, optionally a GGS or GSG linker. 25. The chimeric cytokine modified antibody or antigen-binding fragment of embodiment 23 or embodiment 24, wherein said ascending stalk domain comprises the sequence as set forth in SEQ ID NO:158 or SEQ ID NO:159. 26. The chimeric cytokine modified antibody or antigen-binding fragment of any of aspects 23-25, wherein said descending stalk domain comprises the sequence set forth in SEQ ID NO:161. 27. The chimeric cytokine modified antibody or antigen-binding fragment of any of embodiments 1-4 and 9-26, comprising a variable heavy chain sequence encoded by a sequence of nucleotides set forth in SEQ ID NO:7, or a sequence of nucleotides that exhibits 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%, at least 99%, or at least about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:7, and comprising a modified ultralong CDR3 containing an IL-15 sequence. 28. The chimeric cytokine engineered antibody or antigen-binding fragment of any of embodiments 1 to 4 and 9 to 27, wherein the antibody or antigen-binding fragment is complexed with an extracellular domain of IL15Rα comprising the IL15Rα sushi domain. 29. The chimeric cytokine engineered antibody or antigen-binding fragment of aspect 28, wherein the extracellular domain of the IL15Rα comprising the IL15Rα sushi domain is non-covalently associated with the IL-15 sequence. 30. The chimeric cytokine engineered antibody or antigen-binding fragment of aspect 28, wherein the extracellular domain of said IL15Rα comprising the IL15Rα sushi domain is linked to said variable light chain. 31. The chimeric cytokine modified antibody or antigen-binding fragment of embodiment 30, which is linked via a peptide linker. 32. The chimeric cytokine-modified antibody of aspect 31, wherein said peptide linker is a glycine linker or a glycine-serine linker, and optionally said linker is GS. 33. The chimeric cytokine engineered antibody of any of aspects 28 to 32, wherein the extracellular domain of IL15Rα comprising the IL15Rα sushi domain comprises the sequence shown in SEQ ID NO:2. 34. The chimeric cytokine modified antibody or antigen-binding fragment of any of aspects 30-33, wherein said variable light chain comprises a sequence of amino acids encoded by SEQ ID NO:3. 35. A polynucleotide encoding the chimeric cytokine-modified antibody or antigen-binding fragment of any of embodiments 1 to 34. 36. A polynucleotide encoding the heavy chain or variable region thereof of the chimeric cytokine-modified antibody or antigen-binding fragment of any of aspects 1 to 34. 37. A polynucleotide encoding the light chain or variable region thereof of the chimeric cytokine-modified antibody or antigen-binding fragment of any of aspects 1 to 34. 38. An expression vector comprising the polynucleotide of any one of aspects 35 to 37. 39. A host cell comprising the polynucleotide of any one of aspects 35 to 37 or the expression vector of aspect 37. 40. The host cell of aspect 39, further comprising a polynucleotide or vector expressing the extracellular domain of IL15Rα comprising the IL15Rα sushi domain. 41. The host cell of aspect 40, wherein the extracellular domain of IL15Rα comprising the IL15Rα sushi domain comprises the sequence set forth in SEQ ID NO:2. 42. A method for producing a chimeric cytokine-modified antibody or antigen-binding fragment, comprising culturing a host cell of any of aspects 39-41 under conditions for expression of the antibody or antigen-binding fragment by said cell, and optionally further comprising recovering by purifying said antibody or antigen-binding fragment. 43. A chimeric cytokine-modified antibody or antigen-binding fragment produced by the method of embodiment 42. 44. A pharmaceutical composition comprising the chimeric cytokine-modified antibody or antigen-binding fragment of any of aspects 1-34 or 43. 45. A method of treating cancer in a subject, comprising administering a therapeutically effective amount of the chimeric cytokine-modified antibody or antigen-binding fragment of any of embodiments 1-34 or 43. 46. ​​A method of treating cancer in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of embodiment 44. [Example]

[0162] VI. Working Examples The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0163] Example 1 Generation of Chimeric Interleukin-15 Fusion Antibodies A chimeric BLV1H12-IL-15 (B15) fusion antibody was generated by engineering the ultralong CDR3 region of bovine BLV1H12 antibody by replacing the knob region with interleukin (IL)-15.

[0164] The variable heavy chain (VH) region from the chimeric BLV1H12 bovine heavy chain sequence (SEQ ID NO: 167) was amplified by PCR and subcloned in frame between the signal sequence and the nucleotide sequence encoding the CH1-CH2-CH3 of human IgG1 to generate the sequence shown in SEQ ID NO: 6. The chimeric extra-long bovine heavy chain sequence (SEQ ID NO: 167) contains a stalk sequence from the heavy chain of BLV1H12, with the last serine in the ascending stalk strand changed to threonine for cloning purposes, and a knob sequence from a bovine anti-HIV antibody. To insert the IL-15 cytokine sequence (shown in SEQ ID NO:1) into the CDR3 of the chimeric BLV1H12 heavy chain, a sequence encoding the entire B15 variable region and its signal peptide (SEQ ID NO:7) was designed by replacing the knob sequence (SEQ ID NO:162) with the IL-15 sequence, along with sequences encoding an N-terminal GGS linker (SEQ ID NO:163) and a C-terminal GSG linker (SEQ ID NO:164). Here, IL-15 connects the ascending stalk (SEQ ID NO:157, encoding the sequence shown in SEQ ID NO:159) and the descending stalk (SEQ ID NO:160, encoding the sequence shown in SEQ ID NO:161). This sequence was chemically synthesized with a 5' EcoRI site and cloned into a pUC57 vector by GenScript, Inc. A 3'-terminal NheI site was already present in the synthesized sequence. The synthesized sequence was subcloned into the BLV1H12 expression vector (SEQ ID NO:6) using EcoRI and NheI restriction enzymes.

[0165] The expression vectors encoding each heavy chain were then co-transfected into Freestyle HEK 293 cells (ThermoScientific) in parallel with a pFUSE expression vector encoding the bovine light chain BLV1H12 (SEQ ID NO:168). The cells were grown at 37°C and 8% CO2, and the expressed chimeric BLV1H12-IL-15 (B15) fusion antibodies were secreted into the culture medium and harvested 96 hours posttransfection. The chimeric fusion antibodies were purified using a 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). They were quantified using Nanodrop based on their molecular weight and extinction coefficient.

[0166] To assess whether IL-15 may require its high-affinity receptor α (IL15Rα) for increased transsignaling to the receptor β and γ subunits (IL2 / 15Rβ and γc), two additional molecules were produced by coexpression of an IL-15 chimeric fusion antibody with the IL15Rα sushi domain. Two additional mutant molecules were produced either by coexpressing the IL15Rα sushi domain (SEQ ID NO:2) with a chimeric IgG in freestyle HEK 293 cells (B15_Rαsushi) or by fusing the IL15Rα sushi domain to the light chain via a GS linker (SEQ ID NO:3) (B15_GS_Rαsushi).

[0167] 1A and 1B show a schematic representation of the construct generated.

[0168] The B15 fusion antibody was analyzed by SDS-PAGE gel. Figure 2 shows an SDS-PAGE gel of purified B15 fusion antibody constructs BLV1H12-IL-15 (B15), BLV1H12-IL-15-Rαsushi (B15_Rαsushi), and BLV1H12-IL-15-GS-Rαsushi (B15_GS_Rαsushi) expressed from HEK 293 cells. These results demonstrate that chimeric B15 antibodies or variants containing the IL15-Rαsushi domain can be expressed and purified similarly to typical human antibodies.

[0169] Example 2 Chimeric B15 Fusion Antibody-Receptor Binding Assay The binding of the chimeric BLV1H12-IL-15 (B15) fusion antibody to IL2 receptor α (IL2Rα) and IL15Rα was assessed by enzyme-linked immunosorbent assay (ELISA). 50 ng of IL2Rα or 100 ng of IL15Rα protein (R&D Systems) per well was coated in a 96-well high-binding plate overnight at 4°C. The plate was washed three times with tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST). Unbound sites on the plate were blocked with 1% bovine serum albumin (BSA) prepared in TBST for 1 hour at room temperature. Ten picomoles of B15 (diluted in 1% BSA in TBST) were added per well, and negative control wells containing only BSA were also set up. The plate was incubated for 1 hour at room temperature and then washed four times with TBST to remove unbound B15. The detection antibody used was goat anti-human lambda (Southern Biotech) conjugated to horseradish peroxidase, which was diluted 1:5000 in 1% BSA in TBST, with 50 μl of dilution added per well. After a 30-minute incubation with the secondary antibody, the plate was washed five times with TBST to remove unbound secondary antibody. 50 μl of TMB substrate (TheromoScientific) was added per well, and the horseradish peroxidase-TMB reaction was run for 1 minute and 30 seconds and then stopped by adding 50 μl of 1.0 normal sulfuric acid per well. The plate was read at 450 nm in a Tecan plate reader, and the plotted values ​​were the average of three duplicate wells with background readings subtracted.

[0170] Binding of the chimeric BLV1H12-IL-15 (B15) fusion antibody to the IL2 / 15Rβ receptor was assessed by ELISA assay. Plates were coated overnight at 4°C with 50 ng of IL2 / 15Rβ protein (R&D Systems) per well. Plates were washed three times with tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST). Unbound sites on the plate were blocked with 1% bovine serum albumin (BSA) prepared in TBST for 1 hour at room temperature. Ten picomoles of B15 or premixed equimolar amounts of B15 and IL15Rα-Fc (R&D Systems) were added per well. Negative control wells containing only BSA were also set up. Plates were incubated for 1 hour at room temperature and then washed four times with TBST to remove unbound B15 or premixed B15 and IL15Rα-Fc. The detection antibody used was goat anti-human lambda (Southern Biotech) conjugated to horseradish peroxidase, which was diluted 1:5000 in 1% BSA in TBST, with 50 μl of dilution added per well. After a 30-minute incubation with the secondary antibody, the plate was washed five times with TBST to remove unbound secondary antibody. 50 μl of TMB substrate (TheromoScientific) was added per well, and the peroxidase-TMB reaction was run for 3 minutes and then stopped by adding 50 μl of 1.0 normal sulfuric acid per well. The plate was read at 450 nm in a Tecan plate reader, and the plotted values ​​were the average of three duplicate wells with background readings subtracted.

[0171] As shown in Figures 3A and 3B, chimeric B15 could bind to both the IL15Rα and IL2 / 15Rβ subunits, and the IL15Rα sushi domain subunit could improve the binding of B15 to the IL2 / 15Rβ subunit. No binding between B15 and IL2Rα was detected. These results demonstrated that IL15Rα or its sushi domain is responsible for efficient binding to the IL2 / 15Rβ and γc subunits.

[0172] Example 3 Chimeric B15 Fusion Antibody-Induced Receptor Activation and Signaling Activation of IL2 / 15Rβ and γc receptors and STAT5 signaling by chimeric B15 molecules generated as described in Example 1 was examined using HEK-Blue IL2 reporter cells (InvivoGen) and analyzed through the induction and secretion of the STAT5-inducible alkaline phosphatase (SEAP) reporter gene.

[0173] Because the IL15Rα subunit is not expressed in HEK-Blue IL2 reporter cells, IL15Rα-Fc (R&D Systems) was mixed with IL15 (or B15) to increase its binding to the IL2 / 15Rβ and γc subunits. First, HEK-Blue IL2 reporter cells were prepared in suspension by gently rinsing the cells twice with prewarmed phosphate-buffered saline (PBS), detaching the cells in the presence of PBS using a cell scraper, and resuspending them in fresh prewarmed test medium (DMEM with high glucose and 10% heat-inactivated FBS) to approximately 280,000 cells per ml. IL15 monomer (premixed IL15 & IL15Rα) incubated overnight at 4°C with half-molar IL15Rα-Fc, or IL15 monomer (freshly mixed IL15 & IL15Rα) mixed immediately before the start of the assay, or chimeric B15 (freshly mixed B15 & IL15Rα) mixed with equimolar IL15Rα-Fc immediately before the start of the assay were serially diluted 4-fold in PBS from 64 nM to 0.25 nM. 20 μl of each cytokine dilution was added per well 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 hours. Because the chimeric B15 antibody is bivalent, only half-molar concentrations were used compared to the IL15 monomer. 20 ul of cell culture supernatant from each well containing secreted SEAP was mixed with 180 ul 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 Tecan plate reader.

[0174] As shown in Figure 4, in vitro STAT5 signaling assays demonstrated that the chimeric B15 antibody could associate with the IL2 / 15Rβ receptor much faster than the IL15 monomer.

[0175] We then used HEK-Blue IL2 reporter cells to assess receptor activation and STAT5 signaling in the presence of alternative chimeric B15 molecules associated with the IL15Rα sushi domain. HEK-Blue IL2 reporter cells were prepared as described above and co-cultured with 4-fold serial dilutions (64 nM to 0.25 nM) of chimeric B15 antibody alone, chimeric B15 antibody mixed with IL15Rα-Fc immediately before the start of the assay (freshly mixed B15 & IL15Rα), chimeric B15 variant B15_Rα sushi, or chimeric B15 variant B15_GS_Rα sushi. As shown in Figure 5, the chimeric B15 variants expressed with the IL15Rα sushi domain achieved the same signaling potency as premixed B15 and IL15Rα-Fc, all of which were better than chimeric B15 antibody without the IL15Rα subunit.

[0176] Example 4 Evaluation of the activity of chimeric B15 fusion antibodies by expanding NK-92 cells The activity of the chimeric B15 molecule generated as described in Example 1 was assessed by its ability to expand NK-92 natural killer cells. NK-92 cells express the IL2Rα, IL15Rα, IL2 / 15Rβ, and γc subunits, and their growth and proliferation depend on the exogenous addition of IL2 or IL15 to bind and activate the receptors.

[0177] NK-92 cells were maintained in growth medium supplemented with 200 U / ml IL2. Prior to the expansion assay, NK-92 cells were washed twice with IL2-free growth medium to remove residual cell-bound IL2 and seeded at 10,000 cells per well in tissue-culture-treated 96-well plates. These cells were incubated with 2-fold serial dilutions (1.33 nM to 0.005 nM) of IL2 or IL15 monomer (R&D Systems), or chimeric B15, chimeric mutant B15_Rαsushi, or chimeric B15 mutant B15_GS_Rαsushi antibodies for 48 hours at 37°C and 5% CO2. Only half-molar concentrations of the chimeric B15 antibody and its variants were used compared to IL2 and IL15 monomer. The final NK92 cell number per well was assessed by the reduction of the tetrazolium dye MTT to its insoluble formazan in the presence of metabolically active oxidoreductase enzyme (MTT assay kit, Promega).

[0178] As shown in Figure 6, all B15 constructs were able to expand NK-92 cells, although to a lesser extent than either IL2 or IL15 monomers. The reason why chimeric B15 or its variants bearing IL15Rα sushi were less potent in expanding NK-92 cells was unclear. Without wishing to be bound by theory, one hypothesis is that chimeric B15 or its variants are bivalent but can only bind monovalently to NK-92 cells, whereas only half-molar concentrations of chimeric B15 or its variants were used in these assays. A second hypothesis is that chimeric B15 and its variants are produced in HEK cells and are naturally glycosylated compared to E. coli-produced IL2 and IL15 monomers (R&D Systems), and that glycosylation of IL15 may negatively affect its binding to the IL15 receptor on NK-92 cells. A third hypothesis is that the size of chimeric B15 or its variants is larger than the IL2 or IL15 monomers due to its fusion with the antibody structure, which stabilizes IL15 but reduces its accessibility to the IL15 receptor on NK-92 cells.

[0179] The expansion of NK-92 cells was then used to assess the difference in activity of the chimeric B15 antibody compared to the chimeric B15 mutant B15-Rαsushi or B15-GS-Rαsushi antibody. The experiment was set up in the same manner as in Figure 6. As shown in Figure 7, the presence of the IL15Rαsushi domain improved the ability of the chimeric B15 antibody to expand NK-92 cells.

[0180] Example 5 Generation of Chimeric Interleukin-2 Fusion Antibodies A chimeric BLV1H12-IL-2 (B2) fusion antibody was generated by replacing the IL15 region of the chimeric B15 antibody described above with IL-2 (SEQ ID NO: 165).

[0181] The IL2 coding sequence (SEQ ID NO:166) with a 5'-terminal GGS linker coding sequence (SEQ ID NO:163) and a 3'-terminal GSG linker coding sequence (SEQ ID NO:164) was chemically synthesized by GenScript Inc. A 5'-terminal AgeI site and a 3'-terminal BamHI site were also added. The synthesized sequence was cloned into a pUC57 vector by GenScript, Inc. and subcloned into the chimeric B15 heavy chain variable region (SEQ ID NO:7) using AgeI and BamHI restriction enzymes.

[0182] The heavy chain-encoding expression vector was then co-transfected into Freestyle HEK 293 cells (ThermoScientific) in parallel with a pFUSE expression vector encoding the bovine light chain BLV1H12 (SEQ ID NO:168). Cells were grown at 37°C and 8% CO2, and the expressed chimeric BLV1H12-IL-2 (B2) fusion antibody was secreted into the culture medium and harvested 96 hours posttransfection. The chimeric B2 fusion antibody was purified using a 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). They were quantified using Nanodrop based on their molecular weight and extinction coefficient.

[0183] 8A and 8B show schematic diagrams of the constructs generated.

[0184] The B2 fusion antibody was analyzed by SDS-PAGE gel. Figure 9 shows an SDS-PAGE gel of the purified fusion antibody construct BLV1H12-IL-2(B2) expressed from HEK 293 cells. The results demonstrate that the chimeric B2 antibody can be expressed and purified similarly to a typical human antibody.

[0185] Example 6 Chimeric B2 Fusion Antibody-Receptor Binding Assay The binding of the chimeric BLV1H12-IL-2 (B2) fusion antibody to IL2Rα and IL15Rα was assessed by enzyme-linked immunosorbent assay (ELISA). 50 ng of IL2Rα or 100 ng of IL15Rα protein (R&D Systems) per well was coated in a 96-well high-binding plate overnight at 4°C. The next day, the plate was washed three times with tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST). Unbound sites on the plate were blocked with 1% bovine serum albumin (BSA) prepared in TBST for 1 hour at room temperature. Ten picomoles of B2 (diluted in 1% BSA in TBST) were added per well, and negative control wells containing only BSA were also set up. The plate was incubated for 1 hour at room temperature and then washed four times with TBST to remove unbound B2 antibody. The detection antibody used was goat anti-human lambda (Southern Biotech) conjugated to horseradish peroxidase, which was diluted 1:5000 in 1% BSA in TBST, with 50 μl of dilution added per well. After a 30-minute incubation with the secondary antibody, the plate was washed five times with TBST to remove unbound secondary antibody. 50 μl of TMB substrate (TheromoScientific) was added per well, and the horseradish peroxidase-TMB reaction was run for 1 minute and 30 seconds and then stopped by adding 50 μl of 1.0 normal sulfuric acid per well. The plate was read at 450 nm in a Tecan plate reader, and the plotted values ​​were the average of three duplicate wells with background readings subtracted.

[0186] As shown in Figure 10, chimeric B2 was able to bind to IL2Rα but not to IL15Rα.

[0187] Example 7 Chimeric B2 Fusion Antibody-Induced Receptor Activation and Signaling Activation of IL2 / 15Rβ and γc receptors and STAT5 signaling by chimeric B2 molecules generated as described in Example 5 was tested using HEK-Blue IL2 reporter cells (InvivoGen) against IL2 monomer (R&D systems and Millipore Sigma) and analyzed through the induction and secretion of the STAT5-inducible alkaline phosphatase (SEAP) reporter gene.

[0188] First, HEK-Blue IL2 reporter cells were prepared in suspension by gently rinsing the cells twice with prewarmed phosphate-buffered saline (PBS), detaching the cells in the presence of PBS using a cell scraper, and resuspending them in fresh prewarmed test medium (DMEM with high glucose and 10% heat-inactivated FBS) to approximately 280,000 cells per ml. IL2 monomer or chimeric B2 antibody was serially diluted 4-fold from 64 nM to 0.25 nM in PBS, and 20 μl of each cytokine dilution was added per well to a 96-well tissue culture-treated plate, with triplicates per dilution. 50,000 cells were then added to each well and cultured for 20 hours at 37°C, 5% CO2. Because the chimeric B2 antibody is bivalent, only half-molar concentrations were used compared to the IL2 monomer. 20 ul of cell culture supernatant from each well containing secreted SEAP was mixed with 180 ul 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 Tecan plate reader.

[0189] As shown in Figure 11, in vitro STAT5 signaling assays demonstrated that the chimeric B2 antibody functions similarly to E. coli-derived IL2 monomer (R&D systems and Millipore Sigma).

[0190] Example 8 Evaluation of the activity of chimeric fusion B2 antibodies by expanding NK-92 cells The activity of chimeric B2 molecules generated as described in Example 5 was assessed by their ability to expand NK-92 natural killer cells. NK-92 cells express the IL2Rα, IL15Rα, IL2 / 15Rβ, and γc subunits, and their growth and proliferation depend on the exogenous addition of IL2 or IL15 to bind and activate the receptors.

[0191] NK-92 cells were maintained in growth medium supplemented with 200 U / ml IL2. Prior to the expansion assay, NK-92 cells were washed twice with IL2-free growth medium to remove residual cell-bound IL2 and seeded at 10,000 cells per well in tissue-culture-treated 96-well plates. These cells were incubated with 2-fold serial dilutions (1.33 nM to 0.005 nM) of IL2 monomer (R&D Systems) or chimeric B2 antibody for 48 hours at 37°C and 5% CO2. Only half-molar concentrations of the chimeric B2 antibody were used compared to IL2 monomer. The final NK92 cell number per well was assessed by the reduction of the tetrazolium dye MTT to its insoluble formazan in the presence of metabolically active oxidoreductase enzyme (MTT Assay Kit, Promega).

[0192] As shown in Figure 12, the chimeric B2 antibody was nearly two-fold better than IL2 monomer in expanding NK-92 cells.

[0193] Example 9 Evaluation of in vitro activity of chimeric B15 fusion antibodies in human PBMCs The activity of the chimeric B15 molecules generated as described in Example 1 was assessed by their ability to stimulate NK cells and T cells in human PBMCs in vitro. Both NK cells and T cells express the IL15Rα, IL2 / 15Rβ and γc subunits, and their growth and proliferation depend on endogenous or exogenous IL15 to bind and activate the receptor.

[0194] Human PBMCs were washed twice with PBS, counted using a hemocytometer, and resuspended in RPMI 1640 medium containing 10% FBS. 100,000 cells were seeded in 100 μl per well into tissue-culture-treated 96-well flat-bottom or U-bottom plates (to promote cell contact). B15 and B15_Rαsushi were serially diluted 5-fold in the same medium from 500 nM to 0.032 nM, and 100 μl of each dilution was added to the corresponding cells to achieve final concentrations of 250 nM to 0.016 nM. Controls without B15 or B15_Rαsushi were also set up. These cells were incubated at 37°C, 5% CO2 for 96 hours. After treatment, PBMCs were 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, a marker of cell proliferation, was stained using anti-Ki67-PE-Cy7 (20Raj1) and Foxp3 / Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer's protocol. Subsequently, stained samples were analyzed using a Novocyte Advanteon Flow Cytometer (Agilent, Santa Clara, CA).

[0195] As shown in Figure 13, both B15 and B15_15Rα induced potent proliferation of CD8+ T cells and NK cells in vitro, but to a much lesser extent CD4+ T cells. Proliferation was independent of the different types of 96-well plates used (flat-bottom vs. U-bottom), suggesting that proliferation was induced solely by B15 or B15_15Rα and that effects from cell-to-cell contact were minimal. In these experiments, B15_15Rα performed slightly better than B15 at low concentrations in inducing T cell and NK cell proliferation, suggesting that IL15Rα can enhance IL15 function at low concentrations. NK cell proliferation induced by B15 and B15_15Rα plateaued at 0.4 nM, while CD8+ T cell proliferation plateaued at 10 nM, indicating that B15 and B15_15Rα have higher affinity for NK cells than for CD8+ T cells.

[0196] 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 disclosure and are intended to fall within the scope of the disclosure.

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[0198] Array information SEQUENCE LISTING <110> MINOTAUR THERAPEUTICS, Inc. <120> CHIMERIC CYTOKINE MODIFIED ANTIBODIES AND METHODS OF USE THEREOF <150> US 62 / 925,740 <151> 2019-10-24 <160> 180 <170> PatentIn version 3.5 <210> 1 <211> 114 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> IL-15 <400> 1 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 2 <211> 77 <212> PRT <213> Artificial sequence <220> <223> IL15-Ralphasushi <400> 2 Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val 1 5 10 15 Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30 Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45 Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60 Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro 65 70 75 <210> 3 <211> 912 <212> DNA <213> artificial sequence <220> <223> IL-15 Ralphasushi_GSlinker_BLV1H12 light chain <400> 3 atcacctgcc cacctccaat gagcgtggag cacgcagaca tctgggtgaa gtcttacagc 60 ctgtattccc gggagagata catctgcaac tctggcttca agcggaaggc cggcaccagc 120 tcctgacag agtgcgtgct gaacaaggcc accaatgtgg cccactggac aactccttcc 180 ctgaaatgta ttagagaccc cgccctggtg catcagagac ctgccccccc tggtggaggc 240 ggttcaggcg gaggtggatc ccaggccgtc ctgaaccagc caagcagcgt ctccgggtct 300 ctggggcagc gggtctcaat cacctgtagc gggtcttcct caatgtcgg caacggctac 360 gtgcttggt atcagctgat ccctggcagt gccccacgaa ccctgatcta cggcgacaca 420 tccagagctt ctggggtccc cgatcggttc tcagggagca gatccggaaa cacagctact 480 ctgaccatca gctccctgca ggctgaggac gaagcagatt atttctgcgc atctgccgag 540 gactctagtt caaatgccgt gtttggaagc ggcaccacac tgacagtcct aggtcagccc 600 aaggctgccc cctcggtcac tctgttccg ccctcctctg aggagcttca agccaacaag 660 gccacactgg tgtgtctcat aagtgacttc tacccgggag ccgtgacagt ggcctggaag 720 gcagatagca gccccgtcaa ggcgggagtg gagaccacca caccctccaa acaaagcaac 780 aacaagtacg cggccagcag ctatctgagc ctgacgcctg agcagtggaa gtcccacaga 840 agctacagct gccaggtcac gcatgaaggg agcaccgtgg agaagacagt ggcccctaca 900 gaatgtcat aa 912 <210> 4 <211> 524 <212> PRT <213> artificial sequence <220> <223> IL2 receptor subunit beta <400> 4 Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser Arg Ala Asn 1 5 10 15 Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp Thr Ser Cys 20 25 30 Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln Thr Cys Glu 35 40 45 Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu Gly 50 55 60 Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val Thr Leu Arg 65 70 75 80 Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala Ile Gln Asp 85 90 95 Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile Ser Leu Gln 100 105 110 Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp Glu Ile Ser 115 120 125 Gln Ala Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu Ala Arg Thr 130 135 140 Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu Thr Leu Lys 145 150 155 160 Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro Asp Thr Gln 165 170 175 Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu Phe Thr Thr 180 185 190 Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys Pro Ala Ala 195 200 205 Leu Gly Lys Asp Thr Ile Pro Trp Leu Gly His Leu Leu Val Gly Leu 210 215 220 Ser Gly Ala Phe Gly Phe Ile Ile Leu Val Tyr Leu Leu Ile Asn Cys 225 230 235 240 Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys Asn Thr Pro 245 250 255 Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His Gly Gly Asp 260 265 270 Val Gln Lys Trp Leu Ser Ser Pro Phe Pro Ser Ser Ser Phe Ser Pro 275 280 285 Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu Arg Asp 290 295 300 Lys Val Thr Gln Leu Leu Leu Gln Gln Asp Lys Val Pro Glu Pro Ala 305 310 315 320 Ser Leu Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn Gln Gly 325 330 335 Tyr Phe Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys Gln 340 345 350 Val Tyr Phe Thr Tyr Asp Pro Tyr Ser Glu Glu Asp Pro Asp Glu Gly 355 360 365 Val Ala Gly Ala Pro Thr Gly Ser Ser Pro Gln Pro Leu Gln Pro Leu 370 375 380 Ser Gly Glu Asp Asp Ala Tyr Cys Thr Phe Pro Ser Arg Asp Asp Leu 385 390 395 400 Leu Leu Phe Ser Pro Ser Leu Leu Gly Gly Pro Ser Pro Pro Ser Thr 405 410 415 Ala Pro Gly Gly Ser Gly Ala Gly Glu Glu Arg Met Pro Pro Ser Leu 420 425 430 Gln Glu Arg Val Pro Arg Asp Trp Asp Pro Gln Pro Leu Gly Pro Pro 435 440 445 Thr Pro Gly Val Pro Asp Leu Val Asp Phe Gln Pro Pro Pro Glu Leu 450 455 460 Val Leu Arg Glu Ala Gly Glu Glu Val Pro Asp Ala Gly Pro Arg Glu 465,470,475,480 Gly Val Ser Phe Pro Trp Ser Arg Pro Pro Gly Gln Gly Glu Phe Arg 485 490 495 Ala Leu Donkey Ala Arg Leu Pro Leu Donkey Thr Asp Ala Tyr Leu Ser Leu 500 505 510 Gln Glu Leu Gln Gly Gln Asp Pro Thr His Leu Val 515 520 <210> 5 <211> 513 <212> DNA <213> artificial sequence <220> <223> BLV1H 12 heavy chain <400> 5 caggtccagc tgagagagag cggcccttca ctggtcaagc catcccagac actgagcctg 60 acatgcacag caagcgggtt ttcactgagc gacaaggcag tgggatgggt ccgacaggca 120 ccaggaaaag ccctggaatg gctgggcagc atcgataccg gcgggaacac agggtacaat 180 cccggactga agagcagact gtccattacc aaggacaact ctaaaagtca ggtgtcactg 240 agcgtgagct ccgtcaccac agaggatagt gcaacttact attgcacctc tgtgcaccag 300 gaaactaaga aataccagag ctgtcctgac ggctatcggg agagatctga ttgcagtaat 360 aggccagctt gtggcacatc cgactgctgt cgcgtgtctg tcttcgggaa ctgcctgact 420 accctgcctg tgtcctactc ttatacctac aattatgaat ggcatgtgga tgtctgggga 480 cagggcctgc tggtgacagt ctctagtgct agc 513 <210> 6 <211> 1557 <212> DNA <213> artificial sequence <220> <223> (Sig Seq- VRegion - CH1CH2CH3) BLV1H12 V in human IgG <400> 6 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattcccag 60 gtgcagctgc gggagtcggg ccccagcctg atgaagccgt cacagaccct ctccctcacc 120 tgcacggtct ctggatcttc attgaacgac aagtctgtag gctgggtccg ccaggctcca 180 gggaaggcgc tgcagtggct cggtagtgtg gacactagtg gaaacacaga ctataaccca 240 ggcctgaaat cccggctcag catcaccaag gacaactcca agagccgaat ctctcttaca 300 gtgactggca tgacaactga agactcggcc acatactact gtacttctgt gcaccaggaa 360 acaaaaaaat accaaagttg tccggaggat tatacttata atccacgttg ccctcagcag 420 tatggttgga gtgactgtga ttgtatgggc gataggtttg ggggttactg tcgacaggat 480 ggttgtagta attatagtta tacttacaat tacgaatggc acgtcgatgt ctggggccaa 540 ggactcctgg tcaccgtctc ctcagctagc accaagggcc catcggtctt ccccctggca 600 ccctcctcca agagcacctc tgggggcaca gcggccctgg gctgcctggt caaggactac 660 ttccccgaac ctgtgacggt ctcgtggaac tcaggcgccc tgaccagcgg cgtgcacacc 720 ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt gaccgtgccc 780 tccagcagct tgggcaccca gacctacatc tgcaacgtga atcacaagcc cagcaacacc 840 aaggtggaca agagagttga gcccaaatct tgtgacaaaa ctcacacatg cccaccgtgc 900 ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac 960 accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 1020 gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 1080 aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 1140 caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 1200 gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 1260 accctgcccc catcccggga ggagatgacc aagaaccagg tcagcctgac ctgcctggtc 1320 aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 1380 aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctatagcaag 1440 ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 1500 gaggctctgc acaaccacta cacgcagaag agcctctccc tgtccccggg taaatga 1557 <210> 7 <211> 823 <212> DNA <213> artificial sequence <220> <223> B15 variable region plus signal peptide <400> 7 gaattccacc atgggatggt catgtatcat cctttttcta gtagcaactg caaccggagt acattcccag gtgcagctgc gcgagtcggg ccccagcctg gtgaagccgt cacagaccct 120 ctcgctcacc tgcacggcct ctggattctc attgagcgac aaggctgtag gctgggtccg 180 240. ccaggctcca gggaaggcgc tggagtggct cggtagtata gacactggtg gaaacacagg ctataaccca ggcctgaat cccggctcag catcaccag gacaactcca aggtcaagt ctctctgtca gtgagcagcg tgacaactga ggactcggcc acatactact gtacttctgt gcaccagga acaaaaaaat accaaaccgg tggatcaaac tgggtgaatg taataagtga tttgaaaaaa attgaagatc ttattcaatc tatgcatatt gatgctactt tatacgga aagtgatgtt caccccagtt gcaaagtaac agcaatgaag tgctttctct tggagttaca 540 agttatttca cttgagtccg gagatgcaag tattcatgat acagtagaaa atctgatcat cctagcaaac aacagtttgt cttctaatgg gaatgtaaca gaatctggat gcaaagaatg tgaggaactg gaggaaaaaa attack atttttgcag agttttgtac attacktgtcca aatgttcatc aacacttctg gttcaggatc ctatacttac aattacgaat ggcacgtcga 780 tgtctggggc caaggactcc tggtcaccgt ctcctcagct agc 823 <210> 8 <211> 672 <212> DNA <213> artificial sequence <220> <223> BLV1H12 Light Chain <400> 8 tcacgaattc gcaggccgtc ctgaaccagc caagcagcgt ctccgggtct ctggggcagc 60 gggtctcaat cacctgtagc gggtcttcct ccaatgtcgg caacggctac gtgtcttggt 120 atcagctgat ccctggcagt gccccacgaa ccctgatcta cggcgacaca tccagagctt 180 ctggggtccc cgatcggttc tcagggagca gatccggaaa cacagctact ctgaccatca 240 gctccctgca ggctgaggac gaagcagatt atttctgcgc atctgccgag gactctagtt 300 caaatgccgt gtttggaagc ggcaccacac tgacagtcct ggggcagccc aagagtcccc 360 cttcagtgac tctgttccca ccctctaccg aggaactgaa cggaaacaag gccacactgg 420 tgtgtctgat cagcgacttt taccctggat ccgtcactgt ggtctggaag gcagatggca 480 gcacaattac taggaacgtg gaaactaccc gcgcctccaa gcagtctaat agtaaatacg 540 ccgccagctc ctatctgagc ctgacctcta gtgattggaa gtccaaaggg tcatatagct 600 gcgaagtgac ccatgaaggc tcaaccgtga ctaagactgt gaaaccatcc gagtgctcct 660 aggctagctg gc 672 <210> 9 <211> 12 <212> PRT <213> artificial sequence <220> <223> BLV1H 12 ascending stalk region <400> 9 Thr Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser 1 5 10 <210> 10 <211> 12 <212> PRT <213> artificial sequence <220> <223> BLV1H 12 decending stalk region <400> 10 Ser Tyr Thr Tyr Asn Tyr Glu Trp His Val Asp Val 1 5 10 <210> 11 <211> 11 <212> PRT <213> artificial sequence <220> <223> V2 alternative sequence <400> 11 Trp Gly Gln Gly Leu Leu Val Thr Val Ser Ser 1 5 10 <210> 12 <211> 95 <212> PRT <213> artificial sequence <220> <223> V1 Alternative B sequence of VH4-34_Q5RQ6E <400> 12 Gln Val Gln Leu Arg Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 <210> 13 <211> 95 <212> PRT <213> artificial sequence <220> <223> V1 Alternative B sequence of VH4-34_CDR1-G31DY32K_Q5RQ6E <400> 13 Gln Val Gln Leu Arg Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp Lys 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 <210> 14 <211> 95 <212> PRT <213> artificial sequence <220> <223> V1 Alternative B sequence of VH4-34_CDR2-E50S_Q5RQ6E <400> 14 Gln Val Gln Leu Arg Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Ser Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 <210> 15 <211> 95 <212> PRT <213> artificial sequence <220> <223> synthesized: V1 Alternative B sequence of VH4-34_CDR1-G31DY32K_CDR2-E50S_Q5RQ6E <400> 15 Gln Val Gln Leu Arg Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp Lys 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Ser Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 <210> 16 <211> 95 <212> PRT <213> artificial sequence <220> <223> synthesized: V1 Alternative B sequence of VH4-34_CDR1-Cow_Q5RQ6E <400> 16 Gln Val Gln Leu Arg Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys 20 25 30 Ala Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 <210> 17 <211> 95 <212> PRT <213> artificial sequence <220> <223> synthesized: V1 Alternative B sequence of VH4-34_CDR2-Cow_Q5RQ6E <400> 17 Gln Val Gln Leu Arg Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Leu Gly Ser Ile Asp 20 25 30 Thr Gly Gly Asn Thr Gly Ser Phe Ser Gly Tyr Tyr Trp Ser Trp Ile 35 40 45 Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 <210> 18 <211> 95 <212> PRT <213> artificial sequence <220> <223> synthesized: V1 Alternative B sequence of VH4-34_CDR1-Cow_CDR2-E50S_Q5RQ6E <400> 18 Gln Val Gln Leu Arg Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys 20 25 30 Ala Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Ser Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 <210> 19 <211> 95 <212> PRT <213> artificial sequence <220> <223> synthesized: V1 Alternative N sequence of VH4-34_CDR1-Cow_CDR2-Cow_Q5RQ6E <400> 19 Gln Val Gln Leu Arg Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys 20 25 30 Ala Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Ser Ile Asp Thr Gly Gly Asn Thr Gly Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 <210> 20 <211> 11 <212> PRT <213> artificial sequence <220> <223> V2 alternative sequence <400> 20 Trp Gly His Gly Thr Ala Val Thr Val Ser Ser 1 5 10 <210> 21 <211> 11 <212> PRT <213> artificial sequence <220> <223> V2 alternative sequence <400> 21 Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser 1 5 10 <210> 22 <211> 11 <212> PRT <213> artificial sequence <220> <223> V2 alternative sequence <400> 22 Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser 1 5 10 <210> 23 <211> 11 <212> PRT <213> artificial sequence <220> <223> V2 alternative sequence <400> 23 Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 24 <211> 11 <212> PRT <213> artificial sequence <220> <223> V2 alternative sequence <400> 24 Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser 1 5 10 <210> 25 <211> 61 <212> PRT <213> artificial sequence <220> <223> Synthesized: ultralong CDR3 sequence (BLV1H12) <400> 25 Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser Cys Pro Asp Gly Tyr 1 5 10 15 Arg Glu Arg Ser Asp Cys Ser Asn Arg Pro Ala Cys Gly Thr Ser Asp 20 25 30 Cys Cys Arg Val Ser Val Phe Gly Asn Cys Leu Thr Thr Leu Pro Val 35 40 45 Ser Tyr Ser Tyr Thr Tyr Asn Tyr Glu Trp His Val Asp 50 55 60 <210> 26 <211> 274 <212> PRT <213> artificial sequence <220> <223> BLV1H12 Heavy Chain <400> 26 Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys 20 25 30 Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45 Gly Ser Ile Asp Thr Gly Gly Asn Thr Gly Tyr Asn Pro Gly Leu Lys 50 55 60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Thr 85 90 95 Ser Val His Gln Glu Thr Lys Lys Tyr Gln Ser Cys Pro Asp Gly Tyr 100 105 110 Arg Glu Arg Ser Asp Cys Ser Asn Arg Pro Ala Cys Gly Thr Ser Asp 115 120 125 Cys Cys Arg Val Ser Val Phe Gly Asn Cys Leu Thr Thr Leu Pro Val 130 135 140 Ser Tyr Ser Tyr Thr Tyr Asn Tyr Glu Trp His Val Asp Val Trp Gly 145 150 155 160 Gln Gly Leu Leu Val Thr Val Ser Ser Ala Ser Thr Thr Ala Pro Lys 165 170 175 Val Tyr Pro Leu Ser Ser Cys Cys Gly Asp Lys Ser Ser Ser Thr Val 180 185 190 Thr Leu Gly Cys Leu Val Ser Ser Tyr Met Pro Glu Pro Val Thr Val 195 200 205 Thr Trp Asn Ser Gly Ala Leu Lys Ser Gly Val His Thr Phe Pro Ala 210 215 220 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Met Val Thr Val 225 230 235 240 Pro Gly Ser Thr Ser Gly Gln Thr Phe Thr Cys Asn Val Ala His Pro 245 250 255 Ala Ser Ser Thr Lys Val Asp Lys Ala Val Glu Pro Lys Ser Cys Asp 260 265 270 Gly Ser <210> 27 <211> 216 <212> PRT <213> artificial sequence <220> <223> BLV1H12 Light Chain <400> 27 Gln Ala Val Leu Asn Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln 1 5 10 15 Arg Val Ser Ile Thr Cys Ser Gly Ser Ser Ser Asn Val Gly Asn Gly 20 25 30 Tyr Val Ser Trp Tyr Gln Leu Ile Pro Gly Ser Ala Pro Arg Thr Leu 35 40 45 Ile Tyr Gly Asp Thr Ser Arg Ala Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Arg Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ala Ser Ala Glu Asp Ser Ser 85 90 95 Ser Asn Ala Val Phe Gly Ser Gly Thr Thr Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ser Pro Pro Ser Val Thr Leu Phe Pro Pro Ser Thr Glu Glu 115 120 125 Leu Asn Gly Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ser Val Thr Val Val Trp Lys Ala Asp Gly Ser Thr Ile Thr 145 150 155 160 Arg Asn Val Glu Thr Thr Arg Ala Ser Lys Gln Ser Asn Ser Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Ser Ser Asp Trp Lys Ser Lys 180 185 190 Gly Ser Tyr Ser Cys Glu Val Thr His Glu Gly Ser Thr Val Thr Lys 195 200 205 Thr Val Lys Pro Ser Glu Cys Ser 210 215 <210> 28 <211> 269 <212> PRT <213> artificial sequence <220> <223> BLV5B8 heavy chain <400> 28 Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys 20 25 30 Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45 Gly Ser Ile Asp Thr Gly Gly Ser Thr Gly Tyr Asn Pro Gly Leu Lys 50 55 60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Thr 85 90 95 Thr Val His Gln Glu Thr Arg Lys Thr Cys Ser Asp Gly Tyr Ile Ala 100 105 110 Val Asp Ser Cys Gly Arg Gly Gln Ser Asp Gly Cys Val Asn Asp Cys 115 120 125 Asn Ser Cys Tyr Tyr Gly Trp Arg Asn Cys Arg Arg Gln Pro Ala Ile 130 135 140 His Ser Tyr Glu Phe His Val Asp Ala Trp Gly Arg Gly Leu Leu Val 145 150 155 160 Thr Val Ser Ser Ala Ser Thr Thr Ala Pro Lys Val Tyr Pro Leu Ser 165 170 175 Ser Cys Cys Gly Asp Lys Ser Ser Ser Thr Val Thr Leu Gly Cys Leu 180 185 190 Val Ser Ser Tyr Met Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly 195 200 205 Ala Leu Lys Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 210 215 220 Gly Leu Tyr Ser Leu Ser Ser Met Val Thr Val Pro Gly Ser Thr Ser 225 230 235 240 Gly Gln Thr Phe Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys 245 250 255 Val Asp Lys Ala Val Glu Pro Lys Ser Cys Asp Gly Ser 260 265 <210> 29 <211> 216 <212> PRT <213> artificial sequence <220> <223> BLV5B8 light chain <400> 29 Gln Ala Val Leu Asn Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln 1 5 10 15 Arg Val Ser Ile Thr Cys Ser Gly Ser Ser Ser Asn Val Gly Asn Gly 20 25 30 Tyr Val Ser Trp Tyr Gln Leu Ile Pro Gly Ser Ala Pro Arg Thr Leu 35 40 45 Ile Tyr Gly Asp Thr Ser Arg Ala Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Arg Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ala Ser Ala Glu Asp Ser Ser 85 90 95 Ser Asn Ala Val Phe Gly Ser Gly Thr Thr Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ser Pro Pro Ser Val Thr Leu Phe Pro Pro Ser Thr Glu Glu 115 120 125 Leu Asn Gly Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ser Val Thr Val Val Trp Lys Ala Asp Gly Ser Thr Ile Thr 145 150 155 160 Arg Asn Val Glu Thr Thr Arg Ala Ser Lys Gln Ser Asn Ser Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Ser Ser Asp Trp Lys Ser Lys 180 185 190 Gly Ser Tyr Ser Cys Glu Val Thr His Glu Gly Ser Thr Val Thr Lys 195 200 205 Thr Val Lys Pro Ser Glu Cys Ser 210 215 <210> 30 <211> 56 <212> PRT <213> artificial sequence <220> <223> BLV5B8 CDR3 <400> 30 Thr Val His Gln Glu Thr Arg Lys Thr Cys Ser Asp Gly Tyr Ile Ala 1 5 10 15 Val Asp Ser Cys Gly Arg Gly Gln Ser Asp Gly Cys Val Asn Asp Cys 20 25 30 Asn Ser Cys Tyr Tyr Gly Trp Arg Asn Cys Arg Arg Gln Pro Ala Ile 35 40 45 His Ser Tyr Glu Phe His Val Asp 50 55 <210> 31 <211> 57 <212> PRT <213> artificial sequence <220> <223> BLV5D3 CDR3 <400> 31 Ser Val Thr Gln Arg Thr His Val Ser Arg Ser Cys Pro Asp Gly Cys 1 5 10 15 Ser Asp Gly Asp Gly Cys Val Asp Gly Cys Cys Cys Ser Ala Tyr Arg 20 25 30 Cys Tyr Thr Pro Gly Val Arg Asp Leu Ser Cys Thr Ser Tyr Ser Ile 35 40 45 Thr Tyr Thr Tyr Glu Trp Asn Val Asp 50 55 <210> 32 <211> 58 <212> PRT <213> artificial sequence <220> <223> BLV8C11 CDR3 <400> 32 Thr Val His Gln Lys Thr Thr Arg Lys Thr Cys Cys Ser Asp Ala Tyr 1 5 10 15 Arg Tyr Asp Ser Gly Cys Gly Ser Gly Cys Asp Cys Cys Gly Ala Asp 20 25 30 Cys Tyr Val Phe Gly Ala Cys Thr Phe Gly Leu Asp Ser Ser Tyr Ser 35 40 45 Tyr Ile Tyr Ile Tyr Gln Trp Tyr Val Asp 50 55 <210> 33 <211> 56 <212> PRT <213> artificial sequence <220> <223> BF4E9 CDR3 <400> 33 Thr Val His Gln Ile Phe Cys Pro Asp Gly Tyr Ser Tyr Gly Tyr Gly 1 5 10 15 Cys Gly Tyr Gly Tyr Gly Cys Ser Gly Tyr Asp Cys Tyr Gly Tyr Gly 20 25 30 Gly Tyr Gly Tyr Gly Gly Tyr Gly Gly Tyr Ser Ser Tyr Ser Tyr Ser 35 40 45 Tyr Ser Tyr Glu Tyr Tyr Gly Asp 50 55 <210> 34 <211> 48 <212> PRT <213> artificial sequence <220> <223> BF1H1 CDR3 <400> 34 Thr Val His Pro Ser Pro Asp Gly Tyr Ser Tyr Gly Tyr Gly Cys Gly 1 5 10 15 Tyr Gly Tyr Gly Cys Ser Gly Tyr Asp Cys Tyr Gly Tyr Gly Gly Tyr 20 25 30 Gly Tyr Gly Gly Tyr Gly Gly Tyr Ser Ser Tyr Ser Tyr Ser Tyr Ser 35 40 45 <210> 35 <211> 48 <212> PRT <213> artificial sequence <220> <223> F18 CDR3 <400> 35 Thr Val His Gln Ile Arg Cys Pro Asp Gly Tyr Gly Tyr Gly Tyr Gly 1 5 10 15 Cys Gly Tyr Gly Ser Tyr Gly Tyr Ser Gly Tyr Asp Cys Tyr Gly Tyr 20 25 30 Gly Gly Tyr Gly Gly Tyr Gly Gly Tyr Gly Gly Tyr Ser Ser Tyr Ser 35 40 45 <210> 36 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 36 Thr Thr Val His Gln 1 5 <210> 37 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 37 Thr Ser Val His Gln 1 5 <210> 38 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 38 Ser Ser Val Thr Gln 1 5 <210> 39 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 39 Ser Thr Val His Gln 1 5 <210> 40 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 40 Ala Thr Val Arg Gln 1 5 <210> 41 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 41 Thr Thr Val Tyr Gln 1 5 <210> 42 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 42 Ser Pro Val His Gln 1 5 <210> 43 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 43 Ala Thr Val Tyr Gln 1 5 <210> 44 <211> 20 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 44 Ala Ser Cys Glu Asn Asp Ile Asn Gly Ser Thr Ala Leu Lys Ser Thr 1 5 10 15 Arg Ala Asn Asp 20 <210> 45 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 45 Thr Asn Val His Gln 1 5 <210> 46 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 46 Ala Thr Val His Gln 1 5 <210> 47 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 47 Ser Thr Val Tyr Gln 1 5 <210> 48 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 48 Thr Ile Val His Gln 1 5 <210> 49 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 49 Ala Ile Val Tyr Gln 1 5 <210> 50 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 50 Thr Thr Val Phe Gln 1 5 <210> 51 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 51 Ala Ala Val Phe Gln 1 5 <210> 52 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 52 Gly Thr Val His Gln 1 5 <210> 53 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 53 Ala Ser Val His Gln 1 5 <210> 54 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 54 Thr Ala Val Phe Gln 1 5 <210> 55 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 55 Ala Thr Val Phe Gln 1 5 <210> 56 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 56 Ala Ala Ala His Gln 1 5 <210> 57 <211> 4 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 57 Val Trp Tyr Gln 1 <210> 58 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 58 Gly Thr Val Phe Gln 1 5 <210> 59 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 59 Thr Ala Val His Gln 1 5 <210> 60 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 60 Ile Thr Val His Gln 1 5 <210> 61 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 61 Ile Thr Ala His Gln 1 5 <210> 62 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 62 Val Thr Val His Gln 1 5 <210> 63 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 63 Ala Ala Val His Gln 1 5 <210> 64 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 64 Gly Thr Val Tyr Gln 1 5 <210> 65 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 65 Thr Thr Val Leu Gln 1 5 <210> 66 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 66 Thr Thr Thr His Gln 1 5 <210> 67 <211> 5 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 67 Thr Thr Asp Tyr Gln 1 5 <210> 68 <211> 99 <212> PRT <213> artificial sequence <220> <223> Human heavy chain variable region sequence VH4-39 <400> 68 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg <210> 69 <211> 96 <212> PRT <213> artificial sequence <220> <223> Human heavy chain variable region sequence 4-59*03 <400> 69 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 <210> 70 <211> 97 <212> PRT <213> artificial sequence <220> <223> Human heavy chain variable region sequence 4-34*02 <400> 70 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg <210> 71 <211> 97 <212> PRT <213> artificial sequence <220> <223> Human heavy chain variable region sequence 4-34*09 <400> 71 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg <210> 72 <211> 11 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 72 Thr Ser Val His Gln Glu Thr Lys Lys Tyr Gln 1 5 10 <210> 73 <211> 9 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 73 Val His Gln Glu Thr Lys Lys Tyr Gln 1 5 <210> 74 <211> 6 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 74 Ile His Ser Tyr Glu Phe 1 5 <210> 75 <211> 4 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 75 Ser Tyr Glu Phe 1 <210> 76 <211> 6 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 76 Tyr Thr Tyr Asn Tyr Glu 1 5 <210> 77 <211> 7 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 77 Tyr Thr Tyr Asn Tyr Glu Trp 1 5 <210> 78 <211> 8 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 78 Ser Tyr Thr Tyr Asn Tyr Glu Trp 1 5 <210> 79 <211> 6 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 79 Thr Tyr Asn Tyr Glu Trp 1 5 <210> 80 <211> 4 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 80 Ser Tyr Thr Tyr 1 <210> 81 <211> 14 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 81 Gly Ser Lys His Arg Leu Arg Asp Tyr Phe Leu Tyr Asn Glu 1 5 10 <210> 82 <211> 13 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 82 Gly Ser Lys His Arg Leu Arg Asp Tyr Phe Leu Tyr Asn 1 5 10 <210> 83 <211> 12 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 83 Gly Ser Lys His Arg Leu Arg Asp Tyr Phe Leu Tyr 1 5 10 <210> 84 <211> 11 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 84 Gly Ser Lys His Arg Leu Arg Asp Tyr Phe Leu 1 5 10 <210> 85 <211> 10 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 85 Gly Ser Lys His Arg Leu Arg Asp Tyr Phe 1 5 10 <210> 86 <211> 9 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 86 Gly Ser Lys His Arg Leu Arg Asp Tyr 1 5 <210> 87 <211> 8 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 87 Gly Ser Lys His Arg Leu Arg Asp 1 5 <210> 88 <211> 13 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 88 Glu Ala Gly Gly Pro Asp Tyr Arg Asn Gly Tyr Asn Tyr 1 5 10 <210> 89 <211> 12 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 89 Glu Ala Gly Gly Pro Asp Tyr Arg Asn Gly Tyr Asn 1 5 10 <210> 90 <211> 11 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 90 Glu Ala Gly Gly Pro Asp Tyr Arg Asn Gly Tyr 1 5 10 <210> 91 <211> 10 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 91 Glu Ala Gly Gly Pro Asp Tyr Arg Asn Gly 1 5 10 <210> 92 <211> 9 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 92 Glu Ala Gly Gly Pro Asp Tyr Arg Asn 1 5 <210> 93 <211> 8 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 93 Glu Ala Gly Gly Pro Asp Tyr Arg 1 5 <210> 94 <211> 7 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 94 Glu Ala Gly Gly Pro Asp Tyr 1 5 <210> 95 <211> 6 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 95 Glu Ala Gly Gly Pro Asp 1 5 <210> 96 <211> 13 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 96 Glu Ala Gly Gly Pro Ile Trp His Asp Asp Val Lys Tyr 1 5 10 <210> 97 <211> 12 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 97 Glu Ala Gly Gly Pro Ile Trp His Asp Asp Val Lys 1 5 10 <210> 98 <211> 11 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 98 Glu Ala Gly Gly Pro Ile Trp His Asp Asp Val 1 5 10 <210> 99 <211> 10 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 99 Glu Ala Gly Gly Pro Ile Trp His Asp Asp 1 5 10 <210> 100 <211> 9 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 100 Glu Ala Gly Gly Pro Ile Trp His Asp 1 5 <210> 101 <211> 8 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 101 Glu Ala Gly Gly Pro Ile Trp His 1 5 <210> 102 <211> 7 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 102 Glu Ala Gly Gly Pro Ile Trp 1 5 <210> 103 <211> 6 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 103 Glu Ala Gly Gly Pro Ile 1 5 <210> 104 <211> 11 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 104 Gly Thr Asp Tyr Thr Ile Asp Asp Gln Gly Ile 1 5 10 <210> 105 <211> 10 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 105 Gly Thr Asp Tyr Thr Ile Asp Asp Gln Gly 1 5 10 <210> 106 <211> 9 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 106 Gly Thr Asp Tyr Thr Ile Asp Asp Gln 1 5 <210> 107 <211> 8 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 107 Gly Thr Asp Tyr Thr Ile Asp Asp 1 5 <210> 108 <211> 7 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 108 Gly Thr Asp Tyr Thr Ile Asp 1 5 <210> 109 <211> 6 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 109 Gly Thr Asp Tyr Thr Ile 1 5 <210> 110 <211> 11 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 110 Asp Lys Gly Asp Ser Asp Tyr Asp Tyr Asn Leu 1 5 10 <210> 111 <211> 10 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 111 Asp Lys Gly Asp Ser Asp Tyr Asp Tyr Asn 1 5 10 <210> 112 <211> 9 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 112 Asp Lys Gly Asp Ser Asp Tyr Asp Tyr 1 5 <210> 113 <211> 8 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 113 Asp Lys Gly Asp Ser Asp Tyr Asp 1 5 <210> 114 <211> 7 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 114 Asp Lys Gly Asp Ser Asp Tyr 1 5 <210> 115 <211> 6 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 115 Asp Lys Gly Asp Ser Asp 1 5 <210> 116 <211> 17 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 116 Tyr Gly Pro Asn Tyr Glu Glu Trp Gly Asp Tyr Leu Ala Thr Leu Asp 1 5 10 15 Val <210> 117 <211> 16 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 117 Gly Pro Asn Tyr Glu Glu Trp Gly Asp Tyr Leu Ala Thr Leu Asp Val 1 5 10 15 <210> 118 <211> 15 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 118 Pro Asn Tyr Glu Glu Trp Gly Asp Tyr Leu Ala Thr Leu Asp Val 1 5 10 15 <210> 119 <211> 14 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 119 Asn Tyr Glu Glu Trp Gly Asp Tyr Leu Ala Thr Leu Asp Val 1 5 10 <210> 120 <211> 13 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 120 Tyr Glu Glu Trp Gly Asp Tyr Leu Ala Thr Leu Asp Val 1 5 10 <210> 121 <211> 12 <212> PRT <213> artificial sequence <220> <223> ASCENDING STALK STRAND <400> 121 Glu Glu Trp Gly Asp Tyr Leu Ala Thr Leu Asp Val 1 5 10 <210> 122 <211> 15 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 122 Tyr Asp Phe Tyr Asp Gly Tyr Tyr Asn Tyr His Tyr Met Asp Val 1 5 10 15 <210> 123 <211> 14 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 123 Asp Phe Tyr Asp Gly Tyr Tyr Asn Tyr His Tyr Met Asp Val 1 5 10 <210> 124 <211> 13 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 124 Phe Tyr Asp Gly Tyr Tyr Asn Tyr His Tyr Met Asp Val 1 5 10 <210> 125 <211> 12 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 125 Tyr Asp Gly Tyr Tyr Asn Tyr His Tyr Met Asp Val 1 5 10 <210> 126 <211> 11 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 126 Asp Gly Tyr Tyr Asn Tyr His Tyr Met Asp Val 1 5 10 <210> 127 <211> 10 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 127 Gly Tyr Tyr Asn Tyr His Tyr Met Asp Val 1 5 10 <210> 128 <211> 9 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 128 Tyr Tyr Asn Tyr His Tyr Met Asp Val 1 5 <210> 129 <211> 15 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 129 Tyr Asp Phe Asn Asp Gly Tyr Tyr Asn Tyr His Tyr Met Asp Val 1 5 10 15 <210> 130 <211> 14 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 130 Asp Phe Tyr Asp Gly Tyr Tyr Asn Tyr His Tyr Met Asp Val 1 5 10 <210> 131 <211> 13 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 131 Phe Tyr Asp Gly Tyr Tyr Asn Tyr His Tyr Met Asp Val 1 5 10 <210> 132 <211> 12 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 132 Tyr Asp Gly Tyr Tyr Asn Tyr His Tyr Met Asp Val 1 5 10 <210> 133 <211> 11 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 133 Asp Gly Tyr Tyr Asn Tyr His Tyr Met Asp Val 1 5 10 <210> 134 <211> 10 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 134 Gly Tyr Tyr Asn Tyr His Tyr Met Asp Val 1 5 10 <210> 135 <211> 16 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 135 Gln Gly Ile Arg Tyr Gln Gly Ser Gly Thr Phe Trp Tyr Phe Asp Val 1 5 10 15 <210> 136 <211> 15 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 136 Gly Ile Arg Tyr Gln Gly Ser Gly Thr Phe Trp Tyr Phe Asp Val 1 5 10 15 <210> 137 <211> 14 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 137 Ile Arg Tyr Gln Gly Ser Gly Thr Phe Trp Tyr Phe Asp Val 1 5 10 <210> 138 <211> 13 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 138 Arg Tyr Gln Gly Ser Gly Thr Phe Trp Tyr Phe Asp Val 1 5 10 <210> 139 <211> 12 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 139 Tyr Gln Gly Ser Gly Thr Phe Trp Tyr Phe Asp Val 1 5 10 <210> 140 <211> 11 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 140 Gln Gly Ser Gly Thr Phe Trp Tyr Phe Asp Val 1 5 10 <210> 141 <211> 10 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 141 Gly Ser Gly Thr Phe Trp Tyr Phe Asp Val 1 5 10 <210> 142 <211> 9 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 142 Ser Gly Thr Phe Trp Tyr Phe Asp Val 1 5 <210> 143 <211> 8 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 143 Gly Thr Phe Trp Tyr Phe Asp Val 1 5 <210> 144 <211> 13 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 144 Tyr Asn Leu Gly Tyr Ser Tyr Phe Tyr Tyr Met Asp Gly 1 5 10 <210> 145 <211> 12 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 145 Asn Leu Gly Tyr Ser Tyr Phe Tyr Tyr Met Asp Gly 1 5 10 <210> 146 <211> 11 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 146 Leu Gly Tyr Ser Tyr Phe Tyr Tyr Met Asp Gly 1 5 10 <210> 147 <211> 10 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 147 Gly Tyr Ser Tyr Phe Tyr Tyr Met Asp Gly 1 5 10 <210> 148 <211> 9 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 148 Tyr Ser Tyr Phe Tyr Tyr Met Asp Gly 1 5 <210> 149 <211> 8 <212> PRT <213> artificial sequence <220> <223> DESCENDING STALK STRAND <400> 149 Ser Tyr Phe Tyr Tyr Met Asp Gly 1 5 <210> 150 <211> 2 <212> PRT <213> artificial sequence <220> <223> Linker <400> 150 Gly Ser 1 <210> 151 <211> 3 <212> PRT <213> artificial sequence <220> <223> Linker <400> 151 Gly Gly Ser 1 <210> 152 <211> 6 <212> PRT <213> artificial sequence <220> <223> Linker <400> 152 Gly Gly Ser Gly Gly Ser 1 5 <210> 153 <211> 9 <212> PRT <213> artificial sequence <220> <223> Linker <400> 153 Gly Gly Ser Gly Gly Ser Gly Gly Ser 1 5 <210> 154 <211> 5 <212> PRT <213> artificial sequence <220> <223> Linker <400> 154 Gly Gly Gly Gly Ser 1 5 <210> 155 <211> 662 <212> DNA <213> artificial sequence <220> <223> human light chain lambda region <400> 155 tcacgaattc gcaggccgtc ctgaaccagc caagcagcgt ctccgggtct ctggggcagc 60 gggtctcaat cacctgtagc gggtcttcct ccaatgtcgg caacggctac gtgtcttggt 120 atcagctgat ccctggcagt gccccacgaa ccctgatcta cggcgacaca tccagagctt 180 ctggggtccc cgatcggttc tcagggagca gatccggaaa cacagctact ctgaccatca 240 gctccctgca ggctgaggac gaagcagatt atttctgcgc atctgccgag gactctagtt 300 caaatgccgt gtttggaagc ggcaccacac tgacagtcct aggtcagccc aaggctgccc 360 cctcggtcac tctgttcccg ccctcctctg aggagcttca agccaacaag gccacactgg 420 tgtgtctcat aagtgacttc tacccgggag ccgtgacagt ggcctggaag gcagatagca 480 gccccgtcaa ggcgggagtg gagaccacca caccctccaa acaaagcaac aacaagtacg 540 cggccagcag ctatctgagc ctgacgcctg agcagtggaa gtcccacaga agctacagct 600 gccaggtcac gcatgaaggg agcaccgtgg agaagacagt ggcccctaca gaatgttcat 660 aa 662 <210> 156 <211> 216 <212> PRT <213> artificial sequence <220> <223> human VL1-51 <400> 156 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Ala Glu Asp Ser Ser 85 90 95 Ser Asn Ala Val Phe Gly Ser Gly Thr Thr Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215 <210> 157 <211> 39 <212> DNA <213> artificial sequence <220> <223> BLV1H12 ascending stalk region <400> 157 tgtacttctg tgcaccagga aacaaaaaaa taccaaacc 39 <210> 158 <211> 12 <212> PRT <213> artificial sequence <220> <223> BLV1H12 ascending stalk region <400> 158 Thr Ser Val His Gln Glu Thr Lys Lys Tyr Gln Thr 1 5 10 <210> 159 <211> 13 <212> PRT <213> artificial sequence <220> <223> BLV1H12 ascending stalk region <400> 159 Cys Thr Ser Val His Gln Glu Thr Lys Lys Tyr Gln Thr 1 5 10 <210> 160 <211> 39 <212> DNA <213> artificial sequence <220> <223> Descending stalk region <400> 160 tcctatactt acaattacga atggcacgtc gatgtctgg 39 <210> 161 <211> 13 <212> PRT <213> artificial sequence <220> <223> Descending stalk region <400> 161 Ser Tyr Thr Tyr Asn Tyr Glu Trp His Val Asp Val Trp 1 5 10 <210> 162 <211> 117 <212> DNA <213> artificial sequence <220> <223> BLV1H12 knob sequence <400> 162 tgtccggagg attatactta taatccacgt tgccctcagc agtatggttg gagtgactgt 60 gattgtatgg gcgataggtt tgggggttac tgtcgacagg atggttgtag taattat 117 <210> 163 <211> 9 <212> DNA <213> artificial sequence <220> <223> Coding sequencing for N-terminal GGS linker <400> 163 ggtggatca 9 <210> 164 <211> 9 <212> DNA <213> artificial sequence <220> <223> Coding sequence for C-terminal GSG linker <400> 164 ggttcagga 9 <210> 165 <211> 133 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> IL2 <400> 165 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Ile Phe Cys Gln Ser Ile 115 120 125 There Are Thr Thr Thr 130 <210> 166 <211> 399 <212> DNA <213> artificial sequence <220> <223> IL2 coding sequence <400> 166 gcacctactt caagttctac aaagaaaaaca cagctacac tggagcatttt actgctgat 60 ttacagatga tttgaatg aattaataat tacagaatc ccaactcac caggatgctc 120 acatttaagt tttacatgcc CAagaaggcc acagaactga aacatctca gtgtctagaa 180 gagaactca aacctctgga ggaagtgcta atttagctc aaagcaaaaa ctttcactta 240 agacccagggg acttaatcag caatcaac gtaatagttc tggaactaa gggatctgaa 300 acaacattca tgtgtgaata tgctgatgag acagcaacca tgtagaatt tctgaacaga 360 tggattacct ttgtcaag catcatctca acactgact 399 <210> 167 <211> 513 <212> DNA <213> artificial sequence <220> <223> Chimeric ultralong bovine heavy chain sequence <400> 167 caggtgcagc tgcgggagtc gggccccagc ctgatgaagc cgtcacagac cctctccctc 60 acctgcacgg tctctggatc ttcattgaac gacaagtctg taggctgggt ccgccaggct 120 ccagggaagg cgctgcagtg gctcggtagt gtggacacta gtggaaacac agactataac 180 ccaggcctga aatcccggct cagcatcacc aaggacaact ccaagagccg aatctctctt 240 acagtgactg gcatgacaac tgaagactcg gccacatact actgtacttc tgtgcaccag 300 gaaacaaaaa aataccaaag ttgtccggag gattatactt ataatccacg ttgccctcag 360 cagtatggtt ggagtgactg tgattgtatg ggcgataggt ttgggggtta ctgtcgacag 420 gatggttgta gtaattatag ttatacttac aattacgaat ggcacgtcga tgtctggggc 480 caaggactcc tggtcaccgt ctcctcagct agc 513 <210> 168 <211> 651 <212> DNA <213> artificial sequence <220> <223> BLV1H12 Light Chain- <400> 168 caggccgtcc tgaaccagcc aagcagcgtc tccgggtctc tggggcagcg ggtctcaatc 60 acctgtagcg ggtcttcctc caatgtcggc aacggctacg tgtcttggta tcagctgatc 120 cctggcagtg ccccacgaac cctgatctac ggcgacacat ccagagcttc tggggtcccc 180 gatcggttct cagggagcag atccggaaac acagctactc tgaccatcag ctccctgcag 240 gctgaggacg aagcagatta tttctgcgca tctgccgagg actctagttc aaatgccgtg 300 tttggaagcg gcaccacact gacagtccta ggtcagccca aggctgcccc ctcggtcact 360 ctgttcccgc cctcctctga ggagcttcaa gccaacaagg ccacactggt gtgtctcata 420 agtgacttct acccgggagc cgtgacagtg gcctggaagg cagatagcag ccccgtcaag 480 gcgggagtgg agaccaccac accctccaaa caaagcaaca acaagtacgc ggccagcagc 540 tatctgagcc tgacgcctga gcagtggaag tcccacagaa gctacagctg ccaggtcacg 600 catgaaggga gcaccgtgga gaagacagtg gcccctacag aatgttcata a <210> 169 <211> 311 <212> DNA <213> artificial sequence <220> <223> Human heavy chain variable region sequence 4-39 <400> 169 60. cgctgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc acctgcactg tctctggtgg ctccatcagc actactgggg ctggatccgc 120 cagcccccag ggaaggggct ggagtggatt gggagtatct attatagtgg gagcacctac 240. ccctcaagag tcgagtcacc atatccgtag acacgtccaa gaaccagttc tccctgaagc tgagctctgt gaccgccgca gacacggctg tgtattactg tgcgagacac acagtgaggg g <210> 170 <211> 288 <212> DNA <213> artificial sequence <220> <223> Human heavy chain variable region sequence 4-59*03 <400> 170 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagt agttactact ggagctggat ccggcagccc 120 ccagggaagg gactggagtg gattgggtat atctattaca gtgggagcac caactacaac 180 ccctccctca agagtcgagt caccatatca gtagacacgt ccaagaacca attctccctg 240 aagctgagct ctgtgaccgc tgcggacacg gccgtgtatt actgtgcg 288 <210> 171 <211> 291 <212> DNA <213> artificial sequence <220> <223> Human heavy chain variable region sequence 4-34*09 <400> 171 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcgctg tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc 120 ccagggaagg gactggagtg gattggggaa atcaatcata gtggaagcac caactacaac 180 ccgtccctca agagtcgagt taccatatca gtagacacgt ctaagaacca gttctccctg 240 aagctgagct ctgtgactgc cgcggacacg gccgtgtatt actgtgcgag a 291 <210> 172 <211> 290 <212> DNA <213> artificial sequence <220> <223> Human heavy chain variable region sequence 4-34*02 <400> 172 caggtgcagc tacaacagtg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60 acctgcgctg tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc 120 ccagggaagg ggctggagtg gattggggaa atcaatcata gtggaagcac caactacaac 180 ccgtccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240 aagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag 290 <210> 173 <211> 98 <212> PRT <213> artificial sequence <220> <223> Human germline light chain variable region sequence VL1-47 <400> 173 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Ser Gly <210> 174 <211> 99 <212> PRT <213> artificial sequence <220> <223> Human germline light chain variable region sequence VL1-40*1 <400> 174 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser 85 90 95 Leu Ser Gly <210> 175 <211> 98 <212> PRT <213> artificial sequence <220> <223> Human germline light chain variable region sequence VL1-51*01 <400> 175 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu 85 90 95 Ser Ala <210> 176 <211> 99 <212> PRT <213> artificial sequence <220> <223> Human germline light chain variable region sequence VL2-18*02 <400> 176 Gln Ser Ala Leu Thr Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 20 25 30 Asn Arg Val Ser Trp Tyr Gln Gln Pro Pro Gly Thr Ala Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ser Thr Phe <210> 177 <211> 296 <212> DNA <213> artificial sequence <220> <223> Human germline light chain variable region sequence VL1-47 <400> 177 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgttctg gaagcagctc caacatcgga agtaattatg tatactggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat aggaataatc agcggccctc aggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgag tggtcc 296 <210> 178 <211> 299 <212> DNA <213> artificial sequence <220> <223> Human germline light chain variable region sequence VL1-40*1 <400> 178 cagtctgtgc tgacgcagcc gccctcagtg tctggggccc cagggcagag ggtcaccatc 60 tcctgcactg ggagcagctc caacatcggg gcaggttatg atgtacactg gtaccagcag 120 cttccaggaa cagcccccaa actcctcatc tatggtaaca gcaatcggcc ctcaggggtc 180 cctgaccgat tctctggctc caagtctggc acctcagcct ccctggccat cactgggctc 240 caggctgagg atgaggctga ttattactgc cagtcctatg acagcagcct gagtggttc 299 <210> 179 <211> 296 <212> DNA <213> artificial sequence <220> <223> Human germline light chain variable region sequence VL1-51*01 <400> 179 cagtctgtgt tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc 60 tcctgctctg gaagcagctc caacattggg aataattatg tatcctggta ccagcagctc 120 ccaggaacag cccccaaact cctcatttat gacaataata agcgaccctc agggattcct 180 gaccgattct ctggctccaa gtctggcacg tcagccaccc tgggcatcac cggactccag 240 actggggacg aggccgatta ttactgcgga acatgggata gcagcctgag tgctgg 296 <210> 180 <211> 297 <212> DNA <213> artificial sequence <220> <223> Human germline light chain variable region sequence VL2-18*02 <400> 180 cagtctgccc tgactcagcc tccctccgtg tccgggtctc ctggacagtc agtcaccatc 60 tcctgcactg gaaccagcag tgacgttggt agttataacc gtgtctcctg gtaccagcag 120 cccccaggca cagcccccaa actcatgatt tatgaggtca gtaatcggcc ctcaggggtc 180 cctgatcgct tctctgggtc caagtctggc aacacggcct ccctgaccat ctctgggctc 240 caggctgagg acgaggctga ttattactgc agctcatata caagcagcag cactttc 297

Claims

[Claim 1] A modified ultralong CDR3 comprising an interleukin-15 (IL-15) cytokine sequence or a biologically active portion thereof replacing at least a portion of the ultralong CDR3 region of the heavy chain of a bovine antibody or antigen-binding fragment thereof. A chimeric cytokine-modified antibody or antigen-binding fragment comprising:

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