Bifunctional molecules for treating immune diseases

Abstract

The present invention includes bifunctional molecules comprising an antibody that specifically binds to CD122 and the extracellular domain of CTLA-4. The bifunctional molecules specifically bind to CD122 and the CTLA-4 ligands CD80 and CD86 and inhibit their function in immune activation. The bifunctional molecules can inhibit the interaction of CD122 with its ligands IL-2 and IL-15, and can inhibit the interaction of CD80 and CD86 with their counterreceptor CD28. These bifunctional molecules can suppress signals 2 and 3 of the immune response as a single therapeutic agent for the treatment of immune disorders.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of US63 / 161,713, filed March 16, 2021, which is incorporated by reference in its entirety for all purposes.

[0002] This application was created on Mar. 1, 2022, and contains a sequence listing in file named 574245SEQLIST, which is 118 kilobytes, and is incorporated herein by reference. [Background technology]

[0003] Antigen-specific immune responses are complex biological processes controlled by multiple layers of positive and negative regulators. T cells are initially stimulated through the T cell receptor (TCR) by recognition of cognate peptide antigens presented by major histocompatibility complex (MHC, also called HLA for human proteins) molecules on antigen-presenting cells (APCs). The initial interaction between TCR and MHC (or HLA) for T cell activation is called signal 1. Optimal T cell activation requires a second signal (signal 2) provided by costimulatory molecules such as CD28 and ICOS, which belong to the CD28 superfamily. The immune system is further positively regulated by other costimulatory molecules such as CD40, OX40, GITR, CD27, HVEM, 4-1BB, which belong to the TNF receptor superfamily, and negatively regulated by checkpoint molecules such as PD-1, TIGIT, TIM-3, LAG-3, BTLA, VISTA, CD96, and CD112R. These costimulatory and checkpoint molecules, whose expression is cell type- and developmental stage-dependent, delicately regulate the body's immune response. Pardoll, Nat. Rev. Cancer, 12:252-264, 2012; Mahoney et al., Nat. Rev. Drug Discov. 14:561-584, 2015; Shin et al., Curr. Opin. Immunol. 33:23-35, 2015; Marquez-Rodas et al. Ann. Transl. Med. 3:267, 2015; Mercier et al., Front. Immunol. 6:418, 2015; Topalian et al., Cancer Cell 27: 450-461, 2015; Baumeister et al., Annu. Rev. Immunol. 34:539-573, 2016; Ward-Kavanagh et al., Immunity 44:1005-1019, 2016; Torphy et al., Int. J. Mol. Sci. 18:2642, 2017.

[0004] CTLA-4 (cytotoxic T-lymphocyte-associated protein 4; also known as CD152) is a negative regulator of immune responses. CTLA-4 binds to CD80 (also known as B7-1) and CD86 (also known as B7-2) expressed on the surface of APCs, disrupting the interaction of CD80 and CD86 with CD28 expressed on T cells, and blocking CD28-mediated T cell costimulation. Abatacept is a recombinant protein that contains the extracellular domain of human CTLA-4 fused to the Fc region of the human gamma-1 heavy chain (CTLA4-Ig). Abatacept has been approved for sale in the United States for the treatment of adult rheumatoid arthritis, juvenile polyarticular idiopathic arthritis, and adult psoriatic arthritis. Belatacept, a successor to abatacept, is also a CTLA4-Ig fusion protein with two amino acid substitutions in the CTLA-4 domain (one at position 29, an alanine to tyrosine residue (A29Y), and one at position 104, a leucine to glutamic acid residue (L104E)) to enhance binding to CD80 and CD86 (Larsen et al., Am. J. Transplant. 5:443-453, 2005; Oshima et al., Protein Eng. Des. Sel. 29:159-167, 2016). Belatacept has been approved for sale in the United States for the prevention of renal allograft rejection. For reviews of abatacept and belatacept, see Moreland et al., Nat. Rev. Drug Disc. 5:185-186, 2006; Esensten et al., Immunity 44:973-988, 2016; Perez et al., Transplantation 102:1440-1452, 2008; Gallo et al., PLoS ONE 15:e0240335, 2020.

[0005] Various secreted proteins, such as cytokines and chemokines, also participate in the control of immune responses by promoting the activation, differentiation, proliferation, maintenance, and suppression of specific subsets of immune cells. The action of cytokines on T cells is often referred to as signal 3, and is the third mechanism required for T cell activation, differentiation, and proliferation (Gutcher et al., J. Clin. Invest. 117:1119-1127, 2007; Curtsinger et al., Curr. Opin. Immunol. 22:333-340, 2010; Hurton et al., Proc. Natl. Acad. Sci. 113:E7788-E7797, 2016).

[0006] Interleukin-15 (IL-15) is a proinflammatory cytokine required for the activation, proliferation, and maintenance of natural killer (NK) cells and CD8+ memory T cells. IL-15 is primarily secreted by APCs and is captured with high affinity by the IL-15 receptor α subunit (also called CD215) expressed on the surface of the same or nearby cells. The IL-15 / CD215 complex on the cell surface is then presented to the IL-15 receptor, which contains CD122 (β-chain subunit of IL-2 and IL-15 receptors) and CD132 (common γ-chain) molecules on another cell surface, triggering intracellular signaling to induce cell functions (transpresentation of IL-15). See Waldmann et al., Annu. Rev. Immunol. 17:19-49, 1999; Stonier et al., Immunol. Lett. 127:85-92, 2010; Anthony et al., Research and Reports in Biology, 6:25-37, 2015; Fehniger, J. Immunol. 202:3125-3126, 2019; Yang et al., Cancers 12:3586, 2020.

[0007] Recent studies have demonstrated that various autoimmune diseases are driven by self-reactive CD8+ memory T cells, some of which reside in tissues (tissue-resident memory T cells; Topham et al., Front. Immunol. 9: Article 515, 2018). Anti-CD122 monoclonal antibodies, which block the interaction between IL-15 and CD122 (IL-15 receptor), have been shown to be effective in treating vitiligo (Richmond et al., Sci. Trans. Med. 10:eaam7710, 2018), alopecia areata (Xing et al., Nat. Med. 20:1043-1049, 2014), type 1 diabetes (Chen et al., Proc. Natl. Acad. Sci. 110:13534-13539, 2013; Yuan et al. JCI Insight 3:e96600, 2018), celiac disease (Yokoyama et al., Proc. Natl. Acad. Sci. 106:15849-15854, 2009), and nonalcoholic steatohepatitis (NASH) (Dudek et al., It has been reported to have therapeutic effects in mouse models of cutaneous allograft rejection (Nature 592: 444-449, 2021) and skin allograft rejection (Mathews et al., J. Clin. Invest. 128:4557-4572, 2018).The role of CD8+ memory T cells has been demonstrated in multiple sclerosis (Fransen et al., Brain 143:1714-1730, 2020), polymyositis (Kamiya et al., Rheumatol. 59:224-232, 2020), allergic dermatitis (Gadsboll et al., J. Invest. Dermatol. 140:806-815, 2020), primary biliary cirrhosis (Yang et al., J. Immunol. 186:1259-1267, 2011), Behçet's disease (Yu et al., Clin. Exp. Immunol. 137:437-443, 2004), and ulcerative colitis (Nemeth et al., Cureus 9:e1177, 2017; Boland et al., J. Immunol. 137:137-137, 2017). al., Sci. Immunol. 5:eabb4432, 2020). Summary of the Invention

[0008] The present invention provides bifunctional molecules comprising an antibody that binds to CD122 and inhibits the interaction of CD122 with IL-2 and IL-15, and also binds at its N- or C-terminus to the extracellular domain of CTLA-4.

[0009] Optionally, the antibody comprises a heavy chain comprising a mature heavy chain variable region bound at its C-terminus to a heavy chain constant region that binds to the extracellular domain of CTLA-4, and a light chain comprising a mature light chain variable region bound to a light chain constant region. Optionally, the antibody binds to the extracellular domain of CTLA-4 via the C-terminus of the heavy chain constant region or the C-terminus of the light chain constant region. Optionally, the antibody is bound to the extracellular domain of CTLA-4 via the N-terminus of the mature heavy chain variable region or the N-terminus of the mature light chain variable region. Optionally, the heavy or light chain is bound to the extracellular domain of CTLA-4 via a polypeptide linker. Optionally, the polypeptide linker comprises the amino acid sequence of SEQ ID NO: 11, 36 or 50. Optionally, the bifunctional molecule is in the form of a heterodimer comprising two copies of a heavy chain and two copies of a light chain. Optionally, the antibody comprises three heavy chain CDRs having SEQ ID NO: 17-19, respectively, and three light chain CDRs having SEQ ID NO: 20-22, respectively. Optionally, the antibody comprises a mature heavy chain variable region having SEQ ID NO:37 and a light chain variable region having SEQ ID NO:40. Optionally, the heavy chain comprises SEQ ID NO:8, with the C-terminal lysine optional, and the light chain comprises SEQ ID NO:9. Optionally, the mature heavy chain is attached to the extracellular domain of CTLA-4 as a chain comprising SEQ ID NO:13 or 16, and the mature light chain comprises SEQ ID NO:9. Optionally, the mature light chain is attached to the extracellular domain of CTLA-4 as a chain comprising SEQ ID NO:34, and the mature heavy chain comprises SEQ ID NO:8 or 33, with the C-terminal lysine optional. Optionally, the mature heavy chain is attached to the extracellular domain of CTLA-4 as a chain comprising SEQ ID NO:39, with the C-terminal lysine optional, and the mature light chain comprises SEQ ID NO:9. Optionally, the mature light chain is attached to the extracellular domain of CTLA-4 as a chain comprising SEQ ID NO:42, and the mature heavy chain comprises SEQ ID NO:8 or 33, with the C-terminal lysine optional.

[0010] Optionally, the antibody comprises three heavy chain CDRs having SEQ ID NOs:23-25, respectively, and three light chain CDRs having SEQ ID NOs:26-28, respectively. Optionally, the antibody comprises a mature heavy chain variable region having SEQ ID NO:29, and a mature light chain variable region having SEQ ID NO:30. Optionally, the mature heavy chain comprises SEQ ID NO:53, with the C-terminal lysine optional, and the mature light chain comprises SEQ ID NO:54. Optionally, the mature heavy chain is attached to the extracellular domain of CTLA-4 as a chain comprising SEQ ID NO:57, and the mature light chain comprises SEQ ID NO:54. Optionally, the mature heavy chain is attached to the extracellular domain of CTLA-4 as a chain comprising SEQ ID NO:56, with the C-terminal lysine optional, and the mature light chain comprises SEQ ID NO:54.

[0011] Optionally, the antibody comprises three heavy chain CDRs having SEQ ID NOs: 59-61, respectively, and three light chain CDRs having SEQ ID NOs: 63-65, respectively. Optionally, the mature heavy chain variable region comprises SEQ ID NO: 58, and the mature light chain variable region comprises SEQ ID NO: 62. Optionally, the mature heavy chain comprises SEQ ID NO: 66, with an optional C-terminal lysine, and the mature light chain comprises SEQ ID NO: 67. Optionally, the mature heavy chain is attached to the extracellular domain of CTLA-4 as a chain comprising SEQ ID NO: 71, and the mature light chain comprises SEQ ID NO: 67. Optionally, the mature heavy chain is attached to the extracellular domain of CTLA-4 as a chain comprising SEQ ID NO: 70, with an optional C-terminal lysine, and the mature light chain comprises SEQ ID NO: 67.

[0012] Optionally, the antibody has a human IgG1K isotype. Optionally, the heavy chain constant region has one or more mutations that reduce effector function. Optionally, the mutations are L234A and L235A. Optionally, the extracellular domain is the extracellular domain of human CTLA-4. Optionally, the extracellular domain of CTLA-4 comprises SEQ ID NO: 10. Optionally, the extracellular domain of CTLA-4 comprises SEQ ID NO: 14.

[0013] The present invention further provides a pharmaceutical composition comprising any of the bifunctional molecules described above and a pharma- ceutically acceptable carrier.

[0014] The present invention further provides a method for treating a subject with an immune disorder, comprising administering to the subject any of the above-mentioned bifunctional molecules in an effective regimen.Optionally, the immune disorder is any of vitiligo, alopecia areata, type 1 diabetes, celiac disease, non-alcoholic steatohepatitis, skin allograft rejection, multiple sclerosis, polymyositis, allergic dermatitis, primary biliary cirrhosis, Behcet's disease, and ulcerative colitis.Optionally, the immune disorder is graft rejection or graft-versus-host disease.Optionally, the immune disorder is an autoimmune disease.

[0015] The invention further provides an antibody against human CD122, said antibody comprising three heavy chain CDRs having SEQ ID NOs: 59-61, respectively, and three light chain CDRs having SEQ ID NOs: 63-65, respectively. Optionally, said antibody comprises a mature heavy chain variable region having SEQ ID NO: 58, and a mature light chain variable region having SEQ ID NO: 62. Optionally, said mature heavy chain comprises SEQ ID NO: 66, with the C-terminal lysine optional, and said mature light chain comprises SEQ ID NO: 67. [Brief description of the drawings]

[0016] [Figure 1] 1A and 1B show the schematic structures of the expression vectors pHuABC2p (A) and pHC624 and pHC625 (B).

[0017] [Diagram 2] Schematic structures of the bifunctional molecules HuABC2-CTLA4 and HuABC2-CTLA4YE are shown.

[0018] [Diagram 3] Binding of HuABC2, HuABC2-CTLA4 and HuABC2-CTLA4YE to CD122 is shown. Mean absorbance levels of duplicate analyses are shown with standard deviation bars.

[0019] [Figure 4]Binding of HuABC2, HuABC2-CTLA4 and HuABC2-CTLA4YE to CD80 is shown. Mean absorbance levels of duplicate analyses are shown with standard deviation bars.

[0020] [Diagram 5] Binding of HuABC2, HuABC2-CTLA4 and HuABC2-CTLA4YE to CD86 is shown. Mean absorbance levels of duplicate analyses are shown with standard deviation bars.

[0021] [Figure 6] 6A, B, and C show the schematic structures of expression vectors pHC623 (A), pHC640 (B), and pHC641 (C).

[0022] [Figure 7] 7A, B, C show the schematic structures of the bifunctional molecules HuABC2-Ck-CTLA4YE (A), HuABC2-CTLA4YE-VH (B) and HuABC2-CTLA4YE-Vk (C).

[0023] [Figure 8] Figures 8A, B, and C show the schematic structures of expression vectors pFCm553 (A), pFCm554 (B), and pFCm148 (C).

[0024] [Figure 9] 9A, B, C show flow cytometry analysis of the binding of bifunctional molecules to CHO-K1 / CD80 cells (A), CHO-K1 / CD86 cells (B), and Ramos cells (C).

[0025] [Figure 10] 1 shows flow cytometry analysis of binding of bifunctional molecules to NS0 / CD122 cells.

[0026] [Figure 11] 1 shows the proliferation of human T cells in a mixed lymphocyte reaction.

[0027] [Figure 12] IFN-γ expression in mixed lymphocyte reaction.

[0028] [Figure 13] Figures 13A, B show flow cytometric analysis of the binding of bifunctional molecules to CHO-K1 / CD80 cells (A) and CHO-K1 / CD86 cells (B). Definitions

[0029] The bifunctional molecule or other biological entity is usually provided in an isolated form. This means that the biological entity is usually at least 50% w / w pure interfering proteins and other contaminants resulting from its production or purification, but does not exclude the possibility that the entity may be combined with an excess of pharma- ceutically acceptable carriers or other vehicles intended to facilitate its use. In some cases, the entity may be at least 60, 70, 80, 90, 95 or 99% w / w pure interfering proteins and contaminants resulting from its production or purification. In many cases, the isolated entity is the major macromolecular species remaining after purification.

[0030] The specific binding of the bifunctional molecule to a target protein, such as CD122, CD80, or CD86, is at least 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 M -1 Specific binding refers to an affinity for a molecule that is detectably greater than or equal to the target of interest. Specific binding is detectably greater than or equal to the target of interest, and can be distinguished from nonspecific binding that occurs to at least one unrelated target. Specific binding can be the result of bond formation between specific functional groups or a particular spatial fit (e.g., lock and key type), whereas nonspecific binding is usually the result of van der Waals forces. However, specific binding does not necessarily mean that a molecule binds to only one target. Bifunctional molecules bind to multiple targets.

[0031] The basic antibody structural unit is a tetramer of subunits. Each tetramer contains two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain contains a variable region of about 100-110 or more amino acids that is primarily involved in antigen recognition. This variable region is initially expressed in association with a cleavable signal peptide. The variable region without the signal peptide is sometimes called the mature variable region. Thus, for example, the light chain mature variable region means the light chain variable region without the light chain signal peptide. The carboxy-terminal portion of each chain defines a constant region that is primarily responsible for effector functions.

[0032] Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively. In the light and heavy chains, the variable and constant regions are joined by a "J" region containing about 12 or more amino acids, with the heavy chain containing a "D" region containing about 10 or more amino acids (typically see Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, NY, 1989), Ch. 7) (incorporated by reference in its entirety for all purposes).

[0033] The mature variable regions of each light / heavy chain pair form the antibody binding site. Thus, an intact antibody has two binding sites. In natural antibodies, the binding sites are the same. All variable regions exhibit the same typical structure, with three hypervariable regions, also called complementarity determining regions (CDRs), bound to relatively conserved framework regions (FRs). The CDRs from the two chains of each pair are aligned by the framework regions, allowing binding to a specific epitope. From the N-terminus to the C-terminus, both light and heavy chain variable regions contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain follows the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991), or Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989). Kabat also provides a widely used numbering scheme (Kabat numbering) that assigns the same numbers to corresponding residues between different heavy chains or different light chains.

[0034] The term "antibody" includes intact antibodies and binding fragments thereof. Typically, fragments compete with the intact antibody from which they are derived for specific binding to a target, including separate heavy chains, light chains Fab, Fab', F(ab')2, F(ab)c, Dabs, nanobodies, and Fvs. Fragments can be produced by recombinant DNA techniques or by enzymatic or chemical separation of intact immunoglobulins.

[0035] The term "epitope" refers to a site on an antigen to which an antibody or bifunctional molecule binds. Epitopes can be formed from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also called linear epitopes) are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also called conformational epitopes) are typically lost on treatment with denaturing solvents. Epitopes typically contain at least 3, more commonly at least 5 or 8-10 amino acids, and have a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography and 2D nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0036] Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay showing the ability of one antibody to compete with the binding of another antibody to a target antigen. An antibody's epitope can also be defined by X-ray crystallography of the antibody bound to the antigen to identify contact residues. Two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0037] Competition between antibodies is determined by an assay in which the antibody under test inhibits the specific binding of a reference antibody to a common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). A test antibody competes with a reference antibody if an excess of the test antibody (e.g., at least 2-fold, 5-fold, 10-fold, 20-fold or 100-fold) inhibits the binding of the reference antibody by at least 50%, preferably 75%, 90% or 99%, as measured in a competitive binding assay. Antibodies identified by competitive assays (competitor antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to adjacent epitopes that are sufficiently close to the epitope bound by the reference antibody for steric hindrance to occur.

[0038] The term "subject" includes human and other mammalian subjects receiving prophylactic or therapeutic treatment, and, unless otherwise clear from the context, also includes animal models used to test such treatments.

[0039] For the purpose of classifying amino acid substitutions as conservative or non-conservative, amino acids are grouped as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues that affect chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions are the replacement of amino acids of the same class with each other. Non-conservative substitutions are the exchange of a member of one of these classes for a member of another class.

[0040] The percentage of sequence identity is determined using maximally aligned antibody sequences according to the Kabat numbering convention. After alignment, when a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is compared with the same region of a reference antibody, the percentage of sequence identity between the subject antibody region and the reference antibody region is the number of positions occupied by the same amino acid in both the subject antibody region and the reference antibody region, divided by the total number of aligned positions of these two regions, without considering gaps, and multiplied by 100 to convert to a percentage. For non-antibody sequences, sequence identity can be determined by aligning the sequences and obtaining the best alignment (i.e., the one that results in the highest percentage of sequence similarity over the comparison window) using algorithms such as BESTFIT, FASTA, TFASTA, etc., from Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis., using default gap parameters, or by inspection. Percent sequence identity is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where identical residues occur in both sequences to determine the number of matching positions, dividing the number of matching positions by the total number of matching and mismatching positions not considering gaps in the comparison window (i.e., the window size), and multiplying the result by 100. Unless otherwise specified, the comparison window between two sequences is defined by the longer of: (a) the entire length of the shorter of the two sequences being compared; or (b) at least 25 contiguous nucleotides.

[0041] When a value is characterized as being within a specified factor of a reference value, the specification should be understood to alternately disclose values ​​higher or lower than the reference value within that factor.

[0042] A composition or method "comprising" one or more recited elements may include other elements not specifically recited. For example, a composition that includes a bifunctional molecule may include the bifunctional molecule alone or in combination with other components. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] I. General

[0044] The present invention provides bifunctional molecules comprising antibodies that specifically bind to the extracellular domains of CD122 and CTLA-4. The bifunctional molecules specifically bind to the CD122 and CTLA-4 ligands, CD80 and CD86, and inhibit their function in immune activation. The bifunctional molecules can inhibit the interaction of CD122 with its ligands IL-2 and IL-15, and inhibit the interaction of CD80 and CD86 with their counterreceptor CD28. These bifunctional molecules can suppress signals 2 and 3 of the immune response as a single therapeutic agent for treating immune disorders.

[0045] Exemplary anti-CD122 antibodies for inclusion in the bifunctional molecule are HuABC2 and HuABC101, which are humanized anti-CD122 IgG1 / κ monoclonal antibodies that bind to CD122 and block the interaction of CD122 with IL-2 and IL-15 (US 9,028,830). Another exemplary anti-CD122 antibody for inclusion in the bifunctional molecule is HuABC77, which is a humanized anti-CD122 IgG1 / κ monoclonal antibody that binds to CD122 and blocks the interaction of CD122 with IL-2 and IL-15. The CDRs of HuABC77 are SEQ ID NO: 59-61 for the heavy chain and SEQ ID NO: 63-65 for the light chain. HuABC2 can be considered a signal 3 blocker in the immune system and functions as a suppressor of NK cells and CD8+ memory T cells. HuABC2 had therapeutic effect in preventing renal allograft rejection in non-human primates (Landolfi et al., Abstract No. 185, American Transplant Congress, 2013; Mathews et al. supra). II. CD122 and Antibodies Thereagainst

[0046] Unless otherwise indicated, CD122 refers to human CD122. An exemplary human CD122 sequence has been assigned Swiss Prot accession number P14784. The mature sequence of human CD122 without the signal peptide is shown in SEQ ID NO: 31. Its known natural allelic variants include 10L→V:dbSNP rs57770674, 83S→F:dbSNP rs2228143, and 391D→E:dbSNP rs228942. The complete human CD122 sequence has 551 amino acids, of which amino acids 1-26 are the signal peptide. Approximately residues 27-240 constitute the extracellular domain. Approximately residues 241-265 constitute the transmembrane domain, and approximately residues 266-551 constitute the cytoplasmic domain. The antibodies and bifunctional molecules used in the present invention bind to an epitope within the extracellular domain of CD122.

[0047] CD122, in combination with CD132, CD25, and / or IL-15Rα (also called CD215), constitutes a receptor. Briefly, the combination of CD122 and CD132 constitutes a receptor with intermediate affinity for IL-2 and IL-15. The combination of CD122, CD132, and CD25 constitutes a receptor with high affinity for IL-2. The combination of CD122, CD132, and IL-15Rα constitutes a receptor with high affinity for IL-15.

[0048] Unless otherwise specified, CD132, CD25, and / or IL-15Rα refer to the human forms of these proteins. The human sequence of an exemplary human CD132 is designated Swiss Prot P31785. It is a 369 amino acid protein of which approximately residues 1-22 are a signal peptide, 23-262 are an extracellular domain, 263-283 are a transmembrane domain, and 284-369 are a cytoplasmic domain.

[0049] An exemplary human sequence of CD25 is provided as Swiss Prot P01589. It is an amino acid sequence of 272 residues, of which approximately residues 1-21 are a signal peptide, residues 22-240 are an extracellular domain, residues 241-259 are a transmembrane region, and residues 260-272 are a cytoplasmic domain.

[0050] The human sequence of an exemplary IL-15Rα is designated Swiss Prot Q13261. It is a 267 residue protein, of which approximately 1-30 is the signal peptide, 31-205 is the extracellular domain, 206-228 is the transmembrane region, and 229-267 is the cytoplasmic domain.

[0051] Unless otherwise clear from the context, a reference to one of the above receptors refers to at least the extracellular domain of the protein, and usually refers to the entire protein except for a cleavable signal peptide.

[0052] The above receptors bind IL-2 and / or IL-15, the latter of which may be present in cis or trans. Unless otherwise clear from the context, these molecules refer to the human forms of these proteins. An exemplary human IL-2 sequence is designated Swiss Prot P60568. It is a protein sequence of 153 amino acids, of which amino acids 1-20 are the signal peptide. An exemplary human IL-15 sequence is designated Swiss Prot P40933. It is a protein of 162 amino acids, of which amino acids 1-29 are the signal peptide. Unless otherwise clear from the context, reference to IL-2 or IL-15 refers to the mature protein with the signal sequence removed.

[0053] The present invention uses monoclonal antibodies that bind to an epitope within the CD122 protein. The antibodies designated ABC2 and ABC101 are two such exemplary murine antibodies. Both ABC2 and ABC101 specifically bind human CD122. ABC2 and ABC101 are further characterized by specific binding to cynomolgus monkey CD122 and no significant binding to mouse and dog CD122. Binding to CD122 can be demonstrated by binding to CD122 alone and / or any of the CD122-incorporated receptors described above. Such monoclonal antibodies are characterized by their ability, as single agents, to substantially inhibit the CD122-incorporated receptor from binding to both IL-2 and IL-15 and signaling cell proliferation in response thereto.

[0054] Preferred antibodies and bifunctional molecules incorporating such antibodies are capable of inhibiting the binding of both low and high affinity receptors incorporating CD122 to IL-2 and IL-15, and inhibiting IL-15 binding both in cis and in trans.

[0055] Inhibition can be demonstrated by a binding assay in which a CD122-incorporated receptor is expressed on cells and binds to IL-2 or IL-15 in the presence of the antibody or bifunctional molecule incorporating the antibody being tested. Inhibition can also be demonstrated by expressing a CD122-incorporated receptor on appropriate cells in the presence of IL-2 or IL-15 and evaluating the effect of the monoclonal antibody or bifunctional molecule incorporating the antibody on IL-2- or IL-15-mediated cell proliferation. Inhibition of IL-15 can be tested in cis or trans in such formats. Exemplary assay formats for showing inhibition are described in U.S. Pat. No. 9,028,830. Optionally, inhibition of the test antibody or bifunctional molecule can be demonstrated in comparison to an irrelevant control antibody that does not bind to CD122 or its receptor co-components, IL-2 and IL-15, or a vehicle lacking the antibody.

[0056] Substantial inhibition means at least 25, 30, 40, 50, or 75% (e.g., 25-75% or 30-70%) inhibition of binding, cell proliferation and / or other functional activity mediated by IL-2 or IL-15. Inhibition is typically demonstrated when the antibody or bifunctional molecule is in 100-fold molar excess or less (e.g., 2-50, 2-10, or 2-5-fold molar excess) over the IL-2 or IL-15 ligand. In functional assays, IL-2 or IL-15 is typically present at the minimal level required to stimulate full functional activity in the absence of the antibody or bifunctional molecule being tested. The antibody or bifunctional molecule is typically present at a concentration between 50 nM and 5 μM. Preferred antibodies or bifunctional molecules show at least 40% inhibition of interaction with low or high affinity receptors (or both) for IL-2 and at least 40% inhibition for IL-15 present in cis and trans. The degree of inhibition can also be quantified by the concentration of antibody or bifunctional molecule required for 50% inhibition of proliferation mediated by trans-presentation of IL-15 to TF-1β cells (IC50), as described in Examples 2 and 8 of US Pat. No. 9,028,830. The concentration of antibody or bifunctional molecule is less than 2 μg / ml, preferably less than 1 μg / ml, such as about 0.1-1 or 0.5-1 μg / ml. Furthermore, the concentration of antibody or bifunctional molecule required for 50% inhibition of IL-2-mediated TF-1αβ proliferation on cells with high affinity IL-2 receptors, as described in Example 8 of US Pat. No. 9,028,830, is preferably less than 100 or 50 μg / ml, such as 10-50 μg / ml.

[0057] These results show that the IC50 of the antibodies or bifunctional molecules of the invention depends on the nature of the interaction to be inhibited. In cells with high affinity IL-2 receptors, it is much more difficult for an anti-CD122 antibody or bifunctional molecule to inhibit IL-2 (i.e. higher concentrations of the same antibody or bifunctional molecule are required) than it is to inhibit IL-15 transpresentation. In cells with only intermediate affinity IL-2 receptors, it is much more difficult to inhibit IL-15 transpresentation (i.e. higher concentrations of the same antibody or bifunctional molecule are required) than it is to inhibit soluble IL-2 or IL-15.

[0058] Some of the antibodies bind to the same or overlapping epitopes as an antibody called ABC2 or ABC101. ABC2 and its humanized versions are monoclonal antibodies that bind to CD122 and block the interaction of CD122 with IL-2 and IL-15, and function as immunosuppressants of NK cells and CD8+ memory T cells. Therefore, ABC2 and its humanized versions can be considered as signal 3 blockers in the immune system.

[0059] The sequences of the heavy and light chain CDRs of ABC2 are shown in SEQ ID NOs: 17-19, and the light chain CDRs as defined by Kabat are shown in SEQ ID NOs: 20-22. The heavy and light chain CDRs of ABC101 as defined by Kabat are shown in SEQ ID NOs: 23-25 ​​and 26-28, respectively. The complete sequences of the murine and humanized versions are provided in U.S. Pat. No. 9,028,830. Other antibodies with such binding specificities can be produced by immunizing mice, rats, hamsters, rabbits, or other animal species with CD122 or a portion thereof that contains the desired epitope, and screening the resulting antibodies for binding to CD122 (optionally competing with ABC2 or ABC101). Antibodies identified by such assays can then be screened for the ability to inhibit both IL-2 and IL-15 interactions as described in the Examples or by other methods. Antibodies can also be screened against mutant forms of the CD122 antigen to identify antibodies that exhibit the same or similar binding profile as ABC2 or ABC101 against a collection of mutational changes. The mutations can be systematic substitutions with alanine (or serine, if alanine is already present) one residue at a time, or at more widely spaced intervals, throughout the entire extracellular domain of the CD122 antibody, or through the section where the epitope is known to reside.

[0060] Mutations at residues 42, 43, and 65 of CD122 inhibit specific binding of ABC2 to CD122 (e.g., <10% binding of a positive control anti-FLAG antibody as described in the Examples of U.S. Pat. No. 9,028,830). Similarly, mutations at residues 65, 70, and 133 of SEQ ID NO:31 inhibit specific binding of ABC101 to CD122. Because relatively few residues affect binding and the spacing of the residues is greater than that of a typical linear epitope (e.g., 3-20 contiguous amino acids), these results indicate that ABC101 and possibly ABC2 bind to a conformational epitope. Also, one or more of the residues that affect binding may affect binding allosterically without making direct contact with the antibody. The observation that mutagenesis of residue 65 with threonine rather than alanine inhibits binding suggests that this residue may affect binding allosterically. Other antibodies that bind to epitopes that include one or more of residues 42, 43, 65, 70, and 133 of CD122, particularly epitopes that include one or more of residues 42, 65, and 133, are likely to share useful inhibitory properties with ABC2 and ABC101. Thus, antibodies whose specific binding is inhibited by mutagenesis of one or more of residues 42, 43, and 65 of CD122, particularly residues 42 and 65, are likely to share similar properties as ABC2. Some such antibodies bind to epitopes that include or consist of residues 42, 43, and / or 65 of CD122. Some such antibodies bind to epitopes that include or consist of residues 42 and 43, where residue 65 allosterically affects antibody binding. The epitope may be linear, such as an epitope (e.g., 2-5, 3-5, 3-10, 3-15, 3-20, 5-10, 5-15 or 5-20 consecutive amino acids) that includes or consists of one, two or all three of the specific amino acids (42, 43 and 65), or it may be conformational, that includes or consists of one, two or all three of the specific amino acids. Antibodies whose specific binding is inhibited by mutations at one, two or all three of residues 65, 70 and 133 of CD122 may exhibit inhibitory properties similar to ABC101.Some such antibodies bind to an epitope that includes or consists of residues 65 and / or 133 of CD122. Such epitopes may be linear, such as epitopes that include or consist of one, two or all three of the specific amino acids (65, 70 and 133) (e.g., 2-5, 3-5, 3-10, 3-15, 3-20, 5-10, 5-15 or 5-20 consecutive amino acids), or may be conformational, including or consisting of one, two or all three of the specific amino acids. Some antibodies of the invention specifically bind to CD122 without being substantially inhibited by substitution of alanine at residues 39 or 41 of SEQ ID NO:31 (e.g., binds at least 50% of a positive control antibody, as described in the Examples of U.S. Pat. No. 9,028,830). Any of the above antibody types include antibodies that specifically bind to epitopes that do not include residues 39 or 41 of CD122, in other words, the binding of these antibodies to CD122 is not detectably inhibited by substitution of these residues (binding greater than 50% of that of a positive control anti-FLAG antibody, as described in the Examples).

[0061] Antibodies that bind to epitopes that include one or more specific residues can be generated by immunization with a fragment of CD122 that includes one or more of these residues. The fragment can have, for example, up to 100, 50, 25, 10, or 5 consecutive amino acids from CD122. Such fragments usually have at least 5, 6, 7, 8, or 9 consecutive residues of SEQ ID NO: 31. The fragment can be bound to a carrier that helps induce an antibody response against the fragment and / or can be combined with an adjuvant that helps induce such a response. Antibodies that bind to desired residues can also be obtained by immunization with full-length CD122 (minus the signal sequence) or full-length extracellular domain (minus the signal sequence) of fibronectin III domains 1 and / or 2. Such antibodies can then be screened for differential binding to hybrids of human and mouse CD122 compared to mutants of specific residues, or for differential binding to wild-type CD122. Screening against human and mouse CD122 hybrids maps antibodies that bind to specific domains within CD122, such as fibronectin III domains 1 and / or 2. Screening against mutants allows for more precise definition of binding specificity and identification of antibodies whose binding is inhibited by mutagenesis of residues 43, 43, 65, 70 and / or 133 and that may share the inhibitor properties of the exemplified antibodies.

[0062] Antibodies with the binding specificity of a selected murine antibody (e.g., ABC2 or ABC101) can also be produced using a variant of the phage display method. See Winter, WO 92 / 20791. This method is particularly suitable for producing human antibodies. In this method, either the heavy or light chain variable region of the selected murine antibody is used as the starting material. For example, if a light chain variable region is selected as the starting material, a phage library is constructed in which members display the same light chain variable region (i.e., the murine starting material) and different heavy chain variable regions. The heavy chain variable region can be obtained, for example, from a library of rearranged human heavy chain variable regions. A phage that shows strong specific binding to CD122 (e.g., at least 108 and preferably at least 109 M-1) is selected. The heavy chain variable region from this phage then serves as the starting material for constructing a further phage library, in which each phage displays the same heavy chain variable region (i.e., the region identified from the first display library) and a different light chain variable region. The light chain variable regions can be obtained, for example, from a library of rearranged human variable light chain regions. Again, phage that show strong specific binding to CD122 are selected. The resulting antibodies typically have the same or similar epitopic specificity as the murine starting material.

[0063] Other antibodies can be obtained by mutagenesis of cDNAs encoding the heavy and light chains of exemplary antibodies such as ABC2 or ABC101 and their humanized forms. Also included in the present invention are monoclonal antibodies that are at least 90%, 95% or 99% identical in amino acid sequence to ABC2 or ABC101 and their humanized forms in the mature heavy and / or light chain variable regions and retain their functional properties, and / or that differ from the respective antibodies by a small number of functionally insignificant amino acid substitutions (e.g., conservative substitutions), deletions or insertions. Also included are monoclonal antibodies having at least one, and preferably all six, CDRs according to Kabat's definition that are 90%, 95%, 99% or 100% identical to the corresponding CDRs of ABC2 or ABC101 as defined by Kabat.

[0064] Other non-human monoclonal antibodies, e.g., antibodies against CD122 in mice, guinea pigs, primates, rabbits or rats, can be produced, for example, by immunizing animals with CD122 or a fragment thereof or cells bearing CD122, optionally co-expressing a co-receptor protein as described above. See Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988), incorporated by reference for all purposes. Such immunogens can be obtained from natural sources, by peptide synthesis or recombinant expression. Optionally, they can be administered fused or otherwise conjugated to a carrier protein. Optionally, the immunogens can be administered with an adjuvant. Several types of adjuvants can be used, as described below. Freund's complete adjuvant followed by incomplete adjuvant is preferred for immunizing laboratory animals. Rabbits or guinea pigs are typically used to generate polyclonal antibodies. Mice are typically used to generate monoclonal antibodies. Antibodies are screened for specific binding to CD122. Antibodies are further screened for binding to a particular region of CD122. Such screening can be accomplished by measuring binding of the antibody to a collection of deletion mutants of CD122 and determining which deletion mutants bind to the antibody. Binding can be assessed, for example, by Western blot, FACS or ELISA.

[0065] A humanized antibody is a genetically engineered antibody in which CDRs from a non-human "donor" antibody are grafted onto human "acceptor" antibody sequences (see, e.g., Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539; Carter, US 6,407,213; Adair, US 5,859,205; Foote, US 6,881,557). The acceptor antibody sequences may be, for example, mature human antibody sequences, a composite of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. Thus, a humanized antibody is an antibody having some or all of the CDRs derived completely or substantially from a donor antibody, and constant region (if present) and variable region framework sequences derived completely or substantially from human antibody sequences. Similarly, a humanized heavy chain has at least one, two, and usually all three CDRs entirely or substantially derived from a donor antibody heavy chain, and a heavy chain constant region (if present) and heavy chain variable region framework sequences that are substantially derived from human heavy chain variable region framework and constant region sequences. Similarly, a humanized light chain has at least one, two, and usually all three CDRs entirely or substantially derived from a donor antibody light chain, and a light chain constant region (if present) and light chain variable region framework sequences that are substantially derived from human light chain variable region framework and constant region sequences. Except for nanobodies and dAbs, humanized antibodies include humanized heavy chains and humanized light chains. CDRs in a humanized antibody are substantially derived from corresponding CDRs in a non-human antibody when at least 85%, 90%, 95% or 100% of corresponding residues (as defined by Kabat) between the corresponding CDRs are identical. An antibody chain variable region framework sequence or an antibody chain constant region is substantially derived from a human variable region framework sequence or a human constant region, respectively, when at least 85%, 90%, 95% or 100% of the corresponding residues according to the Kabat definition are identical.

[0066] A humanized antibody often incorporates all six CDRs (preferably according to the Kabat definition) from a non-human (e.g., murine) antibody, although a humanized antibody can also be generated with fewer than all CDRs (e.g., at least three, four or five CDRs from the non-human (e.g., murine) antibody) (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al, Journal of Immunology, 164:1432-1441, 2000).

[0067] In some antibodies, only a portion of the CDRs, i.e., a subset of the CDR residues required for binding, referred to as SDRs, are required to retain binding in the humanized antibody. CDR residues that do not contact the antigen and are not present in the SDRs can be identified by molecular modeling and / or empirical analysis from regions of the Kabat CDRs outside the Chothia hypervariable loops (Chothia, J. Mol. Biol. 196:901, 1987) based on previous studies (e.g., residues H60-H65 in CDR H2 are often not required), or as described in Gonzales et al., Mol. Immunol. 41: 863, 2004. In such humanized antibodies, in positions where one or more donor CDR residues are absent or where the entire donor CDR has been removed, the amino acid occupying that position may be the amino acid occupying the corresponding position (according to Kabat numbering) in the acceptor antibody sequence. The number of such substitutions of acceptor for donor amino acids included in the CDR reflects a balance of competing considerations. Such substitutions are potentially advantageous in that they reduce the number of murine amino acids in the humanized antibody, thereby reducing potential immunogenicity. However, substitutions may also result in changes in affinity, and it is preferable to avoid significant decreases in affinity. The position of substitution within the CDR and the amino acid to be substituted can also be selected empirically.

[0068] The human acceptor antibody sequence can optionally be selected from among the many known human antibody sequences to provide a high degree of sequence identity (e.g., 65-85% identity) between the variable region framework of the human acceptor sequence and the corresponding variable region framework of the donor antibody chain.

[0069] Particular amino acids from the human variable region framework residues can be selected for substitution based on their possible effect on CDR conformation and / or binding to antigen. Study of such possible effects can be by modeling, examination of the characteristics of the amino acid at a particular position, or empirical observation of the effect of substituting or mutagenizing a particular amino acid.

[0070] For example, if an amino acid differs between a non-human variable region framework residue and a selected human variable region framework residue, the human framework amino acid can be replaced with the equivalent framework amino acid from the non-human antibody, where the amino acid is: (1) Direct non-covalent binding to antigens; (2) adjacent to the CDR regions; (3) otherwise interacts with the CDR region (e.g., is within about 6 angstroms of the CDR region). It is reasonable to assume that.

[0071] Other substitution candidates are acceptor human framework amino acids that are unusual for their position in a human immunoglobulin. These amino acids can be substituted with amino acids from the equivalent position in a non-human donor antibody or from the equivalent position in a more typical human immunoglobulin. Other substitution candidates are acceptor human framework amino acids that are unusual for their position in a human immunoglobulin.

[0072] An exemplary humanized antibody of the invention is a humanized form of ABC2, characterized by a mature light chain variable region of residues 23-128 of SEQ ID NO:2 and a mature heavy chain variable region of residues 20-140 of SEQ ID NO:1, and is referred to as HuABC2. The invention also provides variants of HuABC2. Such variants typically differ from the sequence of HuABC2 by a small number of substitutions, deletions, or insertions (e.g., typically no more than 1, 2, 3, 5, or 10). Such differences are usually within the framework, but can also occur within the CDRs. For example, only a subset of substitutions at positions H28, H48, H49, H68, H93, and L71 can be made. Many of the framework residues not in contact with the CDRs of a humanized mAb can accommodate substitutions of amino acids from the corresponding positions of the donor mouse mAb or other mouse or human antibodies, and even many potential CDR contact residues are amenable to substitution, and even amino acids within the CDRs may be altered. One example of a CDR replacement is to replace a residue in a CDR with a residue occupying the corresponding position in the human acceptor sequence used to provide the variable region framework.

[0073] In many cases, the substitutions made in the variant humanized ABC2 sequence are conservative with respect to the HuABC2 amino acids substituted. Preferably, the substitutions in HuABC2 (whether conservative or not) do not substantially affect the binding affinity or potency of the humanized mAb (i.e., its ability to inhibit interactions between receptors such as CD122, IL-2, IL-15, etc. (e.g., potency in some or all of the assays described in this Example or Background of the variant humanized ABC2 antibody is essentially the same as that of HuABC2, i.e., within experimental error). Preferably, the mature variant light and heavy chain V region sequences are at least 90%, more preferably at least 95%, and most preferably at least 98% identical to the respective HuABC2 mature light and heavy chain V regions. Additionally, other human antibody acceptor sequences, particularly sequences with high sequence identity to the variable region framework sequences of ABC2, are also suitable to provide humanized antibody variable region framework sequences.

[0074] In some HuABC2 variants, at least one, two, three, four, five, or all six of the acceptor to donor substitution positions referred to in connection with the exemplified antibodies (i.e., H28, H48, H49, H68, H93, and L71) are preferably occupied by residues T, I, A, I, A, and Y, respectively (residues occupying the corresponding positions in the murine donor antibody heavy chain). In some variants, position H42 is occupied by G. When the heavy chain acceptor sequence is other than the sequence encoded by DA430129 cDNA (GenBank Accession No.), or when the light chain acceptor sequence is other than the sequence encoded by M29469 cDNA (GenBank Accession No.), acceptor to donor substitutions may or may not be required for the designated occupancy of a particular variable framework region position, depending on whether the residue occupying the designated position is already the same between acceptor and donor.

[0075] Another exemplary humanized antibody of the invention is a humanized form of ABC101, characterized by a mature light chain variable region of SEQ ID NO:30 and a mature heavy chain variable region of SEQ ID NO:29, and is referred to as HuABC101. The invention also provides variants of HuABC101. Such variants typically differ from the sequence of HuABC101 by a small number of substitutions, deletions or insertions (e.g., typically no more than 1, 2, 3, 5 or 10). Such differences are usually in the framework, but can also occur in the CDRs. For example, only a subset of substitutions at positions H27, H30, H48, H66, H67, H71 and L49 can be made. Many of the framework residues not in contact with the CDRs of a humanized antibody can accommodate substitutions of amino acids from the corresponding positions of the donor mouse antibody or other mouse or human antibodies, and even many potential CDR contact residues are subject to substitution, and even amino acids within the CDRs can change. One example of a CDR substitution is to replace a residue in a CDR with a residue occupying the corresponding position of the human acceptor sequence used to provide the variable region framework.

[0076] In many cases, the substitutions made in the variant humanized ABC101 sequence are conservative with respect to the HuABC101 amino acid that is substituted. Preferably, the substitutions (whether conservative or not) of HuABC101 do not substantially affect the binding affinity or potency of the humanized mAb (i.e., its ability to inhibit interactions between receptors such as CD122, IL-2, IL-15, etc. (e.g., the potency of the variant humanized ABC101 antibody in some or all of the assays described herein is essentially the same as that of HuABC101, i.e., within experimental error). Preferably, the mature variant light and heavy chain V region sequences are at least 90%, more preferably at least 95%, and most preferably at least 98% identical to the respective HuABC101 mature light and heavy chain V regions. Additionally, other human antibody variable region framework acceptor sequences, particularly those with high sequence identity to the variable region framework sequences of ABC101, are also suitable for providing the humanized antibody framework.

[0077] In some HuABC101 variants, at least one, two, three, four, five, six or all seven of the acceptor to donor substitution positions referred to in connection with the exemplified antibodies (i.e., H27, H30, H48, H66, H67, H71 and L49) are preferably occupied by residues Y, T, M, Q, I, R and K, respectively (residues occupying the corresponding positions in the murine donor antibody heavy chain). If the heavy chain acceptor sequence is other than the sequence encoded by DA936142 cDNA (GenBank Accession No.), or if the light chain acceptor sequence is other than the sequence encoded by Z46622 cDNA (GenBank Accession No.), acceptor to donor substitutions may or may not be required for the specified occupancy of a particular variable framework region position, depending on whether the residue occupying the specified position is already the same between acceptor and donor.

[0078] The present invention may also use non-human antibodies, in particular chimeric and veneered versions of the exemplary ABC2 and ABC101 antibodies.

[0079] Chimeric antibodies are antibodies that combine the mature variable regions of the light and heavy chains of a non-human antibody (e.g., mouse) with human light and heavy chain constant regions. Such antibodies substantially or completely retain the binding specificity of the mouse antibody and contain approximately two-thirds of the human sequence.

[0080] A veneered antibody is a type of humanized antibody that retains some, and usually all, of the CDRs and some of the non-human variable region framework residues of the non-human antibody, but replaces other variable region framework residues that may contribute to B- or T-cell epitopes, e.g., exposed residues (Padlan, Mol. Immunol. 28:489, 1991), with residues from the corresponding positions in a human antibody sequence. The result is an antibody in which the CDRs are completely or substantially derived from a non-human antibody and the variable region framework of the non-human antibody has been made more human-like by the replacement. Veneered versions of the ABC2 or ABC101 antibodies are included in the present invention.

[0081] Human antibodies against CD122 can be obtained by various techniques, as described below. Some human antibodies are selected by the phage display method of Winter, by competitive binding experiments, as described above or otherwise, to have the same epitope specificity as a particular mouse antibody, such as one of the mouse monoclonal antibodies described in the Examples. Human antibodies can also be screened for a particular epitope specificity by using only fragments of CD122 as target antigens and / or by screening antibodies against a collection of deletion mutants of CD122.

[0082] Methods for producing human antibodies include the trioma method of Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, US 4,634,664; and Engleman et al., US 4,634,666, the use of transgenic mice containing human immunoglobulin genes (e.g., Lonberg et al., WO93 / 12227 (1993); US 5,877,397, US 5,874,299, US 5,814,318, US 5,789,650, US 5,770,429, US 5,661,016, US 5,633,425, US 5,625,126, US 5,569,825, US 5,545,806, Nature 148, 1547-1553). (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91 / 10741 (1991)) and phage display methods (e.g., Dower et al., WO 91 / 17271; McCafferty et al., WO 92 / 01047, US 5,877,218, US 5,871,907, US 5,858,657, US 5,837,242, US 5,733,743 and US 5,565,332). III. Selection of constant regions

[0083] The heavy and light chain variable regions of a chimeric, humanized (including veneered) or human antibody can be linked to at least a portion of a human constant region. The choice of constant region depends, in part, on whether antibody-dependent complement and / or cell-mediated cytotoxicity is desired. For example, human isotypes IgG1 and IgG3 have complement-mediated cytotoxicity, whereas human isotypes IgG2 and IgG4 do not. The light chain constant region can be lambda or kappa. Antibodies can be expressed as tetramers containing two light chains and two heavy chains; separate heavy and light chains; Fab, Fab', F(ab')2 and Fv; or as single chain antibodies in which the heavy and light chain variable domains are linked via a spacer.

[0084] Human constant regions exhibit allotypic and isoallotypic variation among different individuals. That is, the constant region may differ in different individuals at one or more polymorphic positions. Isoallotypes differ from allotypes in that sera that recognize an isoallotype bind to non-polymorphic regions of one or more other isotypes. Reference to a human constant region includes constant regions with any naturally occurring allotype or any permutation of residues occupying polymorphic positions in a naturally occurring allotype.

[0085] One or several amino acids at the amino or carboxy termini of the light and / or heavy chains, such as the C-terminal lysine of the heavy chain, may be missing or derivatized in part or all of the molecule. Substitutions may be made in the constant region to reduce or increase effector functions such as complement-mediated cytotoxicity or ADCC (see, for example, Winter et al., US Patent No. 5,624,821; Tso et al., US Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to extend half-life in humans (see, for example, Hinton et al., J. Biol. Chem. 279:6213, 2004). Exemplary substitutions to increase the half-life of the antibody include Gln at position 250 and / or Leu at position 428 (EU numbering of the constant region). Substitutions at any or all of positions 234, 235, 236 and / or 237 reduce affinity for Fcγ receptors, particularly FcγRI receptors (see, e.g., US 6,624,821). An exemplary substitution is a double mutation comprising Leu to Ala substitutions at positions 234 and 235 (L234A and L235A, respectively) (Hazareh et al., J. Virol. 75: 12161-12168, 2001). Optionally, positions 234, 236 and / or 237 in human IgG2 are substituted with alanine and position 235 is substituted with glutamine (see, e.g., US 5,624,821). IV. The extracellular domain of CTLA-4

[0086] CTLA-4 is a negative regulator of immune responses. It specifically binds to CD80 and CD86 expressed on the surface of APCs and inhibits the interaction of CD80 and CD86 with CD28 expressed on T cells, thereby inhibiting CD28-mediated costimulation of T cells.

[0087] An exemplary human form of CTLA-4 has been assigned Swiss Prot P16410. This form has 223 amino acids, of which residues 1-35 are the signal peptide, residues 36-161 are the extracellular domain, residues 162-182 are the transmembrane domain, and residues 173-223 are the cytoplasmic domain. An exemplary extracellular domain is set forth herein in SEQ ID NO: 10, which corresponds to residues 38-160 of the Swiss Prot sequence. Reference to the extracellular domain of CTLA-4 includes SEQ ID NO: 10; variants having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, deletions, or insertions; and variants having at least 90, 95, 95, or 99% identity to SEQ ID NO: 10. Such variants may be natural or induced, examples of which are provided in the annotation of P16410. Variants may also represent alternative amino acids present in CTLA-4 of other species. Systematic mutagenesis of CTLA-4 was performed to identify substitutions that enhance or inhibit binding to CD80 and CD86 (Xu et al., J. Immunol. 189:4470-4477, 2012). Examples of high affinity mutations include K28H, K28T, A29H, A29T, A29Y, A29W, and T30G. These and other mutations are named by the wild type amino acid residue on the left, their position in SEQ ID NO: 10 in the middle, and the mutated amino acid residue on the right. Mutations at T at position 51, M at position 53, M at position 54, L at position 58, and L at position 61 also favorably affect binding to one or both ligands. Motif 97MYPPPY102 was intolerant of mutations, especially for interaction with CD80, whereas beneficial mutations that altered the affinity balance between CD80 and CD86 were clustered adjacent to this conserved motif (K at position 93, L at position 96, L at position 104). CTLA-4 mutants with stronger binding to CD80 and CD86 have also been disclosed by Douthwaite et al. (J. Immunol. 198:528-537, 2017) and Oshima et al. (Protein Eng. Des. Sel. 29:159-167, 2016). Preferred variants that enhance binding include A29Y and L104E.These and other mutations are numbered by their position in SEQ ID NO: 10, or, if present in a sequence having a different number of residues than SEQ ID NO: 10, by their corresponding position in SEQ ID NO: 10 relative to the position of the mutation in the other sequence when the respective sequences are maximally aligned. Additional flanking amino acids from P16410 may or may not be included on either or both sides of SEQ ID NO: 10.

[0088] CTLA-4 and its variants exhibit specific binding to CD80 and CD86. An exemplary form of human CD80, also known as B7-1 or BB1, is provided by Swiss-Prot P33681. An exemplary form of human CD86, also known as B7-2, is provided by Swiss-Prot P42081. CD80 and CD86 interact with CD28. An exemplary form of human CD28 is provided by Swiss-Prot P10747.

[0089] Belatacept is a recombinant protein that contains the extracellular domain of human CTLA-4 (cytotoxic T-lymphocyte-associated protein 4, also known as CD152) fused to the Fc region of the human gamma-1 heavy chain (CTLA4-Ig) (SEQ ID NO: 49). To enhance binding to CD80 (also known as B7-1) and CD86 (also known as B7-2), the CTLA-4 region of belatacept has two amino acid substitutions, one at position 29, where the alanine residue is replaced by a tyrosine residue, and the other at position 104, where the leucine residue is replaced by a glutamic acid residue (L104E) (Larsen et al., Am. J. Transplant. 5:443-453, 2005; Oshima et al., Protein Eng. Des. Sel. 29:159-167, 2016). Belatacept is approved for sale in the United States for the prevention of renal allograft rejection. For reviews of belatacept, see Perez et al., Transplantation 102:1440-1452, 2008; Gallo et al., PLoS ONE 15:e0240335, 2020.

[0090] Abatacept, the predecessor of belatacept, is also a recombinant protein containing the wild-type extracellular domain of human CTLA-4 fused to the Fc region of the human gamma-1 heavy chain. No amino acid substitutions have been introduced into the CTLA-4 region. Abatacept, like belatacept, functions as an immunosuppressant by blocking the interaction of CD80 and CD86 with CD28. Abatacept has been approved for sale in the United States for the treatment of adult rheumatoid arthritis, juvenile polyarticular idiopathic arthritis, and adult psoriatic arthritis. For reviews of abatacept, see Moreland et al., Nat. Rev. Drug Disc. 5:185-186, 2006; Esensten et al., Immunity 44:973-988, 2016. Abatacept has been reported to be effective in suppressing renal allograft rejection (Badell et al., Am. J. Transplant. 19:2342-2349, 2019). Binding of the extracellular domain portion of CTLA-4, such as abatacept and belatacept, to CD80 and CD86 inhibits immune responses by blocking the CD28-mediated signal 2 of the T cell activation pathway. V. Bifunctional molecules

[0091] The bifunctional molecules of the invention combine an antibody against CD122, as further described above, with the extracellular domain of CTLA-4 described above. The extracellular domain of CTLA-4 can be attached to the C-terminus of the heavy chain of the antibody to form a fusion protein. Thus, an exemplary bifunctional molecule comprises an antibody heavy chain comprising a mature heavy chain variable region and a heavy chain constant region fused to the extracellular domain of CTLA-4 at the C-terminus of the heavy chain constant region, and a light chain comprising a mature light chain variable region and a light chain constant region. The extracellular domain of CTLA-4 can also be attached to the C-terminus of the light chain. Thus, another exemplary bifunctional molecule comprises an antibody heavy chain comprising a mature heavy chain variable region and a heavy chain constant region, and a light chain comprising a mature light chain variable region and a light chain constant region fused to the extracellular domain of CTLA-4 at the C-terminus of the light chain constant region. The extracellular domain of CTLA-4 can also be attached to the N-terminus of the heavy or light chain variable region of the antibody. Thus, another exemplary bifunctional molecule comprises an antibody heavy chain comprising a mature heavy chain variable region and a heavy chain constant region, and a light chain comprising a mature light chain variable region and a light chain constant region linked to the extracellular domain of CTLA-4 at the N-terminus of the heavy chain variable region or the light chain variable region. The heavy or light chain can be linked to CTLA-4 directly or indirectly, such as through a flexible peptide linker. The flexible peptide linker can be formed primarily or exclusively from glycine and serine residues (e.g., SEQ ID NOs: 11, 36, and 50). The heavy and light chains can associate with each other in the same manner as a conventional antibody. Also, the two pairs of associated heavy and light chains can dimerize with each other to form a heterodimeric structure similar to a conventional antibody, except for the extracellular domain of CTLA-4 linked to the N-terminus or C-terminus of the heavy or light chain. The heavy chain constant region can include any or all of the regions present in a normal heavy chain constant region (e.g., CH1, hinge, CH2, and CH3) optionally linked via the C-terminus to the extracellular domain of CTLA-4. When the extracellular domain of CTLA-4 is fused to the C-terminus of an antibody heavy chain constant region, the fusion of the extracellular domain of CTLA-4 to the C-terminus of the heavy chain constant region is the opposite of abatacept or belatacept, in which the extracellular domain of CTLA-4 is fused to the N-terminus of the heavy chain constant region.

[0092] In an exemplary bifunctional molecule based on the HuABC2 antibody, the mature heavy chain is attached to the extracellular domain of CTLA-4 as a chain comprising SEQ ID NO: 13 or 16, and the mature light chain comprises SEQ ID NO: 9. In another exemplary bifunctional molecule, the mature light chain is attached to the extracellular domain of CTLA-4 as a chain comprising SEQ ID NO: 34, and the mature heavy chain comprises SEQ ID NO: 8 or 33, with the C-terminal lysine optional. In another exemplary bifunctional molecule, the mature heavy chain is attached to the extracellular domain of CTLA-4 as a chain comprising SEQ ID NO: 39, with the C-terminal lysine optional, and the mature light chain comprises SEQ ID NO: 9. In another exemplary bifunctional molecule, the mature light chain is attached to the extracellular domain of CTLA-4 as a chain comprising SEQ ID NO: 42, and the mature heavy chain comprises SEQ ID NO: 8 or 33, with the C-terminal lysine optional.

[0093] In an exemplary bifunctional molecule based on the HuABC101 antibody, the mature heavy chain is attached to the extracellular domain of CTLA-4 as a chain comprising SEQ ID NO: 57, and the mature light chain comprises SEQ ID NO: 54. In another exemplary bifunctional molecule, the mature heavy chain is attached to the extracellular domain of CTLA-4 as a chain comprising SEQ ID NO: 56, with the C-terminal lysine optional, and the mature light chain comprises SEQ ID NO: 54.

[0094] In an exemplary bifunctional molecule based on the HuABC77 antibody, the mature heavy chain is attached to the extracellular domain of CTLA-4 as a chain comprising SEQ ID NO: 71, and the mature light chain comprises SEQ ID NO: 67. In another bifunctional molecule, the mature heavy chain is attached to the extracellular domain of CTLA-4 as a chain comprising SEQ ID NO: 70, with the C-terminal lysine optional, and the mature light chain comprises SEQ ID NO: 67.

[0095] Such bifunctional molecules exhibit specific binding to each of CD122, CD80, and CD86, inhibit the interaction of CD122 with the ligands IL-2 and IL-15, and inhibit the interaction of CD80 and CD86 with CD28. Optionally, the binding affinity of the bifunctional molecule for CD122 is the same, or within 5-, 3-, or 2-fold, within experimental error, as the binding affinity of an anti-CD122 antibody incorporated into the bifunctional molecule. Optionally, the inhibition of CD122 binding to IL-2 or IL-15 by the bifunctional molecule is the same, or within 5-, 3-, or 2-fold, within experimental error, as the inhibition by an anti-CD122 antibody incorporated into the bifunctional molecule alone. Optionally, the binding affinity of the bifunctional molecule for CD80 and CD86 is the same, or within 5-, 3-, or 2-fold, within experimental error, as the extracellular domain of CTLA-4 (e.g., abatacept or belatacept) incorporated into the bifunctional molecule linked to the heavy chain constant region. Optionally, the inhibition of binding of CD80 and / or CD86 to CD28 by the bifunctional molecule is the same as, or within 5, 3 or 2-fold, the inhibition by the extracellular domain of CTLA-4 bound to the constant or variable region incorporated into the bifunctional molecule, within experimental error. Optionally, the bifunctional molecule exhibits greater inhibition of immune response or greater efficacy in treating immune disorders compared to what its antibody or extracellular domain components can achieve individually at equimolar doses. Optionally, the bifunctional molecule exhibits at least an additive, preferably a synergistic increase in inhibition or efficacy compared to each of its antibody or extracellular domain components. Optionally, the bifunctional molecule exhibits greater inhibition or efficacy at equal moles of each agent compared to when its component antibodies and extracellular domains are co-administered as separate molecules in equimolar ratios. In comparisons involving an extracellular domain that is not part of a bifunctional molecule, the extracellular domain can be used alone or fused to an IgG constant region at either the N- or C-terminus. Optionally, the bifunctional molecule exhibits greater inhibition or efficacy compared to co-administration of its component anti-CD122 antibodies with either abatacept or belatacept, the comparison being made at equimolar amounts of each agent. VI. Expression of bifunctional molecules

[0096] The bifunctional molecules of the invention are typically produced by recombinant expression using techniques similar to the expression of antibodies. The nucleic acids encoding the heavy and light chains bound to the extracellular domain of CTLA-4 or the nucleic acids encoding the heavy and light chains bound to the extracellular domain of CTLA-4 are expressed to produce the encoded polypeptides that associate to form the bifunctional molecule. The recombinant nucleic acid construct typically contains expression control sequences operably linked to the coding sequences of these chains, including naturally associated promoter regions or heterologous promoter regions. Preferably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into a suitable host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and for collection and purification of the bifunctional molecules.

[0097] Mammalian cells can be used to express nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes~Clones, (VCH Publishers, NY, 1987). Several suitable host cell lines capable of secreting intact heterologous proteins have been developed, including CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody producing myelomas including SP2 / 0 and NS0. The cells can be non-human. Expression vectors for these cells can include expression control sequences such as an origin of replication, a promoter, an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), as well as necessary information processing sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination sequences. Expression control sequences can include promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus, and the like (see Co et al., J. Immunol. 148:1149 (1992)).

[0098] Once expressed, the bifunctional molecules can be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, and the like (for a review, see Scopes, Protein Purification (Springer-Verlag, NY, 1982)). VII. Pharmaceutical Compositions and Methods of Treatment

[0099] The bifunctional molecules can be used to suppress a variety of undesirable immune responses, particularly immune responses mediated by any of CD122, CD80 and CD86 and their ligands or receptors, the interactions of which are inhibited by the bifunctional molecules of the invention.

[0100] One category of immune disorders treatable by the bifunctional molecules of the present invention is transplant rejection. When allogeneic cells or organs (e.g., skin, kidney, liver, heart, lung, pancreas, bone marrow, etc.) are transplanted into a host (i.e., the donor and the donee are different individuals of the same species), the host's immune system is likely to mount an immune response against foreign antigens in the graft (host-versus-graft disease) and destroy the transplanted tissue. The bifunctional molecules of the present invention are particularly useful for blocking allogeneic antigen-induced immune responses in the donee.

[0101] A related use of the bifunctional molecules of the invention is to modulate immune responses involved in "graft versus host" disease (GVHD). GVHD is a potentially fatal disease that occurs when immunologically competent cells are transplanted into an allogeneic recipient. In this situation, the donor's immunocompetent cells may attack the recipient's tissues. Tissues of the skin, intestinal epithelium, and liver are frequent targets and may be destroyed during the course of GVHD. The disease poses a particularly severe problem when immune tissues are transplanted, such as in bone marrow transplants, although less severe forms of GVHD have been reported in other cases, such as heart and liver transplants.

[0102] Yet another situation in which immunosuppression is desirable is the treatment of autoimmune diseases such as type 1 diabetes, Crohn's disease, ulcerative colitis, multiple sclerosis, stiff-person syndrome, rheumatoid arthritis, myasthenia gravis, lupus erythematosus, celiac disease, psoriasis, uveitis, alopecia areata, primary biliary cirrhosis, etc. In these autoimmune diseases, the body generates a cellular and / or humoral immune response against one of its own antigens, leading to the destruction of that antigen, with potentially disabling and / or fatal consequences. Autoimmune diseases are treated by administering the bifunctional molecules of the invention.

[0103] Other autoimmune diseases treatable by the bifunctional molecules of the invention include asthma, allergies, vitiligo, polymyositis, allergic dermatitis, and Behcet's disease. Other autoimmune diseases treatable include idiopathic thrombocytopenic purpura, Kawasaki disease, Guillain-Barre syndrome, chronic inflammatory demyelinating systemic lupus erythematosus, autoimmune neutropenic type 1 diabetes, acute disseminated encephalomyelitis, acute motor axonal neuropathy, Addison's disease, steatosis, adult-onset Still's disease, ankylosing spondylitis, anti-glomerular basement membrane nephritis, antineutrophil cytoplasmic antibody-associated vasculitis, anti-N-methyl-D-aspartate receptor encephalitis, antiphospholipid syndrome, antisynthetase syndrome, aplastic anemia, autoimmune angioedema, autoimmune encephalitis, autoimmune enteropathy, autoimmune inflammatory bowel disease, autoimmune enteropathy, autoimmune inflammatory bowel disease, autoimmune encephalopathy, autoimmune inflammatory bowel disease ... Epidemic hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune polyendocrine syndrome type 2, autoimmune polyendocrine syndrome type 3, autoimmune progesterone dermatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura, autoimmune thyroiditis, autoimmune urticaria, autoimmune uveitis, concentric cranial sclerosis, Behcet's disease, Bickerstaff encephalitis, bullous pemphigoid, chronic fatigue syndrome, Churg-Strauss disease, syndrome, cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, complex regional pain syndrome, CREST syndrome, dermatitis herpetiformis, dermatomyositis, discoid lupus erythematosus, endometriosis, enthesitis, enthesitis-associated arthritis, eosinophilic esophagitis, eosinophilic fasciitis, epidermolysis bullosa acquisita, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, Felty syndrome, fibromyalgia, gastritis, pemphigoid of pregnancy, giant cell arteritis, Goodpasture's syndrome, Graves' disease, Graves' ophthalmopathy, Hashimoto's encephalopathy, Hashimoto's thyroiditis, Henoch-Schonlei syndrome purpura, hidradenitis suppurativa, idiopathic dilated cardiomyopathy, idiopathic inflammatory demyelinating disease, IgA nephropathy, IgG4-related systemic disease, inclusion body myositis, inflammatory bowel disease (IBD), intermediate uveitis, interstitial cystitis, juvenile arthritis, Kawasaki disease, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignified conjunctivitis, linear IgA disease, lupus nephritis, lupus vasculitis, Lyme disease, Meniere's disease, microscopic colitis, microscopic polyangiitis, mixed connective tissue disease, Mooren's ulcer, morphea, Mucha-Habermann disease, myocarditis,Myositis, Neuromyelitis optica, Neuromyotonia, Opsoclonus-myoclonus syndrome, Optic neuritis, Ordo thyroiditis, Palindromic rheumatism, Paraneoplastic cerebellar degeneration, Parry-Romberg syndrome, Parsonage-Turner syndrome, Pemphigus vulgaris, Pernicious anemia, Pityriasis lichenoides and acute smallpox-related childhood autoimmune neuropsychiatric disorders, POEMS syndrome, Polyarteritis nodosa, Polymyalgia rheumatica, Polymyositis, Post-myocardial infarction syndrome, Post-pericardiotomy syndrome, Primary biliary cirrhosis, Primary immunodeficiency, Primary sclerosing cholangitis, These include: progressive inflammatory neuropathy, psoriatic arthritis, pure red cell hypoplasia, pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, relapsing polychondritis, restless leg syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatic vasculitis, sarcoidosis, Schnitzler's syndrome, scleroderma, Sjögren's syndrome, subacute bacterial endocarditis, Susac's syndrome, Sydenham's chorea, sympathetic ophthalmia, systemic scleroderma, thrombocytopenia, Tolosa-Hunt syndrome, transverse myelitis, undifferentiated connective tissue disease, urticaria, urticarial vasculitis, vasculitis.

[0104] Although immune disorders can be subdivided into categories as described above, these categories are not necessarily mutually exclusive, nor is treatment dependent on the category into which a disorder falls.

[0105] Other diseases treatable by the bifunctional molecules of the invention include cancers, particularly hematological malignancies such as leukemia (e.g., T-cell large granular lymphocyte leukemia) or lymphoma. Some such cancers exhibit detectable levels of CD122, CD80, and / or CD86, measured at the protein (e.g., by immunoassay using one of the exemplified bifunctional molecules) or mRNA level. Some such cancers preferably exhibit elevated levels of CD122, CD80, and / or CD86 compared to non-cancerous tissue of the same type from the same subject. Optionally, the levels of CD122, CD80, or CD86 in the cancer are measured before administering treatment.

[0106] The bifunctional molecule is administered in an effective regimen, which refers to a dosing plan including a dosage, route of administration, and frequency of administration that delays onset, reduces severity, inhibits further deterioration, and / or improves at least one sign or symptom of the disorder. If the subject is already suffering from the disorder, the regimen may also be referred to as a therapeutically effective regimen. If the subject has a high risk of the disorder compared to the general population, but is not yet symptomatic, the regimen may also be referred to as a prophylactically effective regimen. In some cases, therapeutic or prophylactic efficacy may be observed in an individual subject against historical controls or past experience of the same subject. In other cases, therapeutic or prophylactic efficacy may be demonstrated during preclinical or clinical trials of a treated subject population compared to a control population of untreated subjects.

[0107] Exemplary dosages of the bifunctional molecules are 0.1-20 mg / kg, or 0.5-5 mg / kg body weight (e.g., 0.5, 1, 2, 3, 4 or 5 mg / kg), or as a fixed dose, 10-1500 mg. The dosage will depend, among other things, on the subject's condition and response to previous treatment, if any, whether the treatment is prophylactic or therapeutic, and whether the disorder is acute or chronic.

[0108] Administration may be parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, intranasal or intramuscular. Administration into the systemic circulation is preferred by intravenous or subcutaneous administration. Intravenous administration may be, for example, an infusion over 30 to 90 minutes.

[0109] The frequency of administration depends, inter alia, on the half-life of the bifunctional molecule in circulation, the condition of the subject, and the route of administration. The frequency can be daily, weekly, monthly, quarterly, or at irregular intervals, depending on the change in the condition of the subject or the progression of the disorder being treated. An exemplary frequency for intravenous administration is weekly to quarterly, depending on the cause of the treatment, although more or less frequent administration is possible. For subcutaneous administration, an exemplary frequency of administration is daily to monthly, although more or less frequent administration is possible.

[0110] The number of doses depends on whether the disorder is acute or chronic and the response of the disorder to treatment. For acute disorders or acute exacerbations of chronic disorders, often 1 to 10 doses. Sometimes, for acute disorders or acute exacerbations of chronic disorders, a single rapid dose, optionally in divided form, is sufficient. For recurrence of acute disorders or acute exacerbations, treatment may be repeated. For chronic disorders, the bifunctional molecule may be administered at regular intervals, for example, weekly, biweekly, monthly, every 3 months, every 6 months, for at least 1, 5 or 10 years, or for the life of the subject.

[0111] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., single-administration doses). Pharmaceutical compositions can be formulated using one or more pharmacologically acceptable carriers, diluents, excipients, or auxiliaries. The formulation will vary depending on the route of administration selected. For injection, the bifunctional molecules can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or saline or acetate buffer (to reduce discomfort at the injection site). The solutions can contain formulating agents such as suspending, stabilizing, and / or dispersing agents. Additionally, the bifunctional molecules can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, prior to use.

[0112] Treatment with the bifunctional molecules of the invention can be combined with other treatments that are effective against the disease being treated. In the treatment of immune disorders, conventional treatments include mast cell degranulation inhibitors, corticosteroids, nonsteroidal anti-inflammatory drugs, and more powerful anti-inflammatory drugs such as azathioprine, cyclophosphamide, leukeran, FK506, and cyclosporine. Biological anti-inflammatory agents such as Tysabri® (natalizumab) and Humira® (adalimumab) can also be used. When used to treat cancer, the bifunctional molecules of the invention can be combined with chemotherapy, radiation, stem cell therapy, surgery, or treatment with other biological agents such as Herceptin® (trastuzumab) against the HER2 antigen, Avastin® (bevacizumab) against VEGF, antibodies against the EGF receptor (Erbitux®, cetuximab), and Vectibix® (panitumumab). Chemotherapeutic agents include chlorambucil, cyclophosphamide or melphalan, carboplatinum, daunorubicin, doxorubicin, idarubicin, and mitoxantrone, methotrexate, fludarabine, and cytarabine, etoposide or topotecan, vincristine, and vinblastine. VIII. Other Uses

[0113] The bifunctional molecules can be used to detect CD122, CD80 and / or CD86 in clinical diagnosis or therapy or research. For example, the bifunctional molecules can be used to detect the presence of CD122, CD80 and / or CD86 on T cells as an indication that a subject suffers from a treatable immune-mediated disease. Expression of CD122, CD80 and / or CD86 on a cancer also indicates that the cancer is susceptible to treatment with the bifunctional molecules of the invention. The bifunctional molecules can also be sold as research reagents for laboratory studies to detect T cells and their responses to various stimuli. For such use, the bifunctional molecules can be labeled with fluorescent molecules, spin-labeled molecules, enzymes, or radioisotopes and can be provided in the form of a kit with all the reagents necessary to perform an assay for CD122, CD80, or CD86. The bifunctional molecules can also be used to purify CD122, CD80, and / or CD86, for example by affinity chromatography.

[0114] All patent applications, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item was specifically and individually indicated to be incorporated by reference. Where different versions of a sequence are associated with accession numbers at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date refers to the earlier of the actual filing date or the filing date of the priority application, with reference to the accession number, if applicable. Similarly, where different versions of a publication, website, etc. are published at different times, the version most recently published at the effective filing date of this application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the present invention can be used in combination with others unless otherwise indicated. The present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, but it will be apparent that certain changes and modifications can be practiced within the scope of the appended claims.

[0115] Example 1: General Methods and Materials Gene cloning, mutagenesis, plasmid construction, ELISA, and flow cytometry were performed according to standard laboratory techniques as described by Green and Sambrook (Molecular Cloning, A Laboratory Manual, 4th ed., 2012, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), Greenfield (Antibodies, A Laboratory Manual, 2nd ed., 2014, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), Kostelny et al. (Int. J. Cancer 93:556-565, 2001), Cole et al. (J. Immunol. 159:3613-3621, 1997), and Tsurushita et al. (Methods 36:69-83, 2005) or according to vendor protocols.

[0116] HuABC2 is a humanized anti-CD122 IgG1 / κ monoclonal antibody that (i) binds to the β-chain subunit of the IL-2 and IL-15 receptor (also called CD122) and (ii) blocks the interaction of IL-2 and IL-15 with their respective receptors (US Pat. No. 9,028,830). The amino acid sequence of the heavy chain variable region (VH) of HuABC2 is: MKLWLNWVFLLTLLHGIQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSDFYMEWVRQAPGKGLEWIAASRNKANDYTTEYSASVKGRFIVSRDDSKNSLYLQMNSLKTEDTAVYYCARSYYRYDGMDYWGQGTTVTVSS (SEQ ID NO: 1) The mature HuABC2 VH begins with the glutamic acid residue at position 20 of SEQ ID NO:1.

[0117] The amino acid sequence of the mature light chain variable region (VL) of HuABC2 is: MDFQVQIFSFLLISASVIVSRGEIVLTQSPATLSLSPGERATLSCSAISSVSYMYWYQQKPGQAPRLLIYDTSNLVSGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWNTYPYTFGGGTKVEIK (SEQ ID NO: 2) The mature HuABC2 VL begins at the glutamic acid residue at position 23 of SEQ ID NO:2.

[0118] The schematic structure of the plasmid vector pHuABC2p for the production of HuABC2 in mammalian cells is shown in FIG. 1A. Proceeding clockwise from the SalI site in the diagram, pHuABC2p contains a heavy chain transcription unit beginning with the human cytomegalovirus (CMV) major immediate early promoter and enhancer (CMV-P) to initiate transcription of the antibody heavy chain gene. The CMV promoter is followed by the VH exon encoding HuABC2 VH. HuABC2VH is a genomic sequence containing the human gamma-1 heavy chain constant region including CH1, hinge, CH2, and CH3 exons with an intervening intron, and a polyadenylation site following the CH3 exon. Following the heavy chain gene sequence, the light chain transcription unit begins with the CMV promoter and is followed by a genomic sequence containing the exon encoding HuABC2 VL and the human kappa chain constant region (Ck) exon with a portion of the intron preceding it, and a polyadenylation site following the Ck exon. The light chain gene is followed by the SV40 early promoter (SV40-P), the puromycin N-acetyltransferase gene for puromycin resistance (puro), and a segment containing the SV40 polyadenylation site (SV40-A). Finally, the plasmid contains a portion of the plasmid pUC19, which contains the bacterial origin of replication (pUC ori) and the β-lactamase gene (β-lactamase). The positions of the restriction enzyme sites are indicated on the diagram. The arrow indicates the direction of transcription.

[0119] The amino acid sequences of the CH1, hinge, CH2, and CH3 regions of the human γ-1 heavy chain encoded by pHuABC2p are ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 3), EPKSCDKTHTCPPCP (SEQ ID NO: 4), APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 5), and GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 6) It is. The amino acid sequence of the Ck region encoded by pHuABC2p is TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 7) It is.

[0120] The amino acid sequence of the mature heavy chain encoded in pHuABC2p is: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFYMEWVRQAPGKGLEWIAASRNKANDYTTEYSASVKGRFIVSRDDSKNSLYLQMNSLKTEDTAVYYCARSYYRYDGMDYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8) It is.

[0121] The amino acid sequence of the mature light chain encoded by pHuABC2p is: EIVLTQSPATLSLSPGERATLSCSAISSVSYMYWYQQKPGQAPRLLIYDTSNLVSGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWNTYPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 9) It is. For expression of HuABC2, CHO-K1 cells stably transfected with pHuABC2p (CHO-K1 / pHuABC2p) were grown in SFM4CHO medium (HyClone Laboratories, Logan, UT). For purification of HuABC2, the culture supernatant of CHO-K1 / pHuABC2p cells was purified using a protein-A Sepharose® column (HiTrap® MabSelect SuRe TM The column was washed with PBS, and HuABC2 was eluted with 0.1 M glycine-HCl buffer (pH 3.0) containing 0.1 M NaCl. After neutralization with 1 M Tris-HCl (pH 8.0), the eluted antibody buffer was exchanged into PBS by dialysis. The antibody concentration was determined by measuring the absorbance at 280 nm (1.4 OD = 1 mg / ml).

[0122] Example 2: Bifunctional molecules that bind CD80, CD86, and CD122 Binding of CD80 or CD86 on antigen-presenting cells to CD28 on T cells enhances the immune response. CTLA-4 binds to CD80 and CD86, blocking their interaction with CD28 and suppressing T cell activation (Esensten et al., Immunity 44:973-988, 2016).

[0123] To confer the ability of HuABC2 to block the function of the immune co-stimulatory molecule CD28, the CH3 exon of pHuABC2p was modified to encode the extracellular domain of human CTLA-4 (SEQ ID NO: 10) fused at its N-terminus to the penultimate amino acid residue (Gly) of CH3 (CH3-CTLA4 fusion). A flexible polypeptide linker (SGGGGSG; SEQ ID NO: 11) was placed between CH3 and CTLA-4 in the CH3-CTLA4 fusion. The amino acid sequence of the CH3-CTLA4 fusion is: GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSD (SEQ ID NO: 12) In addition, both leucine residues at positions 234 and 235 (EU numbering) in CH2 were changed to alanine residues to eliminate binding to Fcγ receptors (Hezareh et al., J. Virol. 75:12161-12168, 2001). The resulting pHuABC2p-derived expression vector was named pHC624. The schematic structure of pHC624 is shown in Figure 1B.

[0124] The amino acid sequence of the mature heavy chain of HuABC2 fused at the C-terminus to a linker (underlined), followed by the extracellular domain of CTLA-4, encoded in pHC624 is: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFYMEWVRQAPGKGLEWIAASRNKANDYTTEYSASVKGRFIVSRDDSKNSLYLQMNSLKTEDTAVYYCARSYYRYDGMDYWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SGGGGSG MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSD (SEQ ID NO: 13) It is.

[0125] The amino acid sequence of the mature light chain encoded in pHC624 is the same as that encoded in pHuABC2p (SEQ ID NO: 9). The modified HuABC2 encoded by pHC624 fused to CTLA-4 at the end of the heavy chain was named HuABC2-CTLA4. The schematic structure of HuABC2-CTLA4 is shown in FIG. 2.

[0126] The extracellular domain of CTLA-4 (SEQ ID NO: 10) was modified by the substitution of two amino acid residues. One of them is a substitution of an alanine residue at position 29 of SEQ ID NO: 10 with a tyrosine residue (A29Y). The other is a substitution of a leucine residue at position 104 of SEQ ID NO: 10 with a glutamic acid residue (L104E). The amino acid sequence of the extracellular domain of the resulting modified CTLA-4 (variant CTLA-4) is: MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD (SEQ ID NO: 14) The DNA fragment encoding the variant CTLA-4 was used to replace the region encoding wild-type CTLA-4 in pHC624. The resulting expression vector was named pHC625 (also called pHC622). The schematic structure of pHC625 is shown in Figure 1B.

[0127] The amino acid sequence of the modified CH3-CTLA4 fusion encoded in pHC625 and pHC622 is: GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SGGGGSG MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD (SEQ ID NO: 15) The linker sequences are underlined.

[0128] The amino acid sequence of the mature heavy chain of HuABC2 fused to a linker (underlined) at the C-terminus (followed by the extracellular domain of variant CTLA-4) encoded in pHC625 and pHC622 is: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFYMEWVRQAPGKGLEWIAASRNKANDYTTEYSASVKGRFIVSRDDSKNSLYLQMNSLKTEDTAVYYCARSYYRYDGMDYWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SGGGGSG MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD (SEQ ID NO: 16) It is.

[0129] The amino acid sequence of the mature light chain encoded in pHC625 and pHC622 is the same as the mature light chain sequence encoded in pHuABC2p (SEQ ID NO: 9). The modified HuABC2 fused to variant CTLA-4 at the end of the heavy chain encoded in pHC625 and pHC622 was designated HuABC2-CTLA4YE.

[0130] Example 3: Transient expression and characterization of a bifunctional molecule that binds CD80, CD86, and CD122 The expression vectors pHC624 and pHC625 were transfected separately into the human embryonic kidney cell line HEK293 by the procedure of Durocher et al. (Nucl. Acids Res. 30:e9 2002) for secretion of HuABC2-CTLA4 and HuABC2-CTLA4YE, respectively, in the culture supernatant. HEK293 cells were grown in DME medium containing 10% fetal bovine serum (FBS; HyClone, Logan, UT) at 37°C in a 7.5% CO2 incubator. The expression levels of HuABC2-CTLA4 and HuABC2-CTLA4YE in the culture supernatant were measured by sandwich ELISA. In a typical experiment, wells of an ELISA plate were coated with goat anti-human gamma heavy chain polyclonal antibody in PBS, washed with wash buffer (PBS containing 0.05% Tween 20), and blocked with blocking buffer (PBS containing 2% skim milk and 0.05% Tween 20). After washing the wells with wash buffer, test samples appropriately diluted in ELISA buffer (PBS containing 1% skim milk and 0.025% Tween 20) were applied to the wells. Purified HuABC2 appropriately diluted in ELISA buffer was used as a standard. After 1 h incubation and washing with wash buffer, bound antibody was detected using HRP-conjugated goat anti-human kappa chain polyclonal antibody. After 30 min incubation and washing with wash buffer, color development was initiated by adding ABTS substrate (Sigma-Aldrich, St. Louis, MO) and stopped with 2% oxalic acid. Absorbance was read at 405 nm.

[0131] Binding of HuABC2-CTLA4 and HuABC2-CTLA4YE to human CD122, CD80 and CD86 was analyzed by ELISA. Wells of ELISA plates were coated with CD122 (human IL-2Rβ / CD122 protein, Fc Tag; Cat. No. ILB-H5253, Acro Biosystems, Newark, Del.), CD80 (human B7-1 / CD80 protein, Mouse IgG2a Tag; Cat. No. B71-H52A4, Acro Biosystems) or CD86 (human B7-2 / CD86 protein, Fc Tag; Cat. No. CD6-H5257, Acro Biosystems) at 1 μg / ml in PBS. After blocking with blocking buffer and washing the wells with washing buffer, 0.5 μg / ml of transiently expressed HuABC2-CTLA4 and HuABC2-CTLA4YE and two-fold serial dilutions in ELISA buffer were applied to wells in duplicate. Purified HuABC2 at 0.5 μg / ml and two-fold serial dilutions were used as controls. After 1 h incubation at room temperature and washing with washing buffer, bound antibody was detected using HRP-conjugated goat anti-human kappa chain polyclonal antibody. After 30 min incubation and washing with washing buffer, color development was initiated by adding ABTS substrate and stopped with 2% oxalic acid. Absorbance was read at 405 nm.

[0132] Figures 3, 4 and 5 show the results of ELISA tests for the binding of HuABC2, HuABC2-CTLA4 and HuABC2-CTLA4YE to human CD122, CD80 and CD86, respectively. HuABC2, HuABC2-CTLA4 and HuABC2-CTLA4YE bound to CD122 in a dose-dependent manner (Figure 3). No significant differences were observed between the binding patterns to CD122. For CD80, HuABC2 showed only background levels of binding, whereas HuABC2-CTLA4 and HuABC2-CTLA4YE bound to CD80 in a dose-dependent manner (Figure 4). Similarly, for CD86, HuABC2 showed only background levels of binding, whereas HuABC2-CTLA4 and HuABC2-CTLA4YE bound to CD86 in a dose-dependent manner (Figure 5). Both HuABC2-CTLA4 and HuABC2-CTLA4YE acquired the ability to bind to CD80 and CD86 in addition to the CD122-binding ability derived from HuABC2, but HuABC2-CTLA4YE showed stronger binding to CD80 and CD86, respectively, than HuABC2-CTLA4 (Figures 4 and 5). Example 4: Fusion of CTLA-4 at the C-terminus of the HuABC2 light chain

[0133] To generate another form of HuABC2 fused to CTLA-4, the Ck exon of pHuABC2p (Figure 1A) was modified to encode the extracellular domain of a variant form of human CTLA-4 (variant CTLA-4; SEQ ID NO: 14) fused at its N-terminus to the last amino acid residue (Cys) of Ck (Ck-CTLA4YE fusion). A flexible polypeptide linker (SGGGGSG; SEQ ID NO: 11) was placed between Ck and CTLA-4 in the Ck-CTLA4YE fusion. The resulting pHuABC2p-derived expression vector was named pHC623. Both leucine residues at positions 234 and 235 (EU numbering) of CH2 of pHC623 were changed to alanine residues to eliminate binding to Fcγ receptors (Hezareh et al., J. Virol. 75:12161-12168, 2001). The schematic structure of pHC623 is shown in Figure 6A.

[0134] The amino acid sequence of the Ck-CTLA4YE fusion encoded in pHC623 is: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SGGGGSG MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD (SEQ ID NO: 32) The linker sequences are underlined.

[0135] The amino acid sequence of the mature heavy chain encoded in pHC623 is: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFYMEWVRQAPGKGLEWIAASRNKANDYTTEYSASVKGRFIVSRDDSKNSLYLQMNSLKTEDTAVYYCARSYYRYDGMDYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 33) It is.

[0136] The amino acid sequence of the mature light chain of HuABC2 fused to a linker (underlined) at its C-terminus (followed by the extracellular domain of variant CTLA-4) encoded in pHC623 is: EIVLTQSPATLSLSPGERATLSCSAISSVSYMYWYQQKPGQAPRLLIYDTSNLVSGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWNTYPYTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SGGGGSG MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD (SEQ ID NO: 34) It is.

[0137] The modified HuABC2 fused to the variant CTLA-4 at the light chain end encoded in pHC623 was designated HuABC2-Ck-CTLA4YE. The schematic structure of HuABC2-Ck-CTLA4YE is shown in Figure 7A.

[0138] Example 5: Fusion of CTLA-4 at the N-terminus of the HuABC2 heavy chain An alternative form of HuABC2 fused to CTLA-4 was constructed by modifying the VH exon encoding the HuABC2 VH in pHuABC2p. A variant CTLA-4 (SEQ ID NO: 14) preceded by a synthetic signal peptide (MGWSWIFFFLLSGTASVLS; SEQ ID NO: 35) and followed by a flexible polypeptide linker (SGGGGSGGGGS; SEQ ID NO: 36) was fused to the N-terminus of mature HuABC2 VH (SEQ ID NO: 37) to generate the CTLA-4-HuABC2 VH fusion (CTLA4YE-VH fusion). The amino acid sequence of the CTLA4YE-VH fusion is: MGWSWIFFFLLSGTASVLSMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD SGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFYMEWVRQAPGKGLEWIAASRNKANDYTTEYSASVKGRFIVSRDDSKNSLYLQMNSLKTEDTAVYYCARSYYRYDGMDYWGQGTTVTVSS (SEQ ID NO: 38) The linker sequence is underlined. The mature CTLA4YE-VH fusion begins at position 20 of SEQ ID NO:38.

[0139] The resulting expression vector derived from pHuABC2p was named pHC640. Both leucine residues at positions 234 and 235 (EU numbering) of CH2 of pHC640 were changed to alanine residues to eliminate binding to Fcγ receptors. The schematic structure of pHC640 is shown in Figure 6B.

[0140] The amino acid sequence of the mature heavy chain of HuABC2 fused to the extracellular domain of variant CTLA-4 followed by a linker (underlined) at the N-terminus encoded in pHC640 is: MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD SGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDFYMEWVRQAPGKGLEWIAASRNKANDYTTEYSASVKGRFIVSRDDSKNSLYLQMNSLKTEDTAVYYCARSYYRYDGMDYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 39) It is.

[0141] The amino acid sequence of the mature light chain encoded in pHC640 is identical to the mature light chain sequence encoded in pHuABC2p (SEQ ID NO:9).

[0142] The modified HuABC2 fused to variant CTLA-4 at the N-terminus of the mature heavy chain encoded in pHC640 was designated HuABC2-CTLA4YE-VH. The schematic structure of HuABC2-CTLA4YE-VH is shown in Figure 7B.

[0143] Example 6: Fusion of CTLA-4 at the N-terminus of the HuABC2 light chain An additional form of HuABC2 fused to CTLA-4 was constructed by modifying the Vk exon encoding the HuABC2 VL in pHuABC2p. A variant CTLA-4 (SEQ ID NO:14) preceded by a synthetic signal peptide (SEQ ID NO:35) and followed by a flexible polypeptide linker (SGGGGSGGGGS; SEQ ID NO:36) was fused to the N-terminus of mature HuABC2 VL (SEQ ID NO:40) to generate the CTLA-4-HuABC2 VL fusion (CTLA4YE-Vk fusion). The amino acid sequence of the CTLA4YE-Vk fusion is: MGWSWIFFFLLSGTASVLSMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD SGGGGSGGGGS EIVLTQSPATLSLSPGERATLSCSAISSVSYMYWYQQKPGQAPRLLIYDTSNLVSGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWNTYPYTFGGGTKVEIK (SEQ ID NO: 41) The linker sequence is underlined. The mature CTLA4YE-Vk fusion begins at position 20 of SEQ ID NO:41.

[0144] The resulting expression vector derived from pHuABC2p was named pHC641. Both leucine residues at positions 234 and 235 (EU numbering) of CH2 of pHC641 were changed to alanine residues to eliminate binding to Fcγ receptors. The schematic structure of pHC641 is shown in Figure 6C.

[0145] The amino acid sequence of the mature heavy chain encoded by pHC641 is identical to the mature heavy chain sequence encoded by pHC623 (SEQ ID NO:33).

[0146] The amino acid sequence of the mature light chain of HuABC2 fused at its N-terminus to the extracellular domain of variant CTLA-4, followed by a linker (underlined), encoded in pHC641 is: MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD SGGGGSGGGGS EIVLTQSPATLSLSPGERATLSCSAISSVSYMYWYQQKPGQAPRLLIYDTSNLVSGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWNTYPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 42) It is.

[0147] The modified HuABC2 fused to variant CTLA-4 at the N-terminus of the mature light chain, encoded in pHC641, was designated HuABC2-CTLA4YE-Vk. The schematic structure of HuABC2-CTLA4YE-Vk is shown in Figure 7C.

[0148] Example 7: Expression and purification of HuABC2 fused to CTLA-4 CHO-K1 stable transfectants carrying each of the five expression vectors (pHuABC2p, pHC622, pHC623, pHC640, and pHC641) were generated as described above. Antibody expression in the culture supernatant was analyzed by sandwich ELISA as described above. CHO-K1 stable transfectants producing high levels of each of HuABC2, HuABC2-CTLA4YE (also called HuABC2-CH3-CTLA4YE), HuABC2-Ck-CTLA4YE, HuABC2-CTLA4YE-VH, and HuABC2-CTLA4YE-Vk antibodies were expanded in SFM4CHO. HuABC2 and its CTLA-4 fusion were purified from the culture supernatant by protein A column chromatography as described above.

[0149] Purified HuABC2, HuABC2-CH3-CTLA4YE, HuABC2-Ck-CTLA4YE, HuABC2-CTLA4YE-VH and HuABC2-CTLA4YE-Vk were characterized by SDS-PAGE under reducing conditions according to standard procedures. Each of these antibodies was found to be composed of two major polypeptides. Their approximate sizes were 250 kD and 25 kD for HuABC2, 65 kD and 25 kD for HuABC2-CH3-CTLA4YE, 50 kD and 40 kD for HuABC2-Ck-CTLA4YE, 65 kD and 25 kD for HuABC2-CTLA4YE-VH, and 50 kD and 40 kD for HuABC2-CTLA4YE-Vk.

[0150] Example 8: Binding of HuABC2 fused to CTLA-4 to CD80, CD86 and CD122 To analyze the properties of HuABC2-CH3-CTLA4YE, HuABC2-Ck-CTLA4YE, HuABC2-CTLA4YE-VH and HuABC2-CTLA4YE-Vk, two expression vectors (pFCm553 and pFCm554) were constructed to express recombinant human CD80 and CD86, respectively, on the cell surface.

[0151] The expression vector pFCm553 (FIG. 8A) has the same structure as pHuABC2p, except that (a) the light chain transcription unit has been deleted, and (b) the SpeI-EagI fragment encoding the heavy chain of HuABC2 has been replaced with a DNA fragment (SEQ ID NO:35) encoding a synthetic signal peptide followed by the extracellular domain of human CD80 (SEQ ID NO:43), a FLAG polypeptide (DYKDDDDK; SEQ ID NO:44) and a GPI anchor signal from human CD55 (PNKGSGTTSGTTRLLSGHTCFTLTTGLLGTLVTMGLLT; SEQ ID NO:45) (CD80-FLAG-GPI). The amino acid sequence of mature CD80-FLAG-GPI is: VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNTGGGDYKDDDDKGGGPNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO: 46) It is.

[0152] In another expression vector, pFCm554 (FIG. 8B), the CD80 coding region of pFCm553 is replaced by the coding region for the extracellular domain of human CD86 (SEQ ID NO: 47) to express human CD86 fused to a FLAG peptide and a GPI anchor signal (CD86-FLAG-GPI). The amino acid sequence of mature CD86-FLAG-GPI is: LKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHTGGDYKDDDDKGGGPNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO: 48) It is.

[0153] CHO-K1 stable transfectants expressing CD80-FLAG-GPI (CHO-K1 / CD80) and CD86-FLAG-GPI (CHO-K1 / CD86) were generated by electroporation with pFCm553 and pFCm554, respectively, as described above. Binding of HuABC2-CH3-CTLA4YE, HuABC2-Ck-CTLA4YE, HuABC2-CTLA4YE-VH, and HuABC2-CTLA4YE-Vk to CHO-K1 / CD80 and CHO-K1 / CD86 cells was analyzed by flow cytometry. Belatacept (also known as LEA29Y and Nulojix; SEQ ID NO: 49) is a CTLA4-Ig fusion protein with an alanine to tyrosine substitution at position 29 of SEQ ID NO: 10 (A29Y) and another leucine to glutamic acid substitution at position 104 (L104E) (Larsen et al., Am. J. Transplant. 5:443-453, 2005; Oshima et al., Protein Eng. Des. Sel. 29:159-167, 2016). It was purchased from Creative Biomart (Cat. No. THP-0109, Shirley, NY) and used as a reference in the experiments.

[0154] Purified test samples (HuABC2-CH3-CTLA4YE, HuABC2-Ck-CTLA4YE, HuABC2-CTLA4YE-VH and HuABC2-CTLA4YE-Vk) and belatacept, which were diluted 4-fold starting at 10 μg / ml, were incubated with CHO-K1 / CD80 or CHO-K1 / CD86 cells in FACS buffer (PBS containing 0.5% BSA and 0.05% sodium azide) for 30 min at 4°C. After washing with FACS buffer, cells were incubated with PE-labeled goat anti-human IgG antibody (SouthernBiotech, Birmingham, AL) in FACS buffer for 20 min at 4°C. After washing and suspension in FACS buffer, stained cells were subjected to flow cytometry.

[0155] The binding patterns of HuABC2-CH3-CTLA4YE, HuABC2-Ck-CTLA4YE, HuABC2-CTLA4YE-VH, HuABC2-CTLA4YE-Vk and belatacept to CHO-K1 / CD80 cells are shown in Figure 9 A. The binding of HuABC2-CTLA4YE-VH and HuABC2-CTLA4YE-Vk to CD80 was slightly weaker than that of belatacept, while the binding of HuABC2-CH3-CTLA4YE and HuABC2-Ck-CTLA4YE was much weaker than that of belatacept. For example, the mean channel fluorescence (MCF) values ​​for binding to CHO-K1 / CD80 cells at 625 ng / ml were 1,583 for belatacept, 425 for HuABC2-CH3-CTLA4YE, 307 for HuABC2-Ck-CTLA4YE, 1,313 for HuABC2-CTLA4YE-VH, and 1,310 for HuABC2- CTLA4YE-Vk. According to the package insert for Nulojix (belatacept, Bristol Myers Squibb), the molecular weight of belatacept is approximately 90 kilodaltons (kD), roughly half the molecular weight of HuABC2-CTLA4YE-VH and HuABC2-CTLA4YE-Vk (both approximately 180 kD), and the binding of HuABC2-CTLA4YE-VH and HuABC2-CTLA4YE-Vk to CD80 (where CTLA-4 is fused to the N-terminus of the heavy and light chains, respectively) was comparable to that of belatacept on a molar basis. In contrast, the binding of HuABC2-CH3-CTLA4YE and HuABC2-Ck-CTLA4YE, where CTLA-4 is fused to the C-terminus of the heavy and light chains, respectively, was unexpectedly much weaker than that of belatacept.

[0156] The binding patterns of HuABC2-CH3-CTLA4YE, HuABC2-Ck-CTLA4YE, HuABC2-CTLA4YE-VH, HuABC2-CTLA4YE-Vk and belatacept to CHO-K1 / CD86 cells are shown in Figure 9B. As in the case of CHO-K1 / CD80 cells, the binding of HuABC2-CTLA4YE-VH and HuABC2-CTLA4YE-Vk to CD86 was slightly weaker than that of belatacept, while the binding of HuABC2-CH3-CTLA4YE and HuABC2-Ck-CTLA4YE was much weaker than that of belatacept. For example, the MCF values ​​for binding to CHO-K1 / CD86 cells at 625 ng / ml were 1,179 for belatacept, 221 for HuABC2-CH3-CTLA4YE, 13 for HuABC2-Ck-CTLA4YE, 1,288 for HuABC2-CTLA4YE-VH, and 956 for HuABC2-CTLA4YE-Vk. While the binding of HuABC2-CTLA4YE-VH and HuABC2-CTLA4YE-Vk to CD86 was comparable to that of belatacept on a molar basis, the binding of HuABC2-CH3-CTLA4YE and HuABC2-Ck-CTLA4YE was unexpectedly much weaker than that of belatacept.

[0157] The binding characteristics of HuABC2-CH3-CTLA4YE, HuABC2-Ck-CTLA4YE, HuABC2-CTLA4YE-VH, HuABC2-CTLA4YE-Vk and belatacept were further investigated using the human Burkitt's lymphoma cell line Ramos by flow cytometry as described above. The results are shown in Figure 9C. The MCF values ​​for binding to Ramos cells at 625 ng / ml were 539 for belatacept, 63 for HuABC2-CH3-CTLA4YE, 60 for HuABC2-Ck-CTLA4YE, 611 for HuABC2-CTLA4YE-VH and 715 for HuABC2-CTLA4YE-Vk. As observed with CHO-K1 / CD80 and CHO-K1 / CD86 cells, the binding of HuABC2-CTLA4YE-VH and HuABC2-CTLA4YE-Vk to Ramos cells was comparable to that of belatacept, whereas the binding of HuABC2-CH3-CTLA4YE and HuABC2-Ck-CTLA4YE was much weaker than that of belatacept.

[0158] Example 9: Modification of the linker between CH3 and CTLA-4 HuABC2-CH3-CTLA4YE showed weaker binding to CD80 and CD86 when compared with belatacept and HuABC2-CTLA4YE-VH (Figure 9A and B). To evaluate the effect of a flexible polypeptide linker between CH3 and CTLA-4 on binding to CD80 and CD86, two new expression vectors were generated by modifying the sequence of the polypeptide linker (SGGGGSG; SEQ ID NO: 11) connecting CH3 and CTLA-4 in pHC625, which encodes HuABC2-CH3-CTLA4YE. The sequence of the polypeptide linker between CH3 and CTLA-4 in one of the resulting vectors, pHC634, is SGGGGSGGGGS (SEQ ID NO: 36). The modified HuABC2-CH3-CTLA4YE expressed from pHC634 was named HuABC2-CH3-CTLA4YE-X. The polypeptide linker sequence between CH3 and CTLA-4 in the resulting vector pHC635 was SGGGGSGGGGSGGGGS (SEQ ID NO: 50). The modified HuABC2-CH3-CTLA4YE expressed from pHC635 was designated HuABC2-CH3-CTLA4YE-Y.

[0159] Each of the three expression vectors (pHC625, pHC634, and pHC635) was transfected into HEK293 cells as described above. The expression levels of HuABC2-CH3-CTLA4YE, HuABC2-CH3-CTLA4YE-X, and HuABC2-CH3-CTLA4YE-Y in the culture supernatants were measured by sandwich ELISA as described above. The binding of HuABC2-CH3-CTLA4YE, HuABC2-CH3-CTLA4YE-X, and HuABC2-CH3-CTLA4YE-Y to CHO-K1 / CD80 and CHO-K1 / CD86 cells, respectively, was analyzed by flow cytometry as described above. HuABC2-CH3-CTLA4YE, HuABC2-CH3-CTLA4YE-X and HuABC2-CH3-CTLA4YE-Y showed no obvious differences in binding to CD80 and CD86.

[0160] Example 10: Binding to CD122 To assess the possibility that the binding of CTLA-4 at the N-terminus of the heavy or light chain of HuABC2 may prevent its antigen binding, the binding ability of HuABC2-CTLA4YE-VH and HuABC2-CTLA4YE-Vk to CD122 was compared with HuABC2. The expression vector pFCm148 (Figure 8C) has the same structure as pFCm553 (Figure 8A), except that the coding regions of CD80 and puromycin N-acetyltransferase were replaced by the extracellular domain of human CD122 (SEQ ID NO: 51) and xanthine-guanine phosphoribosyltransferase (shown as gpt in Figure 8C), respectively. The amino acid sequence of mature human CD122 fused to the FLAG peptide and GPI anchor signal (CD122-FLAG-GPI) encoded in pFCm148 is: AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTTGGGDYKDDDDKGGGPNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO: 52) It is.

[0161] The mouse myeloma cell line NS0 was stably transfected with pFCm148 by electroporation and selected for expression of xanthine-guanine phosphoribosyltransferase according to standard procedures. NS0 stable transfectants expressing CD122-FLAG-GPI on their surface (NS0 / CD122) were used to analyze binding of HuABC2, HuABC2-CTLA4YE-VH, and HuABC2-CTLA4YE-Vk to CD122.

[0162] HuABC2, HuABC2-CTLA4YE-VH and HuABC2-CTLA4YE-Vk were each diluted four-fold starting at 2.5 μg / ml and incubated with NS0 / CD122 cells in FACS buffer for 30 min at 4° C. After washing with FACS buffer, the cells were incubated with PE-labeled goat anti-human IgG antibody for 20 min at 4° C. After washing and suspending in FACS buffer, the cells were subjected to flow cytometry. As shown in FIG. 10, HuABC2, HuABC2-CTLA4YE-VH and HuABC2-CTLA4YE-Vk showed no clear difference in binding to CD122. Thus, the antigen binding of HuABC2 was not adversely affected by the fusion of CTLA-4 to the N-terminus of its heavy or light chain.

[0163] Example 11: Immunosuppressive activity of HuABC2 fused to CTLA-4 The suppressive activity of HuABC2 fused to CTLA-4 was examined by mixed lymphocyte reaction (MLR) using CD3+ T cells and allogeneic monocyte-derived dendritic cells (Mo-DCs) (Latek et al., Transplantation,87: 926-933, 2009; Goenka et al., J. Immunol, 206:1102-1113, 2021). CD3+ T cells were purified from human peripheral blood mononuclear cells (PBMCs) using the MojoSort Human CD3 T Cell Isolation Kit (BioLegend, San Diego, CA) and labeled with 2.5 μM CellTrace Far Red (Thermo Fisher Scientific). To generate mature Mo-DCs, CD14+ monocytes were isolated from human PBMCs of different donors using CD14 MicroBeads, human (Miltenyi Biotec, Auburn, CA) and incubated for 6 days at 37°C in a 7.5% CO2 incubator in RPMI 1640 medium supplemented with 10% FBS (Life Technologies, Grand Islands, NY), 10 mM HEPES, 1 mM sodium pyruvate, 250 IU / ml human IL-4 (BioLegend), and 800 IU / ml human GM-CSF (BioLegend), and then incubated for 6 days at 37°C in a 7.5% CO2 incubator in RPMI 1640 medium supplemented with 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, 1,000 IU / ml human IL-6 (BioLegend), 200 IU / ml human IL-1β (BioLegend), and 1,000 IU / ml human TNF-α (BioLegend). Grown in 1640 medium for 1 day.

[0164] MLR was performed by mixing CellTrace Far Red-labeled CD3+ T cells and allogeneic mature Mo-DCs 2:1 in RPMI 1640 medium supplemented with 10% FBS, 10 mM HEPES, and 1 mM sodium pyruvate in the presence or absence of 3 μg / ml of each test sample or their combination. After 6 days of incubation at 37°C in a 7.5% CO2 incubator, cells were stained with Helix NP Green (BioLegend) and subjected to flow cytometry. The fluorescence levels of Helix NP Green and CellTrace Far Red were used to monitor cell viability and proliferation, respectively. Mo-DCs that were not stained by CellTrace Far Red and therefore had negative fluorescence were excluded from the analysis. T cell proliferation was monitored by the fluorescence levels of CellTrace Far Red according to the user guide of the CellTrace Far Red Cell Proliferation Kit (Thermo Fisher Scientific) and was calculated by dividing the number of proliferating T cells by the total number of viable T cells.

[0165] Analysis of T cell proliferation in the MLR is shown in Figure 11. The percentage of proliferating T cells in the viable T cell population was 46.3% without test sample, 7.9% with belatacept, 23.2% with HuABC2, 1.7% with the combination of belatacept and HuABC2, 18.4% with HuABC2-CH3-CTLA4YE, 27.9% with HuABC2-Ck-CTLA4YE, 2.5% with HuABC2-CTLA4YE-VH, and 2.4% with HuABC2-CTLA4-Vk. Both HuABC2-CTLA4YE-VH and HuABC2-CTLA4-Vk suppressed T cell proliferation more potently than belatacept and HuABC2, respectively, and almost as efficiently as the combination of HuABC2 and belatacept. The inhibitory activity of HuABC2-CH3-CTLA4YE and HuABC2-Ck-CTLA4YE on T cell proliferation was much weaker than that of HuABC2-CTLA4YE-VH and HuABC2-CTLA4-Vk.

[0166] The levels of the proinflammatory cytokine IFN-γ in the MLR culture supernatants, measured using the ELISA MAX Deluxe Set Human IFN-γ kit (BioLegend), were 8,217 pg / ml without test samples, 744 pg / ml with belatacept, 5,459 pg / ml with HuABC2, 170 pg / ml with the combination of belatacept and HuABC2, 837 pg / ml with HuABC2-CH3-CTLA4YE, 2,984 pg / ml with HuABC2-Ck-CTLA4YE, 167 pg / ml with HuABC2-CTLA4YE-VH, and 182 pg / ml with HuABC2-CTLA4YE-Vk (Figure 12). Both HuABC2-CTLA4YE-VH and HuABC2-CTLA4YE-Vk suppressed IFN-γ production as potently as the combination of HuABC2 and belatacept.

[0167] Example 12: HuABC101 fused to CTLA-4 HuABC101, expressed from the vector pHuABC101 in mammalian cells, is a humanized anti-CD122 IgG1 / κ antibody comprising HuABC101 VH (SEQ ID NO: 29) and HuABC101 VL (SEQ ID NO: 30). The structure of pHuABC101 is the same as pHuABC2p, except that the coding regions for HuABC2 VH, HuABC2 VL, and puromycin N-acetyltransferase are replaced by HuABC101 VH, HuABC101 VL, and xanthine-guanine phosphoribosyltransferase, respectively (Figure 1A).

[0168] The amino acid sequence of the mature heavy chain encoded in pHuABC101 is: QVQLQESGPGLVKPSQTLSLTCTVSGYSITNDNHWWNWIRQHPGKGLEWMGYIDSSGSSDNNPSLKSQITISRDTSKNQLSLKLSSVTAADTAVYYCARGGGRDYYGMDYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 53) It is.

[0169] The amino acid sequence of the mature light chain encoded in HuABC101 is: DIVMTQSPDSLGVSLGERATINCRASQSVSTSSYSYVHWYQQKPGQPPKLLIKYASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSWDIPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 54) It is.

[0170] Mutant CTLA-4 (SEQ ID NO: 14) was fused to the N-terminus of the mature HuABC101 VH in pHuABC101 to generate a new expression vector, pHC642. The coding region for CTLA-4 fused to HuABC101 VH in pHC642 contains, from N-terminus to C-terminus, a synthetic signal peptide (SEQ ID NO: 35), variant CTLA-4 (SEQ ID NO: 14), a flexible polypeptide linker (SGGGGSGGGGS; SEQ ID NO: 36), and the mature HuABC101 VH (SEQ ID NO: 29) (CTLA4YE-HuABC101 VH fusion). The amino acid sequence of the CTLA4YE-HuABC101 VH fusion is: MGWSWIFFFLLSGTASVLSMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD SGGGGSGGGGS QVQLQESGPGLVKPSQTLSLTCTVSGYSITNDNHWWNWIRQHPGKGLEWMGYIDSSGSSDNNPSLKSQITISRDTSKNQLSLKLSSVTAADTAVYYCARGGGRDYYGMDYWGQGTTVTVSS (SEQ ID NO: 55) The linker sequence is underlined. The mature CTLA4YE-HuABC101 VH fusion begins at position 20 of SEQ ID NO:55.

[0171] The amino acid sequence of the mature light chain encoded in pHC642 is the same as the mature light chain sequence encoded in pHuABC101 (SEQ ID NO:54). The amino acid sequence of the mature heavy chain of HuABC101 fused at its N-terminus to the extracellular domain of variant CTLA-4 (followed by a linker (underlined)) encoded in pHC642 is: MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD SGGGGSGGGGSQVQLQESGPGLVKPSQTLSLTCTVSGYSITNDNHWWNWIRQHPGKGLEWMGYIDSSGSSDNNPSLKSQITISRDTSKNQLSLKLSSVTAADTAVYYCARGGGRDYYGMDYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 56) A modified HuABC101 fused to CTLA-4 at the N-terminus of the heavy chain (designated HuABC101-CTLA4YE-VH) is expressed from pHC642.

[0172] Another expression vector was constructed by fusing a flexible polypeptide linker (SEQ ID NO:11) followed by variant CTLA-4 (SEQ ID NO:14) to the penultimate glycine residue of CH3 in pHuABC101. The amino acid sequence of the mature light chain encoded in the resulting vector, pHC630, is the same as the mature light chain sequence encoded in pHuABC101 (SEQ ID NO:54). The amino acid sequence of the mature heavy chain of HuABC101 fused at its C-terminus to a linker (underlined) (followed by the extracellular domain of variant CTLA-4) encoded in pHC630 is: QVQLQESGPGLVKPSQTLSLTCTVSGYSITNDNHWWNWIRQHPGKGLEWMGYIDSSGSSDNNPSLKSQITISRDTSKNQLSLKLSSVTAADTAVYYCARGGGRDYYGMDYWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SGGGGSG MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD (SEQ ID NO: 57) A modified HuABC101 fused to CTLA-4 at the C-terminus of the heavy chain (called HuABC101-CH3-CTLA4YE) is expressed from pHC630.

[0173] The two expression vectors (pHC642 and pHC630) were transfected separately into HEK293 cells as described above. Their expression levels in the culture supernatants were measured by sandwich ELISA as described above. The binding of transiently expressed HuABC101-CTLA4YE-VH and HuABC101-CH3-CTLA4YE to CD80 and CD86 was analyzed by flow cytometry as described above. Belatacept was used as a reference for binding to CD80 and CD86.

[0174] The MCF values ​​for the binding of HuABC101-CTLA4YE-VH, HuABC101-CH3-CTLA4YE and belatacept to CHO-K1 / CD80 cells at 200 ng / ml were 1163, 291 and 1876, respectively. The MCF values ​​for the binding of HuABC101-CTLA4YE-VH, HuABC101-CH3-CTLA4YE and belatacept to CHO-K1 / CD86 cells at 200 ng / ml were 745, 108 and 917, respectively. The binding of HuABC101-CH3-CTLA4YE to CD80 and CD86, respectively, was much weaker than that of HuABC101-CTLA4YE-VH. As observed with HuABC2 fused to CTLA-4, fusion of CTLA-4 to the C-terminus of the heavy chain of HuABC101 unexpectedly reduced binding to CD80 and CD86.

[0175] Example 13: HuABC77D fused to CTLA-4 A murine hybridoma producing IgG / kappa monoclonal antibody ABC77, which binds and blocks the function of human and cynomolgus monkey CD122, was isolated at JN Biosciences (Mountain View, CA) following standard cell fusion techniques using GenomONE CF EX cell fusion reagent (Cosmo Bio, Carlsbad, CA) as described in U.S. Patent No. 9,028,830. Sequencing and humanization of the VH and VL of ABC77 was performed as described in Trushita et al. (Methods 36:69-83, 2005).

[0176] The amino acid sequence of humanized ABC77 VH (HuABC77D VH) is MDFGLIFFIVALLKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHDYDEGIYFDYWGQGTTVTVSS (SEQ ID NO: 58) The amino acid sequences of CDR1, 2 and 3 of HuABC77D VH based on the definition of Kabat et al. (supra) are RYWMS (SEQ ID NO:59), EINPDSSTINYAPSLKD (SEQ ID NO:60) and HDYDEGIYFDY (SEQ ID NO:61), respectively. The mature HuABC77D VH begins at position 19 of SEQ ID NO:58.

[0177] The amino acid sequence of humanized ABC77 VL (HuABC77D VL) is MDFQVQIFSFLLISASVIISRGDIQMTQSPSSLSASVGDRVTITCSASSRVIYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKVEIK (SEQ ID NO: 62) The amino acid sequences of CDR1, 2 and 3 of HuABC77D VL based on the definition of Kabat et al. (supra) are SASSRVIYMH (SEQ ID NO:63), DTSKLAS (SEQ ID NO:64) and QQWSSNPPT (SEQ ID NO:65), respectively. The mature HuABC77D VL begins at position 23 of SEQ ID NO:62.

[0178] The expression vector pHuABC77D is a derivative of pHuABC2p in which the HuABC2 VH and VL coding regions are replaced by the HuABC77D VH and VL coding regions, respectively. Both leucine residues at positions 234 and 235 (EU numbering) of CH2 of pHuABC77D were changed to alanine residues to abolish binding to Fcγ receptors (Hezareh et al., J. Virol. 75:12161-12168, 2001).

[0179] The amino acid sequence of the mature heavy chain encoded in pHuABC77D is: EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHDYDEGIYFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 66) It is.

[0180] The amino acid sequence of the mature light chain encoded in pHuABC77D is: DIQMTQSPSSLSASVGDRVTITCSASSRVIYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 67) It is.

[0181] CHO-K1 cells stably transfected with pHuABC77D were generated and grown in SFM4CHO medium as described above. Humanized ABC77 IgG1 / kappa antibody (HuABC77D) was purified from culture supernatant as described above. HuABC77D showed (i) specific binding to human and cynomolgus CD122, and (ii) blocking the interaction of human CD122 with human IL-2 and IL-15.

[0182] Variant CTLA-4 (SEQ ID NO: 14) was fused to the N-terminus of the mature HuABC77D VH in pHuABC77D to generate a new expression vector, pHC644. The coding region for CTLA-4 fused to HuABC77D VH in pHC644 comprises, from N-terminus to C-terminus, a synthetic signal peptide (SEQ ID NO: 35), variant CTLA-4 (SEQ ID NO: 14), a flexible polypeptide linker (SGGGGSGGGGS; SEQ ID NO: 36), and the mature HuABC77D VH (SEQ ID NO: 68) (CTLA4YE-HuABC77D VH fusion). The amino acid sequence of the mature CTLA4YE-HuABC77D VH fusion is: MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD SGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHDYDEGIYFDYWGQGTTVTVSS (SEQ ID NO: 69) The linker sequences are underlined.

[0183] The amino acid sequence of the mature light chain encoded in pHC644 is the same as the mature light chain sequence encoded in pHuABC77D (SEQ ID NO: 67). The amino acid sequence of the mature heavy chain of HuABC77D fused at its N-terminus to the extracellular domain of variant CTLA-4 (followed by a linker (underlined)) encoded in pHC644 is: MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD SGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHDYDEGIYFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 70) The modified HuABC77D fused to CTLA-4 at the N-terminus of the heavy chain (designated HuABC77D-CTLA4YE-VH) is expressed from pHC644.

[0184] Another expression vector was constructed by fusing mutant CTLA-4 (SEQ ID NO: 14) to the penultimate glycine residue of CH3 in pHuABC77D. The amino acid sequence of the mature light chain encoded in the resulting vector, pHC645, is the same as the mature light chain sequence encoded in pHuABC77D (SEQ ID NO: 67). The amino acid sequence of the mature heavy chain of HuABC77D fused at the C-terminus to a linker (underlined) (followed by the extracellular domain of variant CTLA-4) encoded in pHC645 is: EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHDYDEGIYFDYWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SGGGGSG MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD (SEQ ID NO: 71) A modified HuABC77D fused to CTLA-4 at the C-terminus of the heavy chain (designated HuABC77D-CH3-CTLA4YE) is expressed from pHC645.

[0185] The two expression vectors (pHC644 and pHC645) were transfected individually into HEK293 cells as described above. Their expression levels in the culture supernatants were measured by sandwich ELISA as described above. The binding of HuABC77D-CH3-CTLA4YE and HuABC77D-CTLA4YE-VH to CHO-K1 / CD80 and CHO-K1 / CD86 cells was analyzed by flow cytometry as described above. Belatacept was used as a reference for binding to CD80 and CD86. Test samples were used at 500 ng / ml in four 3-fold serial dilutions.

[0186] The results of binding to CHO-K1 / CD80 and CHO-K1 / CD86 cells are shown in Figures 13A and B, respectively. Belatacept and HuABC77D-CTLA4YE-VH showed similar levels of binding to CD80 and CD86, respectively, while the binding of HuABC77D-CH3-CTLA4YE was much weaker compared to that of belatacept and HuABC77D-CTLA4YE-VH. The MCF values ​​for the binding of belatacept, HuABC77D-CTLA4YE-VH and HuABC77D-CH3-CTLA4YE to CHO-K1 / CD80 cells at 500 ng / ml were 4390, 4594 and 1809, respectively. The MCF values ​​for binding of 500 ng / ml belatacept, HuABC77D-CTLA4YE-VH and HuABC77D-CH3-CTLA4YE to CHO-K1 / CD86 cells were 723, 794 and 118, respectively. As observed with HuABC2 and HuABC101, fusion of CTLA-4 to the C-terminus of the heavy chain unexpectedly reduced binding of HuABC77D to CD80 and CD86.

[0187] The binding of HuABC77D, HuABC77D-CTLA4YE-VH and HuABC77D-CH3-CTLA4YE to CD122 was analyzed by flow cytometry using NS0 / CD122 cells as described above. No significant differences in the CD122 binding patterns were observed between HuABC77D, HuABC77D-CTLA4YE-VH and HuABC77D-CH3-CTLA4YE. This result indicates that the fusion of CTLA-4 at the N-terminus of the HuABC77D heavy chain does not adversely affect the antigen binding of HuABC77D.

[0188] Sequence Listing SEQ ID NO:1 Amino acid sequence of HuABC2 VH MKLWLNWVFLLTLLHGIQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSDFYMEWVRQAPGKGLEWIAASRNKANDYTTEYSASVKGRFIVSRDDSKNSLYLQMNSLKTEDTAVYYCARSYYRYDGMDYWGQGTTVTVSS SEQ ID NO:2 Amino acid sequence of HuABC2 VL MDFQVQIFSFLLISASVIVSRGEIVLTQSPATLSLSPGERATLSCSAISSVSYMYWYQQKPGQAPRLLIYDTSNLVSGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWNTYPYTFGGGTKVEIK SEQ ID NO:3 Amino acid sequence of the CH1 region of the human gamma-1 heavy chain encoded in pHuABC2p. ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV SEQ ID NO:4 Amino acid sequence of the hinge region of the human gamma-1 heavy chain encoded in pHuABC2p. EPKSCDKTHTCPPCP SEQ ID NO:5 Amino acid sequence of the CH2 region of the human gamma-1 heavy chain encoded in pHuABC2p. APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK SEQ ID NO:6 Amino acid sequence of the CH3 region of the human gamma-1 heavy chain encoded in pHuABC2p. GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:7 Amino acid sequence of the Ck region encoded in pHuABC2p TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:8 Amino acid sequence of the mature heavy chain of HuABC2 encoded in pHuABC2p EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFYMEWVRQAPGKGLEWIAASRNKANDYTTEYSASVKGRFIVSRDDSKNSLYLQMNSLKTEDTAVYYCARSYYRYDGMDYWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:9 Amino acid sequence of the mature light chain of HuABC2 encoded in pHuABC2p EIVLTQSPATLSLSPGERATLSCSAISSVSYMYWYQQKPGQAPRLLIYDTSNLVSGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWNTYPYTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:10 Amino acid sequence of the extracellular domain of human CTLA-4 MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSD SEQ ID NO:11 Amino acid sequence of the flexible polypeptide linker SGGGGSG SEQ ID NO:12 Amino acid sequence of the fusion of CH3 with a linker and the extracellular domain of CTLA-4 (CH3-CTLA4 fusion) encoded in pHC624 GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGMHVAQ PAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSD SEQ ID NO:13 The amino acid sequence of the mature heavy chain of HuABC2 fused to a linker at its C-terminus followed by the extracellular domain of CTLA-4, as encoded in pHC624. EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFYMEWVRQAPGKGLEWIAASRNKANDYTTEYSASVKGRFIVSRDDSKNSLYLQMNSLKTEDTAVYYCARSYYRYDGMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGSGGGGSGMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSD SEQ ID NO:14 Amino acid sequence of the extracellular domain of variant human CTLA-4 carrying the A29Y and L104E mutations (variant CTLA-4) MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD SEQ ID NO:15 Amino acid sequence of CH3 fused to a linker at the C-terminus followed by the extracellular domain of variant CTLA-4 encoded in pHC625 and pHC622 GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGMHVAQ PAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD SEQ ID NO:16 Amino acid sequence of the mature heavy chain of HuABC2 fused to a linker at the C-terminus followed by the extracellular domain of variant CTLA-4 as encoded in pHC625 and pHC622 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFYMEWVRQAPGKGLEWIAASRNKANDYTTEYSASVKGRFIVSRDDSKNSLYLQMNSLKTEDTAVYYCARSYYRYDGMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGSGGGGSGMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD SEQ ID NO:17 Amino acid sequence of ABC2 VH CDR1 DFYME SEQ ID NO:18 ABC2 VH CDR2 amino acid sequence ASRNKANDYTTEYSASVKG SEQ ID NO:19 Amino acid sequence of ABC2 VH CDR3 SYYRYDGMDY SEQ ID NO:20 Amino acid sequence of ABC2 VL CDR1 SAISSVSYMY SEQ ID NO:21 Amino acid sequence of ABC2 VL CDR2 DTSNLVS SEQ ID NO:22 Amino acid sequence of ABC2 VL CDR3 QQWNTYPYT SEQ ID NO:23 Amino acid sequence of ABC101 VH CDR1 NDNHWWN SEQ ID NO:24 Amino acid sequence of ABC101 VH CDR2 YIDSSGSSDNNPSLKS SEQ ID NO:25 Amino acid sequence of ABC101 VH CDR3 GGGRDYYGMDY SEQ ID NO:26 Amino acid sequence of ABC101 VL CDR1 RASQSVSTSSYSYVH SEQ ID NO:27 Amino acid sequence of ABC101 VL CDR2 YASNLES SEQ ID NO:28 Amino acid sequence of ABC101 VL CDR3 QHSWDIPFT SEQ ID NO:29 Amino acid sequence of mature HuABC101 VH QVQLQESGPGLVKPSQTLSLTCTVSGYSITNDNHWWNWIRQHPGKGLEWMGYIDSSGSSDNNPSLKSQITISRDTSKNQLSLKLSSVTAADTAVYYCARGGGRDYYGMDYWGQGTTVTVSS SEQ ID NO:30 Amino acid sequence of mature HuABC101 VL DIVMTQSPDSLGVSLGERATINCRASQSVSTSSYSYVHWYQQKPGQPPKLLIKYASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSWDIPFTFGQGTKLEIK SEQ ID NO:31 Amino acid sequence of mature human CD122 AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQA SHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKF FSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYC TFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV SEQ ID NO:32 Amino acid sequence of Ck fused to a linker (followed by the extracellular domain of variant CTLA-4) encoded in pHC623 (Ck-CTLA4YE fusion) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSGGGGSGMHVAQ PAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD SEQ ID NO:33 Amino acid sequence of the mature heavy chain of HuABC2 encoded in pHC623 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFYMEWVRQAPGKGLEWIAASRNKANDYTTEYSASVKGRFIVSRDDSKNSLYLQMNSLKTEDTAVYYCARSYYRYDGMDYWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:34 Amino acid sequence of the mature light chain of HuABC2 fused to a linker at its C-terminus followed by the extracellular domain of variant CTLA-4, as encoded in pHC623 EIVLTQSPATLLSPGERATLSCSAISSVSYMYWYQQKPGQAPRLLIYDTSNLVSGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWNTYPYTFGGGTKVEIKRTVAAPSVFIFPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSGGGGSGMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD SEQ ID NO:35 Amino acid sequence of synthetic signal peptide MGWSWIFFFLLSGTASVLS SEQ ID NO:36 Amino acid sequence of the flexible polypeptide linker SGGGGSGGGGS SEQ ID NO:37 Amino acid sequence of mature HuABC2 VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFYMEWVRQAPGKGLEWIAASRNKANDYTTEYSASVKGRFIVSRDDSKNSLYLQMNSLKTEDTAVYYCARSYYRYDGMDYWGQGTTVTVSS SEQ ID NO:38 Amino acid sequence of mature HuABC2 VH fused at the N-terminus to a signal peptide followed by a linker and the extracellular domain of variant CTLA-4 (CTLA4YE-VH fusion) MGWSWIFFFLLSGTASVLSMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPE PCPDSDSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDFYMEWVRQAPGKGLEWIAASRNKANDYTTEYSASVKGRFIVSRDDSKNSLYLQMNSLKTEDTAVYYCARSYYRYDGMDYWGQGTTVTVSS SEQ ID NO:39 Amino acid sequence of the mature heavy chain of HuABC2 fused at its N-terminus to the extracellular domain of variant CTLA-4, followed by a linker, encoded in pHC640 MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDSGGGGSGGGGSEVQLVESGGGL VQPGGSLRLSCAASGFTFSDFYMEWVRQAPGKGLEWIAASRNKANDYTTEYSASVKGRFIVSRDDSKNSLYLQMNSLKTEDTAVYYCARSYYRYDGMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:40 Amino acid sequence of mature HuABC2 VL EIVLTQSPATLSLSPGERATLSCSAISSVSYMYWYQQKPGQAPRLLIYDTSNLVSGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWNTYPYTFGGGTKVEIK SEQ ID NO:41 Amino acid sequence of mature HuABC2 VL fused at the N-terminus to the extracellular domain of variant CTLA-4 (followed by a linker) (CTLA4YE-Vk fusion) MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPC PDSDSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSAISSVSYMYWYQQKPGQAPRLLIYDTSNLVSGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWNTYPYTFGGGTKVEIK SEQ ID NO:42 Amino acid sequence of the mature light chain of HuABC2 fused at its N-terminus to the extracellular domain of variant CTLA-4, followed by a linker, encoded in pHC641 MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDSGGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSAISSVSYMYWYQQKP GQAPRLLIYDTSNLVSGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWNTYPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:43 Amino acid sequence of the extracellular domain of human CD80 VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDN SEQ ID NO:44 Amino acid sequence of the FLAG polypeptide DYKDDDDK SEQ ID NO:45 Amino acid sequence of the GPI anchorage signal from human CD55 PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT SEQ ID NO:46 Amino acid sequence of mature CD80-FLAG-GPI VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICST SGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNTGGGDYKDDDDKGGGPNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT SEQ ID NO:47 Amino acid sequence of the extracellular domain of human CD86 LKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLAN FSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSSPFSIELEDPQPPPDH SEQ ID NO:48 Amino acid sequence of mature CD86-FLAG-GPI LKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIH GYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHTGGGDYKDDDDKGGGPNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT SEQ ID NO:49 Belatacept amino acid sequence MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:50 Amino acid sequence of the polypeptide linker SGGGGSGGGGSGGGGS SEQ ID NO:51 Amino acid sequence of the extracellular domain of human CD122 AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRRVLCREGVRWRVMAIQDFKPFENLRLM APISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDT SEQ ID NO:52 Amino acid sequence of CD122-FLAG-GPI encoded in pFCm148 AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASH YFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTTGGGDYKDDDDKGGGPNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT SEQ ID NO:53 Amino acid sequence of the mature heavy chain of HuABC101 encoded in pHuABC101 QVQLQESGPGLVKPSQTLSLTCTVSGYSITNDNHWWNWIRQHPGKGLEWMGYIDSSGSSDNNPSLKSQITISRDTSKNQLSLKLSSVTAADTAVYYCARGGGRDYYGMDYWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:54 Amino acid sequence of the mature light chain of HuABC101 encoded in pHuABC101 DIVMTQSPDSLGVSLGERATINCRASQSVSTSSYSYVHWYQQKPGQPPKLLIKYASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSWDIPFTFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:55 Amino acid sequence of mature HuABC101 VH fused at the N-terminus to the extracellular domain of variant CTLA-4 (followed by a linker) (CTLA4YE-HuABC101 VH fusion) MGWSWIFFFLLSGTASVLSMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPE PCPDSDSGGGGSGGGGSQVQLQESGPGLVKPSQTLSLTCTVSGYSITNDNHWWNWIRQHPGKGLEWMGYIDSSGSSDNNPSLKSQITISRDTSKNQLSLKLSSVTAADTAVYYCARGGGRDYYGMDYWGQGTTVTVSS SEQ ID NO:56 Amino acid sequence of the mature heavy chain of HuABC101 fused at its N-terminus to the extracellular domain of variant CTLA-4, followed by a linker, encoded in pHC642 MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDSGGGGSGGGGSQVQLQESGPGL VKPSQTLSLTCTVSGYSITNDNHWWNWIRQHPGKGLEWMGYIDSSGSSDNNPSLKSQITISRDTSKNQLSLKLSSVTAADTAVYYCARGGGRDYYGMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:57 Amino acid sequence of the mature heavy chain of HuABC101 fused to a linker at its C-terminus followed by the extracellular domain of variant CTLA-4, as encoded in pHC630 QVQLQESGPGLVKPSQTLSLTCTVSGYSITNDNHWWNWIRQHPGKGLEWMGYIDSSGSSDNNPSLKSQITISRDTSKNQLSLKLSSVTAADTAVYYCARGGGRDYYGMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGSGGGGSGMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD SEQ ID NO:58 Amino acid sequence of HuABC77D VH MDFGLIFFIVALLKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEIPDSSTINYAPSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHDYDEGIYFDYWGQGTTVTVSS SEQ ID NO:59 Amino acid sequence of HuABC77D VH CDR1 RYWMS SEQ ID NO:60 Amino acid sequence of HuABC77D VH CDR2 EINPDSSTINYAPSLKD SEQ ID NO:61 Amino acid sequence of HuABC77D VH CDR3 HDYDEGIYFDY SEQ ID NO:62 Amino acid sequence of HuABC77D VL MDFQVQIFSFLLISASVIISRGDIQMTQSPSSLSASVGDRVTITCSASSRVIYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTEFLTISSLQPEDFATYYCQQWSSNPPTFGGGTKVEIK SEQ ID NO:63 Amino acid sequence of HuABC77D VL CDR1 SASSRVIYMH SEQ ID NO:64 Amino acid sequence of HuABC77D VL CDR2 DTSKLAS SEQ ID NO:65 Amino acid sequence of HuABC77D VL CDR3 QQWSSNPPT SEQ ID NO:66 Amino acid sequence of the mature heavy chain of HuABC77D encoded in pHuABC77D EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHDYDEGIYFDYWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:67 Amino acid sequence of the mature light chain of HuABC77D encoded in pHuABC77D DIQMTQSPSSLSASVGDRVTITCSASSRVIYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:68 Amino acid sequence of mature HuABC77D VH EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHDYDEGIYFDYWGQGTTVTVSS SEQ ID NO:69 Amino acid sequence of mature HuABC77D VH fused at the N-terminus to the extracellular domain of variant CTLA-4 followed by a linker (CTLA4YE-HuABC77D VH fusion) MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDSGG GGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHDYDEGIYFDYWGQGTTVTVSS SEQ ID NO:70 Amino acid sequence of the mature heavy chain of HuABC77D fused at its N-terminus to the extracellular domain of variant CTLA-4, followed by a linker, encoded in pHC644 MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDSGGGGSGGGGSEVQLVESGGGL VQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHDYDEGIYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:71 Amino acid sequence of the mature heavy chain of HuABC77D fused to a linker at its C-terminus followed by the extracellular domain of variant CTLA-4, as encoded in pHC645 EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHDYDEGIYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPNSNTKVDKKVEPCSDKTHTCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSGGGSGMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD

Claims

1. A bifunctional molecule comprising an antibody that binds to CD122 and inhibits the interaction of CD122 with IL-2 and IL-15, wherein the antibody is bound to the extracellular domain of CTLA-4 at its N-terminus or C-terminus.

2. The bifunctional molecule of claim 1, wherein the antibody comprises a heavy chain comprising a mature heavy chain variable region bound to a heavy chain constant region and a light chain comprising a mature light chain variable region bound to a light chain constant region.

3. The bifunctional molecule of claim 2, wherein the antibody is linked to the extracellular domain of CTLA-4 via the C-terminus of the heavy chain constant region or the C-terminus of the light chain constant region.

4. The bifunctional molecule of claim 2, wherein the antibody is linked to the extracellular domain of CTLA-4 via the N-terminus of the mature heavy chain variable region or the N-terminus of the mature light chain variable region.

5. The bifunctional molecule of any one of claims 1 to 4, wherein the antibody is linked to the extracellular domain of CTLA-4 via a polypeptide linker.

6. The bifunctional molecule of claim 5 , wherein the polypeptide linker comprises the amino acid sequence of SEQ ID NO: 11, 36 or 50.

7. 7. The bifunctional molecule of any one of claims 2 to 6, in the form of a heterodimer comprising two copies of a heavy chain and two copies of a light chain.

8. The bifunctional molecule of any one of claims 1 to 7, wherein the antibody comprises three heavy chain CDRs comprising SEQ ID NOs: 17-19, respectively, and three light chain CDRs comprising SEQ ID NOs: 20-22, respectively.

9. The bifunctional molecule of claim 8, wherein the antibody comprises a mature heavy chain variable region comprising SEQ ID NO: 37 and a light chain variable region comprising SEQ ID NO:

40.

10. The bifunctional molecule of any one of claims 1 to 9, wherein the heavy chain comprises SEQ ID NO: 8, where the C-terminal lysine can be omitted, and the light chain comprises SEQ ID NO:

9.

11. The bifunctional molecule of claim 3, wherein the mature heavy chain is linked to the extracellular domain of the CTLA-4 as a chain comprising SEQ ID NO: 13 or 16, and the mature light chain comprises SEQ ID NO:

9.

12. The bifunctional molecule of claim 3, wherein the mature light chain is attached to the extracellular domain of the CTLA-4 as a chain comprising SEQ ID NO: 34, and the mature heavy chain comprises SEQ ID NO: 8 or 33, with the C-terminal lysine optional.

13. The bifunctional molecule of claim 4, wherein the mature heavy chain is attached to the extracellular domain of the CTLA-4 as a chain comprising SEQ ID NO: 39, with the C-terminal lysine omitted, and the mature light chain comprises SEQ ID NO:

9.

14. The bifunctional molecule of claim 4, wherein the mature light chain is linked to the extracellular domain of the CTLA-4 as a chain comprising SEQ ID NO: 42, and the mature heavy chain comprises SEQ ID NO: 8 or 33, with the C-terminal lysine optional.

15. The bifunctional molecule of any one of claims 1 to 7, wherein the antibody comprises three heavy chain CDRs comprising SEQ ID NOs: 23-25, respectively, and three light chain CDRs comprising SEQ ID NOs: 26-28, respectively.

16. The bifunctional molecule of claim 15, wherein the antibody comprises a mature heavy chain variable region comprising SEQ ID NO:29 and a mature light chain variable region comprising SEQ ID NO:

30.

17. The bifunctional molecule of claim 15, wherein the antibody comprises a mature heavy chain comprising SEQ ID NO: 53, in which the C-terminal lysine can be omitted, and a mature light chain comprising SEQ ID NO:

54.

18. The bifunctional molecule of claim 3, wherein the mature heavy chain is attached to the extracellular domain of the CTLA-4 as a chain comprising SEQ ID NO:57 and the mature light chain comprises SEQ ID NO:

54.

19. The bifunctional molecule of claim 4, wherein the mature heavy chain is attached to the extracellular domain of the CTLA-4 as a chain comprising SEQ ID NO: 56, with the C-terminal lysine omitted, and the mature light chain comprises SEQ ID NO:

54.

20. The bifunctional molecule of any one of claims 1 to 7, wherein the antibody comprises three heavy chain CDRs comprising SEQ ID NOs: 59-61, respectively, and three light chain CDRs comprising SEQ ID NOs: 63-65, respectively.

21. The bifunctional molecule of claim 20, wherein the mature heavy chain variable region comprises SEQ ID NO: 58 and the mature light chain variable region comprises SEQ ID NO:

62.

22. 21. The bifunctional molecule of claim 20, wherein the mature heavy chain comprises SEQ ID NO: 66, with the C-terminal lysine optional, and the mature light chain comprises SEQ ID NO:

67.

23. The bifunctional molecule of claim 3, wherein the mature heavy chain is attached to the extracellular domain of CTLA-4 as a chain comprising SEQ ID NO: 71 and the mature light chain comprises SEQ ID NO:

67.

24. The bifunctional molecule of claim 4, wherein the mature heavy chain is attached to the extracellular domain of the CTLA-4 as a chain comprising SEQ ID NO: 70, with the C-terminal lysine omitted, and the mature light chain comprises SEQ ID NO:

67.

25. The bifunctional molecule of any one of claims 1 to 24, wherein the antibody comprises a human IgG1κ isotype.

26. 26. The bifunctional molecule of any one of claims 2 to 25, wherein the heavy chain constant region has one or more mutations that reduce effector function.

27. 27. The bifunctional molecule of claim 26, wherein the mutations are L234A and L235A.

28. The bifunctional molecule of claim 1 , wherein the extracellular domain is that of human CTLA-4.

29. The bifunctional molecule of claim 1, wherein the extracellular domain of CTLA-4 comprises SEQ ID NO:

10.

30. The bifunctional molecule of claim 1, wherein the extracellular domain of CTLA-4 comprises SEQ ID NO:

14.

31. 31. A pharmaceutical composition comprising the bifunctional molecule of any one of claims 1 to 30 and a pharma- ceutically acceptable carrier.

32. 31. A pharmaceutical composition for treating a subject having an immune disorder, said treatment comprising administering to said subject an effective regimen of a bifunctional molecule according to any one of claims 1 to 30.

33. 33. The pharmaceutical composition of claim 32, wherein the immune disorder is any one of vitiligo, alopecia areata, type 1 diabetes, celiac disease, nonalcoholic steatohepatitis, skin allograft rejection, multiple sclerosis, polymyositis, allergic dermatitis, primary biliary cirrhosis, Behcet's disease, and ulcerative colitis.

34. 33. The pharmaceutical composition of claim 32, wherein the immune disorder is transplant rejection or graft-versus-host disease.

35. 34. The pharmaceutical composition of claim 33, wherein the immune disorder is an autoimmune disease.

36. An antibody against human CD122, said antibody comprising three heavy chain CDRs comprising SEQ ID NOs: 59-61, respectively, and three light chain CDRs comprising SEQ ID NOs: 63-65, respectively.

37. 37. The antibody of claim 36, comprising a mature heavy chain variable region comprising SEQ ID NO:58 and a mature light chain variable region comprising SEQ ID NO:

62.

38. 37. The antibody of claim 36, wherein the mature heavy chain comprises SEQ ID NO: 66, with the C-terminal lysine optional, and the mature light chain comprises SEQ ID NO: 67.

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