WO2014168715A1 - B7 ligands/cd20 inhibitor composition and use thereof - Google Patents

Abstract

A therapeutic composition is described that can be used for treating or prevention of diseases association with modulation of activity of human CD80 or human CD20. The disclosed invention is based, on engineering of a heterotrimeric protein assembly that is capable of simultaneous binding to human CD80 and human CD20 and attenuating their functions. The heterotrimeric protein assembly comprises an extracellular portions of human CTLA-4 and a heavy and a light chains of an anti-CD20 antibody, or their functional fragments. Each, the CTLA-4 portion and the anti-CD20 antibody portion, is fused to a distinct mutant of Fc portion of the human Ig Gamma- 1. The two distinct Fc mutants in the heterotrimeric protein assembly are engineered as to favor the heteromeric dimer formation between the two Fc mutants over any homomeric assembly. DNA expression vectors and expression systems for overproducing the polypeptides in mammalian cells are also provided for.

Description

B7 LIGANDS/CD20 INHIBITOR COMPOSITION AND USE THEREOF

FIELD OF THE INVENTION

Generally, the invention relates to the field of biological pharmaceuticals as well as their use in conditions associated with inflammatory disorders, e.g rheumatoid arthritis, multiple sclerosis etc. More specifically, the invention relates to a heterodimeric CTLA-4/anti-CD20 antibody -derived composition that is capable of inhibiting B7 ligands (CD80 and CD86) and CD20 protein.

BACKGROUND CTLA-4 (Cytotoxic T-Lymphocyte Antigen 4), also known as CD 152 (Cluster of

differentiation 152), is a cell surface receptor that is found on the surface of T cells which play a central role in cell-mediated immunity. CTLA-4 is a member of the immunoglobulin superfamily, which is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA-4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2 respectively, on antigen-presenting cells (APCs). CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA-4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.

Abatacept is a fusion protein composed of the Fc region of the immunoglobulin IgGl fused to the extracellular domain of CTLA-4. It is a molecule capable of binding to CD80 and CD86. Abatacept is a selective co-stimulation modulator as it inhibits the co-stimulation of T cells.

Abatacept prevents APCs from delivering the co-stimulatory signal to T cells to fully activate them. Ordinarily, full T cell activation requires binding of the T cell receptor to the antigen-MHC complex on the APC and a co-stimulatory signal provided by the binding of CD28 to B7 on the APC. Abatacept acts by binding to B7 molecules on APCs and preventing them from delivering the co-stimulatory signal to T cells, thus preventing the full activation of T cells.

Abatacept under the trade name ORENCIA was approved by the U.S. Food and Drug

Administration (FDA) for the treatment of rheumatoid arthritis (RA). Further clinical trials of rilonacept are currently under way, i.e. for ulcerative colitis, type 1 diabetes, multiple sclerosis, lupus nephritis, etc. This CTLA-4 fusion protein is described, for examples, in U.S. Pat. No. 5,851,795 and U.S. Pat. No. 7,455,835 (all of which are incorporated by reference herein in their entirety).

B-lymphocyte antigen CD20 or CD20 is an activated-glycosylated phosphoprotein expressed on the surface of all B-cells progressively increasing in concentration until maturity. This gene encodes a B-lymphocyte surface molecule that plays a role in the development and differentiation of B-cells into plasma cells. The protein has no known natural ligand and its function is to enable optimal B-cell immune response, specifically against T-independent antigens. CD20 is widely expressed on B cells, from early pre-B cells to later in differentiation, but it is absent on terminally differentiated plasma cells. CD20 does not shed, modulate or internalise.

Rituximab is a chimeric monoclonal antibody against CD20. Rituximab destroys B cells, and is therefore used to treat diseases which are characterized by excessive numbers of B cells, overactive B cells, or dysfunctional B cells. This includes many lymphomas, leukemias, transplant rejection, and autoimmune disorders. Rituximab is described in U.S. Pat. No. 5,843,439, U.S. Pat. No. 5,776,456 and U.S. Pat. No. 5,736,137 (all of which are incorporated by reference herein in their entirety).

Rituximab has been shown to be an effective RA treatment in three randomised controlled trials and is now approved for use in refractory RA. Rituximab under the trade name RETUXAN was approved by the FDA for the treatment of RA for use in combination with methotrexate (MTX) for reducing signs and symptoms in adult patients with moderately to severely active RA who have had an inadequate response to one or more anti-TNF-alpha therapy. There is some evidence for efficacy of rituximab in a range of other autoimmune diseases, and rituximab is widely used off- label to treat difficult cases of multiple sclerosis, systemic lupus erythematosus, and autoimmune anemias.

Other autoimmune diseases that have been treated with rituximab include autoimmune hemolytic anemia, pure red cell aplasia, idiopathic thrombocytopenic purpura (ITP), Evans syndrome, vasculitis (for example Wegener's Granulomatosis), bullous skin disorders (for example pemphigus, pemphigoid), type 1 diabetes mellitus, Sjogren's syndrome, and Devic's disease, and Graves' disease ophthalmopathy.

CD20 is the target of other therapeutic monoclonal antibodies (mAb), e.g. Ibritumomab tiuxetan, and tositumomab, since CD20 is a well established therapeutic molecular target. SUMMARY OF THE INVENTION

This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

In certain aspects, the present invention provides for a heterotrimeric protein composition capable of binding human CD80 protein (NCBI Accession: NP 005182) or human CD20 protein (NCBI Accession: NP 690605). The protein composition comprises a first polypeptide which includes a first amino acid sequence which contains amino acids 38 through 161 of human CTLA- 4 (NCBI Accession: NP_005205.2), and a second amino acid sequence which contains a first mutant of a Fc portion of human immunoglobulin gamma-1 Fc (GenBank: J00228.1). The protein composition also comprises a second polypeptide comprising another first amino acid sequence comprising amino acids of a variable (VH) domain followed by a constant (CHI) domain of a heavy chain of an anti-CD20 human immunoglobulin gamma-1 capable of binding human CD20, and another second amino acid sequence comprising a second mutant of the Fc portion of human immunoglobulin gamma-1 Fc. The protein composition further comprises a third polypeptide comprising a third amino acid sequence comprising a light chain of the anti-CD20 human immunoglobulin gamma-1 capable of binding human CD20. In the protein composition, the first and second mutants are selected as to favor heterodimeric assembly between the first and second mutants over any homodimeric assembly. The protein composition may be capable of exhibiting human IL-Ιβ binding activity in an ELISA assay with an EC50 of about 50 ng/ml. The first polypeptide of the protein composition may contain amino acid sequence of SEQ ID NO. I, while the second polypeptide may contain amino acid sequence of SEQ ID NO. 2, while the third polypeptide may contain amino acid sequence of SEQ ID NO. 3.

In certain aspects, the present invention provides for a therapeutic composition. The therapeutic composition comprises a heterotrimeric protein composition capable of binding human CD80 protein or human CD20 protein. The protein composition comprises a first polypeptide which includes a first amino acid sequence which contains amino acids 38 through 161 of human CTLA- 4, and a second amino acid sequence which contains a first mutant of a Fc portion of human immunoglobulin gamma- 1 Fc. The protein composition also comprises a second polypeptide comprising another first amino acid sequence comprising amino acids of a variable (VH) domain followed by a constant (CHI) domain of a heavy chain of an anti-CD20 human immunoglobulin gamma- 1 capable of binding human CD20, and another second amino acid sequence comprising a second mutant of the Fc portion of human immunoglobulin gamma- 1 Fc. The protein composition further comprises a third polypeptide comprising a third amino acid sequence comprising a light chain of the anti-CD20 human immunoglobulin gamma- 1 capable of binding human CD20. In the protein composition, the first and second mutants are selected as to favor heterodimeric assembly between the first and second mutants over any homodimeric assembly. The therapeutic

composition may exhibit a half-life of the heterotrimeric protein composition in systemic circulation in mice after a subcutaneous administration at a dose of 5 mg/kg of at least about 99 hours, as assayed by human Fc ELISA. The first polypeptide in the protein composition may contain amino acid sequence of SEQ ID NO. 1, while the second polypeptide may contain amino acid sequence of SEQ ID NO. 2, while the third polypeptide may contain amino acid sequence of SEQ ID NO. 3.

In certain aspects, the present invention provides for an isolated nucleic acid encoding a polypeptide comprising amino acid sequence of SEQ ID NO. 4. The codon usage of the nucleic acid may be optimized for high expression of the polypeptide in a mammalian cell. The nucleic acid may contain the sequence of SEQ ID NO. 5. The nucleic acid may comprise an expression vector.

In certain aspects, the present invention provides for an isolated nucleic acid encoding a polypeptide comprising amino acid sequence of SEQ ID NO. 6. The codon usage of the nucleic acid may be optimized for high expression of the polypeptide in a mammalian cell. The nucleic acid may contain the sequence of SEQ ID NO. 7. The nucleic acid may comprise an expression vector.

In certain aspects, the present invention provides for an isolated nucleic acid encoding a polypeptide comprising amino acid sequence of SEQ ID NO. 8. The codon usage of the nucleic acid may be optimized for high expression of the polypeptide in a mammalian cell. The nucleic acid may contain the sequence of SEQ ID NO. 9. The nucleic acid may comprise an expression vector.

In certain aspects, the present invention provides for an isolated nucleic acid of SEQ ID NO. 10. In certain aspects, the present invention provides for a heterologous expression system. The expression system harbors an expression vector comprising a nucleic acid sequence encoding a first polypeptide containing amino acid sequence of SEQ ID NO. 4, another nucleic acid sequence encoding a second polypeptide containing amino acid sequence of SEQ ID NO. 6, and a third nucleic acid sequence encoding a second polypeptide containing amino acid sequence of SEQ ID NO. 8. The expression vector of the expression system may be harbored in a mammalian cell. The mammalian cell may be a HEK293H cell. The expression system may be capable of expressing a heterotrimeric protein comprising a first polypeptide containing amino acid sequence of SEQ ID NO. 1 , a second polypeptide containing amino acid sequence of SEQ ID NO. 2, and a third polypeptide containing amino acid sequence of SEQ ID NO. 3.

In certain aspects the present invention provide for use of a substance for manufacture of a medicament for the treatment or prevention of a disease associated with modulation of activity of human CD80 protein or human CD20 protein. The substance contains a heterotrimeric protein including a first polypeptide comprising amino acid sequence of SEQ ID NO. 1, a second polypeptide comprising amino acid sequence of SEQ ID NO. 2, and a third polypeptide comprising amino acid sequence of SEQ ID NO. 3. The disease, for example, may be a rheumatoid arthritis or a multiple sclerosis.

In certain aspects the present invention provides for a method of treating or preventing a disease or condition associated with modulation of activity of human CD80 protein or human CD20 protein. The method includes administering to a patient in need for treating or preventing a disease associated with modulation of activity of human CD80 protein or human CD20 protein a therapeutically effective amount of a pharmaceutical composition containing a heterotrimeric protein comprising a first polypeptide comprising amino acid sequence of SEQ ID NO. 1, a second polypeptide comprising amino acid sequence of SEQ ID NO. 2, and a third polypeptide comprising amino acid sequence of SEQ ID NO. 3. The disease, for example, may be a rheumatoid arthritis or a multiple sclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings and descriptions are provided to aid in the understanding of the invention:

Figure 1 illustratively shows a heterotrimeric protein assembly of the present teachings comprising an extracellular portion of CTLA-4 fused with an IgG-Fc domain (Fc-II), a heavy chain portion of an anti-CD20 antibody (CD20-AB HC) fused with another IgG-Fc domain (Fc- VI), and a light chain portion of an anti-CD20 antibody (CD20-AB LC);

Figure 2 schematically shows the map of PKN012-CTLA4-RIT plasmid and annotated sequence used in the cloning of the polypeptides of the present teachings;

Figure 3 shows a representative SDS page analysis the sample containing protein A column eluate used in the purification of the polypeptides of the present teachings;

Figure 4 shown a size-exclusion HPLC analytical chromatogram of the sample containing heterotrimer comprising polypeptide of SEQ ID NO. 1, polypeptide of SEQ ID NO. 2, and polypeptide of SEQ ID NO. 3 after the size exclusion chromatography purification step;

Figure 5 shown the results of the CD80 affinity assay comparing the activity of the heterotrimer (Trimer) of the present teachings with that of drug Orencia;

Figure 6 shown the results of the CD20 binding activity assay comparing the activity of the heterotrimer (Trimer) of the present teachings with those of drugs Orencia and Rituximab;

Figure 7 shown the results of the IL-2 production inhibition assay comparing the activity of the heterotrimer (Trimer) of the present teachings with that of drug Orencia; and

Figure 8 shown the results of complement-dependent cytotoxicity (CDC) assay comparing the activity of the heterotrimer (Trimer) of the present teachings with that of drug Rituximab.

DETAILED DESCRIPTION OF THE INVENTION

The teachings disclosed herein are based, in part, upon engineering of a heterotrimeric protein assembly that is capable of binding to human CD80, CD86 and/or CD20 (NCBI Accession: NP_690605) proteins and attenuating their functions. The heterotrimeric protein assembly of the present teachings comprises an extracellular portions of human CTLA-4 (NCBI Accession:

NP 005205.2), a heavy chain portion of an anti-CD20 antibody, and a light chain portion of an anti-CD20 antibody, or functional fragments thereof. Each, the CTLA-4 portion and the anti-CD20 antibody heavy chain portion, is fused to a distinct mutant of Fc portion of the human Ig Gamma- 1 (GenBank: J00228.1). The two distinct Fc mutants in the heterotrimeric protein assembly are engineered as to favor the heteromeric dimer formation between the two Fc mutants over any homomeric assembly. To enable recombinant production of the heterotrimeric protein assembly of the present teachings, a DNA expression vector has been constructed for overproducing the heterotrimeric protein assembly in a heterologous protein expression system, and mammalian cells have been prepared transiently expressing the heterodimeric protein assembly. A protein purification procedure has been devised allowing obtaining a physiologically relevant substantially pure preparation of the heterotrimeric protein assembly of the present teachings. Thus purified protein molecule demonstrates a high degree of specific activity in an in vitro Enzyme -Linked Immunosorbent Assay (ELISA) using human CD80 (GenBank: AAH08678.1), high degree of specific activity against human CD20 in a flow cytometry assay utilizing CD20 presenting human lymphoblast cell line, and high degree of inhibition of human IL-2 production in an in vitro cell based assay. Unexpectedly, the protein molecule exhibits an acceptable pharmacokinetics profile upon subcutaneous animal administration, while not resulting in any body weight loss or adverse clinical events.

The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and in the specific context where each term is used. Certain terms are discussed below or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the invention and how to make and use them. The scope or meaning of any use of a term will be apparent from the specific context in which the term is used. "About" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms "about" and "approximately" may mean values that are within an order of magnitude, preferably within 5- fold and more preferably within 2-fold of a given value.

Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated. The methods of the invention may include steps of comparing sequences to each other, including wild-type sequence to one or more mutants (sequence variants). Such comparisons typically comprise alignments of polymer sequences, e.g., using sequence alignment programs and/or algorithms that are well known in the art (for example, BLAST, FASTA and MEGALIGN, to name a few). The skilled artisan can readily appreciate that, in such alignments, where a mutation contains a residue insertion or deletion, the sequence alignment will introduce a "gap" (typically represented by a dash, or "A") in the polymer sequence not containing the inserted or deleted residue.

The methods of the invention may include statistical calculations, e.g. determination of IC50 or EC50 values, etc.. The skilled artisan can readily appreciate that such can be performed using a variety of commercially available software, e.g. PRISM (GraphPad Software Inc, La Jolla, CA, USA) or similar.

"Homologous," in all its grammatical forms and spelling variations, refers to the relationship between two proteins that possess a "common evolutionary origin," including proteins from superfamilies in the same species of organism, as well as homologous proteins from different species of organism. Such proteins (and their encoding nucleic acids) have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or by the presence of specific residues or motifs and conserved positions. However, in common usage and in the instant application, the term "homologous," when modified with an adverb such as "highly," may refer to sequence similarity and may or may not relate to a common evolutionary origin.

The term "sequence similarity," in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin.

The terms "protein" and "polypeptide" are used interchangeably. The polypeptides described herein may be comprised of more than one contiguous amino acid chain, thus forming dimers or other oligomeric formations. In general, the polypetides of the present teachings for use in mammals are expressed in mammalian cells that allow for proper post-translational modifications, such as CHO or HEK293 cell lines, although other mammalian expression cell lines are expected to be useful as well. It is therefore anticipated that the polypeptides of the present teachings may be post-translationally modified without substantially effecting its biological function. In certain aspects, functional variants of the heterodimeric protein assemblies of the present teachings include fusion proteins having at least a biologically active portion of the human CTLA- 4 or a functional fragment thereof, and one or more fusion domains. Well known examples of such fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (e.g., an Fc), maltose binding protein (MBP), or human serum albumin. A fusion domain may be selected so as to confer a desired property. For example, the CTLA-4 polypeptide portions may be fused with a domain that stabilizes the polypeptides in vivo (a "stabilizer" domain), optionally via a suitable peptide linker. The term "stabilizing" means anything that increases the half life of a polypeptide in systemic circulation, regardless of whether this is because of decreased destruction, decreased clearance, or other pharmacokinetic effect. Fusions with the Fc portion of an

immunoglobulin are known to confer desirable pharmacokinetic properties on certain proteins. Likewise, fusions to human serum albumin can confer desirable properties. Other types of fusion domains that may be selected include multimerizing (e.g., dimerizing, tetramerizing) domains and functional domains that confer an additional biological function, e.g. promoting accumulation at the targeted site of action in vivo.

In certain aspects, the heterotrimeric protein assembly of the present teachings comprises an extracellular portions of human CTLA-4, or a functional fragment thereof, fused with an IgG-Fc domain, a heavy chain portion of an anti-CD20 antibody, or a functional fragment thereof, fused with another IgG-Fc domain, and a light chain portion of an anti-CD20 antibody. The IgG-Fc domain and the another IgG-Fc domain are chosen as to favor a heterodimeric protein assembly over any homodimeric protein assembly. The extracellular portion of CTLA-4 may be fused with the IgG-Fc domain via a flexible linker, while the heavy chain portion of an anti-CD20 antibody, or a functional fragment thereof, may be fused with the another IgG-Fc domain via the flexible linker of the same amino acid sequence or via another flexible linker.

In an example embodiment, illustratively shown in Figure 1, the extracellular portion of CTLA- 4 fused with IgG-Fc domain (Fc-II) may comprise the amino acid sequence of SEQ. ID NO. 1, while heavy chain portion of an anti-CD20 antibody fused with another IgG-Fc domain (Fc-VI) may comprise the amino acid sequence of SEQ. ID NO. 2, and the light chain portion of an anti- CD20 antibody may comprise the amino acid sequence of SEQ. ID NO. 3. hCTLA4-hIgG 1 -Fc polypeptide (SEQ ID NO. 1)

MHVAQPAVVL ASSRGIASFV CEYASPGKAT EVRVTVLRQA DSQVTEVCAA TYMMGNELTF 60

LDDSICTGTS SGNQVNL IQ GLRAMDTGLY ICKVELMYPP PYYLGIGNGT QIYVIDPEPC 120

PDSDQEPKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED 1 80

PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA 2 40

PIEKTI SKAK GQPREPQVYT LPPCRDELTK NQVSLWCLVK GFYPSDIAVE WESNGQPENN 300

YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK 357 hCD20ab-HC-hIgGl-Fc polypeptide (SEQ ID NO. 2)

QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60

NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120

AASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS 1 80

SGLYSLSSW TVPSSSLGTQ TYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPELLG 2 40

GPSVFLFPPK PKDTLMISRT PEVTCVWDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY 300

NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVCTLPPSRD 3 60

ELTKNQVSLS CAVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFKL VSKLTVDKSR 420

WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K 451 hCD20ab-LC polypeptide (SEQ ID NO. 3)

QIVLSQSPAI LSASPGEKVT MTCRASSSVS YIHWFQQKPG SSPKPWIYAT SNLASGVPVR 60

FSGSGSGTSY SL ISRVEAE DAATYYCQQW TSNPPTFGGG TKLEIKRTVA APSVFIFPPS 120

DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL 1 80

SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC 2 13

In certain aspects, the present teachings provides for a recombinant DNA molecule having an open reading frame coding for a polypeptide comprising amino acids 38-161of the human CTLA-4 (NCBI Accession: NP 690605) fused with IgG-Fc domain (Fc-II), for another recombinant DNA molecule having an open reading frame coding for another polypeptide comprising a heavy chain portion of an anti-CD20 antibody fused with another IgG-Fc domain (Fc-VI), and for a third recombinant DNA molecule having an open reading frame coding for a third polypeptide comprising a light chain portion of the anti-CD20 antibody.

In an example embodiment, the polypeptide comprising amino acids 38-161of the human CTLA-4 (NCBI Accession: NP 690605) fused with IgG-Fc domain (Fc-II) comprises the amino acid sequence of SEQ. ID NO. 4. The corresponding to it DNA molecule may comprise the nucleotide sequence of SEQ ID NO. 5. The another polypeptide comprising a heavy chain portion of an anti-CD20 antibody fused with another IgG-Fc domain (Fc-VI) may comprise the amino acid sequence of SEQ. ID NO. 6. The corresponding to it DNA molecule may comprise the nucleotide sequence of SEQ ID NO. 7. The third polypeptide comprising a light chain portion of the anti- CD20 antibody may comprise the amino acid sequence of SEQ. ID NO. 8. The corresponding to it DNA molecule may comprise the nucleotide sequence of SEQ ID NO. 9.

hCTLA4-hIgG 1 -Fc polypeptide (SEQ ID NO. 4)

METDTLLLWV LLLWVPGSTG MHVAQPAVVL AS SRGIAS FV CEYAS PGKAT EVRVTVLRQA 60

DSQVTEVCAA TYMMGNELT F LDDS I CTGTS SGNQVNLT IQ GLRAMDTGLY I CKVELMYPP 1 2 0

PYYLGI GNGT QIYVI DPEPC PDS DQE PKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDT 1 8 0

LMI SRT PEVT CWVDVSHE D PEVKFNWYVD GVEVHNAKTK PREEQYNS TY RWSVLTVLH 2 4 0

QDWLNGKEYK CKVSNKALPA PIEKT I SKAK GQPRE PQVYT LPPCRDELTK NQVSLWCLVK 3 0 0

GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGS FFLYSKL TVDKSRWQQG NVFSC SVMHE 3 60

ALHNHYTQKS LSLSPGK 3 7 7 hCTLA4-hIgGl-Fc DNA (SEQ ID NO. 5)

AAGCTTGCCA CCATGGAAAC CGATACACTG CTGCTCTGGG TCCTGCTGCT CTGGGTGCCC 60

GGATCAACTG GAATGCATGT GGCACAGCCT GCCGTGGTGC TGGCTTCTTC TCGGGGAATT 1 2 0

GCCTCTTTCG TGTGTGAAT CGCCTCACCT G G A A A AG C A A CCGAAGTGAG AGTGACCGTG 1 8 0

CTGAGACAGG CCGATAGTCA GGTCACCGAA GTCTGTGCTG CCACTTACAT GATGGGCAAC 2 4 0

GAACTCACAT TTCTGGACGA CTCCATTTGT ACCGGCACAT CTTCTGGAAA TCAGGTCAAC 3 0 0

CTCACCATTC AGGGACTGAG AGCCATGGAT ACTGGACTGT ACATTTGTAA AGTGGAACTC 3 60

ATGTACCCTC CACCCTACTA CCTGGGCATC GGAAACGGGA CACAGATCTA CGTGATCGAC 4 2 0 CCCGAACCTT GCCCCGACTC CGATCAGGAA CCCAAATCCT CTGACAAAAC TCACACATGC 480

CCACCGTGCC CAGCTCCGGA ACTCCTGGGC GGACCGTCAG TCTTCCTCTT CCCCCCAAAA 540

CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCA CATGCGTGGT GGTGGACGTG 600

AGCCACGAAG ACCCTGAGGT CAAGTTCAAC TGGTACGTGG ACGGCGTGGA GGTGCATAAT 660

GCCAAGACAA AGCCGCGGGA GGAGCAGTAC AACAGCACGT ACCGTGTGGT CAGCGTCCTC 720

ACCGTCCTGC ACCAGGACTG GCTGAATGGC AAGGAGTACA AGTGCAAGGT CTCCAACAAA 780

GCCCTCCCAG CCCCCATCGA GAAAACCATC TCCAAAGCCA AAGGGCAGCC CCGAGAACCA 840

CAGGTGTACA CCCTGCCCCC ATGTCGGGAT GAGCTGACCA AGAACCAGGT CAGCCTGTGG 900

TGCCTGGTCA AAGGCTTCTA TCCCAGCGAC ATCGCCGTGG AGTGGGAGAG CAATGGGCAG 960

CCGGAGAACA ACTACAAGAC CACGCCTCCC GTGTTGGACT CCGACGGCTC CTTCTTCCTC 1020

TACAGCAAGC TCACCGTGGA CAAGAGCAGG TGGCAGCAGG GGAACGTCTT CTCATGCTCC 1080

GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA GCCTCTCCCT GTCTCCGGGT 1140

AAATAATGAA TTC 1153

hCD20ab-HC-hIgGl-Fc polypeptide (SEQ ID NO. 6)

MGWSLILLFL VAVATRVLSQ VQLQQPGAEL VKPGASVKMS CKASGYTFTS YNMHWVKQTP 60

GRGLEWIGAI YPGNGDTSYN QKFKGKATLT ADKSSSTAYM QLSSLTSEDS AVYYCARSTY 120

YGGDWYFNVW GAGTTVTVSA ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS 180

WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP 240

KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW 300

YVDGVEVHNA KTKPREEQYN STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS 360

KAKGQPREPQ VCTLPPSRDE LTKNQVSLSC AVKGFYPSDI AVEWESNGQP ENNYKTTPPV 420

LDSDGSFKLV SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 470 hCD20ab-HC-h!gGl-Fc DNA (SEQ ID NO. 7)

AAGCTTGCCA CCATGGGCTG GAGTCTGATC CTGCTGTTTC TGGTTGCTGT TGCTACCCGT 60

GTTCTGAGCC AAGTTCAACT GCAACAACCG GGCGCTGAAC TGGTGAAACC GGGCGCTTCT 120

GTGAAAATGT CCTGTAAAGC CTCCGGCTAT ACGTTTACCT CCTATAACAT GCACTGGGTC 180

AAACAAACCC CGGGCCGTGG CCTGGAATGG ATTGGTGCCA TCTATCCGGG CAATGGTGAT 240

ACGAGCTATA ATCAAAAATT CAAAGGCAAA GCGACCCTGA CCGCCGACAA ATCTTCTTCT 300

ACGGCGTATA TGCAACTGTC TAGCCTGACG TCTGAAGACT CTGCTGTTTA CTATTGCGCG 360 CGTAGCACCT ATTACGGCGG CGACTGGTAT TTCAATGTGT GGGGCGCTGG CACGACCGTT 420

ACCGTTAGTG CTGCTAGCAC GAAAGGCCCG AGCGTTTTTC CGCTGGCCCC GTCTAGCAAA 480

TCAACGAGCG GTGGCACGGC TGCCCTGGGC TGTCTGGTCA AAGACTATTT TCCGGAACCG 540

GTGACCGTTT CTTGGAACTC TGGTGCCCTG ACGAGTGGTG TTCACACCTT TCCGGCTGTT 600

CTGCAATCAT CTGGCCTGTA TTCACTGTCT AGTGTGGTGA CCGTCCCGTC TAGTAGCCTG 660

GGCACCCAAA CCTATATCTG TAACGTGAAC CACAAACCGA GCAACACGAA AGTGGACAAA 720

AAAGTCG AC CGAAATCCTG TGACAAAACT CACACATGCC CACCGTGCCC AGCTCCGGAA 780

CTCCTGGGCG GACCGTCAGT CTTCCTCTTC CCCCCAAAAC CCAAGGACAC CCTCATGATC 840

TCCCGGACCC CTGAGGTCAC ATGCGTGGTG GTGGACGTGA GCCACGAAGA CCCTGAGGTC 900

AAGTTCAACT GGTACGTGGA CGGCGTGGAG GTGCATAATG CCAAGACAAA GCCGCGGGAG 960

GAGCAGTACA ACAGCACGTA CCGTGTGGTC AGCGTCCTCA CCGTCCTGCA CCAGGACTGG 1020

CTGAATGGCA AGGAGTACAA GTGCAAGGTC TCCAACAAAG CCCTCCCAGC CCCCATCGAG 1080

AAAACCATCT CCAAAGCCAA AGGGCAGCCC CGAGAACCAC AGGTGTGTAC CCTGCCCCCA 1140

TCCCGGGATG AGCTGACCAA GAACCAGGTC AGCCTGAGTT GCGCGGTCAA AGGCTTCTAT 1200

CCCAGCGACA TCGCCGTGGA GTGGGAGAGC AATGGGCAGC CGGAGAACAA CTACAAGACC 1260

ACGCCTCCCG TGTTGGACTC CGACGGCTCC TTCAAGCTCG TCAGCAAGCT CACCGTGGAC 1320

AAGAGCAGGT GGCAGCAGGG GAACGTCTTC TCATGCTCCG TGATGCATGA GGCTCTGCAC 1380

AACCACTACA CGCAGAAGAG CCTCTCCCTG TCTCCGGGTA AATAATAGAA TTC 1433 hCD20ab-LC polypeptide (SEQ ID NO. 8)

MDFQVQIISF LLISASVIMS RGQIVLSQSP AILSASPGEK VTMTCRASSS VSYIHWFQQK 60

PGSSPKPWIY ATSNLASGVP VRFSGSGSGT SYSLTISRVE AEDAATYYCQ QWTSNPPTFG 120

GGTKLEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS 180

QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC 235 hCD20ab-LC DNA (SEQ ID NO. 9)

AAGCTTGCCA CCATGGACTT TCAGGTTCAA ATCATCTCCT TTCTGCTGAT CTCTGCCTCT 60

GTTATCATGT CTCGTGGCCA AATTGTTCTG AGCCAATCTC CGGCTATTCT GTCTGCTTCA 120

CCGGGCGAAA AAGTGACGAT GACGTGTCGT GCTTCTTCTT CTGTGTCCTA TATCCACTGG 180

TTCCAACAAA AACCGGGCTC TTCTCCGAAA CCGTGGATCT ATGCTACGAG CAATCTGGCA 240

TCTGGTGTTC CGGTCCGTTT TAGCGGTAGT GGTTCTGGCA CCTCTTATTC TCTGACCATT 300

TCCCGTGTCG AAGCGGAAGA TGCTGCCACG TATTATTGCC AACAATGGAC CTCAAATCCG 360 CCGACCTTTG GTGGTGGCAC GAAACTGGAA ATCAAACGTA CCGTTGCTGC TCCGAGTGTG 420

TTCATTTTCC CGCCGTCCGA CGAACAACTG AAATCTGGCA CCGCCTCTGT GGTTTGTCTG 480

CTGAACAACT TCTATCCGCG CGAAGCAAAA GTTCAATGGA AAGTGGACAA CGCGCTGCAA 540

TCCGGTAATA GTCAGGAATC CGTCACGGAA CAGGATTCCA AAGATTCCAC CTATAGCCTG 600

TCTAGCACCC TGACCCTGAG TAAAGCCGAC TATGAAAAAC ACAAAGTGTA TGCCTGTGAA 660

GTCACCCATC AAGGCCTGAG TTCTCCGGTC ACGAAATCGT TCAATCGTGG CGAATGCTAA 720 GAATTC 726

In certain aspects, the present invention provides for a recombinant mammalian expression plasmid for high co-expression of a polypeptide comprising amino acids 38- 16 lof the human

CTLA-4 fused with IgG-Fc domain (Fc-II), another polypeptide comprising a heavy chain portion of an anti-CD20 antibody fused with another IgG-Fc domain (Fc-VI), and a third polypeptide comprising a light chain portion of the anti-CD20 antibody. This plasmid comprises two cytomegalovirus (CMV) promoters to drive transcription of the two genes coding for said polypeptide and said another polypeptide, each followed by a transcription termination sequence and a polyadenylation sequence. The plasmid also contains an origin of replication and a gene conferring ampicillin resistance, for supporting plasmid propagation and selection in bacteria. The plasmid further contains a gene for Glutamine synthetase, a selectable marker widely used for establishing stable CHOK1 and NSO cell lines. The plasmid of the present teachings is illustratively shown in Figure 2.

In an example embodiment, the mammalian expression plasmid of the present teachings comprises the nucleotide sequence of SEQ ID NO. 7.

PKN012-CTLA4- IT expression plasmid (SEQ ID NO. 10)

AGCTTGCCAC CATGGGCTGG AGTCTGATCC TGCTGTTTCT GGTTGCTGTT GCTACCCGTG 60

TTCTGAGCCA AGTTCAACTG CAACAACCGG GCGCTGAACT GGTGAAACCG GGCGCTTCTG 120

TGAAAATGTC CTGTAAAGCC TCCGGCTATA CGTTTACCTC CTATAACATG CACTGGGTCA 180

AACAAACCCC GGGCCGTGGC CTGGAATGGA TTGGTGCCAT CTATCCGGGC AATGGTGATA 240

CGAGCTATAA TCAAAAATTC AAAGGCAAAG CGACCCTGAC CGCCGACAAA TCTTCTTCTA 300

CGGCGTATAT GCAACTGTCT AGCCTGACGT CTGAAGACTC TGCTGTTTAC TATTGCGCGC 360

GTAGCACCTA TTACGGCGGC GACTGGTATT TCAATGTGTG GGGCGCTGGC ACGACCGTTA 420

CCGTTAGTGC TGCTAGCACG AAAGGCCCGA GCGTTTTTCC GCTGGCCCCG TCTAGCAAAT 480 CAACGAGCGG TGGCACGGCT GCCCTGGGCT GTCTGGTCAA AGACTATTTT CCGGAACCGG 540

TGACCGTTTC TTGGAACTCT GGTGCCCTGA CGAGTGGTGT TCACACCTTT CCGGCTGTTC 600

TGCAATCATC TGGCCTGTAT TCACTGTCTA GTGTGGTGAC CGTCCCGTCT AGTAGCCTGG 660

GCACCCAAAC CTATATCTGT AACGTGAACC ACAAACCGAG CAACACGAAA GTGGACAAAA 720 AAGTCGAACC GAAATCCTGT GACAAAACTC ACACATGCCC ACCGTGCCCA GCTCCGGAAC 780

TCCTGGGCGG ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT 840

CCCGGACCCC TGAGGTCACA TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA 900

AGTTCAACTG GTACGTGGAC GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG 960

AGCAGTACAA CAGCACGTAC CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC 1020 TGAATGGCAA GGAGTACAAG TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA 1080

AAACCATCTC CAAAGCCAAA GGGCAGCCCC GAGAACCACA GGTGTGTACC CTGCCCCCAT 1140

CCCGGGATGA GCTGACCAAG AACCAGGTCA GCCTGAGTTG CGCGGTCAAA GGCTTCTATC 1200

CCAGCGACAT CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA 1260

CGCCTCCCGT GTTGGACTCC GACGGCTCCT TCAAGCTCGT CAGCAAGCTC ACCGTGGACA 1320 AGAGCAGGTG GCAGCAGGGG AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA 1380

ACCACTACAC GCAGAAGAGC CTCTCCCTGT CTCCGGGTAA ATAGAATTCA TTGATCATAA 1440

TCAGCCATAC CACATTTGTA GAGGTTTTAC TTGCTTTAAA AAACCTCCCA CACCTCCCCC 1500

TGAACCTGAA ACATAAAATG AATGCAATTG TTGTTGTTAA CTTGTTTATT GCAGCTTATA 1560

ATGGTTACAA ATAAAGCAAT AGCATCACAA ATTTCACAAA TAAAGCATTT TTTTCACTGC 1620 ATTCTAGTTG TGGTTTGTCC AAACTCATCA ATGTATCTTA TCATGTCTGG CGGCCGCCGA 1680

TATTTGAAAA TATGGCATAT TGAAAATGTC GCCGATGTGA GTTTCTGTGT AACTGATATC 1740

GCCATTTTTC CAAAAGTGAT TTTTGGGCAT ACGCGATATC TGGCGATAGC GCTTATATCG 1800

TTTACGGGGG ATGGCGATAG ACGACTTTGG TGACTTGGGC GATTCTGTGT GTCGCAAATA 1860

TCGCAGTTTC GATATAGGTG ACAGACGATA TGAGGCTATA TCGCCGATAG AGGCGACATC 1920 AAGCTGGCAC ATGGCCAATG CATATCGATC TATACATTGA ATCAATATTG GCCATTAGCC 1980

ATATTATTCA TTGGTTATAT AGCATAAATC AATATTGGCT ATTGGCCATT GCATACGTTG 2040

TATCCATATC ATAATATGTA CATTTATATT GGCTCATGTC CAACATTACC GCCATGTTGA 2100

CATTGATTAT TGACTAGTTA TTAATAGTAA TCAATTACGG GGTCATTAGT TCATAGCCCA 2160

TATATGGAGT TCCGCGTTAC ATAACTTACG GTAAATGGCC CGCCTGGCTG ACCGCCCAAC 2220 GACCCCCGCC CATTGACGTC AATAATGACG TATGTTCCCA TAGTAACGCC AATAGGGACT 2280

TTCCATTGAC GTCAATGGGT GGAGTATTTA CGGTAAACTG CCCACTTGGC AGTACATCAA 2340

GTGTATCATA TGCCAAGTAC GCCCCCTATT GACGTCAATG ACGGTAAATG GCCCGCCTGG 2400

CATTATGCCC AGTACATGAC CTTATGGGAC TTTCCTACTT GGCAGTACAT CTACGTATTA 2460

GTCATCGCTA TTACCATGGT GATGCGGTTT TGGCAGTACA TCAATGGGCG TGGATAGCGG 2520 TTTGACTCAC GGGGATTTCC AAGTCTCCAC CCCATTGACG TCAATGGGAG TTTGTTTTGG 2580 CACCAAAATC AACGGGACTT TCCAAAATGT CGTAACAACT CCGCCCCATT GACGCAAATG 2640

GGCGGTAGGC GTGTACGGTG GGAGGTCTAT ATAAGCAGAG CTCGTTTAGT GAACCGTCAG 2700

ATCGCCTGGA GACGCCATCC ACGCTGTTTT GACCTCCATA GAAGACACCG GGACCGATCC 2760

AGCCTCCGCG GCCGGGAACG GTGCATTGGA ACGCGGATTC CCCGTGCCAA GAGTGACGTA 2820 AGTACCGCCT ATAGAGTCTA TAGGCCCACC CCCTTGGCTT CTTATGCATG CTATACTGTT 2880

TTTGGCTTGG GGTCTATACA CCCCCGCTTC CTCATGTTAT AGGTGATGGT ATAGCTTAGC 2940

CTATAGGTGT GGGTTATTGA CCATTATTGA CCACTCCCCT ATTGGTGACG ATACTTTCCA 3000

TTACTAATCC ATAACATGGC TCTTTGCCAC AACTCTCTTT ATTGGCTATA TGCCAATACA 3060

CTGTCCTTCA GAGACTGACA CGGACTCTGT ATTTTTACAG GATGGGGTCT CATTTATTAT 3120 TTACAAATTC ACATATACAA CACCACCGTC CCCAGTGCCC GCAGTTTTTA TTAAACATAA 3180

CGTGGGATCT CCACGCGAAT CTCGGGTACG TGTTCCGGAC ATGGGCTCTT CTCCGGTAGC 3240

GGCGGAGCTT CTACATCCGA GCCCTGCTCC CATGCCTCCA GCGACTCATG GTCGCTCGGC 3300

AGCTCCTTGC TCCTAACAGT GGAGGCCAGA CTTAGGCACA GCACGATGCC CACCACCACC 3360

AGTGTGCCGC ACAAGGCCGT GGCGGTAGGG TATGTGTCTG AAAATGAGCT CGGGGAGCGG 3420 GCTTGCACCG CTGACGCATT TGGAAGACTT AAGGCAGCGG CAGAAGAAGA TGCAGGCAGC 3480

TGAGTTGTTG TGTTCTGATA AGAGTCAGAG GTAACTCCCG TTGCGGTGCT GTTAACGGTG 3540

GAGGGCAGTG TAGTCTGAGC AGTACTCGTT GCTGCCGCGC GCGCCACCAG ACATAATAGC 3600

TGACAGACTA ACAGACTGTT CCTTTCCATG GGTCTTTTCT GCAGTCACCG TCCTTGACAC 3660

GAAGCTTGCC ACCATGGACT TTCAGGTTCA AATCATCTCC TTTCTGCTGA TCTCTGCCTC 3720 TGTTATCATG TCTCGTGGCC AAATTGTTCT GAGCCAATCT CCGGCTATTC TGTCTGCTTC 3780

ACCGGGCGAA AAAGTGACGA TGACGTGTCG TGCTTCTTCT TCTGTGTCCT ATATCCACTG 3840

GTTCCAACAA AAACCGGGCT CTTCTCCGAA ACCGTGGATC TATGCTACGA GCAATCTGGC 3900

ATCTGGTGTT CCGGTCCGTT TTAGCGGTAG TGGTTCTGGC ACCTCTTATT CTCTGACCAT 3960

TTCCCGTGTC GAAGCGGAAG ATGCTGCCAC GTATTATTGC CAACAATGGA CCTCAAATCC 4020 GCCGACCTTT GGTGGTGGCA CGAAACTGGA AATCAAACGT ACCGTTGCTG CTCCGAGTGT 4080

GTTCATTTTC CCGCCGTCCG ACGAACAACT GAAATCTGGC ACCGCCTCTG TGGTTTGTCT 4140

GCTGAACAAC TTCTATCCGC GCGAAGCAAA AGTTCAATGG AAAGTGGACA ACGCGCTGCA 4200

ATCCGGTAAT AGTCAGGAAT CCGTCACGGA ACAGGATTCC AAAGATTCCA CCTATAGCCT 4260

GTCTAGCACC CTGACCCTGA GTAAAGCCGA CTATGAAAAA CACAAAGTGT ATGCCTGTGA 4320 AGTCACCCAT CAAGGCCTGA GTTCTCCGGT CACGAAATCG TTCAATCGTG GCGAATGCTA 4380

AGAATTCATT GATCATAATC AGCCATACCA CATTTGTAGA GGTTTTACTT GCTTTAAAAA 4440

ACCTCCCACA CCTCCCCCTG AACCTGAAAC ATAAAATGAA TGCAATTGTT GTTGTTAACT 4500

TGTTTATTGC AGCTTATAAT GGTTACAAAT AAAGCAATAG CATCACAAAT TTCACAAATA 4560

AAGCATTTTT TTCACTGCAT TCTAGTTGTG GTTTGTCCAA ACTCATCAAT GTATCTTATC 4620 ATGTCTGGAT CCTCTACGCC GGACGCATCG TGGCCGGCAT CACCGGCGCC ACAGGTGCGG 4680 TTGCTGGCGC CTATATCGCC GACATCACCG ATGGGGAAGA TCGGGCTCGC CACTTCGGGC 4740 TCATGAGCGC TTGTTTCGGC GTGGGTATGG TGGCAGGCCC CGTGGCCGGG GGACTGTTGG 4800 GCGCCATCTC CTTGCATGCA CCATTCCTTG CGGCGGCGGT GCTCAACGGC CTCAACCTAC 4860 TACTGGGCTG CTTCCTAATG CAGGAGTCGC ATAAGGGAGA GCGTCGACGC GGCCGCCGAT 4920 ATTTGAAAAT ATGGCATATT GAAAATGTCG CCGATGTGAG TTTCTGTGTA ACTGATATCG 4980 CCATTTTTCC AAAAGTGATT TTTGGGCATA CGCGATATCT GGCGATAGCG CTTATATCGT 5040 TTACGGGGGA TGGCGATAGA CGACTTTGGT GACTTGGGCG ATTCTGTGTG TCGCAAATAT 5100 CGCAGTTTCG ATATAGGTGA CAGACGATAT GAGGCTATAT CGCCGATAGA GGCGACATCA 5160 AGCTGGCACA TGGCCAATGC ATATCGATCT ATACATTGAA TCAATATTGG CCATTAGCCA 5220 TATTATTCAT TGGTTATATA GCATAAATCA ATATTGGCTA TTGGCCATTG CATACGTTGT 5280 ATCCATATCA TAATATGTAC ATTTATATTG GCTCATGTCC AACATTACCG CCATGTTGAC 5340 ATTGATTATT GACTAGTTAT TAATAGTAAT CAATTACGGG GTCATTAGTT CATAGCCCAT 5400 ATATGGAGTT CCGCGTTACA TAACTTACGG TAAATGGCCC GCCTGGCTGA CCGCCCAACG 5460 ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT AGTAACGCCA ATAGGGACTT 5520 TCCATTGACG TCAATGGGTG GAGTATTTAC GGTAAACTGC CCACTTGGCA GTACATCAAG 5580 TGTATCATAT GCCAAGTACG CCCCCTATTG ACGTCAATGA CGGTAAATGG CCCGCCTGGC 5640 ATTATGCCCA GTACATGACC TTATGGGACT TTCCTACTTG GCAGTACATC TACGTATTAG 5700 TCATCGCTAT TACCATGGTG ATGCGGTTTT GGCAGTACAT CAATGGGCGT GGATAGCGGT 5760 TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT CAATGGGAGT TTGTTTTGGC 5820 ACCAAAATCA ACGGGACTTT CCAAAATGTC GTAACAACTC CGCCCCATTG ACGCAAATGG 5880 GCGGTAGGCG TGTACGGTGG GAGGTCTATA TAAGCAGAGC TCGTTTAGTG AACCGTCAGA 5940 TCGCCTGGAG ACGCCATCCA CGCTGTTTTG ACCTCCATAG AAGACACCGG GACCGATCCA 6000 GCCTCCGCGG CCGGGAACGG TGCATTGGAA CGCGGATTCC CCGTGCCAAG AGTGACGTAA 6060 GTACCGCCTA TAGAGTCTAT AGGCCCACCC CCTTGGCTTC TTATGCATGC TATACTGTTT 6120 TTGGCTTGGG GTCTATACAC CCCCGCTTCC TCATGTTATA GGTGATGGTA TAGCTTAGCC 6180 TATAGGTGTG GGTTATTGAC CATTATTGAC CACTCCCCTA TTGGTGACGA TACTTTCCAT 6240 TACTAATCCA TAACATGGCT CTTTGCCACA ACTCTCTTTA TTGGCTATAT GCCAATACAC 6300 TGTCCTTCAG AGACTGACAC GGACTCTGTA TTTTTACAGG ATGGGGTCTC ATTTATTATT 6360 TACAAATTCA CATATACAAC ACCACCGTCC CCAGTGCCCG CAGTTTTTAT TAAACATAAC 6420 GTGGGATCTC CACGCGAATC TCGGGTACGT GTTCCGGACA TGGGCTCTTC TCCGGTAGCG 6480 GCGGAGCTTC TACATCCGAG CCCTGCTCCC ATGCCTCCAG CGACTCATGG TCGCTCGGCA 6540 GCTCCTTGCT CCTAACAGTG GAGGCCAGAC TTAGGCACAG CACGATGCCC ACCACCACCA 6600 GTGTGCCGCA CAAGGCCGTG GCGGTAGGGT ATGTGTCTGA AAATGAGCTC GGGGAGCGGG 6660 CTTGCACCGC TGACGCATTT GGAAGACTTA AGGCAGCGGC AGAAGAAGAT GCAGGCAGCT 6720 GAGTTGTTGT GTTCTGATAA GAGTCAGAGG TAACTCCCGT TGCGGTGCTG TTAACGGTGG 6780 AGGGCAGTGT AGTCTGAGCA GTACTCGTTG CTGCCGCGCG CGCCACCAGA CAT ATAGCT 6840 GACAGACTAA CAGACTGTTC CTTTCCATGG GTCTTTTCTG CAGTCACCGT CCTTGACACG 6900 AAGCTTGCCA CCATGGAAAC CGATACACTG CTGCTCTGGG TCCTGCTGCT CTGGGTGCCC 6960 GGATCAACTG GAATGCATGT GGCACAGCCT GCCGTGGTGC TGGCTTCTTC TCGGGGAATT 7020 GCCTCTTTCG TGTGTGAATA CGCCTCACCT GGAAAAGCAA CCGAAGTGAG AGTGACCGTG 7080 CTGAGACAGG CCGATAGTCA GGTCACCGAA GTCTGTGCTG CCACTTACAT GATGGGCAAC 7140 GAACTCACAT TTCTGGACGA CTCCATTTGT ACCGGCACAT CTTCTGGAAA TCAGGTCAAC 7200 CTCACCATTC AGGGACTGAG AGCCATGGAT ACTGGACTGT ACATTTGTAA AGTGGAACTC 7260 ATGTACCCTC CACCCTACTA CCTGGGCATC GGAAACGGGA CACAGATCTA CGTGATCGAC 7320 CCCGAACCTT GCCCCGACTC CGATCAGGAA CCCAAATCCT CTGACAAAAC TCACACATGC 7380 CCACCGTGCC CAGCTCCGGA ACTCCTGGGC GGACCGTCAG TCTTCCTCTT CCCCCCAAAA 7440 CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCA CATGCGTGGT GGTGGACGTG 7500 AGCCACGAAG ACCCTGAGGT CAAGTTCAAC TGGTACGTGG ACGGCGTGGA GGTGCATAAT 7560 GCCAAGACAA AGCCGCGGGA GGAGCAGTAC AACAGCACGT ACCGTGTGGT CAGCGTCCTC 7620 ACCGTCCTGC ACCAGGACTG GCTGAATGGC AAGGAGTACA AGTGCAAGGT CTCCAACAAA 7680 GCCCTCCCAG CCCCCATCGA GAAAACCATC TCCAAAGCCA AAGGGCAGCC CCGAGAACCA 7740 CAGGTGTACA CCCTGCCCCC ATGTCGGGAT GAGCTGACCA AGAACCAGGT CAGCCTGTGG 7800 TGCCTGGTCA AAGGCTTCTA TCCCAGCGAC ATCGCCGTGG AGTGGGAGAG CAATGGGCAG 7860 CCGGAGAACA ACTACAAGAC CACGCCTCCC GTGTTGGACT CCGACGGCTC CTTCTTCCTC 7920 TACAGCAAGC TCACCGTGGA CAAGAGCAGG TGGCAGCAGG GGAACGTCTT CTCATGCTCC 7980 GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA GCCTCTCCCT GTCTCCGGGT 8040 AAATAGAATT CATTGATCAT AATCAGCCAT ACCACATTTG TAGAGGTTTT ACTTGCTTTA 8100 AAAAACCTCC CACACCTCCC CCTGAACCTG AAACATAAAA TGAATGCAAT TGTTGTTGTT 8160 AACTTGTTTA TTGCAGCTTA TAATGGTTAC AAATAAAGCA ATAGCATCAC AAATTTCACA 8220 AATAAAGCAT TTTTTTCACT GCATTCTAGT TGTGGTTTGT CCAAACTCAT CAATGTATCT 8280 TATCATGTCT GGATCCTCTA CGCCGGACGC ATCGTGGCCG GCATCACCGG CGCCACAGGT 8340 GCGGTTGCTG GCGCCTATAT CGCCGACATC ACCGATGGGG AAGATCGGGC TCGCCACTTC 8400 GGGCTCATGA GCGCTTGTTT CGGCGTGGGT ATGGTGGCAG GCCCCGTGGC CGGGGGACTG 8460 TTGGGCGCCA TCTCCTTGCA TGCACCATTC CTTGCGGCGG CGGTGCTCAA CGGCCTCAAC 8520 CTACTACTGG GCTGCTTCCT AATGCAGGAG TCGCATAAGG GAGAGCGTCG ACCTCGGGCC 8580 GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC CCCCCTGACG AGCATCACAA AAATCGACGC 8640 TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGCGTT TCCCCCTGGA 8700 AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA CCGGATACCT GTCCGCCTTT 8760 CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT AGCTCACGCT GTAGGTATCT CAGTTCGGTG 8820 TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG CACGAACCCC CCGTTCAGCC CGACCGCTGC 8880 GCCTTATCCG GTAACTATCG TCTTGAGTCC AACCCGGTAA GACACGACTT ATCGCCACTG 8940

GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATG TAGGCGGTGC TACAGAGTTC 9000

TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGAACAG TATTTGGTAT CTGCGCTCTG 9060

CTGAAGCCAG TTACCTTCGG AAAAAGAGTT GGTAGCTCTT GATCCGGCAA ACAAACCACC 9120 GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG CAGCAGATTA CGCGCAGAAA AAAAGGATCT 9180

CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC AGTGGAACGA AAACTCACGT 9240

TAAGGGATTT TGGTCATGAG ATTATCAAAA AGGATCTTCA CCTAGATCCT TTTAAATTAA 9300

AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA CTTGGTCTGA CAGTTACCAA 9360

TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT TTCGTTCATC CATAGTTGCC 9420 TGACTCCCCG TCGTGTAGAT AACTACGATA CGGGAGGGCT TACCATCTGG CCCCAGTGCT 9480

GCAATGATAC CGCGAGACCC ACGCTCACCG GCTCCAGATT TATCAGCAAT AAACCAGCCA 9540

GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT GCAACTTTAT CCGCCTCCAT CCAGTCTATT 9600

AATTGTTGCC GGGAAGCTAG AGTAAGTAGT TCGCCAGTTA ATAGTTTGCG CAACGTTGTT 9660

GCCATTGCTA CAGGCATCGT GGTGTCACGC TCGTCGTTTG GTATGGCTTC ATTCAGCTCC 9720 GGTTCCCAAC GATCAAGGCG AGTTACATGA TCCCCCATGT TGTGCAAAAA AGCGGTTAGC 9780

TCCTTCGGTC CTCCGATCGT TGTCAGAAGT AAGTTGGCCG CAGTGTTATC ACTCATGGTT 9840

ATGGCAGCAC TGCATAATTC TCTTACTGTC ATGCCATCCG TAAGATGCTT TTCTGTGACT 9900

GGTGAGTACT CAACCAAGTC ATTCTGAGAA TAGTGTATGC GGCGACCGAG TTGCTCTTGC 9960

CCGGCGTCAA TACGGGATAA TACCGCGCCA CATAGCAGAA CTTTAAAAGT GCTCATCATT 10020 GGAAAACGTT CTTCGGGGCG AAAACTCTCA AGGATCTTAC CGCTGTTGAG ATCCAGTTCG 10080

ATGTAACCCA CTCGTGCACC CAACTGATCT TCAGCATCTT TTACTTTCAC CAGCGTTTCT 10140

GGGTGAGCAA AAACAGGAAG GCAAAATGCC GCAAAAAAGG GAATAAGGGC GACACGGAAA 10200

TGTTGAATAC TCATACTCTT CCTTTTTCAA TATTATTGAA GCATTTATCA GGGTTATTGT 10260

CTCATGAGCG GATACATATT TGAATGTATT TAGAAAAATA AACAAATAGG GGTTCCGCGC 10320 ACATTTCCCC GAAAAGTGCC ACCTGACGTC TAAGAAACCA TTATTATCAT GACATTAACC 10380

TATAAAAATA GGCGTATCAC GAGGCCCTGA TGGCTCTTTG CGGCACCCAT CGTTCGTAAT 10440

GTTCCGTGGC ACCGAGGACA ACCCTCAAGA GAAAATGTAA TCACACTGGC TCACCTTCGG 10500

GTGGGCCTTT CTGCGTTTAT AAGGAGACAC TTTATGTTTA AGAAGGTTGG TAAATTCCTT 10560

GCGGCTTTGG CAGCCAAGCT AGATCCGGCT GTGGAATGTG TGTCAGTTAG GGTGTGGAAA 10620 GTCCCCAGGC TCCCCAGCAG GCAGAAGTAT GCAAAGCATG CATCTCAATT AGTCAGCAAC 10680

CAGGTGTGGA AAGTCCCCAG GCTCCCCAGC AGGCAGAAGT ATGCAAAGCA TGCATCTCAA 10740

TTAGTCAGCA ACCATAGTCC CGCCCCTAAC TCCGCCCATC CCGCCCCTAA CTCCGCCCAG 10800

TTCCGCCCAT TCTCCGCCCC ATGGCTGACT AATTTTTTTT ATTTATGCAG AGGCCGAGGC 10860

CGCCTCGGCC TCTGAGCTAT TCCAGAAGTA GTGAGGAGGC TTTTTTGGAG GCCTAGGCTT 10920 TTGCAAAAAG CTAGCTTGGG GCCACCGCTC AGAGCACCTT CCACCATGGC CACCTCAGCA 10980 AGTTCCCACT TGAACAA AA CATCAAGCAA ATGTACTTGT GCCTGCCCCA GGGTGAGAAA 11040

GTCCAAGCCA TGTATATCTG GGTTGATGGT ACTGGAGAAG GACTGCGCTG CAAAACCCGC 11100

ACCCTGGACT GTGAGCCCAA GTGTGTAGAA GAGTTACCTG AGTGGAATTT TGATGGCTCT 11160

AGTACCTTTC AGTCTGAGGG CTCCAACAGT GACATGTATC TCAGCCCTGT TGCCATGTTT 11220 CGGGACCCCT TCCGCAGAGA TCCCAACAAG CTGGTGTTCT GTGAAGTTTT CAAGTACAAC 11280

CGGAAGCCTG CAGAGACCAA TTTAAGGCAC TCGTGTAAAC GGATAATGGA CATGGTGAGC 11340

AACCAGCACC CCTGGTTTGG AATGGAACAG GAGTATACTC TGATGGGAAC AGATGGGCAC 11400

CCTTTTGGTT GGCCTTCCAA TGGCTTTCCT GGGCCCCAAG GTCCGTATTA CTGTGGTGTG 11460

GGCGCAGACA AAGCCTATGG CAGGGATATC GTGGAGGCTC ACTACCGCGC CTGCTTGTAT 11520 GCTGGGGTCA AGATTACAGG AACAAATGCT GAGGTCATGC CTGCCCAGTG GGAACTCCAA 11580

ATAGGACCCT GTGAAGGAAT CCGCATGGGA GATCATCTCT GGGTGGCCCG TTTCATCTTG 11640

CATCGAGTAT GTGAAGACTT TGGGGTAATA GCAACCTTTG ACCCCAAGCC CATTCCTGGG 11700

AACTGGAATG GTGCAGGCTG CCATACCAAC TTTAGCACCA AGGCCATGCG GGAGGAGAAT 11760

GGTCTGAAGC ACATCGAGGA GGCCATCGAG AAACTAAGCA AGCGGCACCG GTACCACATT 11820 CGAGCCTACG ATCCCAAGGG GGGCCTGGAC AATGCCCGTG GTCTGACTGG GTTCCACGAA 11880

ACGTCCAACA TCAACGACTT TTCTGCTGGT GTCGCCAATC GCAGTGCCAG CATCCGCATT 11940

CCCCGGACTG TCGGCCAGGA GAAGAAAGGT TACTTTGAAG ACCGCGGCCC CTCTGCCAAT 12000

TGTGACCCCT TTGCAGTGAC AGAAGCCATC GTCCGCACAT GCCTTCTCAA TGAGACTGGC 12060

GACGAGCCCT TCCAATACAA AAACTAATTA GACTTTGAGT GATCTTGAGC CTTTCCTAGT 12120 TCATCCCACC CCGCCCCAGA GAGATCTTTG TGAAGGAACC TTACTTCTGT GGTGTGACAT 12180

AATTGGACAA ACTACCTACA GAGATTTAAA GCTCTAAGGT AAATATAAAA TTTTTAAGTG 12240

TATAATGTGT TAAACTACTG ATTCTAATTG TTTGTGTATT TTAGATTCCA ACCTATGGAA 12300

CTGATGAATG GGAGCAGTGG TGGAATGCCT TTAATGAGGA AAACCTGTTT TGCTCAGAAG 12360

AAATGCCATC TAGTGATGAT GAGGCTACTG CTGACTCTCA ACATTCTACT CCTCCAAAAA 12420 AGAAGAGAAA GGTAGAAGAC CCCAAGGACT TTCCTTCAGA ATTGCTAAGT TTTTTGAGTC 12480

ATGCTGTGTT TAGTAATAGA ACTCTTGCTT GCTTTGCTAT TTACACCACA AAGGAAAAAG 12540

CTGCACTGCT ATACAAGAAA ATTATGGAAA AATATTCTGT AACCTTTATA AGTAGGCATA 12600

ACAGTTATAA TCATAACATA CTGTTTTTTC TTACTCCACA CAGGCATAGA GTGTCTGCTA 12660

TTAATAACTA TGCTCAAAAA TTGTGTACCT TTAGCTTTTT AATTTGTAAA GGGGTTAATA 12720 AGGAATATTT GATGTATAGT GCCTTGACTA GAGATCATAA TCAGCCATAC CACATTTGTA 12780

GAGGTTTTAC TTGCTTTAAA AAACCTCCCA CACCTCCCCC TGAACCTGAA ACATAAAATG 12840

AATGCAATTG TTGTTGTTAA CTTGTTTATT GCAGCTTATA ATGGTTACAA ATAAAGCAAT 12900

AGCATCACAA ATTTCACAAA TAAAGCATTT TTTTCACTGC ATTCTAGTTG TGGTTTGTCC 12960

AAACTCATCA ATGTATCTTA TCATGTCTGG ATCTAGCTTC GTGTCAAGGA CGGTGACTGC 13020 AGTGAATAAT AAAATGTGTG TTTGTCCGAA ATACGCGTTT TGAGATTTCT GTCGCCGACT 13080 AAATTCATGT CGCGCGATAG TGGTGTTTAT CGCCGATAGA GATGGCGATA TTGGAAAAAT 13140 CGATATTTGA AAATATGGCA TATTGAAAAT GTCGCCGATG TGAGTTTCTG TGTAACTGAT 13200 ATCGCCATTT TTCCAAAAGT GATTTTTGGG CATACGCGAT ATCTGGCGAT AGCGCTTATA 13260 TCGTTTACGG GGGATGGCGA TAGACGACTT TGGTGACTTG GGCGATTCTG TGTGTCGCAA 13320 ATATCGCAGT TTCGATATAG GTGACAGACG ATATGAGGCT ATATCGCCGA TAGAGGCGAC 13380 ATCAAGCTGG CACATGGCCA ATGCATATCG ATCTATACAT TGAATCAATA TTGGCCATTA 13440 GCCATATTAT TCATTGGTTA TATAGCATAA ATCAATATTG GCTATTGGCC ATTGCATACG 13500 TTGTATCCAT ATCATAATAT GTACATTTAT ATTGGCTCAT GTCCAACATT ACCGCCATGT 13560 TGACATTGAT TATTGACTAG TTATTAATAG TAATCAATTA CGGGGTCATT AGTTCATAGC 13620 CCATATATGG AGTTCCGCGT TACATAACTT ACGGTAAATG GCCCGCCTGG CTGACCGCCC 13680 AACGACCCCC GCCCATTGAC GTCAATAATG ACGTATGTTC CCATAGTAAC GCCAATAGGG 13740 ACTTTCCATT GACGTCAATG GGTGGAGTAT TTACGGTAAA CTGCCCACTT GGCAGTACAT 13800 CAAGTGTATC ATATGCCAAG TACGCCCCCT ATTGACGTCA ATGACGGTAA ATGGCCCGCC 13860 TGGCATTATG CCCAGTACAT GACCTTATGG GACTTTCCTA CTTGGCAGTA CATCTACGTA 13920 TTAGTCATCG CTATTACCAT GGTGATGCGG TTTTGGCAGT ACATCAATGG GCGTGGATAG 13980 CGGTTTGACT CACGGGGATT TCCAAGTCTC CACCCCATTG ACGTCAATGG GAGTTTGTTT 14040 TGGCACCAAA ATCAACGGGA CTTTCCAAAA TGTCGTAACA ACTCCGCCCC ATTGACGCAA 14100 ATGGGCGGTA GGCGTGTACG GTGGGAGGTC TATATAAGCA GAGCTCGTTT AGTGAACCGT 14160 CAGATCGCCT GGAGACGCCA TCCACGCTGT TTTGACCTCC ATAGAAGACA CCGGGACCGA 14220 TCCAGCCTCC GCGGCCGGGA ACGGTGCATT GGAACGCGGA TTCCCCGTGC CAAGAGTGAC 14280 GTAAGTACCG CCTATAGAGT CTATAGGCCC ACCCCCTTGG CTTCTTATGC ATGCTATACT 14340 GTTTTTGGCT TGGGGTCTAT ACACCCCCGC TTCCTCATGT TATAGGTGAT GGTATAGCTT 14400 AGCCTATAGG TGTGGGTTAT TGACCATTAT TGACCACTCC CCTATTGGTG ACGATACTTT 14460 CCATTACTAA TCCATAACAT GGCTCTTTGC CACAACTCTC TTTATTGGCT ATATGCCAAT 14520 ACACTGTCCT TCAGAGACTG ACACGGACTC TGTATTTTTA CAGGATGGGG TCTCATTTAT 14580 TATTTACAAA TTCACATATA CAACACCACC GTCCCCAGTG CCCGCAGTTT TTATTAAACA 14640 TAACGTGGGA TCTCCACGCG AATCTCGGGT ACGTGTTCCG GACATGGGCT CTTCTCCGGT 14700 AGCGGCGGAG CTTCTACATC CGAGCCCTGC TCCCATGCCT CCAGCGACTC ATGGTCGCTC 14760 GGCAGCTCCT TGCTCCTAAC AGTGGAGGCC AGACTTAGGC ACAGCACGAT GCCCACCACC 14820 ACCAGTGTGC CGCACAAGGC CGTGGCGGTA GGGTATGTGT CTGAAAATGA GCTCGGGGAG 14880 CGGGCTTGCA CCGCTGACGC ATTTGGAAGA CTTAAGGCAG CGGCAGAAGA AGATGCAGGC 14940 AGCTGAGTTG TTGTGTTCTG ATAAGAGTCA GAGGTAACTC CCGTTGCGGT GCTGTTAACG 15000 GTGGAGGGCA GTGTAGTCTG AGCAGTACTC GTTGCTGCCG CGCGCGCCAC CAGACATAAT 15060 AGCTGACAGA CTAACAGACT GTTCCTTTCC ATGGGTCTTT TCTGCAGTCA CCGTCCTTGA 15120 CACGA 15125 In certain aspects, the present teachings provide for a mammalian expression system for production of a heterotrimeric protein assembly comprising a polypeptide comprising amino acids 38- 16 lof the human CTLA-4 fused with IgG-Fc domain (Fc-II), another polypeptide comprising a heavy chain portion of an anti-CD20 antibody fused with another IgG-Fc domain (Fc-VI), and a third polypeptide comprising a light chain portion of the anti-CD20 antibody..

In an example embodiment, the mammalian expression system of the present invention comprises HEK293H cells harboring a plasmid comprising nucleotide sequence of SEQ ID NO. 10.

EXAMPLES

The following Examples illustrate the forgoing aspects and other aspects of the present teachings. These non-limiting Examples are put forth so as to provide those of ordinary skill in the art with illustrative embodiments as to how the compounds, compositions, articles, devices, and/or methods claimed herein are made and evaluated. The Examples are intended to be purely exemplary of the inventions disclosed herein and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for.

Example 1: Construction of plasmids for expression of polypeptides of the present teachings

Optimized gene sequences coding for extracellular portion of CTLA-4, and anti-CD20 antibody heavy and light chains where chemically synthesized. Recombinant plasmid PKN012- CTLA4-RIT (SEQ ID NO. 10) was then prepared utilizing standard molecular biology techniques. The plasmid map for PKN012-CTLA4-RIT is illustratively shown in Figure 2. Introducing the genes of interest into the plasmid was performed utilizing DNA restriction sites, as indicated in the map.

Recombinant plasmid PKN012- CTLA4-RIT combines expression cassettes for three polypeptides hCTLA4-hIgG 1 -Fc (SEQ ID NO. 4), hCD20ab-HC-h!gGl-Fc (SEQ ID NO. 6) and hCD20ab-LC (SEQ ID NO. 8). The plasmid can be used for co-expressing hCTLA-4-Fc-II, hCD20ab-HC-Fc-VI and hCD20ab-LC proteins in a 1 : 1 : 1 ratio under the control of a CMV promoter. Thus expressed three polypeptides contain leading signal peptides for secreting the proteins. During expression, in the process of protein secretion, the signal peptides are cleaved to yield protein sequences of SEQ ID NO. 1 , SEQ ID NO. 2, and SEQ ID NO. 3, respectfully. The majority of these three proteins were then expected to form hCTLA-4-Fc-II /hCD20ab-HC-Fc-VI/ hCD20ab-LC heterotri ners after secretion and signal peptides' cleavage. The plasmid also expresses Glutamine synthetase (GS) protein via a SV40 promoter, which can be used as a selection marker to generate stable cell lines for hCTLA-4-Fc-II /hCD20ab-HC-Fc-VI/ hCD20ab- LC heterotrimer production.

Example 2: Transient expression to obtain recombinant polypeptides of the present teachings. The chemical-based transfection of PKN012- CTLA4-RIT (SEQ ID NO. 10) construct was performed via polyethylenimine (PEI) to generate transient expression cell lines for hCTLA-4-Fc- II /hCD20ab-HC-Fc-VI/ hCD20ab-LC heterotrimer production. HEK293H cell was used for transient expressions. The following TE condition were used: 50ml Freestyle 293 + 50ml EX 293(+3.8mM VP A) for each plasmid. Post transfection cells were allowed to grown for another week, after which time the grown supernatant was collected for protein purification. The supernatant from transient transfections was analyzed for protein overexpression by 12% SDS- PAGE under non-reducing and reducing conditions. Overexpessed protein was clearly identifiable from the presence of prominent bands of appropriate molecular weights. Example 3; Purification of polypeptides of the present invention.

The supernatant collected from a 1L cell-medium mixture was clarified by filtration. Affinity purification of the supernatant was performed on a protein A column, and the target Fc fusion proteins were eluted at pH3.5. Purified protein was stored at -80°C in the elution buffer. The protein concentration was calculated by measuring OD280. The eluate was analyzed 12% SDS- PAGE. The results are presented in Figure 3. Lane 1 represents the eluate analyzed under non- reducing conditions, and Lane 2 - under reducing conditions. The protein recovery after protein A affinity purification was estimated at 42 mg per 1L of growth culture.

The protein A eluate was further resolved by size-exclusion chromatography on a 1100 series HPLC (Agilent Technologies) utilizing the following buffer as mobile phase: 20 mM phosphate, 300 mM NaCl, pH 7.4. The HPLC operating conditions are summarized in Table 1.

Table 1 : Since-exclusion chromatography conditions for resolving hCTLA-4-Fc-II /hCD20ab-HC-Fc-VI/ hCD20ab-LC protein heterotrimers of the present teachings.

A representative size-exclusion HPLC chromatogram and its analysis results are shown in Figure 4. Reported are all mean (n=3) % peak areas for Aggregates (Multimer), Monomer, and Fragments to one decimal place. Peak Area was calculated as follows:

% Aggregates (%Multimer) = ^Pe^_aT^_e?^ate « 100% %Monomer = ^Pea^[_a ^e^_e ^mer · 100%

· 1 i n 0n0n% /

Wherein:

i. Total Area is the sum of all peak areas.

ii. Peak AreaAggregate is the sum of all peak areas with retention times less than the

Monomer.

iii. Peak AreaFragments is the sum of all peak areas with retention times greater than the Monomer.

iv. Not include are the buffer constituent peaks that elute at the total column volume.

The analysis of the results indicates that the majority of the protein (about 79%) migrates as the major peak with elution time of 10.978 min, having an apparent molecular weight of about 80 kDa, corresponding to hCTLA-4-Fc-II /hCD20ab-HC-Fc-VI/ hCD20ab-LC protein heterotrimer. Example 4: Characterization of hCTLA-4-Fc-II /hCD20ab-HC-Fc-VI/ hCD20ab-LC heterotrimer CD80 binding properties.

Binding affinity of hCTLA-4-Fc-II /hCD20ab-HC-Fc-VI/ hCD20ab-LC heterotrimer to human CD80 was measured by an indirect ELISA method and compared to the binding affinity of Abatacept. The heterotrimer of the present teachings was prepared essentially as described in the foregoing Examples 1-3 and brought to a stock concentration of 16.2 mg/ml. Abatacept (Orencia, Bristol-Myers Squibb) at a stock concentration of 25 mg/ml, CD80-Fc-biotin at a stock concentration of 10 mg/ml, HRP-labeled Streptavidin and BSA were obtained from commercial sources. Following are the buffers and solutions used in the assay:

Coating Buffer: pH 9.6 50mM carbonate buffer

PBS pH 7.4

Washing buffer: 1XPBS containing 0.2%Tween

Blocking buffer: l%BSA in 1XPBS

Sample diluents: (HBS-BSA-T20): pH 7.2 HBS contain 1%BSA, 0.1% Tween20

Stop solution: 2N H₂S0₄ Prior to use, abatacept was diluted to 4ug/mL and hCTLA-4-Fc-II /hCD20ab-HC-Fc-VI/ hCD20ab-LC heterotrimer was diluted to lOug/mL with PBS. CD80-Fc-Biotin was serially diluted 4-fold, 11 times in sample diluents from a starting concentration of 15μg/mL. Microplates were coated with abatacept and hCTLA-4-Fc-II /hCD20ab-HC-Fc-VI/ hCD20ab-LC heterotrimer at 4 μg/mL and l(^g/mL, respectively, in coating buffer, with a ΙΟΟμί per well, and incubate at 2-8°C overnight. Thus coated microplates were washed, blocked and incubated with CD80-Fc-Biotin at room temperature for 2 hours. After wash, streptavidin-HRP was added and incubated for another 2 hours. Finally the assay was stopped and the plates were read at 450 nm, and at 650 nm as a reference, on microplate reader (Molecular Devices). Graphpad prism 4 software was used for the data analysis and representation.

The results of the assay are presented in Figure 5. Taking into account a single functional group (CTLA4) in the heterotrimer of the present teachings (Trimer) and bivalence in abatacept (Orencia), the coating concentration for heterotrimer was doubled. Under these conditions, the binding activity of the heterotrimer of the present teachings to CD80 was comparable to that of abatacept (Log EC50s are 2.177 and 1.757 ng/niL, respectively).

Example 5; Characterization of hCTLA-4-Fc-II /hCD20ab-HC-Fc-VI/ hCD20ab-LC heterotrimer CD20 binding properties. The CD20 binding properties of the heterotrimer of the present teachings were assessed by flow cytometry utilizing CD20 antigen expressing aji lymphoblast cell line. The heterotrimer of the present teachings was prepared essentially as described in the foregoing Examples 1-3 and brought to a stock concentration of 16.2 mg/ml. Abatacept (Orencia, Bristol-Myers Squibb) at a stock concentration of 25 mg/ml, Rituximab at a stock concentration of 10 mg/ml, and FITC Rabbit anti-human IgG were obtained from commercial sources. Following are the buffers and solutions used in the assay:

Growth medium: 10%FBS/l%Pen/Strep/RPMI 1640

Binding buffer: 3%BSA/PBS

Washing buffer: 1%BSA/PBS The samples were serially diluted 4-fold, 10 times in Binding buffer from a starting concentration of 30(^g/mL. All reactions were conducted at 4 deg C. The heterotrimer of the present teachings, Orencia and Rituximab were diluted in various concentrations and incubated with Raji cells for 30 minutes. The cells were then washed and incubated with the FITC- conjugated Rabbit anti human IgG. The mean fluorescence intensity of samples was acquired on a Guava easyCyte. Graphpad prism 4 software was used for data analysis and representation. The results of the assay are presented in Figure 6. The binding activity of the heterotrimer of the present teachings to Raji cells had an EC50 value of about 22 μg/mL compare to that of Rituxan with an EC50 value of about 0.76 μ νηΐ,, respectively.

Example 6: Characterization of hCTLA-4-Fc-II /hCD20ab-HC-Fc-VI/ hCD20ab-LC heterotrimer IL-2 inhibition properties.

The IL-2 production inhibition properties of the heterotrimer of the present teachings were assessed utilizing IL-2 secreting Jurkat Human Leukemic T cells stimulated with Raji cells. The heterotrimer of the present teachings was prepared essentially as described in the foregoing Examples 1-3, the other reagents were obtained from commercial sources. The samples were serially diluted 5-fold, 7 times in a medium containing 10%FBS, l%Pen/Strep/RPMI 1640, beginning with a starting concentration of lOOug/mL. Jurkat T cells were activated for 15 min at a density of 10000 cells/well in culture medium in the presence of anti-CD3. Subsequently Raji cells (20000 cells/well) were added with or without the heterotrimer of the present teachings or Orencia. The cells were co-cultured for 24 hours, and IL-2 secretion from the co-cultured T cells was determined using a Human IL-2 expression assay kit. Pro Softmax software was utilized for data analysis and representation. The results of the assay are presented in Figure 7. The heterotrimer of the present teachings inhibits IL-2 expression with about a 2-fold higher potency compared to that of Orencia (EC50s are 0.167 and 0.0715 ng/mL, respectively).

Example 7; Characterization of hCTLA-4-Fc-II /hCD20ab-HC-Fc-VI/ hCD20ab-LC heterotrimer complement-dependent cytotoxicity (CDC) activity. Tthe CDC activity of the heterotrimer of the present teachings were assessed utilizing human lymphoblast cell line Raji. The heterotrimer of the present teachings was prepared essentially as described in the foregoing Examples 1-3, the other reagents were obtained from commercial sources. The samples were serially diluted 3-fold 9 times in growth medium

(10%FBS/l%Pen/Strep/RPMI 1640) beginning with a starting concentration of 30 μg/mL. Raji cells (5 X 10⁴ cells per well) were incubated for 2 h with the heterotrimer of the present teachings or Rituximab at various concentrations in the presence of 20 % human serum as a complement source. Cell viability was measured with CC -8 assay (Dojindo). The absorbance at 450 nm was measured at 3 hr time points. Graphpad prism 4 software was utilized for data analysis and representation. The results of the assay are presented in Figure 8. The CDC activity of the heterotrimer of the present teachings to Raji cells had an EC50 value of about 1.605 μg/mL compare to that of Rituximab with an EC50 value of about 0.1031 μg/mL.

Example 8: Pharmacokinetics (PK) of hCTLA-4-Fc-II /hCD20ab-HC-Fc-VI/ hCD20ab-LC heterotrimer after subcutaneous administration in mice.

Polypeptides of hCTLA-4-Fc-II /hCD20ab-HC-Fc-VI/ hCD20ab-LC heterotrimer (SEQ ID NO. 1 , SEQ ID NO. 2 and SEQ ID NO. 3) were co-expressed and purified essentially as described in the forgoing examples. For administration into animals, the polypeptides were formulated in the following buffer: 1 % w/v Sucrose, 100 mM Sodium Chloride, 20 mM L-Arginine Hydrochloride, 25 mM Sodium Phosphate, pH 6.3. The dosing stock concentration used was 0.5 mg/mL of the polypeptide.

Fourteen male DBA/1 mice were randomized by body weight into seven groups of two animals on Day 0 of the study. A single treatment of the heterotrimer polypeptide (5 mg/kg) was administered subcutaneously (dorsally) on Day 0 to all groups except mice in Group 1 , which were bled via terminal cardiac puncture for plasma preparation on Day 0 of the study. Blood samples were collected from mice via the orbital sinus or terminal cardiac puncture in the remaining groups at specified times throughout the three-week study for preparation of plasma.

Body weights were recorded for all animals on the treatment day (Day 0) and then three times per week, including the termination day of each group. Body weight change was not measured in groups culled for sample collection at 0 hours and within 36 hours of dose administration. There was mean body weight loss between Day 0 and termination of the groups culled between 96 hours and 21 days post-dose. However, no mice lost body weight exceeding ethical limits.

Following the in-life phase of the study, plasma samples were analyzed by Enzyme Linked

Immunosorbent Assay (ELISA) for Hu-Fc protein. Quantification of Hu-Fc protein in mouse plasma samples by ELISA was used as a read-out for circulating levels of the heterotrimer polypeptide. The assay was performed on samples from all mice in the study.

Peak circulating level of Hu-Fc (Cmax) was determined to be 0.314 μg/mL, and time to peak circulating levels (Tmax) was 8 hours post-dose. Hu-Fc was below the level of detection in the plasma collected at 30 minutes post-dose, and at 7, 14 and 21 days post-dose. The half-life (Tl/2) was approximated at 99 hours and the rate constant (K) was approximately 0.007 hr-1. Hu-Fc was below the level of detection in the plasma of the untreated animals. As Human-Fc protein was below detectable limits in the mouse plasma at all time points investigated in the present study beyond 96-hours post-dose. The results of the study are summarized in Table 2.

Table 2: Mean Human-Fc Protein Concentration ± SEM ^g/mL) at each Time Post- Administration

Bleeding Schedule Mean Human-Fc

Group Treatment (time post- Protein Concentration SEM

administration)

1 No treatment 0 hours * 0 .*

2 30 minutes^* 0 .*

3 polypeptides of 1 hour 0.013 0.009

4 SEQ IDs O. l , No. 2 hours 0.056 0.018

2 and NO. 3 (5

5 4 hours 0.090 0.002

mg/kg, Once only,

6 s.c.) 8 hours^* 0.314 0.011

7 10 hours 0.299 0.023 Bleeding Schedule Mean Human-Fc

Group Treatment (time post- Protein Concentration SEM

administration)

2 24 hours* 0.314 0.031

3 36 hours* 0.287 0.006

4 96 hours* 0.239 0.010

5 7 days* 0 .*

6 14 days* 0 .*

7 21 days* 0 .*

* SEM could not be calculated as the level of Hu-Fc was below detectable limit of ELISA for one or both of the samples in the group.

The Human-Fc Protein Concentration was determined by Prism Software based on the mean absorbance of the triplicate samples

Bleed via orbital sinus

# Bleed via terminal cardiac puncture

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

"Orencia" is a registered trademark of Bristol-Myers Squibb Company Corporation, a Delaware Corporation. "Rituxan" is a registered trademark of Biogen Idee Inc., a Delaware Corporation.

While specific embodiments of the subject matter have been discussed, the above specification is illustrative and not restrictive. Many variations will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

What is claimed is:

1. A heterotrimeric protein composition capable of binding human CD80 and human CD20 proteins, said protein composition comprising:

a first polypeptide comprising

a first amino acid sequence comprising amino acids 38 through 161 of human CTLA-4, and

a second amino acid sequence comprising a first mutant of a Fc portion of human immunoglobulin gamma- 1 Fc;

a second polypeptide comprising

another first amino acid sequence comprising amino acids of a variable (VH) domain followed by a constant (CHI) domain of a heavy chain of an anti- CD20 human immunoglobulin gamma- 1 capable of binding human CD20, and another second amino acid sequence comprising a second mutant of the Fc portion of human immunoglobulin gamma- 1 Fc ;

a third polypeptide comprising a third amino acid sequence comprising a light chain of the anti-CD20 human immunoglobulin gamma- 1 capable of binding human CD20; and

wherein said first and second mutants are selected as to favor heterodimeric assembly between said first and second mutants over any homodimeric assembly.

2. The protein composition of claim 1 , wherein said protein composition exhibits human IL- 1β binding activity in an ELISA assay with an EC50 of about 50 ng/ml.

3. The protein composition of claim 1, said first polypeptide comprises amino acid sequence of SEQ ID NO. 1 , said second polypeptide comprises amino acid sequence of SEQ ID NO. 2, and said third polypeptide comprises amino acid sequence of SEQ ID NO. 3.

4. A therapeutic composition, the composition comprising a heterotrimeric protein

composition capable of binding human CD80 and human CD20 proteins, said heterotrimeric protein composition comprising: a first polypeptide comprising

a first amino acid sequence comprising amino acids 38 through 161 of human CTLA-4, and

a second amino acid sequence comprising a first mutant of a Fc portion of human immunoglobulin gamma- 1 Fc;

a second polypeptide comprising

another first amino acid sequence comprising amino acids of a variable (VH) domain followed by a constant (CHI) domain of a heavy chain of an anti- CD20 human immunoglobulin gamma- 1 capable of binding human CD20, and another second amino acid sequence comprising a second mutant of the Fc portion of human immunoglobulin gamma- 1 Fc;

a third polypeptide comprising a third amino acid sequence comprising a light chain of an anti-CD20 human immunoglobulin gamma- 1 capable of binding human CD20; and

wherein said first and second mutants are selected as to favor heterodimeric assembly between said first and second mutants over any homodimeric assembly.

The therapeutic composition of claim 4, wherein half-life of said heterotrimeric protein composition in systemic circulation in mice after a subcutaneous administration at a dose of 5 mg/kg is at least about 99 hours, as assayed by human Fc ELISA.

A therapeutic composition of claim 4, the composition comprising a heterotrimeric protein composition comprising a first polypeptide comprising amino acid sequence of SEQ ID NO. 1 , a second polypeptide comprising amino acid sequence of SEQ ID NO. 2, and a third polypeptide comprising amino acid sequence of SEQ ID NO. 3.

An isolated nucleic acid encoding a polypeptide comprising amino acid sequence of SEQ ID NO. 4.

An isolated nucleic acid encoding a polypeptide comprising amino acid sequence of SEQ ID NO. 6.

9. An isolated nucleic acid encoding a polypeptide comprising amino acid sequence of SEQ

ID NO. 8. 10. The nucleic acid of claims 7, 8, or 9, wherein the codon usage is optimized for high

expression of said polypeptide in a mammalian cell.

11. The nucleic acid of claim 7, wherein the nucleic acid sequence comprises the sequence of

SEQ ID NO. 5.

The nucleic acid of claim 8, wherein the nucleic acid sequence comprises the sequence of

SEQ ID NO. 7.

13. The nucleic acid of claim 9, wherein the nucleic acid sequence comprises the sequence of

SEQ ID NO. 9.

14. The nucleic acid of claims 11, 12, or 13, wherein said nucleic acid further comprises an expression vector. 15. An isolated nucleic acid of SEQ ID NO. 10.

16. A heterologous expression system, the expression system harboring an expression vector comprising a nucleic acid sequence encoding a first polypeptide comprising amino acid sequence of SEQ ID NO. 4, another nucleic acid sequence encoding a second polypeptide comprising amino acid sequence of SEQ ID NO. 6, and a third nucleic acid sequence encoding a third polypeptide comprising amino acid sequence of SEQ ID NO. 8.

17. The expression system of claim 16, wherein said expression vector is harbored in a

mammalian cell.

18. The expression system of claim 17, wherein said mammalian cell is a HEK293H cell.

19. The expression system of claim 16, 17, or 18, wherein said expression system is capable of expressing a heterotrimeric protein assembly comprising a first polypeptide comprising amino acid sequence of SEQ ID NO. 1, another nucleic acid sequence encoding a second polypeptide comprising amino acid sequence of SEQ ID NO. 2, and a third nucleic acid sequence encoding a third polypeptide comprising amino acid sequence of SEQ ID NO. 3.

20. Use of a substance for manufacture of a medicament for the treatment or prevention of a disease associated with modulation of activity of human CD80 protein or human CD20 protein, the substance comprising a heterotrimeric protein comprising a first polypeptide comprising amino acid sequence of SEQ ID NO. 1, a second polypeptide comprising amino acid sequence of SEQ ID NO. 2, and a third polypeptide comprising amino acid sequence of SEQ ID NO. 3.

21. The use according to claim 20, wherein said disease is a rheumatoid arthritis.

22. The use according to claim 21, wherein said disease is a multiple sclerosis.

23. A method of treating or preventing a disease or condition associated with modulation of activity of human CD80 protein or human CD20 protein, the method comprising administering to a patient in need for treating or preventing a disease associated with modulation of activity of human CD80 protein or human CD20 protein a therapeutically effective amount of a pharmaceutical composition comprising a heterotrimeric protein comprising a first polypeptide comprising amino acid sequence of SEQ ID NO. 1 , a second polypeptide comprising amino acid sequence of SEQ ID NO. 2, and a third polypeptide comprising amino acid sequence of SEQ ID NO. 3.

24. The method according to claim 23, wherein said disease is a rheumatoid arthritis.

25. The method according to claim 23, wherein said disease is a multiple sclerosis.