BR112014027131B1 - TARGETED/IMMUMODULATORY FUSION PROTEINS AND METHODS FOR OBTAINING THEM - Google Patents

Abstract

TARGETED/IMMUMODULATORY FUSION PROTEINS AND METHODS FOR OBTAINING THEM. The present invention relates generally to the field of generating fusion proteins to be used in cancer therapy, and more specifically, to nucleotide sequences that encode the fusion proteins, where the chimeric fusion protein comprises at least one radical of targeting and at least one immunomodulatory moiety that counteracts cancer cell immunotolerance.

Description

CROSS-REFERENCE WITH RELATED ORDERS

[0001] The application claims priority over Indian patent application No. 1689/CHE/2012, filed on April 30, 2012 and Indian patent application No. 1690/CHE/2012, filed on April 30, 2012, the contents of which are incorporated herein by reference for all purposes.

BACKGROUND OF THE INVENTION

[0002] Technical Field

[0003] The present invention relates generally to the field of generating fusion proteins to be used in cancer therapy, and more specifically to nucleotide sequences encoding fusion proteins, where fusion or chimeric polypeptides comprise at least a targeting moiety and at least one immunomodulatory moiety that neutralizes the immunotolerance of cancer cells.

[0004] Related Technique

[0005] The immune system provides the human body with a means to recognize and defend itself against microorganisms and substances recognized as foreign or potentially dangerous. Although passive cancer immunotherapy with monoclonal antibodies and passive transfer of T cells to attack tumor cells have demonstrated clinical efficacy, the goal of active therapeutic vaccination to induce these immune effectors and establish an immunological memory against tumor cells has remained a challenge. Several tumor-specific and tumor-associated antigens have been identified, yet these antigens in general are poorly immunogenic and tumors employ various mechanisms to create an environment that allows them to avoid immune attack. Strategies to overcome such immunotolerance and activate robust levels of antibody and/or T-cell responses are the key to effective cancer immunotherapy. More importantly, individual proteins and how to create an active chimeric polypeptide with an active tertiary structure need to be explored.

SUMMARY OF THE INVENTION

[0006] The present invention provides polynucleotides, as well as polypeptides encoded by them, which are expressed in cancer cells. These expressed polynucleotides and polypeptides are useful in a variety of therapeutic methods for treating cancer. The present invention further provides methods for reducing the growth of cancer cells by neutralizing the immunotolerance of the cancer cells, where the T cell remains active and inhibits the recruitment of regulatory T cells which are known to suppress immune system responses to the tumor. In this way, the chimeric polypeptides generated by the polynucleotide sequences of the present invention are useful in the treatment of cancer, due to the fusion or chimeric polypeptides.

[0007] In one aspect, the present invention provides chimeric polypeptides containing at least one targeting moiety for targeting a cancer cell and at least one immunomodulatory moiety that neutralizes the immunotolerance of the cancer cell, wherein the targeting moiety and the immunomodulatory moiety are linked by a amino acid spacer of sufficient length in amino acid residues such that both radicals can successfully bind to their individual targets. Alternatively, the targeting moiety and the immunomodulatory moiety that neutralizes cancer cell immunotolerance can be linked directly to each other. The chimeric/fusion polypeptides of the invention are useful for binding to a cancer cell receptor and reducing the ability of cancer cells to evade an immune response.

[0008] The present invention is based on the preparation of chimeric/fusion proteins by expressing polynucleotides that encode fusion proteins that neutralize or reverse immunotolerance of cancer cells. Cancer cells are able to evade elimination by chemotherapeutic agents or tumor-directed antibodies through specific immunosuppressive mechanisms in the tumor microenvironment, and this ability of cancer cells is known as immunotolerance. Such immunosuppressive mechanisms include immunosuppressive cytokines (eg, Transforming Growth Factor beta (TGF-β)) and regulatory T cells and/or immunosuppressive myeloid dendritic cells (DCs). By counteracting tumor-induced immunotolerance, the present invention provides effective compositions and methods for treating cancer, in optional combination with another existing cancer treatment. The present invention provides strategies to counteract tumor-induced immunotolerance and enhance the antitumor efficacy of chemotherapy by activating and leveling antitumor adaptive T-cell mediated against resistant or disseminated cancer cells.

[0009] In another aspect, the present invention provides a molecule including at least one targeting moiety fused with at least one immunomodulatory moiety. The targeting moiety specifically binds to a target molecule, and the immunomodulatory moiety specifically binds one of the following molecules: (i) Transforming growth factor-beta (TGF-β): (ii) Programmed death ligand 1 (PD-Ll ) or Programmed Death Ligand 1 (PD-L2); (iii) Nuclear factor-KB activating receptor (RANKL) ligand (RANK); (iv) Transforming growth factor receptor beta (TGF-pR); (v) Programmed Death 1 (PD-1); (vi) 4-1BB Receptor or (vii) Nuclear Factor-KB Activating Receptor (RANK).

[0010] In a further aspect, the targeting moiety includes an antibody, antibody fragment including the light or heavy chains of the antibody, scFv, or Fc-containing polypeptide that specifically binds a component of a tumor cell, tumor antigen, tumor vasculature, micro -tumor environment, or immune cell that infiltrates tumor. Preferably, the targeting moiety is an antibody or fragment thereof having binding affinity for a component on a tumor cell. Notably, each of the heavy and light chains can be individually linked to a separate and distinct immunomodulatory moiety. Furthermore, a heavy or light chain of a targeting moiety of an antibody can be linked to an immunomodulatory moiety which in turn can be further linked to a second immunomodulatory moiety where there is a link between the two immunomodulatory moieties.

[0011] In yet a further aspect, there is provided a chimeric polypeptide comprising a tumor targeting moiety and an immunomodulatory moiety comprising a molecule that binds a transforming growth factor beta (TGF-β), wherein the tumor targeting moiety is a antibody that binds to EGFR1, where the antibody can be the whole antibody, the heavy chain or the light chain. The tumor targeting moiety may include monoclonal antibodies that are targeted to a cancer cell including, but not limited to, cetuximab, trastuzumab, ritubximab, ipilimumab, tremelimumab, Muromonab-CD3, abciximab, daclizumab, basiliximab, palivizumab, infliximab. gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxethane, adalimumab, omalizumab, tositumomab, I-131 tositumomab, efalizumab, bevacizumab, panitumumab, pertuzumab, natalizumab, etanercept, IGN101 (Aphton), volociximab (Biogen Idec and PDL BioPharm), Anti- CD80 mAb ( Biogen Idec), Anti-CD23 mAb (Biogen Idel), CAT-3888 (Cambridge Antibody Technology), CDP-791 (Imclone), eraptuzumab (Immunomedics), 3/IDX-010 (Medarex and BMS), MDX-060 (Medarex ), MDX-070 (Medarex), matuzumab (Merck), CP-675,206 (Pfizer), CAL (Roche), SGN-30 (Seattle Genetics), zanolimumab (Serono and Genmab), adecatumumab (Sereno), oregovomab (United Therapeutics ), nimotuzumab (YM Bioscience), ABT-874 (Abbott Laboratories), denosumab (Amgen), AM 108 (Amgen), AMG 714 (Amgen), fontolizumab (Biogen Idec and PDL BioPhami), daclizumab (Biogent Idec and PDL BioPharm) , golimumab (Centocor and Schering-Plough), CNTO 1275 (Centocor), ocrelizumab (Genetech and Roche), HuMax-CD20 (Genmab), belimumab (HGS and GSK), epratuzumab (Immunomedics), MLN1202 (Millennium Pharmaceuticals), visilizumab ( PDL BioPhami), tocilizumab (Roche), ocrerlizumab (Roche), certolizumab pegal (UCB, formerly Celltech), eculizumab (Alexion Phamiaceuticals), pexelizumab (Alexion Phamiaceuticals and Procter & Gamble), abciximab (Centocor), ranibizimumab (Genetech), mepolizumab (GSK), TNX-355 (Tanox), or MYO-029 (Wyeth).

[0012] In another aspect, the tumor targeting moiety is a monoclonal antibody that binds to HER2/Neu, CD20, CTLA4, EGFR1 and where the antibody may be the whole antibody, the heavy chain or the light chain.

[0013] In yet another aspect, the targeting moiety is a molecule that specifically binds epidermal growth factor receptor (EGFR1, Erb-B l), HER2/neu (Erb-B2), CD20, T lymphocyte antigen 4 cytotoxic (CTLA-4), which is essential for Treg function (CD 152); Interleukin-6 (IL-6) H-land.

[0014] In yet a further aspect, the targeting moiety specifically binds a component of a regulatory T cell (treg), myeloid suppressor cell, or dendritic cell. In another aspect, the targeting moiety specifically binds one of the following molecules: (i) CD4; (ii) CD25 (IL-2α receptor; IL-2aR); (111) transforming growth factor receptor-beta (TGF-pR); (vi) transforming growth factor-beta (TGF-β): (vii) programmed death-1 (PD-1); (viii) programmed death ligand - 1 (PD-LI or PD-L2).

[0015] In another aspect, the immunomodulatory moiety specifically binds one of the following molecules: (i) transforming growth factor-beta (TGF-β): (ii) programmed death ligand-1 (PD-Ll or PD-L2 ); or 4-1BB receiver.

[0016] In yet another aspect, the immunomodulatory moiety includes a molecule that binds TGF-β and inhibits its function. Specifically, the immunomodulatory moiety includes a TGF-βRII, TGF-βRIIb, or extracellular TGF-βRIII transforming growth factor receptor-beta ligand-binding domain. In another aspect the immunomodulatory moiety includes a ligand binding domain (ECD) from extracellular TGF-βRII. Still further, the immunomodulatory moiety can include an H-4-1BB ligand that binds to the 4-1BB receptor to stimulate T cells to aid in tumor eradication.

[0017] In yet a further aspect, the targeting moiety includes an antibody, antibody fragment, or polypeptide that specifically binds to HER2/neu, EGFR1, CD20, or cytotoxic T lymphocyte antigen-4 (CTLA-4) and where the immunomodulatory moiety includes an extracellular TGF-βRIIm ligand-binding domain.

[0018] In yet another aspect, the immunomodulatory moiety includes a molecule that specifically binds and inhibits the activity of programmed death ligand-1 (PD-L1) or programmed death ligand-2 (PD-L2). In another aspect, the immunomodulatory moiety includes an extracellular ligand-binding domain or programmed death-1 (PD-1) ectodomain.

[0019] In a further aspect, the targeting moiety includes an antibody, antibody fragment, or polypeptide that specifically binds to HER2/neu, EGFR1, CD20, cytotoxic T lymphocyte antigen-4 (CTLA-4), CD25 (receptor of IL-2a; IL-2aR), or CD4 and where the immunomodulatory moiety includes an extracellular ligand-binding domain or ectodomain of programmed death-1 (PD-1).

[0020] In yet a further aspect, the targeting moiety includes an antibody or antibody fragment that specifically binds to CD20, and the immunomodulatory moiety includes a transforming growth factor-B (TGF-β) sequence.

[0021] In one aspect, the present invention provides optimized genes encoding a fusion polypeptide comprising at least one targeting moiety and at least one immunomodulatory moiety for treating cancer in a human subject, where the optimized genes have been modified to increase expression in a human individual. preferably the optimized genes comprise sequences for encoding a targeting moiety or an immunomodulatory moiety selected from SEQ ID NOs: 12 to 28.

[0022] In another aspect, the present invention provides a vector comprising genes optimized for treating cancer in a human subject, where the optimized genes have been modified to increase CG sequences. Preferably, the vector includes sequences for encoding at least one targeting moiety and at least one immunomodulatory moiety selected from SEQ ID NOs: 12 to 28.

[0023] In yet another aspect, the present invention provides a method of treating cancer in a subject, the method comprising: a. provision of at least one recombinant vector comprising nucleotide sequences encoding at least one targeting moiety and at least one immunomodulatory moiety selected from SEQ ID NOs: 12 to 28; and b. administering the recombinant vector to the subject under conditions such that said nucleotide sequences are expressed at a level which produces a therapeutically effective amount of the encoded fusion proteins in the subject.

[0024] In an alternative aspect, the present invention provides an expression vector comprising optimized gene polynucleotides encoding at least one targeting moiety and at least one immunomodulatory moiety selected from SEQ ID NOs: 12 to 28.

[0025] In yet another aspect, the present invention provides a recombinant host cell transfected with a polynucleotide encoding a fusion protein peptide of the present invention.

[0026] In yet a further aspect, the present invention contemplates a process for preparing a fusion protein of the present invention comprising: a. transfecting a host cell with polynucleotide sequences encoding chimeric fusion proteins to produce a transformed host cell, wherein the polynucleotide sequences encode at least one targeting moiety and at least one immunomodulatory moiety selected from SEQ ID NOs: 12 to 28; and b. maintaining the transformed host cell under biological conditions sufficient for expression of the peptide.

[0027] In another aspect, the present invention relates to the use of a chimeric fusion protein, as shown in Figures 1 to 15, in the use of a medicament for the treatment of cancer. Preferably, the fusion protein is expressed in a host cell and such expressed proteins are administered in a therapeutic amount to reduce the effects of cancer in an individual in need thereof.

[0028] In yet a further aspect, the present invention provides a method for preventing or treating a neoplastic disease. The method includes administering to a subject in need of such treatment one or more fusion proteins of the invention, in various aspects, administering to the subject one or more molecules of the invention in combination with another anti-cancer therapy, in one aspect, anti-cancer therapy includes a chemotherapeutic molecule, antibody, small molecule kinase inhibitor, hormonal agent, or cytotoxic agent. Anticancer therapy may also include ionizing radiation, ultraviolet radiation, cryoablation, thermal ablation, or radiofrequency ablation.

[0029] In yet another aspect, the present invention provides a method for preparing therapeutically active antibody-peptide fusion proteins, the method comprising: a. preparing a codon-optimized sequence of said fusion protein; B. cloning the optimized sequence of said fusion protein into a host cell capable of transient or continuous expression; w. growing the host cell in a medium under conditions suitable for growth allowing the host cell to express the cloned protein; and d. purifying the expressed protein and optionally verifying the bi-specific binding capacity of the protein to its target.

[0030] In a preferred embodiment, the therapeutically active antibody-peptide fusion proteins are a targeting antibody fused to one or more immunomodulatory moieties that neutralize the immunotolerance of a cancer cell. In one aspect, the immunomodulatory moiety can be linked by an amino acid spacer of sufficient length to allow bispecific binding of the molecule. The immunomodulatory moiety can be attached to the C-terminus of either the heavy chain or the light chain of the antibody.

[0031] In a preferred method as described above, the immunomodulatory moiety is (i) Transforming Growth Factor-beta (TGF-β), (ii) Programmed Death 1 (PD-1), (iii) CTLA-4 or (iv) 4-1BB or parts thereof and the targeting antibody binds epidermal growth factor receptor (EGFR1, Erb-B 1), HER2/neu (Erb-B2), CD20, CD6, CTLA-4, Mucin 1 (MUC-1), Interleukin-2 (IL-2) or Interleukin-6 (IL-6).

[0032] The method of the present invention provides nucleotide sequences encoding therapeutically active antibody-peptide fusion proteins, and such expression can be conducted in a transient cell line or in a stable cell line. Transient expression is achieved by transfecting or transforming the host with vectors containing the fusion proteins in mammalian host cells.

[0033] Once the fusion peptides are expressed, they are preferably subjected to purification and in-vitro tests to verify their bi-specificity, which, if present, presents the ability to bind both the target fraction and the immunomodulatory fraction. Such tests may include in-vitro tests such as ELISA or NK/T cell binding assays in order to validate bi-functional target binding or immune cell stimulation.

[0034] Notably, once specific fusion peptides demonstrate the desired bi-specificity, such fusion peptides are selected for subcloning into a stable cell line for expression and purification on a larger scale. Such stable cell lines have been previously described, such as a mammalian cell line, including, but not limited to, HEK293, CHO or NSO.

[0035] In a further aspect, the culture medium can be improved by additions to such medium. For example, the culture medium can include a divalent transition metal salt that is added to the cell culture either initially or in batch-feed mode to reduce lactate accumulation during culture and/or reduce heterogeneity of the fusion proteins. . A desirable transition metal salt includes a zinc ion and the addition of the metal ion can be conducted during different stages of production.

[0036] Other features and advantages of the invention will become apparent from the detailed description, drawings and claims that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 shows the amino acid sequences with the amino acid sequence of the Anti-HER2/neu-TGFβRII fusion protein in the LC constant region with the amino acid sequence of the heavy chain of anti-HER2/neu (SEQ ID NO: 1) and anti-HER2/neu light chain (SEQ ID NO: 2) fixed at amino residues for TGF-βRII (immunomodulatory radical) (SEQ ID NO: 4) identified in bold letters and where a linker (SEQ ID NO : 3) is positioned between the light chain of anti-HER2/neu and TGF-βRII and shown in italics.

[0038] Figure 2 shows the amino acid sequences of the Anti-EGFR1-TGFβRII fusion protein in the LC constant region with the amino acid sequence of the Anti-EGFR1 heavy chain (SEQ ID NO: 5) and the amino acid sequence of the Anti-EGFR1 chain Anti-EGFR1 light (SEQ ID NO: 6) linked to amino acid residues for TGF-βRII (immunomodulatory radical) (SEQ ID NO: 4) identified in bold letters and where a linker (SEQ ID NO: 3) is positioned between Anti-EGFR1 light chain and TGF-βRII and shown in italics.

[0039] Figure 3 shows the amino acid sequences of the Anti-CTLA4-TGFβRII fusion protein in the LC constant region with the amino acid sequences of the anti-CTLA4 heavy chain (SEQ ID NO: 7) and the amino acid sequence of the anti-CTLA4 chain anti-CTLA4 light (SEQ ID NO: 8) linked at the amino acid residues for TGF-βRII (immunomodulatory radical) (SEQ ID NO: 4) identified in bold letters where a linker (SEQ ID NO: 3) is positioned between the light chain of anti-CTLA4 and TGF-βRII and shown in italics.

[0040] Figure 4 shows the amino acid sequences of the fusion protein Anti-HER2/neu HC-4-1BB and LC-TGFβRII with the amino acid sequences of the fusion protein Anti-HER2/neu/HC-4-1BB where the amino acid sequence for the heavy chain of Anti-HER2/neu (SEQ ID NO: 1) is linked to a linker (SEQ ID NO: 3) shown in italics and the sequence for 4-1BB (immunomodulatory radical) (SEQ ID NO: 9) is in handwritten font and the amino acid sequence of anti-HER2/neu light chain (SEQ ID NO: 2) fixed at amino residues for TGF-βRII (immunomodulatory radical) (SEQ ID NO: 4) identified in bold letters and where a linker (SEQ ID NO: 3) is positioned between the anti-HER2/neu light chain and TGF-βRII and shown in italics.

[0041] Figure 5 shows the amino acid sequence of the fusion protein Anti-EGFR1 HC-4-1BB and LC-TGFβRII with the amino acid sequence of the fusion protein Anti-EGFR1 heavy chain-4-1BB where the amino acid sequence for Anti-EGFR1 heavy chain (SEQ ID NO: 5) is attached to a linker (SEQ ID NO: 3) and is shown in italics and the sequence for 4-1BB (immunomodulatory radical) (SEQ ID NO: 9) is in handwritten font and the amino acid sequence of the Anti-EGFR1 light chain (SEQ ID NO: 6) fixed at the amino residues for TGF-βRII (immunomodulatory radical) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3) between them.

[0042] Figure 6 shows the amino acid sequence of the fusion protein Anti-CTLA4 HC-4-1BB and LC-TGFβRII with the amino acid sequence of the fusion protein Anti-CTLA4 heavy chain-4-1BB where the amino acid sequence for the Anti-CTLA4 heavy chain (SEQ ID NO: 7) is linked to a linker (SEQ ID NO: 3) is shown in italics and the sequence for the 4-1BB (immunomodulatory radical) (SEQ ID NO: 9) is in handwritten font and the amino acid sequence of the Anti-CTLA4 light chain (SEQ ID NO: 8) is fixed at amino residues for TGF-βRII (radical immunomodulatory) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3) between them.

[0043] Figure 7 shows the amino acid sequence of the fusion protein Anti-HER2/neu HC-PD1 and LC-TGFβRII with the amino acid sequence of the fusion protein Anti-HER2/neu heavy chain-PD1 where the amino acid sequence for Anti-HER2/neu heavy chain (SEQ ID NO: 1) is attached to a linker (SEQ ID NO: 3) is shown in italics and the sequence for PD1 (immunomodulatory radical) (SEQ ID NO: 10) is shown in in handwritten font and the amino acid sequence of the Anti-HER2/neu light chain (SEQ ID NO: 2) is attached to amino residues for TGF-βRII (immunomodulatory radical) (SEQ ID NO: 4) identified in letters in bold with a linker (SEQ ID NO: 3) between them.

[0044] Figure 8 shows the amino acid sequence of the fusion protein Anti-EGFR1 HC-PD1 and LC-TGFβRII with the amino acid sequence of the heavy chain fusion protein of Anti-EGFR1-PD1 where the amino acid sequence of the heavy chain of Anti-EGFR1 (SEQ ID NO: 5) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for PD1 (immunomodulatory radical) (SEQ ID NO: 10) is in handwritten font and the Anti-EGFR1 light chain amino acid sequence (SEQ ID NO: 6) attached to amino residues for TGF-βRII (immunomodulatory radical) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3 ) between them.

[0045] Figure 9 shows the amino acid sequence of the fusion proteins Anti-CTLA4 HC-PD1 and LC-TGFβRII with the amino acid sequence of the heavy chain fusion protein of Anti-CTLA4-PD1 where the amino acid sequence of the heavy chain of Anti-CTLA4 (SEQ ID NO: 7) is linked to a linker (SEQ ID NO: 3) shown in italics and the sequence for PD1 (immunomodulatory radical) (SEQ ID NO: 10) is in handwritten font and the amino acid sequence of Anti-CTLA4 light chain (SEQ ID NO: 8) attached to amino residues for TGF-βRII (immunomodulatory radical) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3 ) between them.

[0046] Figure 10 shows the amino acid sequence of the Anti-HER2/neu HC-TGFβRII-4-1BB fusion protein with the amino acid sequence of the Anti-HER2/neu-TGFβRII-4-1BB heavy chain fusion protein where the amino acid sequence for the Anti-HER2/neu heavy chain (SEQ ID NO: 1 with an additional C-terminal Lys) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for TGFβRII ( immunomodulatory radical) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for 4-1BB (immunomodulatory radical) (SEQ ID NO: 9) is in handwritten font with a linker between the (SEQ ID NO: 9) No: 11) and including the light chain amino acid sequence of Anti-HER2/neu (SEQ ID NO: 2).

[0047] Figure 11 shows the amino acid sequence of the Anti-EGFR1 HC-TGFβRII-4-1BB fusion protein with the amino acid sequence of the Anti-EGFR1-TGFβRII-4-1BB heavy chain fusion protein where the sequence of amino acid sequence for the Anti-EGFR1 heavy chain (SEQ ID NO: 5 with an additional C-terminal Lys) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for TGFβRII (immunomodulatory radical) ( SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for 4-1BB (immunomodulatory radical) (SEQ ID NO: 9) is in handwritten font with a linker between them (SEQ ID NO: 11) and including the Anti-EGFR1 light chain amino acid sequence (SEQ ID NO: 6).

[0048] Figure 12 shows the amino acid sequence of the Anti-CTLA4 HC-TGFβRII-4-1BB fusion protein with the amino acid sequence of the Anti-CTLA4-TGFβRII-4-1BB heavy chain fusion protein where the sequence of Anti-CTLA4 heavy chain amino acids (SEQ ID NO: 7 with an additional C-terminal Lys) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for TGFβRII (immunomodulatory radical) (SEQ ID NO : 4) is identified in bold letters and the amino acid sequence for 4-1BB (immunomodulatory radical) (SEQ ID NO: 9) is in handwritten font with a linker between (SEQ ID NO: 11) and including the sequence of Anti-CTLA4 light chain amino acids (SEQ ID NO: 8).

[0049] Figure 13 shows the amino acid sequence of the Anti-HER2/neu HC-TGFβRII-PD1 fusion protein with the amino acid sequence of the Anti-HER2/neu-TGFβRII-PD1 heavy chain fusion protein where the sequence of Anti-HER2/neu heavy chain amino acids (SEQ ID NO: 1 with an additional C-terminal Lys) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for TGFβRII (immunomodulatory radical) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for PD-1 (immunomodulatory radical) (SEQ ID NO: 10) is in handwritten font with the linker between (SEQ ID No: 11) and including the Anti-HER2/neu light chain amino acid sequence (SEQ ID NO: 2).

[0050] Figure 14 shows the amino acid sequence of the fusion protein Anti-EGFR1 HC-TGFβRII-PD1 with the amino acid sequence of the heavy chain fusion protein of Anti-EGFR1-TGFβRII-PD1 where the amino acid sequence of the heavy chain of Anti-EGFR1 (SEQ ID NO: 5 with an additional C-terminal Lys) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for TGFβRII (immunomodulatory stem) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for PD-1 (immunomodulatory radical) (SEQ ID NO: 10) is in handwritten font with the linker enclosed (SEQ ID No: 11) and including the chain amino acid sequence anti-EGFR1 light (SEQ ID NO: 6).

[0051] Figure 15 shows the sequence of the Anti-CTLA4 HC-TGFβRII-PD1 fusion protein with the amino acid sequence of the Anti-CTLA4-TGFβRII-PD1 heavy chain fusion protein where the amino acid sequence of the Anti-CTLA4 heavy chain -CTLA4 (SEQ ID NO: 7 with an additional C-terminal Lys) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for TGFβRII (immunomodulatory radical) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for PD-1 (immunomodulatory radical) (SEQ ID NO: 8) is in handwritten font with the linker enclosed (SEQ ID NO: 11) and including the light chain amino acid sequence of Anti-CTLA4 (SEQ ID NO: 8).

[0052] Figure 16 shows the nucleotide sequence of the heavy chain constant region of Anti-HER2/neu with the linker (SEQ ID NO: 12) and TGFβRII ECD (SEQ ID NO: 13) which has been codon-optimized for expression in CHO cell.

[0053] Figure 17 shows the nucleotide sequence of the heavy chain variable region of Anti-HER2/neu (SEQ ID NO: 14), light chain variable region of Anti-HER2/neu (SEQ ID NO: 15) and Anti-EGFR1 heavy chain constant region with linker (SEQ ID NO: 12) that has been codon-optimized for expression in CHO cell.

[0054] Figure 18 shows the nucleotide sequence of Anti-EGFR1 heavy chain variable region (SEQ ID NO: 17), Anti-EGFR1 light chain variable region (SEQ ID NO: 18), Anti-EGFR1 light chain variable region Anti-CTLA4 heavy chain (SEQ ID NO: 19) and the Anti-CTLA4 light chain variable region (SEQ ID NO: 20) which has been codon-optimized for expression in CHO cell.

[0055] Figure 19 shows the nucleotide sequence of Anti CD20 IgG1 molecule (SEQ ID NO: 21), heavy chain variable region of Anti-CD20 (SEQ ID NO: 22) and light chain variable region of Anti-CD20 (SEQ ID NO: 23) which has been codon-optimized for expression in CHO cell.

[0056] Figure 20 shows the nucleotide sequence of the heavy chain of 4-1BB (SEQ ID NO: 24) and Anti-IL6R (SEQ ID NO: 25) that was codon-optimized for expression in CHO cell.

[0057] Figure 21 shows the nucleotide sequence of the light chain variable region of Anti-IL6R (SEQ ID NO: 26), and heavy chain variable region of Anti-4-1BB (SEQ ID NO: 27) and chain Anti-4-1BB light (SEQ ID NO: 28) that has been codon-optimized for expression in CHO cell.

[0058] Figure 22 shows the analysis of purified protein A Anit-HER2/neu-TGFβRII and Anti-EGFR1-TGFβRII in 12% PAGE.

[0059] Figure 23A shows Anti-HER2/neu-TGFβRII samples analyzed by chromatography on Protein A/SEC samples and Figure 23B Anti-EGFR1-TGFβRII samples analyzed by chromatography on Protein A/SEC.

[0060] Figure 24A shows that Anti-HER2/neu-TGFβRII and Anti-EGFR1-TGFβRII molecules bind to TGFB indicating that the fusion protein is functional and Figure 24B shows that Anti-HER2-TGFβRII inhibits lineage proliferation BT474 cell similar to Bmab200 (Herceptin).

[0061] Figure 25 shows that Anti-EGFR1-TGFβRII inhibits the proliferation of the A431 cell line similarly to Cetuximab.

[0062] Figure 26 shows that the ADCC activity of Anti-HER2-TGFβRII in BT474 cells is similar to that of Bmab200 (Herceptin).

[0063] Figure 27 shows the ADCC activity of Anti-EGFR1-TGFβRII in A431 cells where the ADCC activities are similar to that of Cetuximab.

[0064] Figure 28 shows the ADCC activity of Anti-EGFR1-4-1BB compared to Anti-EGFR1-TGFβRII and cetuximab.

[0065] Figure 29A shows that Anti-CTLA4-TGFβRII binding activity to TGFB1 is comparable to Anti-EGFR1-TGFβRII and B shows Anti-CTLA4-TGFβRII binding activity to CTLA4.

[0066] Figure 30A shows the binding activity of Anti-CTLA4-TGFβRII for determining the level of PD1-Fc binding and B shows the binding activity of Anti-EGRF1-4-1BB for determining the binding of 4 -1BBL.

[0067] Figure 31 shows the binding activity of Anti-EGFR1-4-1BB to EGFR and B shows the binding activity of PD1-Fc-4-1BB to find PDL1-Fc.

[0068] Figure 32 shows the binding activity of Anti-EGFR1-PD1 to EGFR and PD1.

[0069] Figure 33 shows photographs of expressed proteins and their alkylation reduction.

[0070] Figure 34A shows the mass spectrum of the light chain (LC) (Reduced) of the Anti-HER2/neu-TGFβRII ECD fusion and B shows the deconvoluted mass spectrum of LC (Reduced) of the Anti-HER2/neu fusion -TGFβRII ECD.

[0071] Figure 35 shows the mass spectrum of the heavy chain (HC) (Reduced) of the Anti-HER2/neu-TGFβRII ECD fusion.

[0072] Figure 36A shows the mass spectrum of LC (Reduced) of Anti-EGFR1-TGFβRII ECD and B shows the deconvoluted mass spectrum of LC (Reduced) of Anti-EGFR1-TGFβRII ECD.

[0073] Figure 37 shows the HC (Reduced) Mass Spectrum of Anti-EGFR1-TGFβRII ECD.

[0074] Figure 38A shows the UV chromatogram of tryptic peptides of the Anti-HER2/neu- TGFβRII ECD fusion protein and B shows the total ion chromatogram (TIC) of tryptic peptides of the Anti-HER2/neu-TGFβRII fusion protein TGFβRII ECD.

[0075] Figures 39, 40 and 41 provide lists of expected/observed tryptic peptides of the lead chain, heavy chain and linked motif of the Anti-HER2/neu-TGFβRII ECD fusion protein, respectively.

[0076] Figure 42A shows the UV chromatogram of tryptic peptides of Anti-EGFR1-TGFβRII ECD fusion protein and B shows the total ion chromatogram (TIC) of tryptic peptides of Anti-EGFR1-TGFβRII ECD fusion protein.

[0077] Figure 43 provides a list of expected/observed tryptic peptides from the light chain of the Anti-EGFR1-TGFβRII ECD fusion protein.

[0078] Figure 44 shows the list of expected/observed tryptic peptides from the heavy chain of the Anti-EGFR1-TGFβRII ECD fusion protein.

[0079] Figure 45 shows the list of expected/observed tryptic peptides from the heavy chain of the Anti-EGFR1-TGFβRII ECD fusion protein.

[0080] Figure 46 shows the amino acid sequence of the Cantuzumab-TGFβRII fusion protein in the LC constant region with the amino acid sequence of the heavy chain of Cantuzumab (SEQ ID NO: 29) and the amino acid sequence of the light chain of Cantuzumab ( SEQ ID NO: 30) attached to amino acid residues for TGF-βRII (immunomodulatory radical) (SEQ ID NO: 4) identified in bold letters and where a linker (SEQ ID NO: 3) is positioned between the Cantuzumab light chain and TGF-βRII and shown in italics.

[0081] Figure 47 shows the amino acid sequence of the Cixutumumab-TGFβRII fusion protein in the constant region of the LC with the amino acid sequence of the heavy chain of Cixutumumab (SEQ ID NO: 31) and the amino acid sequence of the light chain of Cixutumumab (SEQ ID NO: 32) attached to amino acid residues for TGFβRII (immunomodulatory radical) (SEQ ID NO: 4) identified in bold letters and where a linker (SEQ ID NO: 3) is positioned between the light chain of Cixutumumab and TGF-βRII and shown in italics.

[0082] Figure 48 shows the amino acid sequences of the Clivatuzumab-TGFβRII fusion protein in the constant region of the LC with the amino acid sequence of the heavy chain of Clivatuzumab (SEQ ID NO: 33) and the amino acid sequence of the light chain of Clivatuzumab (SEQ ID NO: 34) attached to amino acid residues for TGF-βRII (immunomodulatory radical) (SEQ ID NO: 4) identified in bold letters and where a linker (SEQ ID NO: 3) is positioned between the light chain of Clivatuzumab and TGF-βRII and shown in italics.

[0083] Figure 49 shows the amino acid sequences of the Pritumumab-TGFβRII fusion protein in the constant region of the LC with the amino acid sequence of the heavy chain of Pritumumab (SEQ ID NO: 35) and the amino acid sequence of the light chain of Pritumumab (SEQ ID NO: 36) attached to amino acid residues for TGFβRII (immunomodulatory radical) (SEQ ID NO: 4) identified in bold letters and where a linker (SEQ ID NO: 3) is positioned between the Pritumumab light chain and TGF-βRII and shown in italics.

[0084] Figure 50 shows the amino acid sequence of the fusion protein Cantuzumab HC-4-1BB and LC-TGFβRII where the amino acid sequence for the heavy chain of Cantuzumab (SEQ ID NO: 29) is attached to a linker (SEQ ID NO: 3) which is shown in italics and the sequence for 4-1BB (immunomodulatory radical) (SEQ ID NO: 9) is in handwritten font and the amino acid sequence of Cantuzumab light chain (SEQ ID NO: 30 ) is attached to amino residues for TGF-βRII (immunomodulatory radical) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3) between them.

[0085] Figure 51 shows the amino acid sequence of the fusion protein Cixutumumab HC-4-1BB and LC-TGFβRII where the amino acid sequence for the heavy chain of Cixutumumab (SEQ ID NO: 31) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for 4-1BB (immunomodulatory radical) (SEQ ID NO: 9) is in handwritten font and the Cixutumumab light chain amino acid sequence (SEQ ID NO: 32) is attached to amino residues for TGF-βRII (immunomodulatory radical) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3) between them.

[0086] Figure 52 shows the amino acid sequence of the fusion protein Clivatuzumab HC-4-1BB and LC-TGFβRII where the amino acid sequence for the heavy chain of Clivatuzumab (SEQ ID NO: 33) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for 4-1BB (immunomodulatory radical) (SEQ ID NO: 9) is in handwritten font and the amino acid sequence of the light chain of Clivatuzumab (SEQ ID NO: 34) is attached to amino residues for TGF-βRII (immunomodulatory radical) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3) between them.

[0087] Figure 53 shows the amino acid sequence of the fusion protein Pritumumab HC-4-1BB and LC-TGFβRII where the amino acid sequence for the heavy chain of Pritumumab (SEQ ID NO: 35) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for 4-1BB (immunomodulatory radical) (SEQ ID NO: 9) is in handwritten font and the Pritumumab light chain amino acid sequence (SEQ ID NO: 36) is attached to amino residues for TGF-βRII (immunomodulatory radical) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3) between them.

[0088] Figure 54 shows the amino acid sequence of the fusion protein Cantuzumab HC-PD1 and LC-TGFβRII where the amino acid sequence for the heavy chain of Cantuzumab (SEQ ID NO: 29) is attached to a linker (SEQ ID NO : 3) shown in italics and the sequence for PD1 (radical immunomodulator) (SEQ ID NO: 10) is in handwritten font and the amino acid sequence of Cantuzumab light chain (SEQ ID NO: 30) is fixed to amino residues for TGF-βRII (immunomodulatory radical) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3) between them.

[0089] Figure 55 shows the amino acid sequence of the fusion protein Cixutumumab - HC-PD1 and LC-TGFβRII where the amino acid sequence for the heavy chain of Cixutumumab (SEQ ID NO: 31) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for PD1 (radical immunomodulatory) (SEQ ID NO: 10) is in handwritten font and the Cixutumumab light chain amino acid sequence (SEQ ID NO: 32) is attached to residues amino to TGF-βRII (immunomodulatory radical) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3) between them.

[0090] Figure 56 shows the amino acid sequence of the fusion protein Clivatuzumab - HC-PD1 and LC-TGFβRII where the amino acid sequence for the heavy chain of Clivatuzumab (SEQ ID NO: 33) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for PD1 (radical immunomodulatory) (SEQ ID NO: 10) is in handwritten font and the amino acid sequence of Clivatuzumab light chain (SEQ ID NO: 34) is attached to residues amino to TGF-βRII (immunomodulatory radical) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3) between them.

[0091] Figure 57 shows the amino acid sequence of the fusion protein Pritumumab - HC-PD1 and LC-TGFβRII where the amino acid sequence for the heavy chain of Pritumumab (SEQ ID NO: 35) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for PD1 (immunomodulatory radical) (SEQ ID NO: 10) is in handwritten font and the Pritumumab light chain amino acid sequence (SEQ ID NO: 36) is attached to residues amino to TGF-βRII (immunomodulatory radical) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3) between them.

[0092] Figure 58 shows the amino acid sequence of the fusion protein Cantuzumab HC-TGFβRII-4-1BB where the amino acid sequence for the Cantuzumab heavy chain (SEQ ID NO: 29) is attached to a linker (SEQ ID NO : 3) shown in italics and the sequence for TGFβRII (immunomodulatory stem) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for 4-1BB (immunomodulatory stem) (SEQ ID NO: 9) is in handwritten text source with a linker between (SEQ ID NO: 11) and including the Cantuzumab light chain amino acid sequence (SEQ ID NO: 30).

[0093] Figure 59 shows the amino acid sequence of the fusion protein Cixutumumab HC-TGFβRII-4-1BB where the amino acid sequence for the heavy chain of Cixutumumab (SEQ ID NO: 31) is attached to a linker (SEQ ID NO : 3) shown in italics and the sequence for TGFβRII (immunomodulatory stem) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for 4-1BB (immunomodulatory stem) (SEQ ID NO: 9) is in handwritten text source with a linker between (SEQ ID NO: 11) and including the Cixutumumab light chain amino acid sequence (SEQ ID NO: 32).

[0094] Figure 60 shows the amino acid sequence of the fusion protein Clivatuzumab HC-TGFβRII-4-1BB where the amino acid sequence for the heavy chain of Clivatuzumab (SEQ ID NO: 33) is attached to a linker (SEQ ID NO : 3) shown in italics and the sequence for TGFβRII (immunomodulatory stem) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for 4-1BB (immunomodulatory stem) (SEQ ID NO: 9) is in handwritten text source with a linker between (SEQ ID NO: 11) and including the light chain amino acid sequence of Clivatuzumab (SEQ ID NO: 34).

[0095] Figure 61 shows the amino acid sequence of the Pritumumab HC-TGFβRII-4-1BB fusion protein where the amino acid sequence for the Pritumumab heavy chain (SEQ ID NO: 35) is attached to a linker (SEQ ID NO: 35) : 3) shown in italics and the sequence for TGFβRII (immunomodulatory stem) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for 4-1BB (immunomodulatory stem) (SEQ ID NO: 9) is in handwritten text source with a linker between (SEQ ID NO: 11) and including the Pritumumab light chain amino acid sequence (SEQ ID NO: 36).

[0096] Figure 62 shows the amino acid sequence of Cantuzumab HC-TGFβRII-PD1 fusion protein where the amino acid sequence for Cantuzumab heavy chain (SEQ ID NO: 29) is attached to a linker (SEQ ID NO: 3 ) shown in italics and the sequence for TGFβRII (immunomodulatory stem) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for PD1 (immunomodulatory stem) (SEQ ID NO: 10) is in handwritten font with a linker between (SEQ ID NO: 11) and including the Cantuzumab light chain amino acid sequence (SEQ ID NO: 30).

[0097] Figure 63 shows the amino acid sequence of the fusion protein Cixutumumab HC-TGFβRII-PD1 where the amino acid sequence for the heavy chain of Cixutumumab (SEQ ID NO: 31) is attached to a linker (SEQ ID NO: 3 ) shown in italics and the sequence for TGFβRII (immunomodulatory stem) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for PD1 (immunomodulatory stem) (SEQ ID NO: 10) is in handwritten font with a linker between (SEQ ID NO: 11) and including the Cixutumumab light chain amino acid sequence (SEQ ID NO: 32).

[0098] Figure 64 shows the amino acid sequence of the fusion protein Clivatuzumab HC-TGFβRII-PD1 where the amino acid sequence for the heavy chain of Clivatuzumab (SEQ ID NO: 33) is attached to a linker (SEQ ID NO: 3 ) shown in italics and the sequence for TGFβRII (immunomodulatory stem) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for PD1 (immunomodulatory stem) (SEQ ID NO: 10) is in handwritten font with a linker between (SEQ ID NO: 11) and including the light chain amino acid sequence of Clivatuzumab (SEQ ID NO: 34).

[0099] Figure 65 shows the amino acid sequence of the Pritumumab HC-TGFβRII-PD1 fusion protein where the amino acid sequence for the heavy chain of Pritumumab (SEQ ID NO: 35) is attached to a linker (SEQ ID NO: 3 ) shown in italics and the sequence for TGFβRII (immunomodulatory stem) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for PD1 (immunomodulatory stem) (SEQ ID NO: 10) is in handwritten font with a linker between (SEQ ID NO: 11) and including the Pritumumab light chain amino acid sequence (SEQ ID NO: 36).

DETAILED DESCRIPTION OF THE INVENTION

[0100] The practice of the present invention will be employed, unless otherwise indicated, in conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are in the state of the art. See, for example, Sambrook et al. “MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); "CURRENT PROTOCOLS IN MOLECULAR BIOLOGY" (F.M. Ausubel, et al. eds., (1987)); the METHODS IN ENZYMOLOGY series (Academic Press, Inc.): “PCR 2: A PRACTICAL APPROACH (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) “ANTIBODIES, A LABORATORY MANUAL”, and “ANIMAL CELL CULTURE” (R.I. Freshney, ed. (1987)).

[0101] Definitions

[0102] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one skilled in the art to which this invention belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and in the appended claims, the singular forms "a", "a" and "the" also include the plural forms, unless the context clearly indicates otherwise. The following terms present the data meanings:

[0103] The term "polynucleotide" as used herein means a sequence of nucleotides connected by phosphodiester bonds. Polynucleotides are shown here in the 5' to 3' direction. A polynucleotide of the present invention can be a deoxyribonucleic acid (DNA) molecule or a ribonucleic acid (RNA) molecule. Where a polynucleotide is a DNA molecule, this molecule can be a gene or a cDNA molecule. Nucleotide bases are indicated here by a single letter code: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I), and uracil (U). A polynucleotide of the present invention can be prepared using standard techniques well known in the art.

[0104] The term "optimized" as used herein means that a nucleotide sequence has been altered to encode an amino acid sequence using codons that are preferred in the productive cell or organism, usually a eukaryotic cell, for example, a Pichia cell , a Trichodemia cell, a Chinese Hamster Ovary (CHO) cell or a human cell. The optimized nucleotide sequence is engineered to completely or as much as possible retain the amino acid sequence originally encoded by the initial nucleotide sequence, which is also known as the "parent" sequence. The optimized sequences here. They have been engineered to display codons that are preferred in mammalian CHO cells; however, the optimized expression of these sequences in other eukaryotic cells is also envisaged here. The amino acid sequences encoded by the optimized nucleotide sequences are also called considered optimized. The term "expression", as used herein, is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

[0105] The term "transfection" of a cell, as used herein, means that genetic material is introduced into a cell for the purpose of genetically modifying the cell. Transfection can be achieved by a variety of means known in the art, such as transduction or electroporation.

[0106] The term "cancer", as used herein, is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, eye cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and others.

[0107] The term "transgenic" is used herein in a broad sense to mean any heterologous nucleotide sequence incorporated into a vector for expression in a target cell and associated expression control sequences, such as promoters. It should be noted by those skilled in the art that expression control sequences will be selected on the basis of ability to promote transgene expression in the target cell. An example of a transgene is a nucleic acid encoding a chimeric fusion protein of the present invention.

[0108] The term "expression vector", as used herein, means a vector containing a nucleic acid sequence that encodes at least part of a gene product capable of being transcribed. The expression vector can contain a variety of control sequences, which refer to the nucleic acid sequences necessary for the transcription and possible translation of operably linked coding sequence in a particular host organism. In addition to the control sequences that govern transcription and translation, vectors and expression vectors can contain nucleic acid sequences that also serve other functions. The term also includes a recombinant plasmid or virus comprising a polynucleotide to be placed into a host cell, either in vitro or in-vivo. Preferably the host cell is a transient cell line or a stable cell line and more preferably it is a mammalian host cell selected from the group consisting of HEK293, CHO and NSO.

[0109] The term "subject" as used herein means a human or vertebrate animal including a dog, cat, horse, cow, pig, sheep, goat, chicken, monkey, rat and mouse.

[0110] The term "therapeutically effective amount", as used herein, means the amount of the compound in question that will produce the biological or medical response of a tissue, system, animal or human being treated by the researcher, veterinarian, physician or another clinician.

[0111] The term "pharmaceutically acceptable" as used herein means that the vehicle, diluent or excipient must be compatible with the other ingredients of the formulation and not harmful to the recipient.

[0112] The term "recombinant", as used herein, means a genetic entity distinct from that generally found in nature. As applied to a polynucleotide or gene, this means that the polynucleotide is the product of various combinations of cloning, restriction and/or ligation steps, and other procedures that result in the production of a construct that is distinct from that of a polynucleotide found in nature.

[0113] The term "substantial identity" or "substantial similarity", as used herein, when referring to a nucleic acid or fragment thereof, indicates that when optimally aligned with appropriate nucleotide insertions or deletions of another nucleic acid ( or its complementary strand), there is a nucleotide sequence identity of at least about 95 to 99% of the sequence.

[0114] The terms "peptide", "polypeptide" and "protein" are used interchangeably to indicate a polymeric sequence of at least two amino acids covalently linked by an amide bond.

[0115] The term "homologous", as used herein in relation to peptides, refers to the similarity of an amino acid sequence between two peptides. When an amino acid position in both peptides is occupied by identical amino acids, they are homologous at that position. Accordingly, "substantially homologous" means an amino acid sequence that is largely, but not entirely, homologous, and which retains most or all of the activity of the sequence to which it is homologous. As used herein, "substantially homologous" means that a sequence is at least 50% identical, and preferably exhibits at least 75% and most preferably 95% homology to the reference peptide. Additional peptide sequence modifications, such as minor amino acid sequence variations, deletions, substitutions or derivatizations of the sequences described herein, are included, provided that the peptide exhibits substantially the same activity or function as the unmodified peptides. Notably, a modified peptide will retain the activity or function associated with the unmodified peptide, the modified peptide will generally have an amino acid sequence "substantially homologous" to the amino acid sequence of the unmodified sequence.

[0116] The term "administration" as used herein is defined as the actual physical introduction of the composition into the host. Any and all methods for introducing the composition to the subject are contemplated in accordance with the present invention; the method is not dependent on any particular means of introduction and should not be considered as such. Means of introduction are well known to those skilled in the art, and preferably, the composition is administered subcutaneously or intratumorally. One skilled in the art will recognize that, although more than one route may be used for administration, a particular route may provide a more immediate and effective response than another route. Local or systemic administration can be carried out by an administration comprising the application or instillation of immunovaccines into body cavities, inhalation or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, intraportal, intrahepatic, peritoneal, subcutaneously or intradermally. In case the tumor is in the central nervous system, the composition must be administered intratumorally, in view of the fact that there is no entry of the immune system into the central nervous system.

[0117] Although chemotherapeutic agents can induce "immunogenic" tumor cell death and facilitate cross-presentation of antigens by dendritic cells, tumors create a tolerogenic environment that allows them to suppress the activation of innate and adaptive immune responses and prevent the immune attack by immune effector cells. The present invention provides strategies to counteract tumor-induced immune tolerance in the tumor microenvironment and can enhance the antitumor efficacy of chemotherapy by activating and leveling adaptive cell-mediated antitumor immunity against disseminated cancer cells.

[0118] The present invention is based on the discovery that targeted immunomodulatory antibodies or fusion proteins of the present invention can neutralize or reverse immunotolerance of cancer cells. Cancer cells are able to evade elimination by chemotherapeutic agents or tumor-directed antibodies through specific immunosuppressive mechanisms in the tumor microenvironment, and this ability of cancer cells is known as immunotolerance. By counteracting tumor-induced immune tolerance, the present invention provides effective compositions and methods for treating cancer, optionally in combination with another existing cancer treatment.

[0119] The present invention provides compositions and methods for producing fusion proteins that counteract immunotolerance in the tumor microenvironment and promote T cell-mediated adaptive antitumor immunity for the maintenance of long-term durable protection against recurrent or disseminated cancers. These fusion proteins are designed to facilitate effective long-term T-cell mediated immune responses against tumor cells by at least one of the following: a. promoting tumor cell death by enhancing antibody-dependent cellular cytotoxicity (ADCC); and b. increased activation and proliferation of antitumor CD8+ T cells by negation of T cell-mediated immunosuppression and regulatory myeloid suppressor cells. These antitumor immune responses can be activated in tandem with the sensitization of tumor cells to effector immune-mediated cytotoxicity, thereby establishing a positive feedback loop that enhances cytoreduction and enhances adaptive antitumor immunity.

[0120] In addition, the fusion proteins of the present invention are distinct and superior to existing therapeutic molecules in at least one of the following aspects: (i) To counteract immunotolerance in the tumor microenvironment and promote T cell-mediated adaptive antitumor immunity to the maintenance of long-term protection against recurrent or disseminated cancers (for the prevention or treatment of various cancers); (ii) To produce immune cell compositions for adaptive cell therapy of various cancers; and (iii) To function as immune adjuvants or vaccines for the prophylaxis of various cancers or infectious diseases.

[0121] The targeted immunostimulatory antibodies and/or fusion proteins of the invention provide the ability to disrupt immunosuppressive networks in the tumor microenvironment. Tumors employ a wide array of regulatory mechanisms to prevent or suppress the immune response. Cancer cells actively promote immunotolerance in the tumor microenvironment through the expression of cytokines and molecules that inhibit the differentiation and maturation of antigen-presenting dendritic cells (DC). Immunosuppressive cytokines and ligands produced by tumor cells include the following: (i) Transforming growth factor-beta (TGF-β); (ii) Programmed Death Ligand 1 (PD-L1; B7-H1); (iii) Vascular endothelial growth factor (VEGF); and (iv) Interleukin-10 (IL-10).

[0122] In addition to blocking dendritic cell (DC) maturation, these molecules promote the development of specialized subsets of CD4+ immunosuppressive T cells (regulatory T cells; Treg cells) and myeloid-derived suppressor cells (MDSC). Tregs are a minority subpopulation of CD4 + T cells that constitutively express CD25 [the cc chain of the interleukin-2 (IL-2) receptor] and the transcription factor Forkhead box P3 (FOXP3). Tregs (CD4+CD25+FoxP3+cells) maintain immune tolerance by restricting the activation, proliferation, and effector functions of a wide range of immune cells, including CD4 and CD5 T cells, natural killer (NK) and NK T cells, B cells and antigen presenting cells (APCs) in vitro and in vivo.

[0123] Accumulation of Treg cells in the tumor microenvironment enhances tumor immunotolerance and facilitates tumor progression and metastasis. Increased expression of immunosuppressive cytokines (TGF-β; PD-L1) and Tregs that infiltrate tumors is correlated with reduced survival in patients with several types of cancers. The fusion proteins of the present invention inhibit key immunosuppressive molecules expressed by targeted tumor cell or Treg cells that infiltrate tumor and myeloid suppressor cells (DCs or MDSC). As such, they provide the desired ability to inhibit the development or function of Tregs in the tumor microenvironment.

[0124] The present invention provides a method for preventing or treating a neoplastic disease. The method includes administering to a subject in need thereof one or more fusion proteins of the present invention in combination with another anti-cancer therapy, where the anti-cancer therapy is a chemotherapeutic molecule, antibody, small molecule kinase inhibitor, hormonal agent , cytotoxic agent, targeted therapeutic agent, anti-angiogenic agent, ionizing radiation, ultraviolet radiation, cryoablation, thermal ablation, or radiofrequency ablation.

[0125] As used herein, the term "antibody" includes mono or polyvalent natural or artificial antibodies, including, but not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, fragments of Fab, F(abi) fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The antibody can be from any animal including birds and mammals. In one aspect, the antibody is, or is derived from, a human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken. Furthermore, such an antibody may be a humanized version of an antibody. The antibody may be monospecific, bispecific, trispecific, or of greater multispecificity. Antibody specifically herein includes a "chimeric" antibody in which a part of the heavy chain and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibody, while that the remainder of the chain(s) is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another class or subclass, as well as fragments of such antibodies, provided they exhibit the desired biological activity .

[0126] Examples of antibodies that can be incorporated into the compositions and methods described herein include, but are not limited to, antibodies such as trastuzumab (anti-HER2/neu antibody); Pertuzumab (anti-HER2 mAb); cetuximab (monoclonal chimeric antibody to epidermal growth factor receptor EGFR): panitumumab (anti-EGFR antibody); nimotuzumab (anti-EGFR antibody); Zalutumumab (anti-EGFR mAb); Necitumumab (anti-EGFR mAb); MDX-210 (bispecific humanized anti-HER-2 antibody); MDX-210 (bispecific humanized anti-HER-2 antibody); MDX-447 (humanized bispecific anti-EGF antibody receptor); Rituximab (chimeric murine/human anti-CD20 mAb); Obinutuzumab (anti-CD20 mAb); Ofatumumab (anti-CD20 mAb); Tositumumab-1131 (anti-CD20 mAb); ibritumomab tiuxetan (anti-CD20 mAb); Bevacizumab (anti-VEGF mAb); Ramucirumab (anti-VEGFR2 mAb); Ranibizumab (anti-VEGF mAb); Aflibercept (extracellular domains of YEGFR1 and VEGFR2 fused to IgGl Fc): AMG386 (peptide fused to IgG1 Fc binding to angiopoietin-1 and -2); Dalotuzumab (anti-IGF-IR mAb): Gemtuzumab ozogamicin (anti-CD33 mAb); Alemtuzumab (anti-Campath-1/CD52 mAb); Brentuximab vedotin (anti-CD30 mAb); Catumaxomab (bispecific mAb targeting epithelial cell adhesion molecule and CD3); Naptumomab (anti-5T4 mAb); Girentuximab (anti-carbonic anhydrase ix): or Farletuzumab (anti-folate receptor). Other examples include antibodies such as PanorexTM (17-1A) (murine monoclonal antibody); Panorex (@(17-1A) (murine chimeric monoclonal antibody); BEC2 (anti-idiotypic mAb, mimics GD epitope) (with BCG): Oncolym (Lym-1 monoclonal antibody); SMART M 1 95 Ab, 13 ' 1 humanized LYM-1 (Oncolym), Ovarex (B43.13, mouse anti-idiotypic mAb); 3622W94 mAb that binds to EGP40 ( 17-1 A) pancarcinoma antigen on adenocarcinomas; Zenapax (SMART Anti-Tac - IL-2 receptor);SMART M1 95 Ab, humanized Ab, humanized); NovoMAb-G2 (pancarcinoma specific Ab): TNT (chimeric mAb to histone antigens); TNT (chimeric mAb to histone antigens); GJiomab-H (Monoclonal Abs - humanized); GN1-250 Mab; EMD-72000 (chimeric EGF antagonist); LymphoCide (humanized IL.L.2 antibody); and bispecific MDX-260, targeted GD-2, Ab ANA, Ab SMART IDiO, Ab SMART ABL 364 or ImmuRAIT-CEA.

[0127] Various methods have been employed to produce the antibodies. Hybridoma technology, which refers to a cloned cell line that produces a unique type of antibody, uses cells from a variety of species, including mice (murine), hamsters, rats, and humans. Another method for making an antibody utilizes genetic engineering including recombinant DNA techniques. For example, antibodies made using these techniques include, but are not limited to, chimeric antibodies and humanized antibodies. A chimeric antibody combines DNA coding regions from more than one type of species. For example, a chimeric antibody can contain the variable region from a mouse and the constant region from a human. A humanized antibody is predominantly from a human, even though it contains non-human parts. Like a chimeric antibody, a humanized antibody can contain a fully human constant region. However, unlike a chimeric antibody, the variable region can be partially derived from a human. The synthetic non-human parts of a humanized antibody often come from CDRs in murine antibodies. In all cases, these regions are crucial for allowing the antibody to recognize and bind to a specific antigen.

[0128] In one embodiment, a hybridoma can produce a targeted fusion protein comprising a targeting moiety and an immunomodulatory moiety. In one embodiment, a targeting moiety comprising an antibody, antibody fragment, or polypeptide is linked or fused to an immunomodulatory moiety consisting of a polypeptide, with a linker or without a linker. The linker can be an amino acid linker. In one embodiment, a linker is (GGGGS)n, where n is 1, 2, 3, 4, 5, 6, 7, or 8. For example, GGGGSGGGGSSGGGGS (SEQ ID NO: 3). In another embodiment, a linker is EPKSCDK (SEQ ID NO: 11). In various aspects, the length of the linker can be modified in order to optimize binding of the target moiety or the function of the immunomodulatory moiety. In various aspects, the immunomodulatory moiety is a polypeptide that is fused to the C-terminus of the Fc region of the heavy chain of a targeted antibody or Fc-containing fusion protein. In another aspect, the immunomodulatory moiety is a polypeptide that is fused to the C-terminus of the light chain of a targeting antibody.

[0129] An antibody fragment may include a part of an intact antibody, for example including antigen binding or its variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; Fc fragments or Fc fusion products; diabodies (bispecific bivalent antibodies); linear antibodies; single chain antibody molecules; and multispecific antibodies formed from antibody fragment(s). An intact antibody is one that includes an antigen-binding variable region, as well as a light chain constant domain (CL) and a heavy chain constant domain, CH1, CH2, and CH3. The constant domains can be native sequence constant domains (e.g., the human native sequence constant domains) or amino acid sequence variants thereof for any other modified Fc (e.g., glycolysis Fc or other engineered Fc).

[0130] The fusion proteins of the present invention can be synthesized by conventional techniques known in the field, for example, by chemical synthesis such as solid phase peptide synthesis. Such methods are known to those skilled in the art. In general, these methods employ solid phase or solution synthesis, well known in the art. Specifically, the methods comprise sequentially adding one or more amino acids or suitably protected amino acids to a growing peptide chain. Usually, either the amino group or the carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then be either attached to an inert support or used in solution by adding the next amino acid in the sequence having the complementary group (amino or carboxyl) suitably protected, under conditions suitable for amide bond formation. The protecting group is then removed from this newly added amino acid residue and the next (suitably protected) amino acid is then added, and so on. After all the desired amino acids have been linked in the proper sequence any remaining protecting groups and any solid support are removed either sequentially or concurrently to produce the final polypeptide. By simply modifying this general procedure, it is possible to add more than one amino acid at the time of chain growth, for example, by coupling (under conditions that do not cause racemization of the chiral centers) a protected tripeptide with an appropriately protected dipeptide to form, after deprotection, a pentapeptide.

[0131] Typical protecting groups include t-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), p-toluenesulfonyl (Tos); 2,4-dinitrophenyl, benzyl (Bzl), biphenylisopropyloxycarboxycarbonyl, cyclohexyl, isopropyl, acetyl, o-nitrophenylsulfonyl, and the like. Of these, Boc and Fmoc are preferred.

[0132] Typical solid supports are generally cross-linked polymeric materials. These include styrene-based polymers crosslinked with divinylbenzene, for example, divinylbenzene-hydroxymethylstyrene copolymers, divinylbenzene-chloromethylstyrene copolymers, and divinylbenzene-benzhydrylaminopolystyrene copolymers. Divinylbenzene-benzhydrylaminopolystyrene copolymers, as illustrated here using p-methyl-benzhydrylamine resin, have the advantage of directly introducing a terminal amido functional group into the polypeptide chain, which function is maintained by the chain when it is cleaved from the support.

[0133] In one method, polypeptides are prepared by conventional solid-phase chemical synthesis in, for example, an Applied Biosystems, Inc. peptide synthesizer. (ABI) 430A using a resin that allows the synthesis of the amido peptide form and using t-Boc derivatives of the amino acid (Peninsula Laboratories, Inc.) with standard solvents and reagents. Polypeptides containing L- or D-amino acids can be synthesized in this way. Polypeptide composition is confirmed by quantitative amino acid analysis and the specific sequence of each peptide can be determined by sequence analysis.

[0134] Preferably, the polypeptides can be produced by recombinant DNA techniques by synthesizing the DNA encoding the desired polypeptide. Once sequences for the desired polypeptides have been synthesized or isolated, they can be cloned into any suitable expression vector. Numerous cloning vectors are known to those skilled in the art, and selection of the appropriate cloning vector is a matter of choice. The gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively called "control" elements herein), such that the DNA sequence it encodes the desired polypeptide is transcribed into RNA in the host cell transformed by the vector containing this expression construct. The coding sequence may or may not contain a signal peptide or leader sequence. Heterologous leader sequences can be added to the coding sequence that causes secretion of the expressed polypeptide from the host organism. Other regulatory sequences may also be desirable which allow regulation of the expression of the protein sequences related to the growth of the host cell. Such regulatory sequences are known to the skilled artisan, and examples include those that cause expression of a gene to be turned on and off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example amplifier sequences.

[0135] Control sequences and other regulatory sequences may be linked to the coding sequence prior to insertion into a vector, such as the cloning vectors described above. Alternatively, the cloning sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.

[0136] The expression vector can then be used to transform an appropriate host cell. A number of mammalian cell lines are known in the art and include immortalized cell lines made available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells , baby hamster kidney cells (BHK), monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney cells ("MDBK"), NOS cells derived from carcinoma, such as sarcoma, as well as others. Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp. find use with the present expression constructs. Yeast hosts useful in the present invention include, inter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for use as a baculovirus expression vector include, inter alia, Aedes aegypti, Autographa califomica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni. Proteins can also be expressed in Trypanoses.

[0137] Depending on the expression system and host selected, proteins of the present invention are produced by growing host cells transformed by an expression vector described above under conditions by which the protein of interest is expressed. The protein is then isolated from the host cell and purified. If the expression system secretes the protein into the growth medium, the protein can be purified directly from the medium. If the protein is not secreted, it is isolated from cell lysates. Selection of appropriate growing conditions and recovery methods are within the skill of the art. Once purified, the amino acid sequences of the proteins can be determined, i.e., by repeated cycles of Edman degradation, followed by amino acid analysis by HPLC. Other amino acid sequencing methods are also known in the art.

[0138] Once synthesized or otherwise produced, a candidate polypeptide can be tested for inhibitory activity by assessing the candidate's ability to inhibit lipopolysaccharide-induced nuclear translocation of NF-.kappa.B, for example, by using of murine endothelial cells.

[0139] The fusion proteins of the present invention can be formulated into therapeutic compositions in a variety of dosage forms, such as, but not limited to, liquid solutions or suspensions, tablets, pills, powders, suppositories, microcapsules or polymeric microvesicles , liposomes, and injectable or infusion solutions. The preferred form depends on the mode of administration and the particular type of target cancer. The compositions also preferably include pharmaceutically acceptable carriers or adjuvants, well known in the art, such as human serum albumin, ion exchangers, alumina, lecithin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, and salts or electrolyte such as protamine sulfate. Suitable carriers are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. Methods of preparing such compositions are known, or will be obvious to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 18th edition, 1990.

[0140] The above compositions can be administered using conventional modes of administration including, but not limited to, intravenous, intraperitoneal, oral, intralymphatic, or subcutaneous administration. Local administration to a tumor in question, or to the site of inflammation, for example direct injection into an arthritic joint, will also find use with the present invention.

[0141] Therapeutically effective doses will be easily determined by one skilled in the art and will depend on the severity and course of the disease, the patient's health and response to treatment, and the judgment of the physician.

[0142] Experimental Part

[0143] Below are examples of specific embodiments leading to the present invention. The examples are provided for illustrative purposes only, and are not intended to limit the scope of the present invention. Efforts have been made to ensure accuracy with regard to the numbers used (eg quantities, temperatures, etc.), however some errors and experimental deviations must indeed be allowed for.

[0144] Example 1

[0145] Fusion proteins consisting of the heavy chain of IgG linked to immunomodulatory ligands (suppressors or activators) were expressed by genes optimized for the expression of CHO cells. The codon-optimized nucleotide sequences defined by SEQ ID NOs: 12 to 28 were expressed in cells (CHO) and the chimeric/fusion proteins expressed are shown in Table 1

[0146] The expressed proteins were characterized using SDS PAGE and the expressed Anit-HER2/neu-TGFβRII and Anti-EGFR1-TGFβRII fusion proteins were purified from culture supernatants using the Protein A column and the results are shown in Figure 22 . Notably the mass of the light chain of Anti-EGFR1-TGFβRII is greater and this may be due to the presence of two glycosylation sites in the variable regions of the light and heavy chain. Both the heavy chain mass of Anti-HER2/neu-TGFβRII and Anti-EGFR1-TGFβRII are larger because of TGFβRII. The heavy chain of Anti-HER2/neu-TGFβRII also has four glycosylation sites, while that of Anti-EGFR1-TGFβRII has five N-glycosylation sites.

[0147] Example 2

[0148] Protein A/SEC chromatography. Anti-HER2/neu-TGFβRII and Anti-EGFR1-TGFβRII samples were analyzed by Protein A/SEC chromatography and the results are shown in Figure 23. Figure 23A shows a sharp elution peak of Bmab200 (Herceptin) vs. broader elution peak, which is believed to be a measure of the heterogeneity due to the presence of glycosylation, as there are three additional N-glycosylation sites that are present in the region. Notably, storage at -80°C did not cause aggregation. The change in position or appearance of the peak right at the beginning of the SEC column indicates that the increase in molecular weight is due to the fusion pair. This again confirms that the full length of the molecule is being expressed. Figure 23B shows a sharp Bmab200 (Herceptin) elution peak vs a broader elution peak which is believed to be a measure of heterogeneity due to the presence of glycosylation sites, as there are three additional N-glycosylation sites present in the TGFβRII region. Again, storage at -80°C did not cause aggregation. The change in position or appearance of the peak right at the beginning of the SEC column indicates that the increase in molecular weight is due to the fusion pair. This again confirms that the full length of the molecule is being expressed.

[0149] Example 3

[0150] Functional assays for fusion proteins. An ELISA experiment was conducted to verify the binding capacity of Anti-HER2/neu-TGFβRII and Anti-EGFR1-TGFβRII to TGFβ. Figure 24A shows that Anti-HER2/neu-TGFβRII and Anti-EGFR1-TGFβRII molecules bind to TGFβ indicating that the fusion protein is functional. Figure 24B shows that Anti-HER2-TGFβRII inhibits proliferation of the BT474 cell line to Bmab200 (Herceptin). Figure 25 shows that Anti-EGFR1-TGFβRII inhibits A431 cell line proliferation similarly to Cetuximab.

[0151] Example 4

[0152] Anti-HER2/neu-TGFβRII fusion protein antibody-dependent cellular cytotoxicity ADCC activity was conducted to determine whether the protein binds to target receptors on cells. The results are shown in Figure 26, where the activity is determined in BT474 cells and it is evident that the ADCC activity (% cell lysis) of Anti-HER2-TGFβRII on BT474 cells is similar to that of Bmab200 (Herceptin). Figure 27 shows the ADCC activity of Anti-EGFR1-TGFβRII on A431 cells, where the ADCC activities are similar to that of Cetuximab. Figure 28 shows the ADCC activity of Anti-EGFR1-4-1BB compared to Anti-EGFR1-TGFβRII and Cetuximab.

[0153] Example 5

[0154] Binding activity of expressed proteins. The purpose of this assay is to test the functionality of fusion proteins to bind to target receptors on cells in a dose-dependent manner. Figure 29A shows that Anti-CTLA4-TGFβRII binding activity to TGFβ1 is comparable to Anti-EGFR1-TGFβRII and Figure 29B shows Anti-CTLA4-TGFβRII binding activity to CTLA4. Figure 30A shows Anti-CTLA4-TGFβRII binding activity to determine the level of PD1-Fc binding and Figure 30B shows Anti-EGRF1-4-1BB binding activity to determine 4-1BBL binding. Figure 31A shows the binding activity of Anti-EGFR1-4-1BB to EGFR and Figure 31B shows the binding activity of PD1-Fc-4-1BB to find PDL1-Fc. Figure 32 shows the binding activity of Anti-EGFR1-PD1 to EGFR and PD1.

[0155] Example 6

[0156] Confirmation of the primary structure of the molecule. As shown in Figure 33, the expressed proteins are evaluated for molecular weight and the presence of glycosylation. Samples were analyzed by reducing and non-reducing SDS PAGE. The heavy chain and light chain of the antibody are separated by reductive alkylation such that the reduced structures can be assessed. Tryptic digestion of fusion proteins provides for primary sequence identification. MS/MS analysis of proteins is performed.

[0157] Mass Spectrometry Analysis of Anti-HER2/neu-TGFβRII and Anti-EGFR1-TGFβRII. The fusion protein shown in Figure 1 was expressed and tested. Figure 34A shows the light chain (LC) mass spectrum (Reduced) of the Anti-HER2/neu-TGFβRII ECD fusion protein and Figure 34B shows the deconvolution mass spectrum of the LC (Reduced) of the Anti-HER2 fusion protein. -HER2/neu-TGFβRII ECD. Figure 35 shows the heavy chain (HC) mass spectrum (Reduced) of the Anti-HER2/neu-TGFβRII ECD fusion protein.

[0158] The fusion protein shown in Figure 2 expressed and tested. Figure 36A shows the LC (Reduced) mass spectrum of Anti-EGFR1-TGFβRII ECD and Figure 36B shows the deconvolution mass spectrum of the LC (Reduced) of Anti-EGFR1-TGFβRII ECD. Figure 37 shows the HC (Reduced) mass spectrum of Anti-EGFR1-TGFβRII ECD.

[0159] Example 7

[0160] The fusion proteins presenting the amino acid sequences as described in Figures 1 and 2 were inspected using UV chromatography and providing chromatograms resulting from the chromatographic separation of the tryptic digestion of the fusion proteins and tested with a UV wavelength of 218 -222 nm. The Total Ionic Current (TIC) corresponding to the UV trace was also evaluated. Figure 38A shows the UV chromatogram of tryptic peptides from the Anti-HER2/neu-TGFβRII ECD fusion protein and Figure 38B shows the total ion chromatogram (TIC) of the tryptic peptides from the Anti-HER2/neu-TGFβRII ECD fusion protein . Figures 39, 40 and 41 provide lists of expected/observed tryptic peptides from the light chain, heavy chain and linked motif of the Anti-HER2/neu-TGFβRII ECD fusion protein, respectively. Notably, all the expected peptides of the molecules were identified including the light and heavy chain peptides and the linked motif peptides (TGF BRII).

[0161] Figure 42A shows the UV chromatogram of tryptic peptides of the Anti-EGFR1-TGFβRII ECD fusion protein and Figure 42B shows the total ion chromatogram (TIC) of the tryptic peptides of the Anti-EGFR1-TGFβRII ECD fusion protein. Figures 43, 44, and 45 provide lists of expected/observed tryptic peptides from the light chain, heavy chain, and linked motif of the Anti-EGFR1-TGFβRII ECD fusion protein, respectively. Again the expected peptides of the molecules were identified including the light and heavy chain peptides and the linked motif peptides (TGF BRII).

[0162] Example 8

[0163] The host cell line used for recombinant fusion protein expression are CHO cells or CHO cell derivatives. CHO cells are referred to here as CHO-S Freedom cells; CHO-S cells are CHO-derived cells adapted for high-density serum-free suspension culture in chemically defined medium that are capable of producing high levels of secreted recombinant protein, or CHO K1 cells; showing accession number ATCC No. CCL-61. Basically it's an adherent cell line. The vectors used for the stable cell line are:

[0164] The pCHO Freedom 1.0 vector, designed by ProBioGen AG, to express one or two genes of interest downstream of the two different hybrid CMV promoters of the vector. This vector contains the selection marker for dihydrofolate reductase (DHFR) and a puromycin resistance gene, allowing selection using MTX and puromycin simultaneously.

[0165] The nucleic acid sequences encoding the light chain or light chain fusion protein are cloned into the restriction enzyme sites AvrII and BstZl7 under the control of the EF2/CMV promoter. The nucleic acid sequences encoding the light chain or light chain fusion protein are cloned into the EcoRV and Pad restriction enzyme sites under the control of the CMV/EF1 promoter.

[0166] The construct(s) is(are) transfected into CHO-S Freedom cells/CHOK1 cells. The unique high producing clonal cell strain is selected to produce the recombinant fusion protein. Prepare MCB and characterize viability, productivity, cell stability and other parameters. Cells are used for cultivation followed by purification.

[0167] Example 9

[0168] Cell culture is performed in batch feed mode. In cell culture, the mammalian host cells used, which are Chinese Hamster Ovary (CHO) cells, and the culture medium are provided initially. CHO cells are genetically engineered to produce the antibody-peptide fusion protein. The zinc sulfate heptahydrate salt is added to the medium at a concentration of 0.4 mM. In contrast, no zinc salt is added to the control medium. The fermentation process starts with an initial cell count of 0.3-0.45 x 106 cells/ml at 37 ± 1°C, the first 3-4 days are dedicated to growing the cells in batch phase. The next step involves reducing the temperature to 31 ± 1 C° and the process is maintained until the 7th day. Lactate is reduced by almost 10-40% throughout the race. The produced fusion protein is then collected from the medium using the affinity chromatography technique.

[0169] Example 10

[0170] Cell culture is performed in batch feed mode. In cell cultures mammalian host cells and culture medium which is Hyclone CDM4Mab are supplied initially. Salt (zinc) is also added to the medium (0.3 mM). The fermentation process starts with an initial cell count of 0.3-0.45 x 106 cells/ml at 37 ± 1°C, the first 3-4 days are dedicated to growing the cells in batch phase. The next step involves reducing the temperature to 31 ± 1°C and the process is maintained until the 7th day.

[0171] Example 11

[0172] Purification of immunostimulatory antibody-peptide fusion molecules using protein A column. The culture supernatant secreted by the recombinant CHO cell line containing the fusion monoclonal antibodies is tested for titer and endotoxins under sterile conditions. The supernatant is subjected to affinity chromatography using Mab Select Xtra Protein A affinity resin, washed and equilibrated with binding buffer. The pH of the supernatant is adjusted using 0.5 M phosphate at the same pH as the column; the supernatant is allowed to bind to the column/pass through the column at a flow rate of 0.5 ml/minute to obtain maximum binding All antibody-protein fusion molecules are bound via the Fc region, while impurities are eliminated as an outflow. The column is washed with equilibration buffer and bound fusion molecules are eluted using 0.1 M glycine at pH 3.0. The pH of the eluted proteins is adjusted to neutral pH or the pH of the stable formulation and the purified protein is stored at -20°C or 2-8°C.

[0173] Example 12

[0174] Differentiation of Trastuzumab from the Trastuzumab-TGF receptor βRII fusion molecule.

[0175] A breast cancer overexpressing the ErbB2 receptor, either by constitutive activation or by heterodimerization with other members of the ErbB family of receptors, leads to tumor progression. This will involve binding of growth factors associated with the ErbB signaling pathway. In addition to this, the tumor creates an environment in which the immune system is suppressed by activating TGF β and specific cytokines involved in moderate immune response. A new molecule is generated where Trastuzumab (anti ErbB2) is fused with the TGF receptor βRII as a fusion protein. Although it is hypothesized that Trastuzumab will act as a migrating target molecule for breast cancer cells overexpressing ErbB2, the TGFβRII receptor will sequester TGFβ leading to immune activation. The experiment will utilize the growth of cell lines expressing ErbB2 resistant to Herceptin (selected by growing BT474 cells in the presence of Herceptin) in the presence of TGFβ, CD8 positive cells and cytotoxic NK cells. Although Trastuzumab is not effective in inducing cytotoxicity, the Trastuzumab TGFβRII receptor fusion molecule will sequester TGFβ, thereby preventing inhibition of cytotoxic CD8 and NK cells. This will lead to the increased cytotoxicity seen in cells treated with the Trastuzumab-TGFβRII receptor fusion molecule relative to cells treated with Trastuzumab alone. The readout for the experiment will use Alamar Blue a resazurin dye that will be activated directly proportional to the number of live cells present. Another method can be to measure cytotoxicity by using cytotox glo which measures protease release which corresponds directly proportional to the number of dead cells. Yet another method can be the use of flow cytometry directly measuring the population of apoptotic and necrotic cells by using Annexin V and propidium iodide. Results from these multiple experiments will further our understanding of the activity of the conjugated molecule compared to Trastuzumab alone.

[0176] While the invention has been described with reference to the above examples, it is to be understood that modifications and variations are encompassed within the spirit and scope of the invention. Likewise, the invention is limited only by the following claims.

Claims (8)

1. A chimeric fusion protein comprising a targeting moiety for targeting a cancer cell and an immunomodulatory moiety that counteracts immunotolerance characterized in that the targeting moiety and the immunomodulatory moiety are linked by an amino acid spacer of sufficient amino acid residue length so that both fractions can successfully bind to their individual targets, wherein the immunomodulatory fraction is TGF-βRII SEQ ID NO: 4; wherein the amino acid spacer is selected from SEQ ID NO: 3 and SEQ ID NO: 11; and wherein a targeting moiety is an antibody selected from Anti-HER2/Neu, consisting of heavy chain SEQ ID NO: 1 and light chain SEQ ID NO: 2; Anti-EGFR1, consisting of heavy chain SEQ ID NO: 5 and light chain SEQ ID NO: 6; anti-CTLA4, consisting of heavy chain SEQ ID NO: 7 and light chain SEQ ID NO: 8; wherein SEQ ID NO: 4 is joined through the amino acid spacer to the C-terminus of SEQ ID NO: 1 or SEQ ID NO: 2 of Anti-HER2/Neu; to the C-terminus of SEQ ID NO: 5 or SEQ ID NO: 6 of Anti-EGFR1; or to the C-terminus of SEQ ID NO: 7 or SEQ ID NO: 8 of anti-CTLA4. 2. Method for preparing therapeutically active antibody-peptide fusion protein, characterized in that it comprises: a) preparing a codon-optimized sequence of said antibody-peptide fusion protein for expression in a Chinese hamster ovary (CHO) host cell, in that the antibody-peptide fusion protein comprises a targeting moiety and an immunomodulatory moiety, wherein the targeting moiety and the immunomodulatory moiety are linked by an amino acid spacer selected from SEQ ID NO: 3 or SEQ ID NO: 11, wherein the immunomodulatory fraction is TGF-βRII comprising an amino acid sequence of SEQ ID NO: 4; wherein the targeting moiety is selected from the group consisting of an Anti-EGFR1 antibody, consisting of heavy chain SEQ ID NO: 5 and light chain SEQ ID NO: 6; an Anti-HER2/Neu antibody, consisting of heavy chain SEQ ID NO: 1 and light chain SEQ ID NO: 2; and anti-CTLA4 antibody, consisting of heavy chain SEQ ID NO: 7 and light chain SEQ ID NO: 8, wherein SEQ ID NO: 4 is joined through the amino acid spacer to the C-terminus of SEQ ID NO: 1 or SEQ Anti-HER2/Neu ID NO: 2; to the C-terminus of SEQ ID NO: 5 or SEQ ID NO: 6 of Anti-EGFR1; or to the C-terminus of SEQ ID NO 7 or SEQ ID NO: 8 of Anti-CTLA-4; b) cloning the optimized sequence of said antibody-peptide fusion protein into the Chinese hamster ovary (CHO) host cell capable of transient or continuous expression; c) culturing the CHO host cell in a fermentation medium under conditions suitable for growth and which allow the CHO host cell to express the antibody-peptide fusion protein, wherein the fermentation medium comprises a bivalent transition metal salt, the what is zinc sulfate salt heptahydrate; and d) purifying the antibody-peptide fusion protein expressed in step c) and, optionally, verifying the bispecific binding capacity of the antibody-peptide fusion protein to its targets. 3. Method according to claim 2, characterized in that the bivalent transition metal salt is introduced into the cell culture initially or in batch feeding mode. 4. Method according to claim 2, characterized in that the bivalent transition metal salt is in an amount sufficient to reduce the accumulation of lactate during cultivation, at a concentration of about 0.3 mM to about 0.4 mM. 5. Method, according to claim 4, characterized by the fact that the lactate decreases about 10 to 40% throughout the race. 6. Method, according to claim 2, characterized by the fact that the fermentation medium is kept at a temperature of 37 ± 1°C in the first 3 to 4 days and reduced to 31 ± 1°C until the 7th day . 7. Method according to claim 2, characterized in that the fermentation medium comprises an initial cell count of about 0.3 to 0.45 x 106. 8. Method according to claim 2, characterized in that the expressed antibody-peptide fusion protein is subjected to affinity chromatography using a MabSelect Xtra protein A column with a specific pH.

Images