WO2017134592A1

WO2017134592A1 - Anti-cd20/immunomodulatory fusion proteins and methods for making same

Info

Publication number: WO2017134592A1
Application number: PCT/IB2017/050562
Authority: WO
Inventors: Nagaraja GOVINDAPPA; Praveen Kumar Reddy MOOLE; Sreesha P SRINIVASA
Original assignee: Biocon Limited
Priority date: 2016-02-03
Filing date: 2017-02-02
Publication date: 2017-08-10

Abstract

The present invention relates generally to the field of generating recombinant chimeric fusion proteins to be used in the cancer therapy, and more specifically, fusion protein comprises of at least one targeting moiety and at least one immunomodulatory moiety 5 that counteracts the immune tolerance of cancer cells. The present invention provides fusion molecules of Anti-CD20-TGFβRII, Anti-CD20-PD1 and Anti-CD20-TIM3 and methods of generating same, wherein the methods reduce production costs and increase homogeneity of the recombinant chimeric fusion proteins.

Description

ANTI-CD20/IMMUNOMODULATORY FUSION PROTEINS AND METHODS

FOR MAKING SAME

The present application claims priority to Indian provisional patent application no. 201641003849 filed on February 03, 2016 the contents of which are hereby incorporated by reference herein for all purposes.

BACKGROUND OF THE INVENTION

Technical Field

The present invention relates to creation of novel bifunctional fusion antibodies as therapeutic agents and more specifically, to fusion molecules of anti-CD20 and immunomodulatory proteins such as PD1, TGF RII and TIM3 and methods of generating the same.

Related Art

In spite of numerous advances in medical research, cancer remains the second leading cause of death in the United States. Traditional modes of clinical care, such as surgical resection, radiotherapy and chemotherapy, have a significant failure rate, especially for solid tumors. Failure occurs either because the initial tumor is unresponsive, or because of recurrence due to regrowth at the original site or metastasis. Cancer remains a central focus for medical research and development.

Immunotherapy of cancer has been explored for over a century, but it is only in the last decade that various antibody-based products have been introduced into the management of patients with diverse forms of cancer. At present, this is one of the most active areas of clinical research, with numerous antibody therapeutic products already approved in oncology.

Using specific antibodies as therapeutic agents offers advantages over the non-targeted therapies such as systemic chemotherapy via oral or intravenous administration of drugs or radiation therapy. There are two types of antibody-based therapies. The more common type is to identify a tumor antigen (i.e., a protein expressed on tumors and cancer cells and not in normal tissues) and develop an antibody, preferably a monoclonal antibody (mAb) directed to the tumor antigen. One can then conjugate any therapeutic agent, such as a chemotherapeutic agent, a radionuclide, modified toxin, etc., to this antibody to achieve targeted therapy by the therapeutic agent to the tumor. The other kind of antibody based therapy is by providing an antibody which in itself has therapeutic properties against the tumor/cancer cells it targets. The added advantage of this second form of antibody-based therapy is that one may additionally conjugate another therapeutic agent to the therapeutic antibody to achieve a more effective treatment. The major advantage with any antibody-directed therapy and of therapy using monoclonal antibodies (mAbs) in particular, is the ability to deliver increased doses of a therapeutic agent to a tumor, with greater sparing of normal tissue from the side effects of the therapeutic agent.

Despite the identification of several antibodies for cancer therapies, there is still a need to identify new and more effective therapeutics to overcome immune tolerance and activate T cell responses. Further, even though molecular engineering has improved the prospects for such antibody-based therapeutics issues still remain regarding continuity in the generated recombinant products.

SUMMARY OF THE INVENTION

The present invention provides a novel and consistent synthesis method for generating homogeneous recombinant fusion immunomodulatory molecules, and more specifically novel bi-functional fusion antibodies including targeting antibodies linked to immunomodulatory proteins.

Programmed Death Ligand-1 (PDL1), one of the B7 ligands discussed above, obstructs anti-tumor immunity by

(i) tolerizing tumor-reactive T cells by binding to its receptor PD1 (CD279) on T cells;

(ii) rendering tumor cells resistant to CD8+ T cell and FasL-mediated lysis by PD-

1 signaling through tumor cell-expressed PDL1 ; and

(iii) promoting the development and maintenance of induced T regulatory cells.

Therefore, PDL1 is a major obstacle to natural anti-tumor immunity and to cancer immunotherapies requiring activation of host T cell-mediated anti- tumor immunity. This concept is supported by studies demonstrating that antibody blocking of PDL 1 -PD1 interactions improves T cell activation and reduces tumor progression. Although antibodies to PDL1 or PD1 have shown therapeutic efficacy in a subset of cancer patients, the majority of patients do not benefit from antibody treatment. Thus, there is needed a mechanism for regulating PD-L1 function that will lead to a new universally applicable treatment for minimizing PD-L1 -mediated immune suppression in cancer patients and that is more effective than currently available mAbs to PD-1 or PD-L1 . The present invention further provides methods of reducing growth of cancer cells by counteracting immune tolerance of cancer cells, wherein T cells remain active and inhibit the recruitment of T-regulatory that are known to suppress the immune system's response to the tumor. Thus, the chimeric polypeptides generated by the polynucleotides sequences of the present invention are useful for treating cancer because of the expressed fusion or chimeric polypeptides.

In one aspect, the present invention provides for chimeric polypeptides containing at least one targeting moiety to target a cancer cell and at least one immunomodulatmg moiety that counteracts immune tolerance of cancer cell, wherein the targeting moiety and the immunomodulating moiety are linked by an amino acid spacer of sufficient length of amino acid residues so that both moieties can successfully bond to their individual target. In the alternative, the targeting moiety and the immunomodulating moiety that counteract immune tolerance of cancer cell may be bound 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 avoid an immune response. Preferably the targeting moiety is an antibody having binding affinity for CD20, wherein the antibody is transcribed from a polynucleotide sequence lacking nucleotides for expression of the C-terminal lysine of the heavy chain of the expressed antibody. It has been discovered that by removing the C-terminal lysine of the heavy chain of an antibody during transcription that the end product exhibits increased homogeneity, thereby reducing the need and costs for further purification. It is known that during the process of transcription and translation of an IgG molecule in CHO cells, the lysine (K) at the C-terminal of the heavy chain will be expressed. In the commercial product such expressed lysine residues have to be removed to increase purity. There is much heterogeneity in the produced product. This occurs because the CHO cell has an endogenous enzyme Carboxypeptidase B (CPB) which will cleave the C-terminal lysine as long as the expressed antibody is still available intracellularly. However, this enzyme will not cleave the lysine once the antibody is secreted into the medium. Thus, the cleavage efficiency of this endogenous CPB is based on the availability within the cell. As such, some of the antibodies will be secreted with the lysine and some will not, and such combination will cause significant heterogeneity in the secreted product, that being some antibodies with the C-terminal lysine and some without. As the recombinant product is being used for the therapeutic use, one needs to purify to homogeneity. Thus, the recombinant products of the prior art requires additional purification steps wherein the recombinant product need to be treated with the enzyme CPB first and purified once again using an additional step to remove any lysine and the enzyme CPB from the final product. These additional steps add a significant cost to the manufacturing process.

The present invention avoids the shortcomings of previous methods of synthesizing recombinant anti-CD20 antibodies by transcribing an expressed protein from a polynucleotide sequence lacking nucleotides for expression of the C-terminal lysine at the heavy chain of the expressed antibody.

The present invention is based on preparing chimeric/fusion proteins by expression of polynucleotides encoding the fusion proteins that counteract or reverse immune tolerance of cancer cells. Cancer cells are able to escape elimination by chemotherapeutic agents or tumor-targeted antibodies via specific immunosuppressive mechanisms in the tumor microenvironment and such ability of cancer cells is recognized as immune tolerance. Such immunosuppressive mechanisms include immunosuppressive cytokines (for example, Transforming growth factor beta (TGF-β)) and regulatory T cells and/or immunosuppressive myeloid dendritic cells (DCs). By counteracting tumor-induced immune tolerance, the present invention provides effective compositions and methods for cancer treatment, optional in combination with another existing cancer treatment. The present invention provides strategies to counteract tumor-induced immune tolerance and enhance the antitumor efficacy of chemotherapy by activating and leveraging T cell- mediated adaptive antitumor against resistant or disseminated cancer cells.

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 a target molecule, and the immunomodulatory moiety specifically binds one of the following molecules: (i) Transforming growth factor-beta (TGF-β) and or (ii) Programmed death- 1 ligand 1 (PD-L1) (iii) T cell immunoglobulin and mucin domain-3 (TIM3).

In a further aspect, the targeting moiety includes an antibody, including both heavy chains and light chains, wherein the antibody specifically binds a component of a tumor cell, tumor antigen, tumor vasculature, tumor microenvironment, or tumor-infiltrating immune cell. Notably the heavy chain and/or light chain may individually be linked to a same type immunomodulatory moiety or a separate and distinct immunomodulatory moiety. Further, a heavy or light chain of an antibody targeting moiety may be linked to an immunomodulatory moiety which in turn can be further linked to a second immunomodulatory moiety wherein there is a linker between the two immunomodulatory moieties.

In a still further aspect, there is provided a chimeric polypeptide that comprised a tumor targeting moiety and an immunomodulatory moiety comprising a molecule that binds transforming growth factor beta (TGF-β), wherein the tumor targeting moiety is an antibody that binds to CD20, where in the antibody can be the full antibody, heavy chain or light chain.

The tumor targeting moiety may include monoclonal antibodies that target a cancer cell, including but not limited to cetuximab, trastuzumab, ritubximab, ipilimumab, tremelimumab, muromonab-CD3, abciximab, daclizumab, basiliximab, palivizumab, infliximab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalimumab, omalizumab, tositumomab, 1-131 tositumomab, efalizumab, bevacizumab, panitumumab, pertuzumab, natalizumab, etanercept, IGNlOl (Aphton), volociximab (Biogen Idee and PDL BioPharm), Anti-CD80 mAb (Biogen Idee), Anti-CD23 mAb (Biogen Idel), CAT- 3888 (Cambridge Antibody Technology), CDP-791 (Imclone), eraptuzumab (Immunomedics), MDX-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 Idee and PDL BioPharm), daclizumab (Biogent Idee 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 BioPharm), tocilizumab (Roche), ocrerlizumab (Roche), certolizumab pegol (UCB, formerly Celltech), eculizumab (Alexion Pharmaceuticals), pexelizumab (Alexion Pharmaceuticals and Procter & Gamble), abciximab (Centocor), ranibizimumab (Genetech), mepolizumab (GSK), TNX-355 (Tanox), or MYO-029 (Wyeth).

In a preferred embodiment, the tumor targeting moiety is a monoclonal antibody that binds to CD20 generated by the methods of the present invention, wherein the method comprises the following steps: a. preparing a codon optimized nucleotide sequence encoding the fusion protein, wherein the codon optimized sequence for the antibody is lacking nucleotides for expression of a lysine at the C-terminal end of the heavy chains of the antibody; b. cloning the optimized sequence of said fusion protein in a host cell capable of transient or continued expression;

c. growing the host cell in a media under suitable conditions for growing and allowing the host cell to express the fusion protein; and

d. collecting secreted fusion proteins. In yet another aspect, the immunomodulatory moiety includes a molecule that binds TGF- β and inhibits the function thereof. Specifically the immunomodulatory moiety includes an extracellular ligand-binding domain of Transforming growth factor-beta receptor TGF- RII, TGF- RIIb or TGF- RIII. In another aspect the immunomodulatory moiety includes an extracellular ligand-binding domain (ECD) of TGF- RII. In a still further aspect, the targeting moiety includes an antibody that specifically binds to, CD20, or cytotoxic T-lymphocyte antigen-4 (CTLA-4), or HER2/neu, EGFRl and wherein the immunomodulatory moiety includes an extracellular ligand-binding domain of TGF- Rn. In yet another aspect, the immunomodulatory moiety includes a molecule that specifically binds to and inhibits the activity of Programmed death- 1 ligand 1 (PD-L1).

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

In a still further aspect, the targeting moiety includes an antibody that specifically binds to CD20, and the immunomodulatory moiety includes a sequence from interacts with transforming growth factor-β (TGF-β). In a still further aspect, the targeting moiety includes an antibody that specifically binds to CD20, and the immunomodulatory moiety includes a sequence from interacts with T cell immunoglobulin and mucin domain-3 (TIM3).

In a still further aspect, the targeting moiety includes an antibody that specifically binds to CD 20 and the immunomodulatory moiety selected from PD1, TGF RII and TIM3. The amino acid sequences are selected from SEQ ID NOs: 1 to 18 wherein SEQ ID 6 is used as a connecting linker.

In one aspect, the present invention provides for optimized genes encoding for a fusion polypeptide comprising at least one targeting moiety and at least one immunomodulatory moiety for treating cancer in a human subject wherein the genes have been optimized to increase expression in a human subject and/or cells.

In another aspect, the present invention provides for a vector comprising optimized genes for treating cancer in a human subject wherein the optimized genes have been modified to increase CG sequences. Preferably, the vector includes nucleotide sequences for encoding at least one targeting moiety, at least one immunomodulatory moiety and a linking moiety, wherein the optimized nucleotide sequences are selected from SEQ ID NOs: 19 to 32.

In yet another aspect, the present invention provides for a method of treating cancer in a subject, the method comprising:

Providing at least one recombinant vector comprising nucleotide sequences that encode at least one targeting moiety, at least one immunomodulatory moiety and a linking moiety positioned between the targeting moiety and immunomodulatory moiety, and 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.

In yet another aspect, the present invention provides a recombinant host cell transfected with a polynucleotide sequence that encodes a fusion protein peptide of the present invention, wherein the polynucleotide sequences are selected from SEQ ID NOs: 19 to 32.

In a still further aspect, the present invention contemplates a process of preparing a chimeric fusion protein of the present invention comprising: transfecting a host cell with a polynucleotide sequence that encodes a chimeric fusion protein to produce a transformed host cell, wherein the polynucleotide sequence encodes at least one targeting moiety and at least one immunomodulatory moiety, wherein the polynucleotide sequence comprises a combination of sequences selected from SEQ ID NOs: 21 to 32; and maintaining the transformed host cell under biological conditions sufficient for expression of the chimeric fusion protein.

In another aspect, the present invention relates to the use of a chimeric fusion protein, wherein the chimeric fusion protein comprises anti-CD20 linker PD1 (SEQ ID NOs: 7, 8, 9, 10 or SEQ ID NOs: 21, 22, 23, 24); anti-CD20 -linker-TGF RII (SEQ ID NOs: 11, 12, 13, 14 or SEQ ID NOs: 25, 26, 27, 28); Anti-CD20-linker-TIM3 (SEQ ID NOs: 15, 16, 17, 18 or SEQ ID NOs: 29, 30, 31, 32), as shown in Figures 2, 3, 4 and 5, 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 a subject in need thereof.

In a still further aspect, the present invention provides a method of treating a neoplastic disease. The method includes administration to a subject in need thereof one or more fusion proteins of the present invention, in various aspects, the subject is administered one or more fusion protein of the present invention in combination with another anticancer therapy. In one aspect, the anticancer therapy includes a chemotherapeutic molecule, antibody, small molecule kinase inhibitor, hormonal agent or cytotoxic agent. The anticancer therapy may also include ionizing radiation, ultraviolet radiation, cryoablation, thermal ablation, or radiofrequency ablation.

In a preferred embodiment the therapeutically active antibody-peptide fusion proteins is a targeting antibody fused to one or more immunomodulating moiety that counteracts immune tolerance of a cancer cell. In one aspect, the immunomodulating moiety may be linked by an amino acid spacer of sufficient length to allow bi-specific binding of the molecule. The immunomodulating moiety may be bound to either the N- terminus or C- terminus of the heavy chain or the N- terminus or C-terminus of the light chain of the antibody.

The method of the present invention provides nucleotide sequences that encode the therapeutically active antibody-peptide fusion proteins and such expression may be conducted in a transient cell line or a stable cell line. The transient expression is accomplished by transfecting or transforming the host cell with vectors carrying the encoded fusion proteins into mammalian host cells.

Once the fusion peptides are expressed, they are preferably subjected to purification and in-vitro tests to check its bi-specificity, that being, having the ability to bind to both the target moiety and immunomodulating moiety. Such tests may include in-vitro tests such as ELISA or NK/T-cell binding assays to validate bi-functional target binding or immune cell stimulation.

Notably once the specific fusion peptides demonstrate the desired bi-specificity, the polynucleotide sequences encoding such fusion peptides are selected for sub-cloning into a stable cell line for larger scale expression and purification. Such stable cell lines are previously disclosed, such as a mammalian cell line, including but not limited to HEK293, CHO or NSO.

In another aspect the present invention provides for a method to inhibit and/or reduce binding of PDl thereby increasing immune response against tumor cells, the method comprising: a. providing a chimeric polypeptide comprising PDl and an anti-CD20 antibody; and

b. contacting a tumor cell with the chimeric polypeptide wherein the chimeric polypeptide binds with at least PDl of the tumor cell.

In yet another aspect, the present invention provides for a method of preparing therapeutically active antibody-peptide fusion proteins, the method comprising; a. preparing a codon optimized sequence of the said fusion protein, wherein the codon optimized sequences for anti-CDC20 antibody is lacking nucleotides for expression of a lysine at the C-terminal end of the heavy chains of the antibodies; b. cloning the optimized sequence of said fusion protein in a host cell capable of transient or continued expression;

d. collecting secreted fusion proteins.

In a still further aspect the present invention provides for a nucleic acid sequence encoding a chimeric fusion protein, wherein the chimeric fusion protein comprises at least one targeting moiety having affinity for a cancer cell and at least one immunomodulatory moiety that counteract immune tolerance of the cancer cell, wherein targeting moiety is an antibody and the nucleic acid sequence of the targeting moiety is lacking nucleotides for expression of a lysine at the C-terminal end of the heavy chains of the antibody. The nucleic acid sequence encoding the antibody preferably includes SEQ ID NO: 19 or NO: 20. The nucleic acid sequence encoding the chimeric fusion proteins preferably comprises a sequence selected from the group consisting of SEQ ID NOs: 21, 22, 23 and 24; SEQ ID NOs: 25, 26, 27 and 28; and SEQ ID NOs: 29, 30, 31 and 32.

In yet another aspect the present invention provides for a method of treating cancer in a subject, the method comprising:

A. preparing a preparing therapeutically active fusion protein, wherein the fusion protein comprises a tumor targeting moiety and at least one immunomodulatory molecule, wherein the tumor targeting moiety is an antibody that binds to CD20 and wherein the fusion protein is prepared by the following steps:

1. preparing a codon optimized nucleotide sequence encoding the fusion protein, wherein the codon optimized nucleotide sequence for the antibody is lacking nucleotides for expression of a lysine at the C-terminal end of the heavy chains of the antibody;

2. cloning the optimized sequence of said fusion protein in a host cell capable of transient or continued expression;

3. growing the host cell in a media under suitable conditions for growing and allowing the host cell to express the fusion protein; and

4. collecting secreted fusion proteins;

B. administering a therapeutically active amount of the secreted fusion proteins to the subject.

The fusion protein is selected from the group of amino acid sequences consisting of SEQ ID NOs: 1 and 3; SEQ ID NOs: 2 and 3; SEQ ID NOs: 1 and 4; SEQ ID NOs: 2 and 4; SEQ ID NOs: 1 and 5; SEQ ID NOs: 2 and 5; SEQ ID NOs: 7, 8, 9, and 10; SEQ ID NOs: 11, 12, 13 and 14; SEQ ID NOs: 15, 16, 17 and 18 wherein the SEQ ID 6 is used as a connecting linker

In another aspect, the present invention provides for a method of treating a neoplastic disease, the method comprising administration to a subject in need thereof one or more fusion proteins encoded by at least one polynucleotide sequence selected from the group consisting of SEQ ID NOs: 21, 22, 23, 24; SEQ ID NOs: 25, 26, 27, 28; SEQ ID NOs: 29, 30, 31, 32. Notably by using the above defined polynucleotide sequences, the following combination of fusion proteins can be expressed including Anti-CD20mAb+PDl (SEQ ID NOs: 21, 22, 23, 24), Anti-CD20mAb+TGF RII (SEQ ID NOs: 25, 26, 27, 28), Anti- CD20mAb+TIM3 (SEQ ID NOs: 29, 30, 31, 32). Other features and advantages of the invention will be apparent from the following detailed description, drawings and claims.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is agarose gel image of the endotoxin free DNA used for Transient Gene Expression (TGE) for material generation Figure 2 is analysis of purified FmAb 21, FmAb 22, FmAb 26 and FmAb 28 on non- reducing SDS-PAGE along with control Anti-CD20 mAb

Figure 3 is analysis of FmAb 21, FmAb 22, FmAb 26, FmAb 28 and FmAb 32 on reducing SDS-PAGE along with control Anti-CD20 mAb

Figure 4 is analysis of FMab 23, 24, 25, 27 29, 30 and 31 on non-reducing SDS-PAGE Figure 5 is analysis of FMab 23, 24, 25, 27 29, 30 and 31 on 4-20% reducing SDS-PAGE

Figure 6 is TGF i Target Binding of the purified proteins for Anti-CD 20 constructs- FmAb 25, FmAb 26, FmAb 27, FmAb 28, TGF RII Fc and IgG Control

Figure 7 is TIM3 Target Binding of the purified proteins for Anti-CD 20 constructs- FmAb 29, FmAb 30, FmAb 31 and FmAb 32 Figure 8 is ADCC activity of the purified proteins for Anti-CD 20 constructs- FmAb 21, FmAb 22, FmAb 23, FmAb 24, FmAb 25, FmAb 27, FmAb 29, FmAb 30, FmAb 31 and Assay control (Rituximab)

Figure 9 is ADCC activity of the purified proteins for Anti-CD 20 constructs- FMab 26, FMab 28, FMab 32, anti-CD 20 control (WT) and Assay control (Rituximab) DETAILED DESCRIPTION OF THE INVENTION

In order to facilitate review of the various embodiments of the invention and provide an understanding of the various elements and constituents used in making and using the present invention, the following terms used in the invention description have the following meanings.

As used herein, the terms "polypeptide," "protein" and "peptide" are used interchangeably to denote a sequence polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristilation, ubiquitination, etc.). D- and L-amino acids, and mixtures of D- and L-amino acids are also included.

Chimeric polypeptide refers to an amino acid sequence having two or more parts which generally are not found together in an amino acid sequence in nature.

The term "spacer/linker" as used herein refers to a molecule that connects two monomeric protein units to form a chimeric molecule and still provides for binding of the parts to the desired receptors. Particular examples of spacer/linkers may include an amino acid spacer, wherein thee amino acid sequence can essentially be any length, for example, as few as 5 or as many as 200 or more preferably from about 5 to 30 amino acid residues.

The term "therapeutic," as used herein, means a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing or eliminating those signs. The term "therapeutically effective amount," as used herein means an amount of the chimeric protein that is sufficient to provide a beneficial effect to the subject to which the chimeric protein is administered.

Another example of a modification is the addition of a heterologous domain that imparts a distinct functionality upon the chimeric polypeptide. A heterologous domain can be any small organic or inorganic molecule or macromolecule, so long as it imparts an additional function. Particular examples of heterologous domains that impart a distinct function include an amino acid sequence that imparts targeting (e.g., receptor ligand, antibody, etc.), immunopotentiating function (e.g., immunoglobulin, an adjuvant), enable purification, isolation or detection (e.g., myc, T7 tag, polyhistidine, avidin, biotin, lectins, etc.).

As exemplified herein, polypeptide sequences may include substitutions, variations, or derivitizations of the amino acid sequence of one or both of the polypeptide sequences that comprise the chimeric polypeptide, so long as the modified chimeric polypeptide has substantially the same activity or function as the unmodified chimeric polypeptide.

As used herein, the term "substantially the same activity or function," when used in reference to a chimeric polypeptide so modified, means that the polypeptide retains most, all or more of the activity associated with the unmodified polypeptide, as described herein or known in the art.

Modified chimeric polypeptides that are "active" or "functional" included herein can be identified through a routine functional assay. For example, by using antibody binding assays or co-receptor binding assays one can readily determine whether the modified chimeric polypeptide has activity. As the modified chimeric polypeptides will retain activity or function associated with unmodified chimeric polypeptide, modified chimeric polypeptides will generally have an amino acid sequence "substantially identical" or "substantially homologous" with the amino acid sequence of the unmodified polypeptide.

As used herein, the term "substantially identical" or "substantially homologous," when used in reference to a polypeptide sequence, means that a sequence of the polypeptide is at least 50% identical to a reference sequence. Modified polypeptides and substantially identical polypeptides will typically have at least 70%>, alternatively 85%, more likely 90%, and most likely 95% homology to a reference polypeptide.

As set forth herein, substantially identical or homologous polypeptides include additions, truncations, internal deletions or insertions, conservative and non-conservative substitutions, or other modifications located at positions of the amino acid sequence which do not destroy the function of the chimeric polypeptide (as determined by functional assays, e.g., as described herein). A particular example of a substitution is where one or more amino acids are replaced by another, chemically or biologically similar residue. As used herein, the term "conservative substitution" refers to a substitution of one residue with a chemically or biologically similar residue. Examples of conservative substitutions include the replacement of a hydrophobic residue, such as isoleucine, valine, leucine, or methionine for another, the replacement of a polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. Those of skill in the art will recognize the numerous amino acids that can be modified or substituted with other chemically similar residues without substantially altering activity.

Modified polypeptides further include "chemical derivatives," in which one or more of the amino acids therein have a side chain chemically altered or derivatized. Such derivatized polypeptides include, for example, amino acids in which free amino groups form amine hydrochlorides, p-toluene sulfonyl groups, carobenzoxy groups; the free carboxy groups form salts, methyl and ethyl esters; free hydroxyl groups that form O-acyl or O-alkyl derivatives, as well as naturally occurring amino acid derivatives, for example, 4-hydroxyproline, for proline, 5-hydroxylysine for lysine, homoserine for serine, ornithine for lysine, and so forth. Also included are D-amino acids and amino acid derivatives that can alter covalent bonding, for example, the disulfide linkage that forms between two cysteine residues that produces a cyclized polypeptide.

As used herein, the terms "isolated" or "substantially pure," when used as a modifier of invention chimeric polypeptides, sequence fragments thereof, and polynucleotides, means that they are produced by human intervention and are separated from their native in vivo - cellular environment. Generally, polypeptides and polynucleotides so separated are substantially free of other proteins, nucleic acids, lipids, carbohydrates or other materials with which they are naturally associated.

Polypeptides of the present invention may be prepared by standard techniques well known to those skilled in the art. Such techniques include, but are not limited to, isolation and purification from tissues known to contain that polypeptide, and expression from cloned DNA that encodes such a polypeptide using transformed cells. Chimeric polypeptides can be obtained by expression of a polynucleotide encoding the polypeptide in a host cell, such as a bacteria, yeast or mammalian cell, and purifying the expressed chimeric polypeptide by purification using typical biochemical methods (e.g., immunoaffinity purification, gel purification, expression screening etc.). Other well- known methods are described in Deutscher et al., 1990. Alternatively, the chimeric polypeptide can be chemically synthesized. Purity can be measured by any appropriate method, e.g., polyacrylamide gel electrophoresis, and subsequent staining of the gel (e.g., silver stain) or by HPLC analysis.

The present invention further provides polynucleotide sequences encoding chimeric polypeptides, fragments thereof, and complementary sequences. As used herein, the terms "nucleic acid," "polynucleotide," "oligonucleotide," and "primer" are used interchangeably to refer to deoxyribonucleic acid (DNA) or ribonucleic (RNA), either double- or single-stranded, linear or circular. RNA can be unspliced or spliced mRNA, rRNA, tRNA, or antisense RNAi. DNA can be complementary DNA (cDNA), genomic DNA, or an antisense. Specifically included are nucleotide analogues and derivatives, such as those that are resistant to nuclease degradation, which can function to encode an invention chimeric polypeptide. Nuclease resistant oligonucleotides and polynucleotides are particularly useful for the present nucleic acid vaccines described herein.

An "isolated" or "substantially pure" polynucleotide means that the nucleic acid is not immediately contiguous with the coding sequences with either the 5 ' end or the 3 ' end with which it is immediately contiguous in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment produced during cloning), as well as a recombinant DNA incorporated into a vector, an autonomously replicating plasmid or virus, or a genomic DNA of a prokaryote or eukaryote.

The polynucleotides sequences of the present invention can be obtained using standard techniques known in the art (e.g., molecular cloning, chemical synthesis) and the purity can be determined by polyacrylamide or agarose gel electrophoresis, sequencing analysis, and the like. Polynucleotides also can be isolated using hybridization or computer-based techniques that are well known in the art. Such techniques include, but are not limited to: (1) hybridization of genomic DNA or cDNA libraries with probes to detect homologous nucleotide sequences; (2) antibody screening of polypeptides expressed by DNA sequences (e.g., using an expression library); (3) polymerase chain reaction (PCR) of genomic DNA or cDNA using primers capable of annealing to a nucleic acid sequence of interest; (4) computer searches of sequence databases for related sequences; and (5) differential screening of a subtracted nucleic acid library. The invention also includes substantially homologous polynucleotides. As used herein, the term "homologous," when used in reference to nucleic acid molecule, refers to similarity between two nucleotide sequences. When a nucleotide position in both of the molecules is occupied by identical nucleotides, then they are homologous at that position. "Substantially homologous" nucleic acid sequences are at least 50% homologous, more likely at least 75% homologous, and most likely 90%> or more homologous. As with substantially homologous invention chimeric polypeptides, polynucleotides substantially homologous to invention polynucleotides encoding chimeric polypeptides encode polypeptides that retain most or all of the activity or function associated with the sequence to which it is homologous. For polynucleotides, the length of comparison between sequences will generally be at least 30 nucleotides, alternatively at least 50 nucleotides, more likely at least 75 nucleotides, and most likely 110 nucleotides or more. Algorithms for identifying homologous sequences that account for polynucleotide sequence gaps and mismatched oligonucleotides are known in the art, such as BLAST (see Altschul, 1990). The polynucleotides of the present invention can, if desired: be naked or be in a carrier suitable for passing through a cell membrane (e.g., polynucleotide-liposome complex or a colloidal dispersion system), contained in a vector (e.g., retrovirus vector, adenoviral vectors, and the like), linked to inert beads or other heterologous domains (e.g., antibodies, ligands, biotin, streptavidin, lectins, and the like), or other appropriate compositions disclosed herein or known in the art. Thus, viral and non-viral means of polynucleotide delivery can be achieved and are contemplated. The polynucleotides of the present invention can also contain additional nucleic acid sequences linked thereto that encode a polypeptide having a distinct functionality, such as the various heterologous domains set forth herein. The polynucleotides of the present invention can also be modified, for example, to be resistant to nucleases to enhance their stability in a pharmaceutical formulation. The described polynucleotides are useful for encoding chimeric polypeptides of the present invention, especially when such polynucleotides are incorporated into expression systems disclosed herein or known in the art. Accordingly, polynucleotides including an expression vector are also included.

For propagation or expression in cells, polynucleotides described herein can be inserted into a vector. The term "vector" refers to a plasmid, virus, or other vehicle known in the art that can be manipulated by insertion or incorporation of a nucleic acid. Such vectors can be used for genetic manipulation (i.e., "cloning vectors") or can be used to transcribe or translate the inserted polynucleotide (i.e., "expression vectors"). A vector generally contains at least an origin of replication for propagation in a cell and a promoter. Control elements, including promoters present within an expression vector, are included to facilitate proper transcription and translation (e.g., splicing signal for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA and stop codons). In vivo or in vitro expression of the polynucleotides described herein can be conferred by a promoter operably linked to the nucleic acid.

"Promoter" refers to a minimal nucleic acid sequence sufficient to direct transcription of the nucleic acid to which the promoter is operably linked (see Bitter 1987). Promoters can constitutively direct transcription, can be tissue-specific, or can render inducible or repressible transcription; such elements are generally located in the 5' or 3' regions of the gene so regulated.

As used herein, the term "operably linked" means that a selected polynucleotide (e.g., encoding a chimeric polypeptide) and regulatory sequence(s) are connected in such a way as to permit transcription when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s). Typically, a promoter is located at the 5' end of the polynucleotide and may be in close proximity of the transcription initiation site to allow the promoter to regulate expression of the polynucleotide.

When cloning in bacterial systems, constitutive promoters, such as T7 and the like, as well as inducible promoters, such as pL of bacteriophage gamma, plac, ptrp, ptac, may be used. When cloning in mammalian cell systems, constitutive promoters, such as SV40, RSV and the like, or inducible promoters derived from the genome of mammalian cells (e.g., the metallothionein promoter) or from mammalian viruses (e.g., the mouse mammary tumor virus long terminal repeat, the adenovirus late promoter), may be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences of the invention.

Mammalian expression systems that utilize recombinant viruses or viral elements to direct expression may be engineered. For example, when using adenovirus expression vectors, the nucleic acid sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. Alternatively, the vaccinia virus 7.5K promoter may be used (see Mackett 1982; Mackett 1984; Panicali 1982).

For yeast expression, a number of vectors containing constitutive or inducible promoters may be used (see Ausubel 1988; Grant 1987; Glover 1986; Bitter 1987; and Strathem 1982). The polynucleotides may be inserted into an expression vector for expression in vitro (e.g., using in vitro transcription/translation kits, which are available commercially), or may be inserted into an expression vector that contains a promoter sequence that facilitates expression in either prokaryotes or eukaryotes by transfer of an appropriate nucleic acid into a suitable cell, organ, tissue, or organism in vivo. As used herein, a "transgene" is any piece of a polynucleotide inserted by artifice into a host cell, and becomes part of the organism that develops from that cell. A transgene can include one or more promoters and any other DNA, such as introns, necessary for expression of the selected DNA, all operably linked to the selected DNA, and may include an enhancer sequence. A transgene may include a polynucleotide that is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism. Transgenes may integrate into the host cell's genome or be maintained as a self -replicating plasmid.

As used herein, a "host cell" is a cell into which a polynucleotide is introduced that can be propagated, transcribed, or encoded polypeptide expressed. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell, since there may be mutations that occur during replication. Host cells include but are not limited to bacteria, yeast, insect, and mammalian cells. For example, bacteria transformed with recombinant bacteriophage polynucleotide, plasmid nucleic acid, or cosmid nucleic acid expression vectors; yeast transformed with recombinant yeast expression vectors; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV), or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid), insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus), or animal cell systems infected with recombinant virus expression vectors (e.g., retroviruses, adenovirus, vaccinia virus), or transformed animal cell systems engineered for stable expression.

As used herein, the term "transformation" means a genetic change in a cell following incorporation of a polynucleotide (e.g., a transgene) exogenous to the cell. Thus, a "transformed cell" is a cell into which, or a progeny of which, a polynucleotide has been introduced by means of recombinant techniques. Transformation of a host cell may be carried out by conventional techniques known to those skilled in the art. When the host cell is a eukaryote, methods of DNA transformation include, for example, calcium phosphate, microinjection, electroporation, liposomes, and viral vectors. Eukaryotic cells also can be co-transformed with invention polynucleotide sequences or fragments thereof, and a second DNA molecule encoding a selectable marker, as described herein or otherwise known in the art. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells, and express the protein (see Gluzman 1982). When the host is prokaryotic (e.g., E. coli), competent cells that are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaC12 method using procedures well-known in the art. Transformation of prokaryotes also can be performed by protoplast fusion of the host cell.

Chimeric polypeptides, polynucleotides, and expression vectors containing same of the present invention can be encapsulated within liposomes using standard techniques and introduced into cells or whole organisms. Cationic liposomes are preferred for delivery of polynucleotides. The use of liposomes for introducing various compositions in vitro or in vivo, including proteins and polynucleotides, is known to those of skill in the art. Liposomes can be targeted to a cell type or tissue of interest by the addition to the liposome preparation of a ligand, such as a polypeptide, for which a corresponding cellular receptor has been identified. Monoclonal antibodies can also be used for targeting; many such antibodies specific for a wide variety of cell surface proteins are known to those skilled in the art and are available. The selected ligand is covalently conjugated to a lipid anchor in either preformed liposomes or are incorporated during liposome preparation (see Lee 1994 and Lee 1995).

As the chimeric polypeptides or polynucleotides of the present invention will be administered to humans, the present invention also provides pharmaceutical formulations comprising the disclosed chimeric polypeptides or polynucleotides. The compositions administered to a subject will therefore be in a "pharmaceutically acceptable" or "physiologically acceptable" formulation.

As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" refer to carriers, diluents, excipients, and the like that can be administered to a subject, preferably without excessive adverse side effects (e.g., nausea, headaches, etc.). Such preparations for administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions, or suspensions, including saline and buffered media. Vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antimicrobial, anti-oxidants, chelating agents, and inert gases and the like. Various pharmaceutical formulations appropriate for administration to a subject known in the art are applicable in the methods of the invention (e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, PA (1990); and The Merck Index, 12th ed., Merck Publishing Group, Whitehouse, NJ (1996)). Controlling the duration of action or controlled delivery of an administered composition can be achieved by incorporating the composition into particles or a polymeric substance, such as polyesters, polyamine acids, hydrogel, polyvinyl pyrrolidone, ethylene- vinylacetate, methylcellulose, carboxymethylcellulose, protamine sulfate or lactide/glycolide copolymers, polylactide/glycolide copolymers, or ethylenevinylacetate copolymers. The rate of release of the composition may be controlled by altering the concentration or composition of such macromolecules. Colloidal dispersion systems include macromolecule complexes, nano-capsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The compositions administered by a method of the present invention can be administered parenterally by injection, by gradual perfusion over time, or by bolus administration or by a microfabricated implantable device. The composition can be administered via inhalation, intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity (e.g., vaginal or anal), transdermally, topically, or intravascularly. The compositions can be administered in multiple doses. An effective amount can readily be determined by those skilled in the art.

The present invention further provides methods of reducing growth of cancer cells by counteracting immune tolerance of cancer cells, wherein T cells remain active and inhibit the recruitment of T-regulatory that are known to suppress the immune system's response to the tumor. Thus, the chimeric polypeptides generated by the polynucleotides sequences of the present invention are useful for treating cancer because of the expressed fusion or chimeric polypeptides.

In one aspect, the present invention provides for chimeric polypeptides containing at least one targeting moiety to target a cancer cell and at least one immunomodulatmg moiety that counteracts immune tolerance of cancer cell, wherein the targeting moiety and the immunomodulating moiety are linked by an amino acid spacer of sufficient length of amino acid residues so that both moieties can successfully bond to their individual target. The fusion proteins bind to CD20 on neoplastic B-cells as well as Immune modulators in the tumor microenvironment and immune cells. Binding of fusion antibody to CD20 on B-cells is expected to cause Antibody Dependent Cellular Cytotoxicity (ADCC) and binding to immune modulators is expected to activate immune response to tumor cells. The chimeric/fusion polypeptides of the invention are useful for binding to a cancer cell receptor and reducing the ability of cancer cells to avoid an immune response.

The Fusion proteins comprising of IgG heavy or light chains linked to immunomodulator (either suppressor or activator) ligands at the N-terminus or C-terminus or both N- and C- terminus for the expression in CHO or NSO cells. The codon optimized nucleotide sequences were expressed in CHO cells and its derivatives. The recombinant expression in CHO cells resulted in the secretion of homogeneous mAb fusion proteins to the medium after relevant cellular modification as per the design of the molecule. The expressed proteins were purified from culture supernatants using Protein A column and characterized by using SDS PAGE, mass spectroscopy and in vitro binding assays to demonstrate the molecular structure and function. These were also tested for biological activity by carrying out inhibition of proliferation and ADCC assays.

In the present invention the fusion protein comprises a known monoclonal antibody which is approved for the treatment of cancer and other disease conditions and proved clinical efficacy by binding to a specific target and a suitable immunomodulators which can mop up a harmful cytokines/immune suppressive molecules secreted from the tumor or it can help activate the immune system through T-cell activation. The monoclonal antibody is mainly tumor targeting antibody. The monoclonal antibody and immunomodulator have been linked through the flexible linker comprises of "GGGGSGGGGSGGGGS" amino acids which does not have a defined secondary structure. Use of this linker in between the mAb and fusion partner is to ensure the functionality of both the fused moieties.

The immune system provides the human body with a means to recognize and defend itself against microorganisms and substances recognized as foreign or potentially harmful. While passive immunotherapy of cancer 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 immunological memory against tumor cells has remained challenging. Several tumor-specific and tumor- associated antigens have been identified, yet these antigens are generally weakly immunogenic and tumors employ diverse mechanisms to create a tolerogenic environment that allows them to evade immunologic attack. Strategies to overcome such immune tolerance and activating robust levels of antibody and/or T cell responses hold the key to effective cancer immunotherapy. One approach to circumvent immune tolerance and increase tumor specific immune response is to combine the activity of an anti-tumor antibody with an immune modulator.

Development of Anti-CD 20 fusion protein constructs and transient expression in CHO cells

The current invention discloses the recombinant bi-functional fusion proteins to be used in the cancer therapy.

More specifically, Anti-CD20 monoclonal antibody was fused with three immunomodulatory ligands, Programmed cell death protein- 1 ligand binding domain (PD-1 LBD), Transforming Growth forming factor beta receptor II extracellular domain (TGF RII ECD) and T cell immunoglobin mucin-3 extracellular domain (TIM3 ECD). The fusion sites are at the C- and N-terminus of HC and LC of the mAb through a 15 amino acid linker.

The nucleotide sequences were codon-optimized for the expression in CHO cells. Synthetic genes were obtained from Geneart. The construct were assembled in the mammalian expression vector suitable for transient expression in CHO cells. Nucleic acid sequences of the individual ant-CD 20 fusion expression constructs used for co- transfection to generate transiently expressed material.

The details of the constructs are presented in the table 1 that mentions fusion of immunomodulatory proteins such as PD-1 LBD, TGF RII ECD and TIM3 ECD at heavy chain C-terminal, heavy chain N-terminal, light chain C-terminal and light chain N- terminal of anti-CD20 monoclonal antibody (targeting moiety) and molecular weight, isoelectric point and SEQ ID No. of these fusion proteins.

Different target nucleotide sequences of anti-CD20 mAb (SEQ ID NO: 19-20) and Anti CD 20+Fusion Partner immunomodulatory amino acid and nucleotide sequences of PD-1 LBD (SEQ ID NO: 7-10, 21-24), TGFp¾n ECD (SEQ ID NO: 11-14, 25-28) and TIM3 ECD (SEQ ID NO: 15-18, 29-32) used for manufacturing of fusion proteins are listed below.

Table 1: details of anti-CD 20 constructs with fusion partners and fusion details Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. The invention is further described in the following examples, which do not limit the scope of the invention(s) described in the claims.

Example 1: Transient gene expression (TGE) to generate material for POC studies The genes corresponding HC, LC and their fusion were cloned into pMBL-CE-GS expression vector. Plasmid was isolated and carried out co-transfection into CHO cell line using Expi CHO kit based TGE Protocol using 1 : 1 ratio of HC: LC. The fusion protein secreted to the medium was purified using protein A affinity chromatography. The experimental results are given below. The final expression plasmids are developed and purified using Endotoxin free plasmid isolation kit. The quality of the plasmid was analysed on Agarose gel. All the plasmids were showing the expected band size on the agarose gel as seen in figure 1.

Legend:

1. Gel-1

1. Based on the agarose gel image, Lane No. 1 represents plasmid FMab27-HC-N- terminal fusion running at an expected size of 10788 bp.

2. Lane No. 2: 1 Kb DNA ladder (molecular weight marker) with top band corresponds to 10 kb in size.

3. Based on the agarose gel image, Lane No. 3 represents plasmid FMab23 HC-N- terminal fusion running at an expected size of 10827 bp.

4. Based on the agarose gel image, Lane No. 4 represents plasmid FMab31 HC-N- terminal fusion running at an expected size of 10920 bp.

2. Gel-2 1. Based on the agarose gel image, Lane No.l represents plasmid FMab21 HC-N- terminal fusion running at the expected size of 10827 bp.

2. Based on the agarose gel image, Lane No.2 represents plasmid FMab22 LC-N- terminal fusion running at an expected size of 10116 bp.

3. Based on the agarose gel image, Lane No.3 represents plasmid FMab24 LC-N- terminal fusion running at an expected size of 10116 bp.

4. Based on the agarose gel image, Lane No.4 represents plasmid FMab29 HC-C- terminal fusion running an expected size of 10920 bp.

5. Based on the agarose gel image, Lane No.5 represents plasmid FMab30 LC-N- terminal fusion running at an expected size of 10209 bp.

6. Based on the agarose gel image, Lane No.6 represents plasmid FMab32 LC-N- terminal fusion running at an expected size of 10209 bp.

7. Lane no. 7: 1 Kb DNA ladder (molecular weight marker) with top band corresponds to 10 kb in size.

8. Based on the agarose gel image, Lane No.8 represents plasmid Anti-CD 20 HC running at an expected size of 10332 bp.

9. Based on the agarose gel image, Lane No.9 represents plasmid Anti-CD 20 LC running at an expected size of 9621 bp.

10. Based on the agarose gel image, Lane No.10 represents plasmid FMab27 HC-C- terminal fusion running at an expected size of 10788 bp.

11. Based on the agarose gel image, Lane No.ll represents plasmid FMab26 LC-C- terminal fusion running at an expected size of 10077 bp.

12. Based on the agarose gel image, Lane No.12 represents plasmid FMab26 LC-N- terminal fusion running at an expected size of 10077 bp.

Example 2: Confirmation of integrity of the expressed fusion proteins by Non- reducing and reducing using SDS-PAGE

The transiently expressed fusion proteins, FmAb 21, 22, 26, 28 and 32 were purified using Protein A affinity chromatography. 1 μg equivalent of each of the above mentioned proteins are loaded onto a 4-20% non-reducing and reducing SDS-PAGE in 10 μΐ volume. The proteins bands of all the fusion proteins FMab 21, 22, 26, 28 and 32 have found to in correct location in the gel (Figure 2 and 3). This indicates that protein expressed in intact and correct.

The proteins bands of all the fusion proteins FMab 23, 24, 25, 27 29, 30 and 31 have found to in correct location in the gel (Figure 4). This indicates that protein expressed in intact and correct.

The proteins bands of all the fusion proteins FMab 23, 24, 25, 27 29, 30 and 31 have found to in correct location in the gel (Figure 5). This indicates that protein expressed in intact and correct.

Legend: Figure 2.

Lane 1 and 8: No protein loaded on the gel (blank)

Lane 2: FMab21 (Anti-CD20 mAB+ PD-1 LBD fusion at HC C-terminal). The molecular weight is 179.68 kDa and protein band observed at expected position on the gel.

Lane 3: FMab22, (Anti-CD20 mAB+ PD-1 LBD fusion at LC C-terminal). The molecular weight is 179.68 kDa and protein band observed at expected position on the gel.

Lane 4, 7 and 9: Protein molecular weight marker

Lane 5: FMab26 (Anti-CD20 mAB + TGF RII ECD fusion at LC C-terminal). The molecular weight is 177.20 kDa and protein band observed at expected position on the gel.

Lane 6: FMab28 (Anti-CD20 mAB + TGF RII ECD fusion at LC N-terminal). The molecular weight is 177.20 kDa and protein band observed at expected position on the gel.

Lane 10: Anti-CD20 mAB control. The molecular weight is -150 kDa and protein band observed at expected position on the gel. Figure 3.

Lane 1: No protein loaded on the gel (blank)

Lane 2: FMab21 (Anti-CD20 mAB+ PD-1 LBD fusion at HC C-terminal). The molecular weight HC fusion is -80 kDa & that of LC is 25 kDA. The protein bands observed are at expected position on the gel.

Lane 3:FMab22, (Anti-CD20 mAB+ PD-1 LBD fusion at LC C-terminal). The HC the LC bands at same place because LC is fused to a partner protein. The bands observed are at expected position on the gel.

Lane 4, 7 and 9: Protein molecular weight marker

Lane 5: FMab26 (Anti-CD20 mAB + TGF RII ECD fusion at LC C-terminal). The HC the LC bands at same place because LC is fused to a partner protein. The bands observed are at expected position on the gel.

Lane 6: FMab28 (Anti-CD20 mAB + TGF RII ECD fusion at LC N-terminal). The HC the LC bands at same place because LC is fused to a partner protein. The bands observed are at expected position on the gel.

Lane 8: FMab32 (Anti-CD20 mAB + TIM3 ECD fusion at LC N-terminal). The HC the LC bands at same place because LC is fused to a partner protein. The bands observed are at expected position on the gel.

Lane 9:. The molecular weight is HC is -50 kDa and that of LC is -25 kDa. The protein bands observed at expected position on the gel.

Lane 10: Anti-CD20 mAB control

Legend: Figure 4. Lane 1: FMab23 (Anti-CD20 mAB+ PD-1 LBD fusion at HC N-terminal). The molecular weight is 179.68 kDa and protein band observed at expected position on the gel.

Lane 2:FMab24, (Anti-CD20 mAB+ PD-1 LBD fusion at LC N-terminal). The molecular weight is 179.68 kDa and protein band observed at expected position on the gel. Lane 3: FMab25 (Anti-CD20 mAB + TGF RII ECD fusion at HC C-terminal). The molecular weight is 177.20 kDa and protein band observed at expected position on the gel.

Lane 4: FMab27 (Anti-CD20 mAB + TGF RII ECD fusion at HC N-terminal). The molecular weight is 177.20 kDa and protein band observed at expected position on the gel.

Lane 5: FMab29 (Anti-CD20 mAB+ TIM3 ECD fusion at HC C-terminal). The molecular weight is 186.26 kDa and protein band observed at expected position on the gel.

Lane 6: FMab30 (Anti-CD20 mAB+ TIM3 ECD fusion at LC C-terminal). The molecular weight is 186.26 kDa and protein band observed at expected position on the gel.

Lane 7: FMab30 (Anti-CD20 mAB+ TIM3 ECD fusion at HC N-terminal). The molecular weight is 186.26 kDa and protein band observed at expected position on the gel.

Lane 8: Protein molecular weight marker

Lane 9: Anti-CD20 mAB control. The molecular weight is -150 kDa and protein band observed at expected position on the gel. Legend: Figure 5.

Lane 1: FMab23 (Anti-CD20 mAB+ PD-1 LBD fusion at HC N-terminal). The molecular weight HC fusion is -80 kDa & that of LC is 25 kDA. The protein band observed at expected position on the gel.

Lane 2: FMab24, (Anti-CD20 mAB+ PD-1 LBD fusion at LC N-terminal). The HC the LC bands at same place because LC is fused to a partner protein. The bands observed are at expected position on the gel.

Lane 3: FMab25 (Anti-CD20 mAB + TGF RII ECD fusion at HC C-terminal). The molecular weight HC fusion is -78 kDa & that of LC is 25 kDA. The protein bands observed are at expected position on the gel. Lane 4: FMab27 (Anti-CD20 mAB + TGF RII ECD fusion at HC N-terminal). The molecular weight HC fusion is -78 kDa & that of LC is 25 kDA. The protein bands observed are at expected position on the gel.

Lane 5: FMab29 (Anti-CD20 mAB+ TIM3 ECD fusion at HC C-terminal). The molecular weight HC fusion is -86 kDa & that of LC is 25 kDA. The protein bands observed are at expected position on the gel.

Lane 6: FMab30 (Anti-CD20 mAB+ TIM3 ECD fusion at LC C-terminal). The HC the LC bands at same place because LC is fused to a partner protein. The bands observed are at expected position on the gel.

Lane 7: FMab30 (Anti-CD20 mAB+ TIM3 ECD fusion at HC N-terminal). The molecular weight HC fusion is -86 kDa & that of LC is 25 kDA. The protein bands observed are at expected position on the gel.

Lane 8: Protein molecular weight marker

Lane 9: Anti-CD20 mAB control. The molecular weight is -50 kDa HC & 25 kDA LC. The protein bands observed are at expected position on the gel.

The expression constructs developed were transfected in the following combination into CHO cells (Table 2) to produce fusion proteins. The expression yields (titer) obtained for each constructs are mentioned in the last column in Table 2.

Sr. Fusion Titer in

Anti CD -20 construct details Fusion location

No Mab codes mg /L

Fusion at HC C-

1 FMab 21 Anti-CD20 mAB+ PD-1 LBD 270

terminal

Fusion at LC C-

2 FMab 22 Anti-CD20 mAB+ PD-1 LBD 160

terminal

Fusion at HC N-

3 FMab 23 Anti-CD20 mAB+ PD-1 LBD 70

terminal

Fusion at LC N-

4 FMab 24 Anti-CD20 mAB+ PD-1 LBD 70

terminal

5 FMab 25 Anti-CD20 mAB+ TGFpRII Fusion at HC C- 250 ECD terminal

Anti-CD20 mAB+ TGF RII Fusion at LC C-

6 FMab 26 260

ECD terminal

Anti-CD20 mAB+ TGF RII Fusion at HC N-

7 FMab 27 200

ECD terminal

Anti-CD20 mAB+ TGF RII Fusion at LC N-

8 FMab 28 260

ECD terminal

Fusion at HC C-

9 FMab 29 Anti-CD20 mAB+ TIM3 ECD 60

terminal

Fusion at LC C-

10 FMab 30 Anti-CD20 mAB+ TIM3 ECD 90

terminal

Fusion at HC N-

11 FMab 31 Anti-CD20 mAB+ TIM3 ECD 100

terminal

Fusion at LC N-

12 FMab 32 Anti-CD20 mAB+ TIM3 ECD 140

terminal

Anti CD 20

13 Anti-CD 20 HC and LC Heavy chain 250

control

Table-2: Details of the Transient expression constructs and productivity

The observations are summarized below:

• Expression levels of FMab 21, 25, 26, 27 and 28 were more than 200 mg/L and similar to Anti-CD20 control.

· Expression levels of FMab 22, 31 and 32 were more than 100 mg/L and showing

<50% lower when compared to Anti-CD20 control.

• Expression levels of FMab 23, 24, 29 and 30 were less than 100 mg/L and may be difficult to difficult to express in large quantity

Example 3: Ability to bind to its targets in two different ELISAs Binding ELISAs-Procedure:

The fusion Mab was tested for its ability to bind to its targets in two different ELISAs: 1) TGF -target binding ELISA and

2) Galectin 9-target binding ELISA

For target binding ELISAs, the targets (TGF and Galectin 9) were coated onto NUNC maxisorb plates overnight at 4°C. The plates were washed and then blocked with superblock at room temperature for 2 hours. Different dilutions of the fusion Mab or positive or the negative control antibody was added to the plate. The plate was incubated at room temperature for 1 hour. Binding of the fusion Mab was detected by the addition of a Biotinylated anti- TGF or anti-TIM3 secondary antibody, followed by a 1 hour incubation with peroxidase-conjugated streptavidin at room temperature. TMB substrate solution was added and the reaction stopped with IN H2SO4. The absorbance was measured at 450nm on a BioTek Synergy H4 Hybrid reader.

Results:

The binding of the anti-CD20 HC C-TGF RII (Fmab 25), anti-CD20 LC C-TGF RII (Fmab 26), anti-CD20 HC N-TGF RII (Fmab 27), and anti-CD20 LC N-TGF RII (Fmab 28) (Figure 6) was comparable with TGF RII FC. Suggests that the immunomodulatory arm (TGF RII) of the fusion construct is binding to its ligand (TGFP).

The binding of anti-CD20 HC C-TIM3 (Fmab 29), anti-CD20 LC C- TIM3 (Fmab 30), anti-CD20 HC N- TIM3 (Fmab 31), and anti-CD20 LC N- TIM3 (Fmab 32) (Figure 7) to its ligand Galectin 9 was evaluated. Fmab's 30, 31 and 32 showed binding at the starting concentration of 10 μg/mL, whereas Fmab 29 showed no binding activity. Suggests that the immunomodulatory arm (TIM3 ECD) of the fusion construct is binding to its ligand (Galectin 9).

Example 4: Antibody-dependent cytotoxicity Activity

Procedure: The primary mechanism of cytotoxicity of anti-CD20 mab is ADCC. The functional activity of ant-CD20 moiety of all the fusion mabs (21-32) were assessed using this assay.

WIL2S cells were grown in flasks until they reached 70-80% confluency. The cells were trypsinized, harvested and plated into 96-well plates. The cells were labelled with different dilutions of the Fmab's 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 or control IgG at 2-8°C for 30 minutes. The labelled cells were co-incubated with ADCC Bioassay Effector cells at 37°C, 5% C(¾ for 24 hours. The assay plates were equilibrated to ambient temperature (22-25 °C) on the bench for 15 minutes. Bio-Glo™ Luciferase Assay reagent was added and incubated at ambient temperature for 10 minutes. Luminescence was measured using plate reader.

Results:

The ADCC of the anti-CD20 HC C-PDL1 (Fmab 21), anti-CD20 LC C- PDL1 (Fmab 22), anti-CD20 HC N- PDL1 (Fmab 23), anti-CD20 LC N- PDL1 (Fmab 24), anti-CD20 HC C-TGF PJI (Fmab 25), anti-CD20 LC C-TGF RII (Fmab 26), anti-CD20 HC N- TGF PJI (Fmab 27), anti-CD20 LC N-TGF RII (Fmab 28), anti-CD20 HC C-TIM3 (Fmab 29), anti-CD20 LC C- TIM3 (Fmab 30), anti-CD20 HC N- TIM3 (Fmab 31), and anti-CD20 LC N- TIM3 (Fmab 32) (Figure 8 and 9) was assessed using CD20 overexpressing WIL2S cells. Except Fmabs 24, 28 and 32, all the other Fambs 21, 22, 23, 25, 26, 27, 29, 30 and 31 showed ADCC. Suggests that the anti-CD20 mab is functionally active in these fusions.

The sequences of fusion protein are mentioned below.

1. Fmab 21:

Amino acid sequence of Anti-CD20 Heavy chain C-terminal PD-1 fusion protein (SEQ ID No. 7)

QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNM HWVKQTPG GLEWIGAIYPGNGDTSYNQKF KGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVV VDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KAL PAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM H EALH NHYTQKSLSLSPGGGGGSGGGGSGGGGS PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPE DRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRA EVPTAHPSPSPRPAGQFQTLV

Amino acid sequence of Anti-CD20 light chain (SEQ ID No. 1)

QIVLSQSPAI LSASPGEKVTMTC ASSSVSYI HWFQQKPGSSPKPWIYATSNLASGVPV FSGSGSGT SYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEI KRTVAAPSVFI FPPSDEQLKSGTASVVCLLN N FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPV TKSFNRGEC

2. Fmab22:

Amino acid sequence of Anti-CD20 heavy chain (SEQ ID No. 2)

QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNM HWVKQTPGRGLEWIGAIYPGNGDTSYNQKF KGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVV VDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KAL PAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM H EALH NHYTQKSLSLSPG

Amino acid sequence of Anti-CD20 light chain C-terminal PD-1 fusion protein: (SEQ ID NO. 8)

QIVLSQSPAI LSASPGEKVTMTCRASSSVSYI HWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGT SYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEI KRTVAAPSVFI FPPSDEQLKSGTASVVCLLN N FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPV TKSFNRGECGGGGSGGGGSGGGGSPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTS ESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLC GAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV 3. Fmab23:

Amino acid sequence of Anti-CD20 Heavy chain N-terminal PD-1 fusion protein: (SEQ ID NO. 9)

PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPE DRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRA

EVPTAHPSPSPRPAGQFQTLVGGGGSGGGGSGGGGSQVQLQQPGAELVKPGASVKMSCKASGY TFTSYNM HWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAV YYCARSTYYGGDWYFNVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVH NAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVM H E ALH N H YTQKSLSLSPG Amino acid sequence of Anti-CD20 light chain (SEQ ID No. 1)

QIVLSQSPAILSASPGEKVTMTCRASSSVSYI HWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGT SYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEI KRTVAAPSVFI FPPSDEQLKSGTASVVCLLN N FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPV TKSFNRGEC

4. Fmab24:

Amino acid sequence of Anti-CD20 heavy chain (SEQ ID No. 2)

Amino acid sequence of Anti-CD20 light chain N-terminal PD-1 fusion protein: (SEQ ID NO. 10)

EVPTAHPSPSPRPAGQFQTLVGGGGSGGGGSGGGGSQIVLSQSPAI LSASPGEKVTMTCRASSSVS YIHWFQQKPGSSPKPWIYATSN LASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSN PPTF GGGTKLEI KRTVAAPSVFI FPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFN RGEC

5. Fmab25:

Amino acid sequence of Anti-CD20 Heavy chain C-terminal TGF RII ECD fusion protein: (SEQ ID NO. 11)

QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNM HWVKQTPGRGLEWIGAIYPGNGDTSYNQKF KGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVV VDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KAL PAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM H EALH NHYTQKSLSLSPGGGGGSGGGGSGGGGS TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYN TSNPD Amino acid sequence of Anti-CD20 light chain (SEQ ID No. 1)

QIVLSQSPAI LSASPGEKVTMTCRASSSVSYI HWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGT SYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEI KRTVAAPSVFI FPPSDEQLKSGTASVVCLLN N FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPV TKSFNRGEC

6. FmAb 26:

Amino acid sequence of Anti-CD20 heavy chain (SEQ ID No. 2) QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNM HWVKQTPGRGLEWIGAIYPGNGDTSYNQKF KGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVV VDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM H EALH NHYTQKSLSLSPG

Amino acid sequence of Anti-CD20 light chain C-terminal TGF RII ECD fusion protein: (SEQ ID NO. 12)

QIVLSQSPAI LSASPGEKVTMTCRASSSVSYI HWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGT SYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEI KRTVAAPSVFI FPPSDEQLKSGTASVVCLLN N FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPV TKSFNRGECGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCD NQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPD

7. Fmab27:

Amino acid sequence of Anti-CD20 heavy chain N-terminal TGF RII ECD fusion protein: (SEQ ID NO. 13)

TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYN

TSNPDGGGGSGGGGSGGGGSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYN MHWVKQTPG RGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFN VWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSN KALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALH NHYTQKSLS LSPG

Amino acid sequence of Anti-CD20 light chain (SEQ ID No. 1)

8. FmAb 28:

Amino acid sequence of Anti-CD20 heavy chain (SEQ ID NO. 2)

Amino acid sequence of Anti-CD20 light chain N-terminal TGF RII ECD fusion protein (SEQ ID NO. 14)

TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYN TSNPDGGGGSGGGGSGGGGSQIVLSQSPAI LSASPGEKVTMTCRASSSVSYI HWFQQKPGSSPKP WIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSN PPTFGGGTKLEI KRTVAAPS VFI FPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYE KH KVYAC EVTHQG LSS PVTKS F N RG EC

9. FmAb 29:

Amino acid sequence of Anti-CD20 heavy chain C-terminal TIM3 ECD fusion protein: (SEQ ID NO. 15)

QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNM HWVKQTPGRGLEWIGAIYPGNGDTSYNQKF KGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVV VDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM H EALH NHYTQKSLSLSPGGGGGSGGGGSGGGGS SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNWLRTDERDVNYWTSRYWL NGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPR MLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIG

Amino acid sequence of Anti-CD20 light chain (SEQ ID NO. 1)

10. FmAb 30:

Amino acid sequence of Anti-CD20 heavy chain: (SEQ ID NO. 2)

QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNM HWVKQTPGRGLEWIGAIYPGNGDTSYNQKF KGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVV VDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM H EALH NHYTQKSLSLSPG

Amino acid sequence of Anti-CD20 light chain C-terminal TIM3 ECD fusion protein: (SEQ ID NO. 16)

QIVLSQSPAI LSASPGEKVTMTCRASSSVSYI HWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGT SYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEI KRTVAAPSVFI FPPSDEQLKSGTASVVCLLN N FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPV TKS F N RG ECGGGGSGGGGSGGGGSSEVEYRAEVGQNAYLPCFYTPAAPG N LVPVCWG KG ACP V FECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNL KLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDL RDSGATIRIG

11. FmAb 31:

Amino acid sequence of Anti-CD20 heavy chain N-terminal TIM3 ECD fusion protein: (SEQ ID NO. 17) SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNWLRTDERDVNYWTSRYWL NGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPR MLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIGGGGGSGGGGSGGG

GSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYN M HWVKQTPGRGLEWIGAIYPGNGDTSYNQ KFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTC VVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN K ALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM H EALHNHYTQKSLSLSPG Amino acid sequence of Anti-CD20 light chain: (SEQ ID NO. 1)

12. FmAb 32:

Amino acid sequence of Anti-CD20 heavy chain: (SEQ ID No. 2) QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNM HWVKQTPGRGLEWIGAIYPGNGDTSYNQKF KGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVV VDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KAL PAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM H EALH NHYTQKSLSLSPG

Amino acid sequence of Anti-CD20 light chain N-terminal TIM3 ECD fusion protein: (SEQ ID No. 18)

SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNWLRTDERDVNYWTSRYWL NGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPR MLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIGGGGGSGGGGSGGG

GSQIVLSQSPAILSASPGEKVTMTCRASSSVSYI HWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGS GTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLL N N FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSS PVTKSFNRGEC

Note:

1. Bold font: Immunomodulatory ligand

2. Underlined: Linker

3. Black: mAB sequences Nucleotide sequence of Anti-CD20 fusion constructs: The nucleotide sequences were codon-optimized for the expression in CHO cells. Synthetic genes were obtained from Geneart. The construct were assembled in the mammalian expression vector suitable for transient expression in CHO cells.

Nucleic acid sequences of the individual ant-CD 20 fusion expression constructs used for co-transfection to generate transiently expressed material are given below.

13. FMab 21:

Nucleotide sequence of Anti-CD20 Heavy chain C-terminal PD-1 fusion protein: (SEQ ID NO. 21)

ATG G AC ATG AG AGTG CCTG CTC AG CTG CTG G G CCTG CTG CTG CTGTG G CTG AG AG G CG CC AG

ATGCCAGGTGCAGCTGCAGCAGCCTGGCGCCGAACTGGTCAAGCCAGGCGCCTCCGTGAAGAT GTCCTGCAAGGCCTCCGGCTACACCTTCACCAGCTACAACATGCACTGGGTCAAGCAGACCCCC GGCAGAGGCCTGGAATGGATCGGCGCCATCTACCCCGGCAACGGCGACACCTCCTACAACCAG A AGTTCA AG GG C AAG G CC ACCCTG ACCG CCG AC AAGTCCTCCTCC ACCG CCT AC ATG C AG CTGT CCTCCCTGACCTCCGAGGACTCCGCCGTGTACTACTGCGCCCGGTCCACCTACTACGGCGGCGA CTGGTACTTCAACGTGTGGGGCGCTGGCACCACCGTGACCGTGTCCTCTGCCTCCACCAAGGGC CCCTCCGTGTTCCCTCTGGCCCCCTCCAGCAAGTCCACCTCTGGCGGCACCGCTGCCCTGGGCTG CCTGGTCAAGGACTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACCAGC GGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCAGCGTCGTGAC CGTGCCTTCCAGCTCCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACA CCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCCTGCCC TGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGA TGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAG TGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGG AACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAA CGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCAT CTCCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACCCTGCCCCCTAGCCGGGACGA GCTGACCAAGAACCAGGTGTCCCTGACCTGCCTCGTGAAGGGCTTCTACCCCTCCGATATCGCC GTGGAATGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGAC TCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCA ACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCC CTGAGCCCTGGCGGAGGCGGAGGAAGTGGTGGCGGAGGTTCTGGCGGCGGAGGATCCCCTGG CTGGTTCCTGGACTCCCCCGACCGGCCTTGGAACCCCCCAACCTTCTCTCCCGCCCTGCTGGTGG TCACCGAGGGCGACAACGCCACCTTCACCTGTTCCTTCAGCAACACCTCCGAGTCCTTCGTGCTG AACTGGTACAGAATGTCCCCCAGCAACCAGACCGACAAGCTGGCCGCCTTCCCCGAGGACAGAT CCCAGCCTGGCCAGGACTGCCGGTTCAGAGTGACCCAGCTGCCCAACGGCCGGGACTTCCACAT GTCCGTCGTGCGGGCCAGACGGAACGACTCCGGCACCTACCTGTGCGGCGCCATCTCTCTGGCC CCCAAGGCCCAGATCAAAGAGTCCCTGCGGGCCGAGCTGAGAGTGACCGAGAGAAGGGCCGA GGTGCCCACCGCCCACCCTAGCCCATCTCCAAGACCTGCCGGCCAGTTCCAGACCCTGGTGTAGT GATGA

14. FMab 22:

Nucleotide sequence of Anti-CD20 light chain C-terminal PD-1 fusion protein: (SEQ ID NO. 22)

ATGCCAGATCGTGCTGTCCCAGTCCCCCGCCATCCTGTCTGCCAGCCCTGGCGAGAAAGTGACA ATGACCTGCCGGGCCTCCTCCTCCGTGTCCTACATCCACTGGTTCCAGCAGAAGCCCGGCTCCAG CCCCAAGCCCTGGATCTACGCCACCTCCAACCTGGCCTCTGGCGTGCCCGTGCGGTTCTCCGGCT CTGGCTCTGGCACCTCCTACTCCCTGACCATCTCCCGGGTGGAAGCCGAGGACGCCGCCACCTA CTACTGCCAGCAGTGGACCTCCAACCCCCCCACCTTCGGCGGAGGCACCAAGCTGGAAATCAAG CGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCCTCCGACGAGCAGCTGAAGTCCGGCAC CGCCTCCGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTG GACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACAGC ACCTACAGCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACG CCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACCGGGGCGAATG CGGCGGAGGCGGATCTGGTGGCGGAGGTTCTGGCGGCGGAGGATCCCCTGGCTGGTTCCTGG ACTCCCCCGACCGGCCTTGGAACCCCCCAACCTTCTCTCCCGCCCTGCTGGTGGTCACCGAGGGC GACAACGCCACCTTCACCTGTTCCTTCAGCAACACCTCCGAGTCCTTCGTGCTGAACTGGTACAG AATGTCCCCCAGCAACCAGACCGACAAGCTGGCCGCCTTCCCCGAGGACAGATCCCAGCCTGGC CAGGACTGCCGGTTCAGAGTGACCCAGCTGCCCAACGGCCGGGACTTCCACATGTCCGTCGTGC GGGCCAGACGGAACGACTCCGGCACCTACCTGTGCGGCGCCATCTCTCTGGCCCCCAAGGCCCA GATCAAAGAGTCCCTGCGGGCCGAGCTGAGAGTGACCGAGAGAAGGGCCGAGGTGCCCACCG CCCACCCTAGCCCATCTCCAAGACCTGCCGGCCAGTTCCAGACCCTGGTGTAGTGATGATGA 15. FmAb 23:

Nucleotide sequence of Anti-CD20 Heavy chain N-terminal PD-1 fusion protein (SEQ ID NO. 23)

ATGGAATTCGCGGCCGCGCCACCATGGACATGAGAGTGCCTGCTCAGCTGCTGGGCCTGCTGC

TGCTGTGGCTGAGAGGCGCCAGATGCCCCGGCTGGTTCCTGGACTCCCCTGACCGGCCTTGGAA CCCCCCTACCTTCAGCCCTGCCCTGCTGGTGGTCACCGAGGGCGACAACGCCACCTTCACCTGTT CCTTCAGCAACACCTCCGAGTCCTTCGTGCTGAACTGGTACAGAATGTCCCCCAGCAACCAGACC GACAAGCTGGCCGCCTTCCCCGAGGACAGATCCCAGCCTGGCCAGGACTGCCGGTTCAGAGTG ACCCAGCTGCCCAACGGCCGGGACTTCCACATGTCCGTCGTGCGGGCCAGACGGAACGACTCC GGCACCTACCTGTGCGGCGCCATCTCTCTGGCCCCCAAGGCCCAGATCAAAGAGTCCCTGCGGG CCGAGCTGAGAGTGACCGAGAGAAGGGCCGAGGTGCCCACCGCCCACCCTAGCCCATCTCCAA GACCTGCCGGCCAGTTCCAGACCCTCGTGGGAGGCGGAGGATCTGGCGGAGGTGGTAGTGGC GGAGGCGGATCCCAGGTGCAGCTGCAGCAGCCTGGCGCCGAACTGGTCAAGCCAGGCGCCTCC GTGAAGATGTCCTGCAAGGCCTCCGGCTACACCTTCACCAGCTACAACATGCACTGGGTCAAGC AGACCCCCGGCAGAGGCCTGGAATGGATCGGCGCCATCTACCCCGGCAACGGCGACACCTCCT ACAACCAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGTCCTCCTCCACCGCCTACAT GCAGCTGTCCAGCCTGACCTCCGAGGACTCCGCCGTGTACTACTGCGCCCGGTCCACCTACTAC GGCGGCGACTGGTACTTCAACGTGTGGGGCGCTGGCACCACCGTGACCGTGTCCTCTGCCTCCA CCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCCTCCAGCAAGTCCACCTCTGGCGGCACCGCTGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCC TGACCAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCC GTCGTGACCGTGCCTTCCAGCTCCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGC CCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCC CCCCTGCCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGG ACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGA CCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCC CAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGA CTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGA AAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACCCTGCCCCCTAGC CGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTCGTGAAGGGCTTCTACCCCTCCG ATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGT GCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAG CAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGT CCCTGTCCCTGAGCCCCGGCTGATGATGA

16. FMab 24:

Nucleotide sequence of Anti-CD20 light chain N-terminal PD-1 fusion protein (SEQ ID NO. 24)

ATGGAATTCGCGGCCGCGCCACCATGGACATGAGAGTGCCTGCTCAGCTGCTGGGCCTGCTGC TGCTGTGGCTGAGAGGCGCCAGATGCCCCGGCTGGTTCCTGGACTCCCCTGACCGGCCTTGGAA CCCCCCTACCTTCAGCCCTGCCCTGCTGGTGGTCACCGAGGGCGACAACGCCACCTTCACCTGTT CCTTCAGCAACACCTCCGAGTCCTTCGTGCTGAACTGGTACAGAATGTCCCCCAGCAACCAGACC GACAAGCTGGCCGCCTTCCCCGAGGACAGATCCCAGCCTGGCCAGGACTGCCGGTTCAGAGTG ACCCAGCTGCCCAACGGCCGGGACTTCCACATGTCCGTCGTGCGGGCCAGACGGAACGACTCC GGCACCTACCTGTGCGGCGCCATCTCTCTGGCCCCCAAGGCCCAGATCAAAGAGTCCCTGCGGG CCGAGCTGAGAGTGACCGAGAGAAGGGCCGAGGTGCCCACCGCCCACCCTAGCCCATCTCCAA GACCTGCCGGCCAGTTCCAGACCCTCGTGGGAGGCGGAGGATCTGGCGGAGGTGGTAGTGGC GGAGGCGGATCCCAGATCGTGCTGTCCCAGTCCCCCGCCATCCTGTCTGCCAGCCCTGGCGAGA AAGTGACAATGACCTGCCGGGCCTCCTCCTCCGTGTCCTACATCCACTGGTTCCAGCAGAAGCCC GGCTCCAGCCCCAAGCCCTGGATCTACGCCACCTCCAACCTGGCCTCTGGCGTGCCCGTGCGGT TCTCCGGCTCTGGCTCTGGCACCTCCTACAGCCTGACCATCTCCCGGGTGGAAGCCGAGGACGC CGCCACCTACTACTGCCAGCAGTGGACCTCCAACCCCCCCACCTTCGGCGGAGGCACCAAGCTG GAAATCAAGCGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCCTCCGACGAGCAGCTGA AGTCCGGCACCGCCTCCGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCA GTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTCACCGAGCAGGACTC CAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCAC AAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACC GGGGCGAGTGCTGATGATGA

17. FmAb 25:

Nucleotide sequence of Anti-CD20 Heavy chain C-terminal TGFpRII ECD fusion protein (SEQ ID NO. 25)

ATG G AC ATG AG AGTG CCTG CTC AG CTG CTG G G CCTG CTG CTG CTGTG G CTG AG AG G CG CC AG ATGCCAGGTGCAGCTGCAGCAGCCTGGCGCCGAACTGGTCAAGCCAGGCGCCTCCGTGAAGAT GTCCTGCAAGGCCTCCGGCTACACCTTCACCAGCTACAACATGCACTGGGTCAAGCAGACCCCC GGCAGAGGCCTGGAATGGATCGGCGCCATCTACCCCGGCAACGGCGACACCTCCTACAACCAG A AGTTCA AG GG C AAG G CC ACCCTG ACCG CCG AC AAGTCCTCCTCC ACCG CCT AC ATG C AG CTGT CCTCCCTGACCTCCGAGGACTCCGCCGTGTACTACTGCGCCCGGTCCACCTACTACGGCGGCGA CTGGTACTTCAACGTGTGGGGCGCTGGCACCACCGTGACCGTGTCCTCTGCCTCCACCAAGGGC CCCTCCGTGTTCCCTCTGGCCCCCTCCAGCAAGTCCACCTCTGGCGGCACCGCTGCCCTGGGCTG CCTGGTCAAGGACTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACCAGC GGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCAGCGTCGTGAC CGTGCCTTCCAGCTCCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACA CCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCCTGCCC TGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGA TGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAG TGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGG AACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAA CGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCAT CTCCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACCCTGCCCCCTAGCCGGGACGA GCTGACCAAGAACCAGGTGTCCCTGACCTGCCTCGTGAAGGGCTTCTACCCCTCCGATATCGCC GTGGAATGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGAC TCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCA ACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCC CTGAGCCCTGGCGGAGGCGGAGGAAGTGGTGGCGGAGGTTCTGGCGGCGGAGGATCCACCAT CCCCCCACACGTGCAGAAATCCGTGAACAACGACATGATCGTGACCGACAACAACGGCGCCGT GAAGTTCCCCCAGCTGTGCAAGTTCTGCGACGTGCGGTTCTCTACCTGCGACAACCAGAAATCCT GCATGTCCAACTGCTCCATCACCTCCATCTGCGAGAAGCCCCAGGAAGTGTGCGTCGCCGTCTG GCGGAAGAACGACGAGAACATCACCCTGGAAACCGTGTGCCACGACCCCAAGCTGCCCTACCA CGACTTCATCCTGGAAGATGCCGCCTCCCCCAAGTGCATCATGAAGGAAAAGAAGAAGCCCGG CGAGACATTCTTCATGTGCTCTTGCTCCTCCGACGAGTGCAACGACAACATCATCTTCTCCGAAG AGTACAACACCTCCAACCCCGACTGATGATGA 18. FMab 26:

Nucleotide sequence of Anti-CD20 light chain C-terminal TGFpRII ECD fusion protein (SEQ ID NO. 26)

ATGCCAGATCGTGCTGTCCCAGTCCCCCGCCATCCTGTCTGCCAGCCCTGGCGAGAAAGTGACA ATGACCTGCCGGGCCTCCTCCTCCGTGTCCTACATCCACTGGTTCCAGCAGAAGCCCGGCTCCAG CCCCAAGCCCTGGATCTACGCCACCTCCAACCTGGCCTCTGGCGTGCCCGTGCGGTTCTCCGGCT CTGGCTCTGGCACCTCCTACTCCCTGACCATCTCCCGGGTGGAAGCCGAGGACGCCGCCACCTA CTACTGCCAGCAGTGGACCTCCAACCCCCCCACCTTCGGCGGAGGCACCAAGCTGGAAATCAAG CGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCCTCCGACGAGCAGCTGAAGTCCGGCAC CGCCTCCGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTG GACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACAGC ACCTACAGCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACG CCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACCGGGGCGAATG CGGCGGAGGCGGATCTGGTGGCGGAGGTTCTGGCGGCGGAGGATCCACCATCCCCCCACACGT GCAGAAATCCGTGAACAACGACATGATCGTGACCGACAACAACGGCGCCGTGAAGTTCCCCCA GCTGTGCAAGTTCTGCGACGTGCGGTTCTCTACCTGCGACAACCAGAAATCCTGCATGTCCAACT GCTCCATCACCTCCATCTGCGAGAAGCCCCAGGAAGTGTGCGTCGCCGTCTGGCGGAAGAACG ACGAGAACATCACCCTGGAAACCGTGTGCCACGACCCCAAGCTGCCCTACCACGACTTCATCCT GGAAGATGCCGCCTCCCCCAAGTGCATCATGAAGGAAAAGAAGAAGCCCGGCGAGACATTCTT CATGTGCTCTTGCTCCTCCGACGAGTGCAACGACAACATCATCTTCTCCGAAGAGTACAACACCT CCAACCCCGACTGATGATGA

19. FMab 27:

Nucleotide sequence of Anti-CD20 heavy chain N-terminal TGFpRII ECD fusion protein (SEQ ID NO. 27)

ATGCACCATCCCCCCACACGTGCAGAAATCCGTGAACAACGACATGATCGTGACCGACAACAAC GGCGCCGTGAAGTTCCCCCAGCTGTGCAAGTTCTGCGACGTGCGGTTCTCTACCTGCGACAACC AGAAATCCTGCATGTCCAACTGCTCCATCACCTCCATCTGCGAGAAGCCCCAGGAAGTGTGCGT CGCCGTCTGGCGGAAGAACGACGAGAACATCACCCTGGAAACCGTGTGCCACGACCCCAAGCT GCCCTACCACGACTTCATCCTGGAAGATGCCGCCTCCCCCAAGTGCATCATGAAGGAAAAGAAG AAGCCCGGCGAGACATTCTTCATGTGCAGCTGCTCCTCCGACGAGTGCAACGACAACATCATCTT CTCCGAAGAGTACAACACCTCCAACCCCGACGGCGGAGGCGGATCTGGTGGCGGAGGTTCTGG CGGCGGAGGATCCCAGGTGCAGCTGCAGCAGCCTGGCGCCGAACTGGTCAAGCCAGGCGCCTC CGTG A AG ATGTCCTG C A AG G CCTCCG G CTAC ACCTTC ACC AG CT AC AAC ATG C ACTG G GTC A AG CAGACCCCCGGCAGAGGCCTGGAATGGATCGGCGCCATCTACCCCGGCAACGGCGACACCTCC TACAACCAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGTCCTCCTCCACCGCCTACA TGCAGCTGTCCAGCCTGACCTCCGAGGACTCCGCCGTGTACTACTGCGCCCGGTCCACCTACTAC GGCGGCGACTGGTACTTCAACGTGTGGGGCGCTGGCACCACCGTGACCGTGTCCTCTGCCTCCA CCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCCTCCAGCAAGTCCACCTCTGGCGGCACCGCTGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCC TGACCAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCC GTCGTGACCGTGCCTTCCAGCTCCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGC CCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCC CCCCTGCCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGG ACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGA CCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCC CAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGA CTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGA AAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACCCTGCCCCCTAGC CGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTCGTGAAGGGCTTCTACCCCTCCG ATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGT GCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAG CAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGT CCCTGTCCCTGAGCCCCGGCTGATGATGA

20. FMab 28:

Nucleotide sequence of Anti-CD20 light chain N-terminal TGFpRII ECD fusion protein (SEQ ID NO. 28)

ATGCACCATCCCCCCACACGTGCAGAAATCCGTGAACAACGACATGATCGTGACCGACAACAAC GGCGCCGTGAAGTTCCCCCAGCTGTGCAAGTTCTGCGACGTGCGGTTCTCTACCTGCGACAACC AGAAATCCTGCATGTCCAACTGCTCCATCACCTCCATCTGCGAGAAGCCCCAGGAAGTGTGCGT CGCCGTCTGGCGGAAGAACGACGAGAACATCACCCTGGAAACCGTGTGCCACGACCCCAAGCT GCCCTACCACGACTTCATCCTGGAAGATGCCGCCTCCCCCAAGTGCATCATGAAGGAAAAGAAG AAGCCCGGCGAGACATTCTTCATGTGCAGCTGCTCCTCCGACGAGTGCAACGACAACATCATCTT CTCCGAAGAGTACAACACCTCCAACCCCGACGGCGGAGGCGGATCTGGTGGCGGAGGTTCTGG CGGCGGAGGATCCCAGATCGTGCTGTCCCAGTCCCCCGCCATCCTGTCTGCCAGCCCTGGCGAG AAAGTGACAATGACCTGCCGGGCCTCCTCCTCCGTGTCCTACATCCACTGGTTCCAGCAGAAGCC CGGCTCCAGCCCCAAGCCCTGGATCTACGCCACCTCCAACCTGGCCTCTGGCGTGCCCGTGCGG TTCTCCGGCTCTGGCTCTGGCACCTCCTACAGCCTGACCATCTCCCGGGTGGAAGCCGAGGACG CCGCCACCTACTACTGCCAGCAGTGGACCTCCAACCCCCCCACCTTCGGCGGAGGCACCAAGCT GGAAATCAAGCGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCCTCCGACGAGCAGCTG AAGTCCGGCACCGCCTCCGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGC AGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTCACCGAGCAGGACT CCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCA CAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAAC CGGGGCGAGTGCTGATGATGA 21. FMab 29:

Nucleotide sequence of Anti-CD20 heavy chain C-terminal TIM3 ECD fusion protein (SEQ ID NO. 29)

ATG G AC ATG AG AGTG CCTG CTC AG CTG CTG G G CCTG CTG CTG CTGTG G CTG AG AG G CG CC AG ATGCCAGGTGCAGCTGCAGCAGCCTGGCGCCGAACTGGTCAAGCCAGGCGCCTCCGTGAAGAT GTCCTGCAAGGCCTCCGGCTACACCTTCACCAGCTACAACATGCACTGGGTCAAGCAGACCCCC GGCAGAGGCCTGGAATGGATCGGCGCCATCTACCCCGGCAACGGCGACACCTCCTACAACCAG A AGTTCA AG GG C AAG G CC ACCCTG ACCG CCG AC AAGTCCTCCTCC ACCG CCT AC ATG C AG CTGT CCTCCCTGACCTCCGAGGACTCCGCCGTGTACTACTGCGCCCGGTCCACCTACTACGGCGGCGA CTGGTACTTCAACGTGTGGGGCGCTGGCACCACCGTGACCGTGTCCTCTGCCTCCACCAAGGGC CCCTCCGTGTTCCCTCTGGCCCCCTCCAGCAAGTCCACCTCTGGCGGCACCGCTGCCCTGGGCTG CCTGGTCAAGGACTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACCAGC GGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCAGCGTCGTGAC CGTGCCTTCCAGCTCCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACA CCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCCTGCCC TGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGA TGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAG TGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGG AACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAA CGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCAT CTCCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACCCTGCCCCCTAGCCGGGACGA GCTGACCAAGAACCAGGTGTCCCTGACCTGCCTCGTGAAGGGCTTCTACCCCTCCGATATCGCC GTGGAATGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGAC TCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCA ACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCC CTGAGCCCTGGCGGAGGCGGAGGAAGTGGTGGCGGAGGTTCTGGCGGCGGAGGATCCAGCG AGGTGGAATACCGGGCCGAAGTGGGCCAGAACGCCTACCTGCCCTGCTTCTACACCCCTGCCGC CCCTGGCAACCTGGTGCCTGTGTGTTGGGGCAAGGGCGCCTGCCCTGTGTTCGAGTGCGGCAA CGTGGTGCTGCGGACCGACGAGCGGGACGTGAACTACTGGACCTCCCGGTACTGGCTGAACGG CGACTTCCGGAAGGGCGACGTGTCCCTGACAATCGAGAACGTGACCCTGGCCGACTCCGGCATC TACTGCTGCAGAATCCAGATCCCCGGCATCATGAACGACGAGAAGTTCAACCTGAAGCTGGTCA

TCAAGCCCGCCAAAGTGACCCCTGCCCCTACCCGGCAGAGAGACTTCACCGCCGCCTTCCCCCG

GATGCTGACCACCAGAGGCCACGGCCCTGCCGAGACTCAGACCCTGGGCTCCCTGCCCGACATC

AACCTGACCCAGATCTCCACCCTGGCCAACGAGCTGCGGGACTCCCGGCTGGCCAATGACCTGA

GAGACTCCGGCGCCACCATCCGGATCGGCTGATGATGA

22. FMab 30:

Nucleotide sequence of Anti-CD20 light chain C-terminal TIM3 ECD fusion protein (SEQ ID NO. 30)

ATGCCAGATCGTGCTGTCCCAGTCCCCCGCCATCCTGTCTGCCAGCCCTGGCGAGAAAGTGACA ATGACCTGCCGGGCCTCCTCCTCCGTGTCCTACATCCACTGGTTCCAGCAGAAGCCCGGCTCCAG CCCCAAGCCCTGGATCTACGCCACCTCCAACCTGGCCTCTGGCGTGCCCGTGCGGTTCTCCGGCT CTGGCTCTGGCACCTCCTACTCCCTGACCATCTCCCGGGTGGAAGCCGAGGACGCCGCCACCTA CTACTGCCAGCAGTGGACCTCCAACCCCCCCACCTTCGGCGGAGGCACCAAGCTGGAAATCAAG CGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCCTCCGACGAGCAGCTGAAGTCCGGCAC CGCCTCCGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTG GACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACAGC ACCTACAGCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACG CCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACCGGGGCGAATG CGGCGGAGGCGGATCTGGTGGCGGAGGTTCTGGCGGCGGAGGATCCAGCGAGGTGGAATACC GGGCCGAAGTGGGCCAGAACGCCTACCTGCCCTGCTTCTACACCCCTGCCGCCCCTGGCAACCT GGTGCCTGTGTGTTGGGGCAAGGGCGCCTGCCCTGTGTTCGAGTGCGGCAACGTGGTGCTGCG GACCGACGAGCGGGACGTGAACTACTGGACCTCCCGGTACTGGCTGAACGGCGACTTCCGGAA GGGCGACGTGTCCCTGACAATCGAGAACGTGACCCTGGCCGACTCCGGCATCTACTGCTGCAGA ATCCAGATCCCCGGCATCATGAACGACGAGAAGTTCAACCTGAAGCTGGTCATCAAGCCCGCCA AAGTGACCCCTGCCCCTACCCGGCAGAGAGACTTCACCGCCGCCTTCCCCCGGATGCTGACCAC CAGAGGCCACGGCCCTGCCGAGACTCAGACCCTGGGCTCCCTGCCCGACATCAACCTGACCCAG ATCTCCACCCTGGCCAACGAGCTGCGGGACTCCCGGCTGGCCAATGACCTGAGAGACTCCGGCG CCACCATCCGGATCGGCTGATGATGA

23. FMab 31:

Nucleotide sequence of Anti-CD20 heavy chain N-terminal TIM3 ECD fusion protein (SEQ ID NO. 31)

ATGGACATGAGAGTGCCTGCTCAGCTGCTGGGCCTGCTGCTGCTGTGGC

TGAGAGGCGCCAGATGCTCCGAGGTGGAATACCGGGCCGAAGTGGGCCAG AACGCCTACCTGCCCTGCTTCTACACCCCTGCCGCCCCTGGCAACCTGGTGCC TGTGTGTTGGGGCAAGGGCGCCTGCCCTGTGTTCGAGTGCGGCAACGTGGTG CTGCGGACCGACGAGCGGGACGTGAACTACTGGACCTCCCGGTACTGGCTGA ACGGCGACTTCCGGAAGGGCGACGTGTCCCTGACCATCGAGAACGTGACCCT GGCCGACTCCGGCATCTACTGCTGCAGAATCCAGATCCCCGGCATCATGAAC GACGAGAAGTTCAACCTGAAGCTGGTCATCAAGCCCGCCAAAGTGACCCCTG CCCCTACCCGGCAGAGAGACTTCACCGCCGCCTTCCCCCGGATGCTGACCACC AGAGGCCACGGCCCTGCCGAGACTCAGACCCTGGGCTCCCTGCCCGACATCA ACCTGACCCAGATCTCCACCCTGGCCAACGAGCTGCGGGACTCCCGGCTGGC CAATGACCTGAGAGACTCCGGCGCCACCATCCGGATTGGCGGAGGCGGAGGA AGTGGTGGCGGAGGTTCTGGCGGCGGAGGATCCCAGGTGCAGCTGCAGCAGC CTGGCGCCGAACTGGTCAAGCCAGGCGCCTCCGTGAAGATGTCCTGCAAGGC CTCCGGCTACACCTTCACCAGCTACAACATGCACTGGGTCAAGCAGACCCCC GGCAGAGGCCTGGAATGGATCGGCGCCATCTACCCCGGCAACGGCGACACCT CCTACAACCAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGTCCTC CTCCACCGCCTACATGCAGCTGTCCAGCCTGACCTCCGAGGACTCCGCCGTGT ACTACTGCGCCCGGTCCACCTACTACGGCGGCGACTGGTACTTCAACGTGTGG GGCGCTGGCACCACCGTGACCGTGTCCTCTGCCTCCACCAAGGGCCCCTCCGT GTTCCCTCTGGCCCCCTCCAGCAAGTCCACCTCTGGCGGCACCGCTGCCCTGG GCTGCCTGGTCAAGGACTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCT GGCGCCCTGACCAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGG CCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCCCTGGGCACCC AGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAA GAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCCTGCCCT GCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAA GGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGAC GTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGG AAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTACAACTCCACCT ACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAA AGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAG ACCATCTCCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACCCTGC CCCCTAGCCGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTCGT GAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAG CCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCAT TCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAA CGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGA AGTCCCTGTCCCTGAGCCCCGGCTGATGATGA

24. FMab 32:

Nucleotide sequence of Anti-CD20 light chain N-terminal TIM3 ECD fusion protein (SEQ ID NO. 32)

ATGGACATGAGAGTGCCTGCTCAGCTGCTGGGCCTGCTGCTGCTGTGGC

TGAGAGGCGCCAGATGCTCCGAGGTGGAATACCGGGCCGAAGTGGGCCAG AACGCCTACCTGCCCTGCTTCTACACCCCTGCCGCCCCTGGCAACCTGGTGCC TGTGTGTTGGGGCAAGGGCGCCTGCCCTGTGTTCGAGTGCGGCAACGTGGTG CTGCGGACCGACGAGCGGGACGTGAACTACTGGACCTCCCGGTACTGGCTGA ACGGCGACTTCCGGAAGGGCGACGTGTCCCTGACCATCGAGAACGTGACCCT GGCCGACTCCGGCATCTACTGCTGCAGAATCCAGATCCCCGGCATCATGAAC GACGAGAAGTTCAACCTGAAGCTGGTCATCAAGCCCGCCAAAGTGACCCCTG CCCCTACCCGGCAGAGAGACTTCACCGCCGCCTTCCCCCGGATGCTGACCACC AGAGGCCACGGCCCTGCCGAGACTCAGACCCTGGGCTCCCTGCCCGACATCA ACCTGACCCAGATCTCCACCCTGGCCAACGAGCTGCGGGACTCCCGGCTGGC CAATGACCTGAGAGACTCCGGCGCCACCATCCGGATTGGCGGAGGCGGAGGA AGTGGTGGCGGAGGTTCTGGCGGCGGAGGATCCCAGATCGTGCTGTCCCAGT CCCCCGCCATCCTGTCTGCCAGCCCTGGCGAGAAAGTGACAATGACCTGCCG GGCCTCCTCCTCCGTGTCCTACATCCACTGGTTCCAGCAGAAGCCCGGCTCCA GCCCCAAGCCCTGGATCTACGCCACCTCCAACCTGGCCTCTGGCGTGCCCGTG CGGTTCTCCGGCTCTGGCTCTGGCACCTCCTACAGCCTGACCATCTCCCGGGT GGAAGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGACCTCCAACCCC CCCACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGCGGACCGTGGCCGCTC CCTCCGTGTTCATCTTCCCACCCTCCGACGAGCAGCTGAAGTCCGGCACCGCC TCCGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTG GAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTCACCGAG CAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAA GGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGC CTGTCCAGCCCCGTGACCAAGTCCTTCAACCGGGGCGAGTGCTGATGATGA

Note:

1. Bold font: VK012 signal sequence

2. Black: Mature gene coding sequences

3. Underlined: Stop codons

Claims

A method for preparing therapeutically active fusion protein, wherein the fusion protein comprises a tumor targeting moiety and at least one immunomodulatory moiety, wherein the tumor targeting moiety is an antibody against the protein CD20 and wherein the fusion protein is prepared by the following steps:

a. preparing a codon optimized nucleotide sequence encoding the fusion protein, wherein the codon optimized nucleotide sequence for the antibody is lacking nucleotides for expression of a lysine at the C-terminal end of the heavy chains of the antibody;

b. cloning the optimized sequence of said fusion protein in a host cell capable of transient or stable expression;

d. collecting secreted fusion proteins and optionally for further purification. The method of claim 1, wherein the immunomodulatory moiety counteracts immune tolerance of a cancer cell.

The method of claim 1, wherein the immunomodulatory moiety is linked to the antibody by an amino acid sequence of sufficient length to allow bi-specific binding of the fusion protein.

The method of claim 1, wherein the immunomodulatory molecule is linked directly or through a linker to the heavy chain of the antibody, light chain of the antibody or both chains.

The method of claim 1, wherein the immunomodulatory molecule is linked directly or through a linker to the N or C terminus of the heavy chain of the antibody, N or C terminus of the light chain of the antibody or both N and C terminus of both chains.

The method of claim 1, wherein the immunomodulatory molecule binds to

(i) Programmed death- 1 ligand 1 (PD-L1) and/or

(ii) Transforming growth factor-beta (TGF-β) and/or

(iii) T cell immunoglobulin and mucin domain-3 (TIM-3)

7. The method of claim 1, wherein the antibody binds to protein CD20 and the immunomodulatory moiety binds to Programmed death- 1 ligand 1 (PD-L1).

8. The method of claim 7, wherein the codon optimized nucleotide sequences comprise SEQ ID NOs: 21, 22, 23 and 24.

9. The method of claim 1, wherein the antibody binds to CD20 and the immunomodulatory moiety binds to TGF-β.

10. The method of claim 9, wherein the codon optimized nucleotide sequences comprise SEQ ID NOs: 25, 26, 27 and 28.

11. The method of claim 1, wherein the antibody binds to CD20 and the immunomodulatory moiety binds to TIM3.

12. The method of claim 11, wherein the codon optimized nucleotide sequences comprise SEQ ID NOs: 29, 30, 31 and 32.

13. A preparation comprising homogeneous therapeutically active fusion proteins, wherein the fusion proteins comprise a tumor targeting moiety and at least one immunomodulatory molecule, wherein the tumor targeting moiety is an antibody that binds to CD20 and wherein the fusion proteins are prepared by the following steps:

a. preparing a codon optimized nucleotide sequence encoding the fusion protein, wherein the codon optimized sequence for the antibody is lacking nucleotides for expression of a lysine at the C-terminal end of the heavy chains of the antibody;

b. cloning the codon optimized sequence of said fusion protein in a host cell capable of transient or stable expression;

d. collecting secreted fusion proteins for optional purification.

14. A nucleic acid sequence encoding a chimeric fusion protein, wherein the chimeric fusion protein comprises at least one targeting moiety having affinity for a cancer cell and at least one immunomodulatory moiety that counteracts immune tolerance of the cancer cell, wherein the targeting moiety is an antibody and the nucleic acid sequence of the antibody is lacking nucleotides for expression of a lysine at the C-terminal end of the heavy chains of the antibody.

15. The nucleic acid sequence of claim 14, wherein the nucleic acid sequence encoding the heavy chain of the antibody is SEQ ID NO: 20; and light chain of the antibody is SEQ ID NO: 19.

16. The nucleic acid sequence of claim 14, wherein the nucleic acid sequence for chimeric fusion protein comprises SEQ ID NOs: 21, 22, 23 and 24.

17. The nucleic acid sequence of claim 14, wherein the nucleic acid sequence for chimeric fusion protein comprises SEQ ID NOs: 25, 26, 27 and 28.

18. The nucleic acid sequence of claim 14, wherein the nucleic acid sequence for chimeric fusion protein comprises SEQ ID NOs: 29, 30, 31 and 32.

19. A vector comprising optimized genes for treating cancer in a human subject wherein the vector includes nucleotide sequences for encoding at least one targeting moiety, at least one immunomodulatory moiety and a linking moiety, wherein the optimized nucleotide sequences are selected from SEQ ID NOs: 21 to 32.

20. The vector of claim 19, wherein the nucleotide sequences comprise SEQ ID NOs:

21, 22, 23 and 24.

21. The vector of claim 19, wherein the nucleotide sequences comprise SEQ ID NOs:

25, 26, 27 and 28.

22. The vector of claim 19, wherein the nucleotide sequences comprise SEQ ID NOs:

29, 30, 31 and 32.

23. A method of treating cancer in a subject, the method comprising:

a) preparing a therapeutically active fusion protein, wherein the fusion protein comprises a tumor targeting moiety and at least one immunomodulatory molecule, wherein the tumor targeting moiety is an antibody that binds to PD-L1, TGF-β and TIM-3 and wherein the fusion protein is prepared by the following steps:

i) preparing a codon optimized nucleotide sequence encoding the fusion protein, wherein the codon optimized nucleotide sequence for the antibody is lacking nucleotides for expression of a lysine at the C-terminal end of the heavy chains of the antibody; ii) cloning the optimized sequence of said fusion protein in a host cell capable of transient or stable expression;

iii) growing the host cell in a media under suitable conditions for growing and allowing the host cell to express the fusion protein; and

iv) collecting secreted fusion proteins for optional purification;

b) administering a therapeutically active amount of the fusion proteins to the subject.

24. The method of claim 23, wherein the fusion protein comprises the amino acid sequences of SEQ ID NOs: 7, 8, 9 and 10. 25. The method of claim 23, wherein the fusion protein comprises the amino acid sequences of SEQ ID NOs: 11, 12, 13 and 14.

26. The method of claim 23, wherein the fusion protein comprises the amino acid sequences of SEQ ID NOs: 15, 16, 17 and 18.