Highly expressed EGFR and PD-L1 bispecific binding protein

Abstract

Disclosed herein are bispecific Fabs-In-Tandem immunoglobulin (FIT-Ig) binding proteins that simultaneously bind to EGFR and PD-L1. Such bispecific EGFR / PD-L1 FIT-Ig binding proteins are effectively expressed and can be used to block EGFR signaling, block PD-L1 signaling, and treat cancer.

Description

Technical Field

[0001] The present invention relates to a novel engineered bispecific binding protein that recognizes epidermal growth factor receptor (EGFR) and programmed death ligand 1 (PD-L1). The bispecific binding protein can be used to treat cancer. Background Art

[0002] Programmed death ligand 1 (PD-L1) is a type I transmembrane glycoprotein of approximately 40 kilodaltons (kD) in size. In humans, PD-L1 is expressed on a variety of immune cell types, including activated and inactive / exhausted T cells, naive and activated B cells, myeloid dendritic cells (DCs), monocytes, mast cells, and other antigen presenting cells (APCs). It is also expressed on non-immune cells, including pancreatic islets, Kupffer cells of the liver, vascular endothelium, and selected epithelia, such as airway epithelium and renal tubular epithelium, and its expression is enhanced during inflammatory episodes. PD-L1 is also present at elevated levels in many malignancies, including but not limited to breast cancer, colon cancer, colorectal cancer, lung cancer, kidney cancer (including renal cell carcinoma), gastric cancer, bladder cancer, non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), pancreatic cancer, and melanoma. It has also been shown that PD-L1 expression on the cell surface can be upregulated by stimulation with IFN-γ (interferon gamma).

[0003] PD-L1 (CD274, B7-H1) binds to programmed cell death protein 1 (PD-1, CD279), which is a member of the CD28 receptor family, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. PD-1 is typically expressed in immune cells, such as T cells, B cells, monocytes, and natural killer (NK) cells. Both PD-L1 and PD-L2 (CD273, B7-DC) are cell surface glycoprotein ligands for PD-1. The binding of PD-1 to PD-L1 or PD-L2 initiates signals that inhibit T cell activation and cytokine secretion. This downregulation of T cell activation in turn leads to reduced T cell proliferation, IL-2 secretion, IFN-γ secretion, and secretion of other growth factors and cytokines. Freeman et al., J. Exp. Med., 192: 1027-1034 (2000); Latchman et al., Nat. Immunol., 2: 261-8 (2001); Carter et al., Eur. J. Immunol., 32: 634-43 (2002); Ohigashi et al., Clin. Cancer Res., 11: 2947-53 (2005). Signaling through PD-1 / PD-L1 interactions is believed to play a vital, non-redundant function within the immune system by negatively regulating T cell responses. This regulation is involved in the development of T cells in the thymus, the regulation of chronic inflammatory responses, and the maintenance of peripheral tolerance and immune pardon. The critical nature of these functions is exemplified in PD-1-deficient mice, which exhibit an autoimmune phenotype. PD-1 deficiency in C57BL / 6 mice leads to chronic progressive lupus-like glomerulonephritis and arthritis. In Balb / c mice, PD-1 deficiency leads to severe cardiomyopathy due to the presence of cardiac tissue-specific autoreactive antibodies.

[0004] It has been proposed that PD-L1 plays a role in tumor immunity by increasing apoptosis of antigen-specific T cell clones. Dong et al., Nat. Med., 8:793-800 (2002). In addition, it has been shown that PD-L1 may be involved in intestinal mucosal inflammation, and PD-L1 inhibition is associated with wasting diseases such as colitis. Kanai et al., J. Immunol., 171:4156-63 (2003). In general, it has been proposed that inhibition of PD-L1 signaling can be used as a means to enhance T cell immunity to treat cancer (e.g., tumor immunity) and infection, including acute and chronic (e.g., persistent) infection.

[0005] Because PD-L1, PD-L2, and PD-1 are involved in downregulating immune responses, including inhibiting anti-tumor immune responses, they are called "immune checkpoint" proteins. Pardoll, Nat. Rev. Cancer, 12: 252-264 (2012). Clinical studies using immune checkpoint inhibitors (e.g., antibodies targeting PD-1, PD-L1, or CTLA-4) have achieved promising results, but it has been observed that only a portion of patients initially respond to current inhibitors, and there is growing clinical evidence that a significant proportion of initial responders eventually relapse, months or years later, becoming fatal drug-resistant diseases. Syn et al., The Lancet Oncology, 18 (12): e731–e741 (2017).

[0006] Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein and a member of the ErbB superfamily of receptor tyrosine kinases. It has been shown that EGFR plays an important role in a complex signal transduction cascade that promotes the development, survival and metastasis of epithelial cancers. When EGFR binds to its cognate ligand epidermal growth factor (EGF) and then forms a dimer with another EGFR (homodimerization) or another receptor tyrosine kinase (heterodimerization), EGFR signaling is triggered. Thereafter, the dimer is internalized to degrade or accumulate in the nucleus, where EGFR can regulate the transcription of multiple genes involved in cancer transformation. Therefore, EGFR has been considered an attractive therapeutic target for anti-tumor therapy. Approved anti-cancer therapies targeting EGFR include the monoclonal antibody cetuximab (a human-mouse chimeric anti-EGFR monoclonal antibody) and panitumumab (a human anti-EGFR monoclonal antibody). Many small molecule tyrosine kinase inhibitors that inhibit EGFR and other tyrosine kinase receptors have been approved for use as anticancer therapies, including gefitinib, erlotinib, lapatinib, and canertinib. These approved drugs have been used alone or in various combinations to treat various cancers. For a review of targeting EGFR in anticancer therapy, see Seshacharyulu et al., Expert Opin. Ther. Targets, 16(1): 15-31 (2012).

[0007] PD-L1 and EGFR are involved in the regulation of different signaling pathways, both of which are known to contribute to the initiation, growth, maintenance, and spread of cancer cells in the human body. However, in some cancer cells, activation of EGFR has been shown to upregulate PD-L1 expression, indicating a certain degree of "crosstalk" between the two pathways (Chen et al., J. Thorac. Oncol., 10(6): 910-923 (2015). Therapies that inhibit these two proteins to block their respective regulatory functions may provide an effective treatment for a variety of cancers. Summary of the invention

[0008] The present invention satisfies the above needs by providing an engineered bispecific protein that binds EGFR and PD-L1. In particular, the present invention provides a bispecific, multivalent binding protein that binds human EGFR and human PD-L1. The preferred bispecific binding protein of the present invention is a "Fabs-In-Tandem immunoglobulin" (FIT-Ig) binding protein that binds EGFR and PD-L1. As shown herein, this "EGFR / PD-L1" FIT-Ig binding protein according to the present invention is produced in mammalian cell culture at a significantly high yield and does not exhibit significant aggregate formation. Low productivity and significant aggregate formation are problems that make it impossible for FIT-Ig binding proteins previously prepared for EGFR and PD-L1 to be evaluated in preclinical and clinical stages, and these evaluations are necessary to determine whether such binding proteins can be used as therapeutic anticancer drugs.

[0009] In one embodiment, the present invention provides an EGFR / PD-L1 FIT-Ig binding protein that binds to EGFR and PD-L1, comprising a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, wherein

[0010] The first polypeptide chain ("heavy chain") comprises, from amino terminus to carboxyl terminus, VL EGFR -CL-VH PD-L1 -CH1-Fc, where VL EGFR is the antibody light chain variable domain of the first parent antibody that binds to EGFR, CL is the antibody light chain constant domain, VH PD-L1 is the antibody heavy chain variable domain of the second parent antibody that binds to PD-L1, CH1 is the first constant region of the antibody heavy chain, and Fc is the antibody Fc region (including hinge-CH2-CH3); wherein CL is directly fused to VH PD-L1 , wherein no artificial linker is inserted between the variable domain and the constant domain, and wherein:

[0011] V L EGFR comprising amino acid residues 1-107 of SEQ ID NO: 1,

[0012] VH PD-L1 comprising amino acid residues 215-331 of SEQ ID NO: 1;

[0013] The second polypeptide chain ("first light chain") includes VH from amino terminus to carboxyl terminus. EGFR -CH1, where VH EGFR is the antibody heavy chain variable domain of the first parent antibody that binds to EGFR, wherein CH1 is the constant domain of the first antibody heavy chain, wherein in VH EGFRand CH1 without an artificial joint inserted therebetween, and wherein:

[0014] VH EGFR comprising amino acid residues 1-119 of SEQ ID NO:2;

[0015] The third polypeptide chain ("second light chain") comprises VL from amino terminus to carboxyl terminus. PD-L1 -CL, where VL PD-L1 is the light chain variable domain of the second parent antibody that binds to PD-L1, wherein CL is an antibody light chain constant domain, wherein in VL PD-L1 There is no artificial linker inserted between and CL, and wherein:

[0016] V L PD-L1 Comprising amino acid residues 1-107 of SEQ ID NO:3.

[0017] In a preferred embodiment, the EGFR / PD-L1 FIT-Ig binding protein is a six-polypeptide chain FIT-Ig binding protein, which comprises two of the first polypeptide chains, two of the second polypeptide chains, and two of the third polypeptide chains, wherein the polypeptide chains are combined to form four Fab binding units, wherein two Fab binding units bind to EGFR and two Fab binding units bind to PD-L1.

[0018] Preferably, the CL domain present in one or more polypeptide chains (e.g., the first polypeptide chain and the third polypeptide chain described above) of the FIT-Ig binding protein of the present invention is a human CL kappa domain (hCκ). Preferably, the CL domain of the FIT-Ig binding protein of the present invention is derived from a human IgG1 (hIgG1) antibody. The preferred hIgG1 CL kappa domain present in one or more polypeptide chains of the EGFR / PD-L1 FIT-Ig binding protein of the present invention comprises amino acid residues 108-214 of SEQ ID NO: 1.

[0019] Preferably, the CH1 domain present in one or more polypeptide chains (e.g., the first polypeptide chain and the second polypeptide chain described above) of the FIT-Ig binding protein of the present invention is derived from a human IgG1 antibody. The preferred hIgG1 CH1 domain present in one or more polypeptide chains of the EGFR / PD-L1 FIT-Ig binding protein of the present invention comprises amino acid residues 332-434 of SEQ ID NO: 1.

[0020] Preferably, the Fc in the polypeptide chain present in the first polypeptide chain (or "heavy chain") of the FIT-Ig binding protein of the present invention comprises an antibody Fc region comprising a hinge-CH2-CH3 domain. Preferably, the Fc is derived from a human IgG1 antibody. The preferred hIgG1 Fc region present in the first polypeptide chain of the EGFR / PD-L1 FIT-Ig binding protein of the present invention comprises amino acid residues 435-661 of SEQ ID NO: 1.

[0021] The present invention also provides an EGFR / PD-L1 FIT-Ig binding protein that binds to EGFR and PD-L1, comprising:

[0022] a first polypeptide chain comprising a sequence of amino acid residues according to SEQ ID NO: 1;

[0023] a second polypeptide chain comprising the sequence of amino acid residues according to SEQ ID NO: 2; and

[0024] a third polypeptide chain comprising a sequence of amino acid residues according to SEQ ID NO: 3;

[0025] The EGFR / PD-L1 FIT-Ig contains the Fab binding unit of EGFR and the Fab binding unit of PD-L1.

[0026] In a preferred embodiment, the above-mentioned EGFR / PD-L1 FIT-Ig binding protein is a six-polypeptide chain FIT-Ig binding protein, which comprises two first polypeptide chains comprising a sequence of amino acid residues according to SEQ ID NO: 1, two second polypeptide chains comprising a sequence of amino acid residues according to SEQ ID NO: 2, and two third polypeptide chains comprising a sequence of amino acid residues according to SEQ ID NO: 3, wherein the polypeptide chains are combined to form four Fab binding units, wherein two Fab binding units bind to EGFR and two Fab binding units bind to PD-L1.

[0027] Preferably, the EGFR / PD-L1 FIT-Ig binding protein described herein binds to EGFR and PD-L1 simultaneously. In another embodiment, the EGFR / PD-L1 FIT-Ig binding protein of the present invention binds to two EGFR proteins and two PD-L1 proteins. In a more preferred embodiment, the EGFR / PD-L1 FIT-Ig binding protein described herein binds to two EGFR proteins and two PD-L1 proteins simultaneously.

[0028] In one embodiment, the EGFR / PD-L1 FIT-Ig binding protein according to the present invention binds to EGFR and PD-L1, wherein the affinity for EGFR and PD-L1 is substantially the same (i.e., the same or within 30%) as the affinity of the respective parent antibodies for EGFR and PD-L1, and each EGFR and PD-L1 antigen binding site of the FIT-Ig binding protein is derived from the parent antibodies.

[0029] In one embodiment, the EGFR / PD-L1 FIT-Ig binding protein of the present invention binds to EGFR, and the binding rate constant (k on ) is at least 1×10 5 M -1 s -1 , more preferably at least 2×10 5 M -1 s -1 , as determined by biolayer interferometry. In a further embodiment, the k of the EGFR / PD-L1 FIT-Ig binding protein according to the present invention to human EGFR on Compared with the parent antibody to human EGFR on The anti-EGFR specificity of the EGFR / PD-L1 FIT-Ig binding protein is approximately 40% lower than that of the parent antibody.

[0030] In one embodiment, the EGFR / PD-L1 FIT-Ig binding protein of the present invention binds to human EGFR, and the dissociation rate constant (k off ) is less than 1.1×10 -4 sec -1 , as determined by biolayer interferometry. In a further embodiment, the EGFR / PD-L1 FIT-Ig binding protein of the invention has a k of 1.1k for human EGFR. off Compared with the parent antibody to human EGFR off The anti-EGFR specificity of the EGFR / PD-L1 FIT-Ig binding protein is approximately 50% lower than that of the parent antibody.

[0031] In one embodiment, the EGFR / PD-L1 FIT-Ig binding protein of the present invention binds to human EGFR, and the dissociation constant (KD) for human EGFR is less than 1×10 -9 M, preferably less than 7×10 -10 M, more preferably less than 6×10 -10 M, and still more preferably less than or equal to 5×10 -10M, as determined by biolayer interferometry. In a further embodiment, the EGFR / PD-L1 FIT-Ig binding protein of the invention has a K of 1.17kDa for human EGFR. D The K of the parental antibody against human EGFR D Substantially identical (ie, identical or within 25%), the anti-EGFR specificity of the EGFR / PD-L1 FIT-Ig binding protein is derived from the parent antibody.

[0032] In one embodiment, the EGFR / PD-L1 FIT-Ig binding protein of the present invention binds to PD-L1, and the binding rate constant (k on ) is at least 5×10 5 M -1 s -1 , more preferably at least 7×10 5 M -1 s -1 , even more preferably at least 8×10 5 M -1 s -1 , as determined by biolayer interferometry. In a further embodiment, the k of the EGFR / PD-L1 FIT-Ig binding protein according to the present invention for human PD-L1 is on The k of the parental antibody against human PD-L1 on The anti-PD-L1 specificity of the EGFR / PD-L1 FIT-Ig binding protein is identical to or within about 90% of that of the parent antibody.

[0033] In one embodiment, the EGFR / PD-L1 FIT-Ig binding protein of the present invention binds to human PD-L1, and the dissociation rate constant (k off ) is less than 2×10 -2 sec -1 , more preferably less than 1.5×10 -2 sec -1 , as determined by biolayer interferometry. In a further embodiment, the EGFR / PD-L1 FIT-Ig binding protein of the invention has a k of 1. off Compared with the parental antibody for human PD-L1 off The anti-PD-L1 specificity of the EGFR / PD-L1 FIT-Ig binding protein is approximately 20% higher than that of the parent antibody.

[0034] In one embodiment, the EGFR / PD-L1 FIT-Ig binding protein of the present invention binds to human PD-L1, and the dissociation constant (k D ) is less than 2×10-8 M, more preferably less than 1.7×10 -8 M, as determined by biolayer interferometry. In a further embodiment, the K of the EGFR / PD-L1 FIT-Ig binding protein of the invention for PD-L1 is D K of parental antibody against PD-L1 D Substantially identical (ie, identical or within 30%), the anti-PD-L1 specificity of the EGFR / PD-L1 FIT-Ig binding protein is derived from the parent antibody.

[0035] In one embodiment, the EGFR / PD-L1 FIT-Ig binding protein according to the present invention is expressed at a level greater than 10 mg / L in mammalian cell culture.

[0036] The fully assembled six-polypeptide chain EGFR / PD-L1 FIT-Ig binding protein "monomer" can be purified from the cell culture medium using protein A affinity chromatography. A solution or suspension of the EGFR / PD-L1 FIT-Ig binding protein that has been purified using protein A affinity chromatography can be further analyzed for possible aggregates using size exclusion chromatography (SEC), wherein protein aggregates are detected as molecular species with a molecular weight greater than that of the six-chain EGFR / PD-L1 FIT-Ig binding protein monomer, which has a molecular weight of approximately 240,000 Daltons. In one embodiment, the present invention provides a composition (e.g., a solution or suspension) comprising the EGFR / PD-L1 FIT-Ig binding protein described herein, which has been purified using protein A affinity chromatography (preferably, a chromatography column) and has less than or equal to 0.1% (≤0.1%) of FIT-Ig protein aggregates.

[0037] In another embodiment, the EGFR / PD-L1 FIT-Ig binding protein described herein is glycosylated. Preferably, the glycosylation is a human glycosylation pattern.

[0038] In one embodiment, the EGFR / PD-L1 FIT-Ig binding protein described herein inhibits or blocks EGFR signaling or PD-L1 signaling. Preferably, the EGFR / PD-L1 FIT-Ig binding protein of the present invention inhibits or blocks EGFR signaling and PD-L1 signaling.

[0039] In one embodiment, the EGFR / PD-L1 FIT-Ig binding protein described herein inhibits the growth or survival of cancer cells.

[0040] The present invention also provides one or more isolated nucleic acids encoding one or more polypeptide chains of the above-mentioned EGFR / PD-L1 FIT-Ig binding protein.

[0041] In a preferred embodiment, the isolated nucleic acid molecule of the present invention encodes the first polypeptide chain (heavy chain), the second polypeptide chain (first light chain) or the third polypeptide chain (second light chain) of the EGFR / PD-L1FIT-Ig binding protein, wherein:

[0042] The first polypeptide chain (heavy chain) comprises the amino acid sequence according to SEQ ID NO: 1;

[0043] The second polypeptide chain (first light chain) comprises the amino acid sequence according to SEQ ID NO: 2; and

[0044] The third polypeptide chain (second light chain) comprises the amino acid sequence according to SEQ ID NO:3.

[0045] In one embodiment, the present invention provides an expression vector comprising one or more isolated nucleic acid molecules encoding one or more polypeptide chains of the EGFR / PD-L1 FIT-Ig binding protein, wherein the one or more isolated nucleic acid molecules are operably linked to suitable transcription and / or translation sequences in a host cell compatible with the expression vector, which transcription and / or translation sequences are required for expressing one or more encoded polypeptide chains of the EGFR / PD-L1 FIT-Ig binding protein. Preferably, a single expression vector comprises a single nucleic acid encoding only one of the three component polypeptide chains of the FIT-Ig binding protein described herein, so that three separate expression vectors (each encoding and expressing only one of the three component polypeptides) must be present in the host cell to produce the FIT-Ig binding protein described herein.

[0046] Preferred vectors for cloning and expressing nucleic acids described herein include, but are not limited to, pcDNA, pcDNA3.1, pTT (Durocher et al. Nucleic Acids Res., 30(2e9):1-9 (2002)), pTT3 (pTT with additional multiple cloning sites), pEFBOS (Mizushima and Nagata, Nucleic Acids Res., 18(17):5322 (1990)), pBV, pJV, pcDNA3.1 TOPO, pEF6 TOPO and pBJ.

[0047] The vector of the present invention may be an autonomously replicating vector or may be a vector incorporated into the genome of a host cell.

[0048] In another embodiment, the present invention provides an isolated host cell comprising one or more vectors described above. Such an isolated host cell can be an isolated prokaryotic cell or an isolated eukaryotic cell.

[0049] In one embodiment of the invention, the isolated prokaryotic host cell comprising one or more vectors described herein is a bacterial host cell. The bacterial host cell can be a Gram-positive, Gram-negative or Gram-variable bacterial cell. Preferably, the bacterial host cell comprising one or more vectors described herein is a Gram-negative bacterium. Even more preferably, the bacterial host cell comprising one or more vectors described herein is an Escherichia coli cell.

[0050] In one embodiment of the invention, the host cell comprising the separation of one or more vectors described herein is a eukaryotic host cell. Examples of eukaryotic host cells that can comprise the separation of one or more vectors described herein include, but are not limited to, mammalian host cells, insect host cells, plant host cells, fungal host cells, eukaryotic algae host cells, nematode host cells, protozoan host cells, and fish host cells.

[0051] The isolated fungal host cell that may contain one or more vectors described herein is selected from: Aspergillus, Neurospora, Saccharomyces, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces, Yarrowia and Candida. Preferred fungal host cells are Saccharomyces cerevisiae host cells. More preferably, the Saccharomyces cerevisiae host cell is a Saccharomyces cerevisiae cell. The insect cell used as a host cell according to the present invention is an insect Sf9 cell.

[0052] In a preferred embodiment, the host cell according to the present invention is an isolated mammalian host cell comprising one or more expression vectors as described herein, wherein the mammalian host cell expresses three polypeptide chains encoded on the one or more expression vectors, and wherein the polypeptide chains combine to form a FIT-Ig binding protein comprising two Fab binding units that bind to EGFR and two Fab binding units that bind to PD-L1. Particularly preferred are mammalian host cells selected from the following: Chinese hamster ovary (CHO) cells, COS cells, Vero cells, SP2 / 0 cells, NS / 0 myeloma cells, human embryonic kidney (HEK293) cells, baby hamster kidney (BHK) cells, HeLa cells, human B cells, CV-1 / EBNA cells, L cells, 3T3 cells, HEPG2 cells, PerC6 cells and MDCK cells.

[0053] More preferably, the isolated mammalian host cell according to the present invention comprises three expression vectors, wherein each expression vector encodes and expresses one of the three component polypeptide chains of the FIT-Ig binding protein described herein, and wherein the three expressed polypeptide chains are combined to form a FIT-Ig binding protein comprising two Fab binding units that bind to EGFR and two Fab binding units that bind to PD-L1.

[0054] The present invention also provides a method for producing the EGFR / PD-L1 FIT-Ig binding protein described herein, the method comprising culturing an isolated host cell comprising one or more expression vectors described herein under conditions sufficient to produce the EGFR / PD-L1 FIT-Ig binding protein.

[0055] Another aspect of the invention is an EGFR / PD-L1 FIT-Ig binding protein produced by the following method, which comprises culturing an isolated host cell comprising one or more expression vectors described herein under conditions sufficient to produce the EGFR / PD-L1 FIT-Ig binding protein.

[0056] The EGFR / PD-L1 FIT-Ig binding protein described herein can be conjugated to another compound, for example, along or at the carboxyl terminus of the CH3 domain of the Fc region of one or two first (heavy) polypeptide chains in a manner similar to other conjugated antibodies. Such compounds that can be conjugated to the EGFR / PD-L1 FIT-Ig binding protein include, but are not limited to, imaging agents and therapeutic agents. Preferred imaging agents that can be conjugated to the EGFR / PD-L1 FIT-Ig binding protein include, but are not limited to: radioactive labels, enzymes, fluorescent labels, luminescent labels, bioluminescent labels, magnetic labels, biotin, streptavidin and avidin. Radioactive labels that can be conjugated to the EGFR / PD-L1 FIT-Ig binding protein described herein include, but are not limited to 3 H. 14 C. 35 S. 90 Y. 99 Tc, 111 In, 131 I. 177 Lu, 166 Ho and 153 Preferred therapeutic compounds that can be conjugated to the EGFR / PD-L1 FIT-Ig binding proteins described herein include, but are not limited to, antibiotics, antiviral agents, small molecule receptor tyrosine kinase inhibitors, and cytokines.

[0057] In another embodiment, the EGFR / PD-L1 FIT-Ig binding protein described herein may be a crystallized EGFR / PD-L1 FIT-Ig binding protein that retains the binding affinity of the non-crystallized EGFR / PD-L1 FIT-Ig binding protein to EGFR and PD-L1. When administered to an individual, such a crystallized EGFR / PD-L1 FIT-Ig binding protein may also provide carrier-free controlled release of the EGFR / PD-L1 FIT-Ig binding protein. Compared to the non-crystalline form, the crystallized EGFR / PD-L1 FIT-Ig binding protein of the present invention may also exhibit a greater in vivo half-life when administered to an individual. The crystallized binding protein of the present invention may be produced according to methods known in the art, such as disclosed in International Publication No. WO 02 / 072636 (Shenoy et al.), which is incorporated herein by reference.

[0058] One embodiment of the present invention provides a composition for releasing a crystallized EGFR / PD-L1 FIT-Ig binding protein, wherein the composition comprises the crystallized EGFR / PD-L1 FIT-Ig binding protein described herein, an excipient component, and at least one polymer carrier. Preferably, the excipient component is selected from the group consisting of albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-β-cyclodextrin, methoxypolyethylene glycol, and polyethylene glycol. Preferably, the polymer carrier is a polymer selected from one or more of the following: poly(acrylic acid), poly(cyanoacrylate), poly(amino acid), poly(anhydride), poly(depsipeptide), poly(ester), poly(lactic acid), poly(lactic-co-glycolic acid) or PLGA, poly(β-hydroxybutyrate), poly(caprolactone), poly(dioxanone); polyethylene glycol, poly(hydroxypropyl)methacrylamide, poly[(organo)phosphazene], poly(orthoester), polyvinyl alcohol, poly(vinyl pyrrolidone), maleic anhydride / alkyl vinyl ether copolymer, pluronic polyol, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycosaminoglycans, sulfated polysaccharides, blends thereof and copolymers thereof.

[0059] The pharmaceutical composition of the present invention comprises the EGFR / PD-L1 FIT-Ig binding protein described herein and one or more pharmaceutically acceptable components, such as a pharmaceutically acceptable carrier (vehicle, buffer), a pharmaceutically acceptable excipient and / or other pharmaceutically acceptable ingredients.

[0060] Preferred pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and combinations thereof.

[0061] The pharmaceutical composition of the present invention may further comprise an isotonic agent. In the pharmaceutical composition of the present invention, the preferred isotonic agent useful is selected from the group consisting of sugars, polyols (such as mannitol or sorbitol), sodium chloride and combinations thereof.

[0062] The pharmaceutical composition comprising the EGFR / PD-L1 FTI-Ig binding protein described herein may further comprise one or more other therapeutically active compounds (therapeutic agents). Examples of such other therapeutic agents that may be incorporated into the pharmaceutical composition of the present invention include, but are not limited to, anticancer agents different from the EGFR / PD-L1 FTI-Ig binding protein described herein (e.g., anticancer compounds containing cytotoxic metals or anticancer compounds based on cytotoxic radioisotopes and combinations thereof), antibiotics, antiviral compounds, sedatives, stimulants, local anesthetics, anti-inflammatory steroids (e.g., natural or synthetic anti-inflammatory steroids and combinations thereof), analgesics (e.g., acetylsalicylic acid, acetaminophen, naproxen, ibuprofen, COX-2 inhibitors, morphine, oxycodone and combinations thereof), antihistamines, nonsteroidal anti-inflammatory drugs ("NSAIDs", e.g., acetylsalicylic acid, ibuprofen, naproxen, COX-2 inhibitors and combinations thereof), and combinations thereof.

[0063] In another embodiment, a pharmaceutical composition of the present invention comprises an EGFR / PD-L1 FIT-Ig binding protein as described herein, a pharmaceutically acceptable carrier, and an adjuvant, wherein the adjuvant provides a general stimulation of the patient's immune system.

[0064] In one embodiment, the present invention provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an EGFR / PD-L1 FIT-Ig binding protein as described herein.

[0065] The present invention also provides a method for inhibiting or blocking EGFR signal transduction in a cell, the method comprising contacting a cell expressing EGFR with the EGFR / PD-L1 FIT-Ig binding protein described herein.

[0066] In another embodiment, the present invention provides a method for inhibiting or blocking PD-L1 signaling in a cell, the method comprising contacting a cell expressing PD-L1 with the EGFR / PD-L1 FIT-Ig binding protein described herein.

[0067] In one embodiment, the present invention provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an EGFR / PD-L1 FIT-Ig binding protein as described herein.

[0068] In another embodiment, the invention provides a method for treating cancer in a cancer patient, wherein the EGFR / PD-L1 FIT-Ig binding protein as described herein is administered to the subject, and wherein the cancer is a cancer that is usually responsive to immunotherapy. In another embodiment, the cancer is a cancer that is not associated with immunotherapy. In another embodiment, the cancer is a refractory or recurrent malignancy.

[0069] Preferably, the cancer treated using the method according to the invention is an epithelial cancer.

[0070] In another embodiment, the cancer treated using the method according to the invention is selected from: melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell renal cell carcinoma, "CCRCC"), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignancies.

[0071] In another embodiment, the invention provides a method for treating a human subject having a disease in which EGFR and / or PD-L1 activity is detrimental, the method comprising administering to the subject an EGFR / PD-L1 FIT-Ig binding protein of the invention such that activity mediated by PD-L1 / PD1 binding and / or EGFR / EGF binding in the subject is reduced or blocked.

[0072] In one embodiment, the present invention provides a method for detecting EGFR and / or PD-L1 in a sample, wherein the sample contains or is suspected of containing EGFR or PD-L1 or a cell expressing EGFR or PD-L1, wherein the sample is contacted with a FIT-Ig binding protein as described herein. For example, the EGFR / PD-L1 FIT-Ig binding protein of the present invention can be used to detect EGFR or PD-L1 or both in a conventional immunoassay, such as an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), or tissue immunohistochemistry, wherein a FIT-Ig binding protein is used instead of an anti-EGFR antibody or an anti-PD-L1 antibody. The present invention provides a method for detecting EGFR or PD-L1 in a biological sample, the method comprising contacting a biological sample with an EGFR / PD-L1 FIT-Ig binding protein of the present invention, and detecting whether binding to a target antigen (EGFR or PD-L1) occurs, thereby detecting whether a target is present in the biological sample. The FIT-Ig binding protein can be directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound FIT-Ig binding proteins. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of suitable radioactive materials include 3 H. 14 C. 35 S. 90 Y. 99 Tc, 111 In, 125 I. 131 I. 177 Lu, 166 Ho or 153 Sm. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 is a diagram illustrating the general procedure for constructing three expression vectors for expressing the three types of polypeptide chains of each FIT-Ig binding protein described in Example 1.

[0074] like Figure 1 As shown in FIG, to express the first polypeptide chain (“heavy chain”) of the FIT-Ig binding protein, a VL encoding the first polypeptide chain was synthesized. A -CL-VH BThe DNA molecule was then inserted ("inserted") into the multiple cloning site (MCS) of the pcDNA3.1 expression vector so that the inserted DNA molecule was positioned downstream of the strong cytomegalovirus (CMV) enhancer promoter of the vector, downstream of and in the reading frame of the DNA fragment encoding the amino-terminal signal peptide (SP), and upstream and in the reading frame of the inserted DNA molecule encoding the antibody CH1 domain, which is connected to the antibody Fc region (designated "h-CH2-CH3") comprising the native continuous hinge-CH2-CH3 domains.

[0075] like Figure 1 As shown, in order to express the second polypeptide chain ("light chain #1") of the FIT-Ig binding protein, the encoding antibody VH A The DNA fragment encoding the antibody CH1 domain is then inserted into the multiple cloning site (MCS) of the pcDNA3.1 expression vector so that the inserted DNA molecule is positioned downstream of the strong CMV enhancer promoter of the vector, downstream of the DNA fragment encoding the amino-terminal signal peptide (SP) and within its reading frame, and upstream and within the reading frame of the inserted DNA molecule encoding the antibody CH1 domain.

[0076] To express the third polypeptide chain ("light chain #2"), a synthetic protein encoding antibody VL B The DNA fragment encoding the antibody CL domain is then inserted into the multiple cloning site (MCS) of the pcDNA3.1 expression vector so that the inserted DNA molecule is positioned downstream of the strong CMV enhancer promoter of the vector, downstream of the DNA fragment encoding the amino-terminal signal peptide (SP) and within its reading frame, and upstream and within the reading frame of the inserted DNA molecule encoding the antibody CL domain.

[0077] See Example 1 for more details.

[0078] Figure 2 The size exclusion chromatography elution profile of a FIT-Ig1 sample previously purified by protein A affinity chromatography is shown. The elution profile is complex, indicating a significant proportion of FIT-Ig1 aggregates. The FIT-Ig1 six-chain monomers account for less than 30% of the purified protein. For details, see Example 1.7.

[0079] Figure 3 The size exclusion chromatography elution profile of a FIT-Ig2 sample previously purified by protein A affinity chromatography is shown. The elution profile shows a major peak of the FIT-Ig2 six-chain monomer and several peaks indicating aggregates. The table below the figure provides the analysis results of several peaks. The FIT-Ig2 six-chain monomer accounts for less than 75% of the purified protein. For more details, see Example 1.7.

[0080] Figure 4 The size exclusion chromatography elution profile of a FIT-Ig3 sample previously purified by protein A affinity chromatography is shown. The elution profile is complex, indicating a significant proportion of aggregates. The FIT-Ig3 six-chain monomers account for less than 40% of the purified protein. For more details, see Example 1.7.

[0081] Figure 5 The size exclusion chromatography elution profile of a FIT-Ig4 sample previously purified by protein A affinity chromatography is shown. The elution profile shows a major peak of the FIT-Ig4 six-chain monomer and several peaks indicating low-proportion aggregates. The table below the figure provides the analysis results of several peaks. The FIT-Ig4 six-chain monomer accounts for about 91% of the purified protein. For more details, see Example 1.7.

[0082] Figure 6 The size exclusion chromatography elution profile of a FIT-Ig5 sample previously purified by protein A affinity chromatography is shown. The elution profile shows a major peak of FIT-Ig5 six-chain monomers and a small peak indicating a very low proportion of aggregates. The table below the figure provides the analysis results of several peaks. FIT-Ig5 six-chain monomers account for more than 98% of the purified protein. For more details, see Example 1.7.

[0083] Figure 7 The size exclusion chromatography elution profile of a FIT-Ig6 sample previously purified by protein A affinity chromatography is shown. The elution profile shows a major peak of FIT-Ig6 six-chain monomers and a barely detectable peak, which indicates that even if aggregates are present, they are present in very low proportions. The table below the figure provides the results of the analysis of several peaks. FIT-Ig6 six-chain monomers surprisingly account for 99.9% of the purified protein. For more information, see Example 1.7.

[0084] Figure 8 A graph showing the serum concentration of FIT-Ig6 over time in three male Sprague-Dawley rats is shown. FIT-Ig6 was administered to each rat at an intravenous dose of 5 mg / kg body weight. See Example 3 for details. DETAILED DESCRIPTION

[0085] The Fabs-In-Tandem immunoglobulin ("FIT-Ig") binding protein format has been shown to be highly adaptable to provide bispecific, multivalent binding proteins for a variety of different target antigen pairs or for different epitopes on the same antigen. See, for example, International Publication Nos. WO 2015 / 103072 A1 and WO 2017 / 136820 A2. In a preferred form, the FIT-Ig binding protein comprises four Fab binding units (rather than two in a natural IgG antibody), wherein each of two Fab units binds to a first antigen (or epitope) and each of the other two Fab units binds to a second antigen (or epitope). Despite the successful use of the FIT-Ig format to produce a variety of bispecific binding proteins that bind to therapeutically relevant target antigen pairs, FIT-Ig binding proteins that bind to PD-L1 and EGFR have not previously been produced in a suitable quantity and quality for routine preclinical evaluation as candidate anticancer therapeutics. For example, as shown herein, many FIT-Ig binding proteins that bind to PD-L1 and EGFR cannot be expressed at sufficiently high levels (i.e., greater than 10 mg / L) in standard mammalian cell cultures to provide sufficient amounts of protein required to support preclinical evaluations, including, for example, standard chemistry, manufacturing, and control ("CMC") phase evaluations. Another problem is that previously produced FIT-Ig binding proteins that bind to PD-L1 and EGFR have shown extensive aggregate formation, which greatly reduces the amount of functional hexapeptide chains ("monomers" of EGFR and PD-L1 binding proteins) necessary for drug development. Unacceptable levels of FIT-Ig aggregates are evident in the fractions of FIT-Ig binding proteins eluted from protein A affinity chromatography. Cost analysis shows that no existing FIT-Ig binding protein constructs can be obtained in sufficient quantity and quality to enable preclinical anticancer evaluations.

[0086] The present invention is based on the discovery of an EGFR / PD-L1 FIT-Ig binding protein that is expressed in mammalian cell culture at sufficiently high levels and without significant levels of aggregate formation to enable preclinical and clinical evaluation as a therapeutic anticancer drug.

[0087] The FIT-Ig binding proteins described herein contain two or more antigen binding sites and are typically tetravalent (four antigen binding site) proteins. Preferred FIT-Ig binding proteins according to the present invention bind to EGFR and PD-L1 and are therefore bispecific. Schematically, in a FIT-Ig binding protein, two first (heavy) polypeptide chains (each having a general structure of "VCVC-Fc", where "V" is an antibody variable domain and C is an antibody constant domain) and four "light" polypeptide chains (each having a general structure of "VC") are combined to form a hexamer with four Fab binding units (VH-CH1 paired with VL-CL, sometimes referred to as VH-CH1::VL-CL). Each Fab binding unit contains an antigen binding site comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, with a total of six CDRs per antigen binding site. Thus, each half of the FIT-Ig binding protein comprises one heavy polypeptide chain and two light polypeptide chains, and the complementary immunoglobulin pairing of the VH-CH1 and VL-CL elements of the three chains produces two Fab binding units, which are arranged in tandem. In the present invention, in the heavy chain polypeptide, the immunoglobulin domains of the Fab binding units are directly fused without the use of an artificial inter-domain linker. That is, the N-terminal VC element of each heavy polypeptide chain is directly fused at its C-terminus to the N-terminus of another VC element, which in turn is connected to the C-terminal antibody Fc region. In the bispecific FIT-Ig binding protein, the tandem Fab binding units will react with different antigens.

[0088] International Publication No. WO2015 / 103072 provides a description of the design, expression and characterization of FIT-Ig binding proteins. Preferred examples of such FIT-Ig molecules described herein include one heavy polypeptide chain and two different light polypeptide chains. The heavy chain comprises the structural formula VL A -CL-VH B -CH1-Fc, where CL is directly attached to VH B Fusion, or heavy chain containing the structural formula VH B -CH1-VL A -CL-Fc, in which CH1 is directly attached to VL A Fusion, where VL A is the variable light chain domain from the parent antibody that binds to antigen A, VH B is the variable heavy chain domain from the parent antibody that binds to antigen B, CL is the human IgG1 light chain kappa constant domain, CH1 is the first human IgG1 antibody heavy chain constant domain, and Fc is the immunoglobulin Fc region (e.g., the C-terminal hinge-CH2-CH3 portion of the human IgG1 antibody heavy chain). The two light polypeptide chains of FIT-Ig have the formula VH A -CH1 and VLB -CL. In the embodiment of the bispecific FIT-Ig, antigen A and antigen B are different antigens, or different epitopes of the same antigen. In the present invention, one of A and B is human EGFR, and the other is human PD-L1. Most preferably, in the present invention, A is human EGFR and B is human PD-L1.

[0089] Using the above scheme, each V domain of each polypeptide chain of the FIT-Ig binding protein can be named with the antigen (or epitope) specificity of the antigen binding site from which it is derived. For example, using this "antigen specific" naming scheme, the structure of the first polypeptide chain (polypeptide chain #1) of the FIT-Ig6 binding protein described in Example 1.6 and Table 6 can be named "VL EGFR -CL-VH PD-L1 -CH1-Fc, where "VL EGFR " indicates that the VL domain is derived from the EGFR-specific antigen binding site of the anti-EGFR parent antibody, and "VH PD-L1 " indicates that the VH domain is derived from the PD-L1-specific antigen binding site of the anti-PD-L1 parent antibody. The structure of the second polypeptide chain (polypeptide chain #2) of FIT-Ig6 can be named "VH EGFR -CH1", where VH EGFR The VH domain is derived from the EGFR-specific antigen binding site of the anti-EGFR parent antibody. Similarly, the third polypeptide chain (polypeptide chain #3) of FIT-Ig6 can be named "VL PD-L1 -CL", where "VL PD-L1 ” indicates that the VL domain is derived from the PD-L1-specific antigen binding site of the anti-PD-L1 parent antibody. Therefore, this antigen-specific naming scheme indicates whether the particular antigen-binding specificity is located in the corresponding external or internal Fab binding unit of the FIT-Ig binding protein.

[0090] In an alternative naming scheme, rather than naming the antigen-specificity of each variable domain of the antigen-binding site (e.g., VL EGFR , VH EGFR , VL PD-L1 , VH PD-L1 ), but rather the abbreviated names of the parent antibody used as the source of the domains are designated as subscripts to the corresponding VL and VH domains of the antigen binding site of the parent antibody. For example, referring again to the FIT-Ig6 binding protein of the present invention described in Example 1.6 and Table 6 below, the structure of the first polypeptide chain (polypeptide chain #1) can be named VL pani -CL-VH 3G10 -CH1-Fc, where "VL pani " indicates that the VL domain is derived from the anti-EGFR monoclonal antibody panitumumab, and "VH3G10 " indicates that the VH domain is derived from the anti-PD-L1 monoclonal antibody 3G10. The structure of the second polypeptide chain (polypeptide chain #2) of FIT-Ig6 can be named "VH pani -CH1", where "VH pani ” indicates that the VH domain is derived from panitumumab. Similarly, the third polypeptide chain (polypeptide chain #3) of FIT-Ig6 can be named “VL 3G10 -CL", where "VL 3G10 " indicates that the VL domain is derived from monoclonal antibody 3G10. Therefore, this alternative naming scheme indicates the source of the antigen binding specificity of the outer and inner Fab binding units of the FIT-Ig binding protein. This source naming scheme is particularly useful when trying to compare the properties of multiple FIT-Ig binding proteins that bind to the same two antigens (or epitopes) but one or both of the parent antibodies used as the source of antigen binding specificity are different. See the examples below.

[0091] As described above, the heavy polypeptide chain binds to each of the two different light polypeptide chains to form two complete Fab binding units, each of which contains a typical antibody VH::VL antigen binding site. As with native IgG antibodies, the Fc region on the heavy chain will bind to the Fc region on the other heavy chain to form a homodimer, thereby providing a FIT-Ig binding protein containing 6 polypeptide chains and 4 Fab binding units. Each arm of the FIT-Ig binding protein has an amino-terminal or "outer" Fab binding unit and a carboxyl-proximal or "inner" Fab binding unit. In the FIT-Ig nomenclature adopted herein, a specific bispecific FIT-Ig binding protein can be assigned a prefix that first indicates the antigen specificity of the outer Fab binding unit and then the antigen specificity of the inner Fab binding unit. Therefore, "EGFR / PD-L1 FIT-Ig binding protein" refers to a FIT-Ig binding protein having two outer Fab binding units that bind to EGFR and two inner Fab binding units that bind to PD-L1.

[0092] Generally, the nomenclature and technology used in connection with cell and tissue culture, molecular biology, immunology, microbiology, oncology, genetics and biochemistry are well-known and commonly used in the art. Unless otherwise indicated, the methods and technologies of the present invention are usually carried out according to conventional methods well known in the art, and are described in various general and more specific references cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as usually completed in the art or as described herein. The nomenclature and experimental procedures and technology used in connection with analytical chemistry, synthetic organic chemistry and medical and pharmaceutical chemistry described herein are well-known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, drug preparation, preparation, delivery and patient treatment.

[0093] In order to more readily understand the present invention, selected terms are defined below.

[0094] A "tumor" is an abnormal mass of tissue.

[0095] A "benign" tumor is an abnormal mass of tissue that grows slowly and is self-limited because it does not have the ability to invade nearby tissues and spread beyond its original site. A benign tumor is not cancer.

[0096] The term "cancer" has a known meaning in the fields of medicine and oncology, and includes definitions according to the National Cancer Institute ("NCI", a division of the National Institutes of Health, Bethesda, Maryland). Thus, according to the NCI, the term "cancer" is a term for a disease in which abnormal cells divide uncontrollably and may invade and damage or destroy nearby tissues. Cancer cells may also break off from a primary tumor and spread (metastasize) to other parts of the body through the blood and / or lymphatic system and form "secondary tumors", also referred to as "metastatic tumors" or "metastatic cancer". Thus, the term "cancer" refers to a malignant tumor in which cells grow uncontrollably and can penetrate and damage or destroy adjacent tissues, and can metastasize to distant parts of the body through the circulation and form new tumors.

[0097] An "anti-cancer" compound or drug is one that blocks, inhibits or stops the growth of cancer cells. Preferred anti-cancer compounds are cytotoxic to cancer cells.

[0098] Unless otherwise distinguished, the terms "intravenous" (or "intravenously") and "systemic" (or "systemically") are used interchangeably with respect to the route of introducing the compounds or compositions of the present invention into the circulatory system of a cancer patient.

[0099] As used herein, the terms "treat" and "treatment" generally refer to any regimen that alleviates one or more symptoms or manifestations of cancer, inhibits the progression of cancer, arrests the progression of cancer, or reverses the progression of cancer, prevents the occurrence of secondary (metastatic) cancers, provides significant killing of metastatic cancer cells, reduces the size of primary or secondary (metastatic) cancer tumors, increases the remission of one or more secondary (metastatic) tumors over a period of time, slows the progression of primary or secondary (metastatic) tumors, reduces the number of secondary (metastatic) tumors over a period of time, reduces the number of new secondary (metastatic) tumors over a period of time, increases organ or tissue function in a cancer patient, increases the vitality of a cancer patient, prolongs the life of a patient, or a combination thereof.

[0100] "Metastasis" has the same meaning known and used by oncology or medical field technicians, and refers to the process of cancer cells spreading from primary tumors to another position in the patient's body. "Metastatic" cancer cells are cancer cells that have fallen off or otherwise detached from the primary tumor, and are in the process of moving from the primary tumor to another position in the patient's body by blood or lymph, or have moved from the primary tumor to another position in the patient's body by blood or lymph. In this case, cancer or its cells are referred to as "metastasis". Therefore, "metastatic" tumors are tumors developed from metastatic cancer cells that are transferred from primary tumors to different positions in the patient's body, and cancer cells at this position establish another ("secondary", "metastatic") tumor, which is the same type as the primary tumor. For example, metastatic intestinal tumors in the liver are initiated and composed of intestinal cancer cells that are transferred from primary intestinal tumors and moved to the liver by blood, and then cancer cells establish secondary (metastatic) intestinal tumors in the liver. It should also be understood that metastatic tumors can also be another source of metastatic cancer cells, which can move to other tissues and organs and establish other metastatic tumors.

[0101] Unless otherwise indicated, when the terms "about" and "approximately" are used in combination with an amount, number, or value, the combination describes the amount, number, integer, or value itself as well as the amount, number, or value plus or minus 5%. For example, the phrases "about 40" and "about 40" disclose "40" and "38 to 42, inclusive".

[0102] The term "polypeptide" refers to any polymeric chain of amino acids. The terms "peptide" and "protein" are used interchangeably with the term polypeptide, and also refer to a polymeric chain of amino acids. The term "polypeptide" encompasses polypeptide analogs of natural or artificial proteins, protein fragments, and protein amino acid sequences. Unless the context is contradictory, the term "polypeptide" encompasses fragments and variants thereof (including fragments of variants). For antigenic polypeptides, a fragment of a polypeptide optionally comprises at least one continuous or non-linear polypeptide epitope. The precise boundaries of at least one epitope fragment can be confirmed using common techniques in the art. The fragment comprises at least about 5 continuous amino acids, for example, at least about 8 continuous amino acids, at least about 10 continuous amino acids, at least about 15 continuous amino acids, or at least about 20 continuous amino acids.

[0103] The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that, depending on the origin or source from which it is derived, is separated from naturally associated components with which it is associated in its native state, is substantially free of other proteins from the same species, is expressed by cells from a different species, or does not exist in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cell system different from the cell of its natural source is "isolated" from its naturally associated components. A protein consisting of one or more polypeptide chains may also be rendered substantially free of naturally associated components by separation using protein purification techniques well known in the art.

[0104] The term "recovering" refers to the process of rendering a chemical substance (eg, a polypeptide) substantially free of naturally associated components by isolation (eg, using protein purification techniques well known in the art).

[0105] The term "biological activity" of PD-L1 or EGFR refers to any or all of the intrinsic biological properties of PD-L1 or EGFR, respectively.

[0106] The term "specific binding" or "specific binding" in relation to the interaction of an antibody, binding protein or peptide with a second chemical substance means that the interaction depends on a specific structure (e.g., an antigenic determinant or epitope) present on the second chemical substance. For example, an antibody recognizes and binds to a specific protein structure, rather than the entire protein. If the antibody is specific for epitope "A", then in a reaction comprising labeled "A" and the antibody, the presence of a molecule comprising epitope A (or free unlabeled A) will reduce the amount of labeled A bound to the antibody. The bispecific FIT-Ig binding protein described herein comprises two Fab binding units that specifically bind to EGFR and two Fab binding units that specifically bind to PD-L1.

[0107] The term "antibody" broadly refers to any immunoglobulin (Ig) molecule consisting of four polypeptide chains (two heavy (H) chains and two light (L) chains), or any functional fragment, mutant, variant or derivative thereof that retains the basic epitope binding characteristics of the Ig molecule. Such mutants, variants or derivatives in the form of antibodies are known in the art. Non-limiting examples thereof are discussed below.

[0108] In a full-length antibody, each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The first, second, and third CDRs of the VH domain are usually listed as CDR-H1, CDR-H2, and CDR-H3; similarly, the first, second, and third CDRs of the VL domain are usually listed as CDR-L1, CDR-L2, and CDR-L3. The immunoglobulin molecule can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass.

[0109] In general, the term "Fc region" or "Fc" for short refers to the C-terminal region of the heavy chain of an antibody, which can be produced by papain digestion of a complete antibody. The Fc region can be a native sequence Fc region or a variant Fc region. The Fc region generally comprises two constant domains, i.e., a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain, such as in the case of the Fc region of IgM and IgE antibodies. The Fc region of IgG, IgA and IgD antibodies comprises a hinge region, a CH2 domain and a CH3 domain. In contrast, the Fc region of IgM and IgE antibodies lacks a hinge region, but comprises a CH2 domain, a CH3 domain and a CH4 domain. Variant Fc regions having amino acid residues substituted in the Fc portion to change antibody effector functions are known in the art (see, e.g., Winter et al., U.S. Patent Nos. 5,648,260 and 5,624,821). Unless otherwise specified, the "Fc region" of the FIT-Ig binding protein described herein is an Fc region derived from a human IgG1 antibody, comprising a hinge region, a CH2 domain, and a CH3 domain, and having the amino acids in any one of Tables 1-6 of the following Examples disclosed herein (SEQ ID NO: 8).

[0110] The Fc region of an antibody mediates several important effector functions, e.g., cytokine induction, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, complement-dependent cytotoxicity (CDC), and half-life / clearance of antibodies and antigen-antibody complexes. In some cases, these effector functions are desirable for therapeutic antibodies, but in other cases may be unnecessary or even harmful, depending on the therapeutic purpose. Certain human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC by binding to Fc gamma receptors (FcγRs) and complement C1q, respectively. Unless otherwise specified, the Fc region used in the FIT-Ig binding proteins described herein retains at least one or more or all of the same functional properties as those possessed by the Fc region in its original donor antibody.

[0111] In one embodiment, at least one amino acid residue is substituted in the Fc region, thereby changing one or more effector functions of the antibody. As in IgG antibodies, the dimerization of two identical heavy chains of the FIT-Ig binding protein described herein is mediated by the dimerization of the CH3 domain and stabilized by disulfide bonds in the hinge region of the Fc constant domain (e.g., CH2 and CH3) by connecting the CH1 or CL domain of the FIT-Ig heavy chain. The anti-inflammatory activity of IgG depends entirely on the sialylation of the N-linked glycans of the IgG Fc fragment. The precise glycan requirements for anti-inflammatory activity have been determined, so that suitable IgG1 Fc fragments can be produced, thereby producing fully recombinant sialylated IgG1 Fc with greatly enhanced efficacy (see, Anthony et al., Science, 320: 373-376 (2008)). Such sialylated Fc regions can be used in the FIT-Ig binding proteins described herein.

[0112] The terms "antigen binding portion" and "antigen binding fragment" or "functional fragment" of an antibody are used interchangeably and refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen, i.e., bind to the same antigen (e.g., EGFR, PD-L1) as the full-length antibody from which the portion or fragment is derived. It has been shown that the antigen binding function of an antibody can be performed by a fragment of a full-length antibody. Embodiments of such antibodies may also be in the form of bispecific, bispecific, or multispecific; which specifically bind to two or more different antigens. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment (Fab binding unit), which is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fd fragment, which consists of the VH and CH1 domains; (iv) a Fv fragment, which consists of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al., Nature, 341:544-546 (1989); International Publication No. WO90 / 05144), which contains a single variable domain; and (vi) isolated complementarity determining regions (CDRs). In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be connected by synthetic linkers using recombinant methods to make them into a single protein chain, in which the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv); see, e.g., Bird et al., Science, 242: 423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988)). Such single-chain antibodies are also intended to be included in the term "antigen-binding portion" of an antibody and the equivalent terms given above. Other forms of single-chain antibodies, such as diabodies, are also included. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain, creating two antigen-binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993). Such antibody binding portions are known in the art (Kontermann and Dübel, eds., Antibody Engineering(Springer-Verlag, New York, 2001), page 790 (ISBN 3-540-41354-5). In addition, single-chain antibodies also include "linear antibodies" that contain a pair of tandem Fv fragments (VH-CH1-VH-CH1) that form a pair of antigen binding regions together with complementary light chain polypeptides (Zapata et al., Protein Eng., 8(10): 1057-1062 (1995); and U.S. Pat. No. 5,641,870).

[0113] Unless otherwise indicated, the terms "donor" and "parent" refer to any antibody or antigen-binding fragment that is a source of antibody variable domains, antibody constant domains, Fab binding units or Fc regions for preparing the FIT-Ig binding proteins described herein. Non-natural or engineered antibodies can also be used as donors or parent antibodies for preparing the FIT-Ig binding proteins described herein.

[0114] Antibody (or immunoglobulin) constant domain (C) refers to the antibody heavy chain constant domain (CH) or light chain constant domain (CL). Mouse and human immunoglobulin heavy chain and light chain constant domain amino acid sequences are known in the art.

[0115] The term "monoclonal antibody" or "mAb" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for possible small amounts of mutations that may occur naturally. Monoclonal antibodies are highly specific for a single antigenic determinant (epitope). Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. The modifier "monoclonal" should not be construed as requiring the antibody to be produced by any particular method.

[0116] The term "human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention may, for example, include amino acid residues not encoded by human germline immunoglobulin sequences in CDR, particularly in CDR3 (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human framework sequences.

[0117] The term "recombinant human antibody" includes all human antibodies prepared, expressed, generated or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library (Hoogenboom, HR, Trends Biotechnol., 15:62-70 (1997); Azzazy and Highsmith, Clin. Biochem., 35:425-445 (2002); Gavilondo and Larrick, BioTechniques, 29:128-145 (2000); Hoogenboom and Chames, Immunol. Today, 21:371-378 (2000)), antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes (see, e.g., Taylor et al., Nucl. Acids Res., 20: 6287-6295 (1992); Kellermann and Green, Curr. Opin. Biotechnol., 13: 593-597 (2002); Little et al., Immunol. Today, 21: 364-370 (2000)); or antibodies prepared, expressed, produced or isolated by any other method (including splicing human immunoglobulin gene sequences to other DNA sequences). Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when transgenic animals of human Ig sequences are used, in vivo somatic cell mutagenesis), so that the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences derived from and related to human germline VH and VL sequences, but may not be naturally present in the human antibody germline repertoire in vivo.

[0118] The term "multivalent binding protein" refers to a binding protein comprising two or more antigen binding sites. Multivalent binding proteins are preferably engineered to have three or more antigen binding sites and are generally not naturally occurring antibodies. The term "bispecific binding protein" refers to a binding protein capable of binding to two different specific targets.

[0119] The term "activity" includes properties such as the ability to specifically bind to a target antigen, the affinity of an antibody or binding protein for an antigen, the ability to neutralize the biological activity of a target antigen, the ability to inhibit the interaction of a target antigen with its natural receptor or natural ligand, etc. The activity of the EGFR / PD-L1 FIT-Ig binding protein of the present invention may include, but is not limited to, inhibiting the binding of EGFR to its cognate ligand (EGF), inhibiting EGFR signaling, inhibiting the binding of PD-L1 to PD-1, inhibiting PD-1 / PD-L1 signaling, upregulating T cell responses to cancer, killing cancer cells, inhibiting cancer cell growth, inhibiting cancer cell survival, and inhibiting cancer cell spread.

[0120] As used herein, the term "kon" (also referred to as "Kon", "kon") refers to the association rate constant for binding of a binding protein (e.g., an antibody) to an antigen to form a binding complex (e.g., an antibody / antigen complex as known in the art). As used interchangeably herein, "kon" has also been referred to as the term "association rate constant" or "ka". For example, this value can represent the binding rate of an antibody to its target antigen or the rate of complex formation between an antibody and an antigen, as shown in the following equation:

[0121] Antibody ("Ab") + Antigen ("Ag") → Ab-Ag.

[0122] As used herein, the term "koff" (also referred to as "Koff", "koff") refers to the dissociation rate constant for a binding protein (e.g., an antibody) to dissociate from a binding complex (e.g., an antibody / antigen complex) as known in the art, or the "dissociation rate constant". For example, this value can represent the dissociation rate of an antibody from its target antigen, or the dissociation rate of an Ab-Ag complex to separate into free antibody and antigen over time, as shown in the following equation:

[0123] Ab+Ag←Ab-Ag.

[0124] As used herein, the term "K D ” (also called “Kd”) means “equilibrium dissociation constant”, which refers to the dissociation rate constant (k off ) divided by the binding rate constant (k on ) The binding rate constant (k on ), dissociation rate constant (k off ) and equilibrium dissociation constant (K D ) is used to express the binding affinity of an antibody or binding protein to an antigen. Methods for determining association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques provides high sensitivity and the ability to examine samples in physiological buffers at equilibrium. Other experimental methods and instrumentation may be used, for example (Biomolecular Interaction Analysis) assay (e.g., instruments available from BIAcore International AB, GE Healthcare, Uppsala, Sweden). Using e.g. Biolayer interferometry (BLI) using the RED96 system (Pall ForteBio LLC) is another affinity measurement technique. (Kinetic Exclusion Assay) assay (available from Sapidyne Instruments, Boise, Idaho) .

[0125] The term "isolated nucleic acid" refers to a polynucleotide (e.g., a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof) that has been separated by human intervention from all or a portion of the polynucleotides with which it occurs in nature, is operably linked to a polynucleotide with which it is not naturally linked, or does not exist as part of a larger sequence in nature.

[0126] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop, in which other DNA segments can be connected. Another type of vector is a viral vector, in which other DNA segments can be connected to the viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors and free mammalian vectors with bacterial replication origins). After being introduced into the host cell, other vectors (e.g., non-free mammalian vectors) can be integrated into the genome of the host cell, thereby replicating with the host genome. In addition, some vectors can guide the expression of genes operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA technology are usually in the form of plasmids. In this specification, "plasmid" and "vector" can be used interchangeably because plasmids are the most commonly used vector forms. However, the present invention is intended to include such other forms of expression vectors with equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0127] The term "operably linked" refers to a juxtaposition in which the components described are in a relationship that allows them to function in their intended manner. A control sequence is "operably linked" to a coding sequence in a manner that achieves expression of the coding sequence under conditions compatible with the control sequence. "Operably linked" sequences include expression control sequences that are continuous with a gene of interest and expression control sequences that control a gene of interest in trans or at a distance. As used herein, the term "expression control sequence" refers to polynucleotide sequences necessary for achieving expression and processing of coding sequences to which they are linked. Expression control sequences include appropriate transcription initiation, terminator, promoter, and enhancer sequences; effective RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance protein secretion when necessary. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences typically include promoters, ribosome binding sites, and transcription termination sequences; in eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term "control sequences" is intended to include components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0128] The term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which exogenous DNA has been introduced. In one embodiment, the host cell comprises two or more (e.g., multiple) nucleic acids encoding antibodies, such as the host cell described in U.S. Patent No. 7,262,028. Such terms are intended not only to refer to a specific subject cell, but also to the offspring of such cells. Because certain modifications may occur in offspring due to mutations or environmental influences, such offspring may actually be different from parental cells, but are still included in the scope of the term "host cell" used herein. In one embodiment, host cells include prokaryotes and eukaryotic cells selected from any kingdom of life. In another embodiment, eukaryotic cells include protists, fungi, plants, and animal cells. In another embodiment, host cells include, but are not limited to, prokaryotic cell lines Escherichia coli; mammalian cell lines CHO, HEK293, COS, NS0, SP2, and PER.C6; insect cell lines Sf9; and fungal cell Saccharomyces cerevisiae.

[0129] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, cell culture, tissue culture, and transformation (e.g., transfection, electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can generally be performed according to conventional methods known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning:A Laboratory Manual , 2nd edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0130] As used herein, the term "agonist" refers to a modulator that, when in contact with a molecule of interest, causes an increase in the magnitude of a certain activity or function of a molecule compared to the magnitude of the activity or function observed in the absence of the agonist. As used herein, the terms "antagonist" and "inhibitor" refer to a modulator that, when in contact with a molecule of interest, causes a decrease in the magnitude of a certain activity or function of a molecule compared to the magnitude of the activity or function observed in the absence of the antagonist. A specific antagonist of the present invention is an EGFR / PD-L1 FIT-Ig described herein that blocks or inhibits the binding of EGFR to EGF, blocks or inhibits the binding of PD-L1 to PD-1, blocks or inhibits EGFR signaling, blocks or inhibits PD-L1 signaling, blocks or inhibits the cancer-promoting activity of EGFR-dependent signaling, prevents or inhibits the cancer-promoting activity of PD-L1-dependent signaling, and one or more combinations thereof.

[0131] As used herein, the term "effective amount" refers to an amount of a therapy sufficient to reduce or lessen the severity and / or duration of a disorder or one or more symptoms thereof; prevent the progression of a disorder; cause regression of a disorder; prevent the recurrence, development, or progression of one or more symptoms associated with a disorder; detect a disorder; or enhance or improve the prophylactic or therapeutic effect of other therapies (e.g., prophylactic or therapeutic agents).

[0132] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In addition, unless the context otherwise requires, singular terms shall include pluralities, and plural terms shall include singulars. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "include" and other forms, such as "comprises" and "comprising", is not restrictive. Similarly, terms such as "element" or "component" include elements and components comprising one unit, and also include elements and components comprising more than one subunit, unless otherwise specifically stated.

[0133] The compositions or methods described herein as "comprising" one or more named elements or steps are open-ended, meaning that the named elements or steps are required, but other elements or steps may be added within the scope of the composition or method. To avoid redundancy, it is also understood that any composition or method described herein as "comprising" (or "it comprises") one or more named elements or steps also describes a corresponding, more restrictive "substantially consisting of the same named elements or steps" or "substantially consisting of...") composition or method, indicating that the composition or method includes the named necessary elements or steps, and may also include other elements or steps that do not substantially affect the basic and novel features of the composition and method. It is also understood that any composition or method described herein as "comprising" or "substantially consisting of one or more named elements or steps" also describes a corresponding, more restrictive and closed "consisting of" (or consisting of) composition or method to exclude any other unnamed elements or steps. In any composition or method disclosed herein, a known or disclosed equivalent of any named necessary element or step may replace the element or step.

[0134] Characteristics of the preferred EGFR / PD-L1 FIT-Ig binding proteins of the present invention

[0135] The preferred EGFR / PD-L1 FIT-Ig binding protein of the present invention, referred to as "FIT-Ig6" (or "EGFR / PD-L1FIT-Ig6"), binds to EGFR and PD-L1 and comprises:

[0136] a first polypeptide chain (heavy chain) comprising the amino acid sequence according to SEQ ID NO: 1;

[0137] a second polypeptide chain (first light chain) comprising the amino acid sequence according to SEQ ID NO: 2;

[0138] A third polypeptide chain (second light chain) comprising the amino acid sequence according to SEQ ID NO:3.

[0139] The combination of the first, second, and third polypeptide chains described above provides a "half FIT" molecule comprising an amino-terminal or "outer" Fab binding unit specific for EGFR, which is tandemly connected to a carboxyl-proximal or "inner" Fab binding unit specific for PD-L1. As in native IgG1 antibodies, the Fc (hinge-CH2-CH3) of the carboxyl-terminal region of the first polypeptide chain can bind to the Fc of the other half of the FIT molecule to form a fully assembled six-polypeptide chain FIT-Ig binding protein, which comprises two outer EGFR-specific Fab binding units and two inner PD-L1-specific Fab binding units.

[0140] The specificity of the Fab binding unit of the FIT-Ig binding protein is derived from the parent antibody, which is used as the source of antibody heavy and light chain variable domains (VH, VL) that form the specific antigen binding site.

[0141] The EGFR / PD-L1 FIT-Ig binding protein of the present invention binds to EGFR and PD-L1 simultaneously. In another embodiment, the EGFR / PD-L1 FIT-Ig binding protein binds to two EGFR proteins and two PD-L1 proteins simultaneously.

[0142] The EGFR / PD-L1 FIT-Ig binding protein according to the present invention binds to EGFR and PD-L1 with similar affinity as each of the parent antibodies from which the EGFR and PD-L1 specificities are derived.

[0143] The affinity of the EGFR / PD-L1 FIT-Ig binding proteins according to the present invention for EGFR and PD-L1 can be measured using any of a variety of systems, including biolayer interferometry (e.g., using RED96 system, Pall ForteBio LLC), surface plasmon resonance (e.g., using (Biomolecular Interaction Analysis) assay system, BIAcore International AB, GE Healthcare, Uppsala, Sweden) or kinetic exclusion assays (e.g., using Analytical Systems, Sapidyne Instruments, Boise, ID).

[0144] The EGFR / PD-L1 FIT-Ig binding protein of the present invention binds to EGFR, and the binding rate constant (k on ) is at least 1×10 5 M -1s -1 , more preferably at least 2×10 5 M -1 s -1 , as determined by biolayer interferometry. In a further embodiment, the k of the EGFR / PD-L1 FIT-Ig binding protein according to the present invention to human EGFR on Compared with the parent antibody to human EGFR on The anti-EGFR specificity of the EGFR / PD-L1 FIT-Ig binding protein is approximately 40% lower than that of the parent antibody.

[0145] The EGFR / PD-L1 FIT-Ig binding protein of the present invention binds to human EGFR, and the dissociation rate constant (k off ) is less than 1.1×10 -4 sec -1 , as determined by biolayer interferometry. In a further embodiment, the EGFR / PD-L1 FIT-Ig binding protein of the invention has a k of 1.1k for human EGFR. off Compared with the parent antibody to human EGFR off The anti-EGFR specificity of the EGFR / PD-L1 FIT-Ig binding protein is approximately 50% lower than that of the parent antibody.

[0146] The EGFR / PD-L1 FIT-Ig binding protein of the present invention binds to human EGFR, and the dissociation constant (KD) for human EGFR is less than 1×10 -9 M, preferably less than 7×10 -10 M, more preferably less than 6×10 -10 M, and still more preferably less than or equal to 5×10 -10 M, as determined by biolayer interferometry. In a further embodiment, the EGFR / PD-L1 FIT-Ig binding protein of the invention has a K of 1.17kDa for human EGFR. D The K of the parental antibody against human EGFR D Substantially identical (ie, identical or within 25%), the anti-EGFR specificity of the EGFR / PD-L1 FIT-Ig binding protein is derived from the parent antibody.

[0147] The EGFR / PD-L1 FIT-Ig binding protein of the present invention binds to PD-L1, and the binding rate constant (k on ) is at least 5×10 5 M -1 s -1 , more preferably at least 7×10 5 M -1 s-1 , even more preferably 8×10 5 M -1 s -1 , as determined by biolayer interferometry. In a further embodiment, the k of the EGFR / PD-L1 FIT-Ig binding protein according to the present invention for human PD-L1 is on The k of the parental antibody against human PD-L1 on The anti-PD-L1 specificity of the EGFR / PD-L1 FIT-Ig binding protein is identical to or within about 90% of that of the parent antibody.

[0148] The EGFR / PD-L1 FIT-Ig binding protein of the present invention binds to human PD-L1, and the dissociation rate constant (k off ) is less than 2×10 -2 sec -1 , more preferably less than 1.5×10 -2 sec -1 , as determined by biolayer interferometry. In a further embodiment, the EGFR / PD-L1 FIT-Ig binding protein of the invention has a k of 1. off Compared with the parental antibody for human PD-L1 off The anti-PD-L1 specificity of the EGFR / PD-L1 FIT-Ig binding protein is approximately 20% higher than that of the parent antibody.

[0149] In one embodiment, the EGFR / PD-L1 FIT-Ig binding protein of the present invention binds to human PD-L1, and the dissociation constant (k D ) is less than 2×10 -8 M, more preferably less than 1.7×10 -8 M, as determined by biolayer interferometry. In a further embodiment, the K of the EGFR / PD-L1 FIT-Ig binding protein of the invention for PD-L1 is D K of parental antibody against PD-L1 D Substantially identical (ie, identical or within 30%), the anti-PD-L1 specificity of the EGFR / PD-L1 FIT-Ig binding protein is derived from the parent antibody.

[0150] After one-step purification from cell culture medium using protein A affinity chromatography, the preferred EGFR / PD-L1FIT-Ig binding protein of the present invention does not show significant aggregate formation. As shown herein, after purification by protein A affinity chromatography, size exclusion chromatography (e.g., size exclusion chromatography using a high performance liquid chromatography (HPLC) column) can be used to analyze the presence of aggregates in the column eluate containing the EGFR / PD-L1 FIT-Ig binding protein. Size exclusion chromatography (SEC) will separate molecules according to size, and therefore, molecules with the expected molecular weight of a fully assembled six-polypeptide chain EGFR / PD-L1 FIT-Ig binding protein (also referred to as a six-chain "monomer") are separated from other substances with higher or lower molecular weights. The molecular weight of a six-chain monomer is approximately 240,000 Daltons. The preferred EGFR / PD-L1 FIT-Ig binding protein of the present invention, which has been purified from the culture medium using protein A affinity chromatography, has less than or equal to 0.1% aggregates. That is, at least 99.9% of the EGFR / PD-L1 FIT-Ig binding proteins of the present invention produced in mammalian cell culture will exist as fully assembled six-chain monomers. It is believed that the level of protein aggregates less than or equal to 0.1% (≤0.1%) is a non-significant amount, which does not hinder the effective preclinical and clinical evaluation of EGFR / PD-L1 FIT-Ig binding proteins as anticancer drugs. In contrast, as shown herein, the amount of aggregates found in other previously produced EGFR / PD-L1 and PD-1 / EGFR FIT-Ig binding proteins is 1.2% to greater than 70%. Therefore, in terms of aggregate formation, the EGFR / PD-L1 FIT-Ig binding proteins of the present invention are more stable and have a significantly lower percentage of aggregates (e.g., at least 10 times lower) than the previously produced FIT-Ig binding proteins that bind to EGFR and PD-L1. See Table 7 in Example 1.7 below.

[0151] Producing the EGFR / PD-L1 FIT-Ig binding protein of the present invention

[0152] The present invention provides a method for producing the EGFR / PD-L1 FIT-Ig binding protein described herein, comprising culturing an isolated host cell under conditions sufficient to produce the EGFR / PD-L1 FIT-Ig binding protein, the host cell comprising one or more vectors encoding three polypeptide chains of the EGFR / PD-L1 FIT-Ig binding protein. The desired EGFR / PD-L1 FIT-Ig binding protein is expressed as a six-polypeptide chain FIT-Ig binding protein comprising two outer EGFR-specific Fab binding units and two inner PD-L1-specific Fab binding units.

[0153] A variety of expression systems comprising expression vectors and compatible prokaryotic or eukaryotic host cells can be used to express recombinant heterologous proteins. An example of a prokaryotic host cell often used to express recombinant proteins is an Escherichia coli cell. Eukaryotic host cells that can be used to express recombinant proteins include, but are not limited to, mammalian host cells, insect host cells, plant host cells, fungal host cells, algae host cells, nematode host cells, protozoan host cells, and fish host cells. Fungal host cells that can be used to express recombinant proteins include, but are not limited to: Aspergillus, Neurospora, Saccharomyces cerevisiae, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces, Yarrowia, and Candida. The preferred Saccharomyces cerevisiae host cell for expressing recombinant proteins is a Saccharomyces cerevisiae cell. The insect cell used as a host cell according to the present invention is an insect Sf9 cell.

[0154] The FIT-Ig binding protein is preferably produced using a mammalian cell expression system. The construction and expression of the FIT-Ig binding protein has been previously described in International Publication Nos. WO 2015 / 103072 A1 and WO 2017 / 136820 A2. Similar materials and methods can be used to produce the EGFR / PD-L1 FIT-Ig binding protein of the present invention. Such methods are generally used to express recombinant antibodies and engineered binding proteins in selected host cells.

[0155] Typically, each polypeptide chain of the FIT-Ig binding protein is encoded on a separate nucleic acid molecule together with an amino-terminal signal sequence (signal peptide), which directs the nascent polypeptide chain to the lumen of the endoplasmic reticulum (ER) and then to the Golgi apparatus for secretion. Individual nucleic acid molecules encoding the polypeptide chains of the FIT-Ig binding protein described herein can be prepared using chemical DNA synthesis methods, using recombinant DNA methods, or using a combination of both methods.

[0156] Each nucleic acid molecule encoding one of the polypeptide chains of the FIT-Ig binding protein is then inserted into a separate expression vector, operably linked to appropriate transcription and / or translation sequences to allow expression of the polypeptide chain in a host cell compatible with the expression vector.

[0157] The vector may be an autonomously replicating vector or a vector that incorporates the isolated nucleic acid present in the vector into the host cell genome. Preferred vectors for expressing nucleic acids described herein include, but are not limited to, pcDNA, pTT (Durocher et al. Nucleic Acids Res., 30(2e9): 1-9 (2002)), pTT3 (pTT with additional multiple cloning sites), pEFBOS (Mizushima and Nagata, Nucleic Acids Res., 18(17): 5322 (1990)), pBV, pJV, pcDNA3.1TOPO, pEF6 TOPO and pBJ, as well as modifications thereof as needed to express the EGFR / PD-L1 FIT-Ig binding protein described herein in a specific host cell.

[0158] In a preferred embodiment of producing the FIT-Ig binding protein, the FIT-Ig binding protein is expressed in mammalian host cells transfected with three expression vectors, wherein each expression vector comprises a nucleic acid encoding one of the three component polypeptide chains of the FIT-Ig binding protein.

[0159] Preferably, the isolated mammalian host cell comprising the vector described herein is selected from the group consisting of Chinese hamster ovary (CHO) cells, COS cells, Vero cells, SP2 / 0 cells, NS / 0 myeloma cells, human embryonic kidney (HEK293) cells, baby hamster kidney (BHK) cells, HeLa cells, human B cells, CV-1 / EBNA cells, L cells, 3T3 cells, HEPG2 cells, PerC6 cells and MDCK cells.

[0160] Transfected HEK293 cells are often used as a transient transfection system, which can provide short-term production of recombinant proteins, including engineered antibodies and binding proteins, such as 4 to 10 days after transfection. Transfected HEK293 cells are often used for initial laboratory cloning, production and analysis of recombinant proteins, thereby avoiding the time and labor required to separate stably transfected production cell lines (e.g., stable transfected CHO cell lines). For technicians familiar with the production of engineered antibodies and binding proteins, the expression level of less than 10 mg / L in the culture of transiently transfected cells is too low to expect sufficient amounts of binding proteins to be used for preliminary preclinical evaluations, such as bioactivity studies, preliminary stability studies, pharmacokinetic (PK) studies, and efficacy in animal models. In addition, technicians in the field also recognize that the expression level of less than 10 mg / L in the culture of transiently transfected HEK293 cells indicates that even if a lot of time and effort is spent, it is unlikely to successfully separate CHO cells with high expression (e.g., greater than 1 g / L). In contrast, it is believed that the expression levels of FIT-Ig binding protein above 10 mg / L in cultures of transiently transfected HEK293 cells are high enough to provide a certain amount of binding protein for evaluation in the early discovery stage before generating stably transfected CHO cells, and also suggests that it may be possible to successfully isolate stably transfected CHO cells to generate higher quantities required for later preclinical and clinical stage evaluations.

[0161] As shown herein, the EGFR / PD-L1 FIT-Ig binding proteins according to the present invention can be expressed in transfected HEK293 cells at levels greater than 10 mg (>10 mg / L) of EGFR / PD-L1 FIT-Ig binding protein per liter of cell culture. This expression level is unexpected given that previously produced EGFR / PD-L1 and PD-1 / EGFR FIT-Ig binding proteins were only expressed at levels of about 1 mg / L to about 8 mg / L. In addition, compared to other FIT-Ig binding proteins, more than just a step-up in production levels, the expression levels of the EGFR / PD-L1 FIT-Ig binding proteins of the present invention in transfected HEK293 cell cultures ensure that the binding proteins can be used in sufficient quantities for preclinical and clinical evaluation as new anti-cancer therapeutics.

[0162] Pharmaceutical composition

[0163] The pharmaceutical composition of the present invention comprises the EGFR / PD-L1 FIT-Ig binding protein described herein and one or more pharmaceutically acceptable components, such as pharmaceutically acceptable carriers (carriers, buffers), excipients and / or other ingredients. "Pharmaceutically acceptable" means that the carrier, compound, component or other ingredient of the composition is compatible with the physiology of the human subject, and is harmless to the binding specificity of the desired EGFR / PD-L1 FIT-Ig binding protein, or any other desired properties or activities of any other component present in the composition administered to the human subject. Examples of pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In some cases, it is preferred to include isotonic agents, including but not limited to sugars; polyols, such as mannitol or sorbitol; sodium chloride; and combinations thereof.

[0164] The pharmaceutically acceptable compositions of the present invention may further comprise one or more excipients, small amounts of auxiliary substances, such as wetting agents or emulsifiers, fillers, preservatives or buffers, to extend the shelf life or effectiveness of the pharmaceutical composition. An excipient is generally any compound or combination of compounds that provides a pharmaceutical composition with beneficial properties or characteristics other than the main therapeutic compound or activity. For pharmaceutical compositions comprising the EGFR / PD-L1 FIT-Ig binding protein of the present invention (the main therapeutic compound), the excipient provides the desired beneficial characteristics other than the desired binding specificity of the EGFR / PD-L1 FIT-Ig binding protein or the anti-cancer activity caused by the EGFR / PD-L1 FTI-Ig binding protein.

[0165] In another embodiment, a pharmaceutical composition of the present invention comprises an EGFR / PD-L1 FIT-Ig binding protein as described herein, a pharmaceutically acceptable carrier, and an adjuvant, wherein the adjuvant provides a general stimulation of the human immune system.

[0166] The pH of a pharmaceutical composition may be adjusted as desired, for example, to promote or maintain solubility of a component ingredient, to maintain stability of one or more component ingredients in the formulation, and / or to prevent the growth of undesirable microorganisms that may have been introduced into the composition.

[0167] Pharmaceutical compositions comprising the EGFR / PD-L1 FTI-Ig binding proteins of the present invention can be prepared to provide sustained or delayed release of the binding proteins. Various methods for preparing such controlled-release or delayed compositions are known to those skilled in the art, including but not limited to implants, transdermal patches, and microcapsule delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and combinations thereof can also be used to prepare controlled-release or delayed compositions comprising the EGFR / PD-L1 FIT-Ig binding proteins of the present invention.

[0168] The pharmaceutical composition comprising the EGFR / PD-L1 FTI-Ig binding protein described herein may further comprise one or more other therapeutically active compounds (therapeutic agents). Examples of such other therapeutic agents that may be incorporated into the pharmaceutical composition of the present invention include, but are not limited to, anticancer agents different from the EGFR / PD-L1 FTI-Ig binding protein described herein (e.g., anticancer compounds containing cytotoxic metals or anticancer compounds based on cytotoxic radioisotopes and combinations thereof), antibiotics, antiviral compounds, sedatives, stimulants, local anesthetics, anti-inflammatory steroids (e.g., natural or synthetic anti-inflammatory steroids and combinations thereof), analgesics (e.g., acetylsalicylic acid, acetaminophen, naproxen, ibuprofen, COX-2 inhibitors, morphine, oxycodone and combinations thereof), antihistamines, nonsteroidal anti-inflammatory drugs ("NSAIDs", e.g., acetylsalicylic acid, ibuprofen, naproxen, COX-2 inhibitors and combinations thereof), and combinations thereof.

[0169] Preparation includes, but is not limited to, parenteral, intravenous (systemic), subcutaneous, intramuscular, oral (i.e., gastrointestinal), sublingual, cheek, intranasal (e.g., inhalation), transdermal (e.g., local), intratumoral, transmucosal, intraarticular, intrabronchial, intracapsular, intracartilaginous, intracavitary, intracervical, intrahepatic, intramyocardial, intraosseous, intrapelvic, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, vaginal, and rectal.

[0170] Preferably, the pharmaceutical composition according to the present invention is formulated for intravenous administration to a human subject with cancer. Intravenous administration of a pharmaceutical composition comprising an EGFR / PD-L1 FTI-Ig binding protein can provide the EGFR / PD-L1 FTI-Ig binding protein throughout the circulatory system, thereby reaching tissues and organs through the circulating blood. Typically, the composition for intravenous administration is a solution in a sterile, isotonic, aqueous buffer. If necessary, the composition may also include a solubilizer and a local anesthetic, such as lidocaine, to relieve pain at the injection site.

[0171] Subcutaneous administration of the pharmaceutical composition of the present invention is a route by which the EGFR / PD-L1 FTI-Ig binding protein can be provided to the lymphatic system. Therefore, a pharmaceutical composition comprising the EGFR / PD-L1 FTI-Ig binding protein of the present invention can be formulated for subcutaneous administration.

[0172] The pharmaceutical composition of the present invention can be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). The preparation for injection can be in unit dosage form (e.g., in an ampoule or in a multi-dose container) with an added preservative. The composition can be in the form of a suspension or solution or emulsion in an oily or aqueous vehicle, and can include formulation agents such as suspending agents, stabilizers and / or dispersants. Alternatively, the active ingredient (i.e., the EGFR / PD-L1 FIT-Ig binding protein of the present invention) can be in powder form (e.g., lyophilized form) and reconstituted with a suitable carrier (e.g., sterile, pyrogen-free water) before use.

[0173] The pharmaceutical composition of the present invention can be formulated into a reservoir preparation of a long-acting preparation type for delivery. Such long-acting preparations can be applied by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection. Therefore, for example, the pharmaceutical composition can be prepared with suitable polymerization or hydrophobic substances (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as an insoluble derivative (e.g., as an insoluble salt).

[0174] In another embodiment, the EGFR / PD-L1 FIT-Ig binding protein described herein may be a crystallized EGFR / PD-L1 FIT-Ig binding protein that retains the binding activity of the non-crystallized EGFR / PD-L1 FIT-Ig binding protein to EGFR and PD-L1. When administered to an individual, this crystallized EGFR / PD-L1 FIT-Ig binding protein may also provide carrier-free controlled release of the EGFR / PD-L1 FIT-Ig binding protein. Compared to the non-crystalline form, the crystallized EGFR / PD-L1 FIT-Ig binding protein of the present invention may also exhibit a greater in vivo half-life when administered to an individual. The crystallized binding protein of the present invention may be produced according to methods known in the art, such as disclosed in International Publication No. WO 02 / 072636 (Shenoy et al.), which is incorporated herein by reference.

[0175] A pharmaceutical composition for releasing a crystallized EGFR / PD-L1 FIT-Ig binding protein, wherein the composition comprises the crystallized EGFR / PD-L1 FIT-Ig binding protein described herein, an excipient component, and at least one polymer carrier. Preferably, the excipient component is selected from the group consisting of albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-β-cyclodextrin, methoxypolyethylene glycol, and polyethylene glycol. Preferably, the polymer carrier is a polymer selected from one or more of the following: poly(acrylic acid), poly(cyanoacrylate), poly(amino acid), poly(anhydride), poly(depsipeptide), poly(ester), poly(lactic acid), poly(lactic-co-glycolic acid) or PLGA, poly(β-hydroxybutyrate), poly(caprolactone), poly(dioxanone); polyethylene glycol, poly(hydroxypropyl)methacrylamide, poly[(organo)phosphazene], poly(orthoester), polyvinyl alcohol, poly(vinyl pyrrolidone), maleic anhydride / alkyl vinyl ether copolymer, pluronic polyol, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycosaminoglycans, sulfated polysaccharides, blends thereof and copolymers thereof.

[0176] Methods and uses of the EGFR / PD-L1 FIT-Ig binding protein of the present invention

[0177] The ability of EGFR / PD-1 FIT-Ig binding proteins to bind to EGFR and PD-L1 is of major interest in providing anticancer therapy. It is speculated that the binding of EGFR / PD-L1 FIT-Ig binding proteins to EGFR and PD-L1 will inhibit or block the binding of EGFR and PD-L1 to their respective ligands (e.g., EGFR and PD-1), thereby inhibiting or blocking their respective independent signaling pathways (i.e., EGFR / EGF signaling and PD-L1 / PD-1 signaling), which are involved in carcinogenesis, cancer cell growth, and cancer cell spread (metastasis). The EGFR / PD-L1 FIT-Ig binding proteins of the present invention are preferably capable of blocking human EGFR or human PD-L1 signaling activity in vitro and in vivo. Therefore, the EGFR / PD-L1 FIT-Ig binding proteins of the present invention can be used to inhibit or block human EGFR and / or human PD-L1 signaling in human subjects or in other mammalian subjects with EGFR and PD-L1 (with which the EGFR / PD-L1 FIT-Ig binding proteins of the present invention cross-react).

[0178] The method of inhibiting or blocking EGFR signaling in a cell comprises contacting a cell expressing EGFR with an EGFR / PD-L1 FIT-Ig binding protein of the present invention.

[0179] The method of inhibiting or blocking PD-L1 signaling in a cell comprises contacting a cell expressing PD-L1 with an EGFR / PD-L1 FIT-Ig combination.

[0180] The method of inhibiting or blocking EGFR signaling and PD-L1 signaling comprises contacting a cell population comprising cells expressing EGFR and cells expressing PD-L1 with an EGFR / PD-L1 FIT-Ig binding protein of the present invention.

[0181] In the above method, a useful FIT-Ig binding protein is an EGFR / PD-L1 FIT-Ig binding protein, which comprises a first (heavy) polypeptide chain comprising an amino acid sequence according to SEQ ID NO: 1; a second (first light) polypeptide chain comprising an amino acid sequence according to SEQ ID NO: 2; and a third (second light) polypeptide chain comprising an amino acid sequence according to SEQ ID NO: 3.

[0182] The present invention also provides a method for treating cancer in a human subject in need of treatment, the method comprising administering to the subject an EGFR / PD-L1 FIT-Ig binding protein or a pharmaceutical composition comprising the EGFR / PD-L1 FIT-Ig binding protein, wherein the EGFR / PD-L1 FIT-Ig binding protein comprises a first (heavy) polypeptide chain comprising an amino acid sequence according to SEQ ID NO: 1; a second (first light) polypeptide chain comprising an amino acid sequence according to SEQ ID NO: 2; and a third (second light) polypeptide chain comprising an amino acid sequence according to SEQ ID NO: 3.

[0183] In the method of treating cancer in a human subject according to the present invention, the cancer may be an epithelial cancer.

[0184] In another embodiment, the cancer treated in the methods of the invention is selected from the group consisting of melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignancies.

[0185] In one embodiment, the present invention provides a method for restoring the activity of activated T cells (reversing inhibition), the method comprising contacting a cell expressing human PD-L1 with the EGFR / PD-L1 FIT-Ig binding protein of the present invention, so that PD-L1 / PD-1-initiated T cell inhibition is inhibited. In another embodiment, the present invention provides a method for inhibiting carcinogenesis induced by EGFR / EGF binding, comprising contacting a cell expressing human EGFR with the EGFR / PD-L1 FIT-Ig binding protein of the present invention, so that EGFR / EGF-mediated signal transduction is inhibited or blocked.

[0186] In another embodiment, the invention provides a method for treating a human subject having a disease in which EGFR and / or PD-L1 activity is detrimental, the method comprising administering to the subject an EGFR / PD-L1 binding protein of the invention such that activity mediated by PD-L1 / PD1 binding and / or EGFR / EGF binding in the subject is reduced.

[0187] In the above method, a useful FIT-Ig binding protein is an EGFR / PD-L1 FIT-Ig binding protein, which comprises a first (heavy) polypeptide chain comprising an amino acid sequence according to SEQ ID NO: 1; a second (first light) polypeptide chain comprising an amino acid sequence according to SEQ ID NO: 2; and a third (second light) polypeptide chain comprising an amino acid sequence according to SEQ ID NO: 3.

[0188] As used herein, the term "disease in which EGFR and / or PD-L1 activity is detrimental" is intended to include diseases in which the interaction of EGFR with its ligand (EGR) or the interaction of PD-L1 with its ligand (PD-1) in a subject suffering from the disorder is a cause of the pathophysiology of the disease or a factor that contributes to the exacerbation of the disease. Thus, a disease in which EGFR and / or PD-L1 activity is detrimental is a disease in which inhibition of EGFR and / or PD-L1 activity is expected to alleviate the symptoms and / or progression of the disease.

[0189] Considering that the EGFR / PD-L1-FIT-Ig binding protein of the present invention binds to human EGFR and PD-L1, the EGFR / PD-L1 binding protein can also be used, for example, to detect EGFR or PD-L1, or both, in a biological sample containing cells expressing one or both of those target proteins. For example, the EGFR / PD-L1 binding protein of the present invention can be used in conventional immunoassays, such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), or immunohistochemistry of tissues. The present invention provides a method for detecting EGFR or PD-L1 in a biological sample, the method comprising contacting the biological sample with the EGFR / PD-L1 FIT-Ig binding protein of the present invention, and detecting whether binding to the target antigen (EGFR or PD-L1) occurs, thereby detecting whether the target is present in the biological sample. The binding protein can be directly or indirectly labeled with a detectable substance to facilitate the detection of bound or unbound antibodies / fragments / binding proteins. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent substances include luminol; examples of suitable radioactive substances include 3 H. 14 C. 35 S. 90 Y. 99 Tc, 111 In, 125 I. 131 I. 177 Lu, 166 Ho or 153 Sm.

[0190] Having now described the present invention in detail, the invention will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to limit the present invention.

[0191] Example

[0192] Example 1: Generation of FIT-Ig binding protein that binds EGFR and PD-L1.

[0193] Six bispecific Fabs-in-Tandem immunoglobulin (FIT-Ig) binding proteins recognizing human EGFR and human PD-L1 were constructed using the binding sites from anti-PD-L1 and anti-EGFR parental antibodies.

[0194] Anti-PD-L1 monoclonal antibodies (mAbs) 1B12, 10A5, and 3G10 have been previously described. See, e.g., U.S. Pat. No. 7,943,743 B2.

[0195] The use of specific amino acid sequences of the anti-EGFR mAb panitumumab to prepare FIT-Ig binding proteins has been previously described. See, for example, International Publication No. WO 2017 / 136820 A2.

[0196] Example 1.1: FIT-Ig1.

[0197] Using the coding sequences from the immunoglobulin domains of the parental antibody mAb 1B12 and panitumumab, a PD-L1 / EGFR FIT-Ig named "FIT-Ig1" (also referred to as "PD-L1 / EGFR FIT-Ig1") was constructed. FIT-Ig1 is a hexamer composed of three component polypeptide chains:

[0198] Polypeptide chain #1 has the domain formula: VL 1B12 -CL directly fused to VH pani -CH1, while VH pani -CH1 is fused directly to the hinge -CH2-CH3 (human IgG1 Fc region);

[0199] Polypeptide chain #2 has the domain formula: VH 1B12 -CH1; and

[0200] Polypeptide chain #3 has the domain formula: VL pani -CL.

[0201] The amino acid sequences of the three expressed FIT-Ig1 polypeptide chains, including the N-terminal signal sequence, are shown in Table 1 below:

[0202] Table 1 Amino acid sequences of FIT-Ig1 component polypeptide chains

[0203]

[0204]

[0205]

[0206] Example 1.2: FIT-Ig2

[0207] The EGFR / PD-L1 FIT-Ig named "FIT-Ig2" (also referred to as "EGFR / PD-L1 FIT-Ig2") was constructed using the coding sequences of the immunoglobulin domains from the parental antibodies panitumumab and mAb 1B12. FIT-Ig2 is a hexamer composed of three component polypeptide chains:

[0208] Polypeptide chain #1 has the domain formula: VL pani -CL directly fused to VH 1B12 -CH1, while VH 1B12 -CH1 is fused directly to the hinge -CH2-CH3 (human IgG1 Fc region);

[0209] Polypeptide chain #2 has the domain formula: VH pani -CH1; and

[0210] Polypeptide chain #3 has the domain formula: VL 1B12 -CL.

[0211] The amino acid sequences of the three expressed FIT-Ig2 polypeptide chains, including the N-terminal signal sequence, are shown in Table 2 below:

[0212] Table 2 Amino acid sequences of FIT-Ig2 component polypeptide chains

[0213]

[0214]

[0215] Example 1.3: FIT-Ig3

[0216] Using the coding sequences of the immunoglobulin domains from the parental antibodies 10A5 and panitumumab, a PD-L1 / EGFR FIT-Ig named "FIT-Ig3" (also referred to as "PD-L1 / EGFR FIT-Ig3") was constructed. FIT-Ig3 is a hexamer composed of three component polypeptide chains:

[0217] Polypeptide chain #1 has the domain formula: VL 10A5 -CL directly fused to VH pani -CH1, while VH pani -CH1 is fused directly to the hinge -CH2-CH3 (human IgG1 Fc region);

[0218] Polypeptide chain #2 has the domain formula: VH 10A5 -CH1; and

[0219] Polypeptide chain #3 has the domain formula: VL pani -CL.

[0220] The amino acid sequences of the three expressed FIT-Ig3 polypeptide chains, including the N-terminal signal sequence, are shown in Table 3 below:

[0221] Table 3 Amino acid sequences of FIT-Ig3 component polypeptide chains

[0222]

[0223]

[0224] Example 1.4: FIT-Ig4

[0225] The EGFR / PD-L1 FIT-Ig named "FIT-Ig4" (also referred to as "EGFR / PD-L1 FIT-Ig4") was constructed using the coding sequences of the immunoglobulin domains from the parental antibodies panitumumab and mAb 10A5. FIT-Ig4 is a hexamer composed of three component polypeptide chains:

[0226] Polypeptide chain #1 has the domain formula: VL pani -CL directly fused to VH 10A5 -CH1, while VH 10A5 -CH1 is fused directly to the hinge -CH2-CH3 (human IgG1 Fc region);

[0227] Polypeptide chain #2 has the domain formula: VH pani -CH1; and

[0228] Polypeptide chain #3 has the domain formula: VL 10A5 -CL.

[0229] The amino acid sequences of the three expressed FIT-Ig4 polypeptide chains, including the N-terminal signal sequence, are shown in Table 4 below:

[0230] Table 4 Amino acid sequences of FIT-Ig4 component polypeptide chains

[0231]

[0232]

[0233] Example 1.5: FIT-Ig5

[0234] Using the coding sequences of the immunoglobulin domains from the parental antibodies mAb 3G10 and panitumumab, a PD-L1 / EGFR FIT-Ig named "FIT-Ig5" (also referred to as "PD-L1 / EGFR FIT-Ig5") was constructed. FIT-Ig5 is a hexamer composed of three component polypeptide chains:

[0235] Polypeptide chain #1 has the domain formula: VL 3G10 -CL directly fused to VH pani -CH1, while VH pani -CH1 is fused directly to the hinge -CH2-CH3 (human IgG1 Fc region);

[0236] Polypeptide chain #2 has the domain formula: VH 3G10 -CH1; and

[0237] Polypeptide chain #3 has the domain formula: VLpani-CL.

[0238] The amino acid sequences of the three expressed FIT-Ig5 polypeptide chains, including the N-terminal signal sequence, are shown in Table 5 below:

[0239] Table 5 Amino acid sequences of FIT-Ig5 component polypeptide chains

[0240]

[0241]

[0242]

[0243] Example 1.6: FIT-Ig6

[0244] The EGFR / PD-L1 FIT-Ig named "FIT-Ig6" (also referred to as "EGFR / PD-L1 FIT-Ig6") was constructed using the coding sequences of the immunoglobulin domains from the parental antibodies panitumumab and mAb 3G10. FIT-Ig6 is a hexamer composed of three component polypeptide chains:

[0245] Polypeptide chain #1 has the domain formula: VL pani -CL directly fused to VH 3G10 -CH1, while VH 3G10 -CH1 is fused directly to the hinge -CH2-CH3 (human IgG1 Fc region);

[0246] Polypeptide chain #2 has the domain formula: VH pani -CH1; and

[0247] Polypeptide chain #3 has the domain formula: VL 3G10 -CL.

[0248] The amino acid sequences of the three expressed FIT-Ig6 polypeptide chains, including the N-terminal signal sequence, are shown in Table 6 below:

[0249] Table 6 Amino acid sequences of FIT-Ig6 component polypeptide chains

[0250]

[0251]

[0252]

[0253] Example 1.7: Expression of FIT-Ig binding protein

[0254] The six FIT-Ig constructs FIT-Ig1, FIT-Ig2, FIT-Ig3, FIT-Ig4, FIT-Ig5, FIT-Ig6 are a class of bispecific multivalent binding proteins, which are known as linkerless Fabs-in-Tandem immunoglobulins (or linkerless FIT-Ig), generally described in WO 2015 / 103072 and WO 2017 / 136820. The binding protein is produced by co-expressing the three component polypeptide chains in a mammalian host cell transfected with expression vectors for all three chains. The design of the binding protein requires that the first polypeptide chain (or "heavy chain") is paired with the second polypeptide chain (or "first light chain") and the third polypeptide chain (or "second light chain") to form a functional tandem Fab portion, and the heavy chain is also designed to dimerize through the Fc region (hinge-CH2-CH3), thereby forming a six-chain binding protein showing four complete Fab binding sites. No synthetic amino acid linker peptides were used to connect the immunoglobulin domains, hence the name "linkerless FIT-Igs"; such binding proteins were found to express well in host cells, similar to recombinantly produced monoclonal antibodies, and the absence of a linker prevented the introduction of possible immunogenic sites, which could result in faster clearance of FIT-Igs with a linker. It was also found that the linkerless FIT-Ig exhibited binding properties to its target antigen comparable to the parent antibody, wherein the steric hindrance between the "inner" and "outer" binding sites found in previously engineered antibodies with tandemly arranged antigen binding sites was unexpectedly avoided based on VH-CH1 and VL-CL. However, as shown herein, despite the use of linkerless FIT-Ig models to be effectively used in conventional preclinical and clinical assays required to evaluate therapeutic anticancer drugs, previous FIT-Ig protein constructs that bind PD-L1 and EGFR (e.g., FIT-Ig 1-5) exhibited abnormally low levels of expression and / or an undesirably high percentage of aggregates.

[0255] In the binding proteins FIT-Ig1, FIT-Ig3, and Fit-Ig5, the N-terminal or "outer" Fab binding site binds PD-1, while the adjacent "inner" Fab binding site binds EGFR. The outer Fab fragment of (anti-PD-L1 mAb 1B12, 10A5, or 3G10) is connected to the inner Fab fragment of (anti-EGFR panitumumab) only via the heavy chain as follows, i.e., by direct fusion of the VL-CL of (anti-PD-L1 mAb 1B12, 10A5, or 3G10) at its C-terminus to the N-terminus of the VH-CH1 of (anti-EGFR panitumumab), without the use of a linker connecting the immunoglobulin domains.

[0256] In the binding proteins FIT-Ig2, FIT-Ig4, and Fit-Ig6, the N-terminal or "outer" Fab binding site binds EGFR, while the adjacent "inner" Fab binding site binds PD-L1. The outer Fab fragment of (anti-EGFR panitumumab) is linked to the inner Fab fragment of (anti-PD-L1 mAb 1B12, 10A5, or 3G10) via the heavy chain only as follows, i.e., by direct fusion of the VL-CL of (anti-EGFR panitumumab) at its C-terminus to the N-terminus of the VH-CH1 of (anti-PD-L1 mAbs 1B12, 10A5, or 3G10), without the use of a linker connecting the immunoglobulin domains.

[0257] Each FIT-Ig was transiently expressed using transfected human embryonic kidney 293E (HEK293) cells. The HEK293E cell line is a derivative of HEK293 that expresses EBNA-1 and provides increased levels of expression of the recombinant protein encoded by the vector.

[0258] The expression vector allows expression of each of the three polypeptide chains of any FIT-Ig binding protein, wherein the first (heavy) polypeptide chain has the following structural formula: VL A -CL-VH B -CH1-Fc, the second (first light chain) polypeptide chain has the following structural formula: VH A -CH1, and the third (second light) polypeptide chain has the following structural formula: VL B -CL, where VL A and VH A is the variable domain of the antigen binding site of the first parent antibody, and VL B and VH B It is the variable domain of the antigen binding site of the second parent antibody.

[0259] like Figure 1 As shown in FIG, to express the first polypeptide chain (“heavy chain”) of the FIT-Ig binding protein, a VL encoding the first polypeptide chain was synthesized. A -CL-VHB The DNA molecule of the fragment is synthesized ("DNA synthesis"). The DNA molecule is then inserted into the multiple cloning site (MCS) of the pcDNA3.1 expression vector in the E. coli cells using homologous recombination. This homologous recombination method relies on the principle that recombinase-positive E. coli cells can recombine homologous sequences with high specificity and high speed. A linear DNA fragment containing the coding sequence of interest is generated by polymerase chain reaction (PCR) to include sequences on the 5' and 3' ends that are homologous to the terminal sequences on the linearized vector. When the PCR product and the linear vector are mixed and transformed into competent E. coli cells, the endogenous bacterial recombinase activity is able to connect the two DNA fragments to form a circular plasmid. The inserted DNA molecule is then positioned downstream of the strong cytomegalovirus (CMV) enhancer promoter of the vector, downstream of the DNA fragment encoding the amino-terminal signal peptide (SP), and in its reading frame, and upstream and in the reading frame of the inserted DNA molecule encoding the antibody CH1 domain, which is connected to the antibody Fc region containing the hinge region-CH2-CH3 domain (named Figure 1 in the “h-CH2-CH3”).

[0260] Also like Figure 1 As shown, in order to express the second polypeptide chain ("light chain #1") of the FIT-Ig binding protein, the encoding antibody VH A The DNA fragment encoding the antibody CH1 domain is then inserted into the multiple cloning site (MCS) of the pcDNA3.1 expression vector so that the inserted DNA molecule is positioned downstream of the strong CMV enhancer promoter of the vector, downstream of the DNA fragment encoding the amino-terminal signal peptide (SP) and within its reading frame, and upstream and within the reading frame of the inserted DNA molecule encoding the antibody CH1 domain.

[0261] like Figure 1 As shown, in order to express the third polypeptide chain (light chain #3) of the FIT-Ig binding protein, the encoding antibody VH B The DNA fragment encoding the antibody CL domain is then inserted into the multiple cloning site (MCS) of the pcDNA3.1 expression vector so that the inserted DNA molecule is positioned downstream of the strong CMV enhancer promoter of the vector, downstream of the DNA fragment encoding the amino-terminal signal peptide (SP) and within its reading frame, and upstream and within the reading frame of the inserted DNA molecule encoding the antibody CL domain.

[0262] The sequence of the resulting expression vector was confirmed by DNA sequencing.

[0263] The resulting expression vector encoding the three component polypeptide chains of each FIT-Ig was transfected into HEK293E cells using a molar ratio of heavy chain: light chain #1: light chain #2 of 1:3:3. This was designed to express more light chains #1 and #2 in proportion to the heavy chain, which in turn would reduce the appearance of VL-CL and VH-CH1 fragments on the heavy chain that are not paired with the corresponding light chain and thus would not form a functional Fab fragment. See WO 2015 / 103072. HEK293E cells were transfected with the expression vector using polyethyleneimine (PEI) as a transfection agent. In this transfection protocol, FreeStyle TM The expression vector in 293 expression medium was mixed with PEI, where the final concentration ratio of DNA to PEI was 1:2, incubated at room temperature for 15-20 minutes, and then added to HEK293E cells (1.0-1.2×10 6 / ml, cell viability>95%). After 6-24 hours of culture in a shaker, peptone was added to the transfected cells at a final concentration of 5%, at 37°C, under 8% CO2, and shaken at 125rpm / min. On days 6-7, the supernatant was collected by centrifugation and filtration, and the FIT-Ig protein was purified using protein A chromatography (GE healthcare, US) according to the manufacturer's instructions. The protein was analyzed by SDS-PAGE, and its concentration was determined by UV absorbance at 280nm and bicinchoninic acid protein assay (BCA) (Pierce BCA protein assay kit, ThermoFisher Scientific).

[0264] The FIT-Ig protein expression product was purified by protein A chromatography. The composition and purity of the purified FIT-Ig were then analyzed by size exclusion chromatography (SEC). The PBS containing the purified FIT-Ig was applied to a TSKgel SuperSW3000, 300×4.6mm column (TOSOH). HPLC instrument, U3000 type (DIONEX) was used for SEC, using UV detection at 280nm and 214nm. In addition to the six polypeptide chain FIT-Ig6 monomer (molecular weight of 240,000 Daltons), the substances detected by SEC, including larger (higher molecular weight) aggregates and smaller substances (including fragments of FIT-Ig6 monomers) were impurities.

[0265] Figure 2-7 The SEC elution profiles of FIT-Ig1 to FIT-Ig6 are shown respectively.

[0266] Figure 2The SEC elution profile of FIT-Ig1 shown in reveals multiple overlapping peaks of protein aggregates. The profile is too complex to allow for detailed analysis of the material in a single peak.

[0267] Figure 3 The SEC elution profile of FIT-Ig2 shown in shows a major peak at the expected position of the hexapolypeptide FIT-Ig2 monomer, preceded by at least two other peaks of protein aggregates.

[0268] Figure 4 The SEC elution profile of FIT-Ig3 shown in Figure 2 shows multiple overlapping peaks of protein aggregates. The profile is too complex to allow for detailed analysis of the material in a single peak.

[0269] Figure 5 The SEC elution profile of FIT-Ig4 shown in shows a major peak at the expected position of the hexapeptide FIT-Ig4 monomer, preceded by a minor peak of protein aggregates, and followed by another minor peak of an unknown substance (possibly a degradation product).

[0270] Figure 6 The SEC elution profile of FIT-Ig5 shown in Figure 2 shows that there is a major peak at the expected position of the hexapeptide FIT-Ig5 monomer, which is significantly smaller than the previous minor peak of protein aggregates. Figure 2-5 Any minor peaks of aggregates found in the

[0271] Figure 7 The SEC elution profile of FIT-Ig6 shown in shows a major peak at the expected position of the six-polypeptide FIT-Ig6 monomer, preceded by an almost undetectable peak in the region of protein aggregates. After one-step purification using protein A affinity chromatography, the FIT-Ig6 binding protein was clearly obtained as a substantially completely homogeneous product without significant aggregate formation. The percentage of aggregates was estimated to be less than or equal to 0.1% (≤0.1%). FIT-Ig6 clearly outperformed all other FIT-Igs without significant percentages of aggregates.

[0272] The expression and SEC data of FIT-Ig 1-6 are shown in Table 7 below.

[0273] Table 7: Expression and SEC analysis of FIT-Ig binding proteins

[0274]

[0275]

[0276] The above data show that:

[0277] FIT-Ig 1, 2, and 3 exhibited abnormally high percentages of aggregates and unacceptably low levels of expression in transfected HEK293 cell cultures and therefore could not provide the protein amounts (no aggregates or insignificant, low percentages of aggregates) and quantities required for further preclinical evaluation as therapeutics.

[0278] The percentage of aggregates for FIT-Ig4 was lower than that for FIT-Ig 1, 2, and 3, but the level was not insignificant for drug development. In addition, FIT-Ig4 showed unacceptably low levels of expression. Therefore, FIT-Ig4 also failed to provide the protein quantity and quality required for further preclinical evaluation as a therapeutic drug.

[0279] Aggregation and expression levels of FIT-Ig5 were almost acceptable, however, expression levels below 10 mg / ml indicate that efforts to isolate stably transfected CHO cell lines to obtain the quantities required for preclinical and clinical evaluation will not be successful or cost-effective.

[0280] FIT-Ig6 surprisingly showed high levels of expression in transfected HEK293 cell cultures without significant amounts of aggregate formation (≤0.1%). Thus, FIT-Ig6 was more stable than FIT-Ig5 in terms of aggregate formation after one-step purification using protein A affinity chromatography, and the percentage of aggregates was at least 10-fold lower. The expression levels of FIT-Ig6 in mammalian cell culture and the unusually low levels of aggregates qualify this binding protein as a candidate for preclinical and clinical evaluation as an anticancer therapeutic.

[0281] The special properties of FIT-Ig6 are due to:

[0282] 1. Use the anti-PD-L1 mAb 3G10 as the source of VH and VL domains that form the PD-L1-specific antigen binding site in each PD-L1-specific Fab binding unit of FIT-Ig6,

[0283] 2. Use panitumumab anti-EGFR mAb as the source of VH and VL domains that form the EGFR-specific antigen binding site in each EGFR-specific Fab binding unit of FIT-Ig6,

[0284] 3. Positioning the EGFR-specific Fab binding unit as the external Fab binding unit of FIT-Ig6, and

[0285] 4. Position the PD-L1-specific Fab binding unit as the internal Fab binding unit of FIT-Ig6.

[0286] Example 2: Binding affinity of FIT-Ig5 and FIT-Ig6.

[0287] The binding affinities of parental anti-EGFR panitumumab, parental anti-PD-L1 mAb 3G10, FIT-Ig5, and FIT-Ig6 were determined by biolayer interferometry. The affinity and binding kinetics of each parent mAb and FIT-Ig were characterized by RED96 biolayer interferometry (Pall FortéBio LLC). Each parent mAb and FIT-Ig were captured at a concentration of 100nM for 30 seconds by anti-human IgG Fc capture (AHC) biosensor (Pall). The sensor was then immersed in running buffer (1X, pH 7.2, PBS, 0.05% Tween 20, 0.1% BSA) for 60 seconds to check the baseline. Binding was measured by immersing the sensor in a single concentration of recombinant human PD-L1 (Novoprotein) or recombinant human EGFR (SinoBiological Inc) ranging from 1nM to 200nM. Afterwards, the sensor was immersed in running buffer for 1200 seconds to dissociate. The binding and dissociation curves were fitted to a 1: 1 Langmuir binding model using FortéBio Data Analysis software (Pall). The results are shown in Table 8.

[0288] Table 8 Binding affinity of parental mAb and FIT-Ig to PD-L1 and EGFR

[0289]

[0290] The results showed that the binding affinities of FIT-Ig5 and FIT-Ig6 for EGFR and PD-L1 target antigens were similar to those of the parent panitumumab and the parent mAb 3G10, respectively. D K D The K of FIT-Ig6 for PD-L1 was about 25% lower. D Compared with the parental mAb 3G10, the K D The K values ​​of FIT-Ig6 and parental mAb were about 30% higher. D These levels of differences in values ​​may be due to differences in the assays. Thus, FIT-Ig6 has substantially the same affinity for EGFR and PD-L1 as each parent antibody from which the corresponding specificity was derived (ie, the same as or within 30% of the affinity of each affinity antibody).

[0291] Therefore, these data indicate that the FIT-Ig6 binding protein retains the binding affinity of the parent mAb. In addition, the binding affinity of the FIT-Ig6 binding protein is acceptable for continuing the preclinical and clinical evaluation of FIT-Ig6 as an anticancer drug.

[0292] Example 3. Pharmacokinetic study of FIT-Ig6 in male Sprague-Dawley rats.

[0293] The pharmacokinetic properties of FIT-Ig6 were evaluated in male Sprague-Dawley (SD) rats. FIT-Ig protein was administered to male SD rats at a single intravenous dose of 5 mg / kg. Serum samples were collected at different time points during 28 days, with continuous blood sampling through the tail vein at 0 minutes, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 2 days, 4 days, 7 days, 10 days, 14 days, 21 days and 28 days, and analyzed by conventional ELISA. In brief, ELISA plates were coated with 125 ng / well goat anti-human IgG Fc antibody (Rockland, catalog number: 609-101-017) at 4 ° C overnight, with 1X PBS / 1% BSA / 0.05% Tween-20 / 0.05% ProClin TM 300 blocking. All serum samples were first diluted 20 times in blocking buffer. Additional dilutions were made in 5% pooled rat serum and incubated on the plates at 37°C for 60 minutes. Detection was performed with peroxidase-conjugated anti-human IgG (Fab fragment) (Sigma; catalog number A0293), and concentrations were determined by standard curves using a four-parameter logarithmic fit. The values ​​of pharmacokinetic parameters were determined by a non-compartmental model using WinNonlin software (Pharsight Corporation, Mountain View, Calif.).

[0294] Figure 8 Shown is a graph of serum concentration of FIT-Ig6 versus time in three SD rats.

[0295] right Figure 8 Analysis of results from two of the animals shown (Rat #1 and Rat #3) produced the PK parameters shown below in Table 9. (The complete data set for Rat #2 could not be analyzed along with Rat #1 and Rat #3 due to unresolved issues associated with the first two data points, which were excluded by the software despite the remaining time points being within the typical range).

[0296] Table 9 PK parameters of FIT-Ig6 in male Sprague-Dawley rats

[0297] PK parameters unit Rat #1 Rat #3 average value CL mL / day / kg 7.52 6.02 6.77 Vss mL / kg 101 105 103 V1 mL / kg 58.0 56.0 57.0 <![CDATA[Alpha t 1 / 2 ]]> sky 0.148 0.189 0.168 <![CDATA[Beta t 1 / 2 ]]> sky 9.43 12.3 10.9 AUC day μg / mL 665 830 747 MRT sky 13.4 17.5 15.5

[0298] CL (total clearance gap), Vss (steady-state distribution volume), V1 (initial volume distribution), αt 1 / 2 (distribution half-life), βt 1 / 2 (elimination half-life), AUC (area under the curve), MRT (mean residence time).

[0299] The above PK data showed that FIT-Ig6 was stable in SD rats and had similar PK parameters to conventional mAbs.

[0300] Importantly, the relatively long elimination half-life of FIT-Ig6 (βt 1 / 2 =10.9 days) and low clearance (CL = 6.77 mL / day / kg) would allow it to be used for chronic indications with less frequent dosing, similar to therapeutic mAbs.

[0301] The contents of all references (including literature references, patents, patent applications and websites) cited throughout this application are hereby expressly incorporated by reference in their entirety. Unless otherwise indicated, the practice of the present invention will employ conventional techniques of immunology, molecular biology and cell biology well known in the art.

[0302] Without departing from the basic features of the present invention described above, the present invention may be implemented in other specific forms. Therefore, the foregoing embodiments should be considered illustrative rather than limiting the invention described herein. Therefore, the scope of the present invention is indicated by the appended claims rather than by the foregoing description, and it is therefore intended that all changes falling within the equivalent meaning and scope of the claims be included.

Claims

1. A Fabs-In-Tandem immunoglobulin FIT-Ig binding protein, which binds to EGFR and PD-L1, and comprises a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, wherein: The first polypeptide chain has a structure from the amino terminal to the carboxyl terminal of VL EGFR -CL-VH PD-L1 -CH1-Fc, where VL EGFR is the antibody light chain variable domain of the first parent antibody that binds to EGFR, CL is the antibody light chain constant domain, VH PD-L1 It is the antibody heavy chain variable domain of the second parent antibody that binds to PD-L1, CH1 is the first constant region of the antibody heavy chain, and Fc is the antibody Fc region; wherein CL is directly fused to VH PD-L1 , wherein no artificial linker is inserted between the variable domain and the constant domain, and wherein: V L EGFR The sequence is shown as amino acid residues 1-107 of SEQ ID NO: 1, VH PD-L1 The sequence is shown as amino acid residues 215-331 of SEQ ID NO: 1; The second polypeptide chain has a structure from the amino terminus to the carboxyl terminus of VH EGFR -CH1, where VH EGFR is the antibody heavy chain variable domain of the first parent antibody that binds to EGFR, wherein CH1 is the first constant domain of the antibody heavy chain, wherein in VH EGFR There is no artificial joint inserted between CH1 and CH2, and wherein: VH EGFR The sequence is shown as amino acid residues 1-119 of SEQ ID NO: 2, The third polypeptide chain has a structure from the amino terminus to the carboxyl terminus of VL PD-L1 -CL, where VL PD-L1 is the light chain variable domain of the second parent antibody that binds to PD-L1, wherein CL is an antibody light chain constant domain, wherein in VL PD-L1 There is no artificial joint inserted between and CL, and wherein: V L PD-L1 The sequence is shown as amino acid residues 1-107 of SEQ ID NO:

3.

2. The FIT-Ig binding protein according to claim 1, wherein the binding protein is a six-polypeptide chain binding protein, comprising two of the first polypeptide chains, two of the second polypeptide chains, and two of the third polypeptide chains, wherein the polypeptide chains are combined to form four Fab binding units, wherein two Fab binding units bind to EGFR and two Fab binding units bind to PD-L1. 3 . The FIT-Ig binding protein according to claim 1 , wherein the antibody CL domains in the first polypeptide chain and the third polypeptide chain are derived from human IgG1 antibodies. 4 . The FIT-Ig binding protein according to claim 1 , wherein the antibody CL domain in the first polypeptide chain and the third polypeptide chain comprises amino acid residues 108-214 of SEQ ID NO:

1. 5 . The FIT-Ig binding protein according to claim 1 or 2 , wherein the antibody CH1 domain present in the first polypeptide chain and the second polypeptide chain is derived from a human IgG1 antibody. 6 . The FIT-Ig binding protein according to claim 1 or 2 , wherein the antibody CH1 domain present in the first polypeptide chain and the second polypeptide chain comprises amino acid residues 332-434 of SEQ ID NO:

1. 7 . The FIT-Ig binding protein according to claim 1 or 2 , wherein the antibody Fc present in the first polypeptide chain is derived from a human IgG1 antibody.

8. The FIT-Ig binding protein according to claim 1 or 2, wherein the antibody Fc present in the first polypeptide chain comprises amino acid residues 435-661 of SEQ ID NO:

1.

9. The FIT-Ig binding protein according to claim 1 or 2, wherein: The first polypeptide chain is represented by the amino acid sequence of SEQ ID NO: 1; The second polypeptide chain is represented by the amino acid sequence of SEQ ID NO: 2; and The third polypeptide chain is shown in the amino acid sequence of SEQ ID NO:

3.

10. The FIT-Ig binding protein of claim 1, wherein the FIT-Ig binding protein has a human glycosylation pattern. 11 . A composition comprising the FIT-Ig binding protein according to claim 1 , wherein the composition comprises less than or equal to 0.1% of FIT-Ig binding protein aggregates. 12 . A pharmaceutical composition comprising the FIT-Ig binding protein according to claim 1 and a pharmaceutically acceptable carrier.

13. The pharmaceutical composition according to claim 12, further comprising one or more other therapeutically active compounds.

14. The pharmaceutical composition of claim 13, wherein the one or more other therapeutically active compounds are selected from the group consisting of anticancer compounds containing cytotoxic metals, anticancer compounds based on cytotoxic radioisotopes, antibiotics, antiviral compounds, sedatives, stimulants, local anesthetics, anti-inflammatory steroids, analgesics, antihistamines, non-steroidal anti-inflammatory drugs, and combinations thereof.

15. The pharmaceutical composition of claim 14, wherein the anti-inflammatory steroid is a natural anti-inflammatory steroid, a synthetic anti-inflammatory steroid, or a combination thereof.

16. The pharmaceutical composition of claim 14, wherein the analgesic is selected from the group consisting of acetylsalicylic acid, acetaminophen, naproxen, ibuprofen, COX-2 inhibitors, morphine, oxycodone, and combinations thereof.

17. The pharmaceutical composition according to claim 14, wherein the nonsteroidal anti-inflammatory drug is selected from the group consisting of acetylsalicylic acid, ibuprofen, naproxen, COX-2 inhibitors and combinations thereof.

18. A composition for releasing a crystallized FIT-Ig binding protein, comprising the crystallized FIT-Ig binding protein according to any one of claims 1 to 10, an excipient component and at least one polymer carrier.

19. The composition according to claim 18, wherein the excipient component is selected from the group consisting of albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-β-cyclodextrin and polyethylene glycol.

20. The composition of claim 19, wherein the polyethylene glycol is methoxypolyethylene glycol.

21. The composition of claim 18, wherein the polymer carrier is a polymer selected from one or more of the following: polyacrylic acid, polyamino acid, polyanhydride, polydepsipeptide, polyester, polylactic acid, polylactic-glycolic acid copolymer or PLGA, polydioxanone; polyethylene glycol, polyhydroxypropylmethacrylamide, polyorganophosphazene, polyvinyl alcohol, polyvinyl pyrrolidone, maleic anhydride / alkyl vinyl ether copolymer, pluronic polyol, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycosaminoglycans, sulfated polysaccharides, blends thereof, and copolymers thereof.

22. An isolated nucleic acid molecule, It encodes the following polypeptide chain: A first polypeptide chain as shown in the amino acid sequence of SEQ ID NO: 1; A second polypeptide chain as shown in the amino acid sequence of SEQ ID NO: 2; and The third polypeptide chain is shown in the amino acid sequence of SEQ ID NO:

3.

23. A vector comprising one or more isolated nucleic acid molecules according to claim 22.

24. The vector of claim 23, wherein the vector is an expression vector and the one or more isolated nucleic acids are operably linked to transcription and translation sequences that allow expression of the encoded one or more polypeptide chains.

25. The vector according to claim 24, which is selected from the group consisting of pcDNA, pTT, pEFBOS, pBV, pJV, pEF6 TOPO and pBJ.

26. The vector according to claim 25, wherein the pcDNA vector is a pcDNA3.1 vector.

27. The vector according to claim 26, wherein the pcDNA3.1 vector is a pcDNA3.1TOPO vector.

28. The vector according to claim 25, wherein the pTT vector is pTT3.

29. An isolated host cell comprising one or more vectors according to claim 24.

30. An isolated host cell comprising one or more expression vectors, wherein the one or more vectors encode three polypeptide chains forming the FIT-Ig binding protein according to claim 1.

31. The isolated host cell of claim 30, wherein the host cell is an isolated prokaryotic host cell.

32. The isolated host cell of claim 30, wherein the host cell is an isolated eukaryotic host cell.

33. The isolated eukaryotic host cell of claim 32, wherein the isolated eukaryotic host cell is an isolated mammalian host cell.

34. The isolated mammalian host cell of claim 33, wherein the mammalian host cell is selected from the group consisting of: CHO cells, COS cells, Vero cells, SP2 / 0 cells, NS / 0 myeloma cells, HEK293 cells, BHK cells, HeLa cells, human B cells, CV-1 / EBNA cells, L cells, 3T3 cells, HEPG2 cells, PerC6 cells, and MDCK cells.

35. A method of producing an EGFR / PD-L1 FIT-Ig binding protein, comprising culturing the isolated mammalian host cell of claim 33 under conditions sufficient to produce the EGFR / PD-L1 FIT-Ig binding protein.

36. The method according to claim 35, wherein the mammalian host cell is a HEK293 cell.

37. The method of claim 36, wherein the FIT-Ig binding protein is expressed at a level greater than 10 mg / L.

38. An EGFR / PD-L1 FIT-Ig binding protein produced according to the method of claim 35.

39. Use of the FIT-Ig binding protein of claim 1 in the preparation of a medicament for treating cancer; wherein the cancer is selected from the group consisting of melanoma, renal cancer, prostate cancer, pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia and lymphoma.

40. The use according to claim 39, wherein the melanoma is metastatic malignant melanoma.

41. The use according to claim 39, wherein the renal cancer is clear cell renal cell carcinoma.

42. The use according to claim 39, wherein the prostate cancer is hormone-refractory prostate adenocarcinoma.

43. The use according to claim 39, wherein the lung cancer is non-small cell lung cancer.

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