Patent Description:
Overexpression and/or deregulation of members of the ErbB/HER receptor family such as EGFR, HER2, HER3, HER4 have been shown to play an important role in tumorigenesis in cancers. Mutation and amplification of EGFR or HER2 produce aberrant growth signal which activates downstream signaling pathway contributing to tumorigenesis. Therapeutic antibodies and small-molecule inhibitors directed against EGFR and HER2 have been approved for use in the treatment of cancer (<NPL>). Monoclonal antibodies against members of EGFR family such as EGFR and HER2, have demonstrated good clinical responses in colon cancer (<NPL>), squamous cell carcinoma of head and neck (<NPL>), breast and gastric cancers (<NPL>). Several therapeutic anti-EGFR antibodies, including cetuximab, panitumumab and nimotuzumab are approved therapeutics for several cancers including metastatic colorectal cancer, head and neck squamous cell carcinoma and glioma (<NPL>; <NPL>). Unfortunately, many tumors that initially respond to these therapeutic agents eventually progress due to an acquired resistance to the agents (<NPL>). Therefore, there exists a need for better cancer therapeutics. Hu et al describes four-in-one antibodies having superior cancer inhibitory activity against EGFR, HER2, HER3 and VEGF through disruption of HER/MET crosstalk. <CIT> describes dual variable region antibody-like binding proteins having cross-over binding region orientation. <CIT> describes multispecific antibodies comprising full length antibodies and single chain Fab fragments. <CIT> describes tetravalent bispecific antibodies. <CIT> describes compositions and methods for using multispecific-binding proteins comprising an antibody-receptor combination. <CIT> describes bispecific anti ErbB3/ anti cMet antibodies.

The disclosure provides a bispecific tetravalent antibody, said bispecific tetravalent antibody comprising: a first IgG1 heavy chain, connector, and single chain Fv (scFv) domain polypeptide comprising SEQ ID NO: <NUM>; a second IgG1 heavy chain, connector, and scFv domain polypeptide comprising SEQ ID NO: <NUM>; a first kappa light chain polypeptide comprising SEQ ID NO: <NUM>; and a second kappa light chain polypeptide comprising SEQ ID NO: <NUM>; wherein the first and second IgG1 heavy chains and the first and second kappa light chains form an IgG moiety with a binding specificity for EGFR; and wherein the first and second scFv domains each have a binding specificity for HER3. The objectives and advantages of the disclosure will become apparent from the following detailed description of preferred embodiments thereof in connection with the accompanying drawings.

The present disclosure will now be described with reference to the FIGs, in which like reference numerals denote like elements.

The present disclosure provides bispecific tetravalent antibodies with superior therapeutic properties or efficacies over the currently known anti-EGFR antibodies.

The bispecific tetravalent antibodies may inhibit both EGFR and HER3 mediated signaling simultaneously therefore overcome resistance in EGFR inhibitor or monoclonal antibody treatment.

It must be noted that as used herein and in the appended claims, the singular forms "a", "and", and "the" in Throughout this specification and claims, the word "comprise," or variations such as "comprises" or "comprising," will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers include plural referents unless the context clearly dictates otherwise.

"Antibody fragments" comprise a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, Fab', F(ab')<NUM>, Fab'-SH; diabodies; linear antibodies (see <CIT>, Example <NUM>;<NPL>)); single-chain antibody molecules (e.g. scFv). While in the present description, and throughout the specification, reference is made to antibodies and various properties of antibodies, the same disclosure also applies to functional antibody fragments, e.g. dual action Fab fragments.

In one aspect, there is provided a bispecific tetravalent antibody, said bispecific tetravalent antibody comprising: a first IgG1 heavy chain, connector, and single chain Fv (scFv) domain polypeptide comprising SEQ ID NO: <NUM>; a second IgG1 heavy chain, connector, and scFv domain polypeptide comprising SEQ ID NO: <NUM>; a first kappa light chain polypeptide comprising SEQ ID NO: <NUM>; and a second kappa light chain polypeptide comprising SEQ ID NO: <NUM>; wherein the first and second IgG1 heavy chains and the first and second kappa light chains form an IgG moiety with a binding specificity for EGFR; and wherein the first and second scFv domains each have a binding specificity for HER3. The IgG moiety may provide stability to the scFv moiety. The bispecific tetravalent antibody may block signalling for both AKT and MAPK/ERK pathways and may mediate antibody dependent cell-mediated cytotoxicity (ADCC) towards cells expressing either one or both antigens. In one embodiment, the bispecific tetravalent antibody is capable of binding both antigens simultaneously. In some embodiments, the bispecific tetravalent antibody provides stronger tumour inhibition in proliferation assays in vitro and in vivo than the mono-specific antibody parental control or combination of the mono-specific antibody parental controls.

The EGFR family members may include EGFR, HER2, HER3, a fragment or a derivative thereof.

The bispecific tetravalent antibodies have the activity of inhibiting cancer cell growth. An antibody may have a dissociation constant (Kd) of ≦<NUM>, ≦<NUM>, ≦<NUM>, ≦<NUM>, ≦<NUM>, or ≦<NUM> for its target EGFR or HER3. The antibody may bind to both targets simultaneously. the antibody may bind to EGFR and HER3 with a Kd less than <NUM>. The antibody may bind to EGFR and/or HER3 with a Kd less than <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>. In one embodiment, the antibody binds to EGFR with a Kd less than <NUM> and binds to HER3 with a Kd less than <NUM>. In one embodiment, the antibody binds to EGFR with a Kd less than <NUM> and binds to HER3 with a Kd less than <NUM> simultaneously.

In another aspect, the disclosure provides isolated nucleic acids encoding the bispecific tetravalent antibodies as claimed.

In a further aspect, the disclosure provides expression vectors having the isolated nucleic acids encoding the bispecific tetravalent antibody as claimed. The vectors may be expressible in a host cell. The host cell may be prokaryotic or eukaryotic.

In a further aspect, the disclosure provides host cells having the isolated nucleic acids encoding the bispecific tetravalent antibodies as claimed or the expression vectors including such nucleic acid sequences.

A method for producing bispecific tetravalent antibodies is described herein. The method may include culturing the above-described host cells so that the antibody is produced.

In a further aspect, the disclosure provides immunoconjugates including the bispecific tetravalent antibodies as claimed and a cytotoxic agent.

In a further aspect, the disclosure provides pharmaceutical compositions comprising the bispecific tetravalent antibodies as claimed or the immunoconjugates described herein and a pharmaceutically acceptable carrier. In some embodiments, the composition may further include radioisotope, radionuclide, a toxin, a therapeutic agent, a chemotherapeutic agent or a combination thereof.

A bispecific tetravalent antibody may be for use in a method of treating a subject with a cancer. The method may include the step of administering to the subject an effective amount of a bispecific tetravalent antibody described herein. The cancer may include cells expressing at least two members of EGFR family including, for example, EGFR, HER2, HER3, a fragment or a derivative thereof. The cancer may be breast cancer, colorectal cancer, pancreatic cancer, head and neck cancer, melanoma, ovarian cancer, prostate cancer, and non-small lung cell cancer, glioma, esophageal cancer, nasopharyngeal cancer, anal cancer, rectal cancer, gastric cancer, bladder cancer, cervical cancer and brain cancer.

The method may further include co-administering an effective amount of a therapeutic agent. The therapeutic agent may be, for example, an antibody, a chemotherapy agent, a cytotoxic agent, an enzyme, or a combination thereof. The therapeutic agent may be an anti-estrogen agent, a receptor tyrosine inhibitor, or a combination thereof.

The therapeutic agent may be a biologic.

The therapeutic agent may be a checkpoint inhibitor.

The therapeutic agent may include PD1, PDL1, CTLA4, <NUM>-1BB, OX40, GITR, TIM3, LAG3, TIGIT, CD40, CD27, HVEM, BTLA, VISTA, B7H4, a derivative, a conjugate, or a fragment thereof. The therapeutic agent may be capecitabine, cisplatin, trastuzumab, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, aminoglutethimide, testolactone, vorozole, formestane, fadrozole, letrozole, erlotinib, lafatinib, dasatinib, gefitinib, imatinib, pazopinib, lapatinib, sunitinib, nilotinib, sorafenib, nab-palitaxel, or a derivative thereof. The subject in need of such treatment may be a human.

A bispecific tetravalent antibody may be for use in a method for treating a subject by administering to the subject an effective amount of the bispecific tetravalent antibody to inhibit a biological activity of a HER receptor.

A diagram of the general structure of IgG is shown in <FIG>.

A diagram of a bispecific tetravalent antibody is shown in <FIG>. In this example, the bispecific tetravalent antibody includes two human IgG1 heavy chains, two human kappa light chains, and two single chain Fv (scFv) domains. The two human IgG1 heavy chains and human kappa light chains form an IgG moiety with a binding specificity to one member of the EGFR family, and each of the two scFv domains is connected to the C-terminal residue of either of the human IgG1 heavy chains by a connector with an amino acid sequence of gly-gly-gly-gly-ser-gly-gly-gly-gly-ser ((G<NUM>S)<NUM>). Each scFv domain is in the order: N terminus-variable heavy-linker-variable light-C terminus. The linker is comprised of amino acid sequence of gly-gly-gly-gly-ser-gly-gly-gly-gly-ser-gly-gly-gly-gly-ser, also known as (G<NUM>S)<NUM>.

The CH1, CH2, CH3, CL, Connector and Linker amino acid sequences may be identical. Each bispecific tetravalent antibody has a bivalent anti-HER3 binding specificity on one end of the antibody and a bivalent anti-EGFR binding specificity on the other end. One pair of anti-HER3 variable heavy chain and variable light chain is designated as 1C1, and four pairs of anti-EGFR variable heavy chains and variable light chains are designated as 1C3, 1C5, 1C5. <NUM>, 1C6 and 1C6. <NUM>, respectively. The bispecific tetravalent antibodies are designated as 1X1, 1X2, 1X3, 1X4, 1X4. <NUM>, 1X5, 1X5. <NUM>, 1X6, and 1X6.

In addition, a control molecule 1C4 (also designated as SI-1C4) was used in some of the studies. 1C4 is a bispecific antibody against EGFR and HER3 built on the two-in-one platform described by Schaefer et. , <NUM> (<NPL>). IC4 has a similar structure to a monoclonal antibody. The molecule can bind to either EGFR or HER3 on each Fab arm, but cannot engage both targets simultaneously on each Fab arm.

Variable light chain, variable heavy chain and single chain Fv (scFv) DNA fragments were generated by gene synthesis through an outside vendor. Human Gamma-<NUM> heavy chain and human kappa light chain DNA fragments were generated by gene synthesis through an outside vendor. The fragments were assembled together by DNA ligation using restriction sites and cloned into a vector that is designed for transient expression in mammalian cells. The vector contains a strong CMV-derived promoter, and other upstream and downstream elements required for transient expression. The resulting IgG expression plasmids were verified as containing the expected DNA sequences by DNA sequencing.

Transient expression of the antibody constructs was achieved using transfection of suspension-adapted HEK293F cells with linear PEI as described elsewhere (see CSH Protocols; <NUM>; doi:<NUM>/pdb. Antibodies were purified from the resulting transfection supernatants using protein affinity chromatography and size exclusion chromatography if needed. Protein quality is analysed by Superdex <NUM> column. Protein used for all the assays have a purity of greater than <NUM>%.

The bispecific antibody may be used for the treatment of cancer types with EGFR and HER3 co-expressions, including without limitation colon cancer, head and neck squamous cell carcinoma, lung cancer, glioma, pancreatic cancer, nasopharyngeal cancer and other cancer types.

The bispecific antibody is of tetravalent dual specificity. The example antibody may include an IgG and two scFv, which provides two different binding specificities compared to mono-specific antibody IgG. The IgG component provides stability and improved serum half-life over other bispecific antibodies that used only scFv such as BiTE technology (<NPL>) and others (for example, <CIT>). It is also capable of mediating ADCC while those without Fc component cannot (for example, <CIT>). The tetravalent dual specificity nature provides the bispecific antibody a simultaneous binding capability over some other bispecific antibodies, which may only bind one antigen at a time (<NPL>; <CIT>).

For the convenience of narration, the sequences of or related to the bispecific antibodies are summarized in TABLE <NUM> herein below.

While The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results.

<NUM> is a modification of SI-1X4 molecule and contained <NUM> amino acid changes as follows: V71A, T75S, N76S, A93T and S107T using the Kabat numbering system. Some of these changes especially positions <NUM>, <NUM> and <NUM> potentially made interaction with antigen even though these are not in the CDR loops and are essential for binding and activity. <FIG> shows the <NUM> amino acid differences between SI-1X4. <NUM> and SI-1X4.

Monomeric EGFR extracellular domain binding was measured in a biolayer interferometry (BLI) binding assay on a BLItz instrument (ForteBio, Inc. 25µg/mL of SI-1C3, SI-1C4, SI-1C6, SI-1X1, SI-1X2, SI-1X5, and SI-1X6 were diluted in PBS and captured on anti-hulgG Fc BLItz biosensor tips for <NUM> seconds. Tips were washed for <NUM> seconds in PBS and moved to an EGFR (ProSpec Bio, PKA-<NUM>) sample for binding at <NUM>. Binding of EGFR ECD to the tips was recorded as biolayer interferometry signals (Δnm) over an association time of <NUM> seconds. Tips were moved to PBS and dissociation was observed for <NUM> seconds (*SI-1C6 dissociation time of only <NUM> seconds observed). <FIG> and <FIG> report data starting at the association step of EGFR to the antibody-loaded biosensor. Each Figure shows comparison to SI-1C4 as a benchmark antibody.

Since SI-1C3 and SI-1X2 share their EGFR binding domain displayed as a Fab, their binding profiles are similar and stronger than the scFv form displayed on SI-1X1 (<FIG>). Each has a very slow off-rate to EGFR compared to SI-1C4 and is not affected by their on-rate. SI-1X1 may show weaker on-rate binding to EGFR, but stays bound very strongly. The same trend is observed in <FIG>, where the Fab versions of the EGFR binding domains displayed on SI-1C6 and SI-1X6 bind at a faster rate than their representative scFv displayed on SI-1X5. Having the EGFR binding domain on the Fab side of the bispecifics antibody appears to bind with faster on-rates than the scFv versions, yet exhibit similar off-rates. SI-1X3 and SI-1X4 do not exhibit monomeric EGFR binding in this assay (data not shown) and dimeric EGFR binding is investigated in an ELISA below.

Bispecific binding to EGFR and Her3 extracellular domains was measured in a biolayer interferometry (BLI) binding assay on a BLItz instrument (ForteBio, Inc. <NUM> of SI-1C1, SI-1C3, SI-1C4, SI-1C6, SI-1X1, SI-1X2, SI-1X3, SI-1X4, SI-1X5, and SI-1X6 were diluted in 1X Kinetics Buffer (ForteBio, Inc. ) and captured on anti-huIgG Fc BLItz biosensor tips for <NUM> seconds. Tips were washed in KB for <NUM> seconds and moved to an EGFR sample (ProSpec Bio, PKA-<NUM>) for binding at <NUM>. Binding of EGFR ECD to the tips was recorded as biolayer interferometry signals (Δnm) over an association time of <NUM> seconds. Tips were moved to KB and dissociation was observed for <NUM> seconds. The process was repeated with Her3 ECD sample (Sino Biological, <NUM>-H08H-<NUM>) at <NUM> for <NUM> seconds and a similar dissociation step of <NUM> seconds in KB. <FIG> <FIG> report data starting at the association step of EGFR to the antibody-loaded biosensor. Antibodies are able to exhibit simultaneous bispecific binding of EGFR and Her3 while being bound by the Fc to the sensor. As observed in <FIG> and <FIG>, the display of the EGFR binding domain as Fab (SI-1X2, SI-1X6) has stronger on-rate binding than their scFv forms (SI-1X1, SI-1X5, respectively). Here, both EGFR and Her3 exhibit the same Fa>>scFv on-rate trend. SI-1X3 and SI-1X4 do not exhibit binding to monomeric EGFR, however each has the ability to bind Her3, as expected since each molecule uses the same αHer3 binding domain as SI-1X1, SI-1X2, SI-1X5, and SI-1X6. SI-1X3 and SI-1X4 are investigated for dimeric EGFR binding in an ELISA below.

As observed earlier, SI-1X3 and SI-1X4 were unable to bind a monomeric form of EGFR in a BLI assay (<FIG>). It has been suggested that in order for the αEGFR binding domain used in SI-1C5, SI-1X3, and SI-1X4 to bind to EGFR in vitro, bivalent binding is required (Perez et al, Chin Clin Oncol <NUM>;<NUM>(<NUM>):<NUM>). To observe this, we utilized ELISA for antibody binding relative to other EGFR binding antibodies using a dimeric form of EGFR.

ELISA was performed using dimeric EGFR ECD reagent, SI-2C1, fused to rabbit Fc created in house. EGFR was coated onto Maxisorp immunoplates (Nunc) at <NUM>µg/mL in PBS at <NUM> overnight. Plates were blocked in PBS with <NUM>% BSA and <NUM>% Tween20 for <NUM> hours at room temperature. Antibodies were captured at starting at 10ug/mL except for SI-1C5, SI-1X3, and SI-1X4 which started at <NUM>µg/mL for (reported in nM), all with 3X dilutions in PBST (<NUM>% BSA) for <NUM> hour at room temperature. Goat αhuman IgG-HRP antibody (Jackson ImmunoResearch, <NUM>-<NUM>-<NUM>) was used for detection of the Fc portion of the antibodies at <NUM>:<NUM> dilution in PBST (<NUM>% BSA) and developed in TMB (Thermo Scientific) for <NUM> minutes with <NUM> H<NUM>SO<NUM> as a stop solution. <NUM> washes with PBST (<NUM>% BSA) were performed between each step. All data points were performed in triplicate and collected at <NUM> (<FIG>). SI-1C5, SI-1X3, and SI-1X4 all bound to the dimeric EGFR ECD in this ELISA format at high concentrations as compared to the other molecules.

Kinetics determined using ForteBio Octet Red96 instrument with anti-human Fc sensors (ForteBio, AHC #<NUM>-<NUM>). Binding experiments performed at <NUM>° C with <NUM> RPM mixing. EGFR protein is extracellular domain (Met <NUM>-Ser <NUM>) of human EGFR with a C-terminal polyhistidine tag. All samples diluted in 10X Kinetics Buffer (ForteBio #<NUM>-<NUM>). <NUM>, 1X6 and 1X4. <NUM> were loaded onto <NUM> sensors at <NUM>µg/ml each for <NUM> seconds followed by a Baseline for <NUM> seconds in 10X Kinetics Buffer. Association with EGFR protein was performed for <NUM> seconds with each sensor in a single concentration of EGFR protein (<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>). Dissociation was then performed in 10X Kinetics Buffer for <NUM> seconds. A typical association and dissociation trace for 1C5. <NUM> and 1X4. <NUM> is shown in <FIG>.

Data analysis was performed using ForteBio Data Analysis Software v9. Software curve-fitting was performed and the four most optimal curve fits for each 1C5. <NUM> (TABLE <NUM>), 1X4. <NUM> (TABLE <NUM>) and 1X6 (TABLE <NUM>) were used and averaged to determine KD, k(on) and k(dis). The average KD for SI-1C5. <NUM> and SI-1X4. <NUM> were <NUM> and <NUM> respectively. The average KD for SI-1C6 was <NUM> 1C5. <NUM> and 1X4. <NUM> contained five amino acid changes as compared to 1C5 and 1X4 as described in example <NUM>. These changes accounted for improved binding to EGFR ECD when compared to data generated for 1C5 and 1X4 in <FIG>.

The bispecific antibodies SI-1X1, SI-1X2, SI-1X3, SI-1X4, SI-1X5, and SI-1X6, as well as an isotype control were tested for binding to the tumor cell lines, A431 (epidermoid carcinoma, ATCC CRL-<NUM>) and BxPC3 (pancreatic adenocarcinoma, ATCC CRL-<NUM>) by flow cytometry. Cells were grown in RPMI-<NUM> medium containing <NUM>% fetal bovine serum and were harvested for analysis while in exponential growth phase. Aliquots of <NUM> × <NUM><NUM> cells were washed once in PBS, then resuspended in <NUM>µl of PBS + <NUM>% bovine serum albumin (BSA) and incubated at <NUM> for <NUM> minutes to block membranes from non-specific binding. <NUM>µl of antibody, diluted to <NUM>µg/ml in PBS/<NUM>%BSA, was added to each sample for a final antibody concentration of <NUM>µg/ml. Cells were incubated in primary antibody for <NUM> hour at <NUM> with mixing. Cells were then washed twice with <NUM> PBS/<NUM>°/BSA and then resuspended in <NUM>µl of PE-conjugated mouse-anti-human IgG-Fc and incubated at <NUM> with mixing for <NUM> minutes. Samples were again washed twice with <NUM> PBS/<NUM>%BSA, resuspended in <NUM> PBS and analyzed using a FACScalibur flow cytometer. For each sample, <NUM> events were collected in the FL-<NUM> channel. Histograms were generated using FCS Express software and SI-1X histograms were overlaid with histograms from the isotype control staining. All six bispecific antibodies displayed histogram shifts with respect to control staining indicating cell binding. This data is displayed in <FIG> (A431 cell binding) and <FIG> (BxPC3 cell binding).

The bispecific antibody, SI-1X4. <NUM>, monospecific antibodies, SI-1C5. <NUM> and SI-1C1, as well as an isotype control were tested for binding to the tumor cell lines, A431 (epidermoid carcinoma, ATCC CRL-<NUM>) (<FIG>) and FaDu (hypopharyngeal squamous cell carcinoma, ATCC HTB-<NUM>) (<FIG>) by flow cytometry. Cells were grown in RPMI-<NUM> medium containing <NUM>% fetal bovine serum and were harvested for analysis while in exponential growth phase. Cells were washed once in PBS, then resuspended in PBS + <NUM>% fetal bovine serum albumin (FBS) at a concentration of <NUM>×<NUM><NUM> cells/ml and incubated at <NUM> for <NUM> minutes to block membranes from non-specific binding. 100µl aliquots of cells were added to 100µl aliquots of antibody (also diluted in PBS + <NUM>% FBS) in a <NUM>-well plate. Samples were incubated in primary antibody for <NUM> minutes on ice. Cells were then washed twice with 200µl of PBS + <NUM>% FBS and then resuspended in 100µl of PE-conjugated mouse-anti-human IgG-Fc and incubated on ice <NUM> minutes. Samples were again washed twice with 200µl of PBS + <NUM>% FBS, resuspended in 200µl PBS and analyzed using a FACScalibur flow cytometer. For each sample, <NUM> events were collected in the FL-<NUM> channel. Histograms were analyzed using FCS Express software and the geometric mean fluorescence intensity (GMFI) was determined for each data set. EC50 binding values were determined by plotting the GMFI versus antibody concentration using Graphpad Prism software. The bispecific antibody, SI-1X4. <NUM> displayed similar binding profile as the monospecific anti-EGFR antibody, SI-1C5. <NUM> with similar EC50 in both cell lines. The other monospecific anti-Her3 antibody, SI-1C1 binds weakly to the two cell lines probably due to low level of expression of Her3 on the surface of the cells. <NUM> and 1X4. <NUM> contained five amino acid changes as compared to 1C5 and 1X4 as described in example <NUM>. These changes accounted for improved binding to target cells when compared to the parental molecule, 1X4.

To assess the growth inhibitory potential of anti-Her3/EGFR bispecific antibodies, the effect on proliferation of A431 cells (ATCC CRL-<NUM>, Manassas, Va. ) which are an epidermoid carcinoma tumor line was tested. The effect on proliferation of BxPC3 (ATCC CRL-<NUM>, Manassas, Va. ), a pancreatic adenocarcinoma tumor line was also tested. For each line, cells were seeded into <NUM>-well tissue culture plates at a density of <NUM> cells/well in <NUM>µl RPMI-<NUM> medium containing <NUM>% fetal bovine serum. After <NUM> hours, test antibodies were added at various concentrations, ranging from <NUM> to <NUM>. Cells were cultured in the presence of test antibodies for <NUM> hours. To each well, <NUM>µl of MTS reagent (Promega, Madison, WI) was added and cells were incubated at <NUM> for <NUM> hours. MTS is readily taken up by actively proliferating cells, reduced into formazan (which readily absorbs light at <NUM>), and then secreted into the culture medium. Following incubation, OD490 values were measured using a BioTek (Winooski, VT) ELx800 absorbance reader. OD490 values for control cells (treated with medium only) were also obtained in this manner at the time of antibody addition in order to establish baseline metabolic activity. Proliferation may be calculated by subtracting the control baseline OD490 from the <NUM> hour OD490. Data from antibody titrations was expressed at % of control population according to the following formula: % of control proliferation = (test proliferation /control proliferation)*<NUM>.

The effects of various bispecific anti-Her3/anti-EGFR antibodies on A431 cell proliferation are shown in <FIG> and <FIG>. SI-1X2 demonstrated more efficacious antiproliferative effect than the control antibodies SI-1C1 (anti-Her3), SI-1C3 (anti-EGFR), or SI-1C1 and SI-1C3 applied together. SI-1X1 exhibited antiproliferative effects, although not to the degree seen with SI-1C3 and the combination of SI-1C1 and SI-1C3. Inhibition plots as well as IC50 values are shown in <FIG>. Similar results were observed for SI-1X5 and SI-1X6, where SI-1X6 is more potent than SI-1X5 and the control antibody SI-1C1 (anti-Her3), however it displayed similar antiproliferative potential as the control antibody SI-1C6 (anti-EGFR) and the combination of SI-1C1 and SI-1C6. This may be seen along with IC50 values in <FIG>.

These molecules were also tested for antiproliferative effects in the BxPC3 cell line (<FIG> and <FIG>). Again, SI-1X2 demonstrated more efficacious antiproliferative effect than the control antibodies SI-1C1 (anti-Her3), SI-1C3 (anti-EGFR), or SI-1C1 and SI-1C3 applied together. SI-1X1 was more efficacious than SI-1C1, but weaker than SI-1C3 and the combination of SI-1C1 and SI-1C3. Inhibition curves and IC50 values are displayed in <FIG>. BxPC3 proliferation was more strongly inhibited by both SI-1X5 and SI-1X6 than with the control antibodies SI-1C1 (anti-Her3), SI-1C6 (anti-EGFR), or SI-1C1 and SI-1C6 in combination. This data along with IC50 values is shown in <FIG>.

To assess the growth inhibitory potential of anti-Her3/EGFR bispecific antibodies, the effect on proliferation of FaDu (nasopharyngeal squamous cell carcinoma line, ATCC HTB-<NUM>) and A431 (epidermoid carcinoma, ATCC CRL-<NUM>) cells were tested. Cells were seeded into <NUM>-well tissue culture plates at a density of <NUM> cells/well in 100µl RPMI-<NUM> medium containing <NUM>% fetal bovine serum. After <NUM> hours, test antibodies were added at various concentrations, ranging from <NUM> to <NUM>. Cells were cultured in the presence of test antibodies for <NUM> hours. To each well, 11µl of alamar blue reagent (Thermo Scientific) was added and cells were incubated at <NUM> for <NUM> hours. Alamar blue is readily taken up by actively proliferating cells, reduced, and then secreted into the culture medium. The reduced form of alamar blue is strongly fluorescent. Following incubation, fluorescence was measured using a Molecular Devices (Sunnyvale, CA) FilterMax F5 multi-mode plate reader using an excitation wavelength of <NUM> and an emission wavelength of <NUM>. Fluorescence values for control cells (treated with medium only) were also obtained in this manner at the time of antibody addition in order to establish baseline metabolic activity. Proliferation may be calculated by subtracting the control baseline fluorescence from the <NUM>-hour fluorescence values. Data from antibody titrations was expressed at % of control population according to the following formula: % of control proliferation = (test proliferation /control proliferation)*<NUM>.

The effects of SI-1C5. <NUM> and SI-1X4. <NUM> on Fadu and A431 cell proliferation are shown in <FIG> and <FIG> respectively. In both cell lines, SI-1X4. <NUM> demonstrated improved efficacious anti-proliferative effect than the control antibodies, SI-1C5. <NUM> (anti-EGFR Mab), SI-1C1 (anti-Her3 Mab) or SI-1C1 and SI-1C7 applied together.

The ability of SI-1X antibodies to mediate cellular cytotoxicity against several tumor cell lines was tested. Whole blood was obtained from normal, healthy volunteers. Blood was diluted with an equal volume of phosphate buffered saline (PBS). <NUM> aliquots of diluted blood were carefully layered over <NUM> Ficol Pacque PLUS (GE Life Sciences cat# <NUM>-<NUM>-<NUM>; Pittsburgh, PA). Tubes were centrifuged at <NUM> for <NUM> minutes with no brake. Following centrifugation most of the plasma layer was carefully aspirated and the buffy coat (containing PBMC) was carefully removed with a pipet in the smallest possible volume. PBMCs were pooled in <NUM> tubes and PBS added to bring each tube up to <NUM>. Tubes were centrifuged at 1300RPM for <NUM> minutes and the supernatant was carefully aspirated. Cells were resuspended in <NUM> PBS and centrifuged again. The process was repeated for a total of <NUM> washes. Following the final wash, cells were resuspended in <NUM> RPMI-<NUM> + <NUM>% FBS and incubated overnight at <NUM>, <NUM>% CO<NUM>.

Target cells tested were the head and neck squamous cell carcinoma line, FaDu (ATCC HTB-<NUM>, Manassus, VA) and the non-small cell lung adenocarcinoma cell line, NCI-H1975 (ATCC CRL-<NUM>, Manassus, VA). Target cells were labeled with calcein as follows. Cells were grown as monolayers and were detached by incubation with accutase. Cells were washed twice in RPMI with no serum. <NUM> of cells at <NUM>×<NUM><NUM> cells/ml was mixed with <NUM> RPMI (no serum) + <NUM> calcein AM (Sigma cat# C1359; St. Louis, MO). Cells were incubated at <NUM> for 30minutes, with gentle mixing every <NUM> minutes. Following labeling, cells were washed twice with <NUM> RPMI + <NUM>% FBS + <NUM> probenecid (assay medium). Probenecid (Sigma cat# P8761; St. Louis, MO) is an anionic transporter inhibitor and is known to reduce spontaneous release of intracellular calcein. Cells were resuspended in <NUM> assay medium and allowed to recover for <NUM> hours at <NUM>, <NUM>% COz. Cells were then washed once with assay medium and diluted to <NUM>,<NUM> cells/ml. Aliquots of 50µl (<NUM>,<NUM> cells) calcein-labeled cells were aliquoted to <NUM>-well round-bottom plates. 50µl of antibody (at 3X final concentration) was added to cells and allowed to bind for <NUM> minutes on ice. PBMCs from the previous day were centrifuged at <NUM> for <NUM> minutes, resuspended in <NUM> fresh assay medium, counted, and diluted to <NUM>×<NUM><NUM> cells/ml. 50µl PBMC (<NUM>,<NUM>) were added to each well and plates incubated at <NUM>, <NUM>% COz for <NUM> hours. Each antibody was titrated in triplicate via <NUM>-fold serial dilutions, starting at <NUM> and going down to <NUM>. Control wells were also set up containing labeled target cells in the absence of antibody and effector cells in order to measure maximal and spontaneous calcein release.

At the end of the <NUM>-hour incubation, 50µl of assay medium containing <NUM>% IGEPAL CA-<NUM> (Sigma cat# I8896; St. Louis, MO) was added to control wells containing labeled target cells only (to measure the maximal calcein release). 50µl of assay medium was added to all the other wells to bring the total volume to 200µl per well. Plates were centrifuged at 2000RPM for <NUM> minutes and 150µl supernatant was carefully transferred to V-bottom <NUM>-well plates. These plates were centrifuged at 2000RPM for an additional <NUM> minutes and <NUM> supernatant was carefully transferred to black, clear-bottom <NUM>-well plates. Calcein in the supernatant was quantitated by measuring the fluorescence of each sample using an excitation wavelength of <NUM> and an emission wavelength of <NUM>. The percentage of specific lysis was calculated as follows: <MAT>.

The data is shown in <FIG> and <FIG>. For both cell lines, SI-1X6. <NUM> mediated cellular cytotoxicity, but was not particularly more effective than the control antibodies, SI-1C6. <NUM>, SI-1C7, or the combination of SI-1C6. <NUM> + SI-1C7. <NUM> did mediate cytotoxicity with a lower EC50 than our benchmark antibody, SI-1C4. For both cell lines, SI-1X4. <NUM> mediated cellular cytotoxicity at about the same degree as the control antibodies. However, it was not as effective as mediating cellular cytotoxicity as the benchmark, SI-1C4. This is likely due to the lower affinity of SI-1X4.

Protein Thermal Shift Study was performed for protein thermal stability analysis. Protein melt reactions were set up using Protein Thermal Shift Buffer ™ and the Protein Thermal Shift Dye ™ (Applied Biosystems). In brief, the 20ul reaction mixture contains 5ug protein, 5ul Protein Thermal Shift Buffer™ and <NUM>. 5µ 8X diluted Protein Thermal Shift ™ Dye. For the negative control, PBS was used instead. The reaction mixture was added into MicroAmp Optical Reaction Plate and sealed with MicroAmp Optical Adhesive Film. Each sample consisted of <NUM> repeats. The protein melt reactions were run on Applied Biosystem Real-Time PCR System from <NUM> - <NUM> in <NUM>% increment and then analyzed by Protein Thermal Shift Software ™. <FIG> shows the thermal curve of SI-1X2, SI-1X4. <NUM>, SI-1X6. <NUM>, SI-1C3, SI-1C3, SI-1C6. <NUM>, SI-1C5. <NUM> and SI-1C7. TABLE <NUM> shows Tm for these molecules. Tm is defined as the temperature needed to unfold <NUM>% of the protein. The bispecific molecules, 1X2, 1X4. <NUM> and 1X6 all have Tm around <NUM> which are comparable to all the MAbs (1C3, 1C6. <NUM>, 1C5. <NUM>) and the Fc-scFv (1C7) molecules.

Serum stability of the molecules SI-1C5. <NUM>, SI-1C6. <NUM>, SI-1X4. <NUM>, and SI-1X6. <NUM> was determined by comparative binding to monomeric EGFR ECD by ELISA after incubation at 100µg/mL in <NUM>% human serum (Atlanta Biologics, S40110) at <NUM> for Days <NUM>, <NUM>, and <NUM> time points with an extra time point of <NUM> on Day <NUM> to provide a known condition where degradation occurs. ELISA plates were coated with monomeric EGFR ECD (SI-2R4) at 3ug/mL in PBS at <NUM> overnight. Coated ELISA plates were blocked with <NUM>% BSA PBST for <NUM> hours at <NUM> and then washed <NUM> times with PBST. <NUM> and SI-1X6. <NUM> were diluted <NUM>:<NUM> with <NUM>% BSA PBST and diluted 4x across the plate. <NUM> and SI-1X4. <NUM> were diluted <NUM>:<NUM> with <NUM>% BSA PBST and diluted 4x across the plate and incubated at <NUM> for <NUM> hour. <NUM> more washes with PBST were performed before antigen capture with 1µg/mL Her3 ECD Rabbit IgG1 (SI-1R1) for <NUM> hour at <NUM> in <NUM>% BSA PBST. <NUM> more washes with PBST were performed before goat anti-rabbit IgG-HRP (Bio-Rad <NUM>-<NUM>) secondary antibody was applied at <NUM>:<NUM> dilution in <NUM>% BSA PBST at <NUM> for <NUM> hour. <NUM> final washes with PBST before development with 100µl Pierce <NUM>-step Ultra TMB ELISA (Pierce, <NUM>) for <NUM> minutes with a final quench of 100µl <NUM> H<NUM>SO<NUM>. Plates were read at <NUM>. ELISA data was plotted and curves created using GraphPad Prism <NUM>.

Results of the ELISA are reported by EC50 on <FIG> and indicate a favorable profile of minor degradation when held at <NUM>. When placed in <NUM>, the EC50 shifts roughly a log as the molecules are subjected to degradation conditions. EC50 values for SI-1C5. <NUM> shift from <NUM> pM on Day <NUM> to <NUM> pM on Day <NUM> at <NUM> (Δ165. <NUM> pM) with a shift to <NUM> on Day <NUM> at <NUM> (Δ5932. EC50 values for SI-1C6 shift from <NUM> pM on Day <NUM> to <NUM> pM on Day <NUM> at <NUM> (Δ8. <NUM> pM) with a shift to <NUM> on Day <NUM> at <NUM> (Δ1103 pM). EC50 values for SI-1X4. <NUM> shift from <NUM> pM on Day <NUM> to <NUM> pM on Day <NUM> at <NUM> (Δ37. <NUM> pM) with a shift to <NUM> on Day <NUM> at <NUM> (Δ3818. EC50 values for SI-1X6 shift from <NUM> pM on Day <NUM> to <NUM> pM on Day <NUM> at <NUM> (Δ28 pM) with a shift to <NUM> on Day <NUM> at <NUM> (Δ3902.

To test their half-life in vivo, pharmacokinetic experiments were performed in SD rats. A single, intravenous tail vein injection of bispecific Abs ( 1C6 <NUM>/kg, 1X6 <NUM>/kg, 1X2 <NUM>/kg, 1X4 <NUM>/kg ) were given to groups of <NUM> female rats randomized by body weight (<NUM>-<NUM> range). Blood (~150µL) was drawn from the orbital plexus at each time point, processed for serum, and stored at -<NUM> until analysis. Study durations were <NUM> days.

Antibody concentrations were determined using three ELISA assays. In assay <NUM> (EGFR ECD coated ELISA), recombinant EGFR-rabbit Fc was coated to the plate, wells were washed with PBST (phosphate buffered saline with <NUM>% Tween) and blocked with <NUM>% BSA in PBST. Serum or serum diluted standards were then added, followed by PBST washing, addition of HRP labeled rabbit-anti-human IgG (BOSTER), and additional PBST washing. TMB was then added and the plates were incubated <NUM> minutes in the dark. Color reaction was stopped by adding <NUM> sulfuric acid. Plate was read at <NUM> wavelength. For assay <NUM> (Her3 coated ELISA), serum was detected using a similar ELISA , but recombinant HER3-His was used as capture reagent. For assay <NUM> (Sandwich ELISA), recombinant HER3-His was coated, serum or serum diluted standard were added, followed by PBST washing, addition of EGFR-rabbit Fc in PBST, and additional PBST washing. HRP labeled goat-anti-rabbit IgG (BOSTER) was then added. PK parameters were determined with a non-compartmental model.

<FIG> show serum concentration data for four antibodies with three different assays respectively. Fitted PK parameters from in vivo PK studies are provided in TABLE <NUM>. PK data include half-life, which represents the beta phase that characterizes elimination of antibody from serum and Cmax, which represents the maximal observed serum concentration, AUC, which represents the area under the concentration time curve.

The example tested the activity of SI-1X2, SI-1X4. <NUM> and SI-1X6 of concomitant blockade of EGFR, HER3 in preclinical models of Fadu ( head and neck squamous cell carcinoma xenograft model ) and compared their potency with cetuximab and cetuximab in combination with an anti-HER3 antibody.

All mouse studies were conducted through Institutional Animal care and used committee-approved animal protocols in accordance with institutional guidelines. Six-week-old female Balb/c Nude mice were purchased from Beijing Vital River Laboratories and housed in air-filtered laminar flow cabinets with a <NUM> - hour light cycle and food and water ad libitum. The size of the animal groups was calculated to measure means difference between placebo and treatment groups of <NUM>% with a power of <NUM>% and a P value of <NUM>. Host mice carrying xenografts were randomly and equally assigned to either control or treatment groups. Animal experiments were conducted in a controlled and non-blinded manner. For cell line-derived xenograft studies, mice were injected subcutaneously with <NUM>×<NUM> Fadu suspended 150µl of culture medium per mouse.

Once tumors reached an average volume of <NUM>-250mm3, mice were randomized into <NUM> groups, with <NUM> mice per group. Vehicle Control, 1C6 ( <NUM>/kg), 1C4 ( <NUM>/kg), 1C6 + 1C1 ( <NUM>/kg+<NUM>/kg), SI-1X2 ( <NUM>/kg), SI-1X6 ( <NUM>/kg), SI-1X6 ( <NUM>/kg), and SI-1X4. <NUM> ( <NUM>/kg) , SI-1X4 (<NUM>/kg). All test articles were administered once weekly via intravenous injection. Tumors were measured by digital caliper over the entire treatment period every <NUM> days and the volume was determined using the following formula: <NUM>/<NUM>×lenth×width2. The body weight of mice were recorded before the first dose and followed by every week during the treatment period and recovery period.

All the test groups of SI-1X2, SI-1X6 and SI-1X4. <NUM> and SI-1X6 combination yielded significantly tumor growth inhibition compared to positive control of SI-1C6 excluding the low dose SI-1X4. <NUM><NUM>/kg group (<FIG>). Moreover, no relapses were observed <NUM> weeks after treatment cessation excluding the low dose SI-1X4. <NUM><NUM>/kg group.

The term "effective amount" refers to an amount of a drug effective to achieve a desired effect, e.g., to ameliorate disease in a subject. Where the disease is a cancer, the effective amount of the drug may inhibit (for example, slow to some extent, inhibit or stop) one or more of the following example characteristics including, without limitation, cancer cell growth, cancer cell proliferation, cancer cell motility, cancer cell infiltration into peripheral organs, tumor metastasis, and tumor growth. Wherein the disease is a cancer, the effective amount of the drug may alternatively do one or more of the following when administered to a subject: slow or stop tumor growth, reduce tumor size (for example, volume or mass), relieve to some extent one or more of the symptoms associated with the cancer, extend progression free survival, result in an objective response (including, for example, a partial response or a complete response), and increase overall survival time. To the extent the drug may prevent growth and/or kill existing cancer cells, it is cytostatic and/or cytotoxic.

With respect to the formulation of suitable compositions for administration to a subject such as a human patient in need of treatment, the antibodies disclosed herein may be mixed or combined with pharmaceutically acceptable carriers known in the art dependent upon the chosen route of administration. There are no particular limitations to the modes of application of the antibodies disclosed herein, and the choice of suitable administration routes and suitable compositions are known in the art without undue experimentation.

Although many forms of administration are possible, an example administration form would be a solution for injection, in particular for intravenous or intra-arterial injection. Usually, a suitable pharmaceutical composition for injection may include pharmaceutically suitable carriers or excipients such as, without limitation, a buffer, a surfactant, or a stabilizer agent. Example buffers may include, without limitation, acetate, phosphate or citrate buffer. Example surfactants may include, without limitation, polysorbate. Example stabilizer may include, without limitation, human albumin.

Similarly, persons skilled in the art have the ability to determine the effective amount or concentration of the antibodies disclosed therein to effective treat a condition such as a cancer. Other parameters such as the proportions of the various components in the pharmaceutical composition, administration does and frequency may be obtained by person skilled in the art without undue experimentation. For example, a suitable solution for injection may contain, without limitation, from about <NUM> to about <NUM>, from about <NUM> to about <NUM> antibodies per ml. The example dose may be, without limitation, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>/Kg body weight. The example administration frequency could be, without limitation, once per day or three times per week.

Claim 1:
A bispecific tetravalent antibody, said bispecific tetravalent antibody comprising:
a first IgG1 heavy chain, connector, and single chain Fv (scFv) domain polypeptide comprising SEQ ID NO: <NUM>;
a second IgG1 heavy chain, connector, and scFv domain polypeptide comprising SEQ ID NO: <NUM>;
a first kappa light chain polypeptide comprising SEQ ID NO: <NUM>; and
a second kappa light chain polypeptide comprising SEQ ID NO: <NUM>;
wherein the first and second IgG1 heavy chains and the first and second kappa light chains form an IgG moiety with a binding specificity for EGFR; and
wherein the first and second scFv domains each have a binding specificity for HER3.