HK1229359A1

HK1229359A1 - Bispecific antigen-binding constructs targeting her2

Info

Publication number: HK1229359A1
Application number: HK17103205.6A
Authority: HK
Inventors: N．E．韦塞; G.Y.K.吴; G．R．威克曼; S.B.迪克西特; E．埃斯科巴-卡布瑞拉; M.桑彻斯
Original assignee: 酵活英属哥伦比亚有限公司
Priority date: 2013-11-27
Filing date: 2014-11-27
Publication date: 2017-11-17

Description

Bispecific antigen binding constructs targeting HER2

Cross reference to related applications

The present application claims U.S. provisional application No. 61/910,026 filed on 27/11/2013; united states provisional application No. 62/000,908 filed on 20/5/2014; and U.S. provisional application No. 62/009,125 filed 6/2014, which is hereby incorporated by reference in its entirety.

Sequence listing

This application contains a sequence listing that will be filed via EFS-Web and hereby incorporated by reference in its entirety. The ASCII copy created in XX month 20XX is named xxxxus _ sequencing.

Background

Most currently marketed antibody therapeutics are bivalent monospecific antibodies optimized and selected for high affinity binding and avidity conferred by the two antigen binding domains. Afucosylation or enhancement of FcgR binding by mutagenesis has been used to make antibodies more effective via antibody Fc-dependent cellular cytotoxicity mechanisms of action. Afucosylated antibodies or antibodies with enhanced FcgR binding still suffer from incomplete efficacy in clinical trials and a drug status to market remains to be achieved for any of these antibodies. Typical bivalent antibodies conjugated to toxins (antibody drug conjugates) are more effective but the broader clinical utility is limited by dose-limiting toxicity.

Ideally, therapeutic antibodies would have certain minor characteristics, including target specificity, biostability, bioavailability, and biodistribution upon administration to a subject patient, as well as sufficient target binding affinity and high target occupancy to maximize antibody-dependent therapeutic effects. Typically the therapeutic antibody is monospecific. However, monospecific targeting does not target other target epitopes that may be involved in signaling and pathogenesis, allowing drug resistance and escape mechanisms. Some current therapeutic paradigms require the use of a combination of two therapeutic monospecific antibodies targeting two different epitopes of the same target antigen. One example is the use of a combination of Trastuzumab (Trastuzumab) and Pertuzumab (Pertuzumab), both targeting HER2 receptor protein on the surface of some cancer cells, but patients still have disease progression, while other patients with lower HER2 receptor levels (HER 2<3+) by the Hercept test show no therapeutic benefit. Therapeutic antibodies targeting HER2 are disclosed in WO 2012/143523 to GenMab and WO 2009/154651 to Genentech. Antibodies are also described in WO 2009/068625 and WO 2009/068631.

Co-owned patent applications PCT/CA2011/001238 filed on day 11/4 of 2011, PCT/CA2012/050780 filed on day 11/2 of 2012, PCT/CA2013/00471 filed on day 5/10 of 2013, and PCT/CA2013/050358 filed on day 8 of 2013 describe therapeutic antibodies. Each of the commonly owned patent applications is hereby incorporated by reference in its entirety for all purposes.

Summary of The Invention

Described herein are bivalent antigen binding constructs that bind HER 2. The antigen-binding construct comprises a first antigen-binding polypeptide construct that monovalently and specifically binds to HER2 (human epidermal growth factor receptor 2) ECD2 (extracellular domain 2) antigen on a HER 2-expressing cell and a second antigen-binding polypeptide construct that monovalently and specifically binds to HER2ECD4 (extracellular domain 4) antigen on a HER 2-expressing cell, a first and a second linker polypeptide, wherein the first linker polypeptide is operably linked to the first antigen-binding polypeptide construct and the second linker polypeptide is operably linked to the second antigen-binding polypeptide construct; wherein the linker polypeptides are capable of forming a covalent bond with each other, wherein at least one of the ECD 2-binding polypeptide construct or the ECD 4-binding polypeptide construct is an scFv. In certain embodiments, the ECD2 binding polypeptide construct is an scFv and the ECD2 binding polypeptide construct is an Fab. In certain embodiments, the ECD 2-binding polypeptide construct is a Fab and the ECD 4-binding polypeptide construct is a scFv. In some embodiments, both the ECD 2-binding polypeptide construct and the ECD 4-binding polypeptide construct are scfvs. In some embodiments, the antigen binding construct has a dimeric Fc comprising a CH3 sequence. In some embodiments, the Fc is a heterodimer with one or more modifications in the CH3 sequence that promote the formation of heterodimers with stability comparable to a wild-type homodimeric Fc. In some embodiments, the melting temperature (Tm) of the heterodimeric CH3 sequence is 68 ℃ or higher. Nucleic acids encoding the antigen binding constructs, as well as vectors and cells, are also described. Also described are methods of treating disorders, such as cancer, using the antigen binding constructs described herein.

Also provided herein are modified pertuzumab constructs comprising the mutation Y96A in the VL region and the mutation T30A/a49G/L70F (numbering according to Kabat) in the VH region. In one embodiment, the modified pertuzumab construct is monovalent and has 7 to 9-fold higher affinity for HER2ECD2 than pertuzumab. In certain embodiments, the modified pertuzumab construct has the Fab/Fab, Fab/scFv, or scFv/scFv format.

Brief Description of Drawings

FIG. 1A depicts the structure of a biparatopic antibody in the Fab-Fab format. FIGS. 1B to 1E depict the structure of possible versions of a biparatopic antibody in the form of an scFv-Fab. In FIG. 1B, antigen binding domain 1 is scFv fused to the A chain, while antigen binding domain 2 is Fab fused to the B chain. In FIG. 1C, antigen binding domain 1 is Fab fused to the A chain, while antigen binding domain 2 is scFv fused to the B chain. In FIG. 1D, antigen binding domain 2 is Fab fused to the A chain, while antigen binding domain 1 is scFv fused to the B chain. In FIG. 1E, antigen binding domain 2 is scFv fused to the A chain, while antigen binding domain 1 is Fab fused to the B chain. In fig. 1F, both antigen binding domains are scFv.

Figure 2 depicts characterization of expression and purification of an exemplary anti-HER 2 biparatopic antibody. FIGS. 2A and 2B depict SEC chromatograms of protein A purified antibodies, and non-reducing SDS-PAGE analysis of 10L expression and purification of v 5019. FIG. 2C depicts SDS-PAGE analysis of 25L expression and purification of v 10000.

Figure 3 depicts the results of UPLC-SEC analysis of an exemplary anti-HER 2 biparatopic antibody purified by protein a and SEC. The results for v5019 are shown in fig. 3A, where the upper panel shows the results of the purification and the lower panel shows the same results with the y-axis scale expanded. A summary of the data obtained is provided below the UPLC-SEC results. Fig. 3B shows the result of v 10000.

Figure 4 depicts LCMS analysis of heterodimer purity of exemplary anti-HER 2 biparatopic antibodies. Fig. 4A depicts the results from LC-MS analysis of the combined SEC component of v 5019. Figure 4B depicts results from LC-MS analysis of pooled protein a fractions on v 10000.

Figure 5 depicts an analysis of 25L scale preparations of an exemplary anti-HER 2 biparatopic antibody. FIG. 5A is a depiction of MabSelect^TMAnd HiTrap^TMSDS-PAGE profile of the exemplary anti-HER 2 biparatopic antibody after SP FF purification. FIG. 5B depicts pureLCMS analysis of the antibody.

Figure 6, the ability of an exemplary biparatopic anti-HER 2 antibody to bind to HER2+ whole cells displaying different HER2 receptor densities was compared to control antibodies as measured by FACS. Fig. 6A and 6E depict binding to SKOV3 cells; fig. 6B depicts binding to JIMT1 cells; fig. 6C and 6F depict binding to MCF7 cells; FIG. 6D depicts binding to MDA-MB-231 cells; and figure 6G depicts binding to WI-38 cells.

Figure 7 depicts the ability of an exemplary anti-HER 2 biparatopic antibody to inhibit growth of HER2+ cells. Fig. 7A and 7D show growth inhibition in SKOV3 cells; FIG. 7B shows growth inhibition in BT-474 cells; FIG. 7C shows growth inhibition in SKBR3 cells, and FIG. 7E shows growth inhibition in JIMT-1 cells.

Figure 8 depicts SPR binding data on the paratope of an exemplary anti-HER 2 biparatopic antibody. FIG. 8A illustrates the K binding of a monovalent anti-Her 2 antibody (v 1040; representing the antigen binding domain on CH-B of an exemplary anti-Her 2 biparatopic antibody) to immobilized Her2ECD or dimeric Her2-Fc_DValues (nM). FIG. 8B illustrates K binding of a monovalent anti-Her 2 antibody (v 4182; representing the antigen binding domain on CH-A of an exemplary anti-Her 2 biparatopic antibody) to immobilized Her2ECD or dimeric Her2-Fc_DValues (nM).

Figure 9 depicts the ability of an exemplary anti-HER 2 biparatopic antibody to internalize in HER2+ cells. FIG. 9A depicts internalization in BT-474 cells, while FIG. 9b depicts internalization in JIMT-1 cells.

Figure 10 depicts surface binding and internalization of an exemplary anti-HER 2 biparatopic antibody. FIG. 10A (v5019) depicts the results in BT-474 cells; fig. 10B (v5019) and fig. 10F (v5019 and v10000) depict the results in JIMT1 cells; fig. 10C (v5019) and fig. 10E (v5019 and v10000) depict the results in SKOV3 cells, and fig. 10D (v5019) depicts the results in MCF7 cells.

Figure 11 depicts the ability of an exemplary anti-HER 2 biparatopic antibody to mediate ADCC in SKOV3 cells. In FIG. 11A, the assay was performed using an effector to target cell ratio of 5: 1; in FIG. 11B, the assay was performed using a 3:1 ratio of effector to target cells; and in fig. 11C, the assay was performed using an effector to target cell ratio of 1: 1.

Figure 12 depicts the characterization of affinity and binding kinetics of monovalent anti-HER 2(v630 and v4182) and an exemplary biparatopic anti-HER 2 antibody (v5019) to recombinant human HER 2. FIG. 12A shows the measurement of ka (1/Ms). FIG. 12B shows the measurement of kd (1/s). FIG. 12C shows K_D(M) measurement.

Figure 13 depicts the affinity and binding characteristics of an exemplary biparatopic anti-HER 2 antibody with recombinant human HER2 at a range of antibody capture levels. Figure 13A depicts the measurement of kd (1/s) for Her2ECD determined at a range of antibody capture levels for an exemplary biparatopic anti-Her 2 antibody (v 5019). Figure 13B depicts measurements of kd (1/s) for Her2ECD determined at a range of antibody capture levels for a monovalent anti-Her 2 antibody (v 4182). Figure 13C depicts measurement of kd (1/s) for Her2ECD determined at a range of antibody capture levels for a monovalent anti-Her 2 antibody (v 630).

Figure 14 shows a comparison of the binding mechanisms of the monospecific anti-ECD 4HER2 antibody (left) and the Fab-scFv biparatopic anti-ECD 2xECD4HER2 antibody (right). The monospecific anti-ECD 4HER2 antibody is capable of binding one antibody molecule to two HER2 molecules; whereas the biparatopic anti-ECD 2x ECD4HER2 antibody enables one antibody to bind to two HER2 molecules, and 2 antibodies to bind to one HER2 molecule and combinations thereof, resulting in HER2 receptor cross-linking and lattice formation followed by downstream biological effects as indicated by the arrows such as internalization and/or growth inhibition. IEC denotes "immune effector cells". The four extracellular domains of HER2 are numbered 1, 2, 3, or 4, with 1 ═ ECD1, 2 ═ ECD2, 3 ═ ECD3, and 4 ═ ECD 4.

Figure 15 depicts the effect of an exemplary anti-HER 2 biparatopic antibody on AKT phosphorylation in BT-474 cells.

Figure 16 depicts the effect of an exemplary anti-HER 2 biparatopic antibody on cardiomyocyte viability. Figure 16A depicts the effect of v5019 and corresponding ADC v6363 on cardiomyocyte viability; fig. 16B depicts the effect of v5019, v7091 and v10000 and corresponding ADCv6363, 7148, 10553 on cardiomyocyte viability and fig. 16C depicts the effect of v5019, v7091 and v10000 and corresponding ADC v6363, 7148, 10553 on cardiomyocyte viability pretreated with doxorubicin (doxorubicin).

Figure 17 depicts the ability of an exemplary anti-HER 2 biparatopic antibody drug conjugate to inhibit HER2+ cell growth. Figure 17A shows the ability of ADC v6363 to inhibit the growth of JIMT1 cells. Figure 17B shows the ability of ADC v6363 to inhibit the growth of SKOV3 cells. Figure 17C shows the ability of ADC v6363 to inhibit the growth of MCF7 cells. FIG. 17D shows the ability of ADC v6363 to inhibit MDA-MB-231 cell growth. Figure 17E shows the ability of ADCs v6363, v10553 and v1748 to inhibit the growth of SKOV3 cells. FIG. 17F shows the ability of ADC v6363, v10553 and v1748 to inhibit the growth of JIMT-1 cells. FIG. 17G shows the ability of ADC v6363, v10553 and v1748 to inhibit the growth of NCI-N87 cells.

Figure 18 depicts the effect of biparatopic anti-HER 2 antibodies in a human ovarian cancer xenograft model (SKOV 3). Figure 18A shows the effect of antibody on mean tumor volume. Figure 18B shows the effect of antibodies on the percent survival of animals.

Figure 19 depicts the effect in a biparatopic anti-HER 2 Antibody Drug Conjugate (ADC) human ovarian cancer line xenograft model (SKOV 3). Figure 19A shows the effect of antibody on mean tumor volume. Figure 19B shows the effect of antibodies on the percent survival of animals.

Figure 20 depicts the effect of biparatopic anti-HER 2 Antibody Drug Conjugates (ADCs) on mean tumor volume in a human breast primary cell xenograft model (HBCx-13 b).

Figure 21 depicts the effect of biparatopic anti-HER 2 Antibody Drug Conjugates (ADCs) on mean tumor volume in a human breast primary cell xenograft model (T226).

Figure 22 depicts the effect of biparatopic anti-HER 2 Antibody Drug Conjugates (ADCs) on mean tumor volume in a human breast primary cell xenograft model (HBCx-5).

Figure 23 depicts the effect of biparatopic anti-HER 2 Antibody Drug Conjugates (ADCs) on anti-HER 2 treatment resistant tumors in a human cell line xenograft model (SKOV 3).

Figure 24 depicts the effect of biparatopic anti-HER 2 Antibody Drug Conjugates (ADCs) on anti-HER 2 treatment resistant tumors in a human primary cell xenograft model (HBCx-13 b).

Figure 25 depicts the thermostability of an exemplary anti-HER 2 biparatopic antibody. Fig. 25A depicts the thermal stability of v 5019. Fig. 25B depicts the thermal stability of v 10000. Fig. 25C depicts the thermal stability of v 7091.

Figure 26 depicts the thermostability of exemplary anti-HER 2 biparatopic antibody drug conjugates. Fig. 26A depicts the thermal stability of v 6363. Fig. 26B depicts the thermal stability of v 10553. Fig. 26C depicts the thermal stability of v 7148.

Figure 27 depicts the ability of an anti-HER 2 biparatopic antibody to mediate ADCC in HER2+ cells. The legend shown in fig. 27C applies to fig. 27A and 27B. Figure 27A depicts this ability in SKBR3 cells; FIG. 27B depicts this ability in JIMT-1 cells; FIG. 27C depicts this ability in MDA-MB-231 cells; and figure 27D depicts this ability in WI-38 cells.

Figure 28 depicts the effect of afucosylation on the ability of anti-HER 2 biparatopic antibodies to mediate ADCC. The legend shown in fig. 28B also applies to fig. 28A. Figure 28A compares the ability of the afucosylated version of v5019 to mediate ADCC in SKOV3 cells with herceptin^TMThe ability to mediate ADCC. FIG. 28B compares the ability of the afucosylated version of v5019 to mediate ADCC in MDA-MB-231 cells with that of herceptin^TMThe ability to mediate ADCC. FIG. 28C compares the v10000 ability to mediate ADCC of afucosylated versions in ZR-75-1 cells with herceptin^TMThe ability to mediate ADCC.

Figure 29 depicts the ability of v5019 to inhibit BT-474 cell growth in the presence or absence of growth stimulating ligands.

Figure 30 depicts the effect of afucosylated versions of v5019(v7187) on tumor volume in the human breast cancer xenograft model (HBCx 13B).

Figure 31 depicts the ability of anti-HER 2 biparatopic antibodies and anti-HER 2 biparatopic-ADCs to bind to HER2+ tumor cells. FIG. 31A compares the binding of v6363 to T-DM1 analog (v6246) in SKOV3 cells. FIG. 31B compares the binding of v6363 to T-DM1 analog (v6246) in JIMT-1 cells. Figure 31C compares the binding of several exemplary anti-HER 2 biparatopic antibodies and anti-HER 2 biparatopic-ADCs in SKOV3 cells to controls. FIG. 31D compares the binding of several exemplary anti-HER 2 biparatopic antibodies and anti-HER 2 biparatopic-ADCs in JIMT-1 cells to controls.

Figure 32 depicts dose-dependent tumor growth inhibition of exemplary anti-HER 2 biparatopic-ADCs in HER 23 + (ER-PR negative) patient-derived xenograft model (HBCx13 b). Fig. 32A shows the effect of v6363 on tumor volume, while fig. 32B shows the effect on percent survival.

FIG. 33 depicts the effect of biparatopic anti-HER 2-ADC v6363 compared to Standard of Care Combinations (Standard of Care Combinations) in trastuzumab-resistant PDX HBCx-13b xenograft model. Fig. 33A depicts the effect of treatment on tumor volume, while fig. 33B depicts the effect of treatment on survival.

Figure 34 depicts the efficacy of biparatopic anti-HER 2-ADC in HER2+ trastuzumab-resistant breast cancer cell-derived tumor xenograft model (JIMT-1).

Figure 35 depicts the efficacy of an exemplary anti-HER 2 biparatopic antibody in vivo in a trastuzumab-sensitive ovarian cancer cell-derived tumor xenograft model (SKOV 3). Fig. 35A depicts the effect of treatment on tumor volume, while fig. 35B depicts the effect of treatment on survival.

Figure 36 depicts the dose-dependent efficacy of an exemplary anti-HER 2 biparatopic antibody in vivo in a trastuzumab-sensitive ovarian cancer cell-derived tumor xenograft model (SKOV 3).

Figure 37 depicts the ability of anti-HER 2 biparatopic antibodies and anti-HER 2 biparatopic-ADCs to inhibit growth of cell lines expressing HER2 and EGFR and/or HER3 at 3+, 2+, or 1+ levels. Figure 37A depicts the ability of v10000 to inhibit growth of selected cell lines. Figure 37B depicts the ability of v10553 to inhibit growth of selected cell lines.

Figure 38 depicts a summary of the ability of v10000 and v10553 to inhibit growth in a set of cell lines. Values with hyphenation (e.g. 1/2) indicate differential erbb receptor levels as reported in the literature; erbb IHC values are obtained internally or from the literature. The amount of receptor is unknown and/or unreported at unreported values. IHC level estimates were based on erBb2 gene expression data (Crown BioSciences). The numbered references are described below.

Figure 39 depicts the ability of v10000 to mediate ADCC in HER2+ cells. Fig. 39A depicts the results in FaDu cells. Fig. 39B depicts results in a549 cells. Fig. 39C depicts results in BxPC3 cells. Fig. 39D depicts the results in MiaPaca2 cells.

Figure 40 depicts the ability of an anti-HER 2 biparatopic antibody to mediate ADCC in HER2+ cells. Fig. 40A depicts results in a549 cells. FIG. 40B depicts the results in NCI-N87 cells. FIG. 40C depicts results in HCT-116 cells.

Figure 41 depicts the effect of the biparatopic antibody format against HER2 on binding to HER2+ cells. Figure 41A depicts the effect of format on binding to BT-474 cells. FIG. 41B depicts the effect of the format on binding to JIMT-1 cells. Figure 41C depicts the effect of format on binding to MCF7 cells. FIG. 41D depicts the effect of format on binding to MDA-MB-231 cells.

Figure 42 depicts the effect of a biparatopic antibody format against HER2 on antibody internalization in HER2+ cells. Figure 42A depicts the effect on internalization in BT-474 cells. FIG. 42B depicts the effect on internalization in JIMT-1 cells. Fig. 42C depicts the effect on internalization in MCF7 cells.

Figure 43 depicts the effect of a biparatopic antibody format against HER2 on the ability to mediate ADCC in HER2+ cells. FIG. 43A depicts the effect in JIMT-1 cells. Fig. 43B depicts the effect in MCF7 cells. FIG. 43C depicts the effect in HER 20/1 + MDA-MB-231 breast tumor cells.

Figure 44 depicts the effect of a biparatopic antibody format against HER2 on the ability of the antibody to inhibit the growth of HER2+ tumor cells in the presence or absence of growth stimulating ligand in BT-474 cells.

Figure 45 depicts the effect of the anti-HER 2 biparatopic antibody format on the ability of the antibody to inhibit the growth of SKBR3 cells.

Figure 46 depicts the effect of the biparatopic antibody format of anti-HER 2 on the ability of the antibody to inhibit the growth of HER2+ tumor cells. Figure 46A depicts growth inhibition in SKOV3 cells. FIG. 46B depicts growth inhibition in JIMT-1 cells. Fig. 46C depicts growth inhibition in MCF7 cells.

Figure 47 depicts a comparison of the binding characteristics of different forms of biparatopic antibodies to HER2 as measured by SPR. FIG. 47A depicts a scatter plot and linear regression analysis of kd (1/s) with v6903 and v7091 at different antibody capture levels. Fig. 47B depicts scatter plot and linear regression analysis for kd (m) at different antibody capture levels with v6903 and v 7091.

The references found in fig. 38 are as follows: labouret et al 2012, Neopalasia 14: 121-; 2, Ghasemi et al 2014, Oncogenesis doi: 10.1038/oncssis.2014.31; gaborit et al 2011J Bio Chem,286: 1133-11345; kimura et al.2006, Clin Cancer Res; 4925 and 4932; komoto et al 2009, Cane Sci; 101: 468-; cretellaet al.2014, molecular cancer 13: 143-155; bunn et al 2001, Clin Cancer Res; 3239-3250; lewis crucilps et al 2013, Clin Cancer Res,20: 456-; 9, McDonagh et al.2012,11: 582-; 10, Coldren et al 2006, Mol Cancer Res: 521-; cavazzoni et al.2012MolCancer,11: 91-115; li et al 2014, Mol Cancer Res, doi 10.1158/1541-7786, MCR-13-0396; chmielewski et al 2004, Immunology,173: 7647-7653; kuwada et al.2004, Int J Cancer,109:291- > 301; Fujimoto-Ouchi et al 2007, Clin ChemotherPharmacol,59: 795-805; Chavez-Bianco et al 2004, BMC Cancer,4: 59; campglioet al 2004, J Cellular physiology.198: 259-268; lehmann et al.2011, J ClinInvestination, 121: 2750-; collins et al.2011, Annals Oncology,23: 1788-; takai et al 2005, Cancer,104: 2701-; rusnag et al 2007, CellProlif,40: 580-; ma et al.2013, PLOS ONE,8: e73261-e 73261; meira et al 2009, British J Cancer,101: 782-; hayashi MP28-14 potter; wang et al.2005J Huazhong Univ Sci technology Med Sci.25: 326-8; makhjaet al 2010 JCline Oncolo 28: 1215-.

Detailed Description

Described herein is an antigen binding construct comprising a first antigen binding polypeptide construct that monovalently and specifically binds to the HER2 (human epidermal growth factor receptor 2) ECD2 (extracellular domain 2) antigen on a HER2 expressing cell and a second antigen binding polypeptide construct that monovalently and specifically binds to the HER2ECD4 (extracellular domain 4) antigen on a HER2 expressing cell, wherein at least one of the ECD2 binding polypeptide construct or the ECD4 binding polypeptide construct is an scFv. In certain embodiments, the ECD2 binding polypeptide construct is an scFv and the ECD4 binding polypeptide construct is an Fab. In certain embodiments, the ECD 2-binding polypeptide construct is a Fab and the ECD 4-binding polypeptide construct is a scFv. In some embodiments, both the ECD 2-binding polypeptide construct and the ECD 4-binding polypeptide construct are scfvs. In some embodiments, the antigen binding construct has a dimeric Fc comprising a CH3 sequence. In some embodiments, the Fc is a heterodimer with one or more modifications in the CH3 sequence that promote the formation of heterodimers with stability comparable to a wild-type homodimeric Fc. In some embodiments, the melting temperature (Tm) of the heterodimeric CH3 sequence is 68 ℃ or higher.

The antigen-binding constructs exhibit anti-tumor activity in vitro, such as (i) the ability to inhibit growth of cancer cells in the presence or absence of epidermal growth factor or heregulin (heregulin) stimulation, (ii) the ability to internalize in cancer cells, and (iii) the ability to mediate antibody-directed effector cell killing (ADCC). These in vitro activities were obtained both with naked antigen-binding constructs (i.e. not conjugated to a drug or toxin) and with antigen-binding constructs conjugated to maytansine (maytansine), and at different HER2 expression levels (1+, 2+ and 3 +).

The format of the antigen binding construct (scFv/scFv, scFv/Fab or Fab/Fab) is shown herein to be important in determining the characteristics of its functional diagram. In certain embodiments, the anti-HER 2 binding construct exhibits increased ability to be internalized by HER2 expressing tumor cells as compared to a reference biparatopic antigen-binding construct in which both the ECD2 binding polypeptide construct and the ECD4 binding polypeptide construct are Fab. One embodiment in which both the ECD2 binding polypeptide construct and the ECD4 binding polypeptide are scfvs is internalized by tumor cells expressing HER2 at 1+, 2+, or 3+ levels to a greater extent than a construct with equivalent affinity in the Fab/scFv format, which in turn is internalized more efficiently than a construct with equivalent affinity in the Fab/Fab format. Embodiments that are susceptible to internalization are good candidates for antibody-drug conjugates that require internalization by tumor cells to achieve killing.

In certain embodiments, the antigen binding construct exhibits enhanced potency in ADCC killing of tumor cells expressing low levels of HER2 as compared to a construct having equivalent affinity in the Fab/Fab form. In one embodiment, an antigen binding construct with Fab/scFv format is more effective in ADCC killing of tumor cells expressing low levels of HER2(HER 20-1 + or 1+) than an anti-HER 2 construct with Fab/Fab format, which in turn is more effective than an antigen binding construct with scFv/scFv format.

In some embodiments, the anti-HER 2 antigen binding construct is glycosylated.

In some embodiments, the anti-HER 2 binding construct is afucosylated. In some embodiments, the anti-HER 2 binding construct is coupled to a drug. In some embodiments, the anti-HER 2 binding construct is coupled to maytansine (DM1) through a SMCC linker.

Also described herein are methods of treating a subject having a HER2+ tumor by administering to the subject an anti-HER 2 antigen binding construct. In some embodiments, the level of HER2 expression on the tumor is 2+ or less. In some embodiments, the antigen binding construct is coupled to maytansine. In certain embodiments, the tumor is pancreatic cancer, head and neck cancer, gastric cancer, colorectal cancer, breast cancer, renal cancer, cervical cancer, ovarian cancer, endometrial cancer, uterine cancer, malignant melanoma, pharyngeal cancer, oral cancer, or skin cancer. In some embodiments, the tumor is (i) HER 23 + estrogen receptor negative (ER-), progesterone receptor negative (PR-), trastuzumab-resistant, chemotherapy-resistant invasive breast ductal carcinoma, (ii) HER 23 + ER-, PR-, trastuzumab-resistant inflammatory breast cancer, (iii) HER 23 +, ER-, PR-invasive ductal carcinoma, or (iv) trastuzumab and pertuzumab-resistant breast cancer with amplification of the HER 22 + HER2 gene.

Also provided herein are methods of inhibiting tumor cell growth or killing a tumor cell by administering the antigen binding construct.

Bispecific antigen binding constructs

Provided herein are bispecific antigen-binding constructs that bind HER 2. The bispecific antigen-binding construct comprises two antigen-binding polypeptide constructs, each specifically binding to a different epitope of HER 2. In some embodiments, the antigen binding construct is derived from a known antibody or antigen binding construct. As described in more detail below, the antigen-binding polypeptide construct may be, but is not limited to, protein constructs such as Fab (antigen-binding fragment), scFv (single chain Fv), and sdab (single domain antibody). Typically the antigen binding construct comprises an Fc.

The term "antigen-binding construct" refers to any agent, such as a polypeptide or polypeptide complex, that is capable of binding to an antigen. In some aspects the antigen binding construct is a polypeptide that specifically binds to an antigen of interest. The antigen-binding construct may be a monomer, dimer, multimer, protein, peptide, or protein or peptide complex; an antibody, antibody fragment, or antigen-binding fragment thereof; scFv and the like. The antigen-binding construct may be a monospecific, bispecific or multispecific polypeptide construct. In some aspects, an antigen binding construct can include, for example, one or more antigen binding components (e.g., Fab or scFv) linked to one or more Fc. Additional examples of antigen binding constructs are described below and provided in the examples.

The term "bispecific" is intended to include any agent, e.g., an antigen-binding construct, having two antigen-binding portions (e.g., antigen-binding polypeptide constructs), each with a unique binding specificity. For example, a first antigen-binding moiety binds to an epitope on a first antigen, while a second antigen-binding moiety binds to an epitope on a second antigen. The term "biparatopic" as used herein refers to a bispecific antibody, wherein a first antigen-binding moiety and a second antigen-binding moiety bind to different epitopes on the same antigen. A biparatopic bispecific antibody may bind to two epitopes on the same antigenic molecule or may bind to epitopes on two different antigenic molecules.

A monospecific antigen-binding construct refers to an antigen-binding construct having one binding specificity. In other words, both antigen binding portions bind to the same epitope on the same antigen. Examples of monospecific antigen-binding constructs include, for example, trastuzumab, pertuzumab, which binds to HER 2.

The antigen binding construct may be an antibodyAs used herein, "antibody" or "immunoglobulin" refers to a polypeptide or fragment thereof that specifically binds and recognizes an analyte (e.g., an antigen) that is substantially encoded by an immunoglobulin gene or genes₂、IgG₃、IgG₄Igai and IgA₂The heavy chain constant domains corresponding to different classes of immunoglobulins are designated α, γ, and μ, respectively.

An exemplary immunoglobulin (antibody) building block is composed of two pairs of polypeptide chains, each pair having one "light" (about 25kD) and one "heavy" chain (about 50-70 kD). The N-terminal domain of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chain domains, respectively. The heavy chain of IgG1 consists of VH, CH1, CH2, and CH3 domains, respectively, from N-to C-terminus. The light chain consists of VL and CL domains from N to C. The IgG1 heavy chain comprises a hinge between the CH1 and CH2 domains. In certain embodiments, the immunoglobulin construct comprises at least one immunoglobulin domain from an IgG, IgM, IgA, IgD, or IgE linked to a therapeutic polypeptide. In some embodiments, the immunoglobulin domains found in the antigen binding constructs provided herein are derived or derived from immunoglobulin-based constructs such as diabodies or nanobodies. In certain embodiments, the immunoglobulin constructs described herein comprise at least one immunoglobulin domain from a heavy chain antibody, such as a camelid antibody. In certain embodiments, the immunoglobulin construct provided herein comprises at least one immunoglobulin domain from a mammalian antibody such as a bovine antibody, a human antibody, a camelid antibody, a mouse antibody or any chimeric antibody.

As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain which is hypervariable in sequence and/or forms structurally defined loops ("hypervariable loops"). Typically, a native four-chain antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and/or from Complementarity Determining Regions (CDRs) which have the highest sequence variability and/or are involved in antigen recognition. In addition to CDR1 in VH, the CDRs typically comprise amino acid residues that form hypervariable loops. Hypervariable regions (HVRs) are also referred to as "complementarity determining regions" (CDRs), and these terms are used interchangeably herein in reference to the variable region portions that form the antigen-binding regions. This particular region has been described by Kabat et al, U.S. Dept. of Health and Human Services, Sequences of Proteins of immunological Interest (1983) and Chothia et al, J Mol Biol 196:901-917(1987), wherein the definitions include overlapping or subsets of amino acid residues when compared to each other. However, use of either definition to refer to the CDRs of an antibody or variant thereof is intended to fall within the scope of the terms defined and used herein. Suitable amino acid residues encompassing the CDRs defined by each of the above-cited references are listed below in table 1 for comparison. The exact number of residues covering a particular CDR will vary depending on the sequence and size of the CDR. One skilled in the art can routinely determine which residues make up a particular CDR, taking into account the variable region amino acid sequence of the antibody.

As used herein, the term "single-chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain embodiments, one of the antigen binding polypeptide constructs is a single chain Fab molecule, i.e., a Fab molecule in which the Fab light chain and Fab heavy chain are connected by a peptide linker to form a single peptide chain. In such embodiments, the C-terminus of the Fab light chain is linked to the N-terminus of the Fab heavy chain in a single chain Fab molecule. In certain other embodiments, one of the antigen-binding polypeptide constructs is a single chain Fv molecule (scFv). As described in more detail herein, an scFv has a light chain variable domain (VL) linked from its C-terminus to the N-terminus of a heavy chain variable domain (VH) by a polypeptide chain. Alternatively the scFv consists of a polypeptide chain, wherein the C-terminus of the VH is linked to the N-terminus of the VL via the polypeptide chain.

Antigen binding polypeptide constructs

The bispecific antigen-binding construct comprises two antigen-binding polypeptide constructs, each of which binds to a specific domain or epitope of HER 2. In one embodiment, each antigen binding polypeptide construct binds to the extracellular domain of HER2, e.g. ECD2 or ECD 4. The antigen-binding polypeptide construct may be, for example, a Fab or scFv depending on the application.

The form of the bispecific antigen binding construct determines the functional characteristics of the bispecific antigen binding construct. In one embodiment, the bispecific antigen binding construct has a scFv-Fab format (i.e., one antigen binding polypeptide construct is a scFv and the other antigen binding polypeptide construct is a Fab, also referred to as a Fab-scFv format). In another embodiment, the bispecific antigen binding construct has a scFv-scFv format (i.e., both antigen binding polypeptide constructs are scFv).

The "Fab fragment" (also known as antigen binding fragment) contains the constant domain of the light Chain (CL) and the first constant domain of the heavy chain (CH1) together with the variable domains VL and VH on the light and heavy chains, respectively. The variable domain comprises complementarity determining loops (CDRs, also referred to as hypervariable regions) that are involved in antigen binding. Fab' fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region.

"Single chain Fv" or "scFv" include the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In one embodiment, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the structure required for antigen binding. For a review of scFv, see Pluckthun, The pharmacy of monoclonal Antibodies, Vol.113, Rosenburg and Moore eds, Springer-Verlag, New York, pp.269-315 (1994). HER2 antibody scFv fragments are described in WO93/16185, U.S. patent No. 5,571,894, and U.S. patent No. 5,587,458.

A "single domain antibody" or "sdAb" format is an independent immunoglobulin domain. Sdab is fairly stable and readily expressed as a fusion partner with the Fc chain of an antibody (Harmsen MM, De Haard HJ (2007). "Properties, production, and applications of functional single-domain antibody fragments". Appl. Microbiol Biotechnol.77(1): 13-22).

Form and function of antigen binding constructs

Provided herein are biparatopic HER2 antigen-binding constructs having two antigen-binding polypeptide constructs, wherein the first specifically binds to HER2ECD2 and the second specifically binds to HER2ECD 4. The form of the antigen-binding construct is such that at least one of the first or second antigen-binding polypeptides is an scFv. The antigen-binding construct may be in the form of an scFv-scFv or a Fab-scFv or an scFv-Fab (primary antigen-binding polypeptide construct-secondary antigen-binding polypeptide, respectively).

In certain embodiments, the antigen-binding construct exhibits anti-tumor activity in vitro, such as (i) the ability to inhibit growth of a cancer cell in the presence or absence of stimulation by epidermal growth factor or heregulin, (ii) the ability to internalize in a cancer cell (by binding to and internalizing the HER2 antigen), and (iii) the ability to mediate antibody-directed effector cell killing (ADCC). These in vitro activities were obtained with both naked antigen-binding constructs and with antigen-binding constructs conjugated to maytansine, and at different HER2 expression levels (1+, 2+ and 3 +).

The examples herein show that the form of the antigen binding construct (scFv/scFv, scFv/Fab or Fab/Fab) is important in determining its functional properties. In certain embodiments, the anti-HER 2 binding construct exhibits enhanced ability to be internalized by HER2 expressing tumor cells as compared to a reference antigen binding construct in which both the ECD2 binding polypeptide construct and the ECD4 binding polypeptide construct are Fab. It is expected that the degree of internalization of the anti-HER 2 antigen-binding construct may be further increased by enhancing the affinity of one or both antigen-binding polypeptide constructs for ECD2 or ECD 4. One embodiment in which both the ECD2 binding polypeptide and the ECD4 binding polypeptide are scfvs is internalized by tumor cells expressing HER2 at 1+, 2+, or 3+ levels to a greater extent than a construct with equivalent affinity in the Fab/scFv format, which in turn is more efficient than a construct with equivalent affinity in the Fab/Fab format. Embodiments that are susceptible to internalization are good candidates for antibody-drug conjugates that require internalization by tumor cells to achieve killing. Conversely, in certain embodiments, antigen-binding constructs that are not readily internalized exhibit enhanced potency in ADCC killing of tumor cells expressing low levels of HER2 as compared to constructs with equivalent affinity in the Fab/Fab form. In one embodiment, an antigen binding construct with Fab/scFv format is more effective in ADCC killing of tumor cells expressing low levels of HER2(HER 20-1 + or 1+) than an anti-HER 2 construct with Fab/Fab format, which in turn is more effective than an antigen binding construct with scFv/scFv format. The enhanced ADCC potency of some embodiments may be due to 1) its ability to affinity bind with low HER2 receptor density and subsequently to cause HER2 receptor to accumulate at the target cell surface and mediate downstream cell-mediated killing; and/or 2) its ability to remain on the cell surface is enhanced (without causing internalization); they are therefore more suitable for cell-mediated effector killing.

HER2

The antigen binding constructs described herein have antigen binding polypeptide constructs that bind to ECD2 and ECD4 of HER 2.

The expressions "ErbB 2" and "HER 2" are used interchangeably herein and refer to, for example, the human HER2 protein (GenBank accession number X03363) described in Semba et al, PNAS (USA)82: 6497-. The terms "erbB 2" and "neu" refer to genes encoding human erbB2 protein. p185 or p185neu refers to the protein product of the neu gene.

HER2 is a HER receptor. The "HER receptor" is a receptor protein tyrosine kinase belonging to the family of human epidermal growth factor receptors (HER) and includes the EGFR, HER2, HER3 and HER4 receptors. The HER receptor will typically comprise an extracellular domain that can bind a HER ligand; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxy-terminal signaling domain with several tyrosine residues that can be phosphorylated. By "HER ligand" is meant a polypeptide that binds to and/or activates a HER receptor.

The extracellular (outer) domain of HER2 includes four domains, domain I (ECD1, amino acid residues from about 1-195), domain II (ECD2, amino acid residues from about 196-488), domain III (ECD3, amino acid residues from about 320-488), and domain IV (ECD4, amino acid residues from about 489-630) (residue numbering, no signal peptide). See Garrett et al mol.cell.11:495-505(2003), Cho et al Nature 421:756-760(2003), Franklin et al Cancer Cell5:317-328(2004), Tse et al Cancer Treat Rev.2012, month 4; 38(2):133-42(2012), or Plowman et al Proc. Natl. Acad. Sci.90: 1746. 1750 (1993).

The sequence of HER2 is as follows; ECD boundary is domain I: 1 to 165; domain II: 166-322; domain III: 323-488; domain IV: 489-607.

"epitope 2C 4" is the region in the extracellular domain of HER2 that binds to antibody 2C 4. Epitope 2C4 comprises residues from domain II in the extracellular domain of HER 2. 2C4 and pertuzumab bind to the HER2 extracellular domain at the junction of domains I, II and III. Franklin et al Cancer Cell 5: 317-. To screen for Antibodies that bind to the epitope of 2C4, a conventional cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring harbor Laboratory, Harlow and David Lane editors (1988) can be performed. Alternatively, epitope mapping can be performed using methods known in the art to assess whether an antibody binds to the 2C4 epitope of HER2 and/or the antibody-HER 2 structure (Cancer Cell 5:317-328(2004) by Franklin et al) can be studied to understand which domain of HER2 is bound by the antibody.

"epitope 4D 5" is the region of the extracellular domain of HER2 that binds to antibody 4D5(ATCC CRL 10463) and trastuzumab. This epitope is close to the transmembrane domain of HER2 and within domain IV of HER 2. To screen for Antibodies that bind to the 4D5 epitope, a conventional cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring harbor Laboratory, editors Harlow and David Lane (1988) can be performed. Alternatively, epitope mapping can be performed to assess whether the antibody binds to the 4D5 epitope of HER2 (e.g., any one or more residues within the region from about residue 529 to about residue 625 (inclusive), see figure 1 of U.S. patent publication No. 2006/0018899).

By "specifically binds", "specifically binds" or "selectively binds" is meant that binding is selective for the antigen and can be distinguished from interactions that are not required or are non-specific. The ability of an antigen-binding construct to bind to a particular epitope can be measured by enzyme-linked immunosorbent assays (ELISAs) or other techniques familiar to those skilled in the art, such as Surface Plasmon Resonance (SPR) techniques (analyzed on a BIAcore instrument) (Liljeblad et al, Glyco J17, 323-. In one embodiment, the extent of binding of the antigen-binding portion to an unrelated protein is less than about 10% of the binding of the antigen-binding construct to the antigen, as measured, for example, by SPR. In certain embodiments, the antigen binding construct that binds to an antigen, or antigen binding molecule comprising an antigen binding portion, dissociation constant (K)_D)<1μM、<100nM、<10nM、<1nM、<0.1nM、<0.01nM or<0.001nM (e.g., 10)^-8M or less, e.g. from 10^-8M to 10"¹³M, e.g. from 10"⁹M to 10"¹³M)。

"Modulator protein" (HRG) as used herein refers to a polypeptide encoded by the heregulin gene product as disclosed in U.S. Pat. No. 5,641,869 or Marchioni et al, Nature,362: 312-. Examples of heregulins include heregulin-alpha, heregulin-beta 1, heregulin-beta 2, and heregulin-beta 3(Holmes et al, Science,256:1205-1210 (1992); and U.S. Pat. No. 5,641,869); neu Differentiation Factor (NDF) (Peles et al Cell 69:205-216 (1992)); acetylcholine Receptor Inducing Activity (ARIA) (cells 72:801-815(1993)) by Falls et al; glial Growth Factor (GGF) (Marchionni et al, Nature,362:312-318 (1993)); sensory and motor neuron derived factor (SMDF) (Ho et al J.biol.chem.270: 14523-; gamma-heregulin (Schaefer et al Oncogene 15:1385-1394 (1997)). The term includes biologically active fragments and/or amino acid sequence variants of native sequence HRG polypeptides, such as EGF-like domain fragments thereof (e.g., HRG. beta. 1177-244).

"HER activation" or "HER 2 activation" refers to the activation or phosphorylation of any one or more HER receptors or HER2 receptors. Typically, HER activation results in signal transduction (e.g., by the HER receptor or an intracellular kinase domain in a substrate polypeptide in which the HER receptor phosphorylates tyrosine residues). HER activation can be mediated by binding of a HER ligand to a HER dimer comprising the HER receptor of interest. Binding of a HER ligand to a HER dimer may activate the kinase domain of one or more HER receptors in the dimer and thereby cause phosphorylation of tyrosine residues in one or more HER receptors and/or phosphorylation of tyrosine residues in additional substrate polypeptides, such as Akt or MAPK intracellular kinases.

Derivatized antigen-binding polypeptide constructs

The antigen-binding polypeptide construct may be derived from a known anti-HER 2 antibody or anti-HER 2 binding domain, regardless of the type of domain. Examples of domain types include Fab fragments, scFv and sdAb. Furthermore, if the antigen binding portion of the known anti-HER 2 antibody or binding domain is a Fab, the Fab can be converted to a scFv. Likewise, if the antigen binding portion of the known anti-HER 2 antibody or binding domain is an scFv, the scFv can be converted to a Fab. Methods for converting between antigen binding domain types are known in the art (see, e.g., Zhou et al (2012) an exemplary method for converting scFv to Fab form as described by Mol Cancer Ther 11: 1167-1476. the methods described herein are incorporated by reference).

The antigen binding constructs described herein may be derived from known anti-HER 2 antibodies that bind to ECD2 or ECD 4. As described elsewhere herein, antibodies that bind to ECD2 or ECD4 are known in the art and include, for example, 2C4 or pertuzumab (binding to ECD2), 4D5, or trastuzumab (binding to ECD 4). Other antibodies that bind to ECD2 or ECD4 of HER2 have also been described in the art, for example in WO 2011/147982 (GenmabA/S).

In some embodiments, the antigen binding polypeptide construct of the antigen binding construct is derived from an antibody that blocks binding of antibody 4D5 or trastuzumab to ECD4 of HER2 by 50% or more. In some embodiments, the antigen binding polypeptide construct of the antigen binding construct is derived from an antibody that blocks binding of antibody 2C4 or pertuzumab to ECD2 of HER2 by 50% or more. In some embodiments, the antigen binding construct is derived from an antibody that blocks binding of antibody 2C4 or pertuzumab to ECD2 of HER2 by 30% or more.

In one embodiment, the antigen-binding polypeptide construct is derived from a Fab fragment of trastuzumab or pertuzumab. In one embodiment, the antigen binding polypeptide is derived from an scFv.

In certain embodiments the antigen-binding polypeptides are derived from humanized or chimeric versions of these antibodies.

A "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody containing minimal sequences derived from non-human immunoglobulins. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and capacity. In some cases, Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications were made to further improve antibody performance. In general, a humanized antibody will comprise a substantial majority of at least one and typically two variable domains, wherein all or a substantial majority of the hypervariable loops correspond to those of a non-human immunoglobulin and all or a substantial majority of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. For more details, see Jones et al, Nature 321:522-525 (1986); riechmann et al, Nature 332: 323-E329 (1988); and Presta, curr, Op, Structure, biol.2:593-596 (1992).

Humanized HER2 antibodies include huMAb4D5-l, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 or trastuzumab as described in Table 3 of U.S. Pat. No. 5,821,337, which is expressly incorporated herein by referenceHumanized 520C9(WO93/21319) and 20' humanized 2C4 antibodies described in U.S. patent publication No. 2006/0018899.

Affinity maturation

In some embodiments, the antigen binding construct is derived from a known HER2 binding antibody using affinity maturation.

Where it is desired to increase the affinity of an antigen-binding polypeptide for its cognate antigen, the affinity of the antigen-binding polypeptide for its antigen can be increased using methods known in the art. Examples of such methods are described in the following references: birtalan et al (2008) JMB 377, 1518-; gerstner et al (2002) JMB 321, 851-862; kelley et al (1993) Biochem 32(27), 6828-6835; li et al (2010) JBC 285(6),3865-3871, and Vajdos et al (2002) JMB 320, 415-428.

An exemplary method for affinity maturation of the HER2 antigen binding domain is described below. The structures of trastuzumab/HER 2(PDB code 1N8Z) complex and pertuzumab/HER 2 complex (PDB code 1S78) were used for modeling. Molecular Dynamics (MD) can be used to evaluate the intrinsic dynamic properties of WT complexes in aqueous environments. Mean field and dead-end exclusion methods along with flexible scaffolds can be used to optimize and prepare model structures for the mutants to be screened. After packaging, a number of characteristics will be scored including contact density, impact score (close score), hydrophobicity, and static. The generalized Born method will allow accurate modeling of solvent environment effects and calculation of the free energy difference after mutation of specific positions in the protein to change residue type. Contact density and collision score will provide a measure of complementarity, which is a key aspect of efficient protein packaging. The screening program employs knowledge-based potential and coupled analysis schemes and entropy calculations that rely on pairwise residue interaction energy. Literature mutations and combinations thereof known to enhance HER2 binding are summarized in the table below.

Table a4. trastuzumab mutations known to increase binding to HER2 for the trastuzumab-HER 2 system.

Table a5 pertuzumab mutations known to increase binding to HER2 for the pertuzumab-HER 2 system.

Fc of antigen binding constructs.

In some embodiments, an antigen binding construct described herein comprises an Fc, e.g., a dimeric Fc.

The term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described by Kabat et al, Sequences of proteins of Immunological Interest, published Health Service 5 th edition, national institutes of Health, Bethesda, MD, 1991. As used herein, an "Fc polypeptide" of a dimeric Fc refers to one of two polypeptides that form a dimeric Fc domain, i.e., a polypeptide that comprises the C-terminal constant region of an immunoglobulin heavy chain, capable of stable self-association. For example, the Fc polypeptide of dimeric IgG Fc comprises IgG CH2 and IgG CH3 constant domain sequences.

The Fc domain comprises the CH3 domain or the CH3 and CH2 domains. The CH3 domain comprises two CH3 sequences, one from each of two Fc polypeptides of a dimeric Fc. The CH2 domain comprises two CH2 sequences, one from each of two Fc polypeptides of a dimeric Fc.

In some aspects, the Fc comprises at least one or two CH3 sequences. In some aspects, the Fc is coupled to the first antigen-binding construct and/or the second antigen-binding construct with or without one or more linkers. In some aspects, the Fc is a human Fc. In some aspects, the Fc is human IgG or IgG1 Fc. In some aspects, the Fc is a heterodimeric Fc. In some aspects, the Fc comprises at least one or two CH2 sequences.

In some aspects, the Fc comprises one or more modifications in at least one CH3 sequence. In some aspects, the Fc comprises one or more modifications in at least one CH2 sequence. In some aspects, the Fc is a single polypeptide. In some aspects, the Fc is a plurality of polypeptides, e.g., two polypeptides.

In some aspects, the Fc is an Fc described in patent application PCT/CA2011, filed on day 11, 4, 2011 or PCT/CA2012/050780 filed on day 11, 2, 2012, each of which is hereby incorporated by reference in its entirety for all purposes.

Modified CH3 domain

In some aspects, the antigen binding constructs described herein comprise a heterodimeric Fc comprising a modified CH3 domain that has been asymmetrically modified. The heterodimeric Fc can comprise two heavy chain constant domain polypeptides: a first Fc polypeptide and a second Fc polypeptide, which are used interchangeably so long as the Fc comprises one first Fc polypeptide and one second Fc polypeptide. Typically, the first Fc polypeptide comprises a first CH3 sequence and the second Fc polypeptide comprises a second CH3 sequence.

Two CH3 sequences comprising one or more amino acid modifications introduced in an asymmetric manner, when two CH3 sequences dimerize, typically produce a heterodimeric Fc rather than a homodimer. As used herein, "asymmetric amino acid modification" refers to any modification in which the amino acid at a particular position on the first CH3 sequence differs from the amino acid at the same position on the second CH3 sequence, and the first and second CH3 sequences preferentially pair to form a heterodimer, rather than a homodimer. This heterodimerization may be only one modification of two amino acids at each identical amino acid position on each sequence; or the result of modification of both amino acids in each sequence at the same position in each of the first and second CH3 sequences. The first and second CH3 sequences of the heterodimeric Fc can comprise one or more asymmetric amino acid modifications.

Table a provides the amino acid sequence of the human IgG1Fc sequence, corresponding to amino acids 231 to 447 of the full-length human IgG1 heavy chain. The CH3 sequence contained amino acids 341-447 of the full-length human IgG1 heavy chain.

Typically, the Fc may comprise two consecutive heavy chain sequences (a and B) capable of dimerizing. In some aspects, one or both sequences of the Fc comprise one or more mutations or modifications at the following positions: l351, F405, Y407, T366, K392, T394, T350, S400 and/or N390, using EU numbering. In some aspects, the Fc comprises a mutated sequence shown in table X. In some aspects, the Fc comprises a mutation of variant 1A-B. In some aspects, the Fc comprises a mutation of variant 2A-B. In some aspects, the Fc comprises a mutation of variant 3A-B. In some aspects, the Fc comprises a mutation of variant 4A-B. In some aspects, the Fc comprises a mutation of variant 5A-B.

Table a: IgG1Fc sequence

With respect to amino acids 231 to 447 of the full-length human IgG1 heavy chain, the first and second CH3 sequences may comprise amino acid mutations as described herein. In one embodiment, the heterodimeric Fc comprises a modified CH3 domain with the first CH3 sequence having amino acid modifications at positions F405 and Y407 and the second CH3 sequence having amino acid modifications at position T394. In one embodiment, the heterodimeric Fc comprises a modified CH3 domain with a first CH3 sequence having one or more amino acid modifications selected from L351Y, F405A, and Y407V, and a second CH3 sequence having one or more amino acid modifications selected from T366L, T366I, K392L, K392M, and T394W.

In one embodiment, the heterodimeric Fc comprises a modified CH3 domain with the first CH3 sequence having amino acid modifications at positions L351, F405, and Y407 and the second CH3 sequence having amino acid modifications at positions T366, K392, and T394, and one of the first or second CH3 sequences further comprises an amino acid modification at position Q347 and the other CH3 sequence further comprises an amino acid modification at position K360. In another embodiment, a heterodimeric Fc comprises a modified CH3 domain with a first CH3 sequence having amino acid modifications at positions L351, F405, and Y407 and a second CH3 sequence having amino acid modifications at positions T366, K392, and T394, one of the first or second CH3 sequences further comprising an amino acid modification at position Q347 and the other CH3 sequence further comprising an amino acid modification at position K360, and one or both of the CH3 sequences further comprising an amino acid modification T350V.

In one embodiment, the heterodimeric Fc comprises a modified CH3 domain with a first CH3 sequence having amino acid modifications at positions L351, F405 and Y407 and a second CH3 sequence having amino acid modifications at positions T366, K392 and T394 and one of said first and second CH3 sequences further comprising amino acid modifications of D399R or D399K and the other CH3 sequence further comprising one or more of T411E, T411D, K409E, K409D, K392E and K392D. In another embodiment, a heterodimeric Fc comprises a modified CH3 domain having amino acid modifications at positions L351, F405, and Y407 in a first CH3 sequence and amino acid modifications at positions T366, K392, and T394 in a second CH3 sequence, one of said first and second CH3 sequences further comprising amino acid modifications of D399R or D399K and the other CH3 sequence comprising one or more of T411E, T411D, K409E, K409D, K392E, and K59392 392D, and one or both of said CH3 sequences further comprising amino acid modification T350V.

In one embodiment, the heterodimeric Fc comprises a modified CH3 domain with a first CH3 sequence having amino acid modifications at positions L351, F405, and Y407, and a second CH3 sequence having amino acid modifications at positions T366, K392, and T394, wherein one or both of the CH3 sequences further comprise amino acid modifications of T350V.

In one embodiment, the heterodimeric Fc comprises a modified CH3 domain comprising the following amino acid modifications, wherein "a" represents an amino acid modification to the first CH3 sequence and "B" represents an amino acid modification to the second CH3 sequence: a: L351Y _ F405A _ Y407V, B: T366L _ K392M _ T394W, a: L351Y _ F405A _ Y407V, B: T366L _ K392L _ T394W, a: T350V _ L351Y _ F405A _ Y407V, B: T350V _ T366L _ K392L _ T394W, a: T350V _ L351Y _ F405A _ Y407V, B: T350V _ T366L _ K392M _ T394W, a: T350V _ L351Y _ S400E _ F405A _ Y407V, and/or B: T350V _ T366L _ N390R _ K392M _ T394W.

The one or more asymmetric amino acid modifications can promote the formation of heterodimeric Fc, where the heterodimeric CH3 domain has comparable stability to the wild-type homodimeric CH3 domain. In one embodiment, the one or more asymmetric amino acid modifications promote the formation of a heterodimeric Fc domain, wherein the heterodimeric Fc domain has comparable stability to a wild-type homodimeric Fc domain. In one embodiment, the one or more asymmetric amino acid modifications promote the formation of a heterodimeric Fc domain, wherein the heterodimeric Fc domain has the property of being observed via melting temperature (Tm) in a differential scanning calorimetry study And wherein the melting temperature is within 4 ℃ of the melting temperature observed for the corresponding symmetric wild-type homodimeric Fc domain. In some aspects, the Fc is comprised in at least one C_H3One or more modifications in the sequence that promote the formation of heterodimeric Fc with comparable stability to a wild-type homodimeric Fc domain.

In one embodiment, the stability of the CH3 domain may be assessed by measuring the melting temperature of the CH3 domain, for example by Differential Scanning Calorimetry (DSC). Thus, in another embodiment, the CH3 domain has a melting temperature of about 68 ℃ or higher. In another embodiment, the CH3 domain has a melting temperature of about 70 ℃ or higher. In another embodiment, the CH3 domain has a melting temperature of about 72 ℃ or greater. In another embodiment, the CH3 domain has a melting temperature of about 73 ℃ or greater. In another embodiment, the CH3 domain has a melting temperature of about 75 ℃ or greater. In another embodiment, the CH3 domain has a melting temperature of about 78 ℃ or higher. In some aspects, the dimerization CH3 sequence has a melting temperature (Tm) of about 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 77.5, 78, 79, 80, 81, 82, 83, 84, or 85 ℃ or more.

In some embodiments, a heterodimeric Fc comprising a modified CH3 sequence in the expression product can be formed at least about 75% pure compared to a homodimeric Fc. In another embodiment, the heterodimeric Fc can be formed with a purity of greater than about 80%. In another embodiment, the heterodimeric Fc can be formed with a purity of greater than about 85%. In another embodiment, the heterodimeric Fc can be formed with a purity of greater than about 90%. In another embodiment, the heterodimeric Fc can be formed with a purity of greater than about 95%. In another embodiment, the heterodimeric Fc can be formed with a purity of greater than about 97%. In some aspects, Fc is a heterodimer formed at a purity of greater than about 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% when expressed. In some aspects, Fc is a heterodimer formed with a purity of greater than about 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% when expressed via a single cell.

In International patent publication No. WO 96/027011 (knobs int holes), in Gunasekaran ET al (Gunasekaran K. ET al (2010) J Biol chem.285,19637-46, electrostatic design to reach selective chemistrinization), in Davis ET al (Davis, JH. ET al (2010) Prot EngDes Sel; 23(4) 195. times 202, Strand Exchange Engineered Domain (SEED) technology), and in Labrijn ET al [ Efficient generation of stable biochemical IgG1by coordinated-arm exchange. Labrijn AF, Schers JI, Goeij, van Bremer, Neivan J, Strivan MJ, Schedule JJ, K20135, perpendicular Fangn, USA PW 3, W.26, German grade J.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P; 110(13) 5145-50 describes additional methods of modifying monomeric Fc polypeptides to promote heterodimeric Fc formation.

CH2 Domain

In some embodiments, the Fc of the antigen binding construct comprises a CH2 domain. An example of the CH2 domain of Fc is amino acids 231-340 of the sequence shown in Table A. Several effector functions are mediated by Fc receptors (fcrs) that bind to the Fc of antibodies.

The terms "Fc receptor" and "FcR" are used to describe a receptor that binds to the Fc region of an antibody. For example, the FcR may be a native sequence human FcR. Typically, an FcR is one that binds an IgG antibody (gamma receptor) and includes receptors of the Fc γ RI, Fc γ RII, and Fc γ RIII subclasses, including allelic variants and variably cleaved forms of these receptors. Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB (inhibiting receptor), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Immunoglobulins of other isotypes may also be bound by certain FcRs (see, e.g., Janeway et al, immunology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4 th edition, 1999)). The activating receptor Fc γ RIIA contains in its cytoplasmic domainImmunoreceptor tyrosine-based activation motifs (ITAMs). The inhibitory receptor Fc γ RIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain Annu Rev.Immunol.15:203-234 (1997). In ravatch and Kinet, Annu.Rev.Immunol 9:457-92 (1991); capel et al, immunolmethods 4:25-34 (1994); and de Haas et al, J.Lab.Clin.Med.126:330-41(1995) reviews FcRs. Other fcrs, including those identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al, J.Immunol.117:587 (1976); and Kim et al, J.Immunol.24:249 (1994)).

Modifications in the CH2 domain may affect FcR binding to Fc. Many amino acid modifications in the Fc region are known in the art to selectively alter the affinity of Fc for different fey receptors. In some aspects, the Fc comprises one or more modifications that promote selective binding of Fc-gamma receptors.

Exemplary mutations that alter Fcr binding to Fc are listed below:

S298A/E333A/K334A, S298A/E333A/K334A/K326A (JImmunel methods, 2011Feb 28, Lu Y, Vernes JM, Chiang N, etc.; 365(1-2): 132-41);

F243L/R292P/Y300L/V305I/P396L, F243L/R292P/Y300L/L235V/P396L (Cancer Res.2007, 9 and 15 days; 67(18): 8882-90; Breast Cancer Res.2011, 11 and 30 days; 13(6): R123) such as Stavenhagen JB, Gorlatov S, Tuailon N, etc.);

F243L (Stewart R, Thom G, Protein Eng Des Sel.2011 9/24 (9):671-8.), S298A/E333A/K334A (Shields RL, Namenuk AK, Hong K J Biol chem.2001 3/2; 276(9): 6591-;

S239D/I332E/A330L, S239D/I332E (Lazar GA, Dang W, Karki S et al Proc Natl Acadsi USA.2006, 3, 14, 3(11): 4005-10);

S239D/S267E, S267E/L328F (Chu SY, Vostinar I, Karki S, etc. Mol Immunol.2008.9 months; 45(15): 3926-33);

S239/D265/S298/I332, S239/S298/K326/A327, G237/S298/A330/I332, S239/I332/S298, S239/K326/A330/I332/S298, G236/S239/D270/I332, S239/S267/H268, L234/S267/N325, G237/V266/S267 and other mutations listed in WO 2011/and WO 2011/which are incorporated herein by reference. The mutations are listed on page 283 in Therapeutic antibody engineering (William r.strohl and Lila m.strohl, Woodhead Pu blishing series in Biomedicine No 11, ISBN 1907568379, month 10 2012).

In some embodiments the antigen binding constructs described herein comprise antigen binding polypeptide constructs that bind an antigen; and dimeric Fc, which has excellent biophysical properties such as stability and is easy to produce relative to antigen binding constructs that do not include the same dimeric Fc. In some embodiments the CH2 domain comprises one or more asymmetric amino acid modifications. Exemplary asymmetric mutations are described in international patent application No. PCT/CA 2014/050507.

Additional modifications that improve effector function.

In some embodiments the antigen binding constructs described herein comprise modifications that improve their ability to mediate effector functions. Such modifications are known in the art and include afucosylation or engineering of Fc affinity for activated receptors, primarily FCGR3a for ADCC and C1q for CDC. Table B below summarizes the various designs reported in the literature for effector function engineering.

Methods of generating antigen binding constructs with little or no fucose at the Fc glycosylation site (Asn 297EU numbering) without altering the amino acid sequence are known in the art.Technique of(ProBioGen AG) is based on the introduction of genes that bias the cellular pathway of fucose biosynthesis towards cells for antigen binding construct production. This prevents the addition of the sugar "fucose" to the N-linked antibody carbohydrate moiety by the cells that produce the antigen binding construct. (von Horsten et al (2010) glycobiology.2010, 12 months; 20(12): 1607-18. Another method of obtaining an antigen binding construct with a reduced level of fucosylation can be found in U.S. Pat. No. 8,409,572, which teaches the ability to produce a lower level of fucosylation on an antigen binding construct selected for a cell line produced from the antigen binding construct.

Thus, in one embodiment, the constructs described herein may comprise a dimeric Fc comprising one or more amino acid modifications conferring improved effector function as indicated in table B. In another embodiment, the construct may be afucosylated to improve effector function.

Table B: the CH2 domain and effector function were engineered.

Fc modifications that reduce Fc γ R and/or complement binding and/or effector function are known in the art. Recent publications describe strategies that have been used to engineer antibodies with reduced or silenced effector activity (see Strohl, WR (2009), Curropin Biotech 20:685 + 691, and Strohl, WR and Strohl LM, "Antibody Fc engineering for optimal Antibody performance" in Therapeutic Antibody engineering, Cambridge: Woodhead Publishing (2012), p.225 + 249). These strategies include reduction of effector function by modification of glycosylation, use of the IgG2/IgG4 backbone, or introduction of mutations in the hinge or CH2 region of Fc. For example, U.S. patent publication No. 2011/0212087 (Strohl), international patent publication No. WO 2006/105338 (xenocor), U.S. patent publication No. 2012/0225058 (xenocor), U.S. patent publication No. 2012/0251531 (Genentech), and shop et al ((2012) j.mol.biol.420:204-219) describe specific modifications that reduce Fc γ R or complement binding to Fc.

Specific, non-limiting examples of known amino acid modifications that reduce Fc γ R or complement binding to Fc include those identified in the following table:

table C: modifications to reduce Fc γ R or complement binding to Fc

Company(s)	Mutations
		GSK	N297A
Ortho Biotech	L234A/L235A
		Protein Design labs	IGG2 V234A/G237A
Wellcome Labs	IGG4 L235A/G237A/E318A
		GSK	IGG4 S228P/L236E
Alexion	IGG2/IGG4 combination
		Merck	IGG2H268Q/V309L/A330S/A331S
Bristol-Myers	C220S/C226S/C229S/P238S
		Seattle Genetics	C226S/C229S/E3233P/L235V/L235A
Amgen	Coli (e.coli) production, non-glycosylation
		Medimune	L234F/L235E/P331S
Trubion	Hinge mutant, probably C226S/P230S

In one embodiment, the Fc comprises at least one amino acid modification identified in the above table. In another embodiment, the Fc comprises an amino acid modification of at least one of L234, L235, or D265. In another embodiment, Fc comprises amino acid modifications at L234, L235 and D265. In another embodiment, the Fc comprises the amino acid modifications L234A, L235A and D265S.

Linker and linker polypeptide

Each antigen-binding polypeptide construct of the antigen-binding constructs is operably linked to a linker polypeptide, wherein the linker polypeptides are capable of forming covalent bonds with each other. The spatial conformation of the antigen-binding construct comprising the first and second antigen-binding polypeptide constructs and the linker polypeptide is similar to the relative spatial conformation of the paratope of the F (ab') 2 fragment generated by papain digestion, although in the case of the bispecific antigen-binding constructs described herein, the two antigen-binding polypeptide constructs are in the form of a Fab-scFv or a scFv-scFv.

Thus, the linker polypeptide is selected such that it retains the relative spatial conformation of the paratope of the F (ab') fragment and is capable of forming covalent bonds equivalent to disulfide bonds in the IgG core hinge. Suitable linker polypeptides include an IgG hinge region, such as a hinge region from IgG1, IgG2, or IgG 4. Modified versions of these exemplary linkers may also be used. For example, modifications that improve the stability of the IgG4 hinge are known in the art (see, e.g., Labrijn et al (2009) Nature Biotechnology 27, 767-771).

In one embodiment, the linker polypeptide is operably linked to a scaffold described herein, e.g., an Fc. In some aspects, the Fc is coupled to the one or more antigen binding polypeptide constructs via one or more linkers. In some aspects, the Fc is coupled to the heavy chain of each antigen-binding polypeptide by a linker.

In other embodiments, the linker polypeptide is operably linked to a scaffold other than an Fc. Many alternative protein or molecular domains are known in the art and can be used to form selective pairs of two different antigen binding polypeptides. Examples are leucine zipper domains such as Fos and Jun selectively paired together [ S A Kostelny, M S Cole and J YTso. Formation of a biospecific antibody by the use of leucosine zippers. JIMMunol 1992148: 1547-53; beld J.Wranik, Erin L.Christensen, Gabrile Schaefer, Janet K.Jackman, Andrew C.Vendel and Dan Eaton.LUZ-Y, a Novel Platform for the Mammarian Cell Production of Full-length IgG-bispecific antibodies J.biol.chem.2012287: 43331- "43339 ]. Alternatively, other selective partner pairs such as the barnase barstar pair [ Deyev, S.M., Waibel, R., Lebedenko, E.N., Schubiger, A.P., and Pluckthun, A. (2003) Design of multiple compounds using the barnase module. Nat Biotechnol 21,1486-1492], the DNA strand pair [ Zahida N. Chaudri, Michael bartl-Jones, George Panatou, Thomas Klonisch, IvanM. Roitt, Torpen Lund, Peter J. Delvers, Dual specific antibodies binding-stranded antibodies binding-bound, Pepper J. Delvers, Dual specific antibodies binding-bound antibodies, Pasteur 3523, Hakkenkun protein, Past 23, Hakken, Sphings et 23, Pasteur, Past 23, Past, Pasteur, Past 23, Hakken, Spiro et 23, Hakken protein, Pasteur, Hakken, No. 3, Hakken, No. 3, Hakker, Hakken, Hakker, Ha.

Dissociation constant (K) _D ) And maximum binding capacity (Bmax)

In some embodiments, the antigen binding construct is described by functional characteristics including, but not limited to, dissociation constant and maximum binding capacity.

The term "dissociation constant (K) as used herein_D) "is intended to mean the equilibrium dissociation constant for a particular ligand-protein interaction. As used herein, ligand-protein interaction refers to, but is not limited to, protein-protein interaction or antibody-antigen interaction. K_DThe tendency of two proteins (e.g., AB) to reversibly dissociate into smaller components (a + B) is measured and defined as the dissociation rate (also referred to as the "separation rate (k))_off) ") and association rate (or" association rate (k)_on) ") ratio. Thus, K_DIs equal to k_off/k_onAnd is expressed as molar concentration (M). Thus concluding that K_DThe smaller the binding affinity, the stronger. Thus, K at 1mM_DIndicating K with 1nM_DRelatively weak binding affinity. K of antigen binding constructs can be determined using methods well established in the art_DThe value is obtained. Determination of antigen binding construct K_DBy using Surface Plasmon Resonance (SPR), usually with biosensing systems such asProvided is a system. Isothermal Titration Calorimetry (ITC) is another method that can be used for the determination.

Resistance can be determined by various techniquesBinding characteristics of the original binding construct. One of them is the measurement of binding to target cells expressing the antigen by flow cytometry (FACS, fluorescence activated cell sorting). Typically, in such experiments, target cells expressing an antigen of interest are incubated with the antigen binding construct at different concentrations, washed, incubated with an auxiliary reagent for detection of the antigen binding construct, washed and analyzed in a flow cytometer to measure the Median Fluorescence Intensity (MFI) indicative of the intensity of the detection signal on the cells, which in turn is related to the amount of antigen binding construct bound to the cells. The antigen binding construct concentration and MFI data were then fitted to a saturated binding equation to obtain two key binding parameters, Bmax and apparent K_D。

Apparent K_DOr an apparent equilibrium dissociation constant, representing the concentration of antigen-binding construct at which half-maximal cell binding is observed. Obviously, K_DThe smaller the value, the smaller the concentration of antigen binding construct required to achieve maximum cell binding and thus the higher the affinity of the antigen binding construct. Apparent K_DDepending on the conditions of the cell binding assay, such as the level of different receptors expressed on the cell and the incubation conditions, and thus the apparent K _DUsually different from K determined by cell-free molecular assays such as SPR and ITC_DThe value is obtained. However, there is often good agreement between the different methods.

The term "Bmax" or maximum binding amount refers to the highest level of antigen binding construct binding on a cell at the saturating concentration of the antigen binding construct. This parameter can be reported in arbitrary units MFI for relative comparison, or converted by means of a standard curve into an absolute value corresponding to the number of antigen-binding constructs bound to the cells. In some embodiments, the antigen binding construct exhibits a Bmax that is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 times the Bmax of the reference antigen binding construct.

For the antigen binding constructs described herein, the most clear separation of Bmax from FSA occurs at saturating concentrations and where Bmax can no longer increase with FSA. At non-saturated concentrations the significance was lower. In one embodiment the increase in Bmax and KD of the antigen binding construct compared to a reference antigen binding construct is independent of the level of target antigen expression on the target cell.

Described herein in some embodiments are isolated antigen binding constructs, wherein the antigen binding constructs exhibit an increase in Bmax (maximum binding amount) for target cells expressing the antigen compared to a corresponding reference antigen binding construct. In some embodiments the increase in Bmax is at least about 125% of the Bmax of the corresponding reference antigen-binding construct. In certain embodiments, the increase in Bmax is at least about 150% of the Bmax of the corresponding reference antigen-binding construct. In some embodiments, the increase in Bmax is at least about 200% of the Bmax of the corresponding reference antigen-binding construct. In some embodiments, the increase in Bmax is greater than about 110% of the Bmax of the corresponding reference antigen-binding construct.

Enhanced effector function

In one embodiment, the bispecific antigen binding constructs described herein exhibit enhanced effector function compared to each corresponding monospecific bivalent antigen binding construct (i.e. compared to the monospecific bivalent antigen binding construct binding to ECD2 or to ECD 4) and/or compared to the combination of the two monospecific bivalent antigen binding constructs. Antibody "effector functions" refer to those biological activities attributable to the Fc domain of an antibody (either a native sequence Fc domain or an amino acid sequence variant Fc domain). Examples of antibody effector functions include C1q binding; complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP); downregulation of cell surface receptors (e.g., B cell receptors; BCR), and the like.

ADCC

Thus, in one embodiment, the bispecific antigen binding construct is in the form of a Fab-scFv and exhibits higher potency in an ADCC assay in cells expressing a 1+ level of HER2 than the reference antigen binding construct in the form of a Fab-Fab.

In one embodiment, the bispecific antigen-binding construct exhibits higher maximal cell lysis in an ADCC assay than a reference antigen-binding construct that is trastuzumab or an analog thereof. In one embodiment, the bispecific antigen binding construct is in the form of a Fab-scFv and exhibits a higher maximal lysis in an ADCC assay than a reference antigen binding construct that is trastuzumab or an analog thereof, or a combination of trastuzumab or a pertuzumab analog. In one embodiment, the bispecific antigen binding construct is in the form of a Fab-scFv and exhibits higher maximal cell lysis in an ADCC assay in cells expressing 1+ or higher levels of HER2 than a reference antigen binding construct that is trastuzumab or an analog thereof. In one embodiment, the bispecific antigen binding construct is in the form of a Fab-scFv and exhibits higher potency in an ADCC assay in HER22+/3+ cells than a reference antigen binding construct that is trastuzumab or an analog thereof.

Internalization

The bispecific antigen binding constructs described herein are internalized in HER2+ cells by binding to the receptor HER 2. Thus, the bispecific antigen binding constructs described herein are capable of inducing receptor internalization in HER2+ cells. In one embodiment, the bispecific antigen binding construct is in the form of a Fab-scFv and induces greater internalization of HER2 in cells expressing 3+ levels of HER2 than the reference antigen binding construct in the form of a Fab-Fab. In one embodiment, the bispecific antigen binding construct is in the form of a Fab-scFv and induces greater internalization of HER2 in cells expressing HER2 at 2+ or 3+ levels than the reference antigen binding construct in the form of a Fab-Fab. In one embodiment, the bispecific antigen binding construct is in the form of an scFv-scFv and induces stronger internalization of HER2 in cells expressing HER2 at 1+, 2+, or 3+ levels than the reference antigen binding construct in the form of a Fab-Fab.

Cytotoxicity

Bispecific antigen binding constructs can be prepared as ADCs as described elsewhere herein and are toxic to cells. In one embodiment, the bispecific antigen-binding construct ADC exhibits greater potency in a cytotoxicity or cell survival assay in HER2+ breast cancer cells than a reference antigen-binding construct that is trastuzumab or an analog thereof, or in HER 21 +, 2+/3+ or 3+ cells than a reference antigen-binding construct that is a combination of T-DM1 and pertuzumab.

Increased binding capacity to Fc gamma R

In some embodiments, the bispecific antigen binding construct exhibits greater binding capacity (Rmax) for one or more Fc γ rs. In one embodiment, the bispecific antigen binding construct exhibits an increase in Rmax for one or more fcyr that is between about 1.3 to 2 fold greater than a reference antigen binding construct with a homodimeric Fc that is either v506 or v 6246. In one embodiment, the bispecific antigen binding construct exhibits between about 1.3 to 1.8 fold increase in Rmax for CD16Fc γ R over a reference bivalent antigen binding construct. In one embodiment, the bispecific antigen binding construct exhibits between about 1.3 to 1.8 fold increase in Rmax for CD32Fc γ R over a reference bivalent antigen binding construct. In one embodiment, the bispecific antigen binding construct exhibits between about 1.3 to 1.8 fold increase in Rmax for CD64Fc γ R over a reference bivalent antigen binding construct.

Enhanced affinity for Fc γ R

The bispecific antigen-binding constructs provided herein have enhanced affinity for Fc γ R compared to a reference antigen-binding construct such as trastuzumab. The increase in Fc concentration resulting from binding is consistent with enhanced ADCC and/or other immune effector killing mechanisms.

In some embodiments, the bispecific antigen binding construct exhibits enhanced affinity for one or more fcyr. In one embodiment, the bispecific antigen binding construct exhibits enhanced affinity for at least one Fc γ R when the bispecific antigen binding construct comprises an antigen binding polypeptide that binds to HER 2. Consistent with this embodiment, the bispecific antigen binding construct exhibits enhanced affinity for CD 32.

FcRn binding and PK parameters

In some embodiments, the antigen binding constructs described herein are capable of binding FcRn. As is known in the art, binding to FcRn circulates endocytosed antibodies from the endosome back into the bloodstream (Raghavan et al, 1996, Annu Rev CellDev Biol 12: 181-. This process, coupled with the obstruction of renal filtration due to the large size of the full length molecule, results in a favorable antibody serum half-life ranging from 1 to 3 weeks. Binding of Fc to FcRn also plays a key role in antibody transport.

Pharmacokinetic parameters

In certain embodiments, the bispecific antigen-binding constructs provided herein exhibit Pharmacokinetic (PK) properties comparable to commercially available therapeutic antibodies. In one embodiment, the bispecific antigen binding constructs described herein exhibit PK properties similar to known therapeutic antibodies with respect to serum concentration, t1/2, β half-life and/or CL. In one embodiment, the bispecific antigen binding construct exhibits in vivo stability comparable to or higher than the monospecific bivalent antigen binding construct. Such in vivo stability parameters include serum concentration, t1/2, beta half-life, and/or CL.

Testing of bispecific antigen binding constructs. Fc gamma R, FcRn and C1q binding

The effector function of bispecific antigen binding constructs can be tested as follows. In vitro and/or in vivo cytotoxicity assays may be performed to assess ADCP, CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to measure Fc γ R binding. Primary cells mediating ADCC, NK cells, express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. FcR expression on hematopoietic cells is summarized in table 3 on page 464 of ravatch and Kinet, annu.rev.immunol 9:457-92 (1991). Examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 or 5,821,337. Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, ADCC activity of a molecule of interest may be assessed in vivo, for example in an animal model such as that disclosed by Clynes et al PNAS (USA)95: 652-. A C1q binding assay may also be performed to determine whether the bispecific antigen binding construct is capable of binding C1q and thereby activating CDC. To assess complement activation, CDC assays can be performed, for example, as described in Gazzano-Santoro et al, J.Immunol.methods 202:163 (1996). FcRn binding (e.g., by SPR) and in vivo PK assays for antibodies may also be performed using methods well known in the art.

Testing of antigen binding constructs: HER2 binding

The antigen binding constructs or pharmaceutical compositions described herein are tested in vitro and then tested in vivo for their desired therapeutic or prophylactic activity prior to use in humans. For example, in vitro assays demonstrating the therapeutic or prophylactic utility of a compound or pharmaceutical composition include the effect of the compound on a cell line or a patient tissue sample. The effect of a compound or composition on a cell line and/or tissue sample can be determined using techniques known to those skilled in the art, including, but not limited to, rosette formation assays (rosette formation assays) and cell lysis assays. In vitro assays useful for determining whether administration of a specific antigen-binding construct is indicated, according to the invention, include in vitro cell culture assays, or in vitro assays in which a patient tissue sample is grown in culture, exposed to, or otherwise administered an antigen-binding construct, and the effect of such antigen-binding constructs on the tissue sample is observed.

Candidate antigen binding constructs may be assayed using cells expressing HER2, such as a breast cancer cell line. Table D below describes the expression levels of HER2 in several representative cancer cell lines.

TABLE D relative expression levels of HER2 in target cell lines

McDonagh et al Mol Cancer ther.2012 3 months; 582-93 in 11 (3); subik et al (2010) Breastcancer: Basic Clinical Research: 4; 35-41; carter et al PNAS, 1994: 89; 4285-; yarden2000, HER2 Basic Research, Prognosis and Therapy; hendricks et al Mol Cancer Ther 2013; 12:1816-28.

As is known in the art, a number of assays can be employed in order to identify antigen binding constructs suitable for use in the methods described herein. These assays can be performed in cancer cells expressing HER 2. Examples of suitable cancer cells are identified in table a 5. Examples of assays that can be performed are described below.

For example, to identify growth inhibitory candidate antigen binding constructs that bind HER2, antibodies that inhibit the growth of cancer cells expressing HER2 can be screened. In one embodiment, the selected candidate antigen binding construct is capable of inhibiting the growth of cancer cells in cell culture by about 20-100% and preferably about 50-100% as compared to the control antigen binding construct.

To select candidate antigen binding constructs that induce cell death, loss of membrane integrity as indicated by, for example, PI (phosphatidylinositol), trypan blue, or 7AAD uptake can be assessed relative to controls.

To select candidate antigen binding constructs that induce apoptosis, an annexin (annexin) binding assay may be employed. In addition to the annexin binding assay, a DNA staining assay may also be used.

In one embodiment, the candidate antigen-binding construct of interest may block heregulin-dependent association of ErbB2 with ErbB3 in MCF7 and SK-BR-3 cells substantially more effectively than monoclonal antibody 4D5, and preferably substantially more effectively than monoclonal antibody 7F3, as determined in a co-immunoprecipitation experiment.

To screen for antigen binding constructs that bind to an epitope on ErbB2 bound by an antibody of interest, a conventional cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Harlow and DavidLane editors (1988) can be performed. Alternatively or additionally, epitope mapping can be performed by methods known in the art.

Competition between antigen-binding constructs can be determined by assays in which the subject antigen-binding construct inhibits or blocks specific binding of a reference antigen-binding construct to a common antigen (see, e.g., Junghans et al, Cancer Res.50:1495,1990; Fendly et al, Cancer Research 50: 1550-. A test antigen-binding construct competes with a reference antigen-binding construct if an excess of the test antigen-binding construct (e.g., at least 2-fold, 5-fold, 10-fold, 20-fold, or 100-fold) inhibits or blocks binding of the reference antigen-binding construct, e.g., by at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, as measured in a competition binding assay. Antigen-binding constructs identified by competition assays (competitive antigen-binding constructs) include antigen-binding constructs that bind to the same epitope as the reference antigen-binding construct and antigen-binding constructs that are sterically hindered by binding to an adjacent epitope sufficiently close to the epitope bound by the reference antigen-binding construct. For example, a second competitive antigen binding construct that competes with the first antigen binding construct described herein for binding to HER2 can be identified. In certain instances, the second construct may block or inhibit binding of the first construct, e.g., by at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, as measured in a competitive binding assay. In certain instances, the second construct may replace more than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the first construct.

In some embodiments, the function of an antigen binding construct described herein is determined in vivo, e.g., in an animal model. In some embodiments, the animal model is the animal model described in table E. In some embodiments, the antigen binding construct exhibits an increase in therapeutic efficacy in an animal model as compared to a reference antigen binding construct.

Table E: animal model for testing HER2 binding antigen binding constructs

Reference antigen binding constructs

In some embodiments, the functional characteristics of a bispecific antigen binding construct described herein are compared to the functional characteristics of a reference antigen binding construct. The nature of the reference antigen binding construct depends on the functional characteristics measured or the differences made. For example, in comparing the functional characteristics of exemplary bispecific antigen-binding constructs, the reference antigen-binding construct may be a trastuzumab analog, e.g., v506, or may be an antibody such as a combination of trastuzumab and pertuzumab (v 4184). In embodiments that compare the form of the bispecific antigen-binding construct, the reference antigen-binding construct is, for example, a biparatopic anti-HER 2 antibody, wherein both antigen-binding portions are in Fab-Fab form (the form reference antigen-binding construct). Examples of the latter construct include v6902 and v 6903.

Antigen binding constructs and Antibody Drug Conjugates (ADCs)

In certain embodiments the antigen-binding construct is coupled to a drug, such as a toxin, a chemotherapeutic agent, an immunomodulator, or a radioisotope. Several methods of preparing ADCs (antibody drug conjugates or antigen binding construct drug conjugates) are known in the art and are described, for example, in U.S. Pat. nos. 8,624,003 (one pot method), 8,163,888 (one step method), and 5,208,020 (two step method).

In some embodiments, the drug is selected from maytansine, an auristatin (auristatin), a calicheamicin (calicheamicin), or a derivative thereof. In other embodiments, the drug is a maytansine selected from the group consisting of DM1 and DM 4. Further examples are described below.

In some embodiments, the drug is coupled to the isolated antigen binding construct via a SMCC linker (DM1) or an SPDB linker (DM 4). Additional examples are described below. The drug to antigen binding protein ratio (DAR) may be 1.0 to 6.0 or 3.0 to 5.0 or 3.5-4.2.

In some embodiments, the antigen binding construct is conjugated to a cytotoxic agent. The term "cytotoxic agent" as used herein refers to a substance that inhibits or interferes with cellular function and/or causes cellular destruction. The term is intended to include radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, and Lu177), chemotherapeutic agents and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and/or variants thereof. Further examples are described below.

Medicine

Non-limiting examples of drugs or payloads for use in various embodiments of ADCs include DM1 (maytansine, N2 '-deacetyl-N2' - (3-mercapto-1-oxopropyl) -or N2 '-deacetyl-N2' - (3-mercapto-1-oxopropyl) -maytansine), mc-MMAD (6-maleimidocaproyl-monomethyl auristatin-D or N-methyl-L-valyl-N- [ (1S,2R) -2-methoxy-4- [ (2S) -2- [ (1R,2R) -1-methoxy-2-methyl-3-oxo-3- [ [ (1S) -2-phenyl-1- (2-thiazolyl) ethyl ] Amino ] propyl ] -1-pyrrolidinyl ] -1- [ (1S) -1-methylpropyl ] -4-oxobutyl ] -N-methyl- (9C1) -L-valinamide), mc-MMAF (maleimidocaproyl-monomethyl auristatin F or N- [6- (2, 5-dihydro-2, 5-dioxo-1H-pyrrol-1-yl) -1-oxohexyl ] -N-methyl-L-valyl- (3R,4S,5S) -3-methoxy-5-methyl-4- (methylamino) heptanoyl- (α R, β R,2S) - [ β -methoxy- α -methyl-2-pyrrolidinopropionyl [ ] acyl-L-phenylalanine) and mc-Val-Cit-PABA-MMAE (6-maleimidocaproyl-ValcCit- (p-aminobenzyloxycarbonyl) -monomethyl auristatin E or N- [ [ [4- [ [ N- [6- (2, 5-dihydro-2, 5-dioxo-1H-pyrrol-1-yl) -1-oxohexyl ] -L-valyl-N5- (aminocarbonyl) -L-ornithyl ] amino ] phenyl ] methoxy ] carbonyl ] -N-methyl-L-valyl-N- [ (1S,2R) -4- [ (2S) -2- [ (1R,2R) -3- [ [ (1R,2S) -2-hydroxy-1-methyl-2-phenylethyl ] amino ] -1-methoxy-2-methyl-3-oxopropyl ] -1-pyrrolidinyl ] -2-methoxy-1- [ (1S) -1-methylpropyl ] -4-oxobutyl ] -N-methyl-L-valinamide). DM1 is a derivative of the tubulin inhibitor maytansine, while MMAD, MMAE and MMAF are auristatin derivatives.

Maytansinoid (Maytansino) drug moiety

As indicated above, in some embodiments the drug is maytansine. Exemplary maytansinoids include DM1, DM3 (N)²' -Deacetyl-N²' - (4-mercapto-1-oxopentyl) maytansine) and DM4 (N)²' -Deacetyl-N²' - (4-methyl-4-mercapto-l-oxopentyl) methyl maytansine) (see US 20090202536).

It is known that many positions on maytansinoids can be used as attachment positions depending on the type of attachment. For example, in order to form an ester bond, the C-3 position having a hydroxyl group, the C-14 position modified with a hydroxymethyl group, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group are all suitable.

All stereoisomers of the maytansinoid (maytansinoid) drug moiety, i.e., any combination of the R and S configurations at the D chiral carbon, are contemplated for the ADCs described herein.

Auristatin

In some embodiments, the drug is an auristatin, such as auristatin E (also known in the art as a derivative of dolastatin-10) or a derivative thereof. An auristatin may be, for example, an ester formed between auristatin E and a keto acid. For example, auristatin E can be reacted with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other exemplary auristatins include AFP, MMAF, and MMAE. The synthesis and structure of exemplary auristatins are disclosed in U.S. patent nos. 6,884,869, 7,098,308, 7,256,257, 7,423,116, 7,498,298 and 7,745,394, each of which is incorporated by reference herein in its entirety and for all purposes.

Chemotherapeutic agents

In some embodiments, the antigen binding construct is conjugated to a chemotherapeutic agent. Examples include, but are not limited to, cisplatin (Cisplantin) and Lapatinib (Lapatinib). A "chemotherapeutic agent" is a chemical compound used in the treatment of cancer.

Examples of chemotherapeutic agents include alkylating agents such as thiotepa (thiotepa) and Cyclophosphamide (CYTOXAN)^TM) (ii) a Alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines (aziridines) such as benzotepa (benzodopa), carboquone (carboquone), metoclopramide (meturedopa), and uretepa (uredpa); vinyl imines and methyl melamines including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustards, such as chlorambucil (chlorambucil), chlorambucil (chloramphazine), chlorophosphamide, estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), mechlorethamine hydrochloride (mechlorethamine oxydichloride), melphalan (melphalan), neonebivron (novembichin), benzene mustard (phenesterine), prednimustine (prednimustine), triamcinolone (trofosfamide), uracil mustard; nitroureas such as carmustine (carmustine), chlorouretocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ramustine (ranimustine); antibiotics, such as aclacinomycin (acrinomycin), actinomycin (actinomycin), anthranilic (authramycin), azaserine (azaserine), bleomycin (bleomycin), actinomycin C (cactinomycin), calicheamicin, karabicin (carabicin), carminomycin (carminomycin), carzinophilin (carzinophillin), chromomycin (chromomycin), actinomycin D (dactinomycin), daunorubicin (daunorubicin), ditorexin (detorubicin), 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marijumycin (marcellomycin), mitomycin (mitomycin), mycophenolic acid, norubicin (nogalamycin), olivomycin (olivomycin), pelomycin (polyplomycin), pofiromycin (potfiromycin), puromycin (puromycin), griseofibrinomycin (quelamycin), rodorirubicin (streptonigrycin), streptozocin (streptozocin), metrizacin (zotocin), tubercidin (zotocin); antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine (fludarabine), 6-mercaptopurine, thioguanine (thiamiprine), thioguanine (thioguanine); pyrimidine analogs such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine, carmofur (carmofur), cytarabine, dideoxyuridine, doxifluridine, enocitabine (enocitabine), floxuridine, 5-FU; androgens such as testosterone carprofonate (calusterone), dromostanolone propionate (dromostanolone propionate), epithioandrostanol (epithiostanol), mepiquitane (mepiquitazone), and testolactone (testolactone); anti-adrenergic agents, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as folinic acid (frilic acid); acetic acid glucurolactone; an aldehydic phosphoramide glycoside; (ii) aminolevulinic acid; amsacrine (amsacrine); bestrabuucil; bisantrene; edatrexate (edatraxate); desphosphamide (defofamine); dimecorsine (demecolcine); diazaquinone (diaziqutone); isoflurine (elfornithine); ammonium etitanium acetate; etoglut (etoglucid); gallium nitrate; a hydroxyurea; lentinan (lentinan); lonidamine (lonidamine); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidamol (mopidamol); diamine nitracridine (nitrarine); spray washer Statin (pentastatin); methionine mustard (phenamett); pirarubicin (pirarubicin); podophyllinic acid (podophyllinic acid); 2-ethyl hydrazide; procarbazine (procarbazine); PSK 7; razoxane (rizoxane); sizofuran (sizofiran); germanium spiroamines (spirogyranium); alternarionic acid; triimine quinone (triaziquone); 2,2',2 ═ -trichlorotriethylamine; urethane (urethan); vindesine (vindesine); dacarbazine (dacarbazine); mannomustine (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); a polycytidysine; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; taxanes, e.g. paclitaxel (A)Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (Rorer, antonyy, France); chlorambucil (chlorambucil); gemcitabine (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine (vincristine); vinorelbine (vinorelbine); navelbine (navelbine); oncostatin (novantrone); teniposide (teniposide); daunorubicin (daunomycin); aminopterin (aminopterin); (xiloda); ibandronate (ibandronate); CPT-11; topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); tretinoin; esperamicin (esperamicin); capecitabine (capecitabine); and a pharmaceutically acceptable salt, acid or derivative of any of the above alkylating agents. Also included in this definition are anti-hormonal agents which act to modulate or inhibit the hormonal action on tumors, such as anti-estrogens, including, for example, tamoxifen (tamoxifen), raloxifene (raloxifene), aromatase inhibiting 4(5) -imidazoles, 4-hydroxyttamoxifen, trioxifene (trioxifene), raloxifene hydrochloride (keoxifene), LY117018, onapristone (onapristone), and toremifene (toremifene) (Fareston); and antiandrogen Drugs such as flutamide (flutamide), nilutamide (nilutamide), bicalutamide (bicalutamide), leuprolide (leuprolide), and goserelin (goserelin); and pharmaceutically acceptable salts, acids or derivatives of any of the above.

Coupling joint

In some embodiments, the drug is linked to the antigen binding construct, e.g., an antibody, via a linker. Attachment of the linker to the antibody can be achieved in a variety of ways, such as by surface lysine, reductive coupling to oxidized carbohydrates, and by reducing the cysteine residues released by interchain disulfide bonds. A variety of ADC linkage systems are known in the art, including hydrazone, disulfide, and peptide-based linkages.

Suitable linkers include, for example, cleavable and non-cleavable linkers. Cleavable linkers are typically sensitive to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers that are cleavable by intracellular proteases, such as lysosomal proteases or endosomal proteases. In exemplary embodiments, the linker may be a dipeptide linker, such as a valine-citrulline (Val-Cit), phenylalanine-lysine (phe-lys) linker, or a maleimidocaproic acid-valine-citrulline-p-aminobenzyloxycarbonyl (mc-Val-Cit-PABA) linker. Another linker is sulfosuccinimidyl 4- [ N-maleimidomethyl ] cyclohexane-1-carboxylate (SMCC). The sulfo-smcc coupling occurs via a maleimide group that reacts with a thiol (thiol, -SH), while its sulfo-NHS ester is reactive towards primary amines (as found at the N-terminus of lysine and protein or peptide). Yet another linker is Maleimidocaproyl (MC). Other suitable linkers include linkers that are hydrolyzable at a particular pH or pH range, such as hydrazone linkers. Additional suitable linkers include disulfide linkers. The linker may be covalently bound to the antibody to the extent that the antibody must degrade intracellularly to release the drug, e.g., MC linkers and the like.

Preparation of ADC

ADCs can be prepared by several routes using organic chemical reactions, conditions and reagents known to those skilled in the art, including: (1) reacting a nucleophilic group or electrophilic group of an antibody with a divalent linker reagent to form an antibody-linker intermediate Ab-L via a covalent bond, followed by reaction with an activating drug moiety D; and (2) reaction of the nucleophilic or electrophilic group of the drug moiety with a linker reagent to form a drug-linker intermediate D-L via a covalent bond, followed by reaction with the nucleophilic or electrophilic group of the antibody. Conjugation methods (1) and (2) can be used with a variety of antibodies, drug moieties and linkers to prepare antibody-drug conjugates described herein.

Several specific examples of methods of preparing ADCs are known in the art and are described in U.S. Pat. nos. 8,624,003 (one pot process), 8,163,888 (one step process) and 5,208,020 (two step process).

Method for preparing antigen binding construct

The antigen binding constructs described herein can be generated using, for example, recombinant methods and compositions described in U.S. Pat. No. 4,816,567.

In one embodiment, isolated nucleic acids encoding the antigen binding constructs described herein are provided. Such nucleic acids can encode the amino acid sequences of the VL that make up the antigen-binding construct and/or the amino acid sequences of the VH that make up the antigen-binding construct (e.g., the light chain and/or heavy chain of the antigen-binding construct). In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In one embodiment, the nucleic acid is provided in a polycistronic vector. In another embodiment, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence of a VL that makes up the antigen-binding construct and an amino acid sequence of a VH that makes up the antigen-binding polypeptide construct, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence of a VL that makes up the antigen-binding polypeptide construct and a second vector comprising a nucleic acid encoding an amino acid sequence of a VH that makes up the antigen-binding polypeptide construct. In one embodiment, the host cell is eukaryotic, such as a Chinese Hamster Ovary (CHO) cell or a Human Embryonic Kidney (HEK) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of making an antigen-binding construct is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antigen-binding construct as provided above under conditions suitable for expression of the antigen-binding construct, and optionally recovering the antigen-binding construct from the host cell (or host cell culture medium).

For recombinant production of the antigen-binding construct, the nucleic acid encoding the antigen-binding construct as described above, for example, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of the antigen-binding construct).

The term "substantially pure" means that the construct described herein, or a variant thereof, may be substantially or essentially free of components that normally accompany or interact with a protein as found in its naturally occurring environment, i.e., in certain embodiments substantially free of cellular material, or host cells in the case of recombinantly produced heteromultimers include preparations of protein having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) contaminating protein. When the heteromultimer or variant thereof is recombinantly produced by a host cell, in certain embodiments the protein is present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less by dry weight of the cell. When the heteromultimer or variant thereof is recombinantly produced by a host cell, in certain embodiments the protein is present in the culture medium at about 5g/L, about 4g/L, about 3g/L, about 2g/L, about 1g/L, about 750mg/L, about 500mg/L, about 250mg/L, about 100mg/L, about 50mg/L, about 10mg/L, or about 1mg/L or less, by dry weight of the cell. In certain embodiments, a "substantially pure" heteromultimer produced by the methods described herein has a purity level of at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, particularly, a purity level of at least about 75%, 80%, 85%, and more particularly, a purity level of at least about 90%, a purity level of at least about 95%, a purity level of at least about 99% or more, as determined by suitable methods such as SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

Suitable host cells for cloning or expressing a vector encoding an antigen-binding construct include prokaryotic or eukaryotic cells as described herein.

"recombinant host cell" or "host cell" refers to a cell that includes an exogenous polynucleotide, regardless of the method used for insertion, e.g., direct uptake, transduction, f-mating, or other methods known in the art to produce recombinant host cells. The exogenous polynucleotide may remain a non-integrating vector, e.g., a plasmid, or alternatively, may integrate into the host genome.

As used herein, the term "eukaryote" refers to organisms belonging to a eukaryotic phylogenetic domain, such as animals (including but not limited to mammals, insects, reptiles, birds, etc.), ciliates, plants (including but not limited to monocots, dicots, algae, etc.), fungi, yeasts, flagellates, microsporidia, protists, and the like.

As used herein, the term "prokaryote" refers to a prokaryotic organism. For example, the non-eukaryotic organism may belong to the eubacterial species (Eubacteriosis) (including but not limited to Escherichia coli (Escherichia coli), Thermus thermophilus (Thermus thermophilus), Bacillus stearothermophilus (Bacillus stearothermophilus), Pseudomonas fluorescens (Pseudomonas fluorescens), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Pseudomonas putida (Pseudomonas putida), etc.) phylogenetic domains or Archaea (Archaea) (including but not limited to Methanococcus jannaschii), Methanobacterium thermoautotrophicum (Methanobacterium thermoautotrophicum), Halobacterium (Halobacterium) (such as Halobacterium vulgare (Halofax volcanerii) and Halobacterium NRC-1), Pyrococcus fuliginosus (Pyrococcus thermophilus), Pyrococcus thermophilus (Pyrococcus), Archaeoglobus Archaea (Archaeoglobus), Archaeoglobus fulgidus, etc.).

For example, antigen binding constructs can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antigen binding construct fragments and polypeptides in bacteria, see, e.g., U.S. Pat. nos. 5,648,237, 5,789,199, and 5,840,523. (see also Charlton, Methods in Molecular Biology, Vol.248 (B.K.C.Lo, eds., Humana Press, Totowa, N.J.,2003), p.245-254, which describes the expression of antibody fragments in E.coli (E.coli). after expression, the antigen-binding constructs can be isolated from the soluble components of the bacterial cytoplasm and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antigen binding constructs, including fungi and yeast strains in which the glycosylation pathway has been "humanized" resulting in the production of antigen binding constructs with partially or fully human glycosylation patterns. See Gerngross, nat. Biotech.22: 1409-.

Suitable host cells expressing the glycosylated antigen binding construct are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculoviral strains have been identified which can be used in particular for transfection of Spodoptera frugiperda cells, together with insect cells.

Plant cell cultures may also be used as hosts. See, for example, U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIIES for generating antigen binding constructs in transgenic plants^TMA technique).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines are SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells as described in Graham et al, J.Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, biol. reprod.23:243-251 (1980)); monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK; Bufaro rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (e.g., described in Mather et al, Annals N.Y.Acad.Sci.383:44-68 (1982); MRC 5 cells; and FS4 cells other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR ovary (CHO) cells ^-CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp 2/0. A review of certain mammalian cell lines suitable for the generation of antigen binding constructs is found, for example, in Yazaki and Wu, Methods in Molecular Biology, Vol.248 (B.K.C.Lo, eds., Humana Press, Totowa, N.J.), p.255-268 (2003).

In one embodiment, the antigen binding constructs described herein are tested in stable mammalian cells by a method comprising the steps of: transfecting at least one stable mammalian cell with a nucleic acid encoding an antigen-binding construct at a predetermined ratio; and expressing the nucleic acid in the at least one mammalian cell. In some embodiments, the predetermined ratio of nucleic acids is determined in a transient transfection experiment to determine the relative ratio of input nucleic acids that produces the highest percentage of antigen binding constructs in the expression product.

In some embodiments is a method of testing an antigen binding construct in a stable mammalian cell as described herein, wherein the expression product of the at least one stable mammalian cell comprises a greater percentage of the desired glycosylated antigen binding construct as compared to the monomeric heavy or light chain polypeptide or other antibody.

In some embodiments is a method of producing a glycosylated antigen binding construct in a stable mammalian cell described herein, the method comprising identifying and purifying the desired glycosylated antigen binding construct. In some embodiments, the identifying is by one or both of liquid chromatography and mass spectrometry.

If desired, the antigen binding construct may be purified or isolated after expression. Proteins can be isolated or purified in a variety of ways known to those skilled in the art. Standard purification methods include chromatographic techniques including ion exchange, hydrophobic interaction, affinity, fractionation or gel filtration and reverse phase chromatography, performed at atmospheric or elevated pressure using systems such as FPLC and HPLC. Purification methods also include electrophoresis, immunology, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques, as well as protein concentration, are also useful. As is well known in the art, a variety of native proteins bind Fc and antibodies, and these proteins can be used in the present invention for purification of antigen binding constructs. For example, bacterial proteins a and G bind to the Fc region. Similarly, bacterial protein L binds to the Fab region of some antibodies. Purification is often made possible by specific fusion partners. For example, if a GST fusion is used, the antibody can be purified using glutathione resin, and if a His-tag is used, Ni can be used ⁺²The antibody is purified by affinity chromatography or, if a flag-tag is used, may be purified using an immobilized anti-flag antibody. See, for example, Protein Purification, Priniplsa and Practice, 3 rd edition, Scopes, Springer-Verlag, NY,1994, incorporated by reference in its entirety, for general guidance on suitable Purification techniques. The degree of purification necessary will vary depending on the use of the antigen-binding construct. In some embodiments, purification is not necessary.

In certain embodiments, the antigen binding construct is purified using anion exchange chromatography, including, but not limited to, chromatography on Q-Sepharose, DEAE Sepharose, poros HQ, poros DEAF, Toyopearl Q, Toyopearl QAE, toyopearlDEAE, Resource/Source Q and DEAE, Fractogel Q and DEAE columns.

In particular embodiments cation exchange chromatography is used to purify the proteins described herein, including but not limited to SP-Sepharose, CM Sepharose, poros HS, poros CM, Toyopearl SP, Toyopearl CM, Resource/Source S and CM, Fractogel S and CM columns, and equivalents thereof.

In addition, the antigen-binding constructs described herein can be chemically synthesized using techniques known in the art (see, e.g., Creighton, 1983, Proteins: Structures and Molecular Principles, W.H.Freeman & Co., N.Y. and Hunkapiller et al, Nature,310:105-111 (1984)). For example, a polypeptide corresponding to a polypeptide fragment can be synthesized by using a peptide synthesizer. In addition, non-classical amino acids or chemical analogs of amino acids may be introduced as substitutions or additions to the polypeptide sequence, if desired. Non-canonical amino acids include, but are not limited to, the D-isomer of the common amino acid, 2,4 diaminobutyric acid, alpha-aminoisobutyric acid, 4 aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6 aminocaproic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, □ -alanine, fluoro-amino acids, designer amino acids such as □ -methyl amino acid, C □ -methyl amino acid, N □ -methyl amino acid, and amino acid analogs in general. In addition, the amino acid may be D (dextrorotatory) or L (levorotatory).

Post-translational modification:

in certain embodiments the antigen binding constructs described herein are differentially modified during or after translation.

The term "modification", as used herein, refers to any change made to a given polypeptide, such as a change in polypeptide length, amino acid sequence, chemical structure, co-translational modification, or post-translational modification. The term "modified" means that the polypeptide in question is optionally modified, i.e. the polypeptide in question may be modified or unmodified.

The term "post-translational modification" refers to any modification of a natural or unnatural amino acid that occurs after such amino acid has been incorporated into a polypeptide chain. By way of example only, the term encompasses co-translational in vivo modifications, co-translational in vitro modifications (such as in a cell-free translation system), post-translational in vivo modifications, and post-translational in vitro modifications.

In some embodiments, the modification is at least one of: glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, and attachment to antibody molecules or antigen binding constructs or other cellular ligands. In some embodiments, the antigen binding construct is chemically modified by known techniques, including but not limited to, with cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH ₄Carrying out specific chemical lysis; acetylation, formylation, oxidation, reduction and metabolic synthesis in the presence of tunicamycin.

Additional post-translational modifications of the antigen-binding constructs described herein include, for example, N-or O-linked carbohydrate chains, processing of the N-or C-terminus), linkage of chemical moieties to the amino acid backbone, chemical modification of N-or O-linked carbohydrate chains, and addition or deletion of the N-terminal methionine residue due to expression in prokaryotic host cells. The antigen binding constructs described herein are modified with a detectable label, such as an enzymatic, fluorescent, isotopic, or affinity label, to allow for the detection and isolation of the protein. In certain embodiments, examples of suitable enzyme labels include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin biotin and avidin/biotin; examples of suitable fluorescent substances include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride or phycoerythrin; examples of the luminescent material include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include iodine, carbon, sulfur, tritium, indium, technetium, thallium, gallium, palladium, molybdenum, xenon, fluorine.

In particular embodiments, the antigen binding constructs described herein are linked to a macrocyclic chelator associated with a radiometal ion.

In some embodiments, the antigen binding constructs described herein are modified by natural processes, such as post-translational processing, or by chemical modification techniques well known in the art. In certain embodiments, the same or different degrees of homologous modification may be present at several sites in a given polypeptide. In certain embodiments, the polypeptides from the antigen binding constructs described herein are branched, e.g., due to ubiquitination, and in some embodiments are cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides are the result of post-translational natural processes or are made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of riboflavin, covalent attachment of heme moieties, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, covalent attachment of phosphatidylinositols, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor point formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation (selenoylation), sulfation, transport RNA-mediated addition of amino acids to proteins such as arginylation and ubiquitination. (see, e.g., PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES, 2 nd edition, T.E.Creighton, W.H.Freeman AND Company, New York (1993); POST-TRANSLATIONAL COAVALENT MODIFICATION OF PROTEINS, B.C.Johnson editor, Academic Press, New York, pages 1-12 (1983); Seifter et al, meth.enzymol.182: 626. Aconic acid 646 (1990); Rattan et al, Ann.N.Y.Acad.Sci.663:48-62 (1992)).

In certain embodiments, the antigen binding constructs described herein are attached to a solid support that is particularly useful for immunoassay or purification of polypeptides bound by, bound to, or associated with the proteins described herein. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

Pharmaceutical composition

Also provided herein are pharmaceutical compositions comprising the antigen binding constructs described herein. The pharmaceutical composition comprises the construct and a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. In some aspects, the vector is an artificial vector not found in nature. Water may be used as a carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The compositions may be formulated as suppositories with conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable Pharmaceutical carriers are described by e.w. martin in "Remington's Pharmaceutical Sciences". Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with an appropriate amount of carrier so as to provide the patient with a form for proper administration. The formulation should be suitable for the mode of administration.

In certain embodiments, the composition comprising the construct is formulated into a pharmaceutical composition suitable for intravenous administration to a human according to conventional procedures. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If desired, the composition may also include a cosolvent and a local anesthetic such as lidocaine (lignocaine) to reduce pain at the injection site. Typically, the ingredients are provided separately or mixed together in a hermetically sealed container, such as an ampoule or sachet, which indicates the amount of active agent, in unit dosage form, e.g., as a lyophilized powder or an anhydrous concentrate. When the composition is to be administered by infusion, the composition can be dispensed with an infusion bottle containing pharmaceutically grade sterile water or saline. When the composition is administered by injection, an ampoule of sterile water or saline for injection may be provided so that the ingredients may be mixed prior to administration.

In certain embodiments, the compositions described herein are formulated in a neutral or salt form. Pharmaceutically acceptable salts include salts with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like, and salts with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine (procaine), and the like.

Method of treatment

In certain embodiments, a method of treating a disease or disorder is provided, comprising administering to a subject in need of such treatment, prevention, or amelioration an antigen binding construct described herein in an amount effective to treat, prevent, or ameliorate the disease or disorder.

By "disorder" is meant any condition that would benefit from treatment with the antigen binding constructs or methods described herein. This includes chronic and acute disorders or diseases, including those pathological conditions that predispose a mammal to the disorder in question. In some embodiments, the disorder is cancer, as described in more detail below.

The term "subject" refers to an animal, in some embodiments a mammal, that is the subject of treatment, observation or experiment. The animal can be a human, a non-human primate, a pet (e.g., dog, cat, etc.), a livestock animal (e.g., cow, sheep, pig, horse, etc.), or a laboratory animal (e.g., rat, mouse, guinea pig, etc.).

The term "mammal" as used herein includes, but is not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

"treatment" refers to clinical intervention in an attempt to alter the natural course of a subject's disease or cells, and may be for prophylaxis or during the course of clinical pathology. Desirable therapeutic effects include preventing the occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antigen binding constructs described herein are used to delay the development of a disease or disorder. In one embodiment, the antigen binding constructs and methods described herein achieve tumor regression. In one embodiment, the antigen binding constructs and methods described herein effect inhibition of tumor/cancer growth.

Desirable therapeutic effects include, but are not limited to, preventing disease occurrence or recurrence, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, increasing survival, and alleviating or improving prognosis. In some embodiments, the antigen binding constructs described herein are used to delay the progression of a disease or slow the progression of a disease.

The term "effective amount" as used herein refers to the amount of construct administered that will achieve the objectives of the method, e.g., to alleviate one or more symptoms of a treated disease, condition, or disorder to some extent. The amount of the compositions described herein that will be effective in the treatment, inhibition, and prevention of a disease or disorder associated with aberrant expression and/or activity of a therapeutic protein can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration and the severity of the disease or condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses are extrapolated from dose-response curves obtained from in vitro or animal model test systems.

Administering the antigen binding construct to the subject. Various delivery systems are known and can be used to administer the antigen-binding construct formulations described herein, e.g., encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compounds, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.biol. chem.262:4429-4432(1987)), construction of nucleic acids as part of a retrovirus or other vector, and the like. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions can be administered by any convenient route, such as by infusion or bolus injection, absorbed through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered with other bioactive agents. Administration may be systemic or local. Additionally, in certain embodiments, it is desirable to introduce the antigen binding construct compositions described herein into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection can be facilitated, for example, by an intraventricular catheter connected to a reservoir, such as an Ommaya reservoir. Pulmonary administration may also be employed, for example, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a particular embodiment, it is desirable to topically administer an antigen-binding construct or composition described herein to an area in need of treatment; this may be achieved, for example and without limitation, by local infusion during surgery, topical application (e.g. together with a wound dressing after surgery), injection, by means of a catheter, by means of a suppository or by means of an implant which is a porous, non-porous or gel material including membranes such as silicone rubber membranes or fibres. Preferably, when administering proteins, including the antigen binding constructs described herein, care must be taken to use the material into which the protein is absorbed.

In another embodiment, the antigen binding construct or composition may be delivered in vesicles, particularly Liposomes (see Langer, Science 249: 1527-.

In yet another embodiment, the antigen binding construct or composition may be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC crit. Ref. biomed. Eng.14:201 (1987); Buchwald et al, Surgery 88:507 (1980); Saudek et al, N.Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials may be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres, Boca Raton, Fla (1974); Controlled Drug delivery Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem.23:61 (1983); see also Levy et al, Science 228:190 (1985); During et al, Ann. Neurol.25:351 (1989); Howard et al, J. Neurosurg.71:105 (1989)). In yet another embodiment, a controlled Release system can be placed near a target of treatment, such as the brain, so that only a portion of the systemic dose is needed (see, e.g., Goodson, in Medical Applications of controlled Release, Vol.2, pp.115-138 (1984)).

In one particular embodiment comprising a nucleic acid encoding an antigen binding construct described herein, the nucleic acid may be administered in vivo to facilitate expression of the protein it encodes by constructing the nucleic acid as part of an appropriate nucleic acid expression vector and administering it to become intracellular, for example by using a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by using microprojectile bombardment (e.g., gene gun; Biolistic, Dupont) or coating of lipids or cell surface receptors or transfection agents, or by placing it in association with a homologous cassette-like peptide known to enter the nucleus (see, for example, Joliot et al, Proc. Natl. Acad. Sci. USA88: 1864-. Alternatively, the nucleic acid may be introduced intracellularly and incorporated into the host cell DNA by homologous recombination for expression.

In certain embodiments the antigen binding constructs described herein are administered as a combination with antigen binding constructs having non-overlapping binding target epitopes.

The amount of antigen binding construct that will be effective in the treatment, inhibition, and prevention of a disease or disorder can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration and the severity of the disease or condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses are extrapolated from dose-response curves obtained from in vitro or animal model test systems.

The antigen binding constructs described herein can be administered alone or in combination with other types of therapies (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy, and anti-tumor agents). In general, it is preferred to administer a species-derived or species-reactive (in the case of antibodies) product of the same species as the patient. Thus, in one embodiment, a human antigen-binding construct, fragment derivative, analog, or nucleic acid is administered to a human patient for treatment or prevention.

Methods of treating cancer

Described herein are methods of treating HER2+ cancer or tumor, and methods of inhibiting growth of or killing HER2+ tumor cells in a subject using the antigen binding constructs described herein.

By HER2+ cancer is meant a cancer that expresses HER2 such that the antigen binding constructs described herein are capable of binding to the cancer. As is known in the art, HER2+ cancers express different levels of HER 2. To determine ErbB, such as ErbB2(HER2) expression in cancer, various diagnostic/prognostic assays are available. In one embodiment, may be passed through IHC, e.g., using(Dako) analysis for ErbB2 overexpression. Paraffin-embedded tissue sections from tumor biopsies can be subjected to IHC assays and meet the ErbB2 protein staining intensity criteria as follows:

Score 0: no staining was observed or membrane staining was observed in less than 10% of the tumor cells.

Score 1 +: a faint/barely visible membrane staining was detected in more than 10% of the tumor cells. Only in a portion of its membrane the cells are stained.

And the score is 2 +: weak to moderate full membrane staining was observed in more than 10% of tumor cells.

Score 3 +: moderate to intense whole membrane staining was observed in more than 10% of tumor cells.

Those tumors assessed for ErbB2 overexpression that scored 0 or 1+ may be characterized as not overexpressing ErbB2, while those scored 2+ or 3+ may be characterized as overexpressing ErbB 2.

Alternatively, or in addition, formalin (formalin) fixed, paraffin embedded tumor tissue may be subjected to Fluorescence In Situ Hybridization (FISH) assays such as INFORM^TM(sold by Ventana, Ariz.) or PATHVISION^TM(Vysis, Ill.) to determine the extent of ErbB2 overexpression, if any, in tumors. The FISH assay measuring HER2 gene amplification appears to be patient-specific compared to the IHC assayThe response of the treatment is more relevant and is currently considered to be a possible benefit of the identificationPreferred determination of the patient to be treated.

Table D describes the expression levels of HER2 on several representative Breast and other Cancer cell lines (Subik et al (2010) Breast Cancer: Basic Clinical Research: 4; 35-41; Prang et al (2005) British journal of Cancer Research: 92; 342-349). As shown in the table, MCF-7 and MDA-MB-231 cells were considered to be HER2 low expressing cells; JIMT-1 and ZR-75-1 cells were considered to be HER2 intermediate expression cells, and SKBR3 and BT-474 cells were considered to be HER2 high expression cells. SKOV3 (ovarian cancer) cells were considered to be moderately expressing cells of HER 2.

Described herein are methods of treating a subject having a HER2+ cancer or tumor comprising administering to the subject an effective amount of a pharmaceutical composition comprising an antigen binding construct described herein.

Also described herein is the use of the HER2 antigen binding construct described herein for the manufacture of a medicament for the treatment of cancer or a tumor. Also described herein are HER2 antigen binding constructs for use in cancer or tumor therapy.

In particular embodiments, the antigen binding construct is v10000, v7091, v5019, or v 5020. In one embodiment, the antigen binding construct is v 10000. In some embodiments, the antigen binding construct is coupled to maytansine (DM 1). When the antigen binding construct coupled to DM1 was internalized into tumor cells, DM1 was cleaved from the construct intracellularly and killed the tumor cells.

In some embodiments, the subject is afflicted with pancreatic cancer, head and neck cancer, gastric cancer, colorectal cancer, breast cancer, renal cancer, cervical cancer, ovarian cancer, brain cancer, endometrial cancer, bladder cancer, non-small cell lung cancer, or cancer of epithelial origin. In some embodiments, the tumor is metastatic.

In general, tumors in treated subjects express on average 10,000 or more copies of HER2 per tumor cell. In certain embodiments, the tumor is HER 20-1 +, HER 22 +, or HER 23 +, as determined by IHC. In some embodiments the tumor is HER 22 + or less, or HER 21 + or less.

In some embodiments, the tumor of the subject treated with the antigen binding construct is breast cancer. In a specific embodiment, the breast cancer expresses HER2 at a level of 2+ or less. In a specific embodiment, the breast cancer expresses HER2 at a level of 1+ or less. In some embodiments, the breast cancer expresses estrogen receptor (ER +) and/or progesterone receptor (PR +). In some embodiments, the breast cancer is ER-and/or PR-. In some embodiments, the breast cancer has an amplified HER2 gene. In some embodiments, the breast cancer is HER 23 + estrogen receptor negative (ER-), progesterone receptor negative (PR-), trastuzumab-resistant, chemotherapy-resistant invasive breast ductal carcinoma. In another embodiment, the breast cancer is HER 23 + ER-, PR-, trastuzumab-resistant inflammatory breast cancer. In another embodiment, the breast cancer is HER 23 +, ER-, PR-, invasive ductal carcinoma. In another embodiment, the breast cancer is trastuzumab and pertuzumab-resistant breast cancer with HER 22 + HER2 gene amplification. In some embodiments, the breast cancer is triple negative (ER-, PR-and HER2 low expression).

In one embodiment, the tumor is HER 22/3 + ovarian epithelial adenocarcinoma with an amplified HER2 gene.

Provided herein are methods for treating a subject having or becoming resistant to HER2+ tumor of other standard of care therapies comprising administering to the subject a pharmaceutical composition comprising an antigen binding construct described herein. In certain embodiments, the antigen binding constructs described herein are provided to a subject that is non-responsive to current therapy, optionally in combination with one or more current anti-HER 2 therapies. In some embodiments, current anti-HER 2 therapies include, but are not limited to, anti-HER 2 or anti-HER 3 monospecific bivalent antibodies, trastuzumab, pertuzumab, T-DM1, bispecific HER2/HER3scFv, or combinations thereof. In some embodiments, the cancer is resistant to various chemotherapeutic agents, such as taxane. In some embodiments the cancer is resistant to trastuzumab. In some embodiments the cancer is resistant to pertuzumab. In one embodiment, the cancer is resistant to TDM1 (trastuzumab coupled with DM 1). In some embodiments, the subject has been previously treated with an anti-HER 2 antibody such as trastuzumab, pertuzumab, or DM 1. In some embodiments, the subject has not been previously treated with an anti-HER 2 antibody. In one embodiment, the antigen binding construct is provided to the subject to treat the metastatic cancer when the patient has progressed on a previous anti-HER 2 therapy.

Provided herein are methods of treating a subject having a HER2+ tumor comprising providing an effective amount of a pharmaceutical composition comprising an antigen binding construct described herein together with an additional anti-tumor agent. The additional anti-neoplastic agent can be a therapeutic antibody, as described above, or a chemotherapeutic agent. Chemotherapeutic agents for use in combination with the antigen-binding constructs of the invention include cisplatin, carboplatin, paclitaxel, albumin-bound paclitaxel, docetaxel, gemcitabine, vinorelbine, irinotecan (irinotecan), etoposide, vinblastine, pemetrexed (pemetrexed), 5-fluorouracil (with or without folinic acid), capecitabine, carboplatin, epirubicin, oxaliplatin (oxaliplatin), calcium folinate (folfirinox), abraxane, and cyclophosphamide.

In some embodiments, the tumor is non-small cell lung cancer and the additional agent is one or more of cisplatin, carboplatin, paclitaxel, albumin-bound paclitaxel, docetaxel, gemcitabine, vinorelbine, irinotecan, etoposide, vinblastine, or pemetrexed. In embodiments, the tumor is gastric or gastric cancer and the additional agent is one or more of 5-fluorouracil (with or without leucovorin), capecitabine, carboplatin, cisplatin, docetaxel, epirubicin, irinotecan, oxaliplatin, or paclitaxel. In other embodiments, the tumor is pancreatic cancer and the additional agent is one or more of gemcitabine, calcium folinate, abraxane, or 5-fluorouracil. In other embodiments, the tumor is estrogen and/or progesterone positive breast cancer and the additional agent is one or more of (a) a combination of doxorubicin and epirubicin, (b) a combination of paclitaxel and docetaxel, or (c) a combination of 5-fluorouracil, cyclophosphamide and carboplatin. In other embodiments, the tumor is a head and neck cancer and the additional agent is one or more of paclitaxel, carboplatin, doxorubicin, or cisplatin. In other embodiments, the tumor is ovarian cancer and the additional agent may be one or more of cisplatin, carboplatin, or a taxane such as paclitaxel or docetaxel.

The additional agent may be administered to the subject simultaneously or sequentially with the antigen-binding construct.

The subject treated with the antigen-binding construct may be a human, a non-human primate, or other mammal such as a mouse.

In some embodiments, the result of providing an effective amount of the antigen-binding construct to a subject having a tumor is a reduction in tumor, inhibition of tumor growth, an increase in time to tumor progression, an increase in disease-free survival in the subject, a reduction in metastasis, an increase in progression-free survival in the subject, or an increase in overall survival in the subject or an increase in overall survival in a group of subjects receiving treatment.

Also described herein are methods of killing a HER 2-expressing tumor cell or inhibiting the growth of a HER 2-expressing tumor cell comprising contacting the cell with an antigen binding construct provided herein.

In various embodiments, the tumor cell can be a HER 21 + or 2+ human pancreatic cancer cell, a HER 23 + human lung cancer cell, a HER 22 + caucasian bronchioloalveolar carcinoma cell, a human pharyngeal cancer cell, a HER 22 + human tongue squamous cell carcinoma cell, a HER 22 + pharyngeal squamous cell carcinoma cell, a HER 21 + or 2+ human colorectal cancer cell, a HER 23 + human gastric cancer cell, a HER 21 + human breast ductal ER (estrogen receptor positive) cancer cell, a HER 22 +/3+ human ER +, a HER 2-amplified breast cancer cell, a HER20+/1+ human triple-negative breast cancer cell, a HER 22 + human endometrial cancer cell, a HER 21 + lung metastatic malignant melanoma cell, a HER 21 + human cervical cancer cell, a HER 21 + human renal cell cancer cell, or a HER 21 + human ovarian cancer cell.

In embodiments where the antigen binding construct is conjugated to DM1, the tumor cell can be a HER 21 + or 2+ or 3+ human pancreatic cancer cell, a HER 22 + metastatic pancreatic cancer cell, a HER 20 +/1+, +3+ human lung cancer cell, a HER 22 + caucasian bronchioloalveolar carcinoma cell, a HER 20 + anaplastic lung cancer cell, a human non-small cell lung cancer cell, a human pharyngeal cancer cell, a HER 22 + human tongue squamous cell carcinoma cell, a HER 22 + pharyngeal squamous cell carcinoma cell, a HER 21 + or 2+ human colorectal cancer cell, a HER 20 +, 1+ or 3+ human gastric cancer cell, a HER 21 + human breast ductal + (estrogen receptor positive) cancer cell, a HER 22 +/3+ human ER +, a HER2 amplified breast cancer cell, a HER 20 +/1+ human triple negative breast cancer cell, a HER 20 + human ductal carcinoma (basal B type, mesenchymal triple negative) breast cancer cell, a HER 22 + ER + HER 20 + human breast cancer cell, a HER 20 + metastatic breast cancer cell (basal B type, inter-like triple negative) breast cancer cell, HER 2-amplification, luminal a, TN), human uteroblastomere (mixed grade III) cells, 2+ human endometrial cancer cells, HER 21 + human dermal epidermoid cancer cells, HER 21 + lung metastatic malignant melanoma cells, HER 21 + malignant melanoma cells, human cervical epidermoid cancer, HER 21 + human bladder cancer cells, HER 21 + human cervical cancer cells, HER 21 + human renal cell cancer cells, or HER 21 +, 2+, or 3+ human ovarian cancer cells.

In some embodiments the tumor cell may be one or more of the following cell lines (shown in fig. 37 and 38): pancreatic tumor cell lines BxPC3, Capan-1, MiaPaca 2; lung tumor cell lines Calu-3, NCI-H322; head and neck tumor cell lines Detroit562, SCC-25, FaDu; colorectal tumor cell lines HT29, SNU-C2B; gastric tumor cell line NCI-N87; breast tumor cell lines MCF-7, MDAMB175, MDAMB361, MDA-MB-231, BT-20, JIMT-1, SkBr3, BT-474; uterine tumor cell line TOV-112D; the skin tumor cell line Malme-3M; cervical tumor cell lines Caski, MS 751; bladder tumor cell line T24; ovarian tumor cell lines CaOV3 and SKOV 3.

In embodiments where the antigen binding construct is coupled to DM1, the tumor cell may be one or more of the following cell lines (shown in figures 37 and 38): pancreatic tumor cell lines BxPC3, Capan-1, MiaPaca2, SW 1990, Panc 1; lung tumor cell lines A549, Calu-3, Calu-6, NCI-H2126, and NCI-H322; head and neck tumor cell lines Detroit562, SCC-15, SCC-25, FaDu; colorectal tumor cell lines Colo201, DLD-1, HCT116, HT29, SNU-C2B; gastric tumor cell lines SNU-1, SNU-16, NCI-N87; breast tumor cell lines SkBr3, MCF-7, MDAMB175, MDAMB361, MDA-MB-231, ZR-75-1, BT-20, BT549, BT-474, CAMA-1, MDAMB453, JIMT-1, T47D; uterine tumor cell lines SK-UT-1, TOV-112D; skin tumor cell lines a431, Malme-3M, SKEMEL 28; cervical tumor cell lines Caski, MS 751; bladder tumor cell line T24; the renal tumor cell line ACHN; ovarian tumor cell lines CaOV3, Ovar-3, and SKOV 3.

Kit and product

Also described herein are kits comprising one or more antigen binding constructs. The individual components of the kit will be packaged in separate containers and accompanying such containers may be in the form as set forth by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which agency reflects approval for manufacture, use or sale. The kit may optionally be provided with instructions or directions outlining methods of use or administration protocols for the antigen-binding constructs.

When one or more of the components of the kit are provided as a solution, e.g., an aqueous or sterile aqueous solution, the container means may itself be an inhaler, syringe, pipette, dropper, or other similar device from which the solution may be administered to a subject or applied to and mixed with the other components of the kit.

The components of the kit may also be provided in a dried or lyophilized form and the kit may additionally contain a suitable solvent for reconstitution of the lyophilized components. Regardless of the number or type of containers, the kits described herein may also comprise a means to assist in administering the composition to the patient. Such an appliance may be an inhaler, nasal spray device, syringe, pipette, forceps, measuring spoon, eye dropper, or similar medically approved delivery tool.

In another aspect described herein, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the above conditions is provided. The article of manufacture includes a container and a label or package insert on or with the container. Suitable containers include, for example, bottles, vials, syringes, Intravenous (IV) bags, and the like. The container may be formed from a variety of materials such as glass or plastic. The container contains the composition alone or in combination with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (e.g., the container may be an intravenous bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a T cell activating antigen binding construct described herein. The label or package insert indicates that the composition is for use in treating the selected condition. Moreover, the article of manufacture can comprise (a) a first container having a composition therein, wherein the composition comprises an antigen binding construct described herein; and (b) a second container having a composition therein, wherein the composition comprises another cytotoxic or other therapeutic agent. The article of manufacture in this embodiment described herein may also comprise package inserts indicating that the composition may be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as the bacteriostatic agents water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

Polypeptides and polynucleotides

The antigen binding constructs described herein comprise at least one polypeptide. Polynucleotides encoding the polypeptides described herein are also described. The antigen binding construct is typically isolated.

As used herein, "isolated" means that an agent (e.g., a polypeptide or polynucleotide) has been identified and isolated and/or recovered from its native cell culture environment. Contaminant components of their natural environment are materials that would interfere with diagnostic or therapeutic uses of the antigen-binding constructs, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. Isolation also refers to reagents that have been synthetically produced, e.g., via human intervention.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, the description for polypeptides applies equally to the description for peptides and to the description for proteins, and vice versa. The term applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the term encompasses amino acid chains of any length, including full length proteins, in which the amino acid residues are linked by covalent peptide bonds.

The term "amino acid" refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. The naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), and pyrrolysine and selenocysteine. Amino acid analogs refer to compounds having the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon, carboxyl, amino, and R group bound to a hydrogen, such as homoserine, norleucine, methionine sulfoxide, methyl methionine sulfonium salt. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Reference to amino acids includes, for example, naturally occurring proteinogenic (proteinogenic) L-amino acids; d-amino acids, chemically modified amino acids such as amino acid variants and derivatives; naturally occurring non-proteogenic amino acids such as beta-alanine, ornithine, and the like; and chemically synthesized compounds having properties known in the art to be unique to amino acids. Examples of non-naturally occurring amino acids include, but are not limited to, alpha-methyl amino acids (e.g., alpha-methyl alanine), D-amino acids, histidine-like amino acids (e.g., 2-amino-histidine, beta-hydroxy-histidine, homohistidine), amino acids with an additional methylene group on the side chain ("homo" amino acids), and amino acids in which the carboxylic acid functional group on the side chain is replaced with a sulfonic acid group (e.g., cysteic acid). Incorporation of unnatural amino acids, including synthetic unnatural amino acids, substituted amino acids, or one or more D-amino acids into proteins of the invention can be advantageous in a number of different respects. D-amino acid-containing peptides and the like exhibit enhanced stability in vitro or in vivo as compared with L-amino acid-containing counterparts. Thus, the construction of peptides and the like incorporating D-amino acids may be particularly useful when greater intracellular stability is desired or required. More specifically, D-peptides and the like are resistant to endogenous peptidases and proteases, providing increased molecular bioavailability and extended in vivo lifetimes when such properties are desired. In addition, D-peptide and the like cannot be efficiently processed because presentation to T helper cells is limited by major histocompatibility complex class II, and therefore, it is unlikely that a humoral immune response is induced in the whole organism.

Amino acids may be referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted single letter symbols.

Also included in the invention are polynucleotides encoding the polypeptides of the antigen binding constructs. The term "polynucleotide" or "nucleotide sequence" is intended to indicate a contiguous stretch of two or more nucleotide molecules. The nucleotide sequence may be of genomic, cDNA, RNA, semisynthetic or synthetic origin, or any combinations thereof.

The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses known analogs containing natural nucleotides, nucleic acids that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also refers to oligonucleotide analogs, including PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioate, phosphoramide, etc.). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. In particular, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res.19:5081 (1991); Ohtsuka et al, J.biol.chem.260:2605-2608 (1985); Rossolini et al, mol.cell.Probes 8:91-98 (1994)).

"conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those nucleic acids that encode identical or substantially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence as substantially identical. Because of the degeneracy of the genetic code, many functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at each position where an alanine is specified by a codon, the codon can be changed to any of the corresponding desired codons without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, each silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence.

With respect to amino acid sequences, one of ordinary skill in the art will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alters, adds or deletes a single amino acid or a small portion of an amino acid in the coding sequence are "conservatively modified variants" when the alteration results in the deletion, addition or substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are known to those of ordinary skill in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles described herein.

Conservative substitution tables providing functionally similar amino acids are known to those of ordinary skill in the art. The following eight groups of amino acids each contain conservative substitutions for each other: 1) alanine (a), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and [0139]8) cysteine (C), methionine (M) (see, e.g., Creighton, Proteins: Structures and molecular Properties (W H Freeman & Co., 2 nd edition (12 months 1993)).

In the case of two or more nucleic acid or polypeptide sequences, the terms "identical" or percent "identity" refer to two or more sequences or subsequences that are the same. Sequences are "substantially identical" if they have a certain percentage of amino acid residues or nucleotides that are identical (i.e., about 60% identity, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity over a specified region) when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using one of the following sequence comparison algorithms (or other algorithms available to one of ordinary skill in the art) or by manual alignment and visual inspection. This definition also relates to the complement of the test sequence. Identity may exist over the entire sequence of a polynucleotide or polypeptide over a region of at least about 50 amino acids or nucleotides in length, or over a region of 75-100 amino acids or nucleotides in length, or unspecified. Polynucleotides encoding the polypeptides of the invention, including homologues from species other than human, may be obtained by a method comprising the steps of: libraries are screened under stringent hybridization conditions with labeled probes having the polynucleotide sequences described herein, or fragments thereof, and full-length cdnas and genomic clones containing the polynucleotide sequences are isolated. Such hybridization techniques are well known to the skilled artisan.

For sequence comparison, typically one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. Default program parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

As used herein, "comparison window" includes reference to a segment having any number of consecutive positions selected from: 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, in which segment after optimal alignment of the two sequences, the sequences can be compared to reference sequences at the same number of consecutive positions. Methods of sequence alignment for comparison are known to those of ordinary skill in the art. Optimal sequence alignments for comparison can be performed by methods including, but not limited to, local homology algorithms by Smith and Waterman (1970) adv.Appl.Math.2:482c, homology alignment algorithms by Needleman and Wunsch (1970) J.mol.biol.48:443, similarity search methods by Pearson and Lipman (1988) Proc.Nat' l.Acad.Sci.USA 85:2444, execution of these algorithms by Computer (Wisconsin Genetics software package (Molecular, BESTFIT, FASTA and TFASTA in Genetics Computer Group,575Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Aurrebel et al, Current Protocols in biological Biology (1995 supplement)).

One example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST2.0 algorithms described in Altschul et al (1997) Nuc. acids Res.25: 3389-. Software for performing BLAST analysis is publicly available through the National Center for biotechnology Information (National Center for biotechnology Information) available on the world wide web ncbi. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the algorithm. The BLASTN program (for nucleotide sequences) defaults to 11 word length (W), 10 expectation (E), 5M, 4N and two strand comparisons. For amino acid sequences, the BLASTP program defaults to using a word size of 3, an expectation (E) of 10 and a BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) proc. natl. acad. sci. usa 89:10915) for comparisons (B) of 50, expectations (E) of 10, M5, N-4 and both strands. The BLAST algorithm is typically performed with the "low complexity" filter turned off.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873- > 5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which indicates the probability by which a match between two nucleotide or amino acid sequences occurs by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, or less than about 0.01, or less than about 0.001.

The phrase "selectively (or specifically) hybridizes to … …" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence (when that sequence is present in a complex mixture including, but not limited to, total cell or library DNA or RNA) under stringent hybridization conditions.

The phrase "stringent hybridization conditions" refers to hybridization of sequences of DNA, RNA, or other nucleic acids, or combinations thereof, under conditions of low ionic strength and high temperature, as known in the art. Typically, a probe will hybridize under stringent conditions to its target subsequence in a complex mixture of nucleic acids (including but not limited to total cell or library DNA or RNA), but not to other sequences in the complex mixture. Stringent conditions are sequence dependent and will be different under different circumstances. Longer sequences hybridize specifically at higher temperatures. A large number of guidelines for Nucleic acid Hybridization are found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic acids Probes, "Overview of principles of Hybridization and Molecular protocols of Nucleic acid assays" (1993).

As used herein, the term "engineered" is considered to include any manipulation of the peptide backbone or post-translational modification of naturally occurring or recombinant polypeptides or fragments thereof. Engineering includes modification of the amino acid sequence, glycosylation patterns, or amino acid side chain groups alone, as well as combinations of these methods. The engineered proteins are expressed and produced by standard molecular biology techniques.

By "isolated nucleic acid molecule or polynucleotide" is meant a nucleic acid molecule, DNA or RNA, which has been removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. An isolated polynucleotide includes a polynucleotide molecule contained in a cell that normally contains the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. Isolated RNA molecules include RNA transcripts in vivo or in vitro, as well as both positive and negative strand forms and double-stranded forms. Isolated polynucleotides or nucleic acids described herein also include such molecules produced synthetically, e.g., via PCR or chemical synthesis. In addition, the polynucleotide or nucleic acid, in certain embodiments, includes regulatory elements such as a promoter, ribosome binding site, or transcription terminator.

The term "polymerase chain reaction" or "PCR" generally refers to a method of amplifying a desired nucleotide sequence in vitro, for example, as described in U.S. patent No. 4,683,195. In general, PCR methods involve repeated cycles of primer extension synthesis, using oligonucleotide primers that preferentially hybridize to a template nucleic acid.

By a nucleic acid or polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention, it is meant that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may include up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These changes to the reference sequence can occur at the 5 'or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually between residues in the reference sequence or in one or more contiguous groups within the reference sequence. Indeed, it is routinely determined whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention using known computer programs, such as those discussed above for polypeptides (e.g., ALIGN-2).

A derivative or variant of a polypeptide is said to share "homology" or "be homologous" to the peptide if the amino acid sequence of the derivative or variant has at least 50% identity with a sequence of 100 amino acids from the original peptide. In certain embodiments, the derivative or variant has at least 75% identity to a peptide or fragment of a peptide having the same number of amino acid residues as the derivative. In certain embodiments, the derivative or variant has at least 85% identity to a peptide or fragment of a peptide having the same number of amino acid residues as the derivative. In certain embodiments, the amino acid sequence of the derivative has at least 90% identity to a peptide or fragment of a peptide having the same number of amino acid residues as the derivative. In some embodiments, the amino acid sequence of the derivative is at least 95% identical to a peptide or fragment of a peptide having the same number of amino acid residues as the derivative. In certain embodiments, the derivative or variant has at least 99% identity to a peptide or fragment of a peptide having the same number of amino acid residues as the derivative.

In some aspects, the antigen-binding construct comprises an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a related amino acid sequence, or fragment thereof, listed in a table or accession number disclosed herein. In some aspects, an isolated antigen-binding construct comprises an amino acid sequence encoded by a polynucleotide having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a related nucleotide sequence, or fragment thereof, listed in a table or accession number disclosed herein.

It is to be understood that this invention is not limited to the particular protocols, cell lines, constructs, and reagents described herein and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.

All publications and patents mentioned herein are incorporated herein by reference for the purpose of describing and disclosing, for example, the constructs and methodologies described in the publications, which might be used in connection with the presently described invention. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason.

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examples

The following are examples of carrying out specific embodiments of the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental error and deviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., t.e.creighton, Proteins: structures and Molecular Properties (w.h. freeman and company, 1993); l. lehninger, Biochemistry (Worth Publishers, inc., currently increasing); sambrook et al, Molecular Cloning: a Laboratory Manual (1989, 2 nd edition); methods In Enzymology (edited by s.colwick and n.kaplan, Academic Press, Inc.); remington's pharmaceutical sciences, 18 th edition (Easton, Pennsylvania: Mack Publishing Company, 1990); carey and Sundberg Advanced Organic Chemistry, 3 rd edition (Plenum Press), volumes A and B (1992).

Example 1: preparation of exemplary anti-HER 2 bispecific antibodies and controls

Various exemplary anti-HER 2 biparatopic antibodies (or antigen-binding constructs) and controls were prepared as described below. Antibodies and controls in different formats have been prepared, and a schematic of an exemplary biparatopic format is shown in table 1. In all the forms shown in fig. 1, heterodimeric Fc is depicted with one chain shown in black (a chain) and another chain shown in gray (B chain), while one antigen binding domain (1) is shown hatched and the other antigen binding domain (2) is shown in white.

The sequences of the following variants are provided in the sequence listing following the examples. CDR regions were identified using a combination of Kabat and Chothia methods. The region may be slightly different depending on the method used for identification.

Exemplary anti-HER 2 biparatopic antibodies

An exemplary anti-HER 2 biparatopic antibody was prepared as shown in table 1.

Table 1: exemplary anti-HER 2 biparatopic antibodies

Note that:

the CH3 numbering is according to the EU index, as is the numbering of the EU antibody mentioned in Kabat (Edelman et al, 1969, Proc Natl Acad Sci USA 63: 78-85);

fab or variable Domain numbering according to Kabat (Kabat and Wu, 1991; Kabat et al, Sequences of proteins of immunological interest, 5 th edition-department of American Health and Human Services, NIH publication n ° 91-3242, p 647 (1991))

An "epitope-containing domain" ═ the domain of HER2 that binds to the antigen-binding moiety;

the "antibody name" refers to the antibody from which the antibody binding moiety is derived, when present including substitutions as compared to wild type;

"Fab substitutions" are substitutions in Fab that promote correct pairing of the light chain;

"substitution of the CH3 sequence" ═ substitution in the CH3 domain to promote heterodimeric Fc formation

Exemplary anti-HER 2 monovalent control antibodies

v 1040: a monovalent anti-HER 2 antibody, wherein the HER2 binding domain is derived from Fab on trastuzumab a chain, and the Fc region is a heterodimer with the mutation T350V _ L351Y _ F405A _ Y407V in the a chain, the mutation T350V _ T366L _ K392L _ T394W in the B chain, and the hinge region of the B chain has the mutation C226S; the antigen binding domain binds to domain 4 of HER 2.

v 630-monovalent anti-HER 2 antibody, wherein HER2 binding domain is derived from an scFv on trastuzumab a chain and the Fc region is a heterodimer with the mutation L351Y _ S400E _ F405A _ Y407V in the a chain and the mutation T366I _ N390R _ K392M _ T394W in the B chain; and the hinge region has the mutation C226S (EU numbering) on both strands; the antigen binding domain binds to domain 4 of HER 2.

v 4182: a monovalent anti-HER 2 antibody, wherein the HER2 binding domain is derived from Fab on the pertuzumab a chain, and the Fc region is a heterodimer with the mutation T350V _ L351Y _ F405A _ Y407V in the a chain, the mutation T350V _ T366L _ K392L _ T394W in the B chain, and the hinge region of the B chain has the mutation C226S; the antigen binding domain binds to domain 2 of HER 2.

Exemplary anti-HER 2 monospecific bivalent antibody control (full-size antibody, FSA)

v506 is wild-type anti-HER 2 produced inside Chinese Hamster Ovary (CHO) cells as a control. Both HER2 binding domains are derived from trastuzumab in Fab form and Fc is a wild-type homodimer; the antigen binding domain binds to domain 4 of HER 2. Such antibodies are also known as trastuzumab analogs.

v792 is wild-type trastuzumab with an IgG1 hinge, with both HER2 binding domains derived from trastuzumab in Fab form, and the Fc region is a heterodimer with the mutation T350V _ L351Y _ F405A _ Y407V in the a chain and the mutation T350V _ T366L _ K392L _ T394W in the B chain; the antigen binding domain binds to domain 4 of HER 2. Such antibodies are also known as trastuzumab analogs.

v4184 is a bivalent anti-HER 2 antibody, wherein both HER2 binding domains are derived from pertuzumab in Fab form, and the Fc region is a heterodimer with the mutation T350V _ L351Y _ F405A _ Y407V in the a chain and the mutation T350V _ T366L _ K392L _ T394W in the B chain. The antigen binding domain binds to domain 2 of HER 2. Such antibodies are also known as pertuzumab analogs.

hIgG was a control of a commercial non-specific polyclonal antibody (Jackson ImmunoResearch, # 009-.

These antibodies and controls (except for human IgG) were cloned and expressed as follows. Genes encoding antibody heavy and light chains were constructed via gene synthesis using codons optimized for human/mammalian expression. Trastuzumab Fab sequences (Carter P. et al (1992) Humanization of an anti 185HER2antibody for human cancer therapy. Proc Natl Acad Sci 89,4285.) were generated from a known antibody that binds to HER2/neu domain 4 and Fc was of the IgG1 isotype. scFv sequences were generated from the VH and VL domains of trastuzumab using glycine-serine linkers (Carter P. et al (1992) Humanization of an anti p185her2antibody for human cancer therapy. Proc Natl Acad Sci 89,4285.). The Pertuzumab Fab sequence was generated from a known antibody that binds to HER2/neu domain 2 (Adams CW et al (2006) mutagenesis of a recombinant monoclonal antibody to product a therapeutic HER dimerization inhibitor, Pertuzumab.Cancer Immunol Immunother,2006；55(6):717-27)。

The final gene product was subcloned into the mammalian expression vector PTT5(NRC-BRI, Canada) and expressed in CHO cells (Durocher, Y., Perret, S. and Kamen, A. high-level and high-throughput recombinant protein production by transfer transformation of subset-growing CHO cells. nucleic acids research 30, e9 (2002)).

CHO cells were transfected with 1mg/ml 25kDa polyethyleneimine in water (PEI, polysciences) at a PEI: DNA ratio of 2.5:1 during exponential growth phase (150 to 200 ten thousand cells/ml). (Raymond C. et al A simple and aqueous transformation process for large-scale and high-throughput applications. methods.55(1):44-51 (2011)). To determine the optimal concentration range for heterodimer formation, transfected cells were harvested with medium collected after centrifugation at 4000rpm after transfection of DNA at the optimal DNA ratio for heavy chain a (HC-a), Light Chain (LC) and heavy chain B (HC-B) that allowed heterodimer formation (e.g., HC-a/HC-B/LC ratio of 30:30:40(v5019) for 5-6 days and clarified using a 0.45 μm filter.

The clarified medium was loaded onto a MabSelect SuRe (GE Healthcare) protein-a column and washed with 10 column volumes of PBS buffer (pH 7.2). The antibody was eluted with 10 column volumes of citrate buffer (pH 3.6) and the pooled fractions contained TRIS-neutralized antibody at pH 11.

The protein-a antibody eluate was further purified by gel filtration (SEC). For gel filtration, 3.5mg of the antibody mixture was concentrated to 1.5mL and loaded onto a Sephadex 200HiLoad 16/600200 pg column (GE Healthcare) via AKTA Express FPLC at a flow rate of 1 mL/min. PBS buffer (pH 7.4) was used at a flow rate of 1 mL/min. Fractions corresponding to purified antibody were collected and concentrated to about 1 mg/mL.

Exemplary anti-HER 2ECD2x ECD4 biparatopic antibodies (e.g., v6717, scFv-scFv IgG 1; v6903 and v6902Fab-Fab IgG 1; v5019, v7091 and v10000Fab-scFv IgG1) with different molecular formats were cloned, expressed and purified as described above.

To quantify antibody purity and determine the amount of target heterodimeric protein and possible homodimeric and/or hemi-antibody and/or mismatched light chain contaminants, a complete mass analysis of LC-MS was performed. Complete mass analysis of LC-MS was performed as described in example 2, except for the calculation of DAR analysis for ADC molecules.

The data are shown in table 2. Table 2 shows that expression and purification of these biparatopic antibodies produced 100% of the desired product for v6717, 91% of the desired heterodimeric product for v6903, and 62% of the desired product for v 6902. The numbers in parentheses refer to the amount of the main plus side peak of +81 Da. This side peak is typically detected with variants containing a C-terminal HA tag (e.g., variants of v6903 and v 6902). For v6903 and v6903, the main and side peaks were added to give heterodimer purities of approximately 98% and 67%. Based on high heterodimer purity, v6903 was identified as a representative Fab-Fab anti-HER 2 biparatopic variant for direct comparison with scFv-scFv and Fab-scFv formats. V6903 was included in all format comparison assays.

Table 2: expression and purification of antibodies

Variants	Desired heterodimer species (+ side peaks)
		6717	100.0
6903	90.9(97.7)
		6902	62.4(67.4)

Example 2: preparation of an exemplary anti-HER 2 biparatopic Antibody Drug Conjugate (ADC)

The following anti-HER 2 biparatopic antibody drug conjugates (anti-HER 2 biparatopic ADCs) were prepared. ADCs of variants 5019, 7091, 10000 and 506 were prepared. These ADCs were identified as follows:

v6363 (v 5019 coupled to DM 1)

v7148 (v 7091 coupled to DM 1)

v10553 (v 10000 coupled with DM 1)

v6246 (v 506 coupled with DM1, analogous to T-DM1, trastuzumab-emtansine)

v6249 (human IgG conjugated with DM 1)

ADCs were prepared via direct coupling with maytansine. The antibody purified (> 95% purity) by protein a and SEC was used to prepare ADC molecules as described in example 1. According to Kovtun YV, Audette CA, Ye Y, etc., Antibody-drugs designed to an iterative nucleotides with a homology and a heterologous expression of the target antigen, cancer Res 2006; 66:3214-21 to ADC. The ADC had an average molar ratio of 3.0 maytansinoid molecules per antibody, as determined by LC/MS and described below.

Details of the reagents used in the ADC coupling reaction are as follows: coupling buffer 1: 50mM potassium phosphate/50 mM sodium chloride, pH6.5, 2mM EDTA. Coupling buffer 2: 50mM sodium succinate, pH 5.0. Buffer preparation of ADC: 20mM sodium succinate, 6% (w/v) trehalose, 0.02% polysorbate 20, pH 5.0. Dimethylacetamide (DMA); SMCC in DMA (prepared before coupling), DM1-SH in DMA (prepared before coupling), 1mM DTNB in PBS, 1mM cysteine in buffer, 20mM sodium succinate, pH 5.0, 10 mM. UV-VIS Spectrophotometer (Nanodrop 100 from Fisher Scientific), PD-10 column (GE Healthcare).

The ADC was prepared as follows. The original antibody solution was loaded onto a PD-10 column, which was previously equilibrated with 25mL of coupling buffer 1, followed by 0.5mL of coupling buffer 1. Collecting the antibody eluate and washing at A₂₈₀The concentration was measured and adjusted to 20 mg/mL. A10 mM solution of SMCC-DM1 in DMA was prepared. To the antibody solution was added 7.5 molar equivalents of SMCC-DM 1-antibody and DMA to a final DMA volume of 10% v/v. The reaction was simply mixed and incubated at room temperature for 2 h. A second PD-10 column was equilibrated with 25ml of coupling buffer 1 and antibody-MCC-DM 1 solution was added to the column followed by 0.5ml of buffer 1. antibody-MCC-DM 1 eluate was collected and A of the antibody solution was measured₂₅₂And A₂₈₀. antibody-MCC-DM 1 concentration was calculated (□ ═ 1.45mg^-1cm^-1Or 217500M^-1cm^-1). Analysis of the ADC on a SEC-HPLC column for high MW analysis (SEC-HPLC column TOSOH, G3000-SWXL, 7.8mmx30cm, buffer, 100mM sodium phosphate, 300mM sodium chloride, pH 7.0, flow rate: 1 ml/min).

ADC drug to antibody ratio (DAR) was analyzed by HIC-HPLC using a Tosoh TSK gel butyl-NPR column (4.6mmx3.5mmx2.5 mm). Elution was performed at 1ml/min for 25 min using 10-90% buffer B followed by a gradient of 100% buffer B for 4 min. Buffer A contained 20mM sodium phosphate, 1.5M ammonium sulfate (pH 7.0). Buffer B contained 20mM sodium phosphate, 25% v/v isopropanol (pH 7.0).

The ADC drug was determined by LC-MS by antibody ratio (DAR). Antibodies were deglycosylated with peptide N-glycoamidase f (pngase f) prior to loading into LC-MS. Liquid chromatography was performed on an Agilent 1100 series HPLC under the following conditions.

Flow rate: after 1mL/min of column, the solution is split to 100uL/min and enters MS. Solvent: a ═ 0.1% formic acid in ddH2O, B ═ 65% acetonitrile, 25% THF, 9.9% ddH20, 0.1% formic acid. Column: 2.1x30mm PorosR 2. Column temperature: 80 ℃; the solvent is also preheated. Gradient: 20% B (0-3min), 20-90% B (3-6min), 90-20% B (6-7min), 20% B (7-9 min).

Mass Spectrometry (MS) was then performed on an LTQ-Orbitrap XL mass spectrometer under the following conditions: ionization method using Ion MaxElectrospray. Calibration and tuning methods: 2mg/mL CsI solution was infused at a flow rate of 10. mu.L/min. Orbitrap (observed overall CsI ion range: 1690 to 2800) was tuned using the auto-tuning function on m/z 2211. Taper hole voltage: 40V; lens barrel lens: 115V; FT resolution: 7,500; the scanning range m/z is 400-4000; scanning delay: 1.5 min. Molecular weight curves of the data were soft-generated using Thermo's Promass deconvolution. The average DAR for the sample is determined from the DAR observed at each partial peak (calculation used:Σ (DARx partial peak intensity)).

Table 3 summarizes the average DAR of the ADC molecules. The average DAR for the exemplary anti-HER 2 biparatopic antibody and control was approximately 3.

Table 3: average DAR for ADCs

	DAR(LC-MS)	DAR(HIC)	n
				v6246	2.9	3.0	5
v6363	2.6	3.3	5
				v7148	3.4	3.9	1
v10553	4.0	4.0	1

Example 3: expression and laboratory-scale purification of anti-HER 2 biparatopic antibodies

The anti-HER 2 biparatopic antibodies described in example 1 (v5019, v7091 and v10000) were expressed in volumes of 10 and/or 25L and purified by protein a and Size Exclusion Chromatography (SEC) as follows.

The protein-a antibody eluate was further purified by gel filtration (SEC). For gel filtration, 3.5mg of the antibody mixture was concentrated to 1.5mL and loaded onto a Sephadex 200HiLoad 16/600200 pg column (GE Healthcare) via AKTA Express FPLC at a flow rate of 1 mL/min. PBS buffer pH 7.4 was used at a flow rate of 1 mL/min. Fractions corresponding to purified antibody were collected and concentrated to about 1 mg/mL. The purified protein was analyzed by LC-MS as described in example 2.

Results for 10L expression and lab-scale protein a and SEC purification are shown in fig. 2A and 2B. Figure 2A shows the SEC chromatogram of v5019 of protein a purification and figure 2B shows a non-reducing SDS-PAGE gel comparing the relative purity of the protein a combined components and SEC components 15 and 19 and combined SEC components 16-18. These results show that the anti-HER 2 biparatopic antibody is expressed and purified by protein a and SEC to give a pure protein sample. Further quantification was performed by UPLC-SEC and LC-MS analysis and is described in example 4.

The results of 25L expression and laboratory scale protein a purification are shown in fig. 2C. Figure 2C shows an SDS-PAGE gel comparing the relative purity of v10000 of protein a purification. Lane M contains: protein molecular weight standards; lane 1 contains: v10000 under reducing conditions; lane 2 contains v10000 under non-reducing conditions. SDS-PAGE gels show that v10000 is pure and at the correct predicted molecular weight of approximately 125kDa under non-reducing conditions. Two heavy chain bands are visible under reducing conditions, corresponding to the CH-A heavy chain (approximately 49kDa) and the CH-B heavy chain (approximately 52.5 kDa); the CH-A light chain is visible and at the correct predicted molecular weight of approximately 23.5 kDa. These results show that the anti-HER 2 biparatopic antibody is expressed and a pure protein sample is obtained by one-step purification of protein a. Further quantification was performed by UPLC-SEC and LC-MS analysis and is described in example 4.

Example 4: double paratopic anti-HER 2 antibody purity by UPLC-SEC and LC-MS analysis

The purity and percent aggregation of the exemplary protein a and SEC purified biparatopic anti-HER 2 heteromultimer was determined by UPLC-SEC using the methods described.

UPLC-SEC analysis was performed at 0.4mL/min using a Waters BEH200SEC column set at 30 ℃ (2.5mL, 4.6x150mm, stainless steel, 1.7 μm particles). Run time consisted of 7min and total volume per injection was 2.8mL, run buffer 25mM sodium phosphate, 150mM sodium citrate (pH 7.1); and 150mM sodium phosphate (pH 6.4-7.1). Detection by absorbance was facilitated at 190-400nm and with fluorescence excited at 280nm and collected from 300-360 nm. Peak integrals were analyzed with Empower 3 software.

UPLC-SEC results of the combined v5019SEC components are shown in fig. 3A. These results indicate that the exemplary anti-HER 2 biparatopic antibody was purified to > 99% purity by protein a and SEC chromatography, containing less than 1% HMW species.

UPLC-SEC results of pooled v10000 protein a fractions are shown in fig. 3B. These results indicate that the exemplary anti-HER 2 biparatopic antibody was purified by protein a chromatography to > 96% purity, containing less than 1% HMW species.

The purity of an exemplary biparatopic anti-HER 2 antibody was determined under standard conditions using LC-MS by the method described in example 2. The results from LC-MS analysis of the combined SEC component for v5019 are shown in fig. 4A. The data show that the exemplary biparatopic anti-HER 2 heterodimer has 100% heterodimer purity. Results from LC-MS analysis of pooled protein a fractions on v10000 are shown in figure 4B. This data shows that the exemplary biparatopic anti-HER 2 heterodimer has 98% heterodimer purity after one-step protein a purification.

Antibodies purified by protein a chromatography and/or protein a and SEC were used in the assays described in the examples below.

Example 5: Large-Scale expression and purification of Biparatopic anti-HER 2 antibodies by protein A and CEX chromatography Evaluation of Productivity

The exemplary anti-HER 2 biparatopic antibody v5019 described in example 1 was expressed and purified on the 25L scale as follows.

The antibody was obtained from the supernatant, followed by a two-step purification procedure, which was purified from protein A (MabSelect)^TMA resin; GEHealthcare), followed by cation exchange chromatography (HiTrap) according to the protocol^TMSP FF resin; GEHealthcare).

CHO-3E7 cells were maintained in serum-free FreestyleCHO expression medium (Invitrogen, Carlsbad, CA, USA) in Erlenmeyer flasks (Erlenmeyer flash) (Corning Inc., Acton, MA) with 5% CO2 at 37 ℃ on an orbital shaker (VWR Scientific, Chester, Pa.). Two days prior to transfection, cells were seeded at the appropriate density in 50L CellBag at a volume of 25L using a shaking Bioreactor System 20/50(Wave Bioreactor System 20/50) (GE Healthcare Bio-Science Corp). On the day of transfection, DNA and PEI (Polysciences, Eppelheim, Germany) were mixed in optimal ratios and added to the cells using the method described in example 1. Cell supernatants collected on day 6 were used for further purification.

The cell culture broth was centrifuged and filtered, and then packed at 10.0mL/min into 30mL of Mabselect packaged in XK26/20(GEHealthcare, Uppsala, Sweden) ^TMOn a resin. After washing with the appropriate buffer and elution, fractions were collected and neutralized with 1M Tris-HCl (pH 9.0). The target protein was further purified via 20mL of SP FF resin packaged in XK16/20(GE Healthcare, Uppsala, Sweden). Via Mabselect^TMThe purified sample was diluted with 20mM NaAC (pH5.5) to adjust the conductivity to<5ms/cm and 50mM citric acid (pH3.0) was added to adjust the pH of the sample to 5.5. The samples were loaded at 1mL/min into HiTrap^TMSP FF resin (GE Healthcare) and washed with 20mM NaAC. Elution was carried out using a 0-100% gradient of 20mM NaAC, 1M NaCl (pH5.5), 10-foldThe Column Volume (CV) was eluted at 1 mL/min.

The purified proteins were analyzed by SDS-PAGE as described in example 1, and heterodimer purity was analyzed by LC-MS by the method described in example 4. The results are shown in FIGS. 5A and 5B. FIG. 5A shows MabSelect^TMAnd HiTrap^TMSDS-PAGE of v5019 after SP FF purification, lane M contains: protein molecular weight standards; strip 1: v5019 (3. mu.g) under reducing conditions; lane 2: v5019 (2.5. mu.g) under non-reducing conditions. SDS-PAGE gels showed v5019 in MabSelect^TMAnd HiTrap^TMSP FF was relatively pure after purification and under non-reducing conditions at the correct predicted molecular weight of approximately 125 kDa. Two heavy chain bands are visible under reducing conditions, corresponding to the CH-A heavy chain (approximately 49kDa) and the CH-B heavy chain (approximately 52.5 kDa); the CH-A light chain is visible and at the correct predicted mass of approximately 23.5 kDa.

MabSelect Using the method described in example 4^TMAnd HiTrap^TMLC-MS analysis of SP FF purified v5019 to determine heterodimer purity. The results from the LC-MS analysis are shown in fig. 5B. These results show the use of MabSelect^TMAnd HiTrap^TMSP FF purification v5019 to a purified plasmid having>99% heterodimer purity and very little (C:)<1%) or undetectable homodimer or half antibody contaminated protein.

Example 6: bmax of biparatopic anti-HER 2 antibody in cell lines expressing low to high levels of HER2 versus control Comparison of Bmax

The following experiments were performed to measure the ability of exemplary biparatopic anti-HER 2 antibodies to bind to cells expressing different levels of HER2 compared to controls. The cell lines used are SKOV3(HER 22 +/3+), JIMT-1(HER 22 +), MDA-MB-231(HER 20/1 +) and MCF7(HER 21 +). Biparatopic anti-HER 2 antibodies tested included v5019, v7091 and v 10000. As described below, warp B_maxAnd apparent K_D(equilibrium dissociation constant) specific measurements, determination of biparatopic anti-HER 2 antibody and HER2Express (HER2+) cell binding ability.

Binding of the test antibody to HER2+ cell surface was determined by flow cytometry. Cells were washed with PBS and at 1x10⁵The individual cells/100. mu.l were resuspended in DMEM. To each microcentrifuge tube, 100. mu.l of cell suspension was added, followed by 10. mu.l/tube of antibody variant. The tubes were incubated for 2 hours at 4 ℃ on a rotator. The microfuge tube was centrifuged at 2000RPM for 2min at room temperature and the cell pellet was washed with 500 μ l of media. Each cell pellet was resuspended in 100. mu.l of a fluorochrome-labeled secondary antibody diluted to 2. mu.g/sample in culture medium. The samples were then incubated for 1 hour at 4 ℃ on a rotator. After incubation, cells were centrifuged at 2000rpm for 2min and washed in medium. Cells were resuspended in 500. mu.l of medium, filtered in a tube containing 5. mu.l of Propidium Iodide (PI) and analyzed on a BD LSR II flow cytometer according to the manufacturer's instructions. K estimation of exemplary biparatopic anti-HER 2 heterodimer antibodies and control antibodies by FACS and data analysis and curve fitting in GraphPad Prism _D。

The results are shown in FIGS. 6A-6G. The results demonstrate that exemplary biparatopic anti-HER 2 antibodies (v5019, v7091 and v10000) can bind to HER2+ cells with about 1.5-fold higher Bmax compared to anti-HER 2FSA (v 506). The results in figures 6A-6G also show that biparatopic anti-HER 2 antibodies (v5019, v7091 and v10000) can bind to HER2+ cells with similar Bmax compared to the combination of two anti-HER 2 FSAs (v506+ v 4184).

The binding results for HER2+ SKOV3 cells (HER 22/3 +) are shown in fig. 6A, 6E and tables 4 and 5. The results in figure 6A and table 4 show that the exemplary biparatopic anti-HER 2 antibody (v5019) exhibited approximately 1.5-fold higher Bmax in binding to SKOV3 cells compared to two different anti-HER 2 FSAs (v506 or v 4184). The results also show that the exemplary biparatopic anti-HER 2 antibody (v5019) exhibited equal Bmax compared to the combination of the two anti-HER 2 FSAs (v506+ v 4184). v5019 apparent K in combination with SKOV3_DApproximately 2 to 4 times higher than either anti-HER 2FSA alone (v506 or v4184) or the combination of the two anti-HER 2 FSAs (v506+ v 4184).

Table 4: binding to SKOV3 cells

The results in figure 6E and table 5 show that the exemplary biparatopic anti-HER 2 antibodies (v5019, 7091, and v10000) displayed about 1.5 to 1.6 times higher Bmax compared to two different anti-HER 2 FSAs (v506 or v4184) in binding to SKOV3 cells. The results also show that the exemplary biparatopic anti-HER 2 antibodies (v5019, 7091 and v10000) displayed equal Bmax compared to the combination of the two anti-HER 2 FSAs (v506+ v 4184). Apparent K binding to SKOV3 for v5019, 7091 and v10000 and for the combination of two anti-HER 2FSA (v506+ v4184) _DApproximately 2 to 3 times higher than anti-HER 2FSA alone (v506 or v 4184).

Table 5: binding to SKOV3 cells

The binding curves in the JIMT-1 cell line (HER 22 +) are shown in FIG. 6B and Table 6. These results show that the exemplary biparatopic anti-HER 2 antibody (v5019) exhibited about 1.5-fold higher Bmax compared to anti-HER 2FSA (v506) in binding to JIMT-1 cells. The results also show that the exemplary biparatopic anti-HER 2 antibody (v5019) exhibited equal Bmax compared to the combination of the two anti-HER 2 FSAs (v506+ v 4184). v5019 apparent K binding to JIMT-1_DApproximately 2-fold higher compared to anti-HER 2FSA (v506), and similar (approximately 1.2-fold higher) compared to the combination of the two anti-HER 2 FSAs (v506+ v 4184).

Table 6: binding to JIMT-1 cells

Binding profiles in MCF7 cell line (HER 21 +)The lines are shown in fig. 6C, 6F and tables 7 and 8. These results show that the exemplary biparatopic anti-HER 2 antibodies (v5019, 7091 and v10000) displayed about 1.5-fold higher Bmax compared to anti-HER 2FSA (v506) in binding to MCF7 cells. The results in figure 6C also show that the exemplary biparatopic anti-HER 2 antibody (v5019) exhibited equal Bmax compared to the combination of the two anti-HER 2 FSAs (v506+ v 4184). v5019 apparent K in combination with MCF7_DSimilar to the combination of anti-HER 2FSA (v506) and two anti-HER 2 FSAs (v506+ v 4184).

Table 7: binding to MCF7 cells

The results in figure 6F and table 8 show that the exemplary biparatopic anti-HER 2 antibodies (v5019, v7091 and v10000) displayed about 1.6 to 1.7 fold higher Bmax compared to FSA monospecific v 506. Apparent K of v5019, v7091 and v10000_DSimilar to anti-HER 2FSA (v 506).

Table 8: binding to MCF7 cells

The binding curves in the MDA-MB-231 cell line (HER 20/1 +) are shown in FIG. 6D and Table 9. These results show that the exemplary biparatopic anti-HER 2 antibody (v5019) displayed approximately 1.5-fold higher Bmax compared to anti-HER 2FSA (v506) in binding to MDA-MB-231 cells. The results also show that the exemplary biparatopic anti-HER 2 antibody (v5019) exhibited equal Bmax compared to the combination of the two anti-HER 2 FSAs (v506+ v 4184). v5019 apparent K binding to MDA-MB-231_DApproximately 2.4 times lower compared to anti-HER 2FSA (v506) and approximately 1.7 times higher compared to the combination of the two anti-HER 2 FSAs (v506+ v 4184).

Table 9: binding to MDA-MB-231 cells

The binding curves in the WI-38 lung fibroblast cell line are shown in figure 6G and table 10. The WI-38 cell line is a normal lung epithelial cell expressing basal levels (HER 20 +, approximately 10,000 receptors/cell) of HER2 (Carter et al 1992, PNAS, 89: 4285-. These results show that the exemplary biparatopic anti-HER 2 antibodies (v5019, v7091 and v10000) exhibit equivalent cell surface modification (decoration) (Bmax) compared to anti-HER 2FSA (v506) in binding to WI-38 cells; however, it was noted that binding for v506 did not appear to reach saturation, and therefore KD could not be determined. Apparent K between exemplary biparatopic anti-HER 2 antibodies _DAre equal.

Table 10: binding to WI-38 cells

Antibody variants	K_D(nM)	Bmax
			v506	Not determined	About 366
v5019	7.0	380
			v7091	8.3	371
v10000	8.4	418

These results show that the exemplary biparatopic anti-HER 2 antibody can bind to HER 21 +, 2+, and 3+ tumor cells to levels approximately 1.5 to 1.6 fold higher than anti-HER 2 monospecific FSA, and the exemplary biparatopic anti-HER 2 antibody can bind to HER 21 +, 2+, and 3+ tumor cells to levels equivalent to the combination of two unique monospecific anti-HER 2 FSAs with different epitope specificities. These results also show that the biparatopic anti-HER 2 antibody (i.e. compared to the monospecific anti-HER 2 antibody v 506) did not show increased binding to basal HER2 expressing cells expressing about 10,000 HER2 receptors per cell or less, and that the threshold for increased cell surface binding to the biparatopic anti-HER 2 antibody occurred at HER2 receptor levels of about >10,000 receptors per cell. Based on this data, it is expected that the cell surface binding of the exemplary biparatopic anti-HER 2 antibody to HER 23 +, 2+, and 1+ tumor cells will increase, but not to non-tumor cells expressing HER2 receptors at basal levels of about 10,000 receptors or less.

Example 7: the ability of biparatopic anti-HER 2 antibodies to inhibit the growth of HER2+ cells

The ability of an exemplary biparatopic anti-HER 2 antibody to inhibit the growth of cells expressing HER2 at 3+ and 2+ levels was measured. Experiments were performed in HER 23 + cell lines BT-474, SKBr3, SKOV3 and HER 22 + JIMT-1. Biparatopic anti-HER 2 antibodies v5019, v7091 and v10000 were tested. The ability of the biparatopic anti-HER 2 antibody to inhibit BT-474 cells (200nM antibody), SKOV3, SKBr3, and JIMT-1 cells (300nM antibody) was measured as follows.

Test antibodies were diluted in culture medium and added to cells at 10 μ l/well in triplicate. Will boardIncubate at 37 ℃ for 3 days. Using AlamarBlue^TM(Biosource # dal1100) orCell viability was measured and absorbance read according to the manufacturer's instructions. Data were normalized to untreated controls and analyzed in GraphPad prism.

The growth inhibition results are shown in FIGS. 7A-E. A summary of the results is provided in tables 11A and 11B. The results of figures 7A-B and table 11A indicate that an exemplary anti-HER 2 biparatopic antibody (v5019) is able to inhibit the growth of HER2+ SKOV3 and BT-474 cell lines. Figure 10A shows that the anti-HER 2 biparatopic antibody mediates the strongest growth inhibition of SKOV3 when compared to anti-HER 2FSA (v506) and when compared to the combination of two anti-HER 2FSA antibodies (v506+ v 4184).

Table 11A: growth inhibition of HER 23 + cancer cells

The results in figures 7C-E and table 11B indicate that exemplary anti-HER 2 biparatopic antibodies (v5019, v7091, and v10000) can inhibit the growth of HER 23 + SKBR3, HER 22 +/3+ SKOV3, and HER 22 + JIMT-1 tumor cell lines. Figure 7C shows that anti-HER 2 biparatopic antibodies v7091 and v10000 mediate the strongest growth inhibition on HER 23 + SKBr3 breast tumor cells. Figure 7D shows that anti-HER 2 biparatopic antibodies (v7091 and v10000) mediate the strongest growth inhibition of HER 23 + SKOV3 ovarian tumor cells. Figure 7E shows that anti-HER 2 biparatopic antibodies (v7091 and v10000) mediate the strongest growth inhibition of HER 22 + Herceptin (Herceptin) -resistant JIMT-1 tumor cells. The exemplary anti-HER 2 biparatopic antibodies (v7091 and v10000) mediated stronger growth inhibition than the anti-HER 2FSA monospecific antibody (v506) in all cell lines tested.

Table 11B: growth inhibition of HER 23 + cancer cells

These results show that exemplary saturating concentrations of biparatopic anti-HER 2 antibody can inhibit growth of HER 23 + and 2+ breast and ovarian tumor cells and HER 22 + trastuzumab-resistant tumor cells by about 20% more than FSA anti-HER 2 monospecific antibody.

Example 8: paratope of the biparatopic anti-HER 2 antibody preferentially binds dimeric HER2 compared to HER2ECD

This experiment was performed to determine the ability of the paratope alone of the exemplary biparatopic anti-HER 2 antibody to bind to dimeric HER2 and HER2ECD as a surrogate for differential binding between membrane bound HER2(HER2-Fc) and shed HER2 ECD. The experiment was performed as follows.

Surface Plasmon Resonance (SPR) analysis: the affinity of binding of a monovalent anti-HER 2 antibody (v1040 or v4182) to HER2 extracellular domain (sHER-2, EbioscienceBMS362, _ which encodes amino acids 23-652 of the full-length protein) and HER2-Fc (dimeric HER2-Fc fusion, which encodes amino acids 1-652 of the extracellular domain; Sino Biological Inc., 10004-H02H) was measured by SPR using the T200 system from Biacore (GE healthcare). Binding to HER2ECD was determined by the following method. HER2ECD in 10mm Hepes (pH 6.8) was immobilized on CM5 chips by amine coupling up to a level of 44RU (reaction units). A monovalent anti-HER 2 antibody was passed over the surface of HER2 immobilized chip at a concentration ranging from 0.76-60 nM. Binding to HER2-Fc was determined by the following method. HER2-Fc in 10mm hepes (pH 6.8) was immobilized on a CM5 chip by amine coupling to a level of 43 RU. A monovalent anti-HER 2 antibody was passed over the surface of HER2 immobilized chip at a concentration ranging from 0.76-60 nM. Analysis was done in triplicate for the concentration of antibody bound. Determination of equilibrium dissociation association constant (K) using single cycle kinetic method _D) And kinetic parameters (ka and kd). The sensorgram (Sensogram) was globally fit to the 1:1Langmuir binding model. All experiments were performed at room temperature.

The results are shown in fig. 8A, fig. 8B, table 11C, and table 11D. The results in figure 8A and table 11C show SPR binding data for a monovalent anti-HER 2 antibody (v 1040; representing the antigen binding domain on CH-B of an exemplary anti-HER 2 biparatopic antibody). FIG. 8A illustrates K binding of v1040 to immobilized HER2ECD or HER2-Fc_DValues (nM) and shows that binding of a monovalent anti-HER 2 antibody to HER2-Fc has a lower K compared to HER2ECD_D. Table 11C shows ka (1/M s) and kd (1/s) values for monovalent anti-HER 2 antibody (OA) in binding to HER2ECD and HER2-FC ('HER 2 mem') compared to full-size anti-HER 2 antibody (FSA). The data show comparable binding (ka) and dissociation (kd) rates of OA and FSA binding to HER2ECD and HER 2-Fc.

Table 11C: monovalent anti-HER 2 antibody (OA) has ka (1/Ms) and kd (1/s) values in binding to HER2ECD and HER2-FC ('HER 2 mem') compared to full-size anti-HER 2 antibody (FSA)

	ka(1/Ms)	kd(1/s)
			OA and HER2ECD	2.00E+05	6.15E-05
FSA and HER2ECD	4.14F+05	2.01E-05
			OA and HER2mem	1.88E+05	4.38E-05
FSA and HER2mem	3.41E+05	4.94E-06*

The results in figure 8B and table 11D show SPR binding data for a monovalent anti-HER 2 antibody (v 4182; representing the antigen binding domain on CH-a of an exemplary anti-HER 2 biparatopic antibody). FIG. 8B illustrates K binding of v4182 to immobilized HER2ECD or HER2-Fc _DValues (nM) and shows that binding of a monovalent anti-HER 2 antibody to HER2-Fc has a lower K compared to HER2ECD_D. Table 11D shows ka (1/M s) and kd (1/s) values for monovalent anti-HER 2 antibody (OA) in binding to HER2ECD and HER2-FC ('HER 2 mem') compared to full-size anti-HER 2 antibody (FSA). The data show comparable binding (ka) and dissociation (kd) rates of OA and FSA binding to HER2ECD and HER 2-Fc.

Table 11D:

	ka(1/Ms)	kd(1/s)
			OA and HER2ECD	9.08E+04	6.17E-04
FSA and HER2ECD	9.55E+04	3.93E-04
			OA and HER2mem	1.39E+05	2.04E-04
FSA and HER2mem	1.77E+05	6.84E-05

These data show that each paratope of the exemplary anti-HER 2 biparatopic antibody binds to the dimeric HER2 antigen (representative of membrane bound HER 2) with a lower K compared to HER2ECD_D. Based on this data, it is expected that an exemplary anti-HER 2 antibody will have a higher binding affinity for membrane-bound HER2 antigen than shed HER2ECD present in the serum of a patient with the disease and available as a sink for therapeutic antibodies (Brodowicz T et al solvent HER-2/neu neutralizebiologicals effects of anti-HER-2/neu anti-antibody on Breast cancer cells in inner. int J cancer. 1997; 73: 875. 879). For example, a baseline HER2ECD level of ≦ 15 ng/mL; whereas patients with progressive disease have a HER2ECD of > 38 ng/mL.

Example 9: whole cell burden and internalization of biparatopic anti-HER 2 antibodies in HER2+ cells

This experiment was performed to evaluate the ability of an exemplary biparatopic anti-HER 2 antibody to be internalized in HER 22 + cells. The direct internalization method was followed according to the protocol detailed in Schmidt, M.et al, Kinetics of anticancer antibiotic intervention, effects of affinity, bivalincy, and stability. cancer Immunol Immunother (2008)57: 1879-. In particular, use is made ofThe 488 protein labeling kit (Invitrogen, cat. No. a10235), labels the antibodies directly according to the manufacturer's instructions.

For internalization assays, 1 × 10 was used⁵One cell/well was seeded in 12-well plates and incubated overnight at 37 ℃ + 5% CO 2. The next day, labeled antibody was added at 200nM in DMEM + 10% FBS and incubated at 37 ℃ + 5% CO2 for 24 hours. Under dark conditions, the medium was aspirated and the wells were washed with 2 × 500 μ L PBS. To harvest the cells, cell dissociation buffer was added (250 μ L) at 37 ℃. Cells were pelleted and resuspended in 100 μ L DMEM + 10% FBS without or with anti-Alexa Fluor 488, 50 μ g/mL rabbit IgG fraction (Molecular Probes, A11094) and incubated on ice for 30 min. Before analysis, 4. mu.l of propidium iodide was filtered through addition of 300. mu.L DMEM + 10% FBS. Samples were analyzed using a LSRII flow cytometer.

The ability of an exemplary anti-HER 2 biparatopic antibody to internalize in HER2+ cells is shown in fig. 9A and 9B. Figure 9A shows the results of detectable surface and internal antibodies in BT-474 cells after 24 hours incubation with exemplary anti-HER 2 biparatopic antibody and anti-HER 2FSA control. These results show that incubation with the exemplary anti-HER 2 biparatopic antibody (v5019) produced approximately 2-fold more internalizing antibody in BT-474 cells compared to the anti-HER 2FSA control. Figure 9B shows the results of detectable surface and internal antibodies in JIMT-1 cells after 24 hours incubation with exemplary anti-HER 2 biparatopic antibody and anti-HER 2FSA control. These results show that incubation with the exemplary anti-HER 2 biparatopic antibody (v5019) produced approximately 2-fold more internalizing antibody in JIMT-1 cells compared to the anti-HER 2FSA control. The amount of surface staining was comparable after 24 hours between the biparatopic anti-HER 2 and anti-HER 2FSA in BT-474 and JIMT-1 cells.

The results in FIGS. 10A-F show a comparison of detectable antibody bound to the surface of whole cells after 2 hours at 4 ℃ with antibody bound to the surface after 24 hours at 37 ℃; except for the amount of internalizing antibody after 24 hours at 37 ℃. Figure 10A shows results after incubation with exemplary anti-HER 2 biparatopic antibody and anti-HER 2FSA control in BT-474 cells. These results show that incubation of exemplary anti-HER 2 biparatopic antibody with BT-474 cells for 24 hours results in a 15% reduction in antibody detected on the surface of whole cells. Figure 10A also shows that upon incubation with the exemplary anti-HER 2 biparatopic antibody (v5019), approximately 2-fold more internalizing antibody was produced in BT-474 cells compared to the anti-HER 2FSA control.

Figure 10B shows the results after incubation with an exemplary anti-HER 2 biparatopic antibody and anti-HER 2FSA control in JIMT-1 cells. FIG. 10B is a repeat of the experiment shown in FIG. 9B, with an increase in surface staining after 2 hours at 4 ℃. These results show that incubation of the exemplary anti-HER 2 biparatopic antibody with JIMT-1 cells for 24 hours resulted in a 57% reduction in the antibody detected on the surface of whole cells. Figure 10B also shows that incubation with the exemplary anti-HER 2 biparatopic antibody (v5019) produced more internalizing antibody compared to the anti-HER 2FSA control after 24 hours of incubation in BT-474 cells at 37 ℃.

Figure 10C shows results after incubation with exemplary anti-HER 2 biparatopic antibodies in SKOV3 cells. These results show that incubation of the exemplary anti-HER 2 biparatopic antibody with SKOV3 cells for 24 hours resulted in approximately 32% reduction in the antibody detected on the surface of whole cells.

Figure 10D shows results after incubation with exemplary anti-HER 2 biparatopic antibody in MCF7 cells. These results show that incubation of the exemplary anti-HER 2 biparatopic antibody with MCF7 cells for 24 hours resulted in approximately 45% reduction in the antibody detected on the surface of whole cells.

Figure 10E shows the results after incubation with exemplary anti-HER 2 biparatopic antibodies v5019, v7091 and v10000 in SKOV3 cells. These results show that incubation of the exemplary anti-HER 2 biparatopic antibody with SKOV3 cells produced 1.5 to 1.8 fold more internalizing antibody compared to the anti-HER 2FSA control. Incubation with the anti-HER 2FSA control for 24 hours resulted in the most (about 77%) reduction of the antibody detected on the surface of the whole cells.

Fig. 10F shows the results after incubation with exemplary anti-HER 2 biparatopic antibodies v5019, v7091 and v10000 in JIMT-1 cells. These results show that incubation of the exemplary anti-HER 2 biparatopic antibody with JIMT-1 cells produced 1.4 to 1.8 fold more internalizing antibody compared to the anti-HER 2FSA control. Incubation with the anti-HER 2 biparatopic antibodies (v5019 and v10000) for 24 hours resulted in the greatest reduction (about 64%) of the antibodies detected on the surface of whole cells.

These results show that the exemplary anti-HER 2 biparatopic antibody has superior internalization properties in HER2+ cells compared to monospecific anti-HER 2 FSA. The reduction in surface antibody detected after 24 hours incubation at 37 ℃ indicates that the exemplary anti-HER 2 biparatopic antibody is able to reduce the amount of cell surface HER2 receptor after incubation in HER2+ cells and the surface HER2 is most reduced after incubation in HER22+ tumor cells.

Example 10: anti-HER 2 biparatopic antibodies cells at 1, 3 and 16 hours after incubation with HER2+ cells Dyeing and positioning

This experiment was performed to analyze internalization of exemplary anti-HER 2 biparatopic antibodies in HER2+ JIMT-1 cells at different time points and to analyze whole cell burden and internalization according to the orthogonal method of the method presented in example 9.

37 ℃ plus 5% CO in serum-free DMEM₂JIMT-1 cells were incubated with 200nM antibody (v506, v4184, v5019, or a combination of v506 and v4184) for 1 hour, 3 hours, and 16 hours. Cells were gently washed twice with warm sterile PBS (500 ml/well). Cells were fixed with 250ml of 10% formalin/PBS solution for 10min at room temperature. The fixed cells were washed three times with PBS (500. mu.l/well), permeabilized with 250. mu.l/well PBS containing 0.2% Triton X-100 for 5min, and washed three times with 500. mu.l/well PBS. Cells were blocked with 500. mu.l/well PBS + 5% goat serum for 1 hour at room temperature. Blocking buffer was removed and a 300. mu.l/well secondary antibody (Alexa Fluor 488-conjugated AffiniPure Fab fragment goat anti-human IgG (H + L); Jackson ImmunoResearch laboratories, Inc.; 109-. The cells were washed three times with 500. mu.l/well PBS and then the cover slips containing the fixed cells were fixed on slides using a Prolong gold anti-fade containing DAPI (Life technologies; # P36931). A single image was acquired at 60 x using olympus fv1000 confocal microscope.

The results indicate that the exemplary anti-HER 2 biparatopic antibody (v5019) internalized into JIMT-1 cells at 3 hours and was located mainly near the nucleus. Comparison of images at 3 hours of incubation showed a greater amount of internal staining associated with the anti-HER 2 biparatopic antibody compared to the combination of the two anti-HER 2 FSAs (v506+ v4184) and compared to the anti-HER 2FSA alone (v506 or v 4184). A difference in the cellular localization of antibody staining was seen when the anti-HER 2 biparatopic antibody (v5019) results were compared to anti-HER 2FSA (v 4184); of these, anti-HER 2FSA (v4184) showed significant plasma membrane staining at 1, 3 and 16 hour time points. For the anti-HER 2FSA (v506), the combination of two anti-HER 2 FSAs (v506+ v4184) and the anti-HER 2 biparatopic antibody treatment (data not shown), the amount of detectable antibody decreased at 16 hours.

These results show that the exemplary anti-HER 2 biparatopic antibody v5019 internalizes in HER2+ cells and that the internalized antibody is detectable after 3 hours incubation. These results are consistent with the results presented in example 9, showing that the exemplary anti-HER 2 biparatopic antibody can be internalized to a greater amount in HER2+ cells compared to anti-HER 2 FSA.

Example 11: biparatopic anti-HER 2 antibody mediated ADCC of HER2+ cells compared to controls

This experiment was performed to measure the ability of an exemplary biparatopic anti-HER 2 antibody to mediate ADCC in SKOV3 cells (ovarian cancer, HER 22 +/3 +).

Target cells were preincubated with test antibody (10-fold decreasing concentration starting from 45. mu.g/ml) for 30 minutes, followed by addition of effector cells at an effector cell/target cell ratio of 5:1 and 37 ℃ + 5% CO₂Incubation was continued for 6 hours. Samples were tested with 8 concentrations starting at 45. mu.g/ml with a 10-fold decrease. LDH release was measured using an LDH assay kit.

Dose-response studies were performed with samples at different concentrations, with effector/target (E/T) ratios of 5:1, 3:1, and 1: 1. Nonlinear regression simulation with sigmoidal dose response Using GraphPad prismAnalysis of half maximal Effective Concentration (EC)₅₀) The value is obtained.

Cells were maintained at 37 ℃/5% CO ₂McCoy 5a below was in complete medium and periodically subcultured according to the protocol from ATCC with suitable medium supplemented with 10% FBS. Cells with a passage number less than p10 were used for the assay. Samples were diluted to a concentration between 0.3-300nM with phenol red free DMEM media supplemented with 1% FBS and 1% penicillin/streptomycin before use in the assay.

ADCC results in HER2+ SKOV3 cells at an effector/target cell ratio of 5:1 are shown in figure 11A and table 12. These results show that the exemplary biparatopic anti-HER 2 antibody (v5019) mediated a maximal percentage of maximal target cell lysis by ADCC when compared to anti-HER 2FSA (v792) and a combination of two different anti-HER 2 FSAs (v792+ v 4184). The difference in maximal cell lysis mediated by the exemplary biparatopic anti-HER 2 antibody was approximately 1.6 fold higher compared to anti-HER 2FSA and approximately 1.2 fold higher compared to the combination of two different anti-HER 2 FSAs (v792+ v 4184).

Table 12:

ADCC results in HER2+ SKOV3 cells at an effector/target cell ratio of 3:1 are shown in figure 11B and table 13. These results show that the exemplary biparatopic anti-HER 2 antibody (v5019) mediated a maximal percentage of maximal target cell lysis by ADCC when compared to anti-HER 2FSA (v792) and a combination of two different anti-HER 2 FSAs (v792+ v 4184). The difference in maximal cell lysis mediated by the exemplary biparatopic anti-HER 2 antibody was approximately 1.3 fold higher than anti-HER 2FSA and approximately 1.8 fold higher than the combination of two different anti-HER 2 FSAs (v792+ v 4184).

Table 13:

ADCC results in HER2+ SKOV3 cells at an effector/target cell ratio of 1:1 are shown in figure 11C and table 14. These results show that the exemplary biparatopic anti-HER 2 antibody (v5019) mediated a maximal percentage of maximal target cell lysis by ADCC when compared to anti-HER 2FSA (v792) and a combination of two different anti-HER 2 FSAs (v792+ v 4184). The difference in maximal cell lysis mediated by the exemplary biparatopic anti-HER 2 antibody is approximately 1.8 fold higher compared to anti-HER 2FSA and approximately 1.13 fold higher compared to the combination of two different anti-HER 2 FSAs (v792+ v 4184).

Table 14:

the results in figure 11 and tables 12-14 show that the exemplary biparatopic HER2 antibodies all mediated the strongest ADCC of SKOV3 cells at different E: T ratios when compared to anti-HER 2FSA and the combination of two anti-HER 2 FSAs. An increase in anti-HER 2 biparatopic antibody mediated ADCC will be expected in HER2+ diseased patients expressing variable and/or reduced circulating effector cells following chemotherapy (Suzuki E. et al Clin Cancer Res 2007; 13: 1875-. The observations in figure 11 are consistent with the whole cell binding Bmax presented in example 6, which shows an approximately 1.5-fold increase in cell binding with the exemplary anti-HER 2 biparatopic antibody compared to anti-HER 2 FSA.

Example 12: ability of exemplary anti-HER 2 antibodies to bind to HER2ECD

SPR assay to evaluate the mechanism of binding of an exemplary anti-HER 2 biparatopic antibody to HER2 ECD; in particular, it was used to understand whether two paratopes of a biparatopic antibody molecule can bind to one HER2ECD (cis binding; 1:1 antibody: HER2 molecule) or whether each paratope of a biparatopic antibody can bind to two different HER2 ECDs (trans binding; 1:2 antibody: HER2 molecule). A schematic of cis and trans binding is illustrated in fig. 14. A correlation between a decrease in separation rate (slower) and an increase in antibody capture level (surface density) indicates trans-binding (i.e. one antibody molecule binds to two HER2 molecules).

The affinity and binding kinetics parameters of an exemplary biparatopic anti-HER 2 antibody (v5019) and recombinant human HER2 were measured by SPR using the T200 system from biacore (ge healthcare) and compared to the affinity and binding kinetics parameters of a monovalent anti-HER 2 antibody (v630 or v 4182; comprising the individual paratope of v 5019). Anti-human Fc injected at concentrations between 5 and 10. mu.g/ml between 2000 and 4000RU was immobilized on CM5 chips using standard amine coupling. Monovalent anti-HER 2 antibody (v630 or v4182) and exemplary biparatopic anti-HER 2 antibody (v5019) were captured on anti-human Fc (injected at 10 μ l/min for 1 min at concentrations ranging from 0.08 to 8 μ g/ml in PBST) at response levels ranging from 350-15 RU. Recombinant human HER2 was diluted in PBST and 3-fold injected at initial concentrations of 120nM, 200nM or 300nM and injected at a flow rate of 50 μ Ι/min for 3 minutes followed by 30 minutes of dissociation at the end of the last injection. Dilutions of HER2 were analyzed in duplicate. The sensorgram was globally fit to the 1:1Langmuir binding model. All experiments were performed at 25 ℃.

The results are shown in fig. 12 and 13.

The results in figure 12A show ka (1/Ms) of binding of monovalent anti-HER 2(v630 and v4182) and exemplary biparatopic anti-HER 2 antibody (v5019) to recombinant human HER2 on the chip surface over a range of injection and capture antibody concentrations. These results show that ka does not change at different antibody capture levels for v630, v4182 and v 5019.

The results in figure 12B show kd (1/s) for binding of monovalent anti-HER 2(v630 and v4182) and an exemplary biparatopic anti-HER 2 antibody (v5019) to recombinant human HER2 on the chip surface over a range of injection and capture antibody concentrations. These results show a reduction in kd at increasing antibody capture levels only for the exemplary anti-HER 2 biparatopic antibody (v 5019).

The results in figure 12C show the K binding of monovalent anti-HER 2(v630 and v4182) and exemplary biparatopic anti-HER 2 antibody (v5019) to recombinant human HER2 on the chip surface over a range of injection and capture antibody concentrations_D(M). These results show that K at increasing antibody capture levels only for the exemplary anti-HER 2 biparatopic antibody (v5019)_DAnd decreases. This result correlates to the decreasing kd values shown in fig. 15B.

The results in figure 13A show kd (1/s) binding of an exemplary biparatopic anti-HER 2 antibody (v5019) to recombinant human HER2 over a range of antibody capture levels. These results show that kd values are inversely proportional to the higher RU of the captured antibody on the chip surface (i.e. the slower the separation rate at higher antibody capture levels). The results indicate that the exemplary biparatopic anti-HER 2 antibody (v5019) was able to bind to HER2ECD2 and HER2ECD4 on two independent HER2 molecules (i.e. trans binding), as demonstrated by the decreased separation rate at higher antibody capture levels. This data is supported by a similar experiment presented in figure 47 and discussed in example 43, where a bivalent monospecific anti-HER 2FSA (v506) displays cis binding (1:1 antibody: HER2), where kd (1/s) and K are as expected for this molecule _DThe (M) value remained constant at increasing antibody capture levels.

The results in figure 13B show kd (1/s) binding of a monovalent anti-HER 2 antibody (v4182) to recombinant human HER2 over a range of antibody capture levels. These results show no change in kd values over the range of RU for different antibodies captured on the chip surface. These results show that the monovalent anti-HER 2 antibody (v4182) binds 1:1 monovalent (cis-binding).

The results in figure 13C show kd (1/s) for binding of a monovalent anti-HER 2 antibody (v630) to recombinant human HER2 over a range of antibody capture levels. These results show no change in kd values over the range of RU for different antibodies captured on the chip surface. These results show monovalent anti-HER 2 antibody (v630)1:1 monovalent binding (cis binding). This data is supported by the experiments presented in figure 47 and discussed in example 43X, where bivalent monospecific anti-HER 2FSA (v506) showed no change in kd (1/s).

The results in figures 12 and 13 show that the exemplary biparatopic anti-HER 2 antibody (v5019) is capable of simultaneously binding to two HER2 molecules in trans (antibody: HER2 ratio 1: 2). The mechanism of trans-binding detected by SPR is consistent with the higher cell surface saturation binding data (Bmax) presented in example 6, along with the internalization data presented in examples 9 and 10.

Example 13: effect of exemplary biparatopic anti-HER 2 antibody incubation on AKT phosphorylation in BT-474 cells

The ability of an exemplary anti-HER 2 biparatopic antibody to reduce pAKT signaling In BT-474 cells was tested using the AKT Colorimetric In-Cell ELISA kit (Thermo scientific; catalog No. 62215) with the following modifications according to the manufacturer's instructions. Press 5x10³Cells were seeded per well and + 5% CO at 37 ℃ +₂Following incubation for 24 hours cells were incubated with 100nM antibody for 30min followed by incubation with rhHRG- β 1 for 15min, cells were washed, fixed and permeabilized according to the instructions secondary antibodies (1: 5000; Jackson ImmunoReasearch, HRP-donkey anti-mouse IgG, JIR, Cat # 715-.

The results in figure 15 show that incubation with the exemplary anti-HER 2 biparatopic antibody mediates an approximately 1.2-fold reduction in p-Akt levels relative to human IgG Control (CTL) in the presence of HRG β 1. The combination of the two anti-HER 2 FSAs (v506+ v4184) mediated the most reduction in p-Akt levels in the presence of HRG β 1, approximately 1.5-fold lower compared to human IgG controls. A modest decrease in p-Akt was detected in the absence of ligand (HRG β 1) using the exemplary anti-HER 2 biparatopic antibody compared to the human IgG control antibody.

These data show that an exemplary anti-HER 2 biparatopic antibody can block signaling of ligand activation in HER2+ cells.

Example 14: double complementationEffect of anti-HER 2 antibody on myocardial cell viability

Measuring the effect of exemplary biparatopic anti-HER 2 antibodies and ADCs on cardiomyocyte viability is to obtain a preliminary indication of potential cardiotoxic effects.

iCell cardiomyocytes (CMC-100-010) expressing basal levels of HER2 receptor were grown according to the manufacturer's instructions and used as target cells to assess cardiomyocyte health following antibody treatment. The measurement was performed as follows. Cells were seeded in 96-well plates (15,000 cells/well) and maintained for 48 hours. The cell culture medium was changed to maintenance medium and the cells were maintained for 72 hours. To evaluate the effect of antibody-induced cardiotoxicity, cells were treated with 10 and 100nM of the individual or combination variants for 72 hours. To assess the effects of anthracycline-induced cardiotoxicity (alone or in combination with an exemplary biparatopic anti-HER 2 antibody), 3uM (ca. IC) was used₂₀) Cells were treated with doxorubicin for 1 hour, followed by treatment with 10 and 100nM of the individual or combination variants for 72 hours. By usingCell viability was assessed by quantifying cellular ATP levels using a luminescent cell viability assay (Promega, G7570) and/or sulforhodamine (Sigma 230162-5G) according to the manufacturer's instructions.

The results are shown in FIGS. 16A-C. The results in figure 16A show that incubation of cardiomyocytes with therapeutically relevant concentrations of the exemplary anti-HER 2 biparatopic antibody (v5019) and the exemplary anti-HER 2 biparatopic ADC (v6363) did not affect cardiomyocyte viability relative to untreated controls ('mock').

The results in figure 16B show that incubation of cardiomyocytes with therapeutically relevant concentrations of the exemplary anti-HER 2 biparatopic antibodies (v5019, v7091 and v10000) and the exemplary anti-HER 2 biparatopic ADCs (v6363, v7148 and v10553) had no effect on cardiomyocyte viability relative to untreated controls ('mock'). Based on the results in FIGS. 16A and 16B, it is expected that the exemplary anti-HER 2 biparatopic antibody and the exemplary anti-HER 2 biparatopic ADC should not induce cardiomyopathy, e.g., by Mitochondrial Dysfunction, as reported with other anti-HER 2 targeting antibodies (Grazette L.P. et al Inhibition of ErbB2 mice Mitochon fatty functions in Cardiomyces; Journal of the American College of medicine: 2004; 44: 11).

The results in figure 16C show that pretreatment of cardiomyocytes with doxorubicin, followed by incubation with therapeutically relevant concentrations of exemplary anti-HER 2 biparatopic antibodies (v5019, v7091, and v10000) and exemplary anti-HER 2 biparatopic ADCs (v6363, v7148, and v10553) had no effect on cardiomyocyte viability relative to the untreated control + doxorubicin ('Mock + doxorubicin'). Based on the results in FIG. 16C, it is expected that the exemplary anti-HER 2 biparatopic antibody and the exemplary anti-HER 2 biparatopic ADC should not cause an increased risk of Cardiac dysfunction in patients receiving simultaneous treatment with anthracyclines (Seidman A, Hudis C, Pierri MK, etc. cardio dysfunction in the trastuzumab clinical trialisexperience. J Clin Oncol (2002)20: 1215-.

Figures 16A-C show that incubation of cardiomyocytes with the anti-HER 2 biparatopic antibody and ADC had equivalent effects when treated alone or in combination with doxorubicin compared to the monospecific anti-HER 2FSA antibody (v506), anti-HER 2FSA antibody combination (v506+ v4184) and ADC (v 6246). Based on these results, the exemplary anti-HER 2 biparatopic antibody and ADC are not expected to have a greater cardiotoxic effect than the anti-monospecific anti-HER 2FSA, trastuzumab or ADC, T-DM 1.

Example 15: exemplary Biparatopic anti-HER 2-ADC cytotoxicity in HER2+ cells

The ability of exemplary biparatopic anti-HER 2-ADC antibodies (v6363, v7148 and v10553) to mediate cytotoxicity in HER2+ cells was measured. Human IgG conjugated to DM1 (v6249) was used as a control in some cases. Experiments were carried out in HER2+ breast tumor cell line JIMT-1, MCF7, MDA-MB-231, HER2+ ovarian tumor cell line SKOV3 and HER2+ gastric cell line NCI-N87. Evaluation of cytotoxicity of exemplary biparatopic anti-HER 2-ADC antibodies in HER2+ cellsAnd compared to monospecific anti-HER 2FSA-ADC (v6246) and anti-HER 2-FSA-ADC + anti-HER 2-FSA controls (v6246+ v 4184). The method was performed as described in example 7, with the following modifications. anti-HER 2 ADCs were incubated with SKOV3 and JIMT-1 (fig. 17A and B) target cells for 24 hours, cells were washed, medium was changed and cell survival was assessed after 5 days incubation at 37 ℃. anti-HER 2ADC was incubated with target MCF7 and MDA-MB-231 target cells for 6 hours (fig. 17C and D), cells were washed, medium was changed and cell survival was assessed after 5 days incubation at 37 ℃. In FIGS. 17E-G, anti-HER 2 ADCs were incubated for 5 days simultaneously with SKOV3, JIMT-1, NCI-N87 target cells. AlamarBlue was used as described in example 7 ^TM(FIGS. 17A-D) or(FIGS. 17E-G) cell viability was measured.

The results are shown in FIGS. 17A-G and the data are summarized in tables 15 and 16.

The results in figure 17A and tables 15 and 16 show that the exemplary anti-HER 2 biparatopic ADC (v6363) is more cytotoxic in JIMT-1 than the combination of anti-HER 2-FSA-ADC (v6246) and anti-HER 2-FSA-ADC + anti-HER 2-FSA (v6246+ v 4184). The exemplary anti-HER 2 double-paratope ADC had an approximately 13-fold lower superior EC than the anti-HER 2-FSA-ADC control₅₀。

The results in figure 17B and table 15 show that the exemplary anti-HER 2 biparatopic ADC (v6363) is more cytotoxic in SKOV3 than the combination of anti-HER 2-FSA-ADC (v6246) and anti-HER 2-FSA-ADC + anti-HER 2-FSA (v6246+ v 4184). The exemplary anti-HER 2 double-paratope ADC had an approximately 5-fold lower superior EC than the anti-HER 2-FSA-ADC control₅₀。

The results in figure 17C and table 15 show that the exemplary anti-HER 2 biparatopic ADC (v6363) is more cytotoxic in MCF7 than the combination of anti-HER 2-FSA-ADC (v6246) and anti-HER 2-FSA-ADC + anti-HER 2-FSA (v6246+ v 4184). The exemplary anti-HER 2 double-paratope ADC had an approximately 2-fold lower superior EC than the anti-HER 2-FSA-ADC control₅₀。

FIG. 17D and the junction in Table 15The results show that the exemplary anti-HER 2 biparatopic ADC (v6363) is more cytotoxic in MDA-MB-231 than the combination of anti-HER 2-FSA-ADC (v6246) and anti-HER 2-FSA-ADC + anti-HER 2-FSA (v6246+ v 4184). The exemplary anti-HER 2 double-paratope ADC had an approximately 2-fold lower superior EC than the anti-HER 2-FSA-ADC control ₅₀。

Table 15:

the results in figure 17E and table 16 show that the exemplary anti-HER 2 biparatopic ADCs (v6363, v7148 and v10553) are more cytotoxic in SKOV3 ovarian tumor cells than the anti-HER 2-FSA-ADC (v 6246). The exemplary anti-HER 2 double-paratope ADC had a superior EC that was approximately 2 to 7-fold lower than the anti-HER 2-FSA-ADC control₅₀The value is obtained.

The results in figure 17F and table 16 show that the exemplary anti-HER 2 biparatopic ADCs (v6363, v7148 and v10553) are more cytotoxic in JIMT-1 breast tumor cells than the anti-HER 2-FSA-ADC (v 6246). The exemplary anti-HER 2 double-paratope ADC had a superior EC that was approximately 6 to 9-fold lower than the anti-HER 2-FSA-ADC control₅₀The value is obtained.

The results in figure 17G and table 16 show that the exemplary anti-HER 2 biparatopic ADCs (v6363, v7148 and v10553) are cytotoxic in NCI-N87 gastric tumor cells. An exemplary anti-HER 2 double-paratope ADC has an approximately equal EC compared to an anti-HER 2-FSA-ADC control₅₀The value is obtained.

Table 16:

these results show that the exemplary anti-HER 2 biparatopic ADCs (v6363, v7148 and v10553) are more cytotoxic in HER 23 +, 2+ and 1+ breast tumor cells than the anti-HER 2-FSA-ADC control. These results also show that the exemplary anti-HER 2 biparatopic ADCs (v6363, v7148 and v10553) are cytotoxic in HER 22/3 + gastric tumor cells. These results are consistent with the internalization results presented in example 9.

Example 16: effect of biparatopic anti-HER 2 antibodies in human ovarian cancer cell xenograft models

The anti-tumor efficacy of the exemplary biparatopic anti-HER 2 antibodies was evaluated using an established human ovarian cancer cell-derived xenograft model SKOV 3.

Female athymic nude mice were inserted 1mm subcutaneously³Tumor fragments were inoculated with tumors. Tumor was monitored until it reached 220mm³Average volume of (d); animals were then randomized into 3 treatment groups: IgG control, anti-HER 2FSA (v506) and biparatopic anti-HER 2 antibody (v 5019).

Each group included 15 animals. The dose for each group was as follows:

A) IgG controls were administered intravenously at a loading dose of 30mg/kg on study day 1, followed by a maintenance dose of 20mg/kg twice weekly until study day 39.

B) anti-HER 2FSA (v506) was administered intravenously at a loading dose of 15mg/kg on study day 1, followed by a maintenance dose of 10mg/kg twice weekly until study day 18. On days 22 to 39, 5mg/kg of anti-HER 2FSA was administered intravenously twice weekly. anti-HER 2FSA (v4184) was also administered intraperitoneally at 5mg/kg twice weekly.

C) The biparatopic anti-HER 2 antibody was administered intravenously at a loading dose of 15mg/kg on study day 1, followed by a maintenance dose of 10mg/kg twice weekly until study day 39.

Tumor volumes were measured twice weekly during the study, and response numbers and median survival were assessed on day 22. The results are shown in fig. 18 and table 17.

Biparatopic anti-HER 2 and anti-HER 2FSA showed superior tumor growth inhibition compared to IgG controls. The biparatopic anti-HER 2 antibody induced superior tumor growth inhibition compared to the anti-HER 2FSA combination (fig. 18A). Biparatopic anti-HER 2 antibody was associated with an increase in response tumor numbers (11 and 5, respectively) compared to anti-HER 2FSA v506 on day 22 (table 17). The exemplary biparatopic anti-HER 2 antibody and anti-HER 2FSA exhibited superior survival compared to IgG controls. The biparatopic anti-HER 2 antibody had superior median survival (61 days) compared to anti-HER 2FSA (36 days) (fig. 18B and table 17). A second anti-HER 2FSA (v4184) was added in combination with anti-HER 2FSA (v506) on study day 22. The combination of the two anti-HER 2 FSAs induced further tumor growth inhibition compared to the anti-HER 2FSA alone (v 506).

Table 17:

example 17: double paratope anti-HER 2 Antibody Drug Conjugate (ADC) in human ovarian cancer cell line xenograft mould Action in type

The anti-tumor efficacy of an exemplary biparatopic anti-HER 2 antibody conjugated to DM1 (v6363) was evaluated using an established human ovarian cancer cell-derived xenograft model SKOV 3.

Female athymic nude mice were inserted 1mm subcutaneously³Tumor fragments were inoculated with tumors. Tumor was monitored until it reached 220mm³Average volume of (d); animals were then randomized into 3 treatment groups: IgG control, anti-HER 2FSA-ADC and biparatopic anti-HER 2-ADC.

Each group included 15 animals. The dose for each group was as follows:

B) anti-HER 2FSA-ADC (v6246) was administered intravenously at a loading dose of 10mg/kg on study day 1, followed by a maintenance dose of 5mg/kg on days 15 and 29.

C) The biparatopic anti-HER 2 antibody-ADC (v6363) was administered intravenously at a loading dose of 10mg/kg on study day 1, followed by a maintenance dose of 5mg/kg on days 15 and 29.

Tumor volumes were measured throughout the study and response numbers and median survival were assessed on day 22. The results are shown in FIG. 19. A summary of the results is shown in table 18.

Biparatopic anti-HER 2-ADC and anti-HER 2FSA-ADC inhibited tumor growth better than IgG controls (FIG. 19A and Table 18). The biparatopic anti-HER 2-ADC inhibits tumor growth to a greater extent than the anti-HER 2 FSA-ADC. The biparatopic anti-HER 2-ADC group was associated with an increase in response tumor numbers (11 and 9, respectively) compared to the anti-HER 2FSA-ADC group. The biparatopic anti-HER 2-ADC and anti-HER 2FSA-ADC groups exhibited superior survival compared to the IgG control (FIG. 19B and Table 18). The biparatopic anti-HER 2 antibody panel exhibited a median survival of 61 days compared to anti-HER 2FSA-ADC with a median survival of 36 days (fig. 19B and table 18).

Table 18:

example 18: biparatopic anti-HER 2 Antibody Drug Conjugate (ADC) in human primary cell xenograft model (HBCx-13b)

The anti-tumor efficacy of an exemplary biparatopic anti-HER 2 antibody conjugated to DM1 was evaluated using a trastuzumab-resistant patient-derived xenograft model HBCx-13B from human breast cancer.

Female athymic nude mice were inserted 20mm subcutaneously³Tumor fragments were inoculated with tumors. Tumor was monitored until it reached 100mm³Average volume of (d); animals were then randomized into 3 treatment groups: anti-HER 2FSA (v506), anti-HER 2FSA-ADC (v6246) anddouble-paratope anti-HER 2-ADC (v 6363). Each group included 7 animals. The dose for each group was as follows:

A) anti-HER 2FSA was administered intravenously at a loading dose of 15mg/kg on study day 1, followed by a maintenance dose of 10mg/kg on study days 4, 8, 11, 15, 18, 22 and 25.

B) anti-HER 2FSA-ADC was administered intravenously at a loading dose of 10mg/kg on study day 1, followed by a maintenance dose of 5mg/kg on day 22.

C) The biparatopic anti-HER 2 antibody-ADC was administered intravenously at a loading dose of 10mg/kg on study day 1, followed by a maintenance dose of 5mg/kg on day 22.

Tumor volumes were measured throughout the study period and mean tumor volumes, complete response and zero residual disease parameters were assessed on day 50. The results are shown in fig. 20. A summary of the results is shown in table 19.

The biparatopic anti-HER 2-ADC and anti-HER 2FSA-ADC showed stronger tumor growth inhibition than anti-HER 2FSA (v 506). The biparatopic anti-HER 2-ADC inhibits tumor growth better than the anti-HER 2 FSA-ADC. The biparatopic anti-HER 2-ADC group was accompanied by an increase in tumor numbers showing a complete response (more than 10% reduction from baseline) (7 and 4, respectively) and zero residual disease (5 and 2, respectively) compared to the anti-HER 2FSA-ADC group.

Table 19:

example 19: biparatopic anti-HER 2 Antibody Drug Conjugate (ADC) in human primary cell xenograft model (T226) Effect

The antitumor efficacy of an exemplary biparatopic anti-HER 2-ADC was evaluated using a patient-derived trastuzumab-resistant xenograft model T226 from human breast cancer.

Female athymic nude mice were inserted 20mm subcutaneously³Tumor fragments were inoculated with tumors. Tumor was monitored until it reached 100mm³Average volume of (d); animals were then randomized into 4 treatment groups: IgG controls (n ═ 15), anti-HER 2FSA (v 506; n ═ 15), anti-HER 2FSA-ADC (v 6246; n ═ 16) and biparatopic anti-HER 2-ADC conjugates (v 6363; n ═ 16). The dose for each group was as follows:

A) IgG controls were administered intravenously at a loading dose of 15mg/kg on study day 1, and a maintenance dose of 10mg/kg was administered on study days 4, 8, 11, 15, 18, 22, and 25.

B) anti-HER 2FSA was administered intravenously at a loading dose of 15mg/kg on study day 1 and a maintenance dose of 10mg/kg was administered on study days 4, 8, 11, 15, 18, 22 and 25.

C) anti-HER 2FSA-ADC was administered intravenously at 5mg/kg on study days 1 and 15.

D) The biparatopic anti-HER 2-ADC conjugate was administered intravenously at 5mg/kg on study days 1 and 15.

Tumor volumes were measured throughout the study period and mean tumor volumes and complete response parameters were assessed on day 31. The results are shown in FIG. 21. A summary of the results is shown in table 20.

The biparatopic anti-HER 2-ADC and anti-HER 2FSA-ADC showed better tumor growth inhibition compared to anti-HER 2FSA (v506) and IgG controls. The exemplary biparatopic anti-HER 2-ADC induced equivalent tumor growth inhibition and complete baseline regression compared to the anti-HER 2FSA-ADC in this model (figure 21 and table 20).

Table 20:

example 20: biparatopic anti-HER 2 antibody drug conjugates(ADC) model of human primary cell xenograft (HBCx-5) Effect

The anti-tumor efficacy of exemplary biparatopic anti-HER 2-ADC was evaluated using a patient-derived trastuzumab-resistant xenograft model HBCx-5 (invasive ductal carcinoma, luminal B) from human breast cancer.

Female athymic nude mice were inserted 20mm subcutaneously³Tumor fragments were inoculated with tumors. Tumor was monitored until it reached 100mm³Average volume of (d); animals were then randomized into 4 treatment groups: IgG controls (n ═ 15), anti-HER 2FSA (v 506; n ═ 15), anti-HER 2FSA-ADC (v 6246; n ═ 16) and biparate anti-HER 2-ADC (v 6363; n ═ 16). The dose for each group was as follows:

C) anti-HER 2FSA-ADC was administered intravenously at 10mg/kg on study days 1 and 15, 22, 29, 36.

D) The biparatopic anti-HER 2-ADC was administered intravenously at 10mg/kg on study days 1 and 15, 22, 29, 36.

Tumor volumes were measured throughout the study and mean tumor volume, T/C ratio, number of responses, complete response and zero residual disease parameters were assessed on day 43. The results are shown in FIG. 22. A summary of the results is shown in table 21.

The biparatopic anti-HER 2-ADC and anti-HER 2FSA-ADC showed better tumor growth inhibition compared to anti-HER 2FSA (v506) and IgG controls. The exemplary biparatopic anti-HER 2-ADC compared to anti-HER 2FSA-ADC (figure 22 and table 21) induced equivalent tumor growth inhibition and had an increased number of responses in the trastuzumab-resistant HBCx-5 human breast cancer xenograft model.

Table 21:

example 21: biparatopic anti-HER 2 antibody drug conjugates in human cell line xenograft model (SKOV3) (ADC) Effect against HER2 treatment resistant tumors

The anti-tumor efficacy of an exemplary biparatopic anti-HER 2-ADC in treating resistant tumors against HER2 was evaluated using the established human ovarian cancer cell-derived xenograft model described in example 17, SKOV 3.

Following the procedure as described in example 17, the following modifications were made. Animals in a group of 15mg/kg on study day 1 and 10mg/kg on days 4, 8, 15 were dosed intravenously with anti-HER 2 antibody; however, this treatment failed to demonstrate an effective response by day 15 in this model. The treatment group was then switched to treatment with the exemplary biparatopic anti-HER 2 antibody drug conjugate (v6363) and administered at 5mg/kg on study days 19 and 27 and 15mg/kg on study days 34, 41 and 48.

Tumor volumes were measured twice a week throughout the experiment.

The results are shown in figure 23 and indicate that the group treated with exemplary biparatopic anti-HER 2-ADC (v6363) showed tumor regression to less than 220mm³Average tumor volume of the initial average volume of (a).

Example 22: biparatopic anti-HER 2 antibody drug couples in human primary cell xenograft model (HBCx-13b) Effect of conjugate (ADC) on treatment of tumors resistant to anti-HER 2

Following the procedure as described in example 18, the following modifications were made. Animals in a group of 15mg/kg on study day 1 and 10mg/kg on days 4, 8, 15, 18, 22 and 25 were dosed intravenously with the bispecific anti-ErbB family-targeting antibody; however, this treatment also failed to exhibit an effective reaction. The treatment group was then switched to treatment with the exemplary biparatopic anti-HER 2 antibody drug conjugate (v6363) and administered at 10mg/kg on days 31, 52 and 5mg/kg on day 45. Tumor volumes were measured throughout the duration of the study.

The results are shown in FIG. 24. These results show that the exemplary biparatopic anti-HER 2-ADC (v6363) prevents tumor progression. From the first dose to day 57, the tumor volume increased by less than 2% in the v 6363-treated group, while the growth was more than 110% in the v 506-treated group at the same time interval.

Example 23: analysis of fucose content of exemplary biparatopic anti-HER 2 antibodies

Glycopeptide analysis was performed to quantify the fucose content of N-linked glycans of exemplary biparatopic anti-HER 2 antibodies (v5019, v7091, and v 10000).

Glycopeptide analysis was performed as follows. Antibody samples were reduced with 10mM DTT for 1 hour at 56 ℃ and alkylated with 55mM iodoacetamide for 1 hour at room temperature and digested with trypsin solution in 50mM ammonium bicarbonate overnight at 37 ℃. Trypsin digestions were analyzed by nano LC-MS/MS on QTof-Ultima. NCBI databases were searched with Mascot to identify protein sequences. The glycopeptide ions were deconvoluted using MaxEnt3(MassLynx) and the different glycoforms were quantified.

The glycopeptide analysis results are summarized in table 22. The N-linked glycans of exemplary biparatopic anti-HER 2 antibodies (v5019, v7091, and v10000) are approximately 90% fucosylated (10% N-linked glycans are afucose). Monospecific anti-HER 2FSA (v506) N-linked glycans were approximately 96% fucosylated (4%N-linked glycan afucose) andapproximately 87% fucosylated (4% N-linked glycans are afucose).

Table 22: fc N-linked glycopeptide analysis

These results show that the biparatopic anti-HER 2 antibody (with heterodimeric Fc) transiently expressed in CHO cells is compatible with commercial productsHas a fucose content of about 3% higher than in N-glycans. Homodimeric anti-HER 2FSA (v506) transiently expressed in CHO cells had a maximum fucose content of about 96%.

Example 24: thermostability of exemplary biparatopic anti-HER 2 antibodies

The thermostability of exemplary biparatopic anti-HER 2 antibodies (v5019, v7091 and v10000) and ADCs (v6363, v7148 and v10533) was measured by DSC as described below.

In MicroCal^TMDSC was performed in VP-Capillary DSC (GE Healthcare) using pure protein samples (anti-HER 2 biparatopic antibody and anti-HER 2 biparatopic-ADC) adjusted to about 0.3mg/ml in PBS. The samples were scanned from 20 to 100 ℃ at a rate of 60 ℃/hour with low feedback, 8 second filtration, 5 minutes prettstat and 70psi nitrogen pressure. The resulting thermograms were analyzed using Origin7 software.

The thermostability results of exemplary biparatopic anti-HER 2 antibodies (v5019, v7091, and v10000) are shown in FIGS. 25A-C. Fig. 25A shows a thermogram (thermogram) of v 5019; fc and the respective A chain Fab have a T of 75 ℃_mWhereas the B chain scFv of 5019 had a T of 69 ℃_m. Fig. 25B shows a thermogram of v 10000; the Fc CH3 domain has a T of 82 DEG C_mFab A chain with a T of 76.5 ℃_mAnd the B chain scFv has a T of 69.5 ℃_m. Fig. 25C shows a thermogram of v 7091; the Fc CH3 domain has a T of 82 DEG C_mFab A chain with a T of 76.7 ℃_mAnd the B chain scFv has a T of 69.5 ℃_m。

The thermal stability results of exemplary biparatopic anti-HER 2 ADCs (v6363, v7148 and v10533) are shown in figures 26A-C. Fig. 26A shows a thermogram of v 6363; fc has a T of 75 ℃ _mAnd the A chain Fab and Fc CH3 domains have a T of 75 ℃_m. 6363 the B chain scFv has a T of 69 ℃_m. Fig. 26B shows a thermogram of v 10533; the Fc CH3 domain has a T of 83 DEG C_mThe A chain Fab has a T of 75.7 DEG C_mAnd the B chain scFv has a T of 66.2 ℃_m. Fig. 26C shows a thermogram of v 7148; the Fc CH3 domain has a T of 82.6 DEG C_mThe A chain Fab has a T of 74.8 DEG C_mAnd the B chain scFv has a T of 66.6 ℃_m。

Exemplary biparatopic antibodies and ADCs have thermal stability comparable to wild-type IgG.

Example 25: exemplary biparatopic anti-HER 2 antibodies cause breast tumor cells expressing different levels of HER2 ADCC capability of

The ability of the exemplary biparatopic antibody (v5019) to elicit dose-dependent ADCC of HER2 positive 3+, 2+ and 0/1+ HER2 expressing (triple negative) breast cancer cell lines was examined. ADCC experiments were performed as described in example 11, except that the NK effector cell to target cell ratio was kept constant at 5: 1.

ADCC results are shown in figure 27 and table 23. The results in FIGS. 27A-C show exemplary Biparatopic antibodies (v5019) withMaximal cell lysis approximately 1.2 to 1.3 times higher than breast cancer cells causing HER2 positive 3+, 2+, and 0/1+ to express HER 2. The results also show that v5019 (90% of N-glycans with fucose) was used with (86% of N-glycans have fucose; example 23) compared to that, despite having a fucose content in N-glycans of about 4% higher (resulting in a lower binding affinity to CD16 on NK cells), it mediates ADCC of HER2 positive 3+, 2+ and 0/1+ HER2 expressing breast cancer cells more effectively. The higher target cell killing by v5019 was presumably due to increased tumor cell modification as described in example 6.

Table 23: ADCC of HER 23 +, 2+ and 0/1+ HER2 expressing breast cancer cells

The ADCC results in figure 27D show exemplary biparatopic antibodies (v7091 and v10000) withCompared to the similar maximal extent of cell lysis induced in the WI-38 cell line expressing substrate HER 2. ADCC results support cell binding data (example 6), showing that increased binding and ADCC thresholds occur at HER2 receptor levels greater than 10,000 HER2 per cell. Based on this data, it is expected that an exemplary biparatopic anti-HER 2 antibody will increase cell surface binding and ADCC of HER 23 +, 2+, and 1+ tumor cells, but not non-tumor cells expressing HER2 at a basal level of about 10,000 receptors or less.

Example 26: effect of antibody afucosylation on ADCC

Afucosylated exemplary biparatopic antibodies (v 5019-afucosyl, 10000-afucosyl) were examined for their ability to induce dose-dependent ADCC in HER2 positive 2/3+, 2+ and 0/1+ HER2 (triple negative) expressing breast cancer cell lines. ADCC experiments were performed in SKOV3 cells, MDA-MB-231 cells, and ZR75-1 cells as described in example 11, except that a 5:1 constant NK effector or PBMC effector to target (E: T) cell ratio was used. The afucosylated exemplary biparatopic antibodies were transiently produced in CHO cells using transiently expressed RMD enzymes as described in von Horsten et al 2010Glycobiology 20:1607-1618, as described in example 1. The fucose content of v 5019-afucosyl and v 10000-afucosyl was measured as described in example 23 and less than < 2% fucosylation was determined (data not shown). Data using NK effector cells are shown in FIGS. 28A-B, while data using PBMC are shown in FIG. 28C.

Figure 28A, figure 28B and table 24 show that afucosylation v5019(v 5019-afucosyl) causes ADCC of HER 2/3+ and 0/1+ HER2 expressing breast cancer cells with maximal cell lysis ratioApproximately 1.5 to 1.7 times higher.

Table 24: ADCC of HER 22/3 + and expressing basal HER2 (triple negative) breast cancer cells

FIG. 28C and the results in Table 25 show that v10000 causes ADCC of HER 22 + ZR-75-1 breast cancer cells, maximal cell lysis ratioAbout 1.3 times higher and v 10000-afucosyl induction ratioMaximal cell lysis approximately 1.5 times higher.

Table 25: ADCC of HER 22/3 + Breast cancer cells

ADCC results show thatWhen used as a reference, the exemplary afucosylated biparatopic antibodies (v 5019-afucosyl, v 10000-afucosyl) caused about 15-25% higher maximal cell lysis compared to the fucosylated antibody (v5019 example 25, v 10000). These results show that decreasing the fucose content of Fc N-glycans results in an increase in maximal cell lysis by ADCC.

Example 27: exemplary Biparatopic anti-HER 2 antibodies in the presence of exogenous growth stimulating ligands (EGF and HRG) Ability of body to inhibit growth of HER 23 + breast cancer cells

5019 was examined for its ability to inhibit the growth of HER 23 + breast cancer cells in the presence of exogenous growth stimulating ligands (EGF and HRG).

Test antibody and exogenous ligand (10ng/mL HRG or 50ng/mLEGF) were added to BT-474HER 23 + target cells in triplicate and incubated for 5 days at 37 ℃. Using AlamarBlue^TMCell viability was measured (37 ℃ C., 2 hours) and absorbance was read at 530/580 nm. Data were normalized to untreated controls and analyzed using GraphPad prism.

The results are shown in fig. 29 and table 26. The results show that the exemplary biparatopic antibody v5019 inhibited HER 23 + breast cancer cell growth in the absence of growth stimulating ligands (70% inhibition), as well as in the presence of EGF (40% inhibition) or HRG (about 10% inhibition). anti-HER 2 monospecific FSA (v506) did not block EGF or HRG induced tumor cell growth via the other erbB receptors EGFR and HER 3. V5019 outperformed v506 in inhibiting HER2 and ligand-dependent dimerization and growth via the other partner erbB receptors.

Table 26: growth inhibition of HER 23 + cancer cells

These results show that the exemplary biparatopic antibodies are capable of reducing ligand-dependent growth of HER2+ cells, presumably due to binding of the anti-ECD 2A chain Fab arm and subsequent blocking of ligand-stimulated receptor homodimerization and heterodimerization, as well as erbB signaling.

Example 28: trastuzumab-resistant and chemotherapy-resistant HER 23 + patient sources in invasive ductal breast cancer Effect of biparatopic anti-HER 2 antibody in metastatic breast cancer xenograft model (PDX)

The anti-tumor efficacy of the exemplary biparatopic anti-HER 2 antibody v7187 was evaluated using HER 23 + (ER-PR negative) patient-derived xenograft model HBCx-13B from human invasive breast ductal carcinoma. v7187 is an afucosylated version of v 5019. This model is resistant to the single agents trastuzumab, trastuzumab and pertuzumab (see example 31), capecitabine, docetaxel and doxorubicin/cyclophosphamide combinations.

Subcutaneous inoculation of 20mm in female athymic nude mice³Tumor fragments. The tumors were then monitored until an average volume of 140mm3 was reached. Animals were then randomized into 2 treatment groups: vehicle control and v7187, 8 animals per group. The IV dose is as follows. Vehicle controls were administered intravenously at 5ml/kg formulation buffer twice weekly until study day 43. V7187 was administered intravenously at 10mg/kg twice weekly until study day 43. Tumor volumes were measured throughout the study and other parameters were evaluated on day 43 as shown in table 27.

The results are shown in fig. 30 and table 27. The results show that vehicle control treated tumors show persistence Extended and exceeded 1600mm by study day 43³. The v7187 treated mice showed significantly greater tumor growth inhibition (T/C-0.44) with a mean tumor volume of 740mm at 43 th balance³. v7187 induced a response in 5/8 tumors, with individual tumors showing complete regression and zero residual disease at study day 43. Animals treated with v7187 had better response rates, 5/8 tumor response to treatment, than 0/8 mice treated with vehicle control. In addition, treatment with v7187 significantly delayed tumor progression compared to vehicle control, with doubling times of 19 and 11 days, respectively.

Table 27:

these data show that the exemplary anti-HER 2 biparatopic (v7187) is effective in trastuzumab + pertuzumab-resistant HER 23 + metastatic breast cancer tumor xenograft model. v7187 treatment has a high response rate and can significantly impair tumor progression in standard-of-care treatment resistant HER 23 + breast cancer.

Example 29: assessment of binding of biparatopic anti-HER 2ADC to HER2+ tumor cell line

Exemplary biparatopic anti-HER 2 ADCs were analyzed by FACS for their ability to bind to and saturate HER2 positive 3+, 2+, breast and ovarian tumor cell lines as described in example 6.

The data are shown in figure 31. Figure 31A shows that v6363 binds to SKOV3 tumor cell line with bmax (mfi) approximately 2.0-fold higher than T-DM1(v6246) at saturating concentrations. FIG. 31B shows that v6363 binds to the JIMT-1 tumor cell line with Bmax (MFI) approximately 1.6-fold higher than T-DM1(v6246) at saturating concentrations. These data show that v6363(ADC) has similar tumor cell binding properties with increased cell surface binding compared to the unconjugated v5019 parent antibody (example 6). Coupling of v5019 to SMCC-DM1(v6363) did not change the antigen binding properties of the antibody.

FACS binding assays were repeated to include direct comparisons to exemplary biparatopic antibodies (v5019, v7091 and v10000) and ADCs (v6363, v7148 and v 10553). The data are shown in fig. 31C and 31D. Exemplary biparatopic anti-HER 2 ADCs (v6363, v7148 and v10553) had equal cell surface saturation (Bmax) compared to unlabeled biparatopic antibodies (v5019, v7091 and v 10000).

These data show that coupling of exemplary biparatopic antibodies (v5019, v7091 and v10000) to SMCC-DM1 did not alter the binding properties. Exemplary anti-HER 2 biparatopic anti-HER 2 ADCs (v6363, v7148 and v10553) had approximately 1.5-fold (or greater) increased cell surface binding compared to monospecific anti-HER 2 ADCs (v6246, T-DM 1).

Example 30: exemplary anti-HER 2 dual interaction in HER 23 + (ER-PR negative) patient-derived xenograft model Dose-dependent tumor growth inhibition by complementary-ADC

The anti-tumor efficacy of an exemplary biparatopic anti-HER 2ADC (v6363) was evaluated using HER 23 + (ER-PR negative) patient-derived xenograft model HBCx-13B from human invasive ductal carcinoma. This model is resistant to the single agents trastuzumab, trastuzumab and pertuzumab (see example 31), capecitabine, docetaxel and doxorubicin/cyclophosphamide combinations.

Female athymic nude mice were inserted 20mm subcutaneously³Tumor fragments inoculated the tumors. Tumor was monitored until it reached 160mm³Average volume of (d); animals were then randomized into 5 treatment groups: non-specific human IgG control and 4 increasing doses of v 6363. Each group comprised 8-10 animals. The dose for each group was as follows. IgG controls were administered intravenously at 10mg/kg twice weekly until study day 29. V6363 was administered intravenously at 0.3, 1, 3 or 10mg/kg on study days 1, 15 and 29. Tumor volumes were assessed throughout the study and parameter assessments were performed as shown in table 29.

The results are shown in fig. 32 and table 28. These results show that the exemplary anti-HER 2 biparatopic ADC (v6363) mediates dose-dependent tumor growth inhibition in the trastuzumab-resistant HBCx-13b PDX model (fig. 32A). In addition, v6363 improved overall survival in a dose-dependent manner with median survival of 63 days or more for the 3mg/kg and 10mg/kg doses compared to 43 days for the IgG control (fig. 32B and table 28). The 3mg/kg dose was associated with an increased response rate (5/10) compared to the control (0/8). All mice treated with v6363 at the 10mg/kg dose responded not only to treatment (9/9), but also showed prevention of tumor progression. Moreover, most tumors had objective partial responses (7/9) and, at the end of the study, many tumors had zero residual disease (6/9). v6363 was well tolerated at all doses, no adverse events were observed and no weight loss was observed.

Table 28:

these data show that the exemplary anti-HER 2 biparatopic ADC (v6363) is effective in trastuzumab + pertuzumab-resistant HER 23 + metastatic breast cancer tumor xenograft model. V6363 treatment in standard of care resistant HER 23 + breast cancer is associated with high response rates, significantly impairing tumor progression, and extending survival.

Example 31: biparatopic anti-HER 2-ADC in trastuzumab-resistant PDX HBCx-13b with Standard of Care Comparison of results

The efficacy of v6363 in HER 23 +, ER-PR negative trastuzumab resistant patient derived breast cancer xenograft model (HBCx-13b) was evaluated and compared to the following combinations: herceptin^TM+Perjeta^TM(ii) a And Herceptin^TM+ docetaxel.

Female athymic nude mice were tumor-inoculated by subcutaneous insertion of 20mm3 tumor fragments. Monitoring the tumor until itAn average volume of 100mm 3; animals were then randomly assigned to 4 treatment groups (8-10 animals/group): nonspecific human IgG control, Herceptin^TM+ docetaxel, herceptin^TM+Perjeta^TMAnd v 6363. The dose for each group was as follows. IgG controls were administered intravenously at 10mg/kg twice weekly until study day 29. Administering Herceptin at 10mg/kg IV^TM+ docetaxel combination herceptin^TMDocetaxel was administered intravenously, twice weekly until study day 29 and intraperitoneally at 20mg/kg on study days 1 and 22. At 5mg/kg of herceptin ^TM+Perjeta^TMThe combination herceptin was administered intravenously, twice weekly until study day 29 and Perjeta was administered at 5mg/kg^TMAdministered intravenously, twice weekly until study day 29. Herceptin^TMAnd Peijeta^TMThe administration is simultaneous. V6363 was administered intravenously at 10mg/kg on study days 1, 15 and 29.

The results are shown in fig. 33 and table 29. Fig. 33A shows tumor volume over time, and fig. 33B shows a survival map. These results show that Herceptin^TM+Perjeta^TMDoes not produce any tumor growth inhibition compared to control IgG and exceeds 1800mm on day 39³. Herceptin^TMThe combination of + docetaxel did not significantly reduce tumor growth, but did extend median survival to 53 days compared to IgG control at 43 days. v6363 produced significant tumor growth inhibition (T/C-0.04), where all tumors responded to treatment and 7/10 tumors underwent complete regression (zero residual disease). v6363 significantly prolonged survival compared to both combination therapies. The body weight of the group was not significantly affected by treatment.

Table 29:

these results show that the exemplary anti-HER 2 double-paratope ADC (v6363) outperforms the standard of care combination for all parameters tested in this xenograft model.

Example 32: mutual potentiation in HER2+ trastuzumab-resistant breast cancer cell-derived tumor xenograft model Effect of complement anti-HER 2-ADC

The efficacy of v6363 in a xenograft model derived from HER 23 + trastuzumab-resistant breast Cancer cells (JIMT-1, HER 22 +) was evaluated (Tanner et al 2004 Molecular Cancer Therapeutics 3: 1585-1592).

Female RAG2 mice were inoculated subcutaneously with tumors. Tumor was monitored until it reached 115mm³Average volume of (d); animals were then randomized into 2 treatment groups: trastuzumab (n ═ 10) and v 6363. The dose for each group was as follows. Trastuzumab was administered intravenously at 15mg/kg on study day 1 and twice weekly at 10mg/kg until study day 26. V6363 was administered intravenously at 5mg/kg on study days 1 and 15, 10mg/kg on days 23 and 30 and 9mg/kg on days 37 and 44.

The results are shown in fig. 34 and table 30. These results show that v6363 significantly inhibited tumor growth compared to trastuzumab at study day 36 (T/C-0.74). v6363 and trastuzumab treatment did not show changes in body weight. After 7 days of the first 10mg/kg dose, the v6363 serum exposure was 17.9. mu.g/ml.

Table 30:

these results show that the exemplary anti-HER 2 biparatopic ADC (v6363) is effective in trastuzumab-resistant breast cancer and has potential utility in treating breast cancer that is resistant to current standard of care.

Example 33: FcyR with heterodimeric Fc of anti-HER 2 biparatopic antibody and anti-HER 2 biparatopic-ADC Bonding of

The binding of anti-HER 2 biparatopic antibodies (v5019, v7019, v10000) and ADC (v6363, v7148 v10553) with heterodimeric Fc to human Fc γ R was evaluated and compared to anti-HER 2FSA (v506) and ADC (v6246) with homodimeric Fc.

The affinity of Fc γ R for the antibody Fc region was measured by SPR using ProteOn XPR36 (BIO-RAD). HER2 was immobilized (3000RU) on CM5 chips by standard amine coupling. The antibody is captured by the antigen on the surface of HER 2. Purified Fc γ R was injected at various concentrations (20-30 μ l/min) for 2 min, followed by dissociation for 4 min. The sensorgram was globally fit to the 1:1Langmuir binding model. All experiments were performed at 25 ℃.

The results are shown in Table 31. Exemplary heterodimeric anti-HER 2 biparatopic antibodies and ADCs bind with comparable affinity to CD16aF, CD16aV158, CD32aH, CD32aR131, CD32bY163, and CD 64A. Conjugation of the antibody to SMCC-DM1 did not negatively affect Fc γ R binding. The heterodimeric anti-HER 2 biparatopic antibody has approximately 1.3 to 2 times higher affinity for CD16aF, CD32aR131, CD32aH than homodimeric anti-HER 2FSA (v506) and ADC (v 6246). These results show that the heterodimeric anti-HER 2 biparatopic antibody and ADC bind Fc γ rs on immune effector cells in different polymorphic forms with similar or higher affinity than wild-type homodimeric IgG 1.

Table 31: human Fc gamma R binding by SPR assay

Example 34: in vivo exemplification in trastuzumab-sensitive ovarian cancer cell-derived tumor xenograft model Efficacy of sex anti-HER 2 biparatopic antibodies

The anti-tumor efficacy of exemplary biparatopic anti-HER 2 antibodies v5019, v7091 and v10000 was evaluated using the established human ovarian cancer cell-derived xenograft model SKOV3 described in example 17.

Female athymic nude mice were subcutaneously inoculated with 325,000 cells in HBSS tumor suspension on the left ventral side. Tumor was monitored until it reached 190mm³And are organized into 4 treatment groups in a randomly staggered manner: non-specific human IgG control, v5019, v7091 and v 10000. The dose for each group was as follows. Nonspecific human IgG control was administered intravenously at 10mg/kg starting on study day 1, twice weekly until study day 26. V5019, v7091 and v10000 were dosed intravenously at 3mg/kg starting on study day 1, twice weekly until study day 26. Tumor volumes were measured throughout the study and the parameters listed in table 32 were measured on day 29.

The data are presented in figure 35A (tumor growth), figure 35B (survival plot) and table 32 and show that treatment with v5019, v7091 and v10000 produced comparable tumor growth inhibition (T/C: 0.53-0.71), response tumor number, time to progression and survival on study day 29 compared to IgG controls. Serum exposure was similar for v5019, v7091 and v10000 on study day 7 (31-41. mu.g/ml).

Table 32:

these results show that exemplary anti-HER 2 biparatopic antibodies v5019, v7091 and v10000 have potential utility in treating moderately trastuzumab-sensitive HER 2-overexpressing ovarian cancer.

Example 35: exemplary biparatopic anti-her 2 antibody on trastuzumab-sensitive ovarian cancer cell-derived tumors Dose-dependent inhibition of tumor growth in tumor xenografts

Dose-dependent efficacy of an exemplary biparatopic anti-HER 2 antibody v10000 was evaluated using the established human ovarian cancer cell-derived xenograft model SKOV3 described in example 17.

Female athymic nude mice were subcutaneously inoculated with 325,000 cells in HBSS tumor suspension on the left ventral side. Tumor was monitored until it reached 190mm³And are organized into 6 treatment groups in a randomly staggered manner: nonspecific human IgG control and 5 increasing doses of v 10000. Each group comprised 9-13 animals. The dose for each group was as follows. IgG controls were administered intravenously at 10mg/kg twice weekly until study day 26. V10000 was administered intravenously at 0.1, 0.3, 1, 3 or 10mg/kg twice weekly.

The data are presented in figure 36 and table 33 and show that treatment with v10000 induces dose-dependent inhibition of tumor growth compared to control IgG (T/C: 0.28-0.73). In addition, on study day 29 v10000 was dose-dependent with a tumor response (7/9 at 10mg/kg and 3/11 at 0.1 mg/kg) and the time to progression increased (24 days at 10mg/kg and 12 days at 0.1 mg/kg). Serum exposure of v10000 on day 7 was dose dependent and increased from 0.46. mu.g/ml at the 0.1mg/kg dose to 79.3. mu.g/ml at the 10mg/kg dose.

Table 33:

these results show that the exemplary anti-HER 2 biparatopic antibody v10000 inhibited tumor progression in a dose-dependent manner.

Example 36: anti-HER 2 biparatopic antibodies and anti-HER 2 biparatopic-ADCs inhibit expression at 3+, 2+, or 1+ levels HER2 and EGFR and/or HER3

The following experiments were performed to measure the ability of an exemplary biparatopic anti-HER 2 antibody (v10000) and corresponding biparatopic anti-HER 2ADC (v10553) to inhibit the growth of selected breast, colorectal, gastric, lung, skin, ovarian, kidney, pancreas, head and neck, uterine and bladder tumor cell lines expressing 3+, 2+, 1+ or 0+ levels of HER2 and EGFR and/or HER3 as defined by IHC.

The experiment was performed as follows. The optimal seeding density for each cell line was uniquely determined to identify seeding densities that produced approximately 60-90% fusion after the assay lasted 72 hours. Each cell line was seeded at optimal seeding density in 96-well plates, each cell line in appropriate growth medium and at 36 ℃ and 5% CO₂The mixture was incubated at 24 ℃. Three concentrations of antibody (300, 30 and 0.3nM v 10000; 300, 1, 0.1nM v10553) were added along with positive and vehicle controls. The positive control was a chemical cocktail (chemococktail) drug combination of 5-FU (5-fluorouracil), paclitaxel, cisplatin, etoposide (25 μ M), and the vehicle control consisted of PBS. Antibody treatment and control with cells in a cell culture incubator at 36 ℃ and 5% CO ₂The mixture was incubated for 72 hours. The plate was centrifuged at 1200RPM for 10 minutes and the medium was completely removed by aspiration. RPMI SFM medium (200. mu.L) and MTS (20. mu.L) were added to each well and at 36 ℃ and 5% CO₂The mixture was incubated for 3 hours. Optical density was read at 490nM and percent growth inhibition was determined relative to vehicle control.

The results are shown in fig. 37 and a summary of all test results is shown in fig. 38. Fig. 37A shows the growth inhibition result of v 10000. These results show that v10000 can inhibit the growth of breast, colorectal, gastric, lung, skin, ovarian, renal, pancreatic, head and neck, uterine and endometrial tumor cell lines that express HER2 at 3+, 2+, 1+ or 0+ levels and that co-express EGFR and/or HER 3. The activity of v10000 and v10553 at 300nM is summarized in fig. 38, where '+' indicates that the cell line shows a decrease in cell viability at 300nM, > 5% of vehicle control, and '-' indicates ≦ 5% of vehicle control viability.

Fig. 37B shows the growth inhibition results of v 10553. These results show that v10553 can inhibit the growth of breast, colorectal, gastric, lung, skin, ovarian, renal, pancreatic, head and neck, uterine and bladder tumor cell lines that express HER2 at 3+, 2+, 1+, or 0+ levels and that co-express EGFR and/or HER3 (see also fig. 38). The results plotted in fig. 37B are defined by cell lines that showed minimal dose-dependent growth inhibition at 300 and 1nM, and where the growth inhibition at 1nM was equal to or higher than 5% (fig. 37B).

These results show that exemplary biparatopic antibodies v10000 and ADC v10553 can inhibit the growth of tumor cells derived from breast, colorectal, gastric, lung, skin, ovarian, renal, pancreatic, head and neck, uterine and bladder tissues, expressing HER2 at 3+, 2/3+, 2+, 1+ and 0/1+ levels and co-expressing EGFR and/or HER3 at 2+, 1+ levels.

Example 37: the ability of anti-HER 2 biparatopic antibodies to mediate ADCC in HER 22 +, 1+ and 0/1+ cancer cells

The following experiment was performed to determine the ability of anti-HER 2 biparatopic antibodies to mediate ADCC of tumor cells expressing HER2 at 2+, 1+ and/or 0/1+ levels and co-expressing EGFR and/or HER3 at 2+ or 1+ levels. The anti-HER 2 biparatopic antibodies tested were 5019, 10000 and 10154 (afucosylated versions of v 10000), among which are herceptin^TMAnd v506 as a control.

The ADCC experiments were performed with NK-92 effector cells at E/T5: 1 as described in example 11 and example 25 (FIG. 39) and PBMC effector cells at E/T30: 1 as described in example 26.

The results are shown in FIG. 39(NK-92 effector cells) and FIG. 40(PBMC effector cells). Figure 39A shows ADCC results for HER 22 + head and neck tumor cell line (hypopharyngeal carcinoma) FaDu, with anti-HER 2 biparatopic causing approximately 15% maximal cell lysis. Figure 39C shows ADCC results for HER 21 + BxPC3 pancreatic tumor cell line, and figure 39D shows ADCC results for HER 22 + MiaPaca2 pancreatic tumor cell line. Figure 39B shows ADCC results for HER 20/1 + a549NSCLC (non-small cell lung cancer) tumor cell line. In BxPC3, MiaPaca2 and a549 tumor cell lines, v10000 mediated maximal tumor cell lysis of approximately 5%.

FIG. 40 shows ADCC results in A549, NCI-N87, and HCT-116 cells, wherePBMCs were used as effector cells. FIG. 40A shows ADCC results for HER 20/1 + A549NSCLC tumor cell line, where v10000 caused about 28% maximal cell lysis and this was comparable to herceptin with an equivalent level of fucose content in N-linked glycans^TMAnd (4) the equivalent. Exemplary 100% afucosylated (0% fucose) biparatopic v10154 shows an increase in maximal cell lysis (40% maximal cell lysis) and an increase in potency compared to v10000 and herceptin, which has approximately 88% fucose in N-linked glycans.

Figure 40B shows ADCC results for HER 23 + gastric tumor cell line NCI-N87. Figure 40B shows that exemplary biparatopic v5019 (approximately 88% fucosylation) mediated approximately 23% maximal cell lysis and had a lower EC50 compared to trastuzumab v506 (approximately 98% fucosylation).

Figure 40C shows ADCC results for HER 21 + HCT-116 colorectal tumor cell line. Figure 40C shows that exemplary biparatopic v5019 (approximately 88% fucosylation) mediated approximately 25% maximal cell lysis and was more effective compared to trastuzumab v506 (approximately 98% fucosylation).

These results show that an exemplary anti-HER 2 biparatopic antibody can cause ADCC by HER 201/+, 2+ and 3+ tumor cells derived from head and neck, stomach, NSCLC and pancreatic tumor tissues. ADCC has a clear HER 22 + receptor level requirement (i.e. 2+ or higher) in the presence of NK-92 cells as effector cells to show a higher (> 5%) percentage of maximal cell lysis. However, when PBMC cells are used as effector cells, higher levels of maximal cell lysis (> 5% and up to 28% or 40%; v10000 and v10154, respectively) are achieved and are independent of HER2 receptor density, as ADCC > 5% is found at 0/1+, 1+ and 3+ HER2 receptor levels.

Example 38: HER2 binding affinity and kinetic parameters measured by SPR

As shown in example 1, biparatopic antibodies against HER2 with different antigen-binding moiety formats were constructed as described in table 1. Such formats include scFv-scFv formats (v6717), Fab-Fab formats (v6902 and v6903), as well as Fab-scFv formats (v5019, v7091 and v 10000). The following experiments were performed to compare HER2 binding affinity and kinetic parameters of these exemplary anti-HER 2 biparatopic antibody forms.

The affinity and binding kinetics for murine HER2ECD (Nano Biological 50714-M08H) were measured by single cycle kinetics (single cycle kinetics) using a T200SPR system from Biacore (GE healthcare). Anti-human Fc between 2000 and 4000RU was immobilized on CM5 chips using standard amine coupling. 5019 was captured on anti-human Fc surface in 50 RU. Recombinant HER2ECD (1.8-120nM) was injected at 50. mu.l/min for 3 min, followed by 30 min dissociation after the last injection. Dilutions of HER2 were analyzed in duplicate. The sensorgram was globally fit to the 1:1Langmuir binding model. All experiments were performed at room temperature 25 ℃.

The results in Table 34 show that Fab-scFv biparatopic antibodies (v5019 and v7091), Fab-Fab variants (v6902 and v6903), and scFv-scFv variants (v6717) have comparable binding affinities (1-4 nM). The Fab-scFv variant v10000 had a higher binding affinity (lower KD) of approximately 0.6 nM. Monospecific anti-HER 2ECD4 antibody (v506) and anti-HER 2ECD2 antibody (v4184) were included in the assay as controls. These results indicate that the molecular forms including v6717, v6902, v6903, v5019 and/or v7091 have equal binding affinities, and thus it is considered that the functional differences between these antibodies are caused by the differences in form.

Table 34:

example 39: effect of anti-HER 2 biparatopic antibody formats on binding to HER2+ tumor cells

The following experiments were conducted to compareWhole cell binding properties (Bmax and apparent K) of exemplary anti-HER 2ECD2x ECD4 biparatopic antibodies in different molecular formats (e.g., v6717, scFv-scFv IgG 1; v6903 and v6902Fab-Fab IgG 1; v5019, v7091 and v10000Fab-scFv IgG1)_D)。

The experiment was performed as described in example 6. The results are shown in FIG. 41 and tables 35 to 38. Fig. 41A and table 35 show FACS binding results of an exemplary biparatopic antibody to BT474HER 23 + breast tumor cell line. The results show that all anti-HER 2 antibodies had higher Bmax (1.5 to 1.7 fold higher) when compared to the monospecific bivalent anti-HER 2 antibody v 506. The Fab-scFv (v5019, v7091 and v10000) and Fab-Fab (v6903) versions had an approximately 1.7-fold increase in Bmax and the scFv-scFv version (v6717) had an approximately 1.5-fold increase in Bmax compared to v 506. The equimolar combination of FSA v506 and v4184 caused a 1.7-fold increase in Bmax. Apparent K of exemplary anti-HER 2 biparatopic antibody _DApproximately 2 to 3 times higher compared to monospecific v 506.

Table 35: FACS binding of BT-474

Antibody variants	K_D(nM)	Bmax
			v506	9.0	23536
v10000	16	39665
			v506+v4184	16	40320
v5019	21	39727
			v7091	22	36718
v6717	30	36392
			v6903	31	40321

Figure 41B and table 36 show FACS binding results to JIMT-1HER 22 + breast tumor cell line. The results show that all anti-HER 2 antibodies had higher Bmax (1.5 to 1.8 fold higher) when compared to the monospecific bivalent anti-HER 2 antibody v 506. Compared to v506, the Fab-scFv (v7091 and v10000) and Fab-Fab (v6903) versions had an increase of Bmax of about 1.7 fold, the scFv-scFv version (v6717) had an increase of Bmax of about 1.5 fold and the Fab-scFv (v5019) and FSA combination (v506+ v4184) had an increase of Bmax of about 1.8 fold. Apparent K of exemplary anti-HER 2 biparatopic Fab-scFv antibody_DAbout 2 to 4 times higher compared to monospecific v 506; k of Fab-Fab (v6903) and scFv-scFv (v6717)_DApproximately 8 times higher compared to v 506.

Table 36: FACS binding of JIMT-1

Antibody variants	K_D(nM)	Bmax
			v506	3.5	2574
v10000	7.6	4435
			v506+v4184	8.0	4617
v5019	12	4690
			v7091	14	4456
v6717	26	3769
			v6903	28	4452

Fig. 41C and table 37 show FACS binding results of an exemplary biparatopic antibody to HER 21 + MCF7 breast tumor cell line. The results show that the anti-HER 2 antibody v10000 and FSA combination (v506+ v4184) are monospecificThe bivalent anti-HER 2 antibody v506 had 1.6-fold higher Bmax compared to the other antibodies. Fab-scFv (v5019, v7091) have an approximately 1.4 fold increase in Bmax compared to v 506; the scFv-scFv format (v6717) had a 1.3-fold increase in Bmax, and the Fab-Fab format (v6903) had a 1.2-fold increase in Bmax. Apparent K of exemplary anti-HER 2 biparatopic Fab-scFv, Fab-Fab (v6903) and FSA combinations (v506+ v4184) _DAbout 2 to 3 times lower compared to v 506; k of scFv-scFv (v6717)_DApproximately 3 times higher compared to v 506.

Table 37: FACS binding of MCF7

Antibody variants	K_D(nM)	Bmax
			v506+v4184	4.5	1410
v7091	6.1	1216
			v5019	6.3	1201
v10000	6.8	1381
			v6903	7.1	1105
v506	12	889
			v6717	32	1167

Figure 41D and table 38 show FACS binding results of an exemplary biparatopic antibody to HER 20/1 + MDA-MD-231 breast tumor cell line. The results show that the exemplary biparatopic anti-HER 2 antibody has an approximately 1.3 to 1.4 fold increase in Bmax compared to the monospecific bivalent anti-HER 2 antibody v 506. The FSA combination (v506+ v4184) had a Bmax increase of 1.7 fold. Apparent K of exemplary anti-HER 2 biparatopic Fab-scFv antibodies (v5019, v7091, v10000) and FSA combination (v506+ v4184)_DK with v506_DApproximately equal; whereas Fab-Fab (v6903) and scFv-scFv (v6717) have approximately 4 and 16 times higher K than v506_D。

Table 38: FACS binding of MDA-MB-231

Antibody variants	K_D(nM)	Bmax
			v506	4.8	395
v10000	5.6	558
			v506+v4184	7.3	662
v7091	7.9	525
			v5019	8.7	548
v6903	17	534
			v6717	77	524

Tumor cell binding results showed that anti-HER 2 biparatopic antibodies with different molecular forms had increased Bmax on HER 23 +, 2+, 1+ and 0/1+ tumor cells compared to the bivalent monospecific anti-HER 2 antibody. Among the different anti-HER 2 biparatopic antibodies, the scFv-scFv format had the lowest increment of Bmax on HER 23 +, 2+, 1+ and 0/1+ tumor cells relative to v 506. These results also show that the scFv-scFv and Fab-Fab forms have K on HER 23 +, 2+, 1+ and 0/1+ tumor cells compared to monospecific v506 (3-to 16-fold increase) and biparatopic Fab-scFv forms (approximately 2-fold or higher) _DThe increase is maximal. K_DAn increase indicates a decrease in affinity binding and indicates that the different biparatopic forms have a unique mechanism of binding to HER2 on the cell surface.

Example 40: effect of anti-HER 2 biparatopic antibody formats on internalization in HER2+ cells

The following experiments were performed to compare the ability of exemplary anti-HER 2ECD2x ECD4 biparatopic antibodies (e.g., v6717, scFv-scFv IgG 1; v6903 and v6902Fab-Fab IgG 1; v5019, v7091 and v10000Fab-scFv IgG1) with different molecular formats to internalize in HER2+ cells expressing different levels of HER 2.

The experiment was performed as detailed in example 9. The results are shown in FIG. 42 and tables 39-41. Figure 42A and table 39 show internalization results in HER23+ BT-474. These results show that the Fab-scFv format (v10000) and FSA combination (v506+ v4184) have 2.2 times higher intracellular antibodies compared to monospecific anti-HER 2v 506. The scFv-scFv format (v6717) was 1.9 fold higher compared to v 506; the Fab-scFv format (v5019 and v7091) was 1.5 to 1.7 fold higher and the Fab-Fab format (v6902 and v6903) had 1.2 to 1.3 fold higher intracellular antibody accumulation.

Table 39: internalized BT-474

Antibody variants	Surface 4 deg.C	Surface 37 deg.C	Internal 37 deg.C
				v506	2156	1590	3453
v6902	2407	2077	4035
				v6903	2717	986	4573
v7091	2759	2227	5111
				v5019	2867	2675	5710
v6717	2006	1212	6498
				v10000	3355	2851	7528
v506+v4184	3998	2326	7569

FIG. 42B and Table 40 show internalization results in HER 22 + JIMT-1. These results show that the Fab-scFv format (v10000) and FSA combination (v506+ v4184) have 1.8 and 1.9 times higher amounts of intrabodies compared to monospecific anti-HER 2v506, respectively. Compared to v506, the scFv-scFv format (v6717) and the Fab-scFv format (v5019) were 1.4-fold higher; and the Fab-scFv (v7091) and Fab-Fab forms (v6902 and v6903) had 1.2 times higher intracellular antibody accumulation.

Table 40: internalizing JIMT-1

Fig. 42C and table 41 show internalization results in HER 21 + MCF 7. These results show that the scFv-scFv format and the Fab-scFv format have 3.0 and 2.8 times higher intrabodies compared to the monospecific anti-HER 2v 506. The Fab-scFv format (v10000) and FSA combination (v506+ v4184) were approximately 2-fold higher compared to v 506; the Fab-scFv (v7091) and Fab-Fab (v6903) versions had 1.8 times higher intracellular antibody accumulation.

Table 41: internalized MCF7

These results show that anti-HER 2 biparatopic antibodies with different molecular forms have unique degrees of internalization in HER 23 +, 2+, and 1+ tumor cells that differ with respect to the structure and form of the antigen binding domain. In general, the monospecific FSA combinations of v506 and v4184, Fab-scFv (v10000, v7091 and v5019) and scFv-scFv (v6717) biparatopic forms have higher internalization values in HER 23 +, 2+ and 1+ tumor cells. However, the Fab-Fab biparatopic forms (v6902 and v6903) have the lowest internalization values in HER 23 +, 2+ and 1+ tumor cells. These data indicate that the molecular form and geometric spacing of the antigen binding domains has an effect on the ability of biparatopic antibodies to cross-link the HER2 receptor and subsequently internalize in HER2+ tumor cells. The Fab-Fab biparatopic form, which has the greatest distance between the two antigen binding domains, produces the lowest degree of internalization, while the Fab-scFv and scFv-scFv forms, which have shorter distances between the antigen binding domains, have stronger internalization in HER2+ cells. This is consistent with the correlation between potency and shorter linker length described in Jost et al 2013, Structure 21, 1979-1991).

Example 41: effect of anti-HER 2 biparatopic antibody Format on ADCC in HER2+ cells

The following experiments were performed to compare the ability of exemplary anti-HER 2ECD2x ECD4 biparatopic antibodies (e.g., v6717, scFv-scFv IgG 1; v6903 and v6902Fab-Fab IgG 1; v5019, v7091 and v10000Fab-scFv IgG1) with different molecular formats to mediate ADCC in HER2+ cells expressing different levels of HER 2.

Prior to ADCC assays, glycopeptide analysis was performed on antibody samples to quantify the fucose content in N-linked glycopeptides. The procedure was as described in example 23. The results are shown in Table 42; the data show that the exemplary biparatopic variants v5019, v6717, v6903 have equal fucose contents (91-93%) in N-linked glycans. Antibody samples with equivalent fucose levels in N-glycans were selected for ADCC assays to normalize the fucose content in the interpretation of the ADCC assay results.

Table 42: LC-MS tryptic peptide analysis

Variants	Percentage of glycopeptides with fucose observed	Percentage of afucose glycopeptide observed
			v6903	90.7	9.3
v6717	92.8	7.2
			v5019	91.3	8.7

ADCC experiments were performed with NK-92 effector cells at E/T:5:1 as described in example 11. ADCC results are shown in FIG. 43 and tables 43-45. Figure 43A and table 43 show ADCC results in HER 22 + JIMT-1 breast tumor cells. These data show that v5019, v6717 and v6903 caused similar levels of maximal cell lysis and that the scFv-scFv format (v6717) was less potent than v5019 and v6903 when HER 22 + tumor cells were targeted.

Table 43: JIMT-1ADCC

Antibody variants	EC50(nM)	Maximum cell lysis%
			v6903	About 0.03	48
v5019	About 0.16	47
			v6717	About 0.72	51

Figure 43B and table 44 show ADCC results in HER 21 + MCF7 breast tumor cells. These data show that v5019 and v6717 have slightly higher maximal cell lysis (27-30%) compared to v6903 (24%). These data also show that v6717 is less potent, followed by v6903 and v5019, which have lower EC50 values.

Table 44: MCF7ADCC

Antibody variants	EC₅₀(nM)	Maximum cell lysis%
			v5019	About 0.69	27
v6717	109	30
			v6903	0.94	24

Figure 43C and table 45 show ADCC results in HER 20/1 + MDA-MB-231 breast tumor cells. These data show that v5019 has slightly higher maximal cell lysis (77%) compared to v6903 (62%) and v6717 (63%). These data also show that v6717 is less potent, followed by v6903 and v5019, which have lower EC₅₀The value is obtained.

Table 45: MDA-MB-231ADCC

Antibody variants	EC₅₀(nM)	Maximum cell lysis% (highest only)
			v5019	0.20	71
v6717	10	63
			v6903	0.79	62

These data show that the exemplary anti-HER 2ECD2x ECD4 biparatopic antibody causes similar levels of maximal cell lysis by ADCC in HER 22 + and 1+ tumor cells. Although similar in terms of maximal cell lysis, these data also show that different molecular forms have unique ADCC potency. Sc in HER 22 + and HER 21 + Fv-scFv with minimal potency (EC)₅₀Highest value). Differential potency among the three forms was seen in ADCC data against HER 21 + cells, with EC50 values v6717>v6903>v 5019. These data are consistent with the observations (FACS binding) presented in example 40, where K is observed in the case of Fab-Fab and scFv-scFv formats_DIncrease (decrease in affinity).

Example 42: effect of anti-HER 2 biparatopic antibody Format on HER2+ tumor cell growth

The following experiments were performed to compare the effect of the anti-HER 2 biparatopic antibody format on the growth (basal growth or growth stimulated by ligand) of HER 23 +, 2+ and 1+ tumor cells. Basal growth was measured as described in example 15, while growth stimulated by the ligand was measured as described in example 27. In both types of experiments, growth was measured as% survival relative to control treatment.

Figure 44 and table 46 show the effect of an exemplary anti-HER 2ECD2x ECD4 biparatopic antibody on growth of HER 23 + breast cancer cells (BT-474) in the presence of exogenous growth stimulating ligands (EGF and HRG). anti-HER 2 biparatopic antibody is capable of inhibiting the growth of BT-474 cells in the absence of EGF or HRG, with% survival ratings for each treatment group as follows: v6903< v506+ v4184<506< v7091< v5019< v10000< v 6717. Growth inhibition relative to mock control was achieved with the FSA combination of v506+ v4184 alone in the presence of HRG. Growth inhibition was achieved in the presence of EGF relative to mock control, with% survival ranking for each treatment group as follows: v6903< v506+ v4184<7091< v10000< 5019.

TABLE 46

Figure 45 shows the dose-dependent effect of the anti-HER 2 biparatopic antibody format on growth inhibition of SKBr3HER 23 + cell line. The data are consistent with the results presented in figure 44, where the potency/efficacy ranking of the biparatopic forms in HER 23 + tumor cells is as follows Fab-Fab > Fab-scFv > scFv-scFv.

The effect of the anti-HER 2 biparatopic antibody form on HER2+ cell survival is shown in figure 46, where figure 46A shows the results in trastuzumab-sensitive SKOV3HER 22 +/3+ cell line at 300 nM; fig. 46B shows the results in JIMT-1HER 22 + (trastuzumab-resistant) cells at 300nM, and fig. 46C shows the results in MCF7HER 21 + cell line at 300 nM. In the SKOV3 cell line, a slight difference was observed in the degree of growth inhibition between the biparatopic forms, and no growth inhibition was observed with any of the test antibodies in JIMT-1 and MCF7 cells.

The data in figures 44 and 45 show that anti-H ER2ECD2x ECD4 biparatopic antibodies with Fab-scFv and Fab-Fab forms (v5019, v7091, v10000, v6903) are able to inhibit HER 23 + tumor cell growth in the absence and presence of EGF or HRG. The rank order of the inhibition of growth relative to mock control in H ER 23 + cell lines BT-474 and SKBR3 was as follows, where v506+ v4184> v6903> v7091> v10000> v5019> v506> v 6717. The distance between the antigen binding domains (Fab-Fab > Fab-scFv > scFv-sc Fv) correlates with the rank order of growth inhibition in HER 23 + tumor cells. Based on the data in trastuzumab-sensitive tumor cells BT-474 and SKBr3, differences in growth inhibition between forms can be expected to be significant at HER 23 + levels, but less significant at HER 22 + or HER 21 + levels.

Example 43: evaluation of HER2 binding affinity and kinetics at different antibody capture levels

The following experiment was performed to compare HER2 binding kinetics (kd, separation rate) of exemplary anti-HER 2ECD2x ECD4 biparatopic antibodies captured at different surface densities by SPR. A correlation between a decrease in separation rate and an increase in antibody capture levels (surface density) indicates trans-binding (i.e. one antibody molecule binds to two HER2 molecules, as described in example 12). In this experiment, the Fab-Fab format (v6903) was compared to the Fab-scFv format (v7091) to determine potential differences between variants in trans-binding. Due to the large spatial distance between the antigen binding domains, it is hypothesized that the Fab-Fab form is capable of cis binding (engineering ECD2 and 4 on one HER2 molecule); however, Fab-scFv will not bind in cis due to the short distance between its antigen binding domains. anti-HER 2 monospecific v506 was included as a control.

Experiments were performed by SPR as described in example 12. The data are shown in fig. 47. FIG. 47A shows plots of kd (1/s) at different antibody capture levels with v6903 and v7091 and linear regression analysis. Both v7091 and v6093 show a trend towards decreasing separation rates with increasing surface capture levels; however, there was significant correlation with the Fab-scFv variant (v 7091; P value 0.023) rather than the Fab-Fab form (v 6093; P value 0.053). For the anti-HER 2 monospecific control v506, the separation rate remained unchanged as the antibody capture level was varied.

FIG. 47B shows K at different antibody capture levels with v6903 and v7091_DGraph of (M) and linear regression analysis. Similar to the separation rate, both v7091 and v6093 showed increased affinity with increasing surface capture levels (K7091 and v 6093)_DDecreasing in value). However, there was a significant correlation with the Fab-scFv variant (v 7091; P value 0.04) rather than the Fab-Fab form (v 6093; P value 0.51). For the anti-HER 2 monospecific control v506, K varied with antibody capture levels_DRemain unchanged. The data in figure 47 show that the Fab-Fab and Fab-scFv anti-HER 2 biparatopic antibody formats show a trend towards decreasing separation rates with increasing antibody surface capture levels; these trends are unique compared to monospecific anti-Her 2 antibodies.

Example 44: affinity and stability engineering of pertuzumab Fab

As shown in table 1, one variant (v10000) contained mutations in pertuzumab Fab. This Fab is derived from in silico affinity and stability engineering efforts, measured in the laboratory as monovalent or single arm antibodies (OAA).

Variant 9996: a monovalent anti-HER 2 antibody, wherein the HER2 binding domain is derived from a Fab on pertuzumab a chain, with Y96A in the VL region and T30A/a49G/L69F (Rabat numbering) in the VH region and the Fc region is a heterodimer with the mutation T350V _ L351Y _ F405A _ Y407V (EU numbering) in the a chain, the mutation T350V _ T366L _ K392L _ T394W (EU numbering) in the B chain, and the hinge region of the B chain with the mutation C226S; the antigen binding domain binds to domain 4 of HER 2.

Variant 10014: a monovalent anti-HER 2 antibody, wherein the HER2 binding domain is derived from Fab on pertuzumab a chain, with Y96A in the VL region and T30A (Rabat numbering) in the VH region and the Fc region is a heterodimer with the mutation T350V _ L351Y _ F405A _ Y407V (EU numbering) in the a chain, the mutation T350V _ T366L _ K392L _ T394W (EU numbering) in the B chain, and the hinge region of the B chain has the mutation C226S; the antigen binding domain binds to domain 4 of HER 2.

Variant 10013: a monovalent anti-HER 2 antibody, wherein the HER2 binding domain is derived from Fab on the pertuzumab a chain, and the Fc region is a heterodimer with the mutation T350V _ L351Y _ F405A _ Y407V (EU numbering) in the a chain, the mutation T350V _ T366L _ K392L _ T394W (EU numbering) in the B chain, and the hinge region of the B chain has the mutation C226S; the antigen binding domain binds to domain 4 of HER 2.

The following experiments were performed to compare HER2 binding affinity and stability of the engineered pertuzumab variants.

OAA variants were cloned and expressed as described in example 1.

OAA was purified by protein a chromatography and size exclusion chromatography as described in example 1.

Heterodimer purity (i.e., the amount of OAA with heterodimeric Fc) was assessed by a non-reducing high throughput protein expression assay using Caliper LabChip gxi (Perkin Elmer # 760499). The procedure was carried out according to the LabChip GXII instruction Manual (LabChip GXII User Manual) 2 nd edition of the HT Protein expression LabChip User Guide (HT Protein Express LabChip User Guide), with the following modifications. Mu.l or 5. mu.l (concentration range 5-2000 ng/. mu.l) of heterodimer sample was added to individual wells of a 96-well plate along with 7. mu.l of HT protein expression sample buffer (Perkin Elmer # 760328). The heterodimer sample was then denatured at 70 ℃ for 15 min. The LabChip instrument was operated using the HT protein expression chip (Perkin Elmer #760499) and Ab-200 assay device. After use, the chips were cleaned with MilliQ water and stored at 4 ℃.

The stability of the samples was assessed by measuring the melting temperature or Tm as determined by DSC using the protocol shown in example 24. DSC was measured before and after SEC purification.

The affinity of the samples for HER2ECD was measured by SPR according to the protocol from example 12. SPR was measured before and after SEC purification. As summarized in tables 47A and 47B, mutations in the variable domains have enhanced HER2 affinity for Fab while maintaining wild-type stability compared to wild-type pertuzumab. (¹Purity as determined by Caliper LabChip;²KD (wild type)/KD (mutant)

Table 47A:

table 47B:

the reagents employed in the examples are generally commercially available or can be prepared using commercially available equipment, methods, or reagents known in the art. The foregoing embodiments illustrate various aspects described herein and practice of the methods described herein. The examples are not intended to provide an exhaustive description of the many different embodiments of the invention. Thus, although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, those of ordinary skill in the art will readily recognize that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in the specification by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.

Sequence listing

Variants	Clone name H1	Clone name H2	Clone name L1	Clone name L2
					792	1011	1015	-2	-2
5019	3057	720	1811	NA
					5020	719	3041	NA	1811
7091	3057	5244	1811	NA
					10000	6586	5244	3382	NA
6903	5065	3468	5037	3904
					6902	5065	3468	5034	3904
6717	3317	720	NA	NA
					1040	4560	4553	NA	4561
630	719	716	NA	NA
					4182	4560	3057	NA	1811
506	642	642	-2	-2
					4184	3057	3041	1811	1811
9996	4372	6586	NA	3382

Pertuzumab variant CDR-L3: QQYYIYPAT

Clone 3382, variant 10000(SEQ ID NO:347)

Pertuzumab variant CDR-H1: GFTFADYT

Clone 6586, variant 10000(SEQ ID NO:348)

The claims (modification according to treaty clause 19)

1. An antigen binding construct comprising

A first antigen-binding polypeptide construct that binds monovalently and specifically to the HER2 (human epidermal growth factor receptor 2) ECD2 (extracellular domain 2) antigen on a HER 2-expressing cell;

a second antigen-binding polypeptide construct that binds monovalently and specifically to the HER2 ECD4 (extracellular domain 4) antigen on a HER2 expressing cell;

first and second linker polypeptides, wherein the first linker polypeptide is operably linked to the first antigen-binding polypeptide construct and the second linker polypeptide is operably linked to the second antigen-binding polypeptide construct;

Wherein the linker polypeptides are capable of forming covalent bonds with each other,

and wherein one or both of the first or the second antigen-binding polypeptide is an scFv.

2. The antigen-binding construct of claim 1, wherein the first and second linker polypeptides each comprise an immunoglobulin hinge region polypeptide selected from an IgG1, IgG2, or IgG4 hinge region.

3. The antigen binding construct according to claim 1 or 2, wherein the first and/or second linker polypeptide is operably linked to a backbone, optionally an Fc.

4. The antigen-binding construct of claim 1 or 2, wherein the first and second linker polypeptides are operably linked to a dimeric Fc comprising first and second Fc polypeptides, each comprising a CH3 sequence, wherein the first Fc polypeptide is operably linked to the first linker polypeptide and the second linker polypeptide is operably linked to the second linker polypeptide.

5. The antigen-binding construct according to any one of the preceding claims, wherein (i) the first antigen-binding polypeptide construct is an scFv and the second antigen-binding polypeptide construct is a Fab; or (ii) the first antigen-binding polypeptide construct is a Fab and the second antigen-binding polypeptide construct is a scFv; or (iii) both the first antigen-binding polypeptide construct and the second antigen-binding polypeptide construct are scfvs.

6. The antigen binding construct of any one of the preceding claims, wherein

i. The first antigen-binding polypeptide construct is a Fab and the second antigen-binding polypeptide construct is a scFv and the first antigen-binding polypeptide construct Fab comprises

a. A first variable heavy chain polypeptide VH1 comprising the VH and VH of the pertuzumab arm of v5019(SEQ ID NO:221), v5020(SEQ ID NO:205), v7091(SEQ ID NO:221), v6717(SEQ ID NO:267) or v10000(SEQ ID NO:99)

b. A first variable light chain polypeptide VL1 comprising the VL of the pertuzumab arm of v5019(SEQ ID NO:35), v5020(SEQ ID NO:35), v7091(SEQ ID NO:35), v6717(SEQ ID NO:259) or v10000(SEQ ID NO: 71);

and the second antigen-binding polypeptide construct scFv comprises

(a) A second variable heavy chain polypeptide VH2 comprising the VH and VH of the trastuzumab arm of v5019(SEQ ID NO:179), v5020(SEQ ID NO:157), v7091(SEQ ID NO:305), v6717(SEQ ID NO:179) or v10000(SEQ ID NO:305)

(b) A second variable light chain polypeptide VL2 comprising the VL of the trastuzumab arm of v5019(SEQ ID NO:171), v5020(SEQ ID NO:149), v7091(SEQ ID NO:297), v6717(SEQ ID NO:171) or v10000(SEQ ID NO: 297); or

The first antigen-binding polypeptide construct is an scFv and the second antigen-binding polypeptide construct is a Fab and the first antigen-binding polypeptide construct scFv comprises

(a) A first variable heavy chain polypeptide VH1 comprising the VH and VH of the pertuzumab arm of v5019(SEQ ID NO:221), v5020(SEQ ID NO:205), v7091(SEQ ID NO:221), v6717(SEQ ID NO:267) or v10000(SEQ ID NO:99)) and

(b) a first variable light chain polypeptide VL1 comprising the VL of the pertuzumab arm of v5019(SEQ ID NO:35), v5020(SEQ ID NO:35), v7091(SEQ ID NO:35), v6717(SEQ ID NO:259) or v10000(SEQ ID NO:71)

And the second antigen-binding polypeptide construct Fab comprises

(a) A second variable heavy chain polypeptide VH2 comprising the VH and VH of the trastuzumab arm of v5019(SEQ ID NO:179), v5020(SEQ ID NO:157), v7091(SEQ ID NO:305), v6717(SEQ ID NO:179) or v10000(SEQ ID NO:305))

The first antigen-binding polypeptide construct is an scFv and the second antigen-binding polypeptide construct is an scFv and the first antigen-binding polypeptide construct scFv comprises

(a) A first variable heavy chain polypeptide VH1 comprising the VH of the pertuzumab arm of v5019(SEQ ID NO:221), v5020(SEQ ID NO:205), v7091(SEQ ID NO:221), v6717(SEQ ID NO:267) or v10000(SEQ ID NO:99), and

(b) A first variable light chain polypeptide VL1 comprising the VL of the pertuzumab arm of v5019(SEQ ID NO:35), v5020(SEQ ID NO:35), v7091(SEQ ID NO:35), v6717(SEQ ID NO:259) or v10000(SEQ ID NO:71),

and the second antigen-binding polypeptide construct scFv and comprising

(b) A second variable light chain polypeptide VL2 comprising the VL of the trastuzumab arm of v5019(SEQ ID NO:171), v5020(SEQ ID NO:149), v7091(SEQ ID NO:297), v6717(SEQ ID NO:171) or v10000(SEQ ID NO: 297).

7. The antigen-binding construct according to any of the preceding claims, wherein the first antigen-binding polypeptide construct is selected from the group consisting of

i. A polypeptide construct comprising three VH CDR sequences comprising the amino acid sequences SEQ ID No. 335, SEQ ID No. 336 and SEQ ID No. 337, or SEQ ID No. 335, SEQ ID No. 336 and SEQ ID No. 348;

a polypeptide construct comprising three VH CDR sequences comprising amino acid sequences having at least 90% identity to the three VH CDR sequences of SEQ ID No. 335, SEQ ID No. 336 and SEQ ID No. 337, or SEQ ID No. 335, SEQ ID No. 336 and SEQ ID No. 348;

A polypeptide construct comprising three VL CDR sequences comprising the amino acid sequences of the three VL CDR sequences of SEQ ID No. 338, SEQ ID No. 339 and SEQ ID No. 340, or SEQ ID No. 338, SEQ ID No. 347 and SEQ ID No. 340;

a polypeptide construct comprising three VL CDR sequences having at least 90% identity to the amino acid sequences of SEQ ID No. 338, SEQ ID No. 339 and SEQ ID No. 340, or of the three VL CDR sequences having at least 90% identity to SEQ ID No. 338, SEQ ID No. 347 and SEQ ID No. 340;

v. a polypeptide construct comprising six CDR sequences comprising SEQ ID NO 335, 336, 337, 338, 339 and 340; or the amino acid sequences of the six CDR sequences of SEQ ID NO 335, SEQ ID NO 336, SEQ ID NO 348, SEQ ID NO 338, SEQ ID NO 347 and SEQ ID NO 340; or

A polypeptide construct comprising six CDR sequences comprising at least one CDR sequence selected from the group consisting of SEQ ID No. 335, SEQ ID No. 336, SEQ ID No. 337, SEQ ID No. 338, SEQ ID No. 339, and SEQ ID No. 340; or the six CDR sequences of SEQ ID NO 335, 336, 348, 338, 347 and 340 have an amino acid sequence of at least 90% identity and the second antigen binding polypeptide is selected from the group consisting of

A polypeptide construct comprising three VH CDR sequences comprising the amino acid sequences of the three VH CDR sequences of SEQ ID No. 341, SEQ ID No. 342 and SEQ ID No. 343;

a polypeptide construct comprising three VH CDR sequences comprising amino acid sequences having at least 90% identity to the three VH CDR sequences of SEQ ID No. 341, SEQ ID No. 342 and SEQ ID No. 343;

a polypeptide construct comprising three VL CDR sequences comprising the amino acid sequences of the three VL CDR sequences of SEQ ID No. 344, SEQ ID No. 345 and SEQ ID No. 346;

a polypeptide construct comprising three VL CDR sequences having at least 90% identity to the amino acid sequences of the three VL CDR sequences of SEQ ID No. 344, SEQ ID No. 345 and SEQ ID No. 346;

a polypeptide construct comprising six CDR sequences comprising the amino acid sequences of the six CDR sequences of SEQ ID No. 341, SEQ ID No. 342, SEQ ID No. 343, SEQ ID No. 344, SEQ ID No. 345 and SEQ ID No. 346; or

A polypeptide construct comprising six CDR sequences comprising amino acid sequences having at least 90% identity to the six CDR sequences of SEQ ID No. 341, SEQ ID No. 342, SEQ ID No. 343, SEQ ID No. 344, SEQ ID No. 345 and SEQ ID No. 346.

8. The antigen-binding construct according to any one of the preceding claims, wherein the first antigen-binding polypeptide construct: (i) block pertuzumab binding to ECD2 by 50% or more, and/or (ii) the second antigen-binding polypeptide blocks trastuzumab binding to ECD4 by 50% or more.

9. The antigen-binding construct according to any preceding claim, wherein the first antigen-binding polypeptide construct comprises one of said v5019, v10000, v7091, v5020 or v6717 antigen-binding polypeptide constructs specific for HER2 ECD2 and the second antigen-binding polypeptide construct comprises one of said v5019, v10000, v7091, v5020 or v6717 antigen-binding polypeptide constructs specific for HER2 ECD 4.

10. The antigen-binding construct according to any preceding claim, wherein the first antigen-binding polypeptide construct comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the v5019, v10000, v7091, v5020 or v6717 antigen-binding polypeptide construct specific for HER2 ECD2 and the second antigen-binding polypeptide construct comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the v5019, v10000, v7091, v5020 or v6717 antigen-binding polypeptide construct specific for HER2 ECD 4.

11. The antigen binding construct according to any preceding claim, which is selected from v5019, v10000, v7091, v5020 and v 6717.

12. The antigen-binding construct according to any preceding claim, wherein the first antigen-binding polypeptide construct is a Fab and the second antigen-binding polypeptide construct is a scFv, and wherein the antigen-binding construct is compared to a reference biparatopic antigen-binding construct having two fabs

(i) Inducing enhanced receptor internalization and/or in HER 23 + cells

(ii) Higher potency was shown for HER 21 + cells in ADCC (antibody-directed cytotoxicity) assay.

13. The antigen-binding construct according to any preceding claim, wherein the first and second antigen-binding polypeptide constructs are scfvs, and wherein the antigen-binding construct induces enhanced receptor internalization in HER 21 +, 2+, and 3+ cells as compared to a reference antigen-binding construct having two fabs.

14. A modified pertuzumab construct comprising one or more antigen-binding polypeptide constructs that monovalently and specifically bind HER2 ECD2, each polypeptide construct comprising a VH and a VL, wherein the VH comprises three CDR sequences comprising the amino acid sequences of the three VH CDR sequences of SEQ ID NO:335, SEQ ID NO:336, and SEQ ID NO:348, and the VL comprises three CDR sequences comprising the amino acid sequences of the three VL CDR sequences of SEQ ID NO:338, SEQ ID NO:347, and SEQ ID NO: 340.

15. The modified pertuzumab construct of claim 14, wherein the VH comprises the VH of v9996 and the VL comprises the VL of v 9996.

16. The modified pertuzumab construct of claim 14 or 15, wherein the antigen-binding polypeptide construct comprises an amino acid sequence at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the VH of v9996 and the VL of v 9996.

17. The modified pertuzumab construct of claims 14-16, wherein the modified pertuzumab construct is monovalent, bivalent, or multivalent.

18. The modified pertuzumab construct of claims 14-17, wherein the construct is (i) monovalent and comprises a Fab or an scFv, (ii) bivalent and comprises a Fab and an scFv, or (iii) bivalent and comprises two scfvs.

19. The modified pertuzumab construct of claims 14-17, wherein the construct is monovalent and displays a 7-to 9-fold increase in affinity for HER2 ECD2 as compared to pertuzumab.

20. The modified pertuzumab construct of claims 14-19, comprising a dimeric Fc linked to the antigen-binding polypeptide construct with or without a linker.

21. The modified pertuzumab construct of claims 14-20, wherein the modified pertuzumab construct comprises first and second antigen-binding polypeptide constructs, and first and second Fc polypeptides each comprising a CH3 sequence, wherein the first Fc polypeptide is operably linked to the first antigen-binding polypeptide construct with or without a first linker, and the second monomeric Fc polypeptide is operably linked to the second antigen-binding polypeptide construct with or without a second linker.

22. The modified pertuzumab construct of claims 20 and 21, comprising a polypeptide linker.

23. The modified pertuzumab construct of claim 22, wherein the linker comprises an IgG1 hinge region.

24. The modified pertuzumab construct of claims 20 and 21, wherein the Fc is a human Fc.

25. The modified pertuzumab construct of claims 20 and 21, wherein the Fc is human IgG1 Fc.

26. The construct according to any preceding claim, comprising a heterodimeric Fc, wherein the dimerized CH3 sequence has a melting temperature (Tm) of about 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 77.5, 78, 79, 80, 81, 82, 83, 84, or 85 ℃ or higher.

27. The construct of any preceding claim, comprising a heterodimeric Fc formed at greater than about 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% purity upon expression.

28. The construct of any preceding claim, comprising a heterodimeric Fc formed with a purity of greater than about 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% when expressed via a single cell.

29. The construct according to any preceding claim, comprising a heterodimeric Fc comprising one or more modifications in at least one CH3 sequence.

30. The construct of any preceding claim, comprising a heterodimeric Fc comprising one or more modifications in at least one CH3 sequence that promote the formation of heterodimers with stability comparable to a wild-type homodimeric Fc.

31. The construct of any preceding claim, comprising a homodimeric Fc compared to wild-type according to EU numbering

i. Heterodimeric IgG1 Fc with modification T350V _ L351Y _ F405A _ Y407V in the first Fc polypeptide and modification T366I _ N390R _ K392M _ T394W in the second Fc polypeptide; or

A heterodimeric IgG1 Fc with the modification L351Y _ S400E _ F405A _ Y407V in the first Fc polypeptide and the modification T350V _ T366L _ K392L _ T394W in the second Fc polypeptide,

heterodimeric IgG1 Fc with the modification L351Y _ F405A _ Y407V in the first Fc polypeptide and the modification T366L _ K392M _ T394W in the second polypeptide;

heterodimeric IgG1 Fc with the modification L351Y _ F405A _ Y407V in the first Fc polypeptide and the modification T366L _ K392L _ T394W in the second Fc polypeptide;

a heterodimeric IgG1 Fc with the modification T350V _ L351Y _ F405A _ Y407V in the first Fc polypeptide and the modification T350V _ T366L _ K392L _ T394W in the second Fc polypeptide;

heterodimeric IgG1 Fc with the modification T350V _ L351Y _ F405A _ Y407V in the first Fc polypeptide and the modification T350V _ T366L _ K392M _ T394W in the second Fc polypeptide; or

Heterodimeric IgG1 Fc with modification T350V _ L351Y _ S400E _ F405A _ Y407V in the first Fc polypeptide and modification T350V _ T366L _ N390R _ K392M _ T394W in the second Fc polypeptide.

32. The construct of any preceding claim, comprising a heterodimeric Fc comprising at least one CH2 domain.

33. The construct of claim 32, wherein the CH2 domain of the heterodimeric Fc comprises one or more modifications.

34. The construct according to any preceding claim, comprising a heterodimeric Fc comprising one or more modifications that promote selective binding of Fc-gamma receptors.

35. The construct according to any preceding claim, wherein the construct is glycosylated.

36. The construct of any preceding claim, wherein the construct is coupled to a drug.

37. The construct of claim 36, wherein the drug is maytansine (DM 1).

38. The construct of claim 36, wherein the construct is coupled to DM1 via a SMCC linker.

39. A pharmaceutical composition comprising the construct of any preceding claim and a pharmaceutical carrier.

40. The pharmaceutical composition of claim 39, the pharmaceutical carrier comprising a buffer, an antioxidant, a low molecular weight molecule, a drug, a protein, an amino acid, a carbohydrate, a lipid, a chelator, a stabilizer, or an excipient.

41. A pharmaceutical composition for use in medicine, comprising the construct of any one of claims 1-38.

42. A pharmaceutical composition for cancer treatment comprising the construct according to any one of claims 1-38.

43. A method of treating a subject having a HER2 expressing (HER2+) tumor comprising administering to the subject an effective amount of the construct of any one of claims 1-38 or the pharmaceutical composition of any one of claims 39-42.

44. The method of claim 43, wherein the result of said treatment is shrinking the tumor, inhibiting the growth of the tumor, increasing the time to progression of the tumor, prolonging disease-free survival of the subject, reducing metastasis, increasing progression-free survival of the subject, or increasing overall survival of the subject.

45. The method of claim 43 or 44, wherein the tumor is pancreatic cancer, head and neck cancer, gastric cancer, colorectal cancer, breast cancer, renal cancer, cervical cancer, ovarian cancer, endometrial cancer, uterine cancer, malignant melanoma, pharyngeal cancer, oral cancer, or skin cancer.

46. The method of claims 43-45, wherein said tumor comprises cells that express an average of 10,000 or more copies of HER2 per tumor cell.

47. The method of claims 43-46, wherein the tumor is HER 21 +, HER 22 +, or HER 23 + as determined by Immunohistochemistry (IHC).

48. The method of claim 47, wherein the tumor expresses 2+ or lower levels of HER2 as determined by IHC.

49. The method of claim 43, wherein the HER2+ tumor is a HER 22 +/3+ expressing ovarian cancer and is gene amplified and moderately sensitive to trastuzumab as determined by IHC.

50. The method of claim 43, wherein the HER2+ tumor is a breast cancer expressing HER2 at a level of 2+ or less as determined by Immunohistochemistry (IHC).

51. The method of claim 43, wherein the HER2+ tumor is (i) HER 23 + estrogen receptor negative (ER-), progesterone receptor negative (PR-), trastuzumab-resistant, chemotherapy-resistant invasive breast ductal carcinoma, (ii) HER 23 + ER-, PR-, trastuzumab-resistant inflammatory breast cancer, (iii) HER 23 +, ER-, PR-invasive ductal carcinoma, or (iv) trastuzumab and pertuzumab-resistant breast cancer with an amplified HER 22 + HER2 gene.

52. The method of claims 43-51, wherein the subject has not been previously treated with an anti-HER 2 antibody.

53. The method of claims 43-51, wherein the tumor is resistant to or refractory to pertuzumab, trastuzumab, and/or TDM 1.

54. The method of any one of claims 43-51, wherein the subject has been previously treated with pertuzumab, trastuzumab, and/or TDM 1.

55. The method of any one of claims 43-54, wherein the construct is selected from v5019, v10000, v7091, v5020, or v 6717.

56. The method of any one of claims 43-55, wherein administration is by injection or infusion.

57. The method of any one of claims 43-56, further comprising administering to the subject an additional agent, optionally a chemotherapeutic agent.

58. The method of claim 57, wherein

i. The tumor is non-small cell lung cancer and the additional agent is one or more of cisplatin, carboplatin, paclitaxel, albumin-bound paclitaxel, docetaxel, gemcitabine, vinorelbine, irinotecan, etoposide, vinblastine, or pemetrexed;

the tumor is gastric or gastric cancer and the additional agent is one or more of 5-fluorouracil (with or without folinic acid), capecitabine, carboplatin, cisplatin, docetaxel, epirubicin, irinotecan, oxaliplatin or paclitaxel;

The tumor is pancreatic cancer and the additional agent is one or more of gemcitabine, calcium folinate, abraxane, or 5-fluorouracil;

the tumor is estrogen and/or progesterone positive breast cancer and the additional agent is one or more of (a) a combination of doxorubicin and epirubicin, (b) a combination of paclitaxel and docetaxel, or (c) a combination of fluorouracil, cyclophosphamide and carboplatin;

v. the tumour is a head and neck cancer and the further agent is one or more of paclitaxel, carboplatin, doxorubicin or cisplatin;

the tumour is ovarian cancer and the further agent is one or more of cisplatin, carboplatin or a taxane such as paclitaxel or docetaxel.

59. The method of claims 43-58, wherein the subject is human.

60. A method of detecting or measuring HER2 in a sample, comprising contacting the sample with the antigen binding construct of claims 1-38 and detecting or measuring binding complexes.

61. A method of inhibiting, reducing, or blocking HER2 signaling in a cell, comprising administering to the cell an effective amount of the antigen binding construct of claims 1-38.

62. A method of killing a HER 2-expressing tumor cell or inhibiting growth of a HER 2-expressing tumor cell comprising contacting the cell with the antigen binding construct of claims 1-38.

63. The method of claim 62, wherein the tumor cell is a HER 21 + or 2+ human pancreatic cancer cell, HER 23 + human lung cancer cell, HER 22 + caucasian bronchioloalveolar carcinoma cell, human pharyngeal cancer cell, HER 22 + human tongue squamous cell carcinoma cell, HER 22 + pharyngeal squamous cell carcinoma cell, HER 21 + or 2+ human colorectal cancer cell, HER 23 + human gastric cancer cell, HER 21 + human mammary duct ER + (estrogen receptor positive) cancer cell, HER 22 +/3+ human ER +, HER 2-amplified breast cancer cell, HER 20 +/1+ human triple negative breast cancer cell, HER 22 + human endometrial cancer cell, HER 21 + lung metastatic malignant melanoma cell, HER 21 + human cervical cancer cell, HER 21 + human renal cell cancer cell, or HER 21 + human ovarian cancer cell.

64. The method of claim 62, wherein the tumor cell is a HER 21 + or 2+ or 3+ human pancreatic cancer cell, a HER 22 + metastatic pancreatic cancer cell, a HER 20 +/1+, +3+ human lung cancer cell, a HER 22 + caucasian bronchioloalveolar carcinoma cell, a HER 20 + anaplastic lung cancer cell, a human non-small cell lung cancer cell, a human pharyngeal cancer cell, a HER 22 + human tongue squamous cell carcinoma cell, a HER 22 + pharyngeal squamous cell carcinoma cell, a HER 21 + or 2+ human colorectal cancer cell, a HER 20 +, 1+ or 3+ human gastric cancer cell, a HER 21 + human breast ductal ER + (estrogen receptor positive) cancer cell, a HER 22 +/3+ human ER +, a HER2 amplified breast cancer cell, a HER 20 +/1+ human triple negative breast cancer cell, a HER 20 + human breast ductal carcinoma (basal B type, mesenchymal triple negative) cell, a HER 22 + ER + breast cancer cell, a HER 20 + metastatic cell (ER-) -human lung cancer cell, HER 2-amplification, luminal a, TN), human uteroblastomere (mixed grade III) cells, 2+ human endometrial cancer cells, HER 21 + human dermal epidermoid cancer cells, HER 21 + lung metastatic malignant melanoma cells, HER 21 + malignant melanoma cells, human cervical epidermoid cancer cells, HER 21 + human bladder cancer cells, HER 21 + human cervical cancer cells, HER 21 + human renal cell cancer cells, or HER 21 +, 2+ or 3+ human ovarian cancer cells, and wherein the antigen-binding construct is coupled to maytansine (DM 1).

65. The method of claim 62, wherein the tumor cell is a cell selected from the group consisting of: pancreatic tumor cell lines BxPC3, Capan-1, MiaPaca 2; lung tumor cell lines Calu-3, NCI-H322; head and neck tumor cell lines Detroit 562, SCC-25, FaDu; colorectal tumor cell lines HT29, SNU-C2B; gastric tumor cell line NCI-N87; breast tumor cell lines MCF-7, MDAMB175, MDAMB361, MDA-MB-231, BT-20, JIMT-1, SkBr3, BT-474; uterine tumor cell line TOV-112D; the skin tumor cell line Malme-3M; cervical tumor cell lines Caski, MS 751; bladder tumor cell line T24; ovarian tumor cell lines CaOV3 and SKOV 3.

66. The method of claim 62, wherein the tumor cell is a cell selected from the group consisting of: pancreatic tumor cell lines BxPC3, Capan-1, MiaPaca2, SW 1990, Panc 1; lung tumor cell lines A549, Calu-3, Calu-6, NCI-H2126, and NCI-H322; head and neck tumor cell lines Detroit 562, SCC-15, SCC-25, FaDu; colorectal tumor cell lines Colo201, DLD-1, HCT116, HT29, SNU-C2B; gastric tumor cell lines SNU-1, SNU-16, NCI-N87; breast tumor cell lines SkBr3, MCF-7, MDAMB175, MDAMB361, MDA-MB-231, ZR-75-1, BT-20, BT549, BT-474, CAMA-1, MDAMB453, JIMT-1, T47D; uterine tumor cell lines SK-UT-1, TOV-112D; skin tumor cell lines a431, Malme-3M, SKEMEL 28; cervical tumor cell lines Caski, MS 751; bladder tumor cell line T24; the renal tumor cell line ACHN; ovarian tumor cell lines CaOV3, Ovar-3, and SKOV 3; and wherein the antigen binding construct is coupled to maytansine.

67. The method of claim 62, wherein the tumor cell is selected from the group consisting of HER 22/3 +, gene amplified ovarian cancer cell; HER 20 +/1+ triple negative breast cancer cells; ER +, HER 21 + breast cancer cells; trastuzumab-resistant HER 22 + breast cancer cells; ER +, HER2+ breast cancer cells; or HER 23 + breast cancer cells.

68. A method of producing the construct of claims 1-38, comprising culturing a host cell under conditions suitable for expression of the antigen binding construct, wherein the host cell comprises a polynucleotide encoding the antigen binding construct of claims 1-38; and purifying the construct.

69. An isolated polynucleotide or collection of isolated polynucleotides comprising at least one nucleic acid sequence encoding at least one polypeptide of an antigen binding construct of claims 1-38.

70. The polynucleotide of claim 69, wherein the polynucleotide or collection of polynucleotides is a cDNA.

71. A polynucleotide or isolated collection of polynucleotides encoding v5019, v7091, v10000, v5020, or v 6717.

72. A vector or collection of vectors comprising one or more of the polynucleotides or collection of polynucleotides according to claims 69-71.

73. A vector or collection of vectors comprising one or more of the set of nucleotides or polynucleotides according to claim 72 selected from the group consisting of a plasmid, a viral vector, a non-episomal mammalian vector, an expression vector, and a recombinant expression vector.

74. An isolated cell comprising a polynucleotide or set of polynucleotides according to claims 69-71, or a vector or set of vectors according to claim 72 or 73.

75. The isolated cell of claim 74, which is a hybridoma cell, a Chinese Hamster Ovary (CHO) cell, or a HEK293 cell.

76. A kit comprising the construct of any one of claims 1-38 and instructions for use.

77. The construct of claim 35, wherein the construct is afucosylated.

Claims

1. An antigen binding construct comprising

6. The antigen binding construct of any one of the preceding claims, wherein

i. The first antigen-binding polypeptide construct is a Fab and comprises

a. A first variable heavy chain polypeptide VH1 comprising the VH of the pertuzumab arm of v5019, v5020 v7091, v6717 or v10000) and

b. a first variable light chain polypeptide VL1 comprising the VL of the pertuzumab arm of v5019, v5020 v7091, v6717, or v 10000); and is

The second antigen-binding polypeptide construct is an scFv and comprises

(a) A second variable heavy chain polypeptide VH2 comprising the VH of the trastuzumab arm of v5019, v5020 v7091, v6717 or v10000) and

(b) a second variable light chain polypeptide VL2 comprising the VL of the trastuzumab arm of v5019, v5020 v7091, v6717, or v 10000);

The first antigen-binding polypeptide construct is an scFv and comprises

(a) A first variable heavy chain polypeptide VH1 comprising the VH of the pertuzumab arm of v5019, v5020 v7091, v6717 or v10000) and

(b) a first variable light chain polypeptide VL1 comprising the VL of the pertuzumab arm of v5019, v5020 v7091, v6717 or v10000) and

the second antigen-binding polypeptide construct is a Fab and comprises

(b) a second variable light chain polypeptide VL2 comprising the VL of the trastuzumab arm of v5019, v5020 v7091, v6717, or v 10000); or

The first antigen-binding polypeptide construct is an scFv and comprises

(a) A first variable heavy chain polypeptide VH1 comprising the VH of the pertuzumab arm of v5019, v5020 v7091, v6717 or v10000), and

(b) a first variable light chain polypeptide VL1 comprising the VL of the pertuzumab arm of v5019, v5020 v7091, v6717 or v10000), and

the second antigen-binding polypeptide construct is an scFv and comprises

(b) a second variable light chain polypeptide VL2 comprising the VL of the trastuzumab arm of v5019, v5020 v7091, v6717 or v 10000).

(i) Inducing enhanced receptor internalization and/or in HER 23 + cells

A heterodimeric IgG1 Fc with the modification L351Y _ S400E _ F405A _ Y405V in the first Fc polypeptide and the modification T350V _ T366L _ K392L _ T394W in the second Fc polypeptide,

37. The construct of claim 36, wherein the drug is maytansine (DM 1).

40. The pharmaceutical composition of claim 36, the pharmaceutical carrier comprising a buffer, an antioxidant, a low molecular weight molecule, a drug, a protein, an amino acid, a carbohydrate, a lipid, a chelator, a stabilizer, or an excipient.

58. The method of claim 57, wherein

59. The method of claims 43-58, wherein the subject is human.

77. The construct of claim 35, wherein the construct is afucosylated.