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WO2023141714A1 - Methods of using anti-her2 biparatopic antibody-drug conjugates in the treatment of cancer - Google Patents

Methods of using anti-her2 biparatopic antibody-drug conjugates in the treatment of cancer Download PDF

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Publication number
WO2023141714A1
WO2023141714A1 PCT/CA2023/050104 CA2023050104W WO2023141714A1 WO 2023141714 A1 WO2023141714 A1 WO 2023141714A1 CA 2023050104 W CA2023050104 W CA 2023050104W WO 2023141714 A1 WO2023141714 A1 WO 2023141714A1
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Prior art keywords
her2
cancer
seq
set forth
adc
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PCT/CA2023/050104
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French (fr)
Inventor
Joseph Edwin WOOLERY
Neil C. JOSEPHSON
Rupert H. DAVIES
Kevin HAMBLETT
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Zymeworks Bc Inc.
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Publication of WO2023141714A1 publication Critical patent/WO2023141714A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
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    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6863—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from stomach or intestines cancer cell
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6875—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin
    • A61K47/6879—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin the immunoglobulin having two or more different antigen-binding sites, e.g. bispecific or multispecific immunoglobulin
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • C—CHEMISTRY; METALLURGY
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    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
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Definitions

  • the present disclosure relates to the field of cancer therapeutics and, in particular, to methods of treating cancer with antibody-drug conjugates comprising a biparatopic anti-HER2 antibody and an auri statin analogue.
  • HER2 is a transmembrane surface-bound receptor tyrosine kinase that is a member of the ErbB family of receptor tyrosine kinases and is normally involved in the signal transduction pathways leading to cell growth and differentiation.
  • HER2 is overexpressed in about one-quarter of breast cancer patients (Bange et al, Nature Medicine 7:548 (2001)) and, as such, is an important target for treatment of breast cancer.
  • Herceptin® (trastuzumab, U.S. Patent No. 5,821,337) was the first monoclonal antibody developed for the treatment of HER2 -positive breast cancer.
  • Pertuzumab (Perjeta®, U.S. Patent No. 7,862,817) is a humanized monoclonal antibody, which was designed specifically to prevent the HER2 receptor from pairing (dimerizing) with other HER receptors (EGFR/HER1, HER3 and HER4) on the surface of cells, a process that is believed to play a role in tumor growth and survival.
  • Pertuzumab binds to domain II of HER2, essential for dimerization, while trastuzumab binds to extracellular domain IV of HER2.
  • the combination of Perjeta®, Herceptin® and chemotherapy is thought to provide a more comprehensive blockade of HER signaling pathways.
  • Auristatins are synthetic analogues of dolastatin 10, which is a potent microtubule inhibitor with anti -cancer activity.
  • Antibody-drug conjugates comprising auristatin payloads, such as monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF), have been described (U.S. PatentNos. 7,498,298 and 7,659,241; International Patent Application Publication Nos. WO 2002/088172 and WO 2016/041082).
  • International Patent Application Publication No. WO 2106/041082 describes N-acyl sulfonamide modified auristatins and their use as antibody-drug conjugate payloads.
  • the present disclosure relates to a method treating a subject having a HER2-expressing cancer comprising administering to the subject an effective amount of an anti-HER2 biparatopic antibody drug conjugate (ADC), the effective amount comprising a dose of between 1 mg/kg and 2.0 mg/kg administered every week (QW) for 3 weeks of a 4-week cycle, or a dose of between 2 mg/kg and 3 mg/kg administered every 3 weeks (Q3W), wherein the anti-HER2 biparatopic ADC has general Formula (II): wherein: n is the average drug-to-antibody ratio (DAR) and is between about 1.5 and about 2.5, and
  • DAR drug-to-antibody ratio
  • Ab is an anti-HER2 biparatopic antibody comprising a first antigen-binding domain that binds to an epitope on ECD2 of HER2 and a second anti gen -binding domain that binds to an epitope on ECD4 of HER2.
  • the present disclosure relates to a method of treating a subject having a HER2-expressing cancer comprising administering to the subject an effective amount of an anti- HER2 biparatopic antibody drug conjugate (ADC), the effective amount comprising a dose of 1.25 mg/kg administered every week (QW) for 3 weeks of a 4-week cycle, wherein the anti-HER2 biparatopic ADC has general Formula (II):
  • n is the average drug-to-antibody ratio (DAR) and is between about 1.8 and about 2.3
  • Ab is an anti-HER2 biparatopic antibody comprising (a) a first antigen-binding domain comprising the CDR sequences as set forth in SEQ ID NOs: 32, 34 and 33, and in SEQ ID NOs: 22, 24 and 23, and (b) a second antigen-binding domain comprising the CDR sequences as set forth in SEQ ID NOs: 56, 57 and 58, and SEQ ID NOs: 53, 54 and 55.
  • the present disclosure relates to a method of treating a subject having a
  • HER2-expressing cancer comprising administering to the subject an effective amount of an anti- HER2 biparatopic antibody drug conjugate (ADC), the effective amount comprising a dose of 2.5 mg/kg administered every 3 weeks (Q3W), wherein the anti-HER2 biparatopic ADC has general Formula (II): n is the average drug-to-antibody ratio (DAR) and is between about 1.8 and about 2.3, and Ab is an anti-HER2 biparatopic antibody comprising (a) a first antigen-binding domain comprising the CDR sequences as set forth in SEQ ID NOs: 32, 34 and 33, and in SEQ ID NOs: 22, 24 and 23, and (b) a second antigen-binding domain comprising the CDR sequences as set forth in SEQ ID NOs: 56, 57 and 58, and SEQ ID NOs: 53, 54 and 55.
  • ADC anti-HER2 biparatopic antibody drug conjugate
  • DAR drug-to-antibody ratio
  • Ab is an anti-HER2 biparatopic antibody compris
  • the present disclosure relates to a pharmaceutical kit comprising: i) one or more containers containing an anti-HER2 biparatopic antibody drug conjugate (ADC) having general Formula (II): wherein: n is the average drug-to-antibody ratio (DAR) and is between about 1.5 and about 2.5, and
  • ADC anti-HER2 biparatopic antibody drug conjugate
  • Ab is an anti-HER2 biparatopic antibody comprising a first antigen-binding domain that binds to an epitope on ECD2 of HER2 and a second anti gen -binding domain that binds to an epitope on ECD4 of HER2, and ii) a label or package insert on or associated with the one or more containers indicating the anti-HER2 biparatopic ADC is for administration to a subject having a HER2-expressing cancer at a dose of 1.25 mg/kg, 1.5 mg/kg or 1.75 mg/kg administered every week (QW) for 3 weeks of a 4-week cycle, or a dose of 2.5 mg/kg administered every 3 weeks (Q3W).
  • FIGS. 1A-1D show the extracellular ATP levels (as determined by RLU fluorescence) induced by 24 hr treatment of HCC1954 cells (FIG. 1A), SKOV-3 cells (FIG. IB), JIMT-1 cells (FIG. 1C) and MDA-MB-468 cells (FIG. ID) with various ADCs, including the anti-HER2 biparatopic ADC v21252, at EC99 concentration.
  • FIGS. 2A-2C show the extracellular HMGB1 levels (as fold increase over untreated control) induced by 48 hr treatment of SK-BR-3 cells (FIG. 2A), NCI-N87 cells (FIG. 2B) and MDA-MB-468 (FIG. 2C) with various ADCs, including the anti-HER2 biparatopic ADC v21252, at EC99 concentration.
  • FIGS. 3A-3C show the levels of cell surface calreticulin (CRT) (% CRT + cells) induced by 24 hr treatment (FIG. 3A), 48 hr treatment (FIG. 3B) or 72 hr treatment (FIG. 3C) of SK-BR- 3 and MDA-MB-468 cells with various ADCs, including the anti-HER2 biparatopic ADC v21252.
  • CRT cell surface calreticulin
  • FIG. 4 shows the Cmax and Cmin profiles derived by pharmacokinetic modeling for administration of anti-HER2 biparatopic ADC v21252 at 1 mg/kg weekly (QW) (on Days 1, 8 and 15 of a 28 day cycle), 2 mg/kg every 2 weeks (Q2W) and 2.5 mg/kg every 3 weeks (Q3W).
  • FIGS. 5A-5B show the Cmax and Cmin profiles derived by pharmacokinetic modeling for administration of anti-HER2 biparatopic ADC v21252 at 3 mg/kg every 3 weeks (Q3W) vs.
  • FIG. 5A 1.5 mg/kg weekly (QW) (on Days 1 , 8 and 15 of a 28 day cycle)
  • QW Q3 W
  • FIG. 6 presents an overview of the study design for a Phase 1 study of anti-HER2 biparatopic ADC v21252 in HER2-positive cancer.
  • GEA gastroesophageal adenocarcinoma
  • QW once every week
  • Q2W once every 2 weeks
  • Q3W once every 3 weeks
  • MTD maximum tolerated dose
  • RD recommended dose.
  • FIG. 7 presents Table 7.2 showing the treatment related adverse events (TRAEs) observed during the Phase 1 study of anti-HER2 biparatopic ADC v21252 in HER2-positive cancer in 77 patients treated at 2.5 mg/kg every 3 weeks (Q3W). Values are number of patients (%).
  • FIG. 8 presents a waterfall plot showing the change in sum of target lesions observed during the Phase 1 study of anti-HER2 biparatopic ADC v21252 in HER2 -positive cancer in patients treated with the ADC at 2.5 mg/kg every 3 weeks (Q3W).
  • BTC biliary tract cancer
  • CRC colorectal cancer
  • GEA gastroesophageal adenocarcinoma
  • NSCLC non-small cell lung cancer.
  • FIG. 9 presents a swimmer plot showing treatment duration, response, prior treatment and HER2 status by immunohistochemistry (IHC) and/or fluorescent in situ hybridization (FISH) for patients treated with anti-HER2 biparatopic ADC v21252 at 2.5 mg/kg every 3 weeks (Q3W) in the Phase 1 study of the ADC in HER2-positive cancer.
  • IHC immunohistochemistry
  • FISH fluorescent in situ hybridization
  • BTC biliary tract cancer
  • cPR confirmed partial response
  • CRC colorectal cancer
  • D T-DXd
  • FISH fluorescent in situ hybridization
  • GEA gastroesophageal adenocarcinoma
  • I investigational
  • IHC immunohistochemistry
  • K T-DM1
  • L lapatinib
  • M margetuximab
  • N neratinib
  • NE not evaluable
  • NSCLC non-small cell lung cancer
  • P pertuzumab
  • PD progressive disease
  • PR partial response
  • SD stable disease
  • T trastuzumab
  • Tx treatment
  • U tucatinib
  • Z zanidatamab.
  • the present disclosure relates to methods of treating a HER2-expressing (or “HER2- positive”) cancer with an anti-HER2 biparatopic antibody-drug conjugate (ADC) in which the drug is an auristatin analogue conjugated to the anti-HER2 biparatopic antibody at an average drug-to- antibody ratio (DAR) between about 1.5 and about 2.5.
  • the anti-HER2 biparatopic ADC is administered to a subject having a HER2- expressing cancer at a dose between 1 mg/kg and 2 mg/kg weekly (QW) or between 2 mg/kg and 3 mg/kg every 3 weeks (Q3W).
  • the anti-HER2 biparatopic ADC is administered in combination with a PD-1 inhibitor, for example, an anti -PD-1 antibody. Definitions
  • subject refers to a human patient who is the object of treatment and/or observation.
  • the term “about” refers to an approximately +/-10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.
  • compositions, use or method denotes that additional elements and/or method steps may be present, but that these additions do not materially affect the manner in which the recited composition, method or use functions.
  • Consisting of when used herein in connection with a composition, use or method, excludes the presence of additional elements and/or method steps.
  • a composition, use or method described herein as comprising certain elements and/or steps may also, in certain embodiments consist essentially of those elements and/or steps, and in certain embodiments consist of those elements and/or steps, whether or not these embodiments are specifically referred to.
  • the terms “derived from” and “based on” when used with reference to a recombinant amino acid sequence mean that the recombinant amino acid sequence is substantially identical to the sequence of the corresponding reference amino acid sequence.
  • an Ig Fc amino acid sequence that is derived from (or based on) a wild-type Ig Fc sequence is substantially identical to (e.g. shares at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with) the corresponding wild-type Ig Fc sequence.
  • Anti-HER2 biparatopic ADCs for use in the methods of the present disclosure comprise an anti-HER2 biparatopic antibody conjugated to an auristatin analogue having structure 1 (also referred to herein as “Compound 1”) via a linker (L) at a low drug-to-antibody ratio (DAR).
  • Compound 1 an auristatin analogue having structure 1 (also referred to herein as “Compound 1”) via a linker (L) at a low drug-to-antibody ratio (DAR).
  • auristatin analogue having structure 1 may also be referred to herein using the terms “drug,” “toxin,” or “auristatin analogue.”
  • Low DAR is defined as an average DAR of between about 1.5 and about 2.5.
  • the average DAR of the anti-HER2 biparatopic ADCs is between about 1.6 and about 2.5. In some embodiments, the average DAR of the anti-HER2 biparatopic ADCs is between about 1.7 and about 2.5. In some embodiments, the average DAR of the anti- HER2 biparatopic ADCs is between about 1.8 and about 2.5. In some embodiments, the average DAR of the anti-HER2 biparatopic ADCs is between about 1.8 and about 2.4, or between about 1.8 and about 2.3. In some embodiments, the average DAR of the anti-HER2 biparatopic ADCs is between about 1.9 and about 2.2. In some embodiments, the average DAR of the anti-HER2 biparatopic ADCs is about 2.0. In some embodiments, the average DAR of the anti-HER2 biparatopic ADCs is 2.0 + 0.2.
  • anti-HER2 biparatopic ADCs that include a proportion of DAR0 species above a certain threshold may be advantageous. Accordingly, in some embodiments, the anti-HER2 biparatopic ADCs may include between about 5% and about 50% DAR0 species. In some embodiments, the anti-HER2 biparatopic ADCs may include between about 10% and about 50% DAR0 species, for example, between about 10% and about 40%, between about 10% and about 30%, or between about 10% and about 25% DAR0 species. In some embodiments, the anti- HER2 biparatopic ADCs may include between about 12% and about 28% DAR0 species, for example, between about 15% and about 28% DAR0 species, or between about 15% and about 25% DAR0 species.
  • the anti-HER2 biparatopic ADCs have general Formula (I): wherein: L is a cleavable linker; n is the average DAR and is between about 1.5 and about 2.5, and Ab is an anti-HER2 biparatopic antibody.
  • L is a protease-cleavable linker.
  • L is a peptide-containing linker.
  • L comprises a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, He-Cit and Trp-Cit.
  • L comprises a dipeptide and a stretcher.
  • n is between about 1.6 and about 2.5. In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (I), n is between about 1.8 and about 2.5. In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (I), n is between about 1.8 and about 2.4. In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (I), n is between about 1.8 and about 2.3. In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (I), n is between about 1.9 and about 2.2. In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (I), n is about 2.0. In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (I), n is 2.0 + 0.2.
  • linker L in anti-HER2 biparatopic ADCs of Formula (I) is conjugated to the antibody via a cysteine residue on the antibody.
  • linker L in the anti-HER2 biparatopic ADCs of general Formula (I) is conjugated to the antibody via a cysteine residue on the antibody that has been liberated by reduction of an interchain disulfide bond.
  • anti-HER2 biparatopic ADCs of general Formula (I) are also contemplated and each combination forms a separate embodiment for the purposes of the present disclosure.
  • the anti-HER2 biparatopic ADCs have general Formula (II): n is the average DAR, and
  • Ab is an anti-HER2 biparatopic antibody.
  • n is between about 1.6 and about 2.5. In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (II), n is between about 1.8 and about 2.5. In some embodiments, in the anti- HER2 biparatopic ADCs of general Formula (II), n is between about 1.8 and about 2.4. In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (II), n is between about 1.8 and about 2.3. In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (II), n is between about 1.9 and about 2.2. In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (II), n is about 2.0. In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (II), n is 2.0 + 0.2.
  • the anti-HER2 biparatopic antibody is linked to the auristatin analogue by a linker (L).
  • Linkers are bifunctional or multifunctional moieties capable of linking one or more drug molecules to an antibody.
  • a linker may be bifunctional (or monovalent) such that it links a single drug to a single site on the antibody, or it may be multifunctional (or polyvalent) such that it links more than one drug molecule to a single site on the antibody.
  • Linkers capable of linking one drug molecule to more than one site on the antibody may also be considered to be multifunctional.
  • Attachment of a linker to an antibody can be accomplished in a variety of ways, such as through surface lysines on the antibody, reductive-coupling to oxidized carbohydrates on the antibody, or through cysteine residues on the antibody liberated by reducing interchain disulfide linkages.
  • attachment of a linker to an antibody may be achieved by modification of the antibody to include additional cysteine residues (see, for example, U.S. Patent Nos.
  • non-natural amino acids that provide reactive handles, such as selenomethionine, p-acetylphenylalanine, formylglycine or p-azidomethyl-L-phenylalanine (see, for example, Hofer et al., Biochemistry, 48: 12047-12057 (2009); Axup et al., PNAS, 109: 16101- 16106 (2012); Wu et al., PNAS, 106:3000-3005 (2009); Zimmerman et al., Bioconj. Chem., 25:351-361 (2014)), to allow for site-specific conjugation.
  • selenomethionine such as selenomethionine, p-acetylphenylalanine, formylglycine or p-azidomethyl-L-phenylalanine
  • Linkers include at least one functional group capable of reacting with the target group or groups on the antibody, and one or more functional groups capable of reacting with a target group on the drug.
  • Suitable functional groups are known in the art and include those described, for example, in Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press).
  • target groups on the antibody to which a linker may be conjugated include the thiol groups of cysteine residues and the amine groups of lysine residues.
  • functional groups for reacting with free cysteines or thiols include maleimide, haloacetamide, haloacetyl, activated esters (such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters and tetrafluorophenyl esters), anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates.
  • Non-limiting examples of functional groups for reacting with free amines include activated esters (such as N- hydroxysuccinamide (NHS) esters and sulfo-NHS esters), imido esters (such as Traut’s reagent), isothiocyanates, aldehydes and acid anhydrides (such as diethylenetriaminepentaacetic anhydride (DTP A)).
  • activated esters such as N- hydroxysuccinamide (NHS) esters and sulfo-NHS esters
  • imido esters such as Traut’s reagent
  • isothiocyanates such as aldehydes and acid anhydrides (such as diethylenetriaminepentaacetic anhydride (DTP A)).
  • TSTU succinimido-l,l,3,3-tetra- methyluronium tetrafluoroborate
  • PyBOP benzotriazol- 1-yl-oxytripyrrolidinophosphonium hexafluorophosphate
  • Other linkers include those having a functional group that allows for bridging of two interchain cysteines on the antibody, such as a ThioBridgeTM linker (Badescu et al., Bioconjug.
  • a linker may comprise one or more linker components.
  • a linker will comprise two or more linker components.
  • Exemplary linker components include functional groups for reaction with the antibody, functional groups for reaction with the drug, stretchers, peptide components, self-immolative groups, self-elimination groups, hydrophilic moieties, and the like.
  • Various linker components are known in the art, some of which are described below.
  • Certain useful linker components can be obtained from various commercial sources, such as Pierce Biotechnology, Inc. (now Thermo Fisher Scientific Corporation, Waltham, MA) and Molecular Biosciences Inc. (Boulder, Colo.), or may be synthesized in accordance with procedures described in the art (see, for example, Toki et al., J. Org. Chem., 67: 1866-1872 (2002); Dubowchik, et al., Tetrahedron Letters, 38:5257-60 (1997); Walker, M. A., J. Org. Chem., 60:5352-5355 (1995); Frisch, et al., Bioconjugate Chem., 7: 180-186 (1996); U.S. Patent Nos. 6,214,345 and 7,553,816, and International Patent Application Publication No. WO 02/088172).
  • linker components include, but are not limited to, N- (P-maleimidopropyloxy)-N-hydroxy succinimide ester (BMPS), N-(s-maleimidocaproyloxy) succinimide ester (EMCS), N-[y-maleimidobutyryloxy]succinimide ester (GMBS), 1,6-hexane- bis-vinylsulfone (HBVS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxy-(6- amidocaproate) (LC-SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N- maleimidophenyl)butyric acid hydrazide (MPBH), succinimidyl 3-(bromoacetamido)propionate (SBAP), succinimidyl iodoacetate (SIA), succinimidyl
  • Additional examples include bis-maleimide reagents such as dithiobismaleimidoethane (DTME), bis-maleimido-tri oxyethylene glycol (BMPEO), 1,4-bismaleimidobutane (BMB), 1,4 bismaleimidyl-2,3-dihydroxybutane (BMDB), bismaleimidohexane (BMH), bismaleimidoethane (BMOE), BM(PEG)2 and BM(PEG)s; bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as dithi
  • Suitable linkers typically are more chemically stable to conditions outside the cell than to conditions inside the cell, although less stable linkers may be contemplated in certain situations, such as when the drug is selective or targeted and has a low toxicity to normal cells.
  • Linkers may be “cleavable linkers” or “non-cleavable linkers.”
  • a cleavable linker is typically susceptible to cleavage under intracellular conditions, for example, through lysosomal processes. Examples include linkers that are protease-sensitive, acid-sensitive, reduction-sensitive or photolabile.
  • Non- cleavable linkers by contrast, rely on the degradation of the antibody in the cell, which typically results in the release of an amino acid-linker-toxin moiety.
  • linker L comprised by the anti-HER2 biparatopic ADCs of Formula (I) is a cleavable linker.
  • Suitable cleavable linkers include, for example, linkers comprising a peptide component that includes two or more amino acids and is cleavable by an intracellular protease, such as lysosomal protease or an endosomal protease.
  • a peptide component may comprise amino acid residues that occur naturally and/or minor amino acids and/or non- naturally occurring amino acid analogues, such as citrulline.
  • Peptide components may be designed and optimized for enzymatic cleavage by a particular enzyme, for example, a tumor-associated protease, cathepsin B, C or D, or a plasmin protease.
  • linker L comprised by the anti-HER2 biparatopic ADCs of Formula (I) may be a peptide-containing linker.
  • linker L comprised by the anti-HER2 biparatopic ADCs may be a dipeptide-containing linker, such as a linker containing valine-citrulline (Val-Cit) or phenylalanine-lysine (Phe-Lys).
  • suitable dipeptides for inclusion in linker L include Val-Lys, Ala-Lys, Me-Val-Cit, Phe-homoLys, Phe- Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Arg, Ala-Phe, Vai-Ala, Met-Lys, Asn-Lys, Ile-Pro, Ile-Val, Asp-Val, His-Val, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, NorVal-(D)Asp, Ala-(D)Asp, MesLys- Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys and Met-(D)Lys.
  • Cleavable linkers may also include longer peptide components such as tripeptides, tetrapeptides or pentapeptides. Examples include, but are not limited to, the tripeptides Met-Cit-Val, Gly-Cit-Val, (D)Phe-Phe- Lys and (D)Ala-Phe-Lys, and the tetrapeptides Gly-Phe-Leu-Gly and Ala-Leu- Ala-Leu.
  • linker L comprised by the anti-HER2 biparatopic ADCs may be a peptide- containing linker, where the peptide is between two and five amino acids in length.
  • cleavable linkers include disulfide-containing linkers, such as, for example, N-succinimydyl-4-(2-pyridyldithio) butanoate (SPBD) and N-succinimydyl-4-(2- pyridyldithio)-2-sulfo butanoate (sulfo-SPBD).
  • Disulfide-containing linkers may optionally include additional groups to provide steric hindrance adjacent to the disulfide bond in order to improve the extracellular stability of the linker, for example, inclusion of a geminal dimethyl group.
  • linkers include linkers hydrolyzable at a specific pH or within a pH range, such as hydrazone linkers.
  • Linkers comprising combinations of these functionalities may also be useful, for example, linkers comprising both a hydrazone and a disulfide are known in the art.
  • a further example of a cleavable linker is a linker comprising a P -glucuronide, which is cleavable by P-glucuronidase, an enzyme present in lysosomes and tumor interstitium (see, for example, De Graaf et al., Curr. Pharm. Des., 8: 1391-1403 (2002)).
  • Cleavable linkers may optionally further comprise one or more additional components such as self-immolative and self-elimination groups, stretchers or hydrophilic moieties.
  • Self-immolative and self-elimination groups that find use in linkers include, for example, p-aminobenzyloxycarbonyl (PABC) and p-aminobenzyl ether (PABE) groups, and methylated ethylene diamine (MED).
  • PABC p-aminobenzyloxycarbonyl
  • PABE p-aminobenzyl ether
  • MED methylated ethylene diamine
  • Other examples of self-immolative groups include, but are not limited to, aromatic compounds that are electronically similar to the PABC or PABE group such as heterocyclic derivatives, for example 2-aminoimidazol-5-methanol derivatives as described in U.S. Patent No. 7,375,078.
  • Stretchers that find use in linkers for ADCs include, for example, alkylene groups and stretchers based on aliphatic acids, diacids, amines or diamines, such as diglycolate, malonate, caproate and caproamide.
  • Other stretchers include, for example, glycine-based stretchers, polyethylene glycol (PEG) stretchers and monomethoxy polyethylene glycol (mPEG) stretchers. PEG and mPEG stretchers also function as hydrophilic moieties.
  • cleavable linkers examples include, but are not limited to, SPBD, sulfo-SPBD, hydrazone, Val-Cit, maleidocaproyl (MC), MC-Val-Cit, MC-Val- Cit-PABC, Phe-Lys, MC-Phe-Lys, MC-Phe-Lys-PABC, maleimido triethylene glycolate (MT), MT-Val-Cit, MT-Phe-Lys and adipate (AD).
  • linker L included in the anti-HER2 biparatopic ADCs of the present disclosure is a peptide-based linker having Formula (V):
  • Z is a functional group capable of reacting with a target group on the anti-HER2 biparatopic antibody
  • Str is a stretcher
  • AAi and AA2 are each independently an amino acid, wherein AAi-[AA2] r forms a protease cleavage site;
  • X is a self-immolative group; s is 0 or 1; m is 1, 2, 3 or 4; o is 0, 1 or 2;
  • % is the point of attachment to the rest of the molecule.
  • m is 1, 2 or 3.
  • s is 1.
  • o is 0 (z.e. X is absent).
  • Str is selected from:
  • each R is independently H or Ci-Ce alkyl; each p is independently an integer between 2 and 10; each q is independently an integer between 1 and 10, $ is the point of attachment to Z, and
  • Str is: where p, q, $ and * are as defined above.
  • Str is: where $ and * are as defined above, p is an integer between 2 and 6, and q is an integer between 2 and 8.
  • AAi-[AA2] m is selected from Val-Lys,
  • m is 1 (i.e. AAi-[AA2] m is a dipeptide).
  • AAi-[AA2] m is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit.
  • Str is $ — ( CH 2)p— C— or $ — (CH 2 CH 2 O) q — (CH 2 ) p — C— where $ is the point of attachment to Z, * is the point of attachment to the remainder of the linker, p is an integer between 2 and 6, and q is an integer between 2 and 8; m is 1 and AAi-[AA2] m is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit; s is 1, and o is 0.
  • the linker included in the anti-HER2 biparatopic ADCs of the present disclosure has general Formula (VI): where:
  • Y is one or more additional linker components, or is absent, and D is the point of attachment to the auristatin analogue.
  • Y is absent.
  • the linker included in the anti-HER2 biparatopic ADCs of the present disclosure has general Formula (VII):
  • * is the point of attachment to the anti-HER2 biparatopic antibody;
  • Y is one or more additional linker components, or is absent, and
  • D is the point of attachment to the auristatin analogue.
  • Y is absent.
  • the auristatin analogue-linker (or “drug-linker”) has the following structure: where * is the point of attachment to the anti-HER2 biparatopic antibody.
  • the ADCs described herein comprise an anti-HER2 biparatopic antibody that binds to two different epitopes of HER2.
  • the term “antibody,” as used herein, generally refers to a proteinaceous binding molecule with immunoglobulin-like functions. Typical examples of an antibody are immunoglobulins, as well as derivatives or functional fragments thereof which still retain binding specificity. Techniques for the production of antibodies are well known in the art.
  • the term “antibody” may also include immunoglobulins of different classes (i.e.
  • an antibody examples include whole antibodies and antigen-binding fragments thereof, such as Fab fragments, F(ab') 2 , Fv fragments, single-chain Fv fragments (scFv), diabodies, domain antibodies, and combinations thereof.
  • Domain antibodies may be single domain antibodies, single variable domain antibodies or immunoglobulin single variable domain having only one variable domain, which may be a heavy chain variable domain or a light chain variable domain, that specifically bind an antigen or epitope independently of other variable regions or domains.
  • the term “antibody” also includes embodiments such as chimeric, single chain and humanized antibodies.
  • a typical whole antibody comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region comprises three domains: CHI, CH2 and CH3.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are known as a (IgA), 6 (IgD), 8 (IgE), y (IgG) and p (IgM).
  • Each light chain is comprised of a light chain variable region (VL) and a light chain constant region.
  • the light chain constant region comprises just one domain: CL. Light chains are classified as either kappa or lambda.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDR), interspersed with regions that are more conserved, termed framework regions (FW).
  • CDR Complementarity Determining Regions
  • FW framework regions
  • Each VH and VL is composed of three CDRs and four FWs, arranged from amino-terminus to carboxy-terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4.
  • the variable regions of the heavy and light chains contain a binding domain (a paratope) that interacts with an antigen.
  • the constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and Clq, which is a component of the complement system.
  • the anti-HER2 biparatopic antibodies comprised by the ADCs described herein comprise two antigen-binding polypeptide constructs (also referred to as antigenbinding domains), each of which binds to a different epitope of HER2.
  • antigen-binding polypeptide construct and “antigen-binding domain,” as used interchangeably herein, refer to an immunoglobulin-based construct, for example, an antibody fragment.
  • the antigen-binding polypeptide constructs comprised by the anti-HER2 biparatopic antibody are antibody fragments.
  • the antigen-binding polypeptide constructs comprised by the anti-HER2 biparatopic antibody may each independently be a Fab fragment, a Fab’ fragment, an scFv or an sdAb.
  • the antigen -binding polypeptide constructs comprised by the anti-HER2 biparatopic antibody may each independently be a Fab fragment or an scFv.
  • one antigen-binding polypeptide construct comprised by the anti-HER2 biparatopic antibody may be a Fab fragment and the other antigen-binding polypeptide construct may be an scFv.
  • At least one of the antigen-binding polypeptide constructs comprised by the anti-HER2 biparatopic antibody may be a Fab fragment or a Fab’ fragment.
  • a “Fab fragment” contains the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CHI) along with the variable domains of the light and heavy chains (VL and VH, respectively).
  • Fab' fragments differ from Fab fragments by the addition of a few amino acid residues at the C-terminus of the heavy chain CHI domain, including one or more cysteines from the antibody hinge region.
  • an antigen-binding polypeptide construct comprised by the anti-HER2 biparatopic antibody may be a single-chain Fab molecule, i.e. a Fab molecule in which the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain.
  • the C-terminus of the Fab light chain may be connected to the N-terminus of the Fab heavy chain in the single-chain Fab molecule.
  • At least one of the antigen-binding polypeptide constructs comprised by the anti-HER2 biparatopic antibody may be a single-chain Fv (scFv).
  • An “scFv” includes a heavy chain variable domain (VH) and a light chain variable domain (VL) of an antibody in a single polypeptide chain.
  • the scFv may optionally further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form a desired structure for antigen binding.
  • an scFv may include a VL connected from its C-terminus to the N- terminus of a VH by a polypeptide linker.
  • an scFv may comprise a VH connected through its C-terminus to the N-terminus of a VL by a polypeptide chain or linker.
  • the two antigen-binding polypeptide constructs comprised by the anti-HER2 biparatopic antibody each bind to a different epitope of HER2, that is, a first antigen-binding polypeptide construct binds to a first HER2 epitope and a second antigen-binding polypeptide construct binds to a second HER2 epitope.
  • each of the antigen-binding polypeptide constructs specifically binds to its target epitope.
  • binds or “specific binding” mean that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions.
  • the ability of an antigen-binding polypeptide construct to bind to a specific epitope can be measured, for example, through an enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) techniques (analyzed on a BIAcoreTM instrument) (Liljeblad ei al. Glyco J 17, 323-329 (2000)) or traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • the antigen-binding polypeptide construct is considered to specifically bind to its target epitope when the extent of binding of the antigen-binding polypeptide construct to an unrelated protein is less than about 10% of the binding of the antigen-binding polypeptide construct to its target epitope as measured, for example, by SPR.
  • HER2 also known as ErbB2 refers to human HER2 protein described, for example, in Semba et al, PNAS (USA), 82:6497-6501 (1985) and Yamamoto et al., Nature, 319:230-234 (1986) (GenBank accession number X03363).
  • the terms “erbB2” and “neu” refer to the gene encoding human HER2 protein.
  • the terms p 185 or p 185neu may also be used to refer to the protein product of the neu gene.
  • HER2 comprises an extracellular domain, which typically binds a HER ligand, a lipophilic transmembrane domain, a conserved intracellular tyrosine kinase domain and a carboxyterminal signaling domain harboring several tyrosine residues which can be phosphorylated.
  • the extracellular (ecto) domain of HER2 comprises four domains, Domains I-IV.
  • the sequence of HER2 is provided in Table 1 (SEQ ID NO:2).
  • the Extracellular Domain (ECD) boundaries are: Domain I - approximately amino acids 1-165; Domain II - approximately amino acids 166-322; Domain III - approximately amino acids 323-488, and Domain IV - approximately amino acids 489-607.
  • Epitopope 2C4 is the region in the extracellular domain of HER2 to which the antibody 2C4 binds and comprises residues from Domain II in the extracellular domain of HER2 (also referred to as ECD2). 2C4 and Pertuzumab bind to the extracellular domain of HER2 at the junction of Domains I, II and III (Franklin et al. Cancer Cell 5:317-328 (2004)).
  • Epitope 4D5 is the region in the extracellular domain of HER2 to which the antibody 4D5 (ATCC CRL 10463) and trastuzumab bind. This epitope is close to the transmembrane domain of HER2, and within Domain IV of HER2 (also referred to as ECD4).
  • the anti-HER2 biparatopic antibody of the present disclosure will bind to epitopes within the extracellular domains of HER2.
  • the first and second HER2 epitopes bound by the first and second antigen-binding domains of the anti-HER2 biparatopic antibody are non-overlapping epitopes.
  • the first and second HER2 epitopes bound by the first and second antigen-binding domains of the anti-HER2 biparatopic antibody are on different extracellular domains of HER2.
  • a first antigen-binding domain of the anti-HER2 biparatopic antibody binds to a first HER2 epitope on a first domain of HER2, and a second antigen-binding domain binds to a second HER2 epitope on a second domain of HER2.
  • the first domain of HER2 is ECD2 and the second domain of HER2 is ECD4.
  • one of the antigen-binding domains comprised by the anti-HER2 biparatopic antibody competes with trastuzumab for binding to HER2.
  • one of the antigen-binding domains comprised by the anti-HER2 biparatopic antibody competes with pertuzumab for binding to HER2. In some embodiments, one of the antigen -binding domains comprised by the anti-HER2 biparatopic antibody competes with trastuzumab for binding to HER2, and the other antigen-binding domain competes with pertuzumab for binding to HER2.
  • one of the antigen-binding domains comprised by the anti-HER2 biparatopic antibody is in a Fab or scFv format and competes with trastuzumab for binding to HER2, and the other antigen-binding domain is in a Fab or scFv format and competes with pertuzumab for binding to HER2.
  • one of the anti gen -binding domains comprised by the anti-HER2 biparatopic antibody is in an scFv format and competes with trastuzumab for binding to HER2, and the other antigen-binding domain is in a Fab format and competes with pertuzumab for binding to HER2.
  • one of the antigen-binding domains comprised by the anti-HER2 biparatopic antibody binds to the same epitope on HER2 as trastuzumab. In some embodiments, one of the antigen-binding domains comprised by the anti-HER2 biparatopic antibody binds to the same epitope on HER2 as pertuzumab. In some embodiments, one of the anti gen -binding domains comprised by the anti-HER2 biparatopic antibody binds to the same epitope on HER2 as trastuzumab, and the other antigen-binding domain binds to the same epitope on HER2 as pertuzumab.
  • one of the antigen-binding domains comprised by the anti-HER2 biparatopic antibody comprises the CDR sequences of trastuzumab or a variant thereof comprising one or more mutations known to increase HER2 binding
  • the other antigen-binding domain comprises the CDRs of pertuzumab or a variant thereof comprising one or more mutations known to increase HER2 binding.
  • the anti-HER2 biparatopic antibody is one of the biparatopic antibodies described in U.S. Patent Application Publication No. 2016/0289335.
  • the anti-HER.2 biparatopic antibody is one of v5019, v5020, v7091, vlOOOO, v6902, v6903 or v6717 (see Tables 4, 5 and 6, and Sequence Tables).
  • one of the antigen-binding domains of the anti-HER2 biparatopic antibody comprises the CDR sequences from the ECD2-binding arm of one of v5019, v5020, v7091, vlOOOO, v6902, v6903 or v6717.
  • one of the antigen-binding domains of the anti-HER2 biparatopic antibody comprises the CDR sequences from the ECD2- binding arm of one of v5019, v5020, v7091, vlOOOO, v6902, v6903 or v6717
  • the other antigen-binding domain comprises the CDR sequences from the ECD4-binding arm of one of v5019, v5020, v7091, vlOOOO, v6902, v6903 or v6717.
  • one of the antigen- binding domains of the anti-HER2 biparatopic antibody comprises the CDR sequences from the ECD2-binding arm of vlOOOO, and the other antigen-binding domain comprises the CDR sequences from the ECD4-binding arm of vlOOOO.
  • one of the antigen-binding domains of the anti-HER2 biparatopic antibody comprises a VH sequence and a VL sequence from the ECD2-binding arm of one of v5019, v5020, v7091, vlOOOO, v6902, v6903 or v6717.
  • one of the antigenbinding domains of the anti-HER2 biparatopic antibody comprises a VH sequence and a VL sequence from the ECD2-binding arm of one of v5019, v5020, v7091, vlOOOO, v6902, v6903 or v6717
  • the other antigen-binding domain comprises a VH sequence and a VL sequence from the ECD4-binding arm of one of v5019, v5020, v7091, vlOOOO, v6902, v6903 or v6717.
  • one of the antigen-binding domains of the anti-HER2 biparatopic antibody comprises a VH sequence and a VL sequence from the ECD2-binding arm of vlOOOO
  • the other antigen -binding domain comprises a VH sequence and a VL sequence from the ECD4- binding arm of vlOOOO.
  • the anti-HER2 biparatopic antibody comprised by the ADCs comprises (a) a first antigen-binding domain comprising the CDR sequences as set forth in SEQ ID NOs: 32, 34 and 33, and in SEQ ID NOs: 22, 24 and 23, and (b) a second antigen-binding domain comprising the CDR sequences as set forth in SEQ ID NOs: 53, 54 and 55, and SEQ ID NOs: 56, 57 and 58.
  • the anti-HER2 biparatopic antibody comprised by the ADCs comprises (a) a first antigen -binding domain that is a Fab and comprises the CDR sequences as set forth in SEQ ID NOs: 32, 34 and 33, and in SEQ ID NOs: 22, 24 and 23, and (b) a second antigen- binding domain that is an scFv and comprises the CDR sequences as set forth in SEQ ID NOs: 53, 54 and 55, and SEQ ID NOs: 56, 57 and 58.
  • the anti-HER2 biparatopic antibody comprised by the ADCs comprises (a) a first antigen-binding domain comprising a first set of CDRs comprising the CDR1 sequence as set forth in SEQ ID NO: 32, the CDR2 sequence as set forth in SEQ ID NO: 34 and the CDR3 sequence as set forth in SEQ ID NO: 33, and a second set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 22, the CDR2 sequence as set forth in SEQ ID NO: 24 and the CDR3 sequence as set forth in SEQ ID NO: 23, and (b) a second anti gen -binding domain comprising a third set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 53, the CDR2 sequence as set forth in SEQ ID NO: 54 and the CDR3 sequence as set forth in SEQ ID NO: 55, and a fourth set of CDR sequences comprising the CDR1 sequence as set forth in SEQ
  • the anti-HER2 biparatopic antibody comprised by the ADCs comprises (a) a first heavy chain (Hl) comprising the CDR sequences as set forth in SEQ ID NOs: 32, 34 and 33, (b) a second heavy chain (H2) comprising the CDR sequences as set forth in SEQ ID NOs: 53, 54, 55, 56, 57 and 58, and (c) a light chain (LI) comprising the CDR sequences as set forth in SEQ ID NOs: 22, 24 and 23.
  • Hl first heavy chain
  • H2 second heavy chain
  • LI light chain
  • the anti-HER2 biparatopic antibody comprised by the ADCs comprises (a) a first heavy chain (Hl) comprising a first set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 32, the CDR2 sequence as set forth in SEQ ID NO: 34 and the CDR3 sequence as set forth in SEQ ID NO: 33, (b) a second heavy chain (H2) comprising a second set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 53, the CDR2 sequence as set forth in SEQ ID NO: 54, and the CDR3 sequence as set forth in SEQ ID NO: 55, and a third set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 56, the CDR2 sequence as set forth in SEQ ID NO: 57 and the CDR3 sequence as set forth in SEQ ID NO: 58, and a light chain (LI) comprising a fourth set of CDR sequences comprising
  • the anti-HER2 biparatopic antibody comprised by the ADCs comprises (a) a first antigen -binding domain comprising the VH sequence as set forth in SEQ ID NO: 31, and the VL sequence as set forth in SEQ ID NO: 21, and (b) a second antigen-binding domain comprising the VH sequence as set forth in SEQ ID NO: 52, and the VL sequence as set forth in SEQ ID NO: 51.
  • the anti-HER2 biparatopic antibody comprised by the ADCs comprises (a) a first antigen-binding domain that is a Fab and comprises the VH sequence as set forth in SEQ ID NO: 31, and the VL sequence as set forth in SEQ ID NO: 21, and (b) a second antigen-binding domain that is an scFv and comprises the VH sequence as set forth in SEQ ID NO: 52, and the VL sequence as set forth in SEQ ID NO: 51.
  • the anti-HER2 biparatopic antibody comprised by the ADCs comprises a first heavy chain (Hl) comprising the VH sequence as set forth in SEQ ID NO: 31, a second heavy chain (H2) comprising the VH sequence as set forth in SEQ ID NO: 52 and the VL sequence as set forth in SEQ ID NO: 51, and a light chain (LI) comprising the VL sequence as set forth in SEQ ID NO: 21.
  • the anti-HER2 biparatopic antibody comprised by the ADCs comprises (a) a first heavy chain (Hl) comprising the sequence as set forth in SEQ ID NO: 30, a second heavy chain (H2) comprising the sequence as set forth in SEQ ID NO: 50, and a light chain (LI) comprising the sequence as set forth in SEQ ID NO: 20.
  • Hl first heavy chain
  • H2 second heavy chain
  • LI light chain
  • the anti-HER2 biparatopic antibody comprised by the ADCs comprises (a) a first heavy chain (Hl) consisting of the sequence as set forth in SEQ ID NO: 30, a second heavy chain (H2) consisting of the sequence as set forth in SEQ ID NO: 50, and a light chain (LI) consisting of the sequence as set forth in SEQ ID NO: 20.
  • Hl first heavy chain
  • H2 second heavy chain
  • LI light chain
  • the anti-HER2 biparatopic antibodies comprised by the ADCs described herein may have various formats.
  • the minimal components of the anti-HER2 biparatopic antibody are a first antigen-binding domain that binds to a first HER2 epitope and a second antigen-binding domain that binds to a second HER2 epitope, with the first and second HER2 epitopes being different.
  • An antibody that comprises two antigen-binding domains that bind to different HER2 epitopes may be considered to be a bivalent, biparatopic antibody.
  • Antibodies that comprise one or more additional antigen-binding domains, each of which binds to either the first or second HER2 epitope, are also biparatopic, but are considered to be trivalent or tetravalent, for example.
  • the anti-HER2 biparatopic antibody comprised by the ADC is a bivalent, anti-HER2 biparatopic antibody.
  • the anti-HER2 biparatopic antibody comprises a scaffold to which first and second antigen-binding domains are operably linked.
  • the term “operably linked,” as used herein, means that the components described are in a relationship permitting them to function in their intended manner. Suitable scaffolds are described below.
  • the anti-HER2 biparatopic antibody comprises two antigen-binding domains operably linked to a scaffold, and at least one of the antigen-binding domains is an scFv.
  • the anti-HER2 biparatopic antibody comprises two antigen-binding domains operably linked to a scaffold, and at least one of the antigen-binding domains is a Fab.
  • the anti- HER2 biparatopic antibody comprises two antigen-binding domains operably linked to a scaffold, where one of the antigen-binding domains is an scFv and the other antigen-binding domain is a Fab.
  • Suitable scaffolds include, but are not limited to, immunoglobulin Fc regions, albumin, albumin analogs and derivatives, heterodimerizing peptides (such as leucine zippers, heterodimer-forming “zipper” peptides derived from Jun and Fos, IgG CHI and CL domains or barnase-barstar toxins), cytokines, chemokines or growth factors.
  • Other examples include antibodies based on the DOCK-AND-LOCKTM (DNLTM) technology developed by IBC Pharmaceuticals, Inc. and Immunomedics, Inc. (see, for example, Chang, etal., 2007, Clin Cancer Res., 13:5586s-5591s).
  • the anti-HER2 biparatopic antibody comprises a scaffold that is based on an immunoglobulin Fc region, an albumin or an albumin analogue or derivative (such as those described in International Patent Application Publication No. WO 2012/116453 or WO 2014/012082).
  • the anti-HER2 biparatopic antibody comprises a protein scaffold that is based on an immunoglobulin (Ig) Fc region.
  • the anti-HER2 biparatopic antibody comprises a protein scaffold that is based on an IgG Fc region.
  • Fc region refers to a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).
  • Ig Fc regions are typically dimeric and composed of two Fc polypeptides.
  • An “Fc polypeptide” of a dimeric Fc refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising one or more C-terminal constant regions of an immunoglobulin heavy chain that is capable of stable self-association.
  • the terms “first Fc polypeptide” and “second Fc polypeptide” may be used interchangeably to describe the Fc polypeptides comprised by a dimeric Fc region, provided that the Fc region comprises one first Fc polypeptide and one second Fc polypeptide.
  • An Fc region comprises a CH3 domain or both a CH3 and a CH2 domain.
  • an Fc polypeptide of a dimeric IgG Fc region comprises an IgG CH2 and an IgG CH3 constant domain sequence.
  • the CH3 domain comprises two CH3 sequences, one from each of the two Fc polypeptides of the dimeric Fc region.
  • the CH2 domain comprises two CH2 sequences, one from each of the two Fc polypeptides of the dimeric Fc region.
  • the anti-HER2 biparatopic antibody comprised by the ADCs may comprise a scaffold that is based on an IgG Fc region. In some embodiments, the anti-HER2 biparatopic antibody may comprise a scaffold that is based on a human Fc region. In some embodiments, the anti-HER2 biparatopic antibody may comprise a scaffold based on a human IgG Fc region, for example a human IgGl Fc region.
  • the anti-HER2 biparatopic antibody comprised by the ADCs may comprise a scaffold based on an IgG Fc region, which is a heterodimeric Fc region, comprising a first Fc polypeptide and a second Fc polypeptide, each comprising a CH3 sequence, and optionally a CH2 sequence.
  • the anti-HER2 biparatopic antibody comprised by the ADCs may comprise a scaffold based on an Fc region which comprises first and second Fc polypeptides, and the first antigen-binding domain is operably linked to the first Fc polypeptide and the second antigen-binding domain is operably linked to the second Fc polypeptide.
  • the anti-HER2 biparatopic antibody comprised by the ADCs may comprise a scaffold based on an Fc region which comprises first and second Fc polypeptides, in which the first antigen-binding domain is operably linked to the first Fc polypeptide and the second antigen-binding domain is operably linked to the second Fc polypeptide, and in which the first and second antigen-binding domains are independently a Fab fragment or an scFv.
  • the anti-HER2 biparatopic antibody comprised by the ADCs may comprise a scaffold based on an Fc region which comprises two CH3 sequences, at least one of which comprises one or more amino acid modifications.
  • the anti-HER2 biparatopic antibody comprises a heterodimeric Fc region comprising a modified CH3 domain, wherein the modified CH3 domain is an asymmetrically modified CH3 domain.
  • the first Fc polypeptide of the heterodimeric Fc comprises a first CH3 sequence and the second Fc polypeptide comprises a second CH3 sequence.
  • asymmetric amino acid modification refers to a modification where an amino acid at a specific position on a first CH3 sequence is different to the amino acid on a second CH3 sequence at the same position.
  • first and second CH3 sequence will typically preferentially pair to form a heterodimer, rather than a homodimer.
  • These asymmetric amino acid modifications can be a result of modification of only one of the two amino acids at the same respective amino acid position on each sequence, or different modifications of both amino acids on each sequence at the same respective position on each of the first and second CH3 sequences.
  • Each of the first and second CH3 sequence of a heterodimeric Fc may comprise one or more than one asymmetric amino acid modification.
  • the anti-HER2 biparatopic antibody may comprise a scaffold based on a modified Fc region as described in International Patent Application Publication No. WO 2012/058768 or WO 2013/063702.
  • Table 7 provides the amino acid sequence of the human IgGl Fc sequence (SEQ ID NO: 1), corresponding to amino acids 231 to 447 of the full-length human IgGl heavy chain.
  • the CH3 sequence comprises amino acids 341-447 of the full-length human IgGl heavy chain.
  • the anti-HER2 biparatopic antibody may comprise a heterodimeric Fc scaffold comprising a modified CH3 domain that comprises asymmetric amino acid modifications that promote formation of a heterodimeric Fc over formation of a homodimeric Fc.
  • the anti-HER2 biparatopic antibody may comprise a heterodimeric Fc scaffold which includes modifications as described below at one or more of the following positions: L351, F405, Y407, T366, K392, T394, T350, S400 and/or N390, using EU numbering.
  • the anti-HER2 biparatopic antibody comprised by the ADCs may comprise a heterodimeric Fc comprising a modified CH3 domain having a first polypeptide sequence comprising amino acid modifications at positions F405 and Y407, and optionally further comprising an amino acid modification at position L351, and a second polypeptide sequence comprising amino acid modifications at positions T366 and T394, and optionally further comprising an amino acid modification at position K392, where the amino acid modification at position F405 is F405A, F405I, F405M, F405S, F405T or F405V; the amino acid modification at position Y407 is Y407I or Y407V; the amino acid modification at position T366 is T366I, T366L or T366M; the amino acid modification at position T394 is T394W; the amino acid modification at position L351 is L351Y, and the amino acid modification at position K392 is K392F, K
  • the amino acid modification at position F405 is F405A, F405S, F405T or F405V.
  • one or both of the first and second Fc polypeptide sequences further comprises the amino acid modification T350V.
  • the first Fc polypeptide sequence further comprises an amino acid modification at one or both of positions S400 or Q347 and/or the second Fc polypeptide sequence further comprises an amino acid modification at one or both of positions K360 or N390, where the amino acid modification at position S400 is S400E, S400D, S400R or S400K; the amino acid modification at position Q347 is Q347R, Q347E or Q347K; the amino acid modification at position K360 is K360D or K360E, and the amino acid modification at position N390 is N390R, N390K or N390D.
  • the anti-HER2 biparatopic antibody comprised by the ADCs may comprise a heterodimeric Fc scaffold having a modified CH3 domain comprising the modifications of any one of Variant 1, Variant 2, Variant 3, Variant 4 or Variant 5, as shown in Table 7.
  • Table 7 IgGl Fc sequences
  • the anti-HER2 biparatopic antibody comprised by the ADCs may comprise a heterodimeric Fc scaffold having a modified CH3 domain with a first CH3 sequence comprising the amino acid modifications L351Y, F405A, and Y407V, and the second CH3 sequence comprising the amino acid modifications T366L or T366I; K392L or K392M, and T394W, and one or both of the first and second CH3 sequences may optionally further comprise the amino acid modification T350V.
  • the anti-HER2 biparatopic antibody comprised by the ADCs described herein may be produced using standard recombinant methods known in the art (see, for example, "Antibodies: A Laboratory Manual,” 2 nd Edition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014, and International Patent Application Publication No. WO 2015/077891).
  • nucleic acid For recombinant expression, appropriate host cells are transformed with an isolated nucleic acid encoding the anti-HER2 biparatopic antibody and cultured under conditions allowing for expression of the anti-HER2 biparatopic antibody.
  • nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the anti- HER2 biparatopic antibody (for example, the light and/or heavy chains of the antibody).
  • many amino acid acids are encoded by more than one codon and as such multiple nucleic acids may encode a single polypeptide sequence.
  • An exemplary nucleic acid is provided herein for each polypeptide of the anti-HER2 biparatopic antibody, however, it is understood that other nucleic acids encoding the anti-HER2 biparatopic antibody may be employed.
  • the nucleic acid encoding the anti-HER2 biparatopic antibody may be provided in the form of an expression vector or vectors.
  • the nucleic acid is provided in a multi ci str onic vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the anti-HER2 biparatopic antibody and an amino acid sequence comprising the VH of the anti-HER2 biparatopic antibody.
  • the nucleic acid is provided as a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the anti-HER2 biparatopic antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the anti-HER2 biparatopic antibody.
  • the anti-HER2 biparatopic antibody is produced in stable mammalian cells by a method comprising: transfecting stable mammalian cells with nucleic acid encoding the anti-HER2 biparatopic antibody in a predetermined ratio; and expressing the nucleic acid in the mammalian cells.
  • the predetermined ratio of nucleic acid is determined in transient transfection experiments to determine the relative ratio of input nucleic acids that results in the highest percentage of the anti-HER2 biparatopic antibody in the expressed product.
  • the anti-HER2 biparatopic antibody is produced in stable mammalian cells where the expression product of the mammalian cells comprises a larger percentage of the glycosylated anti-HER2 biparatopic antibody as compared to the monomeric heavy or light chain polypeptides, or other antibodies.
  • the anti-HER2 biparatopic antibody is purified after expression.
  • Proteins may be isolated or purified in a variety of ways known to those skilled in the art (see, for example, Protein Purification: Principles and Practice, 3 rd Ed., Scopes, Springer-Verlag, NY, 1994).
  • Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reverse-phase, carried out at atmospheric pressure or at high pressure using systems such as FPLC and HPLC.
  • Additional purification methods include electrophoretic, immunological, precipitation, dialysis and chromatofocusing techniques. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful.
  • the bacterial proteins A and G bind to the Fc region.
  • the bacterial protein L binds to the Fab region of some antibodies.
  • purification of the anti-HER2 biparatopic antibody comprises anion exchange chromatography, for example, using a Q SepharoseTM, diethylaminoethyl (DEAE) SepharoseTM, POROSTM HQ, POROSTM DEAF, Toyopearl® Q, Toyopearl® QAE, Toyopearl® DEAE, Resource/Source Q, Resource/Source DEAE, Fractogel® Q or Fractogel® DEAE column, or equivalent.
  • Q SepharoseTM diethylaminoethyl (DEAE) SepharoseTM
  • POROSTM HQ POROSTM DEAF
  • Toyopearl® Q Toyopearl® QAE
  • Toyopearl® DEAE Toyopearl® DEAE
  • Resource/Source Q Resource/Source DEAE
  • Fractogel® Q or Fractogel® DEAE column or equivalent.
  • purification of the anti-HER2 biparatopic antibody comprises cation exchange chromatography, for example, using an SP SepharoseTM, CM SepharoseTM, POROSTM HS, POROSTM CM, Toyopearl® SP, Toyopearl® CM, Resource/Source S, Resource/Source CM, Fractogel® S or Fractogel® CM column, or equivalent.
  • the anti-HER2 biparatopic ADCs of the present disclosure may be prepared by one of several routes known in the art, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art (see, for example, Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press, and the Examples provided herein).
  • conjugation may be achieved by (1) reaction of a functional group on the antibody with a bifunctional linker to form an antibody -linker intermediate Ab-L, via a covalent bond, followed by reaction with an activated form of the auristatin analogue, or (2) reaction of a functional group on the auristatin analogue with a linker to form linker-toxin, via a covalent bond, followed by reaction with a functional group on the antibody.
  • Suitable functional groups on the antibody include, for example, side-chain amino groups of lysine residues and thiol groups of cysteine residues.
  • Suitable functional groups may also be provided by the side-chains of non-natural amino acids, such as selenomethionine, p-acetylphenylalanine, formylglycine or p-azidomethyl-L-phenylalanine, in those antibodies engineered to include such amino acids.
  • non-natural amino acids such as selenomethionine, p-acetylphenylalanine, formylglycine or p-azidomethyl-L-phenylalanine
  • linker-toxin and “drug-linker” are used interchangeably herein to refer to a moiety comprising the auristatin analogue covalently bonded to a linker.
  • the linker-toxin typically includes a functional group suitable for reaction with the selected target group on the anti-HER2 biparatopic antibody.
  • the anti-HER2 biparatopic ADCs of the present disclosure comprise an auristatin analogue conjugated via an appropriate linker to the thiol group of a cysteine residue on the antibody.
  • the auristatin analogue is conjugated via an appropriate linker to the thiol group of a cysteine residue that has been liberated by reducing an interchain disulfide bond.
  • the anti-HER2 biparatopic antibody is conjugated to the auristatin analogue via a linker at a low average drug-to-antibody ratio (DAR), such as an average DAR of between about 1.5 and about 2.5.
  • DAR drug-to-antibody ratio
  • a partial reduction of the antibody interchain disulfide bonds may be conducted followed by conjugation to linker-toxin.
  • Partial reduction can be achieved by limiting the amount of reducing agent used in the reduction reaction (see, for example, the Examples provided herein, and Lyon et al., Methods in Enzymology, 502: 123-138 (2012), and examples therein).
  • Suitable reducing agents are known in the art and include, for example, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), 2- mercaptoethanol, cysteamine and a number of water soluble phosphines.
  • TCEP tris(2-carboxyethyl)phosphine
  • 2- mercaptoethanol cysteamine
  • cysteamine and a number of water soluble phosphines.
  • fewer equivalents of linker-toxin may be employed in the conjugation reaction in order to obtain a low average DAR.
  • Low average DAR may also be achieved by employing an engineered antibody in which one or more of the cysteine residues that make up the interchain disulfide bonds is replaced with a serine residue resulting in fewer available cysteine residues for conjugation (see McDonagh et al., Protein Eng. Des. Sei. PEDS, 19(7):299-307).
  • the engineered antibody can be treated with reducing agent and conjugated to linker-toxin using standard methods.
  • Another approach is to employ a bis-thiol linker that bridges two cysteines that normally make up an interchain disulfide bond.
  • Use of a bis-thiol linker that carries only one toxin molecule produces an ADC with a maximum DAR4 for a full-size antibody, if all four interchain disulfide bonds are reduced and replaced with the bis-thiol linker. Partial reduction of the interchain disulfide bonds and/or fewer equivalents of linker or linker-toxin may be used in conjunction with a bis-thiol linker in order to further reduce the DAR.
  • Various bis-thiol linkers are known in the art (see, for example, Badescu etal., Bioconjug.
  • Cysteine engineering approaches may also be employed in order to generate ADCs with a low average DAR. Such approaches involve engineering solvent-accessible cysteines into the antibody in order to provide a site-specific handle for conjugation.
  • a number of appropriate sites for introduction of a cysteine residue have been identified with the IgG structure, and include those described in Junutula, et al., J. Immunol Methods, 332(l-2):41-52 (2008); Junutula, et al., Nat. Biotechnol., 26(8), 925-932 (2008), and U.S. Patent Nos. 9,315,581; 9,000,130; 8,455,622; 8,507,654 and 7,521,541.
  • ADCs with a low average DAR may be isolated from an ADC preparation containing a mixture of DAR species using chromatographic separation techniques, such as hydrophobic interaction chromatography (see, for example, Hamblett, et al., Clin. Cancer Res., 10:7063-7070 (2004); Sun, et al., Bioconj Chem., 28: 1371-81 (2017); U.S. Patent Application Publication No. 2014/0286968).
  • ADC preparations with a low average DAR may also be generated by adding unconjugated (i.e. DAR0) antibody to preparations of ADC having an average DAR > 2.5.
  • DAR0 unconjugated antibody
  • the majority of conjugation methods yield an ADC preparation that includes various DAR species, with the reported DAR being the average of the individual DAR species. Accordingly, adding additional DAR0 species into an ADC preparation having a DAR > 2.5 will reduce the average DAR of the preparation.
  • ADCs that include a proportion of DAR0 species may be advantageous.
  • the low average DAR ADCs of the present disclosure may include at least 5% DAR0 species, for example, between about 5% and about 50% DAR0 species.
  • the low average DAR ADCs may include between about 10% and about 50% DAR0 species.
  • the low average DAR ADCs may include between about 10% and about 40%, for example, between about 10% and about 30% DAR0 species.
  • the average DAR for the ADCs may be determined by standard techniques such as UV/VIS spectroscopic analysis, ELISA-based techniques, chromatography techniques such as hydrophobic interaction chromatography (HIC), UV-MALDI mass spectrometry (MS) and MALDI-TOF MS.
  • chromatography techniques such as hydrophobic interaction chromatography (HIC), UV-MALDI mass spectrometry (MS) and MALDI-TOF MS.
  • distribution of drug-linked forms for example, the fraction of DAR0, DARI, DAR2, etc.
  • MS with or without an accompanying chromatographic separation step
  • HPLC reverse-phase HPLC
  • IEF iso-electric focusing gel electrophoresis
  • the average DAR of the ADCs is determined by hydrophobic interaction chromatography (HIC) techniques.
  • HIC hydrophobic interaction chromatography
  • the ADCs may be purified and separated from unconjugated reactants and/or any conjugate aggregates by purification methods known in the art. Such methods include, but are not limited to, size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), ion exchange chromatography, chromatofocusing, ultrafiltration, centrifugal ultrafiltration, and combinations thereof.
  • Certain aspects of the present disclosure relate to methods of treating a HER2-expressing cancer in a subject by administering an effective amount of an anti-HER2 biparatopic ADC described herein.
  • a “HER2-expressing cancer” may also be referred to in the art as a “HER2- positive cancer” and includes cancers that express or overexpress HER2 protein with or without HER2 gene amplification.
  • HER2 protein expression may be identified, for example, by immunohistochemistry (IHC) techniques.
  • HER2 gene amplification may be identified, for example, by in situ hybridization (ISH) techniques.
  • the anti-HER2 biparatopic ADC is administered to the subject in an “effective amount.”
  • the effective amount of the anti-HER2 biparatopic ADC may be a dose of between 1 mg/kg and 2.5 mg/kg administered to the subject every week (QW), or a dose of between 2 mg/kg and 3 mg/kg administered to the subject every 3 weeks (Q3W).
  • the effective amount of the anti-HER2 biparatopic ADC administered to the subject in the disclosed methods may be a dose of between 1 mg/kg and 2.5 mg/kg administered QW for 3 weeks of a 4-week cycle, or a dose of between 2 mg/kg and 3 mg/kg administered Q3W.
  • the effective amount of the anti-HER2 biparatopic ADC administered to the subject in the methods of the present disclosure is a dose of between 1 mg/kg and 2 mg/kg administered QW for 3 weeks of a 4-week cycle. In some embodiments, the effective amount of the anti-HER2 biparatopic ADC administered to the subject in the methods of the present disclosure is a dose of between 1.25 mg/kg and 2 mg/kg administered QW for 3 weeks of a 4-week cycle. In some embodiments, the effective amount of the anti-HER2 biparatopic ADC administered to the subject in the disclosed methods is a dose of 1.25 mg/kg, 1.5 mg/kg, 1.75 mg/kg or 2.0 mg/kg administered QW for 3 weeks of a 4-week cycle.
  • the effective amount of the anti-HER2 biparatopic ADC administered to the subject in the disclosed methods is a dose of 1.25 mg/kg QW for 3 weeks of a 4-week cycle. In some embodiments, the effective amount of the anti-HER.2 biparatopic ADC administered to the subject in the disclosed methods is a dose of 1.5 mg/kg QW for 3 weeks of a 4-week cycle. In some embodiments, the effective amount of the anti-HER.2 biparatopic ADC administered to the subject in the disclosed methods is a dose of 1.75 mg/kg QW for 3 weeks of a 4-week cycle.
  • some embodiments of the present disclosure relate to methods of treating a subject having a HER2-expressing cancer comprising administering to the subject an effective amount of an anti-HER.2 biparatopic antibody drug conjugate (ADC), the effective amount comprising a dose of between 1 mg/kg and 2.0 mg/kg, for example, 1.25 mg/kg,
  • ADC anti-HER.2 biparatopic antibody drug conjugate
  • administering to the subject an anti-HER.2 biparatopic antibody drug conjugate (ADC) at a dose of 1.25 mg/kg administered QW for 3 weeks of a 4-week cycle results in a substantially equivalent Cmax, Cmin and/or exposure than is observed with a dose of 2.5 mg/kg administered Q3W.
  • ADC anti-HER.2 biparatopic antibody drug conjugate
  • the effective amount of the anti-HER.2 biparatopic ADC administered to the subject in the methods of the present disclosure is a dose of between 2 mg/kg and 3 mg/kg administered every 3 weeks (Q3W). In some embodiments, the effective amount of the anti-HER.2 biparatopic ADC administered to the subject in the disclosed methods is a dose of
  • the anti-HER.2 biparatopic ADC is administered to the subject intravenously.
  • the anti-HER.2 biparatopic antibody is administered to the subject by intravenous infusion.
  • the HER2-expressing cancer that may be treated using the methods of the present disclosure is a HER2-expressing solid tumor.
  • HER2-expressing solid tumors include, but are not limited to, breast cancer, endometrial cancer, ovarian cancer, cervical cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, anal cancer, urothelial cancer, pancreatic cancer, salivary gland cancer, bladder cancer and brain cancer.
  • HER2-expressing breast cancers include estrogen receptor negative (ER-) and/or progesterone receptor negative (PR-) breast cancers and triple negative (ER-, PR-, low HER2) breast cancers.
  • HER2-expressing lung cancers include non-small cell lung cancer (NSCLC) and small cell lung cancer.
  • the methods described herein are for the treatment of a HER2- expressing breast cancer, HER2-expressing gastroesophageal adenocarcinoma (GEA), HER2- expressing esophageal cancer, HER2-expressing endometrial cancer, HER2-expressing ovarian cancer, HER2-expressing cervical cancer, HER2-expressing NSCLC, HER2-expressing anal cancer or HER2-expressing colorectal cancer (CRC).
  • GAA gastroesophageal adenocarcinoma
  • HER2-expressing esophageal cancer HER2-expressing endometrial cancer
  • HER2-expressing ovarian cancer HER2-expressing cervical cancer
  • HER2-expressing NSCLC HER2-expressing anal cancer or HER2-expressing colorectal cancer (CRC).
  • the methods described herein are for the treatment of breast cancer, GEA, endometrial cancer, ovarian cancer, NSCLC, anal cancer, pancreatic cancer, biliary tract cancer or bladder cancer. In certain embodiments, the methods described herein are for the treatment of GEA, endometrial cancer, ovarian cancer, NSCLC or CRC.
  • the cancer may be HER2- expressing with or without HER2 gene amplification.
  • the methods described herein are for the treatment of a HER2- expressing cancer that is metastatic or locally advanced. In some embodiments, the methods described herein are for the treatment of a HER2-expressing cancer that has metastasized to the brain.
  • HER2-expressing (or HER2-positive) cancers may be characterized buy the level of HER2 they express (i.e. by “HER2-status”).
  • HER2 status can be assessed, for example, by immunohistochemistry (IHC), fluorescent in situ hybridization (FISH) and/or chromogenic in situ hybridization (CISH) techniques.
  • IHC immunohistochemistry
  • FISH fluorescent in situ hybridization
  • CISH chromogenic in situ hybridization
  • IHC HER2 protein expression on the cell membrane.
  • paraffin- embedded tissue sections from a tumor biopsy may be subjected to the IHC assay and accorded a HER2 staining intensity criteria as follows:
  • Score 0 no staining observed or membrane staining is observed in less than 10% of tumor cells; typically ⁇ 20,000 receptors/cell.
  • Score 1+ a faint/barely perceptible membrane staining is detected in more than 10% of the tumor cells. The cells are only stained in part of their membrane. Typically about 100,000 receptors/cell.
  • Score 2+ a weak to moderate complete membrane staining is observed in more than 10% of the tumor cells; typically about 500,000 receptors/cell.
  • Score 3+ a moderate to strong complete membrane staining is observed in more than 10% of the tumor cells; typically about 2,000,000 receptors/cell.
  • the anti-HER2 biparatopic ADCs described herein may be useful in methods of treating cancers that express HER2 at various levels.
  • the methods of treating HER2-expressing cancers according to the present disclosure comprise administering an anti- HER2 biparatopic ADC as described herein to a subject having a cancer that expresses high levels of HER2 (HER2-high), where HER2-high is defined as IHC 3+
  • the methods of treating HER2-expressing cancers according to the present disclosure comprise administering an anti-HER2 biparatopic ADC as described herein to a subject having a cancerthat expresses high levels of HER2 (HER2-high), where HER2-high is defined as IHC 2+, IHC 2+/3+ or IHC 3+.
  • the methods of treating HER2-expressing cancers according to the present disclosure comprise administering an anti-HER2 biparatopic ADC as described herein to a subject having a cancer that expresses low levels of HER2 (HER2-low), where HER2-low is defined as IHC 1+ or IHC 1+/2+.
  • ISH In situ hybridization
  • the methods of treating HER2-expressing cancers according to the present disclosure comprise administering an anti- HER2 biparatopic ADC as described herein to a subject having a HER2-expressing cancer having a score of IHC 3+ or IHC 2+/FISH+.
  • the methods described herein are for the treatment of a subject having a HER2-expressing cancer that is resistant or becoming resistant to other standard-of-care therapies.
  • the methods described herein are for the treatment of a subject having a HER2-expressing cancer who is unresponsive to, and/or has relapsed after, one or more current therapies, such as another anti-HER2 therapeutic (such as trastuzumab (Herceptin®), pertuzumab (Peijeta®), T-DM1 (Kadcyla® or trastuzumab emtansine), and the like) or a taxane (such as such as paclitaxel, docetaxel, cabazitaxel, and the like).
  • another anti-HER2 therapeutic such as trastuzumab (Herceptin®), pertuzumab (Peijeta®), T-DM1 (Kadcyla® or trastuzumab emtansine), and the like
  • a taxane
  • the methods described herein are for the treatment of a subject having a HER2-expressing cancer that is resistant to trastuzumab. In some embodiments, the methods described herein are for the treatment of a subject having cancer that has progressed after treatment with one or more other anti-HER2 therapeutics. In some embodiments, the methods described herein are for the treatment of a subject having metastatic cancer that has progressed on previous anti-HER2 therapy. In some embodiments, the methods described herein are for the treatment of a subject who has previously undergone treatment with one or more of trastuzumab, pertuzumab and T-DM1.
  • Certain embodiments of the present disclosure relate to a method of treating a HER2- expressing cancer in a subject by administering an effective amount of an anti-HER2 biparatopic ADC to the subject, in which the effective amount is a dose of 2.5 mg/kg administered Q3W, and the HER2-expressing cancer is a HER2-high cancer (IHC 3+, IHC 2+/3+ or IHC 2+).
  • Some embodiments relate to a method of treating a HER2-expressing cancer in a subject by administering an effective amount of an anti-HER2 biparatopic ADC to the subject, in which the effective amount is a dose of 2.5 mg/kg administered Q3W, and the HER2-expressing cancer is a HER2-low cancer (IHC 1+, IHC 1+/2+).
  • Some embodiments relate to a method of treating a HER2-expressing cancer in a subject by administering an effective amount of an anti-HER2 biparatopic ADC to the subject, in which the effective amount is a dose of 2.5 mg/kg administered Q3W, and the HER2-expressing cancer has a score of IHC 3+ or IHC 2+/FISH+.
  • Certain embodiments of the present disclosure relate to a method of treating a HER2- expressing cancer in a subject by administering an effective amount of an anti-HER2 biparatopic ADC to the subject, in which the effective amount is a dose of 1.25 mg/kg, 1.5 mg/kg or 1.75 mg/kg administered QW for 3 weeks of a 4-week cycle, and the HER2-expressing cancer is a HER2-high cancer (IHC 3+, IHC 2+/3+ or IHC 2+).
  • Some embodiments relate to a method of treating a HER2-expressing cancer in a subject by administering an effective amount of an anti- HER2 biparatopic ADC to the subject, in which the effective amount is a dose of 1.25 mg/kg, 1.5 mg/kg or 1.75 mg/kg administered QW for 3 weeks of a 4-week cycle, and the HER2-expressing cancer is a HER2-low cancer (IHC 1+, IHC 1+/2+).
  • Some embodiments relate to a method of treating a HER2-expressing cancer in a subject by administering an effective amount of an anti- HER2 biparatopic ADC to the subject, in which the effective amount is a dose of 1.25 mg/kg, 1.5 mg/kg or 1.75 mg/kg administered QW for 3 weeks of a 4-week cycle, and the HER2-expressing cancer has a score of IHC 3+ or IHC 2+/FISH+.
  • the present disclosure relates to methods of treating a HER2- expressing cancer by administering an effective amount of an anti-HER2 biparatopic ADC having general Formula (I), where the effective amount of the anti-HER2 biparatopic antibody is a dose of between 1 mg/kg and 2.5 mg/kg administered QW for 3 weeks of a 4-week cycle, or a dose of between 2 mg/kg and 3 mg/kg administered Q3W: wherein:
  • L in general Formula (I) is a protease- cleavable linker.
  • L in general Formula (I) is a peptide-containing linker.
  • L in general Formula (I) comprises a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit.
  • L in general Formula (I) comprises a dipeptide and a stretcher.
  • n in general Formula (I) (the average DAR) is between about 1.6 and about 2.5. In some embodiments of the disclosed methods, n in general Formula (I) is between about 1.8 and about 2.5. In some embodiments of the disclosed methods, n in general Formula (I) is between about 1.8 and about 2.4. In some embodiments of the disclosed methods, n in general Formula (I) is between about 1.8 and about 2.3. In some embodiments of the disclosed methods, n in general Formula (I) is between about 1.9 and about 2.2. In some embodiments of the disclosed methods, n in general Formula (I) is about 2.0. In some embodiments of the disclosed methods, n in general Formula (I) is 2.0 + 0.2.
  • linker L in the anti-HER2 biparatopic ADCs of Formula (I) is conjugated to the antibody via a cysteine residue on the antibody.
  • linker L in the anti-HER2 biparatopic ADCs of Formula (I) is conjugated to the antibody via a cysteine residue on the antibody that has been liberated by reduction of an interchain disulfide bond.
  • the anti-HER2 biparatopic ADCs have general Formula (II): n is the average DAR and is between about 1.5 and about 2.5, and Ab is an anti-HER2 biparatopic antibody.
  • n in general Formula (II) (the average DAR) is between about 1.6 and about 2.5. In some embodiments of the disclosed methods, n in general Formula (II) is between about 1.8 and about 2.5. In some embodiments of the disclosed methods, n in general Formula (II) is between about 1.8 and about 2.4. In some embodiments of the disclosed methods, n in general Formula (II) is between about 1.8 and about 2.3. In some embodiments of the disclosed methods, n in general Formula (II) is between about 1.9 and about 2.2. In some embodiments of the disclosed methods, n in general Formula (II) is about 2.0. In some embodiments of the disclosed methods, n in general Formula (II) is 2.0 + 0.2.
  • the anti-HER2 biparatopic ADC has a structure as described herein for any one of the embodiments of general Formula (I) or general Formula (II), and the anti-HER2 biparatopic antibody comprises (a) a first antigen-binding domain comprising the CDR sequences as set forth in SEQ ID NOs: 32, 34 and 33, and in SEQ ID NOs: 22, 24 and 23, and (b) a second antigen -binding domain comprising the CDR sequences as set forth in SEQ ID NOs: 53, 54 and 55, and in SEQ ID NOs: 56, 57 and 58.
  • the anti-HER2 biparatopic ADC has a structure as described herein for any one of the embodiments of general Formula (I) or general Formula (II), and the anti-HER2 biparatopic antibody comprises (a) a first antigen-binding domain that is a Fab and comprises the CDR sequences as set forth in SEQ ID NOs: 32, 34 and 33, and in SEQ ID NOs: 22, 24 and 23, and (b) a second antigen-binding domain that is an scFv and comprises the CDR sequences as set forth in SEQ ID NOs: 53, 54 and 55, and in SEQ ID NOs: 56, 57 and 58.
  • the anti-HER2 biparatopic ADC has a structure as described herein for any one of the embodiments of general Formula (I) or general Formula (II), and the anti-HER2 biparatopic antibody comprises (a) a first antigen-binding domain comprising a first set of CDRs comprising the CDR1 sequence as set forth in SEQ ID NO: 32, the CDR2 sequence as set forth in SEQ ID NO: 34 and the CDR3 sequence as set forth in SEQ ID NO: 33, and a second set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 22, the CDR2 sequence as set forth in SEQ ID NO: 24 and the CDR3 sequence as set forth in SEQ ID NO: 23, and (b) a second antigen-binding domain comprising a third set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 53, the CDR2 sequence as set forth in SEQ ID NO: 54 and the CDR3 sequence
  • the anti-HER2 biparatopic ADC has a structure as described herein for any one of the embodiments of general Formula (I) or general Formula (II), and the anti-HER2 biparatopic antibody comprises (a) a first heavy chain (Hl) comprising the CDR sequences as set forth in SEQ ID NOs: 32, 34 and 33, (b) a second heavy chain (H2) comprising the CDR sequences as set forth in SEQ ID NOs: 53, 54, 55, 56, 57 and 58, and (c) a light chain (LI) comprising the CDR sequences as set forth in SEQ ID NOs: 22, 24 and 23.
  • the anti-HER2 biparatopic ADC has a structure as described herein for any one of the embodiments of general Formula (I) or general Formula (II), and the anti-HER2 biparatopic antibody comprises (a) a first heavy chain (Hl) comprising a first set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 32, the CDR2 sequence as set forth in SEQ ID NO: 34 and the CDR3 sequence as set forth in SEQ ID NO: 33, (b) a second heavy chain (H2) comprising a second set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 53, the CDR2 sequence as set forth in SEQ ID NO: 54, and the CDR3 sequence as set forth in SEQ ID NO: 55, and a third set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 56, the CDR2 sequence as set forth in SEQ ID NO: 57 and the C
  • Hl first heavy chain
  • H2 compris
  • the anti-HER2 biparatopic ADC has a structure as described herein for any one of the embodiments of general Formula (I) or general Formula (II), and the anti-HER2 biparatopic antibody comprises (a) a first antigen-binding domain comprising the VH sequence as set forth in SEQ ID NO: 31, and the VL sequence as set forth in SEQ ID NO: 21, and (b) a second antigen-binding domain comprising the VH sequence as set forth in SEQ ID NO: 52, and the VL sequence as set forth in SEQ ID NO: 51.
  • the anti-HER2 biparatopic ADC has a structure as described herein for any one of the embodiments of general Formula (I) or general Formula (II), and the anti-HER2 biparatopic antibody comprises (a) a first antigen-binding domain that is a Fab and comprises the VH sequence as set forth in SEQ ID NO: 31, and the VL sequence as set forth in SEQ ID NO: 21, and (b) a second antigen -binding domain that is an scFv and comprises the VH sequence as set forth in SEQ ID NO: 52, and the VL sequence as set forth in SEQ ID NO: 51.
  • the anti-HER2 biparatopic ADC has a structure as described herein for any one of the embodiments of general Formula (I) or general Formula (II), and the anti-HER2 biparatopic antibody comprises a first heavy chain (Hl) comprising the VH sequence as set forth in SEQ ID NO: 31, a second heavy chain (H2) comprising the VH sequence as set forth in SEQ ID NO: 52 and the VL sequence as set forth in SEQ ID NO: 51, and a light chain (LI) comprising the VL sequence as set forth in SEQ ID NO: 21.
  • Hl first heavy chain
  • H2 second heavy chain
  • LI light chain
  • the anti-HER2 biparatopic ADC has a structure as described herein for any one of the embodiments of general Formula (I) or general Formula (II), and the anti-HER2 biparatopic antibody comprises (a) a first heavy chain (Hl) comprising the sequence as set forth in SEQ ID NO: 30, a second heavy chain (H2) comprising the sequence as set forth in SEQ ID NO: 50, and a light chain (LI) comprising the sequence as set forth in SEQ ID NO: 20.
  • the anti-HER2 biparatopic ADC has a structure as described herein for any one of the embodiments of general Formula (I) or general Formula (II), and the anti-HER2 biparatopic antibody comprises (a) a first heavy chain (Hl) consisting of the sequence as set forth in SEQ ID NO: 30, a second heavy chain (H2) consisting of the sequence as set forth in SEQ ID NO: 50, and a light chain (LI) consisting of the sequence as set forth in SEQ ID NO: 20.
  • Hl first heavy chain
  • H2 consisting of the sequence as set forth in SEQ ID NO: 50
  • a light chain (LI) consisting of the sequence as set forth in SEQ ID NO: 20.
  • the auristatin analogue, Compound 1 is capable of inducing the release of immune-activating damage associated molecular patterns (DAMPs) from tumor cells at similar levels to the known immunogenic cell death inducer, MMAE.
  • DAMPs immune-activating damage associated molecular patterns
  • MMAE immunogenic cell death inducer
  • the release of DAMPs can induce immune cell activation and modify the tumor microenvironment to support T-cell responses critical for activity of checkpoint inhibitor therapeutics, such as PD-1 inhibitors.
  • Certain aspects of the present disclosure thus relate to methods of treating a HER2- expressing cancer in a subject by administering an effective amount of an anti-HER2 biparatopic ADC as described herein, in combination with a checkpoint inhibitor.
  • the checkpoint inhibitor is a PD-1 inhibitor.
  • the checkpoint inhibitor is an anti- PD-1 antibody.
  • anti -PD-1 antibodies include, but are not limited to, pembrolizumab (Keytruda®), nivolumab (Opdivo®), cemiplimab (Libtayo®), JTX-4014 (Jounce Therapeutics), spartalizumab (PDR001) (Novartis), camrelizumab (SHR1210) (Jiangsu HengRui Medicine Co., Ltd.), sinitilimab (Innovent, Eli-Lilly), tislelizumab (BGB-A317) (Beigene), toripalimab (JS 001) (Junshi Biosciences), dostarlimab (GlaxoSmithKline), INCMGA00012 (MGA012) (Incyte, MacroGenics), AMP -224 (AstraZeneca/Medlmmune and GlaxoSmithKline) and AMP-514 (MEDI0680) (AstraZeneca).
  • Certain embodiments relate to methods of treating a HER2-expressing cancer in a subject by administering an effective amount of an anti-HER2 biparatopic ADC as described herein, in combination with an anti -PD-1 antibody. Some embodiments relate to methods of treating a HER2-expressing cancer in a subject by administering an effective amount of an anti-HER2 biparatopic ADC as described herein, in combination with tisleizumab.
  • Certain embodiments relate to methods of treating a HER2-expressing, PD-L1 + cancer in a subject by administering an effective amount of an anti-HER2 biparatopic ADC as described herein, in combination with a PD-1 inhibitor, for example, an anti-PD-1 antibody as described above.
  • the effective amount of the anti-HER2 biparatopic ADC to be used in the combination therapy with a PD-1 inhibitor may be the same as the amount used when the anti-HER2 biparatopic ADC is administered as a single agent.
  • the effective amount of the anti- HER2 biparatopic ADC to be used in combination with an anti-PD-1 antibody is a dose of between 1 mg/kg and 2.0 mg/kg, for example 1.25 mg/kg, 1.5 mg/kg or 1.75 mg/kg, administered to the subject QW for 3 weeks of a 4-week cycle, or a dose of between 2 mg/kg and 3 mg/kg, for example 2.5 mg/kg, administered to the subject Q3W.
  • the anti-HER2 biparatopic ADCs may be provided in the form of compositions comprising the ADC and a pharmaceutically acceptable carrier or diluent.
  • the compositions may be prepared by known procedures using well-known and readily available ingredients.
  • compositions may be formulated for administration to a subject by, for example, oral (including, for example, buccal or sublingual), topical, parenteral, rectal or vaginal routes, or by inhalation or spray.
  • parenteral as used herein includes subcutaneous injection, and intradermal, intra-articular, intravenous, intramuscular, intravascular, intrasternal, intrathecal injection or infusion.
  • the pharmaceutical composition will typically be formulated in a format suitable for administration to the subject by the selected route, for example, as a syrup, elixir, tablet, troche, lozenge, hard or soft capsule, pill, suppository, oily or aqueous suspension, dispersible powder or granule, emulsion, injectable or solution.
  • pharmaceutical compositions may be provided as unit dosage formulations.
  • the pharmaceutical compositions comprising the anti-HER2 biparatopic ADCs are formulated for parenteral administration in injectable form, for example as a powder or lyophilized formulation for reconstitution, or as an aqueous solution.
  • compositions comprising the anti-HER2 biparatopic ADCs may be in the form of a sterile injectable aqueous or oleaginous solution or suspension.
  • a sterile injectable aqueous or oleaginous solution or suspension Such suspensions may be formulated using suitable dispersing or wetting agents and/or suspending agents that are known in the art.
  • the sterile injectable solution or suspension may comprise the ADC in a non-toxic parentally acceptable diluent or carrier.
  • Acceptable diluents and carriers include, for example, 1,3 -butanediol, water, Ringer’s solution, isotonic sodium chloride solution or dextrose.
  • sterile, fixed oils may be employed as a carrier.
  • various bland fixed oils may be employed, including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid may find use in the preparation of injectables.
  • Adjuvants such as local anaesthetics, preservatives and/or buffering agents may optionally be included in the injectable solution or suspension.
  • the composition comprising the anti-HER2 biparatopic ADC may be formulated for intravenous administration, for example for intravenous infusion.
  • compositions that are administered intravenously are formulated as solutions in sterile pharmaceutical grade water or isotonic aqueous solution, for example, containing sodium chloride or dextrose.
  • the composition may also include a solubilizing agent and/or a local anaesthetic such as lignocaine to ease pain at the site of the injection.
  • a composition for intravenous administration may be provided in a lyophilized or dry form, such as a powder or water free concentrate, for reconstitution in an appropriate carrier prior to administration or it may be provided as an injectable solution.
  • the composition comprising the anti-HER2 biparatopic ADC is a lyophilized or dry composition for reconstitution.
  • compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in “Remington: The Science and Practice of Pharmacy” (formerly “Remingtons Pharmaceutical Sciences”),' Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).
  • kits comprising an anti-HER2 biparatopic ADC as described herein.
  • the anti-HER2 biparatopic ADC may be provided in the kit in one or more containers and the one or more containers may be associated with a label and/or package insert.
  • Suitable containers include, for example, bottles, vials, ampoules, syringes, intravenous solution bags, and the like, which may in some embodiments have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper that may be pierced by a hypodermic injection needle).
  • the container may be formed from a variety of materials such as glass or plastic.
  • the container is a hermetically sealed bottle or vial.
  • the label or package insert contains instructions customarily included in commercial packages of therapeutic products, providing information regarding indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
  • the label or package insert may specify that the anti-HER2 biparatopic ADC is for use to treat a HER2-expressing cancer and is for intravenous administration at a dose of between 1 mg/kg and 2.0 mg/kg, for example 1.25 mg/kg, 1.5 mg/kg or 1.75 mg/kg, administered to the subject QW for 3 weeks of a 4-week cycle, or at a dose of between 2 mg/kg and 3 mg/kg, for example, 2.5 mg/kg, administered to the subject Q3W.
  • the label or package insert may additionally include information regarding the patient population to be treated, for example, an appropriate age range or stipulation that the patient should be an adult; the type and/or stage of the patient’s cancer; whether or not the patient is required to have undergone one or more prior treatment regimens (and the type of prior treatment regimen or therapeutic), and/or HER2-status.
  • the label or package insert of the pharmaceutical kit indicates that the HER2-expressing cancer is breast cancer, gastroesophageal adenocarcinoma (GEA), esophageal cancer, endometrial cancer, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), anal cancer or colorectal cancer (CRC).
  • the label or package insert of the pharmaceutical kit indicates that the HER2-expressing cancer is one of breast cancer, GEA, endometrial cancer, ovarian cancer, NSCLC, anal cancer, pancreatic cancer, biliary tract cancer orbladder cancer.
  • the label or package insert of the pharmaceutical kit indicates that the HER2-expressing cancer is one of GEA, endometrial cancer, ovarian cancer, NSCLC or CRC. In some embodiments, the label or package insert of the pharmaceutical kit indicates that the HER2-expressing cancer is breast cancer or GEA.
  • the label or package insert of the pharmaceutical kit indicates that the patient to be treated has received prior treatment with one or more HER2 -targeting therapy. In some embodiments, the label or package insert of the pharmaceutical kit indicates that the patient to be treated has received prior treatment with one or more of trastuzumab, pertuzumab and T-DM1. [00205] In certain embodiments, the label or package insert of the pharmaceutical kit indicates that the patient to be treated has a HER2-expressing cancer that is metastatic. In some embodiments, the label or package insert of the pharmaceutical kit indicates that the patient to be treated has a HER2-expressing cancer that is locally advanced.
  • the label or package insert of the pharmaceutical kit indicates that the patient to be treated has a HER2-expressing cancer is classed as IHC 2+, IHC 2+/3+ or IHC 3+. In some embodiments, the label or package insert of the pharmaceutical kit indicates that the patient to be treated has a HER2-expressing cancer that is classed as IHC 3+ or IHC 2+/FISH+.
  • the label or package insert of the pharmaceutical kit indicates that the patient should receive ocular prophylaxis, for example, treatment with prednisolone, tetrahydrozoline or naphazoline, and/or cooling masks.
  • the label or package insert may further indicate that the anti-HER2 biparatopic ADC is suitable for use in combination with an anti-PDl agent.
  • the label or package insert may also include a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, for use or sale for human or animal administration.
  • the kit may comprise one or more additional containers comprising other components of the kit.
  • a pharmaceutically acceptable diluent or buffer such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer’s solution, sodium chloride, dextrose solution, or the like.
  • the anti-HER2 biparatopic ADC may be provided in the kit in lyophilized or dry form, such as a powder or granules, and the kit may optionally contain a suitable solvent for reconstitution of the lyophilized or dried ADC.
  • the anti-HER2 biparatopic ADC may be provided in the kit as a sterile liquid.
  • the anti- HER2 biparatopic antibody may be provided in the kit as a sterile liquid in concentrated form that is for dilution prior to use.
  • the kit may further include other materials desirable from a commercial or user standpoint, such as filters, needles, and syringes.
  • Linker-Toxin 001 shown below comprises the auristatin analogue Compound 1 (also shown below) and may be prepared as described in International Patent Application Publication No. WO 2019/173911.
  • Antibody-drug conjugate (ADC) v21252 which comprises the biparatopic anti-HER2 mAb, vlOOOO, conjugated to the Linker-Toxin 001 at an average DAR of 2, may be generated by partial reduction of the antibody interchain disulfide bonds, followed by capping of the free cysteine residues by reaction with the maleimide component of the Linker-Toxin, as described in this Example and in International Patent Application Publication No. WO 2019/173911.
  • a solution (138.9 mL) of the antibody vlOOOO (2.0 g) in 10 mM sodium acetate, 9% (w/v) sucrose, pH 4.5 was pH-adjusted by addition of 200 mM Na2HPC>4, pH 8.9 (15.4 mL).
  • a DTPA solution 44 mL in PBS, pH 7.4, final concentration 1.0 mM
  • reduction of the interchain disulfides was initiated by addition of an aqueous lO mM TCEP solution (1.68 mL, 1.05 eq.).
  • ADC Quenched antibody drug conjugate
  • ADC solution was purified with 9-15 diavolumes of 10 mM sodium acetate, 9% (w/v) sucrose, pH 4.5 on a Millipore LabscaleTM Tangential Flow Filtration instrument using a Pellicon® XL Ultrafiltration Module (Ultracel® 30 kDa 0.005m 2 ; Millipore Sigma).
  • the eluted ADC was sterile filtered (0.22 um).
  • ADCs produced on small scale were purified over 40 KDa MWCO ZebaTM columns (ThermoFisher Scientific, Waltham, MA) preconditioned with either PBS or 10 mM sodium acetate, 9% (w/v) sucrose, pH 4.5.
  • HIC hydrophobic interaction chromatography
  • the average drug to antibody ratio (DAR) of an ADC can vary depending on the number of disulfide bonds liberated during the reduction of the antibody.
  • a single conjugation reaction that yields an ADC with a particular average DAR comprises a mixture of species.
  • v21252 a mixture of four species was generated: unconjugated antibody (i.e. DAR0), ADC with a DAR of 2, ADC with a DAR of 4 and ADC with a DAR of 6.
  • v21252 had an average DAR of 2.07.
  • the DAR distribution for v21252 is shown in Table 2.2.
  • EXAMPLE 3 EFFECT OF ANTI-HER2 BIPARATOPIC ADC ON ATP SECRETION
  • ICD immunogenic cell death
  • T trastuzumab
  • P pertuzumab
  • v22277 Palivizumab, isotype (negative) control
  • Test articles were diluted to the desired treatment concentrations in complete growth medium (RPMI 1640 + 10% FBS). Seeding media was removed from the seeded plates, then wells were washed with 100 uL/well PBS pH 7.4. 50 uL/well of fresh cell line-specific complete growth medium and 20 uL/well of test article (70 uL/well final volume) were then added. Plates were incubated at 37°C/5% CChfor 24 hours. All supematant/media was then removed and transferred to V-bottom 96-well plates. Plates were centrifuged at 400 x g for 5 min to collect cell debris.
  • complete growth medium RPMI 1640 + 10% FBS
  • HER2-expressing cell lines for 24 hr with the Linker-Toxin 001 ADCs and MMAE ADCs resulted in increased levels of ATP secretion compared to untreated cells.
  • the unconjugated antibodies, trastuzumab and vlOOOO did not show increased levels of extracellular ATP (eATP) in HER2 + cell lines.
  • Negative controls (isotype ADCs and untreated) did not show eATP secretion in any cell line, as expected.
  • T-MTvcCompound 1 DAR 8 ADC yielded higher eATP secretion compared to T-MTvcCompound 1 (DAR 4 and DAR 2) ADCs.
  • T-MCvcPABC-MMAE DAR 8 ADC yielded higher eATP secretion compared to T-MCvcPABC-MMAE (DAR 4) ADC.
  • v21252 yielded similar eATP levels to T-MTvcCompound 1 (DAR 4) and higher eATP levels to the DAR-matched T-MTvcCompound 1 (DAR 2) indicating greater internalization, payload delivery and cytotoxicity of v21252 compared to monospecific ADCs utilizing Linker-Toxin 001.
  • v21252 showed comparable eATP secretion to T-Linker-Toxin 001 (DAR 2) + P-Linker-Toxin 001 (DAR 2) 1 :1 mix in HER2 + cell lines tested.
  • Free Compound 1 and MMAE yielded similar and cell line-dependent increases in eATP levels compared to untreated cells, whereas free topoisomerase inhibitor toxin DXd did not yield eATP in any of the cell lines tested.
  • the T-MC-GGFG-DXd (DAR 8) ADC did not show increased eATP secretion compared to untreated in any of the cell lines tested.
  • Example 3 The same test articles as described in Example 3 were employed together with the following cell lines: SK-BR-3 (HER2 + ), NCI-N87 (HER2 + ) and MDA-MB-468 (HER2‘).
  • Cells were seeded in flat-bottom TC-treated 48-well plates at 50,000 cells/well in their respective complete growth medium, using at least 100 uL/well. Plates were left at room temperature for 5-10 minutes after seeding to allow cells to settle at the bottom of wells, then incubated at 37°C/5% CO2 overnight (-18-24 hrs) to allow cells to attach. Test articles were diluted to desired treatment concentrations in complete growth medium (RPMI 1640 + 10% FBS). Seeding media was removed from seeded plates and wells were washed with 200 uL/well PBS pH 7.4. 80 uL/well of fresh cell line-specific complete growth medium and 40 uL/well of test article (120 uL/well final volume) were then added. Plates were incubated at 37°C/5% CChfor 48 hours.
  • ELISA plates were prepared by coating Nunc® MaxisorpTM 96-well plates with rabbit anti-HMGBl antibody (Thermo Fisher Scientific Corporation, Waltham, MA; PAI-16926) at 4 ug/mL in PBS pH 7.4, 100 uL/well, then incubated at 4°C overnight. Plates were washed with deionized (DI) water using BioTek® plate washer reader (BioTek Instruments, Winooski, VT) (3 rounds of washing, 100 uL/well DI water per wash), then blocked with 100 uL/well of blocking buffer (2% skim milk powder in PBS pH 7.4) and incubated at RT for 1 hour. Plates were then washed with DI water using BioTek® plate washer (3 rounds of washing, 100 uL/well DI water per wash).
  • DI deionized
  • Mouse anti-HMGBl 1D5 antibody (Sigma Aldrich, St. Louis, MI; SAB1403925-100UG) was added to the ELISA plates at 3 ug/mL, 80 uL/well, and the plates were incubated at RT for 1 hour. Following incubation, ELISA plates were washed with DI water using BioTek® plate washer (3 rounds of washing, 100 uL/well DI water per wash).
  • Detection antibody goat anti-rabbit IgG Fab2 HRP (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA; 111-035-047) was then added to the ELISA plates at 2 ug/mL, 80 uL/well; the plates were incubated at RT for 1 hour and then washed with DI water using BioTek® plate washer (3 rounds of washing, 100 uL/well DI water per wash).
  • HER2 + cell lines, SK-BR-3 and NCI-N87 a two- to four-fold increase in extracellular HMGB1 (eHMGBl) was observed following a 48-hour incubation with HER2 -targeted ADCs and free toxins at 100 nM.
  • the unconjugated antibodies, trastuzumab and vlOOOO, at 100 nM showed minimal increases in eHMGBl levels compared to the untreated control. Little or no HMGB1 secretion was observed in the HER2" cell line MDA- MB-468 treated with HER2 -targeted ADCs, control ADCs, or unconjugated antibodies.
  • HMGB1 release was observed in MDA-MB-468 cells after treatment with free toxins, Compound 1, MMAE and DXd, for 48-hours.
  • Treatment of HER2 + cells with v21252 resulted in similar levels of HMGB1 compared to treatment with the T- MTvcCompound 1 (DAR 4) ADC or T-MCvcPABC-MMAE (DAR 4) ADC.
  • Treatment of HER2 + cells with a 1 : 1 mix of T-Linker-Toxin 001 (DAR 2) and P-Linker-Toxin 001 (DAR 2) resulted in similar amounts of eHMGBl release as HER2 + cells treated with v21252.
  • EXAMPLE 5 EFFECT OF ANTI-HER2 BIPARATOPIC ADC ON CELL SURFACE CALRETICULIN LEVELS
  • SK-BR-3 and MDA-MB-468 cells were seeded in 48-well cell culture plates at 30,000 cells/well and allowed to attach overnight. Test articles were spiked on cells and incubated at 37°C/5% CO2 for 24 hrs, 48 hrs, or 72 hrs. At each time point, cells were detached with GibcoTM TrpLETM Express cell dissociation buffer (Thermo Fisher Scientific Corporation, Waltham, MA) and stained with a commercial anti-CRT antibody (Abeam pic, Waltham, MA; Cat. No: abl96158).
  • Cells were washed in FACS buffer (PBS + 2% FBS), then resuspended in FACS buffer containing 1 ug/ml 7-aminoactinomycin D (AAD) (dead cell exclusion stain) (BioLegend®, San Diego, CA). Cell surface CRT was analyzed by flow cytometry on a BD LSRFortessaTM Cell Analyzer (BD Biosciences, Franklin Lakes, NJ).
  • AAD 7-aminoactinomycin D
  • T-MTvcCompound 1 For T-MTvcCompound 1, increasing DAR from 2 to 4 resulted in an increase in the percent CRT induced. However, increasing DAR from 4 to 8 resulted in a slight decrease. v21252 induced comparable percent CRT levels to T-MTvcCompound 1 (DAR 2). At DAR4, T- MCvcPABC-MMAE induced a higher percent CRT level than that induced by T-MTvcCompound 1
  • T-MC-GGFG-DXd induced extracellular CRT in both cell-lines tested (HER2 + and HER2'). Free toxins induced cell surface CRT exposure. Negative controls did not induce surface CRT up to 72 hr, as expected.
  • DAMPs immune-activating damage associated molecular patterns
  • Extracellular ATP, extracellular HMGB1, and calreticulin are key DAMPs for cell death to be perceived as immunogenic.
  • the payload utilized in v21252, Compound 1 drives similar responses from tumor cells at similar levels to the known immunogenic cell death inducer, MMAE.
  • ADCs containing Compound 1 induce DAMPs from tumor cells at similar levels to ADCs containing MMAE.
  • the release of DAMPs can induce immune cell activation and modify the tumor microenvironment to support T-cell responses critical for anti-PD-1 activity.
  • Eligible patients include those with locally advanced (unresectable) or metastatic HER2-expressing cancers.
  • the study is using a 3+3 dose-escalation study design to evaluate the safety and tolerability of the investigational ADC and to determine the MTD or RD of the investigational ADC for further study.
  • Selected expansion cohorts have subsequently been opened based upon Safety Monitoring Committee (SMC) recommendation to further evaluate the safety and tolerability of the investigational ADC at the MTD or RD and to assess preliminary anti-tumor activity (see Example 8).
  • SMC Safety Monitoring Committee
  • DLTs dose-limiting toxicities
  • ECG electrocardiogram
  • LVEF left ventricular ejection fraction
  • Serum concentrations of investigational ADC [Time Frame: Up to 7 months]: End of infusion concentration, maximum serum concentration, and trough concentration of investigational ADC.
  • AD As anti-drug antibodies
  • Duration of response [Time Frame: Up to 2 years]: Median duration of response (in months) and range (minimum, maximum).
  • Progression-free survival [Time Frame: Up to 2 years]: Median progression-free survival (in months) and range (minimum, maximum).
  • Ages eligible for study 18 years and older (adult, older adult)
  • Expansion (Cohort 4): HER2-high other non-breast and non-GEA cancers [00260] 2. Progressive disease that has progressed on or been refractory to all standard of care. Patients who were intolerant to or ineligible for standard therapy may be eligible if the reasons are carefully documented and approval is provided by the sponsor medical monitor.
  • T-DM1 trastuzumab, pertuzumab, and ado-trastuzumab emtansine
  • Brain metastases Untreated central nervous system (CNS) metastases, symptomatic CNS metastases, or radiation treatment for CNS metastases within 4 weeks of start of study treatment. Stable, treated brain metastases are allowed (defined as patients who are off steroids and anticonvulsants and are stable for at least 1 month at the time of screening).
  • CNS central nervous system
  • the investigational ADC (v21252) is a humanized biparatopic anti-HER2 ADC.
  • the IgGl-like antibody is directed against two distinct HER2 epitopes: a scFv that binds to the ECD4 and a Fab that binds to ECD2.
  • the antibody is covalently linked to the auristatin-based cytotoxic microtubule inhibitor Linker-Toxin 001, which is comprised of a protease cleavable dipeptide valine-citrulline linker and the toxin Compound 1 (see Example 1).
  • Linker-toxin 001 is conjugated to the antibody via a maleimide to the antibody cysteines that comprise the interchain disulfide bonds.
  • the average DAR of the investigational ADC is approximately 2.
  • the investigational ADC was manufactured according to the relevant regulatory requirements for human trials and diluted in 0.9% sodium chloride or 5% dextrose in water for injection, United States Pharmacopeia (USP) or equivalent, for IV administration.
  • the investigational ADC was formulated for administration as a 10 mg/mL solution in 10 mM sodium acetate, 9% sucrose, 0.01% (w/v) Polysorbate 20, pH 4.5.
  • the investigational ADC was initially administered intravenously (IV) either every 2 weeks (Q2W) on Days 1 and 15 of each 28-day cycle or every 3 weeks (Q3W) on Day 1 of each 21 -day cycle. Subsequently, a third 3+3 dose-escalation cohort was initiated to evaluate the investigational ADC administered IV every week (QW) for 3 weeks in 28-day cycles.
  • PK Pharmacokinetic modeling of weekly (QW) dosing (on Days 1, 8 and 15 of a 28 day cycle) of the anti-HER2 biparatopic ADC v21252 (see Example 2) suggests that a lower Cmax, higher Cmin and/or higher exposure can be achieved with this dosing schedule when compared to dosing every 2 weeks (Q2W) or every 3 weeks (Q3W) (see Fig. 4).
  • modeled exposure was comparable between 1.5 mg/kg QW dosing (on Days 1, 8 and 15 of a 28 day cycle) and 3 mg/kg Q3W dosing with significantly different Cmax and Cmin profiles (see Fig. 5A). This indicates a potential for 1.5 mg/kg QW dosing to be better tolerated than 3 mg/kg Q3W dosing while the increased dose (1.5 mg/kg) should increase anti-tumor activity in both HER2-high and HER2-low cancers.
  • Example 6 The overall study design for this Phase I study is provided in Example 6 and summarized in Fig. 6. Based on the interim results (see Example 6, Section 6.5), indication-specific dose expansion cohorts were opened at 2.5 mg/kg Q3W in HER2+ breast cancer, HER2+ gastroesophageal adenocarcinoma (GEA), and a basket cohort of other HER2+ cancers. In addition, a QW dosing regimen is being evaluated at dose levels of 1.0 mg/kg, 1.25 mg/kg, 1.5 mg/kg and 1.75 mg/kg QW. Initial results from dose-escalation have led to the decision to open a dose-expansion cohort at 1.5 mg/kg QW. During dose expansion at both doses, all subjects must have HER2-high disease, including IHC 3+, or IHC 2+ and FISH +.
  • Primary objectives are to determine the maximum tolerated dose (MTD)/recommended dose (RD) for the investigational ADC and to characterize the safety and tolerability of the investigational ADC. Secondary objectives are to evaluate the anti -tumor activity of the investigational ADC in HER2-expressing cancers.
  • MTD maximum tolerated dose
  • RD recommended dose
  • AEs that resulted in a dose reduction were keratitis (12 (16%) patients), dry eye (2 (3%) patients), and infusion-related reaction, punctuate keratitis, prolonged QT interval by electrocardiogram (prolonged ECG QT) and decreased neutrophils (one patient each).
  • the investigational ADC has a unique mechanism of action including biparatopic- induced internalization with increased toxin-mediated cytotoxicity and immunogenic cell death.
  • This Phase 1 study evaluated multiple tumor types of HER2-expressing cancers with the investigational ADC showing consistent safety and antitumor activity.
  • the investigational ADC has a manageable safety profile (with the majority of AEs being Grade 1 or 2 in severity) and demonstrates encouraging single-agent anti-tumor activity in heavily pretreated patients with HER2+ cancers (conformed ORR of 31%, disease control rate of 72% observed across 29 response-evaluable patients treated at 2.5 mg/kg Q3W).
  • Table A Clone Numbers for Variants v5019, v5020, v7091, vlOOOO, v6903, v6902 and v6717
  • Table B Sequence for Variants v5019, v5020, v7091, vlOOOO, v6903, v6902 and v6717 by

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Abstract

Methods of treating a HER2-expressing cancer with an anti-HER2 biparatopic antibody drug conjugate (ADC) in which the drug is an auristatin analogue conjugated to the antibody at an average drug-to-antibody ratio (DAR) of about 1.5 and about 2.5. Dosing regimens and combination therapy with a PD-1 inhibitor, for example an anti-PD-1 antibody, are also described.

Description

METHODS OF USING ANTI-HER2 BIPARATOPIC ANTIBODY-DRUG
CONJUGATES IN THE TREATMENT OF CANCER
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63/403,632, filed on September 2, 2022, and U.S. Provisional Patent Application No. 63/303,147, filed on January 26, 2022, the entire contents of each of which are incorporated by reference herein for all purposes.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML file copy, created on January 24, 2023, is named ZWI-088WO_SL.xml and is 66,388 bytes in size.
FIELD
[0003] The present disclosure relates to the field of cancer therapeutics and, in particular, to methods of treating cancer with antibody-drug conjugates comprising a biparatopic anti-HER2 antibody and an auri statin analogue.
BACKGROUND
[0004] HER2 (ErbB2) is a transmembrane surface-bound receptor tyrosine kinase that is a member of the ErbB family of receptor tyrosine kinases and is normally involved in the signal transduction pathways leading to cell growth and differentiation. HER2 is overexpressed in about one-quarter of breast cancer patients (Bange et al, Nature Medicine 7:548 (2001)) and, as such, is an important target for treatment of breast cancer.
[0005] Herceptin® (trastuzumab, U.S. Patent No. 5,821,337) was the first monoclonal antibody developed for the treatment of HER2 -positive breast cancer. Pertuzumab (Perjeta®, U.S. Patent No. 7,862,817) is a humanized monoclonal antibody, which was designed specifically to prevent the HER2 receptor from pairing (dimerizing) with other HER receptors (EGFR/HER1, HER3 and HER4) on the surface of cells, a process that is believed to play a role in tumor growth and survival. Pertuzumab binds to domain II of HER2, essential for dimerization, while trastuzumab binds to extracellular domain IV of HER2. The combination of Perjeta®, Herceptin® and chemotherapy is thought to provide a more comprehensive blockade of HER signaling pathways.
[0006] Li et al (Cancer Res., 73:6471-6483 (2013)) describe bispecific, bivalent antibodies to HER2 that are based on the native trastuzumab and pertuzumab sequences and which overcome trastuzumab resistance. Other bispecific anti-HER2 antibodies have been described (International Patent Application Publication Nos. WO 2015/077891 and WO 2016/179707; U.S. Patent Application Publication Nos. 2014/0170148, 2015/0284463, 2017/0029529 and 2017/0291955; U.S. Patent No. 9,745,382). An antibody-drug conjugate comprising a HER2 -targeting biparatopic antibody site-specifically conjugated to a tubulysin derivative has also been described (Li et al., Cancer Cell, 29: 117-129 (2016)).
[0007] Auristatins are synthetic analogues of dolastatin 10, which is a potent microtubule inhibitor with anti -cancer activity. Antibody-drug conjugates comprising auristatin payloads, such as monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF), have been described (U.S. PatentNos. 7,498,298 and 7,659,241; International Patent Application Publication Nos. WO 2002/088172 and WO 2016/041082). International Patent Application Publication No. WO 2106/041082 describes N-acyl sulfonamide modified auristatins and their use as antibody-drug conjugate payloads.
[0008] International Patent Application Publication No. WO 2019/173911 describes anti-HER2 biparatopic antibody-drug conjugates comprising an auristatin analogue.
[0009] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present disclosure. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the claimed invention.
SUMMARY
[0010] Described herein are methods of using anti-HER2 biparatopic antibody drug conjugates in the treatment of cancer and, in particular, HER2 -positive cancer. In one aspect, the present disclosure relates to a method treating a subject having a HER2-expressing cancer comprising administering to the subject an effective amount of an anti-HER2 biparatopic antibody drug conjugate (ADC), the effective amount comprising a dose of between 1 mg/kg and 2.0 mg/kg administered every week (QW) for 3 weeks of a 4-week cycle, or a dose of between 2 mg/kg and 3 mg/kg administered every 3 weeks (Q3W), wherein the anti-HER2 biparatopic ADC has general Formula (II):
Figure imgf000004_0001
wherein: n is the average drug-to-antibody ratio (DAR) and is between about 1.5 and about 2.5, and
Ab is an anti-HER2 biparatopic antibody comprising a first antigen-binding domain that binds to an epitope on ECD2 of HER2 and a second anti gen -binding domain that binds to an epitope on ECD4 of HER2.
[0011] In another aspect, the present disclosure relates to a method of treating a subject having a HER2-expressing cancer comprising administering to the subject an effective amount of an anti- HER2 biparatopic antibody drug conjugate (ADC), the effective amount comprising a dose of 1.25 mg/kg administered every week (QW) for 3 weeks of a 4-week cycle, wherein the anti-HER2 biparatopic ADC has general Formula (II):
Figure imgf000005_0001
n is the average drug-to-antibody ratio (DAR) and is between about 1.8 and about 2.3, and
Ab is an anti-HER2 biparatopic antibody comprising (a) a first antigen-binding domain comprising the CDR sequences as set forth in SEQ ID NOs: 32, 34 and 33, and in SEQ ID NOs: 22, 24 and 23, and (b) a second antigen-binding domain comprising the CDR sequences as set forth in SEQ ID NOs: 56, 57 and 58, and SEQ ID NOs: 53, 54 and 55. [0012] In another aspect, the present disclosure relates to a method of treating a subject having a
HER2-expressing cancer comprising administering to the subject an effective amount of an anti- HER2 biparatopic antibody drug conjugate (ADC), the effective amount comprising a dose of 2.5 mg/kg administered every 3 weeks (Q3W), wherein the anti-HER2 biparatopic ADC has general Formula (II):
Figure imgf000005_0002
n is the average drug-to-antibody ratio (DAR) and is between about 1.8 and about 2.3, and Ab is an anti-HER2 biparatopic antibody comprising (a) a first antigen-binding domain comprising the CDR sequences as set forth in SEQ ID NOs: 32, 34 and 33, and in SEQ ID NOs: 22, 24 and 23, and (b) a second antigen-binding domain comprising the CDR sequences as set forth in SEQ ID NOs: 56, 57 and 58, and SEQ ID NOs: 53, 54 and 55.
[0013] In another aspect, the present disclosure relates to a pharmaceutical kit comprising: i) one or more containers containing an anti-HER2 biparatopic antibody drug conjugate (ADC) having general Formula (II):
Figure imgf000006_0001
wherein: n is the average drug-to-antibody ratio (DAR) and is between about 1.5 and about 2.5, and
Ab is an anti-HER2 biparatopic antibody comprising a first antigen-binding domain that binds to an epitope on ECD2 of HER2 and a second anti gen -binding domain that binds to an epitope on ECD4 of HER2, and ii) a label or package insert on or associated with the one or more containers indicating the anti-HER2 biparatopic ADC is for administration to a subject having a HER2-expressing cancer at a dose of 1.25 mg/kg, 1.5 mg/kg or 1.75 mg/kg administered every week (QW) for 3 weeks of a 4-week cycle, or a dose of 2.5 mg/kg administered every 3 weeks (Q3W).
BRIEF DESCRIPTION OF THE DRAWINGS [0014] FIGS. 1A-1D show the extracellular ATP levels (as determined by RLU fluorescence) induced by 24 hr treatment of HCC1954 cells (FIG. 1A), SKOV-3 cells (FIG. IB), JIMT-1 cells (FIG. 1C) and MDA-MB-468 cells (FIG. ID) with various ADCs, including the anti-HER2 biparatopic ADC v21252, at EC99 concentration.
[0015] FIGS. 2A-2C show the extracellular HMGB1 levels (as fold increase over untreated control) induced by 48 hr treatment of SK-BR-3 cells (FIG. 2A), NCI-N87 cells (FIG. 2B) and MDA-MB-468 (FIG. 2C) with various ADCs, including the anti-HER2 biparatopic ADC v21252, at EC99 concentration.
[0016] FIGS. 3A-3C show the levels of cell surface calreticulin (CRT) (% CRT+ cells) induced by 24 hr treatment (FIG. 3A), 48 hr treatment (FIG. 3B) or 72 hr treatment (FIG. 3C) of SK-BR- 3 and MDA-MB-468 cells with various ADCs, including the anti-HER2 biparatopic ADC v21252.
[0017] FIG. 4 shows the Cmax and Cmin profiles derived by pharmacokinetic modeling for administration of anti-HER2 biparatopic ADC v21252 at 1 mg/kg weekly (QW) (on Days 1, 8 and 15 of a 28 day cycle), 2 mg/kg every 2 weeks (Q2W) and 2.5 mg/kg every 3 weeks (Q3W). The dotted lines represent the estimated exposure range for different tumors at varying HER2 expression levels (top line = high HER2 (low sensitivity), centre line = moderate HER2 (medium sensitivity), lower line = low HER2 (high sensitivity)).
[0018] FIGS. 5A-5B show the Cmax and Cmin profiles derived by pharmacokinetic modeling for administration of anti-HER2 biparatopic ADC v21252 at 3 mg/kg every 3 weeks (Q3W) vs.
1.5 mg/kg weekly (QW) (on Days 1 , 8 and 15 of a 28 day cycle) (FIG. 5A), and at 2.5 mg/kg Q3 W vs. 3 mg/kg Q3W (FIG. 5B). The dotted lines represent the estimated exposure range for different tumors at varying HER2 expression levels (top line = high HER2 (low sensitivity), centre line = moderate HER2 (medium sensitivity), lower line = low HER2 (high sensitivity)).
[0019] FIG. 6 presents an overview of the study design for a Phase 1 study of anti-HER2 biparatopic ADC v21252 in HER2-positive cancer. GEA = gastroesophageal adenocarcinoma; QW = once every week; Q2W = once every 2 weeks; Q3W = once every 3 weeks; MTD = maximum tolerated dose; RD = recommended dose. [0020] FIG. 7 presents Table 7.2 showing the treatment related adverse events (TRAEs) observed during the Phase 1 study of anti-HER2 biparatopic ADC v21252 in HER2-positive cancer in 77 patients treated at 2.5 mg/kg every 3 weeks (Q3W). Values are number of patients (%).
[0021] FIG. 8 presents a waterfall plot showing the change in sum of target lesions observed during the Phase 1 study of anti-HER2 biparatopic ADC v21252 in HER2 -positive cancer in patients treated with the ADC at 2.5 mg/kg every 3 weeks (Q3W). BTC = biliary tract cancer; CRC = colorectal cancer; GEA = gastroesophageal adenocarcinoma; NSCLC = non-small cell lung cancer.
[0022] FIG. 9 presents a swimmer plot showing treatment duration, response, prior treatment and HER2 status by immunohistochemistry (IHC) and/or fluorescent in situ hybridization (FISH) for patients treated with anti-HER2 biparatopic ADC v21252 at 2.5 mg/kg every 3 weeks (Q3W) in the Phase 1 study of the ADC in HER2-positive cancer. BTC = biliary tract cancer; cPR = confirmed partial response; CRC = colorectal cancer; D = T-DXd; FISH = fluorescent in situ hybridization; GEA = gastroesophageal adenocarcinoma; I = investigational; IHC = immunohistochemistry; K = T-DM1; L = lapatinib; M = margetuximab; N = neratinib; NE = not evaluable; NSCLC = non-small cell lung cancer; P = pertuzumab; PD = progressive disease; PR = partial response; SD = stable disease; T = trastuzumab; Tx = treatment; U = tucatinib; Z = zanidatamab.
DETAILED DESCRIPTION
[0023] The present disclosure relates to methods of treating a HER2-expressing (or “HER2- positive”) cancer with an anti-HER2 biparatopic antibody-drug conjugate (ADC) in which the drug is an auristatin analogue conjugated to the anti-HER2 biparatopic antibody at an average drug-to- antibody ratio (DAR) between about 1.5 and about 2.5. In certain aspects of the methods of the present disclosure, the anti-HER2 biparatopic ADC is administered to a subject having a HER2- expressing cancer at a dose between 1 mg/kg and 2 mg/kg weekly (QW) or between 2 mg/kg and 3 mg/kg every 3 weeks (Q3W). In certain aspects of the methods of the present disclosure, the anti-HER2 biparatopic ADC is administered in combination with a PD-1 inhibitor, for example, an anti -PD-1 antibody. Definitions
[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
[0025] The term “subject,” as used herein, refers to a human patient who is the object of treatment and/or observation.
[0026] As used herein, the term “about” refers to an approximately +/-10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.
[0027] The use of the word “a” or “an” when used herein in conjunction with the term “comprising” may mean “one,” but it is also consistent in certain embodiments with the meaning of “one or more,” “at least one” or “one or more than one.”
[0028] As used herein, the terms “comprising,” “having,” “including” and “containing,” and grammatical variations thereof, are inclusive or open-ended and do not exclude additional, unrecited elements and/or method steps. The term “consisting essentially of’ when used herein in connection with a composition, use or method, denotes that additional elements and/or method steps may be present, but that these additions do not materially affect the manner in which the recited composition, method or use functions. The term “consisting of’ when used herein in connection with a composition, use or method, excludes the presence of additional elements and/or method steps. A composition, use or method described herein as comprising certain elements and/or steps may also, in certain embodiments consist essentially of those elements and/or steps, and in certain embodiments consist of those elements and/or steps, whether or not these embodiments are specifically referred to.
[0029] The terms “derived from” and “based on” when used with reference to a recombinant amino acid sequence mean that the recombinant amino acid sequence is substantially identical to the sequence of the corresponding reference amino acid sequence. For example, an Ig Fc amino acid sequence that is derived from (or based on) a wild-type Ig Fc sequence is substantially identical to (e.g. shares at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with) the corresponding wild-type Ig Fc sequence. [0030] Where a range of values is provided herein, for example where a value is defined as being “between” an upper limit value and a lower limit value, it is understood that the range encompasses both the upper limit value and the lower limit value as well as each intervening value.
[0031] It is contemplated that any embodiment discussed herein can be implemented with respect to any method, use or composition disclosed herein.
[0032] Particular features, structures and/or characteristics described in connection with an embodiment disclosed herein may be combined with features, structures and/or characteristics described in connection with another embodiment disclosed herein in any suitable manner to provide one or more further embodiments.
[0033] It is also to be understood that the positive recitation of a feature in one embodiment, serves as a basis for excluding the feature in an alternative embodiment. For example, where a list of options is presented for a given embodiment or claim, it is to be understood that one or more option may be deleted from the list and the shortened list may form an alternative embodiment, whether or not such an alternative embodiment is specifically referred to.
ANTI-HER2 BIPARATOPIC ANTIBODY DRUG CONJUGATES
[0034] Anti-HER2 biparatopic ADCs for use in the methods of the present disclosure comprise an anti-HER2 biparatopic antibody conjugated to an auristatin analogue having structure 1 (also referred to herein as “Compound 1”) via a linker (L) at a low drug-to-antibody ratio (DAR).
Figure imgf000010_0001
[0035] The auristatin analogue having structure 1 may also be referred to herein using the terms “drug,” “toxin,” or “auristatin analogue.”
[0036] As is known in the art, the majority of conjugation methods used in the preparation of ADCs yield a preparation of ADCs that includes various DAR species, with the reported DAR being the average of the individual DAR species in the ADC preparation. “Low DAR” as used herein, is defined as an average DAR of between about 1.5 and about 2.5.
[0037] In some embodiments, the average DAR of the anti-HER2 biparatopic ADCs is between about 1.6 and about 2.5. In some embodiments, the average DAR of the anti-HER2 biparatopic ADCs is between about 1.7 and about 2.5. In some embodiments, the average DAR of the anti- HER2 biparatopic ADCs is between about 1.8 and about 2.5. In some embodiments, the average DAR of the anti-HER2 biparatopic ADCs is between about 1.8 and about 2.4, or between about 1.8 and about 2.3. In some embodiments, the average DAR of the anti-HER2 biparatopic ADCs is between about 1.9 and about 2.2. In some embodiments, the average DAR of the anti-HER2 biparatopic ADCs is about 2.0. In some embodiments, the average DAR of the anti-HER2 biparatopic ADCs is 2.0 + 0.2.
[0038] In certain embodiments, anti-HER2 biparatopic ADCs that include a proportion of DAR0 species above a certain threshold may be advantageous. Accordingly, in some embodiments, the anti-HER2 biparatopic ADCs may include between about 5% and about 50% DAR0 species. In some embodiments, the anti-HER2 biparatopic ADCs may include between about 10% and about 50% DAR0 species, for example, between about 10% and about 40%, between about 10% and about 30%, or between about 10% and about 25% DAR0 species. In some embodiments, the anti- HER2 biparatopic ADCs may include between about 12% and about 28% DAR0 species, for example, between about 15% and about 28% DAR0 species, or between about 15% and about 25% DAR0 species.
[0039] In certain embodiments, the anti-HER2 biparatopic ADCs have general Formula (I):
Figure imgf000011_0001
wherein: L is a cleavable linker; n is the average DAR and is between about 1.5 and about 2.5, and Ab is an anti-HER2 biparatopic antibody.
[0040] In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (I), L is a protease-cleavable linker.
[0041] In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (I), L is a peptide-containing linker. In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (I), L comprises a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, He-Cit and Trp-Cit.
[0042] In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (I), L comprises a dipeptide and a stretcher.
[0043] In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (I), n is between about 1.6 and about 2.5. In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (I), n is between about 1.8 and about 2.5. In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (I), n is between about 1.8 and about 2.4. In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (I), n is between about 1.8 and about 2.3. In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (I), n is between about 1.9 and about 2.2. In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (I), n is about 2.0. In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (I), n is 2.0 + 0.2.
[0044] In certain embodiments, linker L in anti-HER2 biparatopic ADCs of Formula (I) is conjugated to the antibody via a cysteine residue on the antibody. In some embodiments, linker L in the anti-HER2 biparatopic ADCs of general Formula (I) is conjugated to the antibody via a cysteine residue on the antibody that has been liberated by reduction of an interchain disulfide bond.
[0045] Combinations of any of the foregoing embodiments for anti-HER2 biparatopic ADCs of general Formula (I) are also contemplated and each combination forms a separate embodiment for the purposes of the present disclosure. [0046] In certain embodiments, the anti-HER2 biparatopic ADCs have general Formula (II):
Figure imgf000013_0001
n is the average DAR, and
Ab is an anti-HER2 biparatopic antibody.
[0047] In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (II), n is between about 1.6 and about 2.5. In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (II), n is between about 1.8 and about 2.5. In some embodiments, in the anti- HER2 biparatopic ADCs of general Formula (II), n is between about 1.8 and about 2.4. In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (II), n is between about 1.8 and about 2.3. In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (II), n is between about 1.9 and about 2.2. In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (II), n is about 2.0. In some embodiments, in the anti-HER2 biparatopic ADCs of general Formula (II), n is 2.0 + 0.2.
Linker (L)
[0048] In the anti-HER2 biparatopic ADCs described herein, the anti-HER2 biparatopic antibody is linked to the auristatin analogue by a linker (L). Linkers are bifunctional or multifunctional moieties capable of linking one or more drug molecules to an antibody. A linker may be bifunctional (or monovalent) such that it links a single drug to a single site on the antibody, or it may be multifunctional (or polyvalent) such that it links more than one drug molecule to a single site on the antibody. Linkers capable of linking one drug molecule to more than one site on the antibody may also be considered to be multifunctional. [0049] Attachment of a linker to an antibody can be accomplished in a variety of ways, such as through surface lysines on the antibody, reductive-coupling to oxidized carbohydrates on the antibody, or through cysteine residues on the antibody liberated by reducing interchain disulfide linkages. Alternatively, attachment of a linker to an antibody may be achieved by modification of the antibody to include additional cysteine residues (see, for example, U.S. Patent Nos. 7,521,541; 8,455,622 and 9,000,130) or non-natural amino acids that provide reactive handles, such as selenomethionine, p-acetylphenylalanine, formylglycine or p-azidomethyl-L-phenylalanine (see, for example, Hofer et al., Biochemistry, 48: 12047-12057 (2009); Axup et al., PNAS, 109: 16101- 16106 (2012); Wu et al., PNAS, 106:3000-3005 (2009); Zimmerman et al., Bioconj. Chem., 25:351-361 (2014)), to allow for site-specific conjugation.
[0050] Linkers include at least one functional group capable of reacting with the target group or groups on the antibody, and one or more functional groups capable of reacting with a target group on the drug. Suitable functional groups are known in the art and include those described, for example, in Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press).
[0051] Examples of target groups on the antibody to which a linker may be conjugated include the thiol groups of cysteine residues and the amine groups of lysine residues. Non-limiting examples of functional groups for reacting with free cysteines or thiols include maleimide, haloacetamide, haloacetyl, activated esters (such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters and tetrafluorophenyl esters), anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. Also useful in this context are “self-stabilizing” maleimides as described in Lyon et al., 2014, Nat. Biotechnol, 32: 1059-1062. Non-limiting examples of functional groups for reacting with free amines include activated esters (such as N- hydroxysuccinamide (NHS) esters and sulfo-NHS esters), imido esters (such as Traut’s reagent), isothiocyanates, aldehydes and acid anhydrides (such as diethylenetriaminepentaacetic anhydride (DTP A)). Other examples include the use of succinimido-l,l,3,3-tetra- methyluronium tetrafluoroborate (TSTU) or benzotriazol- 1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) to convert a carboxylic acid to an activated ester, which may then be reacted with an amine. [0052] Other linkers include those having a functional group that allows for bridging of two interchain cysteines on the antibody, such as a ThioBridge™ linker (Badescu et al., Bioconjug. Chem., 25:1124-1136 (2014)), a dithiomaleimide (DTM) linker (Behrens et al., Mol. Pharm., 12:3986-3998 (2015)), a dithioarylpyridazinedione-based linker (Lee et al., Chem. Sci., 7:799- 802 (2016)), a dibromopyridazinedione-based linker (Maruani et al., Nat. Commun., 6:6645 (2015)) and others known in the art.
[0053] A linker may comprise one or more linker components. Typically, a linker will comprise two or more linker components. Exemplary linker components include functional groups for reaction with the antibody, functional groups for reaction with the drug, stretchers, peptide components, self-immolative groups, self-elimination groups, hydrophilic moieties, and the like. Various linker components are known in the art, some of which are described below.
[0054] Certain useful linker components can be obtained from various commercial sources, such as Pierce Biotechnology, Inc. (now Thermo Fisher Scientific Corporation, Waltham, MA) and Molecular Biosciences Inc. (Boulder, Colo.), or may be synthesized in accordance with procedures described in the art (see, for example, Toki et al., J. Org. Chem., 67: 1866-1872 (2002); Dubowchik, et al., Tetrahedron Letters, 38:5257-60 (1997); Walker, M. A., J. Org. Chem., 60:5352-5355 (1995); Frisch, et al., Bioconjugate Chem., 7: 180-186 (1996); U.S. Patent Nos. 6,214,345 and 7,553,816, and International Patent Application Publication No. WO 02/088172).
[0055] Examples of linker components include, but are not limited to, N- (P-maleimidopropyloxy)-N-hydroxy succinimide ester (BMPS), N-(s-maleimidocaproyloxy) succinimide ester (EMCS), N-[y-maleimidobutyryloxy]succinimide ester (GMBS), 1,6-hexane- bis-vinylsulfone (HBVS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxy-(6- amidocaproate) (LC-SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N- maleimidophenyl)butyric acid hydrazide (MPBH), succinimidyl 3-(bromoacetamido)propionate (SBAP), succinimidyl iodoacetate (SIA), succinimidyl (4-iodoacetyl)aminobenzoate (STAB), N- succinimidyl-3-(2-pyridyldithio) propionate (SPDP), N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC), succinimidyl 4- (p-maleimidophenyl)butyrate (SMPB), succinimidyl 6-[(P-maleimidopropionamido)hexanoate] (SMPH), iminothiolane (IT), sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB and succinimidyl-(4-vinylsulfone)benzoate (SVSB).
[0056] Additional examples include bis-maleimide reagents such as dithiobismaleimidoethane (DTME), bis-maleimido-tri oxyethylene glycol (BMPEO), 1,4-bismaleimidobutane (BMB), 1,4 bismaleimidyl-2,3-dihydroxybutane (BMDB), bismaleimidohexane (BMH), bismaleimidoethane (BMOE), BM(PEG)2 and BM(PEG)s; bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4- dinitrobenzene).
[0057] Suitable linkers typically are more chemically stable to conditions outside the cell than to conditions inside the cell, although less stable linkers may be contemplated in certain situations, such as when the drug is selective or targeted and has a low toxicity to normal cells. Linkers may be “cleavable linkers” or “non-cleavable linkers.” A cleavable linker is typically susceptible to cleavage under intracellular conditions, for example, through lysosomal processes. Examples include linkers that are protease-sensitive, acid-sensitive, reduction-sensitive or photolabile. Non- cleavable linkers by contrast, rely on the degradation of the antibody in the cell, which typically results in the release of an amino acid-linker-toxin moiety.
[0058] In certain embodiments, linker L comprised by the anti-HER2 biparatopic ADCs of Formula (I) is a cleavable linker. Suitable cleavable linkers include, for example, linkers comprising a peptide component that includes two or more amino acids and is cleavable by an intracellular protease, such as lysosomal protease or an endosomal protease. A peptide component may comprise amino acid residues that occur naturally and/or minor amino acids and/or non- naturally occurring amino acid analogues, such as citrulline. Peptide components may be designed and optimized for enzymatic cleavage by a particular enzyme, for example, a tumor-associated protease, cathepsin B, C or D, or a plasmin protease.
[0059] In certain embodiments, linker L comprised by the anti-HER2 biparatopic ADCs of Formula (I) may be a peptide-containing linker. In some embodiments, linker L comprised by the anti-HER2 biparatopic ADCs may be a dipeptide-containing linker, such as a linker containing valine-citrulline (Val-Cit) or phenylalanine-lysine (Phe-Lys). Other examples of suitable dipeptides for inclusion in linker L include Val-Lys, Ala-Lys, Me-Val-Cit, Phe-homoLys, Phe- Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Arg, Ala-Phe, Vai-Ala, Met-Lys, Asn-Lys, Ile-Pro, Ile-Val, Asp-Val, His-Val, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, NorVal-(D)Asp, Ala-(D)Asp, MesLys- Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys and Met-(D)Lys. Cleavable linkers may also include longer peptide components such as tripeptides, tetrapeptides or pentapeptides. Examples include, but are not limited to, the tripeptides Met-Cit-Val, Gly-Cit-Val, (D)Phe-Phe- Lys and (D)Ala-Phe-Lys, and the tetrapeptides Gly-Phe-Leu-Gly and Ala-Leu- Ala-Leu. In some embodiments, linker L comprised by the anti-HER2 biparatopic ADCs may be a peptide- containing linker, where the peptide is between two and five amino acids in length.
[0060] Additional examples of cleavable linkers include disulfide-containing linkers, such as, for example, N-succinimydyl-4-(2-pyridyldithio) butanoate (SPBD) and N-succinimydyl-4-(2- pyridyldithio)-2-sulfo butanoate (sulfo-SPBD). Disulfide-containing linkers may optionally include additional groups to provide steric hindrance adjacent to the disulfide bond in order to improve the extracellular stability of the linker, for example, inclusion of a geminal dimethyl group. Other suitable linkers include linkers hydrolyzable at a specific pH or within a pH range, such as hydrazone linkers. Linkers comprising combinations of these functionalities may also be useful, for example, linkers comprising both a hydrazone and a disulfide are known in the art.
[0061] A further example of a cleavable linker is a linker comprising a P -glucuronide, which is cleavable by P-glucuronidase, an enzyme present in lysosomes and tumor interstitium (see, for example, De Graaf et al., Curr. Pharm. Des., 8: 1391-1403 (2002)).
[0062] Cleavable linkers may optionally further comprise one or more additional components such as self-immolative and self-elimination groups, stretchers or hydrophilic moieties.
[0063] Self-immolative and self-elimination groups that find use in linkers include, for example, p-aminobenzyloxycarbonyl (PABC) and p-aminobenzyl ether (PABE) groups, and methylated ethylene diamine (MED). Other examples of self-immolative groups include, but are not limited to, aromatic compounds that are electronically similar to the PABC or PABE group such as heterocyclic derivatives, for example 2-aminoimidazol-5-methanol derivatives as described in U.S. Patent No. 7,375,078. Other examples include groups that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, 2:223-227 (1995)) and 2-aminophenylpropionic acid amides (Amsberry, el a!., J. Org. Chem., 55:5867-5877 (1990)).
[0064] Stretchers that find use in linkers for ADCs include, for example, alkylene groups and stretchers based on aliphatic acids, diacids, amines or diamines, such as diglycolate, malonate, caproate and caproamide. Other stretchers include, for example, glycine-based stretchers, polyethylene glycol (PEG) stretchers and monomethoxy polyethylene glycol (mPEG) stretchers. PEG and mPEG stretchers also function as hydrophilic moieties.
[0065] Examples of components commonly found in cleavable linkers include, but are not limited to, SPBD, sulfo-SPBD, hydrazone, Val-Cit, maleidocaproyl (MC), MC-Val-Cit, MC-Val- Cit-PABC, Phe-Lys, MC-Phe-Lys, MC-Phe-Lys-PABC, maleimido triethylene glycolate (MT), MT-Val-Cit, MT-Phe-Lys and adipate (AD).
[0066] Selection of an appropriate linker for a given ADC may be readily made by the skilled person having knowledge of the art and taking into account relevant factors, such as the site of attachment to the antibody, any structural constraints of the payload drug and the hydrophobicity of the payload drug (see, for example, review in Nolting, Chapter 5, Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed.), Springer).
[0067] In certain embodiments, linker L included in the anti-HER2 biparatopic ADCs of the present disclosure is a peptide-based linker having Formula (V):
#_z_ st s AA l- AA2]_[x| %
(V) wherein:
Z is a functional group capable of reacting with a target group on the anti-HER2 biparatopic antibody;
Str is a stretcher;
AAi and AA2 are each independently an amino acid, wherein AAi-[AA2]r forms a protease cleavage site;
X is a self-immolative group; s is 0 or 1; m is 1, 2, 3 or 4; o is 0, 1 or 2;
# is the point of attachment to the anti-HER2 biparatopic antibody, and
% is the point of attachment to the rest of the molecule.
[0068] In some embodiments, in general Formula (V), m is 1, 2 or 3.
[0069] In some embodiments, in general Formula (V), s is 1.
[0070] In some embodiments, in general Formula (V), o is 0 (z.e. X is absent).
[0071] In some embodiments, in general Formula (V):
Figure imgf000019_0001
the point of attachment to the anti-HER2 biparatopic antibody, and * is the point of attachment to the remainder of the linker.
[0072] In some embodiments, in general Formula (V), Str is selected from:
Figure imgf000019_0002
O R O O R O
$-(CH2)p— C-N— (CH2)p— C— * and $-(CH2)P— C-N— (CH2CH2O)q— C— ’ wherein: each R is independently H or Ci-Ce alkyl; each p is independently an integer between 2 and 10; each q is independently an integer between 1 and 10, $ is the point of attachment to Z, and
* is the point of attachment to the remainder of the linker.
[0073] In some embodiments, in general Formula (V), Str is:
Figure imgf000020_0001
where p, q, $ and * are as defined above.
[0074] In some embodiments, in general Formula (V), Str is:
Figure imgf000020_0002
where $ and * are as defined above, p is an integer between 2 and 6, and q is an integer between 2 and 8. [0075] In some embodiments, in general Formula (V), AAi-[AA2]m is selected from Val-Lys,
Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Arg, Ala-Phe, Vai-Ala, Met- Lys, Asn-Lys, Ile-Pro, Ile-Val, Asp-Val, His-Val, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, NorVal- (D)Asp, Ala-(D)Asp, MesLys-Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Met-(D)Lys, Met-Cit-Val, Gly-Cit-Val, (D)Phe-Phe-Lys, (D)Ala-Phe-Lys, Gly-Phe-Leu-Gly and Al a-Leu- Al a-Leu .
[0076] In some embodiments, in general Formula (V), m is 1 (i.e. AAi-[AA2]m is a dipeptide).
[0077] In some embodiments, in general Formula (V), AAi-[AA2]m is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit.
[0078] In some embodiments, in general Formula (V):
Figure imgf000021_0001
the point of attachment to the anti-HER2 biparatopic antibody, and * is the point of attachment to the remainder of the linker;
O O
Str is $— (CH2)p— C— or $— (CH2CH2O)q— (CH2)p— C— where $ is the point of attachment to Z, * is the point of attachment to the remainder of the linker, p is an integer between 2 and 6, and q is an integer between 2 and 8; m is 1 and AAi-[AA2]m is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit; s is 1, and o is 0.
[0079] In certain embodiments, the linker included in the anti-HER2 biparatopic ADCs of the present disclosure has general Formula (VI):
Figure imgf000021_0002
where:
* is the point of attachment to the anti-HER2 biparatopic antibody;
Y is one or more additional linker components, or is absent, and D is the point of attachment to the auristatin analogue.
[0080] In some embodiments, in the linker of Formula (VI), Y is absent.
[0081] In certain embodiments, the linker included in the anti-HER2 biparatopic ADCs of the present disclosure has general Formula (VII):
Figure imgf000022_0001
where:
* is the point of attachment to the anti-HER2 biparatopic antibody; Y is one or more additional linker components, or is absent, and
D is the point of attachment to the auristatin analogue.
[0082] In some embodiments, in the linker of Formula (VII), Y is absent.
[0083] In certain embodiments, the auristatin analogue-linker (or “drug-linker”) has the following structure:
Figure imgf000022_0002
where * is the point of attachment to the anti-HER2 biparatopic antibody.
Anti-HER2 Biparatopic Antibodies
[0084] The ADCs described herein comprise an anti-HER2 biparatopic antibody that binds to two different epitopes of HER2. [0085] The term “antibody,” as used herein, generally refers to a proteinaceous binding molecule with immunoglobulin-like functions. Typical examples of an antibody are immunoglobulins, as well as derivatives or functional fragments thereof which still retain binding specificity. Techniques for the production of antibodies are well known in the art. The term “antibody” may also include immunoglobulins of different classes (i.e. IgA, IgG, IgM, IgD and IgE) and subclasses (such as IgGi, IgG2, IgGs, IgG4, IgAi and IgA2). Illustrative examples of an antibody are whole antibodies and antigen-binding fragments thereof, such as Fab fragments, F(ab')2, Fv fragments, single-chain Fv fragments (scFv), diabodies, domain antibodies, and combinations thereof. Domain antibodies may be single domain antibodies, single variable domain antibodies or immunoglobulin single variable domain having only one variable domain, which may be a heavy chain variable domain or a light chain variable domain, that specifically bind an antigen or epitope independently of other variable regions or domains. The term “antibody” also includes embodiments such as chimeric, single chain and humanized antibodies.
[0086] A typical whole antibody comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region comprises three domains: CHI, CH2 and CH3. The heavy chain constant domains that correspond to the different classes of immunoglobulins are known as a (IgA), 6 (IgD), 8 (IgE), y (IgG) and p (IgM). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region. The light chain constant region comprises just one domain: CL. Light chains are classified as either kappa or lambda. The VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDR), interspersed with regions that are more conserved, termed framework regions (FW). Each VH and VL is composed of three CDRs and four FWs, arranged from amino-terminus to carboxy-terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The variable regions of the heavy and light chains contain a binding domain (a paratope) that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and Clq, which is a component of the complement system.
[0087] In certain embodiments, the anti-HER2 biparatopic antibodies comprised by the ADCs described herein comprise two antigen-binding polypeptide constructs (also referred to as antigenbinding domains), each of which binds to a different epitope of HER2. The terms “antigen-binding polypeptide construct” and “antigen-binding domain,” as used interchangeably herein, refer to an immunoglobulin-based construct, for example, an antibody fragment. In some embodiments, the antigen-binding polypeptide constructs comprised by the anti-HER2 biparatopic antibody are antibody fragments.
[0088] In certain embodiments, the antigen-binding polypeptide constructs comprised by the anti-HER2 biparatopic antibody may each independently be a Fab fragment, a Fab’ fragment, an scFv or an sdAb. In some embodiments, the antigen -binding polypeptide constructs comprised by the anti-HER2 biparatopic antibody may each independently be a Fab fragment or an scFv. In some embodiments, one antigen-binding polypeptide construct comprised by the anti-HER2 biparatopic antibody may be a Fab fragment and the other antigen-binding polypeptide construct may be an scFv.
[0089] In certain embodiments, at least one of the antigen-binding polypeptide constructs comprised by the anti-HER2 biparatopic antibody may be a Fab fragment or a Fab’ fragment. A “Fab fragment” contains the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CHI) along with the variable domains of the light and heavy chains (VL and VH, respectively). Fab' fragments differ from Fab fragments by the addition of a few amino acid residues at the C-terminus of the heavy chain CHI domain, including one or more cysteines from the antibody hinge region. In certain embodiments, an antigen-binding polypeptide construct comprised by the anti-HER2 biparatopic antibody may be a single-chain Fab molecule, i.e. a Fab molecule in which the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain. For example, the C-terminus of the Fab light chain may be connected to the N-terminus of the Fab heavy chain in the single-chain Fab molecule.
[0090] In certain embodiments, at least one of the antigen-binding polypeptide constructs comprised by the anti-HER2 biparatopic antibody may be a single-chain Fv (scFv). An “scFv” includes a heavy chain variable domain (VH) and a light chain variable domain (VL) of an antibody in a single polypeptide chain. The scFv may optionally further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form a desired structure for antigen binding. For example, an scFv may include a VL connected from its C-terminus to the N- terminus of a VH by a polypeptide linker. Alternately, an scFv may comprise a VH connected through its C-terminus to the N-terminus of a VL by a polypeptide chain or linker. See, for example, review in Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
[0091] The two antigen-binding polypeptide constructs comprised by the anti-HER2 biparatopic antibody each bind to a different epitope of HER2, that is, a first antigen-binding polypeptide construct binds to a first HER2 epitope and a second antigen-binding polypeptide construct binds to a second HER2 epitope. In the context of the present disclosure, each of the antigen-binding polypeptide constructs specifically binds to its target epitope.
[0092] “Specifically binds” or “specific binding” mean that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an antigen-binding polypeptide construct to bind to a specific epitope can be measured, for example, through an enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) techniques (analyzed on a BIAcore™ instrument) (Liljeblad ei al. Glyco J 17, 323-329 (2000)) or traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In some embodiments, the antigen-binding polypeptide construct is considered to specifically bind to its target epitope when the extent of binding of the antigen-binding polypeptide construct to an unrelated protein is less than about 10% of the binding of the antigen-binding polypeptide construct to its target epitope as measured, for example, by SPR.
[0093] “HER2” (also known as ErbB2) refers to human HER2 protein described, for example, in Semba et al, PNAS (USA), 82:6497-6501 (1985) and Yamamoto et al., Nature, 319:230-234 (1986) (GenBank accession number X03363). The terms “erbB2” and “neu” refer to the gene encoding human HER2 protein. The terms p 185 or p 185neu may also be used to refer to the protein product of the neu gene.
[0094] HER2 comprises an extracellular domain, which typically binds a HER ligand, a lipophilic transmembrane domain, a conserved intracellular tyrosine kinase domain and a carboxyterminal signaling domain harboring several tyrosine residues which can be phosphorylated. The extracellular (ecto) domain of HER2 comprises four domains, Domains I-IV. The sequence of HER2 is provided in Table 1 (SEQ ID NO:2). The Extracellular Domain (ECD) boundaries are: Domain I - approximately amino acids 1-165; Domain II - approximately amino acids 166-322; Domain III - approximately amino acids 323-488, and Domain IV - approximately amino acids 489-607.
Table 1: Amino Acid Sequence of Human HER2 (SEQ ID NO:2)
Figure imgf000026_0001
[0095] “Epitope 2C4” is the region in the extracellular domain of HER2 to which the antibody 2C4 binds and comprises residues from Domain II in the extracellular domain of HER2 (also referred to as ECD2). 2C4 and Pertuzumab bind to the extracellular domain of HER2 at the junction of Domains I, II and III (Franklin et al. Cancer Cell 5:317-328 (2004)).
[0096] “Epitope 4D5” is the region in the extracellular domain of HER2 to which the antibody 4D5 (ATCC CRL 10463) and trastuzumab bind. This epitope is close to the transmembrane domain of HER2, and within Domain IV of HER2 (also referred to as ECD4).
[0097] In general, the anti-HER2 biparatopic antibody of the present disclosure will bind to epitopes within the extracellular domains of HER2. In some embodiments, the first and second HER2 epitopes bound by the first and second antigen-binding domains of the anti-HER2 biparatopic antibody are non-overlapping epitopes. In some embodiments, the first and second HER2 epitopes bound by the first and second antigen-binding domains of the anti-HER2 biparatopic antibody are on different extracellular domains of HER2. In some embodiments, a first antigen-binding domain of the anti-HER2 biparatopic antibody binds to a first HER2 epitope on a first domain of HER2, and a second antigen-binding domain binds to a second HER2 epitope on a second domain of HER2. In some embodiments, the first domain of HER2 is ECD2 and the second domain of HER2 is ECD4. [0098] In some embodiments, one of the antigen-binding domains comprised by the anti-HER2 biparatopic antibody competes with trastuzumab for binding to HER2. In some embodiments, one of the antigen-binding domains comprised by the anti-HER2 biparatopic antibody competes with pertuzumab for binding to HER2. In some embodiments, one of the antigen -binding domains comprised by the anti-HER2 biparatopic antibody competes with trastuzumab for binding to HER2, and the other antigen-binding domain competes with pertuzumab for binding to HER2.
[0099] In some embodiments, one of the antigen-binding domains comprised by the anti-HER2 biparatopic antibody is in a Fab or scFv format and competes with trastuzumab for binding to HER2, and the other antigen-binding domain is in a Fab or scFv format and competes with pertuzumab for binding to HER2. In some embodiments, one of the anti gen -binding domains comprised by the anti-HER2 biparatopic antibody is in an scFv format and competes with trastuzumab for binding to HER2, and the other antigen-binding domain is in a Fab format and competes with pertuzumab for binding to HER2.
[00100] In some embodiments, one of the antigen-binding domains comprised by the anti-HER2 biparatopic antibody binds to the same epitope on HER2 as trastuzumab. In some embodiments, one of the antigen-binding domains comprised by the anti-HER2 biparatopic antibody binds to the same epitope on HER2 as pertuzumab. In some embodiments, one of the anti gen -binding domains comprised by the anti-HER2 biparatopic antibody binds to the same epitope on HER2 as trastuzumab, and the other antigen-binding domain binds to the same epitope on HER2 as pertuzumab.
[00101] In some embodiments, one of the antigen-binding domains comprised by the anti-HER2 biparatopic antibody comprises the CDR sequences of trastuzumab or a variant thereof comprising one or more mutations known to increase HER2 binding, and the other antigen-binding domain comprises the CDRs of pertuzumab or a variant thereof comprising one or more mutations known to increase HER2 binding. Literature mutations known to enhance HER2 binding by trastuzumab or pertuzumab include those listed in Tables 2 and 3 below (HC = heavy chain; LC = light chain). Combinations of these mutations are also contemplated. Table 2: Trastuzumab Mutations that Increase Binding to HER2
Figure imgf000028_0001
Table 3: Pertuzumab Mutations that Increase Binding to HER2
Figure imgf000028_0002
[00102] In certain embodiments, the anti-HER2 biparatopic antibody is one of the biparatopic antibodies described in U.S. Patent Application Publication No. 2016/0289335. In some embodiments, the anti-HER.2 biparatopic antibody is one of v5019, v5020, v7091, vlOOOO, v6902, v6903 or v6717 (see Tables 4, 5 and 6, and Sequence Tables).
[00103] In some embodiments, one of the antigen-binding domains of the anti-HER2 biparatopic antibody comprises the CDR sequences from the ECD2-binding arm of one of v5019, v5020, v7091, vlOOOO, v6902, v6903 or v6717. In some embodiments, one of the antigen-binding domains of the anti-HER2 biparatopic antibody comprises the CDR sequences from the ECD2- binding arm of one of v5019, v5020, v7091, vlOOOO, v6902, v6903 or v6717, and the other antigen-binding domain comprises the CDR sequences from the ECD4-binding arm of one of v5019, v5020, v7091, vlOOOO, v6902, v6903 or v6717. In some embodiments, one of the antigen- binding domains of the anti-HER2 biparatopic antibody comprises the CDR sequences from the ECD2-binding arm of vlOOOO, and the other antigen-binding domain comprises the CDR sequences from the ECD4-binding arm of vlOOOO.
[00104] In some embodiments, one of the antigen-binding domains of the anti-HER2 biparatopic antibody comprises a VH sequence and a VL sequence from the ECD2-binding arm of one of v5019, v5020, v7091, vlOOOO, v6902, v6903 or v6717. In some embodiments, one of the antigenbinding domains of the anti-HER2 biparatopic antibody comprises a VH sequence and a VL sequence from the ECD2-binding arm of one of v5019, v5020, v7091, vlOOOO, v6902, v6903 or v6717, and the other antigen-binding domain comprises a VH sequence and a VL sequence from the ECD4-binding arm of one of v5019, v5020, v7091, vlOOOO, v6902, v6903 or v6717. In some embodiments, one of the antigen-binding domains of the anti-HER2 biparatopic antibody comprises a VH sequence and a VL sequence from the ECD2-binding arm of vlOOOO, and the other antigen -binding domain comprises a VH sequence and a VL sequence from the ECD4- binding arm of vlOOOO.
Table 4: Exemplary Anti-HER2 Biparatopic Antibodies
Figure imgf000029_0001
Figure imgf000030_0001
* Fab or variable domain numbering according to Kabat (Kabat et al., Sequences of proteins of immunological interest, 5th Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, p.647, 1991)
§ CH3 numbering according to EU index as in Kabat (Edelman et al., 1969, PNAS USA, 63 :78-85) Table 5: CDR Sequences of the ECD2-Binding Arm of Variants v5019, v5020, v7091, vlOOOO, v6902, v6903 and v6717
Figure imgf000031_0001
Table 6: CDR Sequences of the ECD4-Binding Arm of Variants v5019, v5020, v7091, vlOOOO, v6902, v6903 and v6717
Figure imgf000031_0002
[00105] In certain embodiments, the anti-HER2 biparatopic antibody comprised by the ADCs comprises (a) a first antigen-binding domain comprising the CDR sequences as set forth in SEQ ID NOs: 32, 34 and 33, and in SEQ ID NOs: 22, 24 and 23, and (b) a second antigen-binding domain comprising the CDR sequences as set forth in SEQ ID NOs: 53, 54 and 55, and SEQ ID NOs: 56, 57 and 58.
[00106] In certain embodiments, the anti-HER2 biparatopic antibody comprised by the ADCs comprises (a) a first antigen -binding domain that is a Fab and comprises the CDR sequences as set forth in SEQ ID NOs: 32, 34 and 33, and in SEQ ID NOs: 22, 24 and 23, and (b) a second antigen- binding domain that is an scFv and comprises the CDR sequences as set forth in SEQ ID NOs: 53, 54 and 55, and SEQ ID NOs: 56, 57 and 58.
[00107] In certain embodiments, the anti-HER2 biparatopic antibody comprised by the ADCs comprises (a) a first antigen-binding domain comprising a first set of CDRs comprising the CDR1 sequence as set forth in SEQ ID NO: 32, the CDR2 sequence as set forth in SEQ ID NO: 34 and the CDR3 sequence as set forth in SEQ ID NO: 33, and a second set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 22, the CDR2 sequence as set forth in SEQ ID NO: 24 and the CDR3 sequence as set forth in SEQ ID NO: 23, and (b) a second anti gen -binding domain comprising a third set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 53, the CDR2 sequence as set forth in SEQ ID NO: 54 and the CDR3 sequence as set forth in SEQ ID NO: 55, and a fourth set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 56, the CDR2 sequence as set forth in SEQ ID NO: 57 and the CDR3 sequence as set forth in SEQ ID NO: 58.
[00108] In certain embodiments, the anti-HER2 biparatopic antibody comprised by the ADCs comprises (a) a first heavy chain (Hl) comprising the CDR sequences as set forth in SEQ ID NOs: 32, 34 and 33, (b) a second heavy chain (H2) comprising the CDR sequences as set forth in SEQ ID NOs: 53, 54, 55, 56, 57 and 58, and (c) a light chain (LI) comprising the CDR sequences as set forth in SEQ ID NOs: 22, 24 and 23.
[00109] In certain embodiments, the anti-HER2 biparatopic antibody comprised by the ADCs comprises (a) a first heavy chain (Hl) comprising a first set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 32, the CDR2 sequence as set forth in SEQ ID NO: 34 and the CDR3 sequence as set forth in SEQ ID NO: 33, (b) a second heavy chain (H2) comprising a second set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 53, the CDR2 sequence as set forth in SEQ ID NO: 54, and the CDR3 sequence as set forth in SEQ ID NO: 55, and a third set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 56, the CDR2 sequence as set forth in SEQ ID NO: 57 and the CDR3 sequence as set forth in SEQ ID NO: 58, and a light chain (LI) comprising a fourth set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 22, the CDR2 sequence as set forth in SEQ ID NO: 24 and the CDR3 sequence as set forth in SEQ ID NO: 23.
[00110] In certain embodiments, the anti-HER2 biparatopic antibody comprised by the ADCs comprises (a) a first antigen -binding domain comprising the VH sequence as set forth in SEQ ID NO: 31, and the VL sequence as set forth in SEQ ID NO: 21, and (b) a second antigen-binding domain comprising the VH sequence as set forth in SEQ ID NO: 52, and the VL sequence as set forth in SEQ ID NO: 51.
[00111] In certain embodiments, the anti-HER2 biparatopic antibody comprised by the ADCs comprises (a) a first antigen-binding domain that is a Fab and comprises the VH sequence as set forth in SEQ ID NO: 31, and the VL sequence as set forth in SEQ ID NO: 21, and (b) a second antigen-binding domain that is an scFv and comprises the VH sequence as set forth in SEQ ID NO: 52, and the VL sequence as set forth in SEQ ID NO: 51.
[00112] In certain embodiments, the anti-HER2 biparatopic antibody comprised by the ADCs comprises a first heavy chain (Hl) comprising the VH sequence as set forth in SEQ ID NO: 31, a second heavy chain (H2) comprising the VH sequence as set forth in SEQ ID NO: 52 and the VL sequence as set forth in SEQ ID NO: 51, and a light chain (LI) comprising the VL sequence as set forth in SEQ ID NO: 21.
[00113] In certain embodiments, the anti-HER2 biparatopic antibody comprised by the ADCs comprises (a) a first heavy chain (Hl) comprising the sequence as set forth in SEQ ID NO: 30, a second heavy chain (H2) comprising the sequence as set forth in SEQ ID NO: 50, and a light chain (LI) comprising the sequence as set forth in SEQ ID NO: 20.
[00114] In certain embodiments, the anti-HER2 biparatopic antibody comprised by the ADCs comprises (a) a first heavy chain (Hl) consisting of the sequence as set forth in SEQ ID NO: 30, a second heavy chain (H2) consisting of the sequence as set forth in SEQ ID NO: 50, and a light chain (LI) consisting of the sequence as set forth in SEQ ID NO: 20.
[00115] The anti-HER2 biparatopic antibodies comprised by the ADCs described herein may have various formats. The minimal components of the anti-HER2 biparatopic antibody are a first antigen-binding domain that binds to a first HER2 epitope and a second antigen-binding domain that binds to a second HER2 epitope, with the first and second HER2 epitopes being different. An antibody that comprises two antigen-binding domains that bind to different HER2 epitopes may be considered to be a bivalent, biparatopic antibody. Antibodies that comprise one or more additional antigen-binding domains, each of which binds to either the first or second HER2 epitope, are also biparatopic, but are considered to be trivalent or tetravalent, for example. In certain embodiments, the anti-HER2 biparatopic antibody comprised by the ADC is a bivalent, anti-HER2 biparatopic antibody.
[00116] In certain embodiments, the anti-HER2 biparatopic antibody comprises a scaffold to which first and second antigen-binding domains are operably linked. The term “operably linked,” as used herein, means that the components described are in a relationship permitting them to function in their intended manner. Suitable scaffolds are described below. In some embodiments, the anti-HER2 biparatopic antibody comprises two antigen-binding domains operably linked to a scaffold, and at least one of the antigen-binding domains is an scFv. In some embodiments, the anti-HER2 biparatopic antibody comprises two antigen-binding domains operably linked to a scaffold, and at least one of the antigen-binding domains is a Fab. In some embodiments, the anti- HER2 biparatopic antibody comprises two antigen-binding domains operably linked to a scaffold, where one of the antigen-binding domains is an scFv and the other antigen-binding domain is a Fab.
[00117] Examples of suitable scaffolds include, but are not limited to, immunoglobulin Fc regions, albumin, albumin analogs and derivatives, heterodimerizing peptides (such as leucine zippers, heterodimer-forming “zipper” peptides derived from Jun and Fos, IgG CHI and CL domains or barnase-barstar toxins), cytokines, chemokines or growth factors. Other examples include antibodies based on the DOCK-AND-LOCK™ (DNL™) technology developed by IBC Pharmaceuticals, Inc. and Immunomedics, Inc. (see, for example, Chang, etal., 2007, Clin Cancer Res., 13:5586s-5591s).
[00118] In certain embodiments, the anti-HER2 biparatopic antibody comprises a scaffold that is based on an immunoglobulin Fc region, an albumin or an albumin analogue or derivative (such as those described in International Patent Application Publication No. WO 2012/116453 or WO 2014/012082). In some embodiments, the anti-HER2 biparatopic antibody comprises a protein scaffold that is based on an immunoglobulin (Ig) Fc region. In some embodiments, the anti-HER2 biparatopic antibody comprises a protein scaffold that is based on an IgG Fc region.
[00119] The terms “Fc region,” “Fc” or “Fc domain” as used herein refer to a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).
[00120] Ig Fc regions are typically dimeric and composed of two Fc polypeptides. An “Fc polypeptide” of a dimeric Fc refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising one or more C-terminal constant regions of an immunoglobulin heavy chain that is capable of stable self-association. The terms “first Fc polypeptide” and “second Fc polypeptide” may be used interchangeably to describe the Fc polypeptides comprised by a dimeric Fc region, provided that the Fc region comprises one first Fc polypeptide and one second Fc polypeptide.
[00121] An Fc region comprises a CH3 domain or both a CH3 and a CH2 domain. For example, an Fc polypeptide of a dimeric IgG Fc region comprises an IgG CH2 and an IgG CH3 constant domain sequence. The CH3 domain comprises two CH3 sequences, one from each of the two Fc polypeptides of the dimeric Fc region. The CH2 domain comprises two CH2 sequences, one from each of the two Fc polypeptides of the dimeric Fc region.
[00122] In some embodiments, the anti-HER2 biparatopic antibody comprised by the ADCs may comprise a scaffold that is based on an IgG Fc region. In some embodiments, the anti-HER2 biparatopic antibody may comprise a scaffold that is based on a human Fc region. In some embodiments, the anti-HER2 biparatopic antibody may comprise a scaffold based on a human IgG Fc region, for example a human IgGl Fc region.
[00123] In certain embodiments, the anti-HER2 biparatopic antibody comprised by the ADCs may comprise a scaffold based on an IgG Fc region, which is a heterodimeric Fc region, comprising a first Fc polypeptide and a second Fc polypeptide, each comprising a CH3 sequence, and optionally a CH2 sequence.
[00124] In some embodiments, the anti-HER2 biparatopic antibody comprised by the ADCs may comprise a scaffold based on an Fc region which comprises first and second Fc polypeptides, and the first antigen-binding domain is operably linked to the first Fc polypeptide and the second antigen-binding domain is operably linked to the second Fc polypeptide.
[00125] In some embodiments, the anti-HER2 biparatopic antibody comprised by the ADCs may comprise a scaffold based on an Fc region which comprises first and second Fc polypeptides, in which the first antigen-binding domain is operably linked to the first Fc polypeptide and the second antigen-binding domain is operably linked to the second Fc polypeptide, and in which the first and second antigen-binding domains are independently a Fab fragment or an scFv.
[00126] In some embodiments, the anti-HER2 biparatopic antibody comprised by the ADCs may comprise a scaffold based on an Fc region which comprises two CH3 sequences, at least one of which comprises one or more amino acid modifications. In some embodiments, the anti-HER2 biparatopic antibody comprises a heterodimeric Fc region comprising a modified CH3 domain, wherein the modified CH3 domain is an asymmetrically modified CH3 domain. Generally, the first Fc polypeptide of the heterodimeric Fc comprises a first CH3 sequence and the second Fc polypeptide comprises a second CH3 sequence.
[00127] As used herein, “asymmetric amino acid modification” refers to a modification where an amino acid at a specific position on a first CH3 sequence is different to the amino acid on a second CH3 sequence at the same position. For CH3 sequences comprising asymmetric amino acid modifications, the first and second CH3 sequence will typically preferentially pair to form a heterodimer, rather than a homodimer. These asymmetric amino acid modifications can be a result of modification of only one of the two amino acids at the same respective amino acid position on each sequence, or different modifications of both amino acids on each sequence at the same respective position on each of the first and second CH3 sequences. Each of the first and second CH3 sequence of a heterodimeric Fc may comprise one or more than one asymmetric amino acid modification.
[00128] In some embodiments, the anti-HER2 biparatopic antibody may comprise a scaffold based on a modified Fc region as described in International Patent Application Publication No. WO 2012/058768 or WO 2013/063702. [00129] Table 7 provides the amino acid sequence of the human IgGl Fc sequence (SEQ ID NO: 1), corresponding to amino acids 231 to 447 of the full-length human IgGl heavy chain. The CH3 sequence comprises amino acids 341-447 of the full-length human IgGl heavy chain.
[00130] In certain embodiments, the anti-HER2 biparatopic antibody may comprise a heterodimeric Fc scaffold comprising a modified CH3 domain that comprises asymmetric amino acid modifications that promote formation of a heterodimeric Fc over formation of a homodimeric Fc. In some embodiments, the anti-HER2 biparatopic antibody may comprise a heterodimeric Fc scaffold which includes modifications as described below at one or more of the following positions: L351, F405, Y407, T366, K392, T394, T350, S400 and/or N390, using EU numbering.
[00131] In certain embodiments, the anti-HER2 biparatopic antibody comprised by the ADCs may comprise a heterodimeric Fc comprising a modified CH3 domain having a first polypeptide sequence comprising amino acid modifications at positions F405 and Y407, and optionally further comprising an amino acid modification at position L351, and a second polypeptide sequence comprising amino acid modifications at positions T366 and T394, and optionally further comprising an amino acid modification at position K392, where the amino acid modification at position F405 is F405A, F405I, F405M, F405S, F405T or F405V; the amino acid modification at position Y407 is Y407I or Y407V; the amino acid modification at position T366 is T366I, T366L or T366M; the amino acid modification at position T394 is T394W; the amino acid modification at position L351 is L351Y, and the amino acid modification at position K392 is K392F, K392L or K392M. In some embodiments, the amino acid modification at position F405 is F405A, F405S, F405T or F405V. In some embodiments, one or both of the first and second Fc polypeptide sequences further comprises the amino acid modification T350V. In certain embodiments, the first Fc polypeptide sequence further comprises an amino acid modification at one or both of positions S400 or Q347 and/or the second Fc polypeptide sequence further comprises an amino acid modification at one or both of positions K360 or N390, where the amino acid modification at position S400 is S400E, S400D, S400R or S400K; the amino acid modification at position Q347 is Q347R, Q347E or Q347K; the amino acid modification at position K360 is K360D or K360E, and the amino acid modification at position N390 is N390R, N390K or N390D. [00132] In some embodiments, the anti-HER2 biparatopic antibody comprised by the ADCs may comprise a heterodimeric Fc scaffold having a modified CH3 domain comprising the modifications of any one of Variant 1, Variant 2, Variant 3, Variant 4 or Variant 5, as shown in Table 7. Table 7: IgGl Fc sequences
Figure imgf000038_0001
[00133] In certain embodiments, the anti-HER2 biparatopic antibody comprised by the ADCs may comprise a heterodimeric Fc scaffold having a modified CH3 domain with a first CH3 sequence comprising the amino acid modifications L351Y, F405A, and Y407V, and the second CH3 sequence comprising the amino acid modifications T366L or T366I; K392L or K392M, and T394W, and one or both of the first and second CH3 sequences may optionally further comprise the amino acid modification T350V. PREPARATION OF ANTI-HER2 BIPARATOPIC ANTIBODY DRUG CONJUGATES
Anti-HER2 Biparatopic Antibodies
[00134] The anti-HER2 biparatopic antibody comprised by the ADCs described herein may be produced using standard recombinant methods known in the art (see, for example, "Antibodies: A Laboratory Manual,” 2nd Edition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014, and International Patent Application Publication No. WO 2015/077891).
[00135] For recombinant expression, appropriate host cells are transformed with an isolated nucleic acid encoding the anti-HER2 biparatopic antibody and cultured under conditions allowing for expression of the anti-HER2 biparatopic antibody. Such nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the anti- HER2 biparatopic antibody (for example, the light and/or heavy chains of the antibody). As is known in the art, many amino acid acids are encoded by more than one codon and as such multiple nucleic acids may encode a single polypeptide sequence. An exemplary nucleic acid is provided herein for each polypeptide of the anti-HER2 biparatopic antibody, however, it is understood that other nucleic acids encoding the anti-HER2 biparatopic antibody may be employed.
[00136] The nucleic acid encoding the anti-HER2 biparatopic antibody may be provided in the form of an expression vector or vectors. In some embodiments, the nucleic acid is provided in a multi ci str onic vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the anti-HER2 biparatopic antibody and an amino acid sequence comprising the VH of the anti-HER2 biparatopic antibody. In some embodiments, the nucleic acid is provided as a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the anti-HER2 biparatopic antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the anti-HER2 biparatopic antibody.
[00137] In certain embodiments, the anti-HER2 biparatopic antibody is produced in stable mammalian cells by a method comprising: transfecting stable mammalian cells with nucleic acid encoding the anti-HER2 biparatopic antibody in a predetermined ratio; and expressing the nucleic acid in the mammalian cells. In some embodiments, the predetermined ratio of nucleic acid is determined in transient transfection experiments to determine the relative ratio of input nucleic acids that results in the highest percentage of the anti-HER2 biparatopic antibody in the expressed product.
[00138] In some embodiments, the anti-HER2 biparatopic antibody is produced in stable mammalian cells where the expression product of the mammalian cells comprises a larger percentage of the glycosylated anti-HER2 biparatopic antibody as compared to the monomeric heavy or light chain polypeptides, or other antibodies.
[00139] Typically, the anti-HER2 biparatopic antibody is purified after expression. Proteins may be isolated or purified in a variety of ways known to those skilled in the art (see, for example, Protein Purification: Principles and Practice, 3rd Ed., Scopes, Springer-Verlag, NY, 1994). Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reverse-phase, carried out at atmospheric pressure or at high pressure using systems such as FPLC and HPLC. Additional purification methods include electrophoretic, immunological, precipitation, dialysis and chromatofocusing techniques. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. As is well known in the art, a variety of natural proteins bind Fc and antibodies, and these proteins may be used for purification of certain antibody constructs. For example, the bacterial proteins A and G bind to the Fc region. Likewise, the bacterial protein L binds to the Fab region of some antibodies.
[00140] In certain embodiments, purification of the anti-HER2 biparatopic antibody comprises anion exchange chromatography, for example, using a Q Sepharose™, diethylaminoethyl (DEAE) Sepharose™, POROS™ HQ, POROS™ DEAF, Toyopearl® Q, Toyopearl® QAE, Toyopearl® DEAE, Resource/Source Q, Resource/Source DEAE, Fractogel® Q or Fractogel® DEAE column, or equivalent. In certain embodiments, purification of the anti-HER2 biparatopic antibody comprises cation exchange chromatography, for example, using an SP Sepharose™, CM Sepharose™, POROS™ HS, POROS™ CM, Toyopearl® SP, Toyopearl® CM, Resource/Source S, Resource/Source CM, Fractogel® S or Fractogel® CM column, or equivalent.
Anti-HER2 Biparatopic ADCs
[00141] The anti-HER2 biparatopic ADCs of the present disclosure may be prepared by one of several routes known in the art, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art (see, for example, Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press, and the Examples provided herein). For example, conjugation may be achieved by (1) reaction of a functional group on the antibody with a bifunctional linker to form an antibody -linker intermediate Ab-L, via a covalent bond, followed by reaction with an activated form of the auristatin analogue, or (2) reaction of a functional group on the auristatin analogue with a linker to form linker-toxin, via a covalent bond, followed by reaction with a functional group on the antibody. Suitable functional groups on the antibody include, for example, side-chain amino groups of lysine residues and thiol groups of cysteine residues. Suitable functional groups may also be provided by the side-chains of non-natural amino acids, such as selenomethionine, p-acetylphenylalanine, formylglycine or p-azidomethyl-L-phenylalanine, in those antibodies engineered to include such amino acids.
[00142] The terms “linker-toxin” and “drug-linker” are used interchangeably herein to refer to a moiety comprising the auristatin analogue covalently bonded to a linker. The linker-toxin typically includes a functional group suitable for reaction with the selected target group on the anti-HER2 biparatopic antibody.
[00143] In certain embodiments, the anti-HER2 biparatopic ADCs of the present disclosure comprise an auristatin analogue conjugated via an appropriate linker to the thiol group of a cysteine residue on the antibody. In some embodiments, the auristatin analogue is conjugated via an appropriate linker to the thiol group of a cysteine residue that has been liberated by reducing an interchain disulfide bond.
[00144] In the ADCs described herein, the anti-HER2 biparatopic antibody is conjugated to the auristatin analogue via a linker at a low average drug-to-antibody ratio (DAR), such as an average DAR of between about 1.5 and about 2.5.
[00145] Various methods are known in the art to prepare ADCs with a low average DAR (see, for example, review by McCombs and Owen, The AAPS Journal, 17(2):339-351 (2015) and references therein; Boutureira & Bernardes, Chem. Rev., 115:2174-2195 (2015)).
[00146] For example, for conjugation to cysteine residues, a partial reduction of the antibody interchain disulfide bonds may be conducted followed by conjugation to linker-toxin. Partial reduction can be achieved by limiting the amount of reducing agent used in the reduction reaction (see, for example, the Examples provided herein, and Lyon et al., Methods in Enzymology, 502: 123-138 (2012), and examples therein). Suitable reducing agents are known in the art and include, for example, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), 2- mercaptoethanol, cysteamine and a number of water soluble phosphines. Alternatively, or in addition, fewer equivalents of linker-toxin may be employed in the conjugation reaction in order to obtain a low average DAR.
[00147] Low average DAR may also be achieved by employing an engineered antibody in which one or more of the cysteine residues that make up the interchain disulfide bonds is replaced with a serine residue resulting in fewer available cysteine residues for conjugation (see McDonagh et al., Protein Eng. Des. Sei. PEDS, 19(7):299-307). The engineered antibody can be treated with reducing agent and conjugated to linker-toxin using standard methods.
[00148] Another approach is to employ a bis-thiol linker that bridges two cysteines that normally make up an interchain disulfide bond. Use of a bis-thiol linker that carries only one toxin molecule produces an ADC with a maximum DAR4 for a full-size antibody, if all four interchain disulfide bonds are reduced and replaced with the bis-thiol linker. Partial reduction of the interchain disulfide bonds and/or fewer equivalents of linker or linker-toxin may be used in conjunction with a bis-thiol linker in order to further reduce the DAR. Various bis-thiol linkers are known in the art (see, for example, Badescu etal., Bioconjug. Chem., 25(6): 1124-1136 (2014); Behrens etal., Mol. Pharm., 12:3986-3998 (2015); Lee et al., Chem. Sci., 7:799-802 (2016); Maruani et al., Nat. Commun., 6:6645 (2015)).
[00149] Cysteine engineering approaches may also be employed in order to generate ADCs with a low average DAR. Such approaches involve engineering solvent-accessible cysteines into the antibody in order to provide a site-specific handle for conjugation. A number of appropriate sites for introduction of a cysteine residue have been identified with the IgG structure, and include those described in Junutula, et al., J. Immunol Methods, 332(l-2):41-52 (2008); Junutula, et al., Nat. Biotechnol., 26(8), 925-932 (2008), and U.S. Patent Nos. 9,315,581; 9,000,130; 8,455,622; 8,507,654 and 7,521,541. [00150] Alternatively, ADCs with a low average DAR may be isolated from an ADC preparation containing a mixture of DAR species using chromatographic separation techniques, such as hydrophobic interaction chromatography (see, for example, Hamblett, et al., Clin. Cancer Res., 10:7063-7070 (2004); Sun, et al., Bioconj Chem., 28: 1371-81 (2017); U.S. Patent Application Publication No. 2014/0286968).
[00151] ADC preparations with a low average DAR may also be generated by adding unconjugated (i.e. DAR0) antibody to preparations of ADC having an average DAR > 2.5. As is known in the art, the majority of conjugation methods yield an ADC preparation that includes various DAR species, with the reported DAR being the average of the individual DAR species. Accordingly, adding additional DAR0 species into an ADC preparation having a DAR > 2.5 will reduce the average DAR of the preparation. In certain embodiments, ADCs that include a proportion of DAR0 species may be advantageous. In some embodiments, the low average DAR ADCs of the present disclosure may include at least 5% DAR0 species, for example, between about 5% and about 50% DAR0 species. In some embodiments, the low average DAR ADCs may include between about 10% and about 50% DAR0 species. In some embodiments, the low average DAR ADCs may include between about 10% and about 40%, for example, between about 10% and about 30% DAR0 species.
[00152] The average DAR for the ADCs may be determined by standard techniques such as UV/VIS spectroscopic analysis, ELISA-based techniques, chromatography techniques such as hydrophobic interaction chromatography (HIC), UV-MALDI mass spectrometry (MS) and MALDI-TOF MS. In addition, distribution of drug-linked forms (for example, the fraction of DAR0, DARI, DAR2, etc. species) may also be analyzed by various techniques known in the art, including MS (with or without an accompanying chromatographic separation step), hydrophobic interaction chromatography, reverse-phase HPLC or iso-electric focusing gel electrophoresis (IEF) (see, for example, Sun et al., Bioconj Chem., 28: 1371-81 (2017); Wakankar et al., mAbs, 3: 161- 172 (2011)).
[00153] In certain embodiments, the average DAR of the ADCs is determined by hydrophobic interaction chromatography (HIC) techniques. [00154] Following conjugation, the ADCs may be purified and separated from unconjugated reactants and/or any conjugate aggregates by purification methods known in the art. Such methods include, but are not limited to, size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), ion exchange chromatography, chromatofocusing, ultrafiltration, centrifugal ultrafiltration, and combinations thereof.
METHODS OF USE
[00155] Certain aspects of the present disclosure relate to methods of treating a HER2-expressing cancer in a subject by administering an effective amount of an anti-HER2 biparatopic ADC described herein. A “HER2-expressing cancer” may also be referred to in the art as a “HER2- positive cancer” and includes cancers that express or overexpress HER2 protein with or without HER2 gene amplification. HER2 protein expression may be identified, for example, by immunohistochemistry (IHC) techniques. HER2 gene amplification may be identified, for example, by in situ hybridization (ISH) techniques.
[00156] In the methods of treatment according to the present disclosure, the anti-HER2 biparatopic ADC is administered to the subject in an “effective amount.” In certain embodiments, the effective amount of the anti-HER2 biparatopic ADC may be a dose of between 1 mg/kg and 2.5 mg/kg administered to the subject every week (QW), or a dose of between 2 mg/kg and 3 mg/kg administered to the subject every 3 weeks (Q3W). In certain embodiments, the effective amount of the anti-HER2 biparatopic ADC administered to the subject in the disclosed methods may be a dose of between 1 mg/kg and 2.5 mg/kg administered QW for 3 weeks of a 4-week cycle, or a dose of between 2 mg/kg and 3 mg/kg administered Q3W.
[00157] In certain embodiments, the effective amount of the anti-HER2 biparatopic ADC administered to the subject in the methods of the present disclosure is a dose of between 1 mg/kg and 2 mg/kg administered QW for 3 weeks of a 4-week cycle. In some embodiments, the effective amount of the anti-HER2 biparatopic ADC administered to the subject in the methods of the present disclosure is a dose of between 1.25 mg/kg and 2 mg/kg administered QW for 3 weeks of a 4-week cycle. In some embodiments, the effective amount of the anti-HER2 biparatopic ADC administered to the subject in the disclosed methods is a dose of 1.25 mg/kg, 1.5 mg/kg, 1.75 mg/kg or 2.0 mg/kg administered QW for 3 weeks of a 4-week cycle. In some embodiments, the effective amount of the anti-HER2 biparatopic ADC administered to the subject in the disclosed methods is a dose of 1.25 mg/kg QW for 3 weeks of a 4-week cycle. In some embodiments, the effective amount of the anti-HER.2 biparatopic ADC administered to the subject in the disclosed methods is a dose of 1.5 mg/kg QW for 3 weeks of a 4-week cycle. In some embodiments, the effective amount of the anti-HER.2 biparatopic ADC administered to the subject in the disclosed methods is a dose of 1.75 mg/kg QW for 3 weeks of a 4-week cycle.
[00158] Pharmacokinetic modeling indicates that lower Cmax, higher Cmin and/or higher exposure may be achieved with QW dosing with the anti-HER.2 biparatopic ADC than with Q2W or Q3W dosing (see Fig. 4). Accordingly, some embodiments of the present disclosure relate to methods of treating a subject having a HER2-expressing cancer comprising administering to the subject an effective amount of an anti-HER.2 biparatopic antibody drug conjugate (ADC), the effective amount comprising a dose of between 1 mg/kg and 2.0 mg/kg, for example, 1.25 mg/kg,
1.5 mg/kg or 1.75 mg/kg, administered QW for 3 weeks of a 4-week cycle, whereby lower Cmax, higher Cmin and/or higher exposure is achieved than with a dose of 2.5 mg/kg administered Q3W. In some embodiments, in the methods of the present disclosure, administering to the subject an anti-HER.2 biparatopic antibody drug conjugate (ADC) at a dose of 1.25 mg/kg administered QW for 3 weeks of a 4-week cycle results in a substantially equivalent Cmax, Cmin and/or exposure than is observed with a dose of 2.5 mg/kg administered Q3W.
[00159] In certain embodiments, the effective amount of the anti-HER.2 biparatopic ADC administered to the subject in the methods of the present disclosure is a dose of between 2 mg/kg and 3 mg/kg administered every 3 weeks (Q3W). In some embodiments, the effective amount of the anti-HER.2 biparatopic ADC administered to the subject in the disclosed methods is a dose of
2.5 mg/kg administered Q3W.
[00160] In certain embodiments, the anti-HER.2 biparatopic ADC is administered to the subject intravenously. In some embodiments, the anti-HER.2 biparatopic antibody is administered to the subject by intravenous infusion.
[00161] In certain embodiments, the HER2-expressing cancer that may be treated using the methods of the present disclosure is a HER2-expressing solid tumor. Examples of HER2- expressing solid tumors include, but are not limited to, breast cancer, endometrial cancer, ovarian cancer, cervical cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, anal cancer, urothelial cancer, pancreatic cancer, salivary gland cancer, bladder cancer and brain cancer. HER2-expressing breast cancers include estrogen receptor negative (ER-) and/or progesterone receptor negative (PR-) breast cancers and triple negative (ER-, PR-, low HER2) breast cancers. HER2-expressing lung cancers include non-small cell lung cancer (NSCLC) and small cell lung cancer.
[00162] In certain embodiments, the methods described herein are for the treatment of a HER2- expressing breast cancer, HER2-expressing gastroesophageal adenocarcinoma (GEA), HER2- expressing esophageal cancer, HER2-expressing endometrial cancer, HER2-expressing ovarian cancer, HER2-expressing cervical cancer, HER2-expressing NSCLC, HER2-expressing anal cancer or HER2-expressing colorectal cancer (CRC).
[00163] In certain embodiments, the methods described herein are for the treatment of breast cancer, GEA, endometrial cancer, ovarian cancer, NSCLC, anal cancer, pancreatic cancer, biliary tract cancer or bladder cancer. In certain embodiments, the methods described herein are for the treatment of GEA, endometrial cancer, ovarian cancer, NSCLC or CRC. The cancer may be HER2- expressing with or without HER2 gene amplification.
[00164] In certain embodiments, the methods described herein are for the treatment of a HER2- expressing cancer that is metastatic or locally advanced. In some embodiments, the methods described herein are for the treatment of a HER2-expressing cancer that has metastasized to the brain.
[00165] As is known in the art, HER2-expressing (or HER2-positive) cancers may be characterized buy the level of HER2 they express (i.e. by “HER2-status”). HER2 status can be assessed, for example, by immunohistochemistry (IHC), fluorescent in situ hybridization (FISH) and/or chromogenic in situ hybridization (CISH) techniques. A number of commercial kits are available for assessing HER2 status in patients. Examples of FDA-approved commercial kits available for HER2 detection using IHC include HercepTest™ (Dako Denmark A/S); PATHWAY (Ventana Medical Systems, Inc.); InSite™HER2/NEU kit (Biogenex Laboratories, Inc.) and Bond Oracle HER2 IHC System (Leica Biosystems). [00166] IHC identifies HER2 protein expression on the cell membrane. For example, paraffin- embedded tissue sections from a tumor biopsy may be subjected to the IHC assay and accorded a HER2 staining intensity criteria as follows:
Score 0: no staining observed or membrane staining is observed in less than 10% of tumor cells; typically <20,000 receptors/cell.
Score 1+: a faint/barely perceptible membrane staining is detected in more than 10% of the tumor cells. The cells are only stained in part of their membrane. Typically about 100,000 receptors/cell.
Score 2+: a weak to moderate complete membrane staining is observed in more than 10% of the tumor cells; typically about 500,000 receptors/cell.
Score 3+: a moderate to strong complete membrane staining is observed in more than 10% of the tumor cells; typically about 2,000,000 receptors/cell.
[00167] The anti-HER2 biparatopic ADCs described herein may be useful in methods of treating cancers that express HER2 at various levels. In certain embodiments, the methods of treating HER2-expressing cancers according to the present disclosure comprise administering an anti- HER2 biparatopic ADC as described herein to a subject having a cancer that expresses high levels of HER2 (HER2-high), where HER2-high is defined as IHC 3+ In some embodiments, the methods of treating HER2-expressing cancers according to the present disclosure comprise administering an anti-HER2 biparatopic ADC as described herein to a subject having a cancerthat expresses high levels of HER2 (HER2-high), where HER2-high is defined as IHC 2+, IHC 2+/3+ or IHC 3+. In some embodiments, the methods of treating HER2-expressing cancers according to the present disclosure comprise administering an anti-HER2 biparatopic ADC as described herein to a subject having a cancer that expresses low levels of HER2 (HER2-low), where HER2-low is defined as IHC 1+ or IHC 1+/2+.
[00168] In situ hybridization (ISH) techniques such as FISH and CISH identify HER2 gene amplification and chromosome aneuploidy. Usually, cancers that show a positive result by ISH (for example, those that are HER2-amplified) also show a positive result by IHC. For cancers having a score of IHC 2+ or lower, ISH is often used to confirm that the cancer is HER2 -positive. Accordingly, a cancer referred to herein as “HER2-expressing” or “HER2-positive” includes cancers that test positive by IHC and/or by FISH. In certain embodiments, the methods of treating HER2-expressing cancers according to the present disclosure comprise administering an anti- HER2 biparatopic ADC as described herein to a subject having a HER2-expressing cancer having a score of IHC 3+ or IHC 2+/FISH+.
[00169] In certain embodiments, the methods described herein are for the treatment of a subject having a HER2-expressing cancer that is resistant or becoming resistant to other standard-of-care therapies. In some embodiments, the methods described herein are for the treatment of a subject having a HER2-expressing cancer who is unresponsive to, and/or has relapsed after, one or more current therapies, such as another anti-HER2 therapeutic (such as trastuzumab (Herceptin®), pertuzumab (Peijeta®), T-DM1 (Kadcyla® or trastuzumab emtansine), and the like) or a taxane (such as such as paclitaxel, docetaxel, cabazitaxel, and the like). In some embodiments, the methods described herein are for the treatment of a subject having a HER2-expressing cancer that is resistant to trastuzumab. In some embodiments, the methods described herein are for the treatment of a subject having cancer that has progressed after treatment with one or more other anti-HER2 therapeutics. In some embodiments, the methods described herein are for the treatment of a subject having metastatic cancer that has progressed on previous anti-HER2 therapy. In some embodiments, the methods described herein are for the treatment of a subject who has previously undergone treatment with one or more of trastuzumab, pertuzumab and T-DM1.
[00170] Certain embodiments of the present disclosure relate to a method of treating a HER2- expressing cancer in a subject by administering an effective amount of an anti-HER2 biparatopic ADC to the subject, in which the effective amount is a dose of 2.5 mg/kg administered Q3W, and the HER2-expressing cancer is a HER2-high cancer (IHC 3+, IHC 2+/3+ or IHC 2+). Some embodiments relate to a method of treating a HER2-expressing cancer in a subject by administering an effective amount of an anti-HER2 biparatopic ADC to the subject, in which the effective amount is a dose of 2.5 mg/kg administered Q3W, and the HER2-expressing cancer is a HER2-low cancer (IHC 1+, IHC 1+/2+). Some embodiments relate to a method of treating a HER2-expressing cancer in a subject by administering an effective amount of an anti-HER2 biparatopic ADC to the subject, in which the effective amount is a dose of 2.5 mg/kg administered Q3W, and the HER2-expressing cancer has a score of IHC 3+ or IHC 2+/FISH+. [00171] Certain embodiments of the present disclosure relate to a method of treating a HER2- expressing cancer in a subject by administering an effective amount of an anti-HER2 biparatopic ADC to the subject, in which the effective amount is a dose of 1.25 mg/kg, 1.5 mg/kg or 1.75 mg/kg administered QW for 3 weeks of a 4-week cycle, and the HER2-expressing cancer is a HER2-high cancer (IHC 3+, IHC 2+/3+ or IHC 2+). Some embodiments relate to a method of treating a HER2-expressing cancer in a subject by administering an effective amount of an anti- HER2 biparatopic ADC to the subject, in which the effective amount is a dose of 1.25 mg/kg, 1.5 mg/kg or 1.75 mg/kg administered QW for 3 weeks of a 4-week cycle, and the HER2-expressing cancer is a HER2-low cancer (IHC 1+, IHC 1+/2+). Some embodiments relate to a method of treating a HER2-expressing cancer in a subject by administering an effective amount of an anti- HER2 biparatopic ADC to the subject, in which the effective amount is a dose of 1.25 mg/kg, 1.5 mg/kg or 1.75 mg/kg administered QW for 3 weeks of a 4-week cycle, and the HER2-expressing cancer has a score of IHC 3+ or IHC 2+/FISH+.
[00172] In certain embodiments, the present disclosure relates to methods of treating a HER2- expressing cancer by administering an effective amount of an anti-HER2 biparatopic ADC having general Formula (I), where the effective amount of the anti-HER2 biparatopic antibody is a dose of between 1 mg/kg and 2.5 mg/kg administered QW for 3 weeks of a 4-week cycle, or a dose of between 2 mg/kg and 3 mg/kg administered Q3W:
Figure imgf000049_0001
wherein:
L is a cleavable linker; n is the average DAR and is between about 1.5 and about 2.5, and Ab is an anti-HER2 biparatopic antibody. [00173] In some embodiments of the disclosed methods, L in general Formula (I) is a protease- cleavable linker. In some embodiments of the disclosed methods, L in general Formula (I) is a peptide-containing linker. In some embodiments, L in general Formula (I) comprises a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit. In some embodiments of the disclosed methods, L in general Formula (I) comprises a dipeptide and a stretcher.
[00174] In some embodiments of the disclosed methods, n in general Formula (I) (the average DAR) is between about 1.6 and about 2.5. In some embodiments of the disclosed methods, n in general Formula (I) is between about 1.8 and about 2.5. In some embodiments of the disclosed methods, n in general Formula (I) is between about 1.8 and about 2.4. In some embodiments of the disclosed methods, n in general Formula (I) is between about 1.8 and about 2.3. In some embodiments of the disclosed methods, n in general Formula (I) is between about 1.9 and about 2.2. In some embodiments of the disclosed methods, n in general Formula (I) is about 2.0. In some embodiments of the disclosed methods, n in general Formula (I) is 2.0 + 0.2.
[00175] In certain embodiments of the disclosed methods, linker L in the anti-HER2 biparatopic ADCs of Formula (I) is conjugated to the antibody via a cysteine residue on the antibody. In certain embodiments of the disclosed methods, linker L in the anti-HER2 biparatopic ADCs of Formula (I) is conjugated to the antibody via a cysteine residue on the antibody that has been liberated by reduction of an interchain disulfide bond.
[00176] In certain embodiments of the disclosed methods, the anti-HER2 biparatopic ADCs have general Formula (II):
Figure imgf000050_0001
n is the average DAR and is between about 1.5 and about 2.5, and Ab is an anti-HER2 biparatopic antibody.
[00177] In some embodiments of the disclosed methods, n in general Formula (II) (the average DAR) is between about 1.6 and about 2.5. In some embodiments of the disclosed methods, n in general Formula (II) is between about 1.8 and about 2.5. In some embodiments of the disclosed methods, n in general Formula (II) is between about 1.8 and about 2.4. In some embodiments of the disclosed methods, n in general Formula (II) is between about 1.8 and about 2.3. In some embodiments of the disclosed methods, n in general Formula (II) is between about 1.9 and about 2.2. In some embodiments of the disclosed methods, n in general Formula (II) is about 2.0. In some embodiments of the disclosed methods, n in general Formula (II) is 2.0 + 0.2.
[00178] In certain embodiments of the disclosed methods, the anti-HER2 biparatopic ADC has a structure as described herein for any one of the embodiments of general Formula (I) or general Formula (II), and the anti-HER2 biparatopic antibody comprises (a) a first antigen-binding domain comprising the CDR sequences as set forth in SEQ ID NOs: 32, 34 and 33, and in SEQ ID NOs: 22, 24 and 23, and (b) a second antigen -binding domain comprising the CDR sequences as set forth in SEQ ID NOs: 53, 54 and 55, and in SEQ ID NOs: 56, 57 and 58.
[00179] In certain embodiments of the disclosed methods, the anti-HER2 biparatopic ADC has a structure as described herein for any one of the embodiments of general Formula (I) or general Formula (II), and the anti-HER2 biparatopic antibody comprises (a) a first antigen-binding domain that is a Fab and comprises the CDR sequences as set forth in SEQ ID NOs: 32, 34 and 33, and in SEQ ID NOs: 22, 24 and 23, and (b) a second antigen-binding domain that is an scFv and comprises the CDR sequences as set forth in SEQ ID NOs: 53, 54 and 55, and in SEQ ID NOs: 56, 57 and 58.
[00180] In certain embodiments of the disclosed methods, the anti-HER2 biparatopic ADC has a structure as described herein for any one of the embodiments of general Formula (I) or general Formula (II), and the anti-HER2 biparatopic antibody comprises (a) a first antigen-binding domain comprising a first set of CDRs comprising the CDR1 sequence as set forth in SEQ ID NO: 32, the CDR2 sequence as set forth in SEQ ID NO: 34 and the CDR3 sequence as set forth in SEQ ID NO: 33, and a second set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 22, the CDR2 sequence as set forth in SEQ ID NO: 24 and the CDR3 sequence as set forth in SEQ ID NO: 23, and (b) a second antigen-binding domain comprising a third set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 53, the CDR2 sequence as set forth in SEQ ID NO: 54 and the CDR3 sequence as set forth in SEQ ID NO: 55, and a fourth set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 56, the CDR2 sequence as set forth in SEQ ID NO: 57 and the CDR3 sequence as set forth in SEQ ID NO: 58.
[00181] In certain embodiments of the disclosed methods, the anti-HER2 biparatopic ADC has a structure as described herein for any one of the embodiments of general Formula (I) or general Formula (II), and the anti-HER2 biparatopic antibody comprises (a) a first heavy chain (Hl) comprising the CDR sequences as set forth in SEQ ID NOs: 32, 34 and 33, (b) a second heavy chain (H2) comprising the CDR sequences as set forth in SEQ ID NOs: 53, 54, 55, 56, 57 and 58, and (c) a light chain (LI) comprising the CDR sequences as set forth in SEQ ID NOs: 22, 24 and 23.
[00182] In certain embodiments of the disclosed methods, the anti-HER2 biparatopic ADC has a structure as described herein for any one of the embodiments of general Formula (I) or general Formula (II), and the anti-HER2 biparatopic antibody comprises (a) a first heavy chain (Hl) comprising a first set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 32, the CDR2 sequence as set forth in SEQ ID NO: 34 and the CDR3 sequence as set forth in SEQ ID NO: 33, (b) a second heavy chain (H2) comprising a second set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 53, the CDR2 sequence as set forth in SEQ ID NO: 54, and the CDR3 sequence as set forth in SEQ ID NO: 55, and a third set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 56, the CDR2 sequence as set forth in SEQ ID NO: 57 and the CDR3 sequence as set forth in SEQ ID NO: 58, and a light chain (LI) comprising a fourth set of CDR sequences comprising the CDR1 sequence as set forth in SEQ ID NO: 22, the CDR2 sequence as set forth in SEQ ID NO: 24 and the CDR3 sequence as set forth in SEQ ID NO: 23.
[00183] In certain embodiments of the disclosed methods, the anti-HER2 biparatopic ADC has a structure as described herein for any one of the embodiments of general Formula (I) or general Formula (II), and the anti-HER2 biparatopic antibody comprises (a) a first antigen-binding domain comprising the VH sequence as set forth in SEQ ID NO: 31, and the VL sequence as set forth in SEQ ID NO: 21, and (b) a second antigen-binding domain comprising the VH sequence as set forth in SEQ ID NO: 52, and the VL sequence as set forth in SEQ ID NO: 51.
[00184] In certain embodiments of the disclosed methods, the anti-HER2 biparatopic ADC has a structure as described herein for any one of the embodiments of general Formula (I) or general Formula (II), and the anti-HER2 biparatopic antibody comprises (a) a first antigen-binding domain that is a Fab and comprises the VH sequence as set forth in SEQ ID NO: 31, and the VL sequence as set forth in SEQ ID NO: 21, and (b) a second antigen -binding domain that is an scFv and comprises the VH sequence as set forth in SEQ ID NO: 52, and the VL sequence as set forth in SEQ ID NO: 51.
[00185] In certain embodiments of the disclosed methods, the anti-HER2 biparatopic ADC has a structure as described herein for any one of the embodiments of general Formula (I) or general Formula (II), and the anti-HER2 biparatopic antibody comprises a first heavy chain (Hl) comprising the VH sequence as set forth in SEQ ID NO: 31, a second heavy chain (H2) comprising the VH sequence as set forth in SEQ ID NO: 52 and the VL sequence as set forth in SEQ ID NO: 51, and a light chain (LI) comprising the VL sequence as set forth in SEQ ID NO: 21.
[00186] In certain embodiments of the disclosed methods, the anti-HER2 biparatopic ADC has a structure as described herein for any one of the embodiments of general Formula (I) or general Formula (II), and the anti-HER2 biparatopic antibody comprises (a) a first heavy chain (Hl) comprising the sequence as set forth in SEQ ID NO: 30, a second heavy chain (H2) comprising the sequence as set forth in SEQ ID NO: 50, and a light chain (LI) comprising the sequence as set forth in SEQ ID NO: 20.
[00187] In certain embodiments of the disclosed methods, the anti-HER2 biparatopic ADC has a structure as described herein for any one of the embodiments of general Formula (I) or general Formula (II), and the anti-HER2 biparatopic antibody comprises (a) a first heavy chain (Hl) consisting of the sequence as set forth in SEQ ID NO: 30, a second heavy chain (H2) consisting of the sequence as set forth in SEQ ID NO: 50, and a light chain (LI) consisting of the sequence as set forth in SEQ ID NO: 20. Combination Therapy
[00188] As shown in the Examples herein, the auristatin analogue, Compound 1, is capable of inducing the release of immune-activating damage associated molecular patterns (DAMPs) from tumor cells at similar levels to the known immunogenic cell death inducer, MMAE. The release of DAMPs can induce immune cell activation and modify the tumor microenvironment to support T-cell responses critical for activity of checkpoint inhibitor therapeutics, such as PD-1 inhibitors.
[00189] Certain aspects of the present disclosure thus relate to methods of treating a HER2- expressing cancer in a subject by administering an effective amount of an anti-HER2 biparatopic ADC as described herein, in combination with a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is a PD-1 inhibitor. In some embodiments, the checkpoint inhibitor is an anti- PD-1 antibody. Examples of anti -PD-1 antibodies include, but are not limited to, pembrolizumab (Keytruda®), nivolumab (Opdivo®), cemiplimab (Libtayo®), JTX-4014 (Jounce Therapeutics), spartalizumab (PDR001) (Novartis), camrelizumab (SHR1210) (Jiangsu HengRui Medicine Co., Ltd.), sinitilimab (Innovent, Eli-Lilly), tislelizumab (BGB-A317) (Beigene), toripalimab (JS 001) (Junshi Biosciences), dostarlimab (GlaxoSmithKline), INCMGA00012 (MGA012) (Incyte, MacroGenics), AMP -224 (AstraZeneca/Medlmmune and GlaxoSmithKline) and AMP-514 (MEDI0680) (AstraZeneca).
[00190] Certain embodiments relate to methods of treating a HER2-expressing cancer in a subject by administering an effective amount of an anti-HER2 biparatopic ADC as described herein, in combination with an anti -PD-1 antibody. Some embodiments relate to methods of treating a HER2-expressing cancer in a subject by administering an effective amount of an anti-HER2 biparatopic ADC as described herein, in combination with tisleizumab.
[00191] Certain embodiments relate to methods of treating a HER2-expressing, PD-L1+ cancer in a subject by administering an effective amount of an anti-HER2 biparatopic ADC as described herein, in combination with a PD-1 inhibitor, for example, an anti-PD-1 antibody as described above.
[00192] The effective amount of the anti-HER2 biparatopic ADC to be used in the combination therapy with a PD-1 inhibitor may be the same as the amount used when the anti-HER2 biparatopic ADC is administered as a single agent. In some embodiments, the effective amount of the anti- HER2 biparatopic ADC to be used in combination with an anti-PD-1 antibody is a dose of between 1 mg/kg and 2.0 mg/kg, for example 1.25 mg/kg, 1.5 mg/kg or 1.75 mg/kg, administered to the subject QW for 3 weeks of a 4-week cycle, or a dose of between 2 mg/kg and 3 mg/kg, for example 2.5 mg/kg, administered to the subject Q3W.
PHARMACEUTICAL COMPOSITIONS
[00193] For therapeutic use, the anti-HER2 biparatopic ADCs may be provided in the form of compositions comprising the ADC and a pharmaceutically acceptable carrier or diluent. The compositions may be prepared by known procedures using well-known and readily available ingredients.
[00194] Pharmaceutical compositions may be formulated for administration to a subject by, for example, oral (including, for example, buccal or sublingual), topical, parenteral, rectal or vaginal routes, or by inhalation or spray. The term “parenteral” as used herein includes subcutaneous injection, and intradermal, intra-articular, intravenous, intramuscular, intravascular, intrasternal, intrathecal injection or infusion. The pharmaceutical composition will typically be formulated in a format suitable for administration to the subject by the selected route, for example, as a syrup, elixir, tablet, troche, lozenge, hard or soft capsule, pill, suppository, oily or aqueous suspension, dispersible powder or granule, emulsion, injectable or solution. In some embodiments, pharmaceutical compositions may be provided as unit dosage formulations.
[00195] In certain embodiments, the pharmaceutical compositions comprising the anti-HER2 biparatopic ADCs are formulated for parenteral administration in injectable form, for example as a powder or lyophilized formulation for reconstitution, or as an aqueous solution.
[00196] In certain embodiments, the compositions comprising the anti-HER2 biparatopic ADCs may be in the form of a sterile injectable aqueous or oleaginous solution or suspension. Such suspensions may be formulated using suitable dispersing or wetting agents and/or suspending agents that are known in the art. The sterile injectable solution or suspension may comprise the ADC in a non-toxic parentally acceptable diluent or carrier. Acceptable diluents and carriers that may be employed include, for example, 1,3 -butanediol, water, Ringer’s solution, isotonic sodium chloride solution or dextrose. In addition, sterile, fixed oils may be employed as a carrier. For this purpose, various bland fixed oils may be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may find use in the preparation of injectables. Adjuvants such as local anaesthetics, preservatives and/or buffering agents may optionally be included in the injectable solution or suspension.
[00197] In certain embodiments, the composition comprising the anti-HER2 biparatopic ADC may be formulated for intravenous administration, for example for intravenous infusion. Typically, compositions that are administered intravenously are formulated as solutions in sterile pharmaceutical grade water or isotonic aqueous solution, for example, containing sodium chloride or dextrose. Where necessary, the composition may also include a solubilizing agent and/or a local anaesthetic such as lignocaine to ease pain at the site of the injection. A composition for intravenous administration may be provided in a lyophilized or dry form, such as a powder or water free concentrate, for reconstitution in an appropriate carrier prior to administration or it may be provided as an injectable solution. In certain embodiments, the composition comprising the anti-HER2 biparatopic ADC is a lyophilized or dry composition for reconstitution.
[00198] Other pharmaceutical compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in “Remington: The Science and Practice of Pharmacy" (formerly “Remingtons Pharmaceutical Sciences"),' Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).
PHARMACEUTICAL KITS
[00199] Certain embodiments of the present disclosure relate to pharmaceutical kits comprising an anti-HER2 biparatopic ADC as described herein.
[00200] The anti-HER2 biparatopic ADC may be provided in the kit in one or more containers and the one or more containers may be associated with a label and/or package insert. Suitable containers include, for example, bottles, vials, ampoules, syringes, intravenous solution bags, and the like, which may in some embodiments have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper that may be pierced by a hypodermic injection needle). The container may be formed from a variety of materials such as glass or plastic. In certain embodiments, the container is a hermetically sealed bottle or vial. [00201] The label or package insert contains instructions customarily included in commercial packages of therapeutic products, providing information regarding indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. For example, the label or package insert may specify that the anti-HER2 biparatopic ADC is for use to treat a HER2-expressing cancer and is for intravenous administration at a dose of between 1 mg/kg and 2.0 mg/kg, for example 1.25 mg/kg, 1.5 mg/kg or 1.75 mg/kg, administered to the subject QW for 3 weeks of a 4-week cycle, or at a dose of between 2 mg/kg and 3 mg/kg, for example, 2.5 mg/kg, administered to the subject Q3W.
[00202] The label or package insert may additionally include information regarding the patient population to be treated, for example, an appropriate age range or stipulation that the patient should be an adult; the type and/or stage of the patient’s cancer; whether or not the patient is required to have undergone one or more prior treatment regimens (and the type of prior treatment regimen or therapeutic), and/or HER2-status.
[00203] In certain embodiments, the label or package insert of the pharmaceutical kit indicates that the HER2-expressing cancer is breast cancer, gastroesophageal adenocarcinoma (GEA), esophageal cancer, endometrial cancer, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), anal cancer or colorectal cancer (CRC). In certain embodiments, the label or package insert of the pharmaceutical kit indicates that the HER2-expressing cancer is one of breast cancer, GEA, endometrial cancer, ovarian cancer, NSCLC, anal cancer, pancreatic cancer, biliary tract cancer orbladder cancer. In certain embodiments, the label or package insert of the pharmaceutical kit indicates that the HER2-expressing cancer is one of GEA, endometrial cancer, ovarian cancer, NSCLC or CRC. In some embodiments, the label or package insert of the pharmaceutical kit indicates that the HER2-expressing cancer is breast cancer or GEA.
[00204] In certain embodiments, the label or package insert of the pharmaceutical kit indicates that the patient to be treated has received prior treatment with one or more HER2 -targeting therapy. In some embodiments, the label or package insert of the pharmaceutical kit indicates that the patient to be treated has received prior treatment with one or more of trastuzumab, pertuzumab and T-DM1. [00205] In certain embodiments, the label or package insert of the pharmaceutical kit indicates that the patient to be treated has a HER2-expressing cancer that is metastatic. In some embodiments, the label or package insert of the pharmaceutical kit indicates that the patient to be treated has a HER2-expressing cancer that is locally advanced.
[00206] In certain embodiments, the label or package insert of the pharmaceutical kit indicates that the patient to be treated has a HER2-expressing cancer is classed as IHC 2+, IHC 2+/3+ or IHC 3+. In some embodiments, the label or package insert of the pharmaceutical kit indicates that the patient to be treated has a HER2-expressing cancer that is classed as IHC 3+ or IHC 2+/FISH+.
[00207] In certain embodiments, the label or package insert of the pharmaceutical kit indicates that the patient should receive ocular prophylaxis, for example, treatment with prednisolone, tetrahydrozoline or naphazoline, and/or cooling masks.
[00208] The label or package insert may further indicate that the anti-HER2 biparatopic ADC is suitable for use in combination with an anti-PDl agent. The label or package insert may also include a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, for use or sale for human or animal administration.
[00209] In addition to the container holding the composition comprising the anti-HER2 biparatopic ADC, the kit may comprise one or more additional containers comprising other components of the kit. For example, a pharmaceutically acceptable diluent or buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer’s solution, sodium chloride, dextrose solution, or the like.
[00210] In certain embodiments, the anti-HER2 biparatopic ADC may be provided in the kit in lyophilized or dry form, such as a powder or granules, and the kit may optionally contain a suitable solvent for reconstitution of the lyophilized or dried ADC. In certain embodiments, the anti-HER2 biparatopic ADC may be provided in the kit as a sterile liquid. In some embodiments, the anti- HER2 biparatopic antibody may be provided in the kit as a sterile liquid in concentrated form that is for dilution prior to use. [00211] The kit may further include other materials desirable from a commercial or user standpoint, such as filters, needles, and syringes.
[00212] The following Examples are provided for illustrative purposes and are not intended to limit the scope of the invention in any way. EXAMPLES
EXAMPLE 1: SYNTHESIS OF LINKER-TOXIN
[00213] Linker-Toxin 001 shown below comprises the auristatin analogue Compound 1 (also shown below) and may be prepared as described in International Patent Application Publication No. WO 2019/173911. Linker-Toxin 001 (MTvcCompound 1):
Figure imgf000059_0001
Compound 1:
Figure imgf000059_0002
EXAMPLE 2: CONJUGATION OF LINKER-TOXIN TO BIPARATOPIC ANTIBODY [00214] Antibody-drug conjugate (ADC) v21252, which comprises the biparatopic anti-HER2 mAb, vlOOOO, conjugated to the Linker-Toxin 001 at an average DAR of 2, may be generated by partial reduction of the antibody interchain disulfide bonds, followed by capping of the free cysteine residues by reaction with the maleimide component of the Linker-Toxin, as described in this Example and in International Patent Application Publication No. WO 2019/173911.
[00215] Preparation of vlOOOO is described in International Patent Application Publication No. WO 2015/077891. Details of this antibody are provided in Table 2.1 below. Sequences are provided in the Sequence Tables.
Table 2.1
Figure imgf000060_0001
* Fab or variable domain numbering according to Kabat (Kabat et al., Sequences of proteins of immunological interest, 5th Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, p.647, 1991) § CH3 numbering according to EU index as in Kabat (Edelman et al., 1969, PNAS USA, 63 : 78-85)
2.1 Conjugation of vl 0000 to Linker-Toxin 001
[00216] A solution (138.9 mL) of the antibody vlOOOO (2.0 g) in 10 mM sodium acetate, 9% (w/v) sucrose, pH 4.5 was pH-adjusted by addition of 200 mM Na2HPC>4, pH 8.9 (15.4 mL). After addition of a DTPA solution (44 mL in PBS, pH 7.4, final concentration 1.0 mM), reduction of the interchain disulfides was initiated by addition of an aqueous lO mM TCEP solution (1.68 mL, 1.05 eq.). After 90 minutes at 37°C, the reaction was cooled on ice before addition of an excess of Linker-Toxin 001 (4.81 mL; 6 eq) from a 20 mM DMSO stock solution. The conjugation reaction was quenched after 90 minutes by addition of an excess of a 20 mM Macetyl cysteine solution (4.81 mL; 6 eq.).
2.2 Purification of Antibody Drug Conjugate v21252
[00217] Quenched antibody drug conjugate (ADC) solution was purified with 9-15 diavolumes of 10 mM sodium acetate, 9% (w/v) sucrose, pH 4.5 on a Millipore Labscale™ Tangential Flow Filtration instrument using a Pellicon® XL Ultrafiltration Module (Ultracel® 30 kDa 0.005m2; Millipore Sigma). The eluted ADC was sterile filtered (0.22 um). ADCs produced on small scale were purified over 40 KDa MWCO Zeba™ columns (ThermoFisher Scientific, Waltham, MA) preconditioned with either PBS or 10 mM sodium acetate, 9% (w/v) sucrose, pH 4.5.
[00218] Following purification, the concentration of the ADC was determined by a BCA assay with reference to a standard curve generated from vlOOOO. Alternatively, concentrations were estimated by measurement of absorption at 280 nm (s = 195065 M'1 cm'1).
[00219] Samples of the ADCs were assessed by non-reducing and reducing SDS-PAGE. No extraneous bands were observed.
2.3 Hydrophobic Interaction Chromatography
[00220] Antibody and ADCs were analyzed by hydrophobic interaction chromatography (HIC) to estimate the drug-to-antibody ratio (DAR). Chromatography was on a Proteomix® HIC Ethyl column (7.8x50mm, 5pm) (Sepax Technologies Inc., Newark, DE) employing a gradient of 80% MPA/20% MPB to 35% MPA/65% MPB over a period of 13.5 minutes at a flow rate of 1 mL/min (MPA = 1.5 M (NHfhSCh, 25 mM NaxPC>4, and MPB = 75% 25 mM NaxPC>4, 25% isopropanol).
[00221] The average drug to antibody ratio (DAR) of an ADC can vary depending on the number of disulfide bonds liberated during the reduction of the antibody. A single conjugation reaction that yields an ADC with a particular average DAR comprises a mixture of species. For v21252, a mixture of four species was generated: unconjugated antibody (i.e. DAR0), ADC with a DAR of 2, ADC with a DAR of 4 and ADC with a DAR of 6. v21252 had an average DAR of 2.07. The DAR distribution for v21252 is shown in Table 2.2. Table 2.2: DAR Distribution for v21252
Figure imgf000062_0001
EXAMPLE 3: EFFECT OF ANTI-HER2 BIPARATOPIC ADC ON ATP SECRETION
[00222] Induction of immunogenic cell death (ICD) has been shown to sensitize in vivo models of cancer to subsequent immunotherapy, and in particular to immune checkpoint blockade targeting the PD-1/PD-L1 interaction (Kepp, et aL, 2019, Oncoimmunology, 8(10):el637188). This Example and Examples 4 and 5, report the effect of the anti-HER2 biparatopic ADC v21252 (see Example 2) on ATP secretion, HMGB1 (high-mobility group box 1 protein) levels and cell surface calreticulin (CRT) levels. ATP, HMGB1 and CRT are all immune activating molecules and indicators of ICD (Fucikova, J., et al., 2020, Cell Death Disease, 11(1013).
Method
[00223] The following test articles and cell lines were employed.
Test Articles (T = trastuzumab; P=pertuzumab; v22277= Palivizumab, isotype (negative) control):
• MMAE
• Compound 1
• DXd
• T-MCvcPABC-MMAE (DAR 4 and DAR 8)
• v22277-MCvcPABC-MMAE (DAR 4)
• T-Linker-Toxin 001 (DAR 2, DAR 4 and DAR 8)
• v22277- Linker-Toxin 001 (DAR 4) • T-MC-GGFG-DXd (DAR 8)
• v22277-MC-GGFG-DXd (DAR 8)
• v21252
• T -Linker-Toxin 001 (DAR 2) + P-Linker-Toxin 001 (DAR 2) (1 : 1)
• Trastuzumab
• vlOOOO
Cell Lines
• HCC-1954 (HER2+)
• SKOV-3 (HER2+)
• JIMT-1 (HER.2 )
• MDA-MB-468 (HER2’)
[00224] Briefly, cells were seeded in TC-treated plates and treated with test article at EC99 concentrations for 24 hours under standard culturing conditions followed by detection of extracellular ATP (eATP), as described below.
[00225] Cells were seeded in flat-bottom TC-treated 96-well plates at 15,000 cells/well in their respective complete growth medium, using at least 50 uL/well. Plates were left at room temperature for 5-10 minutes after seeding to allow cells to settle at the bottom of wells, then incubated at 37°C/5% CO2 overnight (-18-24 hrs) to allow cells to attach.
[00226] Test articles were diluted to the desired treatment concentrations in complete growth medium (RPMI 1640 + 10% FBS). Seeding media was removed from the seeded plates, then wells were washed with 100 uL/well PBS pH 7.4. 50 uL/well of fresh cell line-specific complete growth medium and 20 uL/well of test article (70 uL/well final volume) were then added. Plates were incubated at 37°C/5% CChfor 24 hours. All supematant/media was then removed and transferred to V-bottom 96-well plates. Plates were centrifuged at 400 x g for 5 min to collect cell debris. Following centrifugation, 70 uL/well were transferred into an opaque-bottom 384-well white walled plate, taking care to not transfer cell debris pelleted at the bottom of the 96-well plate. 20 uL/well of lx Cell Titer-Gio® reagent (Promega Corporation, Madison, WI) (90 uL/well final volume) was added and plates were incubated for 30 min. in the dark at room temperature. After incubation, luminescence was measured using a Synergy® Hl BioTek® plate reader (BioTek Instruments, Winooski, VT).
Results
[00227] The results are shown in Fig. 1. Treatment of HER2-expressing cell lines for 24 hr with the Linker-Toxin 001 ADCs and MMAE ADCs resulted in increased levels of ATP secretion compared to untreated cells. Treatment of HER2+ cell lines and the HER2" cell line, MDA-MB- 468, for 24 hr with free drug payloads, MMAE and Compound 1, resulted in increased levels of ATP secretion compared to untreated cells. The unconjugated antibodies, trastuzumab and vlOOOO, did not show increased levels of extracellular ATP (eATP) in HER2+ cell lines. Negative controls (isotype ADCs and untreated) did not show eATP secretion in any cell line, as expected.
[00228] The secretion of eATP was drug load-dependent in moderate HER2-expressing cell lines (SKOV-3 and JIMT-1) as T-MTvcCompound 1 (DAR 8) ADC yielded higher eATP secretion compared to T-MTvcCompound 1 (DAR 4 and DAR 2) ADCs. Similarly, T-MCvcPABC-MMAE (DAR 8) ADC yielded higher eATP secretion compared to T-MCvcPABC-MMAE (DAR 4) ADC. v21252 yielded similar eATP levels to T-MTvcCompound 1 (DAR 4) and higher eATP levels to the DAR-matched T-MTvcCompound 1 (DAR 2) indicating greater internalization, payload delivery and cytotoxicity of v21252 compared to monospecific ADCs utilizing Linker-Toxin 001. v21252 showed comparable eATP secretion to T-Linker-Toxin 001 (DAR 2) + P-Linker-Toxin 001 (DAR 2) 1 :1 mix in HER2+ cell lines tested. Free Compound 1 and MMAE yielded similar and cell line-dependent increases in eATP levels compared to untreated cells, whereas free topoisomerase inhibitor toxin DXd did not yield eATP in any of the cell lines tested. Similarly, the T-MC-GGFG-DXd (DAR 8) ADC did not show increased eATP secretion compared to untreated in any of the cell lines tested.
EXAMPLE 4: EFFECT OF ANTI-HER2 BIPARATOPIC ADC ON HMGB1 LEVELS
[00229] The same test articles as described in Example 3 were employed together with the following cell lines: SK-BR-3 (HER2+), NCI-N87 (HER2+) and MDA-MB-468 (HER2‘).
Method [00230] Briefly, cells were seeded in TC-treated plates and treated with test article at EC99 concentrations for 48 hours under standard culturing conditions, followed by detection of extracellular HMGB1 (eHMGBl), as described below.
[00231] Cells were seeded in flat-bottom TC-treated 48-well plates at 50,000 cells/well in their respective complete growth medium, using at least 100 uL/well. Plates were left at room temperature for 5-10 minutes after seeding to allow cells to settle at the bottom of wells, then incubated at 37°C/5% CO2 overnight (-18-24 hrs) to allow cells to attach. Test articles were diluted to desired treatment concentrations in complete growth medium (RPMI 1640 + 10% FBS). Seeding media was removed from seeded plates and wells were washed with 200 uL/well PBS pH 7.4. 80 uL/well of fresh cell line-specific complete growth medium and 40 uL/well of test article (120 uL/well final volume) were then added. Plates were incubated at 37°C/5% CChfor 48 hours.
[00232] ELISA plates were prepared by coating Nunc® Maxisorp™ 96-well plates with rabbit anti-HMGBl antibody (Thermo Fisher Scientific Corporation, Waltham, MA; PAI-16926) at 4 ug/mL in PBS pH 7.4, 100 uL/well, then incubated at 4°C overnight. Plates were washed with deionized (DI) water using BioTek® plate washer reader (BioTek Instruments, Winooski, VT) (3 rounds of washing, 100 uL/well DI water per wash), then blocked with 100 uL/well of blocking buffer (2% skim milk powder in PBS pH 7.4) and incubated at RT for 1 hour. Plates were then washed with DI water using BioTek® plate washer (3 rounds of washing, 100 uL/well DI water per wash).
[00233] Supernatant/media was removed from flat-bottom 48-well plates and transferred to V- bottom 96-well plates. Plates were centrifuged at 400 x g for 5 min to collect cell debris. Following centrifugation, 80 uL/well were transferred into ELISA plates (neat), taking care to not transfer cell debris pelleted at the bottom of the 96-well plate. Human HMGB1 protein control (ACROBiosystems, Newark, DE; HM1-H5220) was added alongside cell supernatant samples at 1,000 ng/mL, 80 uL/well. ELISA plates were incubated at RT for 1 hour, then washed with DI water using BioTek® plate washer (3 rounds of washing, 100 uL/well DI water per wash). Mouse anti-HMGBl 1D5 antibody (Sigma Aldrich, St. Louis, MI; SAB1403925-100UG) was added to the ELISA plates at 3 ug/mL, 80 uL/well, and the plates were incubated at RT for 1 hour. Following incubation, ELISA plates were washed with DI water using BioTek® plate washer (3 rounds of washing, 100 uL/well DI water per wash). Detection antibody, goat anti-rabbit IgG Fab2 HRP (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA; 111-035-047) was then added to the ELISA plates at 2 ug/mL, 80 uL/well; the plates were incubated at RT for 1 hour and then washed with DI water using BioTek® plate washer (3 rounds of washing, 100 uL/well DI water per wash). Turbo TMB (3,3 ',5,5' tetramethylbenzidine) (Thermo Fisher Scientific Corporation, Waltham, MA) was added to ELISA plates (neat) at 80 uL/well, the ELISA plates were incubated at RT for 10-30 min (based on visual inspection of colour change), then 1 N HC1 was added at 20 uL/well. Absorbance at 450 nm was immediately measured using Synergy® Hl BioTek® plate reader (BioTek Instruments, Winooski, VT).
Results
[00234] The results are shown in Fig. 2. In HER2+ cell lines, SK-BR-3 and NCI-N87, a two- to four-fold increase in extracellular HMGB1 (eHMGBl) was observed following a 48-hour incubation with HER2 -targeted ADCs and free toxins at 100 nM. The unconjugated antibodies, trastuzumab and vlOOOO, at 100 nM showed minimal increases in eHMGBl levels compared to the untreated control. Little or no HMGB1 secretion was observed in the HER2" cell line MDA- MB-468 treated with HER2 -targeted ADCs, control ADCs, or unconjugated antibodies. An approximately two-fold increase in HMGB1 release was observed in MDA-MB-468 cells after treatment with free toxins, Compound 1, MMAE and DXd, for 48-hours. Treatment of HER2+ cells with v21252 resulted in similar levels of HMGB1 compared to treatment with the T- MTvcCompound 1 (DAR 4) ADC or T-MCvcPABC-MMAE (DAR 4) ADC. Treatment of HER2+ cells with a 1 : 1 mix of T-Linker-Toxin 001 (DAR 2) and P-Linker-Toxin 001 (DAR 2) resulted in similar amounts of eHMGBl release as HER2+ cells treated with v21252.
EXAMPLE 5: EFFECT OF ANTI-HER2 BIPARATOPIC ADC ON CELL SURFACE CALRETICULIN LEVELS
[00235] The same test articles as described in Example 3 were employed together with the following cell lines: SK-BR-3 (HER2+) and MDA-MB-468 (HER2‘). Method
[00236] Briefly, cells were seeded in TC-treated plates and treated with test ADC (20 nM) or free drug (100 nM and 10 nM) for 24, 48 or 72 hours under standard culturing conditions, followed by detection of secreted calreticulin (CRT), as described below.
[00237] SK-BR-3 and MDA-MB-468 cells were seeded in 48-well cell culture plates at 30,000 cells/well and allowed to attach overnight. Test articles were spiked on cells and incubated at 37°C/5% CO2 for 24 hrs, 48 hrs, or 72 hrs. At each time point, cells were detached with Gibco™ TrpLE™ Express cell dissociation buffer (Thermo Fisher Scientific Corporation, Waltham, MA) and stained with a commercial anti-CRT antibody (Abeam pic, Waltham, MA; Cat. No: abl96158). Cells were washed in FACS buffer (PBS + 2% FBS), then resuspended in FACS buffer containing 1 ug/ml 7-aminoactinomycin D (AAD) (dead cell exclusion stain) (BioLegend®, San Diego, CA). Cell surface CRT was analyzed by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lakes, NJ).
Results
[00238] The results are shown in Fig. 3. T-MCvcPABC-MMAE, T-MTvcCompound 1, v21252, and the T-Linker-Toxin 001 + P-Linker-Toxin 001 1 : 1 mixture all induced HER2-dependent cell surface exposure of CRT. The percent CRT levels increased with time, with 72 hr treatment giving the highest level.
[00239] For T-MTvcCompound 1, increasing DAR from 2 to 4 resulted in an increase in the percent CRT induced. However, increasing DAR from 4 to 8 resulted in a slight decrease. v21252 induced comparable percent CRT levels to T-MTvcCompound 1 (DAR 2). At DAR4, T- MCvcPABC-MMAE induced a higher percent CRT level than that induced by T-MTvcCompound 1
[00240] T-MC-GGFG-DXd induced extracellular CRT in both cell-lines tested (HER2+ and HER2'). Free toxins induced cell surface CRT exposure. Negative controls did not induce surface CRT up to 72 hr, as expected. [00241] The results from Examples 3-5 show that v21252 selectively induces the release of immune-activating damage associated molecular patterns (DAMPs) from tumor cells in vitro. Extracellular ATP, extracellular HMGB1, and calreticulin are key DAMPs for cell death to be perceived as immunogenic. The payload utilized in v21252, Compound 1, drives similar responses from tumor cells at similar levels to the known immunogenic cell death inducer, MMAE. As such, ADCs containing Compound 1 induce DAMPs from tumor cells at similar levels to ADCs containing MMAE. The release of DAMPs can induce immune cell activation and modify the tumor microenvironment to support T-cell responses critical for anti-PD-1 activity.
EXAMPLE 6: PHASE I STUDY OF ANTI-HER2 BIPARATOPIC ADC (PART 1)
6.1 Study Design
[00242] A Phase 1, multicenter, open-label, dose-escalation study to establish the maximum- tolerated dose (MTD) or recommended dosage (RD) of the anti-HER2 biparatopic ADC v21252 (see Example 2) (the “investigational ADC”) and to assess the safety and tolerability of v21252 was initiated on April 19, 2019. Eligible patients include those with locally advanced (unresectable) or metastatic HER2-expressing cancers.
[00243] The study is using a 3+3 dose-escalation study design to evaluate the safety and tolerability of the investigational ADC and to determine the MTD or RD of the investigational ADC for further study. Selected expansion cohorts have subsequently been opened based upon Safety Monitoring Committee (SMC) recommendation to further evaluate the safety and tolerability of the investigational ADC at the MTD or RD and to assess preliminary anti-tumor activity (see Example 8).
Table 6.1: Arms and Interventions
Figure imgf000068_0001
6.2 Outcome Measures
6.2.1 Primary outcome measures
[00244] 1. Incidence of dose-limiting toxicities (DLTs) [Time Frame: Up to 4 weeks]: Number of participants who experienced a DLT. DLTs are events that occur following administration of any amount of the investigational ADC and are considered related to the investigational ADC per the investigator. DLTs will include only events considered related to the investigational ADC.
[00245] 2. Incidence of adverse events [Time Frame: Up to 7 months]: Number of participants who experienced an adverse event.
[00246] 3. Incidence of laboratory abnormalities [Time Frame: Up to 7 months]: Number of participants who experienced a maximum severity of Grade 3 or higher post-baseline laboratory abnormality, including either hematology and/or chemistry. Grades are defined using National Cancer Institute’s Common Terminology Criteria for Adverse Events (CTCAE), version 5.0.
[00247] 4. Incidence of electrocardiogram (ECG) and left ventricular ejection fraction (LVEF) abnormalities [Time Frame: Up to 7 months]: Number of participants who experienced an abnormal ECG or LVEF.
[00248] 5. Incidence of dose reductions of investigational ADC [Time Frame: Up to 7 months]: Number of doses reduced and number of participants who require a dose reduction.
6.2.2 Secondary outcome measures
[00249] 1. Serum concentrations of investigational ADC [Time Frame: Up to 7 months]: End of infusion concentration, maximum serum concentration, and trough concentration of investigational ADC.
[00250] 2. Incidence of anti-drug antibodies (AD As) [Time Frame: Up to 7 months]: Number of participants who develop AD As.
[00251] 3. Objective response rate (ORR) [Time Frame: Up to 6 months]: Number of participants who achieved a best response of either complete or partial response during treatment according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. [00252] 4. Disease control rate [Time Frame: Up to 6 months]: Number of participants who achieved a best response of complete response, partial response, or stable disease during treatment according to the RECIST version 1.1.
[00253] 5. Duration of response [Time Frame: Up to 2 years]: Median duration of response (in months) and range (minimum, maximum).
[00254] 6. Progression-free survival [Time Frame: Up to 2 years]: Median progression-free survival (in months) and range (minimum, maximum).
[00255] 7. Overall survival [Time Frame: Up to 2 years]: Median overall survival (in months) and range (minimum, maximum).
6.3 Eligibility Criteria
[00256] Ages eligible for study: 18 years and older (adult, older adult)
[00257] Sexes eligible for study: All
[00258] Healthy volunteers are not accepted for the study.
6.3.1 Inclusion Criteria
[00259] 1. Pathologically-confirmed diagnosis of breast cancer, gastroesophageal adenocarcinoma (GEA), or other HER2-expressing cancer with evidence of locally advanced (unresectable) and/or metastatic disease. During dose escalation, all subjects must have IHC 3+ HER2 overexpression or HER2 (ERBB2) gene amplification based on either FISH///? situ hybridization (ISH) or a fold change of >2 by next-generation sequencing (NGS). During dose expansion, all subjects must have HER2-high disease, including IHC 3+, or IHC 2+ and FISH +.
• Dose-escalation (Cohort 1): HER2-high advanced solid tumors
• Expansion (Cohort 2): HER2-high breast cancer
• Expansion (Cohort 3): HER2-high GEA
• Expansion (Cohort 4): HER2-high other non-breast and non-GEA cancers [00260] 2. Progressive disease that has progressed on or been refractory to all standard of care. Patients who were intolerant to or ineligible for standard therapy may be eligible if the reasons are carefully documented and approval is provided by the sponsor medical monitor.
• Patients with HER2-high breast cancer must have received prior treatment with trastuzumab, pertuzumab, and ado-trastuzumab emtansine (T-DM1).
• Patients with HER2-high GEA must have received prior treatment with trastuzumab.
[00261] 3. Sites of disease assessable per Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1.
• Dose-escalation: measurable or non-measurable disease
• Expansion: measurable disease.
[00262] 4. ECOG performance status score of 0 or 1.
[00263] 5. Adequate organ function.
[00264] 6. Adequate cardiac left ventricular function, as defined by a LVEF >/= institutional standard of normal.
6.3.2 Exclusion Criteria
[00265] 1. History of myocardial infarction or unstable angina within 6 months prior to enrollment, troponin levels consistent with myocardial infarction, or clinically significant cardiac disease, such as ventricular arrhythmia requiring therapy, uncontrolled hypertension, or any history of symptomatic congestive heart failure (CHF).
[00266] 2. Clinically significant infiltrative pulmonary disease not related to lung metastases.
[00267] 3. Active hepatitis B or hepatitis C infection or other known chronic liver disease.
[00268] 4. Acute or chronic uncontrolled renal disease, pancreatitis, or liver disease (with exception of patients with Gilbert’s Syndrome, asymptomatic gall stones, liver metastases, or stable chronic liver disease per investigator assessment). [00269] 5. Known history of human immunodeficiency virus (HIV) infection.
[00270] 6. Brain metastases: Untreated central nervous system (CNS) metastases, symptomatic CNS metastases, or radiation treatment for CNS metastases within 4 weeks of start of study treatment. Stable, treated brain metastases are allowed (defined as patients who are off steroids and anticonvulsants and are stable for at least 1 month at the time of screening).
[00271] 7. Known leptomeningeal disease (LMD).
6.4 Investigational ADC: Dosage and Administration
[00272] The investigational ADC (v21252) is a humanized biparatopic anti-HER2 ADC. The IgGl-like antibody is directed against two distinct HER2 epitopes: a scFv that binds to the ECD4 and a Fab that binds to ECD2. The antibody is covalently linked to the auristatin-based cytotoxic microtubule inhibitor Linker-Toxin 001, which is comprised of a protease cleavable dipeptide valine-citrulline linker and the toxin Compound 1 (see Example 1). Linker-toxin 001 is conjugated to the antibody via a maleimide to the antibody cysteines that comprise the interchain disulfide bonds. The average DAR of the investigational ADC is approximately 2.
[00273] The investigational ADC was manufactured according to the relevant regulatory requirements for human trials and diluted in 0.9% sodium chloride or 5% dextrose in water for injection, United States Pharmacopeia (USP) or equivalent, for IV administration. The investigational ADC was formulated for administration as a 10 mg/mL solution in 10 mM sodium acetate, 9% sucrose, 0.01% (w/v) Polysorbate 20, pH 4.5.
[00274] The investigational ADC was initially administered intravenously (IV) either every 2 weeks (Q2W) on Days 1 and 15 of each 28-day cycle or every 3 weeks (Q3W) on Day 1 of each 21 -day cycle. Subsequently, a third 3+3 dose-escalation cohort was initiated to evaluate the investigational ADC administered IV every week (QW) for 3 weeks in 28-day cycles.
6.5 Interim Results
[00275] As of January, 2021, the interim results of this Phase I trial were as follows.
[00276] Of the initial regimens (Q2W and Q3W) and dose levels evaluated, all showed evidence of anti -tumor activity, including at the lowest dose of 1 mg/kg Q2W. Multiple confirmed responses and stable disease were observed in several tumor types. The majority of treatment-related events were Grade 1 or 2 in severity, reversible and manageable.
[00277] Safety and anti-tumor activity of the initial Q2W and Q3W regimens were evaluated with the objective of selecting a go-forward dose and regimen to advance into disease-specific expansion cohorts. From the initial results, the Q3W schedule (evaluated at dose levels of 2.0 mg/kg, 2.5 mg/kg and 3.0 mg/kg) was considered to have demonstrated a more optimal profile for continued future development.
[00278] Although an MTD for the investigational ADC was not reached in this interim analysis, the anti-tumor activity observed resulted in a decision to continue to advance the program and open the expansion cohorts. Dosing was initiated in indication-specific expansion cohorts at 2.5 mg/kg Q3W in HER2+ breast cancer, HER2+ gastroesophageal adenocarcinoma (GEA), and a basket cohort of other HER2+ cancers.
[00279] In addition, a QW dosing regimen was initiated at dose levels of 1.0 mg/kg, 1.25 mg/kg and 1.5 mg/kg. Initial results from this regimen led to the decision to open a dose-escalation cohort at 1.75 mg/kg QW.
EXAMPLE 7: PHARMACOKINETIC MODELING
[00280] Pharmacokinetic (PK) modeling of weekly (QW) dosing (on Days 1, 8 and 15 of a 28 day cycle) of the anti-HER2 biparatopic ADC v21252 (see Example 2) suggests that a lower Cmax, higher Cmin and/or higher exposure can be achieved with this dosing schedule when compared to dosing every 2 weeks (Q2W) or every 3 weeks (Q3W) (see Fig. 4).
[00281] In addition, modeled exposure (AUC) was comparable between 1.5 mg/kg QW dosing (on Days 1, 8 and 15 of a 28 day cycle) and 3 mg/kg Q3W dosing with significantly different Cmax and Cmin profiles (see Fig. 5A). This indicates a potential for 1.5 mg/kg QW dosing to be better tolerated than 3 mg/kg Q3W dosing while the increased dose (1.5 mg/kg) should increase anti-tumor activity in both HER2-high and HER2-low cancers.
[00282] PK modeling of 2.5 mg/kg Q3W dosing and 3 mg/kg Q3W dosing shows similar Cmax and Cmin profiles (see Fig. 5B). EXAMPLE 8: PHASE I STUDY OF ANTI-HER2 BIPARATOPIC ADC (PART 2)
[00283] This Example provides preliminary results from the Phase I study described in Example 6. Data cut-off was June 9, 2022.
Overview
[00284] The overall study design for this Phase I study is provided in Example 6 and summarized in Fig. 6. Based on the interim results (see Example 6, Section 6.5), indication-specific dose expansion cohorts were opened at 2.5 mg/kg Q3W in HER2+ breast cancer, HER2+ gastroesophageal adenocarcinoma (GEA), and a basket cohort of other HER2+ cancers. In addition, a QW dosing regimen is being evaluated at dose levels of 1.0 mg/kg, 1.25 mg/kg, 1.5 mg/kg and 1.75 mg/kg QW. Initial results from dose-escalation have led to the decision to open a dose-expansion cohort at 1.5 mg/kg QW. During dose expansion at both doses, all subjects must have HER2-high disease, including IHC 3+, or IHC 2+ and FISH +.
[00285] Primary objectives are to determine the maximum tolerated dose (MTD)/recommended dose (RD) for the investigational ADC and to characterize the safety and tolerability of the investigational ADC. Secondary objectives are to evaluate the anti -tumor activity of the investigational ADC in HER2-expressing cancers.
[00286] The key eligibility criteria can be summarized as follows:
• Refractory HER2-expressing or amplified cancers o Patients with HER2+ breast cancer must have received trastuzumab, pertuzumab, and T-DM1 o Patients with HER2+ GEA must have received trastuzumab
• ECOG status 0 or 1
Preliminary Results
[00287] As of June 9, 2022, a total of 77 patients were treated across dose escalation (DE) and dose expansion (DX) (at 2.5 mg/kg Q3W) parts of the study. Of the 77 patients, 9 (12%) continue on treatment with the investigational ADC. The baseline disease characteristics and disposition of the 77 patients are shown in Table 8.1. Table 8.1: Baseline Disease Characteristics and Disposition of Patients
Figure imgf000075_0001
* Other included: Black or African American, Not Reported/Unknown/Multiple.
# IHC: immunohistochemistry; 11 FISH: fluorescent in situ hybridization
Safety Summary [00288] Treatment related adverse events (AEs) observed during the study period are shown in
Table 8.2 (Fig. 7). Treatment safety can be summarized as follows:
• The MTD has not been reached
• Two dose-limiting toxicities (Grade 2 keratitis > 14 days) were observed in 1 patient each at the 1.75 mg/kg QW (DE) and 2.5 mg/kg Q3W (DX) cohorts • No interstitial lung disease (ILD) or pneumonitis were reported • There were no treatment-related deaths
• Keratitis was reported in 33 (43%) patients. All keratitis events decreased to Grade 1 or resolved
• Dose reduction was required in 16 (21%) patients due to a treatment-related AE (10 [ 19%] patients in DE and 6 [24%] patients in DX). These patients continued receiving the investigational ADC at a reduced dose level.
[00289] After observation of keratitis, patients were provided with mandatory ocular prophylaxis consisting of prednisolone, tetrahydrozoline (0.05%) or naphazoline (0.012%) (or equivalent), and cooling masks. [00290] AEs that resulted in a dose reduction were keratitis (12 (16%) patients), dry eye (2 (3%) patients), and infusion-related reaction, punctuate keratitis, prolonged QT interval by electrocardiogram (prolonged ECG QT) and decreased neutrophils (one patient each).
Anti-Tumor Activity
[00291] Anti -turn or activity for patients treated with the investigational ADC at 2.5 mg/kg Q3W (in both the DE and DX cohorts) is shown in Table 8.3, the change in sum of target lesions for this patient population is shown in Fig. 8, and treatment duration is shown in Fig. 9. Prior treatment information for these patients is shown in Table 8.4.
Table 8.3: Anti-Tumor Activity
Figure imgf000076_0001
Figure imgf000077_0001
2 cORR = confirmed objective response rate
3 DCR = CR, PR or SD
4 CBR =SD > 24 weeks or best overall response of CR or PR Table 8.4: Prior Treatments
Figure imgf000077_0002
Conclusions
[00292] The investigational ADC has a unique mechanism of action including biparatopic- induced internalization with increased toxin-mediated cytotoxicity and immunogenic cell death. This Phase 1 study evaluated multiple tumor types of HER2-expressing cancers with the investigational ADC showing consistent safety and antitumor activity.
[00293] The investigational ADC has a manageable safety profile (with the majority of AEs being Grade 1 or 2 in severity) and demonstrates encouraging single-agent anti-tumor activity in heavily pretreated patients with HER2+ cancers (conformed ORR of 31%, disease control rate of 72% observed across 29 response-evaluable patients treated at 2.5 mg/kg Q3W).
[00294] Recommended dose(s): 2.5 mg/kg Q3W. [00295] QW dosing is still being evaluated. Interim results indicate that for this dosing regimen, fewer DLTs were observed in the cohort of patients receiving a 1.25 mg/kg dose than in the cohort of patients receiving a 1.5 mg/kg dose. [00296] The disclosures of all patents, patent applications, publications and database entries referenced in this specification are hereby specifically incorporated by reference in their entirety to the same extent as if each such individual patent, patent application, publication and database entry were specifically and individually indicated to be incorporated by reference.
[00297] Modifications of the specific embodiments described herein that would be apparent to those skilled in the art are intended to be included within the scope of the following claims.
SEQUENCE TABLES
Table A: Clone Numbers for Variants v5019, v5020, v7091, vlOOOO, v6903, v6902 and v6717
Figure imgf000079_0001
Table B: Sequence for Variants v5019, v5020, v7091, vlOOOO, v6903, v6902 and v6717 by
Clone Number
Figure imgf000079_0002
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001

Claims

WE CLAIM:
1. A method of treating a subject having a HER2-expressing cancer comprising administering to the subject an effective amount of an anti-HER2 biparatopic antibody drug conjugate (ADC), the effective amount comprising a dose of between 1 mg/kg and 2.0 mg/kg administered every week (QW) for 3 weeks of a 4-week cycle, or a dose of between 2 mg/kg and 3 mg/kg administered every 3 weeks (Q3W), wherein the anti-HER2 biparatopic ADC has general Formula (II):
Figure imgf000085_0001
wherein: n is the average drug-to-antibody ratio (DAR) and is between about 1.5 and about 2.5, and
Ab is an anti-HER2 biparatopic antibody comprising a first antigen-binding domain that binds to an epitope on ECD2 of HER2 and a second anti gen -binding domain that binds to an epitope on ECD4 of HER2.
2. The method according to Claim 1, wherein the effective dose comprises a dose of 1.25 mg/kg, 1.5 mg/kg or 1.75 mg/kg administered QW for 3 weeks of a 4-week cycle.
3. The method according to Claim 2, wherein the effective dose comprises a dose of 1.25 mg/kg administered QW for 3 weeks of a 4-week cycle.
4. The method according to Claim 1, wherein the effective amount comprises a dose of between 2 mg/kg and 3 mg/kg administered Q3W.
5. The method according to Claim 4, wherein the effective amount comprises a dose of 2.5 mg/kg administered Q3W.
6. The method according to any one of Claims 1 to 5, wherein the average DAR is between about 1.8 and about 2.5.
7. The method according to any one of Claims 1 to 5, wherein the average DAR is between about 1.8 and about 2.3.
8. The method according to any one of Claims 1 to 5, wherein the average DAR is about 2.0.
9. The method according to any one of Claims 1 to 8, wherein the first antigen-binding domain is a Fab and the second antigen-binding domain is an scFv.
10. The method according to any one of Claims 1 to 9, wherein the first antigen-binding domain of the anti-HER2 biparatopic antibody comprises the CDR sequences as set forth in SEQ ID NOs: 32, 34 and 33, and in SEQ ID NOs: 22, 24 and 23, and the second antigen-binding domain of the anti-HER2 biparatopic antibody comprises the CDR sequences as set forth in SEQ ID NOs: 56, 57 and 58, and SEQ ID NOs: 53, 54 and 55.
11. A method of treating a subject having a HER2-expressing cancer comprising administering to the subject an effective amount of an anti-HER2 biparatopic antibody drug conjugate (ADC), the effective amount comprising a dose of 1.25 mg/kg administered every week (QW) for 3 weeks of a 4-week cycle, wherein the anti-HER2 biparatopic ADC has general Formula (II):
Figure imgf000086_0001
n is the average drug-to-antibody ratio (DAR) and is between about 1.8 and about 2.3, and
Ab is an anti-HER2 biparatopic antibody comprising (a) a first antigen-binding domain comprising the CDR sequences as set forth in SEQ ID NOs: 32, 34 and 33, and in SEQ ID NOs: 22, 24 and 23, and (b) a second antigen-binding domain comprising the CDR sequences as set forth in SEQ ID NOs: 56, 57 and 58, and SEQ ID NOs: 53, 54 and 55.
12. A method of treating a subject having a HER2-expressing cancer comprising administering to the subject an effective amount of an anti-HER2 biparatopic antibody drug conjugate (ADC), the effective amount comprising a dose of 2.5 mg/kg administered every 3 weeks (Q3W), wherein the anti-HER2 biparatopic ADC has general Formula (II):
Figure imgf000087_0001
n is the average drug-to-antibody ratio (DAR) and is between about 1.8 and about 2.3, and
Ab is an anti-HER2 biparatopic antibody comprising (a) a first antigen-binding domain comprising the CDR sequences as set forth in SEQ ID NOs: 32, 34 and 33, and in SEQ ID NOs: 22, 24 and 23, and (b) a second antigen-binding domain comprising the CDR sequences as set forth in SEQ ID NOs: 56, 57 and 58, and SEQ ID NOs: 53, 54 and 55.
13. The method according to any one of Claims 1 to 12, wherein the first antigen -binding domain of the anti-HER2 biparatopic antibody comprises a VH sequence as set forth in SEQ ID NO: 31 and a VL sequence as set forth in SEQ ID NO: 21, and the second anti gen -binding domain of the anti-HER2 biparatopic antibody comprises a VH sequence as set forth in SEQ ID NO: 52 and a VL sequence as set forth in SEQ ID NO: 51.
14. The method according to any one of Claims 1 to 12, wherein the anti-HER2 biparatopic antibody comprises a first heavy chain (Hl) comprising the sequence as set forth in SEQ ID NO: 30, a second heavy chain (H2) comprising the sequence as set forth in SEQ ID NO: 50, and a light chain (LI) comprising the sequence as set forth in SEQ ID NO: 20.
15. The method according to any one of Claims 1 to 14, wherein the HER2-expressing cancer is a solid tumor.
16. The method according to any one of Claims 1 to 15, wherein the HER2-expressing cancer is breast cancer, gastroesophageal adenocarcinoma, endometrial cancer, ovarian cancer, non-small cell lung cancer, anal cancer, pancreatic cancer, biliary tract cancer or bladder cancer.
17. The method according to any one of Claims 1 to 15, wherein the HER2-expressing cancer is gastroesophageal adenocarcinoma, endometrial cancer, ovarian cancer, non-small cell lung cancer or colorectal cancer.
18. The method according to any one of Claims 1 to 17, wherein the subject has received prior treatment with one or more other anti-HER2 therapeutics.
19. The method according to Claim 18, wherein the one or more other anti-HER2 therapeutics are one or more of trastuzumab, pertuzumab and T-DM1.
20. The method according to any one of Claims 1 to 19, wherein the cancer is metastatic.
21. The method according to any one of Claims 1 to 19, wherein the cancer is locally advanced.
22. The method according to any one of Claims 1 to 21, wherein the cancer is immunohistochemistry (IHC) 2+, IHC 2+/3+ or IHC 3+
23. The method according to any one of Claims 1 to 21, wherein the cancer is IHC 3+ or IHC 2+/FISH+.
24. The method according to any one of Claims 1 to 23, wherein the anti-HER2 biparatopic ADC is administered in combination with a PD-1 inhibitor.
25. The method according to Claim 24, wherein the PD-1 inhibitor is an anti-PD-1 antibody.
26. The method according to any one of Claims 1 to 25, wherein the anti-HER2 biparatopic ADC is administered intravenously.
27. The method according to Claim 26, wherein the anti-HER2 biparatopic ADC is administered by intravenous infusion.
28. A pharmaceutical kit comprising: i) one or more containers containing an anti-HER2 biparatopic antibody drug conjugate (ADC) having general Formula (II):
Figure imgf000089_0001
wherein: n is the average drug-to-antibody ratio (DAR) and is between about 1.5 and about 2.5, and
Ab is an anti-HER2 biparatopic antibody comprising a first antigen-binding domain that binds to an epitope on ECD2 of HER2 and a second anti gen -binding domain that binds to an epitope on ECD4 of HER2, and ii) a label or package insert on or associated with the one or more containers indicating the anti-HER2 biparatopic ADC is for administration to a subject having a HER2-expressing cancer at a dose of 1.25 mg/kg, 1.5 mg/kg or 1.75 mg/kg administered every week (QW) for 3 weeks of a 4-week cycle, or a dose of 2.5 mg/kg administered every 3 weeks (Q3W).
29. The pharmaceutical kit according to Claim 28, wherein the label or package insert indicates the anti-HER2 biparatopic ADC is for administration at a dose of 1.25 mg/kg administered QW for 3 weeks of a 4-week cycle.
30. The pharmaceutical kit according to Claim 28, wherein the label or package insert indicates the anti-HER2 biparatopic ADC is for administration at a dose of 2.5 mg/kg administered Q3W.
31. The pharmaceutical kit according to any one of Claims 28 to 30, wherein the label or package insert indicates the anti-HER2 biparatopic ADC is for intravenous administration.
32. The pharmaceutical kit according to Claim 31, wherein the intravenous administration is intravenous infusion.
33. The pharmaceutical kit according to any one of Claims 28 to 32, wherein the average DAR is between about 1.8 and about 2.5.
34. The pharmaceutical kit according to any one of Claims 28 to 32, wherein the average DAR is between about 1.8 and about 2.3.
35. The pharmaceutical kit according to any one of Claims 28 to 32, wherein the average DAR is about 2.0.
36. The pharmaceutical kit according to any one of Claims 28 to 35, wherein the first antigenbinding domain is a Fab and the second antigen-binding domain is an scFv.
37. The pharmaceutical kit according to any one of Claims 28 to 36, wherein the first antigenbinding domain of the anti-HER2 biparatopic antibody comprises the CDR sequences as set forth in SEQ ID NOs: 32, 34 and 33, and in SEQ ID NOs: 22, 24 and 23, and the second antigen-binding domain of the anti-HER2 biparatopic antibody comprises the CDR sequences as set forth in SEQ ID NOs: 56, 57 and 58, and SEQ ID NOs: 53, 54 and 55.
38. The pharmaceutical kit according to any one of Claims 28 to 37, wherein the first antigenbinding domain of the anti-HER2 biparatopic antibody comprises a VH sequence as set forth in SEQ ID NO: 31 and a VL sequence as set forth in SEQ ID NO: 21, and the second antigen-binding domain of the anti-HER2 biparatopic antibody comprises a VH sequence as set forth in SEQ ID NO: 52 and a VL sequence as set forth in SEQ ID NO: 51.
39. pharmaceutical kit according to any one of Claims 28 to 37, wherein the anti-HER2 biparatopic antibody comprises a first heavy chain (Hl) comprising the sequence as set forth in SEQ ID NO: 30, a second heavy chain (H2) comprising the sequence as set forth in SEQ ID NO: 50, and a light chain (LI) comprising the sequence as set forth in SEQ ID NO: 20.
40. The pharmaceutical kit according to any one of Claims 28 to 39, wherein the label or package insert further indicates that the HER2-expressing cancer is one of breast cancer, gastroesophageal adenocarcinoma, endometrial cancer, ovarian cancer, non-small cell lung cancer, anal cancer, pancreatic cancer, biliary tract cancer or bladder cancer.
41. The pharmaceutical kit according to any one of Claims 28 to 39, wherein the label or package insert further indicates that the HER2-expressing cancer is one of gastroesophageal adenocarcinoma, endometrial cancer, ovarian cancer, non-small cell lung cancer or colorectal cancer.
42. The pharmaceutical kit according to any one of Claims 28 to 41, wherein the label or package insert further indicates that the subject has received prior treatment with one or more other anti-HER2 therapeutics.
43. The pharmaceutical kit according to Claim 42, wherein the one or more other anti-HER2 therapeutics are one or more of trastuzumab, pertuzumab and T-DM1.
44. The pharmaceutical kit according to any one of Claims 28 to 43, wherein the label or package insert further indicates that the HER2-expressing cancer is metastatic.
45. The pharmaceutical kit according to any one of Claims 28 to 43, wherein the label or package insert further indicates that the HER2-expressing cancer is locally advanced.
46. The pharmaceutical kit according to any one of Claims 28 to 45, wherein the label or package insert further indicates that the HER2-expressing cancer is H4C 2+, H4C 2+/3+ or H4C 3+
47. The pharmaceutical kit according to any one of Claims 28 to 45, wherein the label or package insert further indicates that the HER2-expressing cancer is H4C 3+ or H4C 2+/FISH+.
48. The pharmaceutical kit according to any one of Claims 28 to 47, wherein the label or package insert further indicates that the anti-HER2 biparatopic ADC is suitable for administration in combination with a PD-1 inhibitor.
49. The pharmaceutical kit according to Claim 48, wherein the PD-1 inhibitor is an anti-PD-1 antibody.
50. The pharmaceutical kit according to any one of Claims 28 to 49, wherein the anti-HER2 biparatopic ADC is provided as a sterile liquid.
PCT/CA2023/050104 2022-01-26 2023-01-26 Methods of using anti-her2 biparatopic antibody-drug conjugates in the treatment of cancer WO2023141714A1 (en)

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