Patent Application
20250122303
This application claims priority from SG 10202109667X filed 3 Sep. 2021, the contents and elements of which are herein incorporated by reference for all purposes.
The present invention relates to the fields of molecular biology, more specifically antibody technology and methods of medical treatment and prophylaxis.
Increased HER3 expression is linked to poor prognosis in multiple solid tumors, including breast, gastric, head & neck, pancreatic, ovarian, and lung cancers. HER3-mediated signalling has adverse consequences for tumour progression; HER3 upregulation is associated with resistance to anti-HER2 and anti-EGFR therapy, and solid tumors refractory to anti-PD-1 therapy have been shown to have higher HER3 expression compared to responders to anti-PD-1 therapy.
HER3-binding antibodies are described e.g. in Zhang et al., Acta Biochimica et Biophysica Sinica (2016) 48(1): 39-48. The anti-HER3 antibody LJM-716 binds to an epitope on subdomains II and IV of the HER3 extracellular domain, locking HER3 in the inactive conformation (Garner et al., Cancer Res (2013) 73: 6024-6035). MM-121 (also known as seribantumab) has been shown to inhibit HER3-mediated signalling by blocking binding of heregulin (HRG) to HER3 (Schoeberl et al., Sci. Signal. (2009) 2(77): ra31). Patritumab (also known as U-1287 and AMG-888) also blocks binding of heregulins to HER3 (see e.g. Shimizu et al. Cancer Chemother Pharmacol. (2017) 79(3):489-495. RG7116 (also known as lumretuzumab and RO-5479599) recognises an epitope in subdomain I of the HER3 extracellular domain (see e.g. Mirschberger et al. Cancer Research (2013) 73(16) 5183-5194). KTN3379 binds to HER3 through interaction with amino acid residues in subdomain III (corresponding to the following positions of SEQ ID NO:1: Gly476, Pro477, Arg481, Gly452, Arg475, Ser450, Gly420, Ala451, Gly419, Arg421, Thr394, Leu423, Arg426, Gly427, Lys356, Leu358, Leu358, Lys356, Ala330, Lys329 and Gly337), and Met310, Glu311 and Pro328 of subdomain II (see Lee et al., Proc Natl Acad Sci USA. 2015 Oct. 27; 112(43):13225). AV-203 (also known as CAN-017) has been shown to block binding of NRG1 to HER3 and to promote HER3 degradation (see Meetze et al., Eur J Cancer 2012; 48:126). REGN1400 also inhibits binding of ligand to HER3 (see Zhang et al., Mol Cancer Ther (2014) 13:1345-1355). RG7597 (duligotuzumab) is a dual action Fab (DAF) capable of binding to both HER3 and EGFR, and binds to subdomain III of HER3 (see Schaefer et al., Cancer Cell (2011) 20(4):472-486). MM-111 and MM-141 are bispecific antibodies having HER3-binding arms which inhibit HRG ligand binding to HER3 (see McDonagh et al. Mol Cancer Ther (2012) 11:582-593 and Fitzgerald et al., Mol Cancer Ther (2014) 13:410-425).
The present disclosure relates, in part, to compositions comprising antigen-binding molecules which are capable of binding to HER3. The compositions find use in the treatment of cancers and cancer cells that express HER3.
Thus, in one aspect, the disclosure provides a composition comprising an antigen-binding molecule which is capable of binding to HER3.
In some embodiments, the antigen-binding molecule comprises:
In some embodiments, the antigen-binding molecule comprises:
In some embodiments, the composition comprises:
In some embodiments, the composition comprises:
In some embodiments, the composition comprises 20 mM histidine, 8% (w/v) sucrose; 0.02% (w/v) polysorbate-80, and has a pH 5.8.
In some embodiments, the composition comprises at least 1.2 mg/mL of the antigen-binding molecule. In some embodiments, the composition comprises up to 50 mg/mL of the antigen-binding molecule. In some embodiments, the composition comprises 1.2 mg/mL to 50 mg/mL of the antigen-binding molecule.
In some embodiments, the antigen-binding molecule comprises:
In some embodiments, the antigen-binding molecule comprises:
In some embodiments, the antigen-binding molecule comprises:
In some embodiments, the antigen-binding molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:171. In some embodiments, the antigen-binding molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO:177.
Also provided is a composition according to the present disclosure, for use as a medicament.
Also provided is a composition according to the present disclosure, for use in a method of treating or preventing a cancer in a subject.
Also provided is the use of a composition according to the present disclosure in the manufacture of a medicament for treating or preventing a cancer in a subject.
Also provided is a method of treating or preventing a cancer in a subject, the method comprising administering a therapeutically- or prophylactically-effective amount of a composition according to the present disclosure.
In some embodiments, the cancer comprises cells that express HER3, EGFR, HER2, HER4, NRG1, NRG2, and/or a ligand for HER3. In some embodiments, the cancer comprises cells having a mutation resulting in increased expression of a ligand for HER3. In some embodiments, the cancer comprises cells having an NRG gene fusion. In some embodiments, the NRG gene fusion is selected from CLU-NRG1, CD74-NRG1, DOC4-NRG1, SLC3A2-NRG1, RBPMS-NRG1, WRN-NRG1, SDC4-NRG1, RAB2IL1-NRG1, VAMP2-NRG1, KIF13B-NRG1, THAP7-NRG1, SMAD4-NRG1, MDK-NRG1, TNC-NRG1, DIP2B-NRG1, MRPL13-NRG1, PARP8-NRG1, ROCK1-NRG1, DPYSL2-NRG1, ATP1B1-NRG1, CDH6-NRG1, APP-NRG1, AKAP13-NRG1, THBS1-NRG1, FOXA1-NRG1, PDE7A-NRG1, RAB31L1-NRG1, CDK1-NRG1, BMPRIB-NRG1, TNFRSF10B-NRG1, MCPH1-NRG1 and SLC12A2-NRG2.
In some embodiments, the cancer derives from the lung, breast, head, neck, kidney, ovary, cervix, pancreas, stomach, liver, oesophagus, prostate, uterus, gallbladder, colon, rectum, bladder, soft tissue or nasopharynx.
In some embodiments, the cancer is selected from lung cancer, non-small cell lung cancer, lung adenocarcinoma, invasive mucinous lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, triple negative breast cancer, breast carcinoma, breast invasive carcinoma, head and neck cancer, head and neck squamous cell carcinoma, renal cancer, renal clear cell carcinoma, ovarian cancer, ovarian serous cystadenocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, prostate cancer, prostate adenocarcinoma, castration resistant prostate cancer, endometrial cancer, uterine carcinosarcoma, gallbladder cancer, cholangiocarcinoma, colorectal cancer, RAS wild type colorectal cancer, gastric cancer, hepatocellular carcinoma (HCC), oesophageal cancer, bladder cancer, urothelial bladder cancer, cervical cancer, endometrial cancer, sarcoma, soft tissue sarcoma, neuroendocrine tumor and neuroendocrine tumor of the nasopharynx.
In some embodiments, the method comprises a step of detecting cancer cells expressing HER3, EGFR, HER2, HER4, NRG1, NRG2, a ligand for HER3 and/or an NRG gene fusion in the subject. In some embodiments, the method comprises a step of obtaining the cells from the subject. In some embodiments, the cells have been obtained from the subject. In some embodiments, the detection step is performed on an in vitro sample and/or is performed in vitro.
In some embodiments, the subject is selected for treatment with the composition when cancer cells expressing HER3, EGFR, HER2, HER4, NRG1, NRG2, a ligand for HER3 and/or an NRG gene fusion are detected.
In some embodiments, the composition is administered in combination with one or more of: a HER2-targeted therapy, an EGFR-targeted therapy, and/or an androgen receptor-targeted therapy. In some embodiments, the composition is administered in combination with one or more of cetuximab, enzalutamide and/or trastuzumab.
Also provided as part of the present disclosure is an antigen-binding molecule which is capable of binding to HER3 for use in a method of treating or preventing a cancer in a subject, wherein the antigen-binding molecule comprises:
Also provided is the use of an antigen-binding molecule which is capable of binding to HER3 in the manufacture of a medicament for treating or preventing a cancer in a subject, wherein the antigen-binding molecule comprises:
Also provided is a method of treating or preventing a cancer in a subject, wherein the method comprises administering a therapeutically or prophylactically effective amount of an antigen-binding molecule which is capable of binding to HER3 to the subject, and wherein the antigen-binding molecule comprises:
In some embodiments, the antigen-binding molecule comprises:
In some embodiments, the antigen-binding molecule comprises:
In some embodiments, the antigen-binding molecule comprises:
In some embodiments, the antigen-binding molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:171. In some embodiments, the antigen-binding molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO:177.
In some embodiments the cancer comprises cells that express HER3, EGFR, HER2, HER4, NRG1, NRG2, and/or a ligand for HER3. In some embodiments, the cancer comprises cells having a mutation resulting in increased expression of a ligand for HER3. In some embodiments, the cancer comprises cells having an NRG gene fusion. In some embodiments, the NRG gene fusion is selected from CLU-NRG1, CD74-NRG1, DOC4-NRG1, SLC3A2-NRG1, RBPMS-NRG1, WRN-NRG1, SDC4-NRG1, RAB2IL1-NRG1, VAMP2-NRG1, KIF13B-NRG1, THAP7-NRG1, SMAD4-NRG1, MDK-NRG1, TNC-NRG1, DIP2B-NRG1, MRPL13-NRG1, PARP8-NRG1, ROCK1-NRG1, DPYSL2-NRG1, ATP1B1-NRG1, CDH6-NRG1, APP-NRG1, AKAP13-NRG1, THBS1-NRG1, FOXA1-NRG1, PDE7A-NRG1, RAB3IL1-NRG1, CDK1-NRG1, BMPRIB-NRG1, TNFRSF10B-NRG1, MCPH1-NRG1 and SLC12A2-NRG2.
In some embodiments, the cancer derives from the lung, breast, head, neck, kidney, ovary, cervix, pancreas, stomach, liver, oesophagus, prostate, uterus, gallbladder, colon, rectum, bladder, soft tissue or nasopharynx.
In some embodiments, the cancer is selected from lung cancer, non-small cell lung cancer, lung adenocarcinoma, invasive mucinous lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, triple negative breast cancer, breast carcinoma, breast invasive carcinoma, head and neck cancer, head and neck squamous cell carcinoma, renal cancer, renal clear cell carcinoma, ovarian cancer, ovarian serous cystadenocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, prostate cancer, prostate adenocarcinoma, castration resistant prostate cancer, endometrial cancer, uterine carcinosarcoma, gallbladder cancer, cholangiocarcinoma, colorectal cancer, RAS wild type colorectal cancer, gastric cancer, hepatocellular carcinoma (HCC), oesophageal cancer, bladder cancer, urothelial bladder cancer, cervical cancer, endometrial cancer, sarcoma, soft tissue sarcoma, neuroendocrine tumor and neuroendocrine tumor of the nasopharynx.
In some embodiments, the method comprises a step of detecting cancer cells expressing HER3, EGFR, HER2, HER4, NRG1, NRG2, a ligand for HER3 and/or an NRG gene fusion in the subject. In some embodiments, the method comprises a step of obtaining the cells from the subject. In some embodiments, the cells have been obtained from the subject. In some embodiments, the detection step is performed on an in vitro sample and/or is performed in vitro. In some embodiments, the subject is selected for treatment with the antigen-binding molecule when cancer cells expressing HER3, EGFR, HER2, HER4, NRG1, NRG2, a ligand for HER3 and/or an NRG gene fusion are detected.
In some embodiments, the antigen-binding molecule is administered in combination with one or more of: a HER2-targeted therapy, an EGFR-targeted therapy, and/or an androgen receptor-targeted therapy. In some embodiments, the antigen-binding molecule is administered in combination with one or more of cetuximab, enzalutamide and/or trastuzumab.
In some embodiments, a composition or antigen-binding molecule according to the present disclosure is administered once every 7 days, once every 14 days, once every 21 days or once every 28 days.
In some embodiments, a composition or antigen-binding molecule according to the present disclosure is administered four times every 28 days, twice every 28 days, or three times every 21 days, e.g. for one or multiple periods of 21 or 28 days. In some embodiments, a composition or antigen-binding molecule according to the present disclosure is administered over 1, 2, 3, 4, 5, 6 or more periods of 21 or 28 days.
In some embodiments, treatment according to the present disclosure comprises administering 150 mg to 3000 mg of antigen-binding molecule per administration and/or over each period of 21 or 28 days.
In some embodiments, treatment according to the present disclosure comprises administering 1800-2500 mg of antigen-binding molecule per administration.
In some embodiments, treatment according to the present disclosure comprises administering at least 600 mg, at least 900 mg, at least 1200 mg, at least 1500 mg, at least 1800 mg, at least 2100, at least 2400 mg, at least 2700 mg, at least 3000 mg, at least 3300 mg, at least 3600 mg, at least 3900 mg, at least 4200 mg, at least 4500 mg, at least 4800 mg, at least 5100 mg, at least 5400 mg, at least 5700 mg, at least 6000 mg, at least 6300 mg, at least 6600 mg, at least 6900 mg, at least 7200 mg, at least 7500 mg, at least 7800 mg, at least 8100 mg, at least 8400 mg, at least 8700 mg, at least 9000 mg, at least 9300 mg, at least 9600 mg, at least 9900 mg, at least 10200 mg, at least 10500 mg, at least 10800 mg, at least 11100 mg, at least 11400 mg, at least 11700 mg or at least 12000 mg of antigen-binding molecule in total per administration cycle, e.g. every 21 or 28 days.
In some embodiments, treatment according to the present disclosure comprises administering about 3600 mg of antigen-binding molecule in total every 21 days or about 4800 mg of antigen-binding molecule in total every 28 days. In some embodiments, treatment according to the present disclosure comprises administering about 5400 mg of antigen-binding molecule in total every 21 or about 7200 mg of antigen-binding molecule in total every 28 days. In some embodiments, treatment according to the present disclosure comprises administering about 6300 mg of antigen-binding molecule in total every 21 or about 8400 mg of antigen-binding molecule in total every 28 days. In some embodiments, treatment according to the present disclosure comprises administering about 9000 mg of antigen-binding molecule in total every 21 or about 12000 mg of antigen-binding molecule in total every 28 days.
The antigen-binding molecule may be administered in multiple doses (e.g. once per week) to reach a total amount of antigen-binding molecule per 21 or 28 days.
In some embodiments, treatment according to the present disclosure comprises administering at least 150 mg, at least 300 mg, at least 600 mg, at least 900 mg, at least 1200 mg, at least 1500 mg, at least 1800 mg, at least 2100 mg, at least 2400, or at least 2800 mg of antigen-binding molecule every 7 or 14 days (once every week or once every 2 weeks). In some embodiments, treatment according to the present disclosure comprises administering 1500 mg to 3000 mg of antigen-binding molecule every 7 or 14 days (once every week or once every 2 weeks). In some embodiments, treatment according to the present disclosure comprises administering 1800 mg to 2500 mg of antigen-binding molecule every 7 or 14 days (once every week or once every 2 weeks). In some embodiments, treatment according to the present disclosure comprises administering about 1200 mg of antigen-binding molecule every 7 or 14 days (once every week or once every 2 weeks). In some embodiments, treatment according to the present disclosure comprises administering about 1500 mg of antigen-binding molecule every 7 or 14 days (once every week or once every 2 weeks). In some embodiments, treatment according to the present disclosure comprises administering about 1800 mg of antigen-binding molecule every 7 or 14 days (once every week or once every 2 weeks). In some embodiments, treatment according to the present disclosure comprises administering about 2100 mg of antigen-binding molecule every 7 or 14 days (once every week or once every 2 weeks).
In some embodiments, administration of antigen-binding molecule every 7 days (e.g. as above) is performed for at least 21 or at least 28 days. In some embodiments, administration of antigen-binding molecule every 7 days (e.g. as above) is performed for 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or more weeks, or for 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 or more weeks.
Known anti-HER3 antibodies fall broadly into two classes. Antibodies of the first class bind to domains I and/or III of HER3, and thereby competitively inhibit ligand binding to HER3. Seribantumab (MM-121) is a representative member of this class, and other members include patritumab (U3-1287 or AMG-888), lumretuzumab (RG-7116), AV-203, GSK2849330 and REGN1400. Antibodies of the second class lock HER3 in an inactive conformation, through binding to the interface between domains II and IV, or between domains II and III. LJM-716 is a representative example of this class, as is KTN3379. Overexpression of HER3 is frequently observed in multiple tumour types and is associated with a poorer clinical outcome. Enhanced expression of HER3 is found in colorectal carcinoma, head and neck squamous cell carcinoma, melanoma, and breast, gastric, ovarian, prostate, and bladder cancers. The impact of HER3 overexpression is greater in cancers where HER2 is also overexpressed e.g. breast, gastric and ovarian. HER3 is the preferred heterodimeric partner for EGFR in melanoma and pancreatic carcinoma. Up-regulation of HER3 expression and activity is associated with resistance to multiple pathway inhibitors and associated with a poor prognosis.
The present invention relates to novel HER3-binding molecules having improved properties as compared to known anti-HER3 antibodies.
The inventors undertook the targeted generation of antigen-binding molecules which bind to particular regions of interest in the extracellular region of HER3. The HER3-binding molecules of the present invention are provided with combinations of desirable biophysical and/or functional properties as compared to antigen-binding molecules disclosed in the prior art.
In embodiments of the present invention the antigen binding molecules are capable of binding to the subdomain II of the extracellular region of HER3 (SEQ ID NO:16), and inhibit association of the bound HER3 molecule with interaction partners.
In particular, HER3-binding antigen-binding molecules described herein are demonstrated to bind to an epitope of HER3 providing for (i) potent inhibition of association of HER3 with interaction partners (e.g. EGFR, HER2) and (ii) high-affinity binding to HER3 both in the presence and absence of NRG ligand. This unique combination of properties provides for strong inhibition of downstream signalling and exceptional anti-cancer activity against a wide range of cancers.
HER3 (also known e.g. as ERBB3 LCCS2, MDA-BF-1) is the protein identified by UniProt P21860. Alternative splicing of mRNA encoded by the human ERBB3 gene yields five different isoforms: isoform 1 (UniProt: P21860-1, v1; SEQ ID NO:1); isoform 2 (UniProt: P21860-2; SEQ ID NO:2), which comprises a different sequence to SEQ ID NO:1 from position 141, and which lacks amino acid sequence corresponding to positions 183 to 1342 of SEQ ID NO:1; isoform 3 (UniProt: P21860-3; SEQ ID NO:3), which comprises the substitution C331F relative to SEQ ID NO:1, and which lacks the amino acid sequence corresponding to positions 332 to 1342 of SEQ ID NO:1; isoform 4 (UniProt: P21860-4; SEQ ID NO:4), which lacks the amino acid sequence corresponding to positions 1 to 59 of SEQ ID NO:1; and isoform 5 (UniProt: P21860-5; SEQ ID NO:5), which lacks the amino acid sequence corresponding to positions 1 to 643 of SEQ ID NO:1.
The N-terminal 19 amino acids of SEQ ID NOs:1 to 3 constitute a signal peptide, and so the mature form of HER3 isoforms 1, 2 and 3 (i.e. after processing to remove the signal peptide) have the amino acid sequences shown in SEQ ID NOs:6, 7 and 8, respectively.
The structure and function of HER3 is described e.g. in Cho and Leahy Science (2002) 297 (5585):1330-1333, Singer et al., Journal of Biological Chemistry (2001) 276, 44266-44274, Roskoski et al., Pharmacol. Res. (2014) 79: 34-74, Bazley and Gullick Endocrine-Related Cancer (2005) S17-S27 and Mujoo et al., Oncotarget (2014) 5(21):10222-10236, each of which are hereby incorporated by reference in their entirety. HER3 is a single-pass transmembrane ErbB receptor tyrosine kinase having an N-terminal extracellular region (SEQ ID NO:9) comprising two leucine-rich subdomains (domains I and III, shown in SEQ ID NOs:15 and 17, respectively) and two cysteine-rich subdomains (domains II and IV, shown in SEQ ID NOs:16 and 18, respectively). Domain II comprises a 3 hairpin dimerisation loop (SEQ ID NO:19) which is involved in intermolecular interaction with other HER receptor molecules. The extracellular region is linked via a transmembrane region (SEQ ID NO:10) to a cytoplasmic region (SEQ ID NO:11). The cytoplasmic region comprises a juxtamembrane segment (SEQ ID NO:12), a protein kinase domain (SEQ ID NO:13), and a C-terminal segment (SEQ ID NO:14).
Signalling through HER3 involves receptor homodimerisation (i.e. with other HER3 receptors) or heterodimerisation (with other HER receptors, e.g. HER2) and consequent autophosphorylation by the protein kinase domain of tyrosines of the cytoplasmic region. The phosphorylated tyrosine residues recruit adaptor/effector proteins (e.g. Grb2 and phospholipase Cy (PLCγ), containing src homology domain 2 (SH2) or phosphotyrosine binding (PTB) domains.
Signalling through HER3 can be activated in a ligand-dependent or ligand-independent manner. In the absence of ligand, HER3 receptor molecules are normally expressed at the cell surface as monomers with a conformation which prevents receptor dimerisation in which the dimerisation loop of subdomain II makes intramolecular contact with a pocket on subdomain IV. Binding of a HER3 ligand such as a neuregulin (NRG), e.g. NRG1 (also known as heregulin, HRG) or NRG2 to subdomains I and III of the extracellular region causes a conformational change which results in the exposure of the dimerisation loop of subdomain II, facilitating receptor dimerisation and signalling. Some cancer-associated mutations in HER3 may disrupt interaction of subdomains II and IV required for the formation of the inactive ‘closed’ conformation and thereby cause constitutive presentation of the dimerisation loop and activation of HER3-mediated signalling in the absence of ligand binding (see e.g. in Jaiswal et al., Cancer Cell (2013) 23(5): 603-617).
In this specification “HER3” refers to HER3 from any species and includes HER3 isoforms, fragments, variants (including mutants) or homologues from any species.
As used herein, a “fragment”, “variant” or “homologue” of a protein may optionally be characterised as having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of the reference protein (e.g. a reference isoform). In some embodiments fragments, variants, isoforms and homologues of a reference protein may be characterised by ability to perform a function performed by the reference protein.
A “fragment” generally refers to a fraction of the reference protein. A “variant” generally refers to a protein having an amino acid sequence comprising one or more amino acid substitutions, insertions, deletions or other modifications relative to the amino acid sequence of the reference protein, but retaining a considerable degree of sequence identity (e.g. at least 60%) to the amino acid sequence of the reference protein. An “isoform” generally refers to a variant of the reference protein expressed by the same species as the species of the reference protein (e.g. HER3 isoforms 1 to 5 are all isoforms of one another). A “homologue” generally refers to a variant of the reference protein produced by a different species as compared to the species of the reference protein. For example, human HER3 isoform 1 (P21860-1, v1; SEQ ID NO:1) and Rhesus macaque HER3 (UniProt: F7HEH3-1, v2; SEQ ID NO:20) are homologues of one another. Homologues include orthologues.
A “fragment” of a reference protein may be of any length (by number of amino acids), although may optionally be at least 20% of the length of the reference protein (that is, the protein from which the fragment is derived) and may have a maximum length of one of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the reference protein.
A fragment of HER3 may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200 amino acids, and may have a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids.
In some embodiments, the HER3 is HER3 from a mammal (e.g. a primate (rhesus, cynomolgous, non-human primate or human) and/or a rodent (e.g. rat or murine) HER3). Isoforms, fragments, variants or homologues of HER3 may optionally be characterised as having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of an immature or mature HER3 isoform from a given species, e.g. human.
Isoforms, fragments, variants or homologues may optionally be functional isoforms, fragments, variants or homologues, e.g. having a functional property/activity of the reference HER3 (e.g. human HER3 isoform 1), as determined by analysis by a suitable assay for the functional property/activity. For example, an isoform, fragment, variant or homologue of HER3 may display association with one or more of: HER2, NRG1 (type I, II, III, IV, V or VI) or NRG2 (α or β).
In some embodiments, the HER3 comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to one of SEQ ID NOs:1 to 8.
In some embodiments, a fragment of HER3 comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to one of SEQ ID NOs:9 to 19, e.g. one of 9, 16 or 19.
The antigen-binding molecules of the present invention were specifically designed to target regions of HER3 of particular interest. In a two-step approach, HER3 regions to be targeted were selected following analysis for predicted antigenicity, function and safety. Antibodies specific for the target regions of HER3 were then prepared using peptides corresponding to the target regions as immunogens to raise specific monoclonal antibodies, and subsequent screening identified antibodies capable of binding to HER3 in the native state. This approach provides exquisite control over the antibody epitope.
The antigen-binding molecules of the present invention may be defined by reference to the region of HER3 to which they bind. The antigen-binding molecules of the present invention may bind to a particular region of interest of HER3. In some embodiments the antigen-binding molecule may bind to a linear epitope of HER3, consisting of a contiguous sequence of amino acids (i.e. an amino acid primary sequence). In some embodiments, the antigen-binding molecule may bind to a conformational epitope of HER3, consisting of a discontinuous sequence of amino acids of the amino acid sequence.
In some embodiments, the antigen-binding molecule of the present invention binds to HER3. In some embodiments, the antigen-binding molecule binds to the extracellular region of HER3 (e.g. the region shown in SEQ ID NO:9). In some embodiments, the antigen-binding molecule binds to subdomain II of the extracellular region of HER3 (e.g. the region shown in SEQ ID NO:16).
In some embodiments, the antigen-binding molecule binds to the region of HER3 shown in SEQ ID NO:229. In some embodiments the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 shown in SEQ ID NO:229. In some embodiments, the antigen-binding molecule binds to the regions of HER3 shown in SEQ ID NOs:230 and 231. In some embodiments the antigen-binding molecule contacts one or more amino acid residues of the regions of HER3 shown in SEQ ID NOs:230 and 231. In some embodiments, the antigen-binding molecule binds to the region of HER3 shown in SEQ ID NO:230. In some embodiments the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 shown in SEQ ID NO:230. In some embodiments, the antigen-binding molecule binds to the region of HER3 shown in SEQ ID NO:231. In some embodiments the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 shown in SEQ ID NO:231.
In some embodiments, the antigen-binding molecule binds to the region of HER3 shown in SEQ ID NO:23. In some embodiments the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 shown in SEQ ID NO:23. In some embodiments, the antigen-binding molecule binds to the region of HER3 shown in SEQ ID NO:21. In some embodiments the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 shown in SEQ ID NO:21. In some embodiments the antigen-binding molecule binds to the region of HER3 shown in SEQ ID NO:19. In some embodiments the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 shown in SEQ ID NO:19. In some embodiments, the antigen-binding molecule binds to the region of HER3 shown in SEQ ID NO:22. In some embodiments the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 shown in SEQ ID NO:22.
In some embodiments, the antigen-binding molecule does not bind to the region of HER3 corresponding to positions 260 to 279 of SEQ ID NO:1. In some embodiments the antigen-binding molecule does not contact an amino acid residue of the region of HER3 corresponding to positions 260 to 279 of SEQ ID NO:1. In some embodiments, the antigen-binding molecule does not bind to the region of HER3 shown in SEQ ID NO:23. In some embodiments the antigen-binding molecule does not contact an amino acid residue of the region of HER3 shown in SEQ ID NO:23.
The region of a peptide/polypeptide to which an antibody binds can be determined by the skilled person using various methods well known in the art, including X-ray co-crystallography analysis of antibody-antigen complexes, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange analysis by mass spectrometry, phage display, competition ELISA and proteolysis-based ‘protection’ methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21(3):145-156, which is hereby incorporated by reference in its entirety.
In some embodiments the antigen-binding molecule is capable of binding the same region of HER3, or an overlapping region of HER3, to the region of HER3 which is bound by an antibody comprising the VH and VL sequences of one of antibody clones 10D1, 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c89, 10D1_c90, 10D1_c91, 10D1_c92, 10D1_c93, 10A6, 4-35-B2 or 4-35-B4 described herein. In some embodiments the antigen-binding molecule is capable of binding the same region of HER3, or an overlapping region of HER3, to the region of HER3 which is bound by an antibody comprising the VH and VL sequences of one of antibody clones 10D1_c89, 10D1_c90 or 10D1_c91. In some embodiments the antigen-binding molecule is capable of binding the same region of HER3, or an overlapping region of HER3, to the region of HER3 which is bound by an antibody comprising the VH and VL sequences of antibody clone 10D1_c89.
As used herein, a “peptide” refers to a chain of two or more amino acid monomers linked by peptide bonds. A peptide typically has a length in the region of about 2 to 50 amino acids. A “polypeptide” is a polymer chain of two or more peptides. Polypeptides typically have a length greater than about 50 amino acids.
In some embodiments, the antigen-binding molecule of the present invention is capable of binding to a polypeptide comprising, or consisting of, the amino acid sequence of one of SEQ ID NOs:1, 3, 4, 6 or 8.
In some embodiments, the antigen-binding molecule is capable of binding to a polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:9. In some embodiments, the antigen-binding molecule is capable of binding to a polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:16.
In some embodiments, the antigen-binding molecule is capable of binding to a peptide/polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:229. In some embodiments, the antigen-binding molecule is capable of binding to a peptide/polypeptide comprising, or consisting of, the amino acid sequences of SEQ ID NOs:230 and 231. In some embodiments, the antigen-binding molecule is capable of binding to a peptide/polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:230. In some embodiments, the antigen-binding molecule is capable of binding to a peptide/polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:231. In some embodiments, the antigen-binding molecule is capable of binding to a peptide/polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:23. In some embodiments, the antigen-binding molecule is capable of binding to a peptide/polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:21. In some embodiments, the antigen-binding molecule is capable of binding to a peptide/polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:19. In some embodiments, the antigen-binding molecule is capable of binding to a peptide/polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:22.
In some embodiments, the antigen-binding molecule is not capable of binding to a peptide consisting of the amino acid sequence corresponding to positions 260 to 279 of SEQ ID NO:1. In some embodiments, the antigen-binding molecule is not capable of binding to a peptide consisting of the amino acid sequence of SEQ ID NO:23.
The ability of an antigen-binding molecule to bind to a given peptide/polypeptide can be analysed by methods well known to the skilled person, including analysis by ELISA, immunoblot (e.g. western blot), immunoprecipitation, Surface Plasmon Resonance (SPR; see e.g. Hearty et al., Methods Mol Biol (2012) 907:411-442) or Bio-Layer Interferometry (see e.g. Lad et al., (2015) J Biomol Screen 20(4): 498-507).
In embodiments where the antigen binding molecule is capable of binding to a peptide/polypeptide comprising a reference amino acid sequence, the peptide/polypeptide may comprise one or more additional amino acids at one or both ends of the reference amino acid sequence. In some embodiments the peptide/polypeptide comprises e.g. 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 5-10, 5-20, 5-30, 5-40, 5-50, 10-20, 10-30, 10-40, 10-50, 20-30, 20-40 or 20-50 additional amino acids at one or both ends of the reference amino acid sequence.
In some embodiments the additional amino acid(s) provided at one or both ends (i.e. the N-terminal and C-terminal ends) of the reference sequence correspond to the positions at the ends of the reference sequence in the context of the amino acid sequence of HER3. By way of example, where the antigen-binding molecule is capable of binding to a peptide comprising the sequence of SEQ ID NO:23 and an additional two amino acids at the C-terminal end of SEQ ID NO:23, the additional two amino acids may be threonine and lysine, corresponding to positions 278 and 279 of SEQ ID NO:1.
In some embodiments the antigen-binding molecule is capable of binding to a peptide/polypeptide which is bound by an antibody comprising the VH and VL sequences of one of antibody clones 10D1, 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c89, 10D1_c90, 10D1_c91, 10D1_c92, 10D1_c93, 10A6, 4-35-B2 or 4-35-B4 described herein. In some embodiments the antigen-binding molecule is capable of binding to a peptide/polypeptide which is bound by an antibody comprising the VH and VL sequences of one of antibody clones 10D1_c89, 10D1_c90 or 10D1_c91. In some embodiments the antigen-binding molecule is capable of binding to a peptide/polypeptide which is bound by an antibody comprising the VH and VL sequences of antibody clone 10D1_c89.
The present invention provides antigen-binding molecules capable of binding to HER3. An “antigen-binding molecule” refers to a molecule which is capable of binding to a target antigen, and encompasses monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g. Fv, scFv, Fab, scFab, F(ab′)2, Fab2, diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g. VhH), etc.), as long as they display binding to the relevant target molecule(s).
The antigen-binding molecule of the present invention comprises a moiety capable of binding to a target antigen(s). In some embodiments, the moiety capable of binding to a target antigen comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) of an antibody capable of specific binding to the target antigen. In some embodiments, the moiety capable of binding to a target antigen comprises or consists of an aptamer capable of binding to the target antigen, e.g. a nucleic acid aptamer (reviewed, for example, in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3):181-202). In some embodiments, the moiety capable of binding to a target antigen comprises or consists of a antigen-binding peptide/polypeptide, e.g. a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody (i.e. a single-domain antibody (sdAb)) affilin, armadillo repeat protein (ArmRP), OBody or fibronectin—reviewed e.g. in Reverdatto et al., Curr Top Med Chem. 2015; 15(12): 1082-1101, which is hereby incorporated by reference in its entirety (see also e.g. Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48).
The antigen-binding molecules of the present invention generally comprise an antigen-binding domain comprising a VH and a VL of an antibody capable of specific binding to the target antigen. The antigen-binding domain formed by a VH and a VL may also be referred to herein as an Fv region.
An antigen-binding molecule may be, or may comprise, an antigen-binding polypeptide, or an antigen-binding polypeptide complex. An antigen-binding molecule may comprise more than one polypeptide which together form an antigen-binding domain. The polypeptides may associate covalently or non-covalently. In some embodiments the polypeptides form part of a larger polypeptide comprising the polypeptides (e.g. in the case of scFv comprising VH and VL, or in the case of scFab comprising VH-CH1 and VL-CL).
An antigen-binding molecule may refer to a non-covalent or covalent complex of more than one polypeptide (e.g. 2, 3, 4, 6, or 8 polypeptides), e.g. an IgG-like antigen-binding molecule comprising two heavy chain polypeptides and two light chain polypeptides.
The antigen-binding molecules of the present invention may be designed and prepared using the sequences of monoclonal antibodies (mAbs) capable of binding to HER3. Antigen-binding regions of antibodies, such as single chain variable fragment (scFv), Fab and F(ab′)2 fragments may also be used/provided. An “antigen-binding region” is any fragment of an antibody which is capable of binding to the target for which the given antibody is specific.
Antibodies generally comprise six complementarity-determining regions CDRs; three in the heavy chain variable (VH) region: HC-CDR1, HC-CDR2 and HC-CDR3, and three in the light chain variable (VL) region: LC-CDR1, LC-CDR2, and LC-CDR3. The six CDRs together define the paratope of the antibody, which is the part of the antibody which binds to the target antigen.
The VH region and VL region comprise framework regions (FRs) either side of each CDR, which provide a scaffold for the CDRs. From N-terminus to C-terminus, VH regions comprise the following structure: N term-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C term; and VL regions comprise the following structure: N term-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-C term.
There are several different conventions for defining antibody CDRs and FRs, such as those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and VBASE2, as described in Retter et al., Nucl. Acids Res. (2005) 33 (suppl 1): D671-D674. The CDRs and FRs of the VH regions and VL regions of the antibody clones described herein were defined according to the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue):D413-22), which uses the IMGT V-DOMAIN numbering rules as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77.
In some embodiments, the antigen-binding molecule comprises the CDRs of an antigen-binding molecule which is capable of binding to HER3. In some embodiments, the antigen-binding molecule comprises the FRs of an antigen-binding molecule which is capable of binding to HER3. In some embodiments, the antigen-binding molecule comprises the CDRs and the FRs of an antigen-binding molecule which is capable of binding to HER3. That is, in some embodiments the antigen-binding molecule comprises the VH region and the VL region of an antigen-binding molecule which is capable of binding to HER3. In some embodiments the antigen-binding molecule comprises a VH region and a VL region which is, or which is derived from, the VH/VL region of a HER3-binding antibody clone described herein (i.e. anti-HER3 antibody clones 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c89, 10D1_c90, 10D1_c91, 10D1_c92, 10D1_c93, 10D1, 10A6, 4-35-B2 or 4-35-B4; e.g. 10D1_c89, 10D1_c90 or 10D1_c91; e.g. 10D1_c89).
In some embodiments the antigen-binding molecule comprises a VH region according to one of (1) to (10) below:
In some embodiments the antigen-binding molecule comprises a VH region according to one of (11) to (24) below:
In some embodiments the antigen-binding molecule comprises a VH region comprising the CDRs according to one of (1) to (10) above, and the FRs according to one of (11) to (24) above.
In some embodiments the antigen-binding molecule comprises a VH region according to one of (25) to (41) below:
In some embodiments the antigen-binding molecule comprises a VH region according to one of (42) to (61) below:
In some embodiments the antigen-binding molecule comprises a VL region according to one of (62) to (71) below:
In some embodiments the antigen-binding molecule comprises a VL region according to one of (72) to (86) below:
In some embodiments the antigen-binding molecule comprises a VL region comprising the CDRs according to one of (62) to (71) above, and the FRs according to one of (72) to (86) above. In some embodiments the antigen-binding molecule comprises a VL region according to one of (87) to (102) below:
In some embodiments the antigen-binding molecule comprises a VL region according to one of (103) to (119) below:
In some embodiments the antigen-binding molecule comprises a VH region according to any one of (1) to (61) above, and a VL region according to any one of (62) to (119) above.
In embodiments in accordance with the present invention in which one or more amino acids are substituted with another amino acid, the substitutions may be conservative substitutions, for example according to the following Table. In some embodiments, amino acids in the same block in the middle column are substituted. In some embodiments, amino acids in the same line in the rightmost column are substituted:
In some embodiments, substitution(s) may be functionally conservative. That is, in some embodiments the substitution may not affect (or may not substantially affect) one or more functional properties (e.g. target binding) of the antigen-binding molecule comprising the substitution as compared to the equivalent unsubstituted molecule.
The VH and VL region of an antigen-binding region of an antibody together constitute the Fv region. In some embodiments, the antigen-binding molecule according to the present invention comprises, or consists of, an Fv region which binds to HER3. In some embodiments the VH and VL regions of the Fv are provided as single polypeptide joined by a linker region, i.e. a single chain Fv (scFv). In some embodiments the antigen-binding molecule of the present invention comprises one or more regions of an immunoglobulin heavy chain constant sequence. In some embodiments the immunoglobulin heavy chain constant sequence is, or is derived from, the heavy chain constant sequence of an IgG (e.g. IgG1, IgG2, IgG3, IgG4), IgA (e.g. IgA1, IgA2), IgD, IgE or IgM.
In some embodiments the immunoglobulin heavy chain constant sequence is human immunoglobulin G 1 constant (IGHG1; UniProt: P01857-1, v1; SEQ ID NO:171). Positions 1 to 98 of SEQ ID NO:171 form the CH1 region (SEQ ID NO:172). Positions 99 to 110 of SEQ ID NO:171 form a hinge region between CH1 and CH2 regions (SEQ ID NO:173). Positions 111 to 223 of SEQ ID NO:171 form the CH2 region (SEQ ID NO:174). Positions 224 to 330 of SEQ ID NO:171 form the CH3 region (SEQ ID NO:175).
The exemplified antigen-binding molecules may be prepared using pFUSE-CHIg-hG1, which comprises the substitutions D356E, L358M (positions numbered according to EU numbering) in the CH3 region. The amino acid sequence of the CH3 region encoded by pFUSE-CHIg-hG1 is shown in SEQ ID NO:176. It will be appreciated that CH3 regions may be provided with further substitutions in accordance with modification to an Fc region of the antigen-binding molecule as described herein.
In some embodiments a CH1 region comprises or consists of the sequence of SEQ ID NO:172, or a sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:172. In some embodiments a CH1-CH2 hinge region comprises or consists of the sequence of SEQ ID NO:173, or a sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:173. In some embodiments a CH2 region comprises or consists of the sequence of SEQ ID NO:174, or a sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:174. In some embodiments a CH3 region comprises or consists of the sequence of SEQ ID NO:175 or 176, or a sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:175 or 176.
In some embodiments the antigen-binding molecule of the present invention comprises one or more regions of an immunoglobulin light chain constant sequence. In some embodiments the immunoglobulin light chain constant sequence is human immunoglobulin kappa constant (IGKC; Cκ; UniProt: P01834-1, v2; SEQ ID NO:177). In some embodiments the immunoglobulin light chain constant sequence is a human immunoglobulin lambda constant (IGLC; Cλ), e.g. IGLC1, IGLC2, IGLC3, IGLC6 or IGLC7. In some embodiments a CL region comprises or consists of the sequence of SEQ ID NO:177, or a sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:177.
The VL and light chain constant (CL) region, and the VH region and heavy chain constant 1 (CH1) region of an antigen-binding region of an antibody together constitute the Fab region. In some embodiments the antigen-binding molecule comprises a Fab region comprising a VH, a CH1, a VL and a CL (e.g. Cκ or Cλ). In some embodiments the Fab region comprises a polypeptide comprising a VH and a CH1 (e.g. a VH-CH1 fusion polypeptide), and a polypeptide comprising a VL and a CL (e.g. a VL-CL fusion polypeptide). In some embodiments the Fab region comprises a polypeptide comprising a VH and a CL (e.g. a VH-CL fusion polypeptide) and a polypeptide comprising a VL and a CH (e.g. a VL-CH1 fusion polypeptide); that is, in some embodiments the Fab region is a CrossFab region. In some embodiments the VH, CH1, VL and CL regions of the Fab or CrossFab are provided as single polypeptide joined by linker regions, i.e. as a single chain Fab (scFab) or a single chain CrossFab (scCrossFab).
In some embodiments, the antigen-binding molecule of the present invention comprises, or consists of, a Fab region which binds to HER3.
In some embodiments, the antigen-binding molecule described herein comprises, or consists of, a whole antibody which binds to HER3. As used herein, “whole antibody” refers to an antibody having a structure which is substantially similar to the structure of an immunoglobulin (Ig). Different kinds of immunoglobulins and their structures are described e.g. in Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202): S41-S52, which is hereby incorporated by reference in its entirety.
Immunoglobulins of type G (i.e. IgG) are ˜150 kDa glycoproteins comprising two heavy chains and two light chains. From N- to C-terminus, the heavy chains comprise a VH followed by a heavy chain constant region comprising three constant domains (CH1, CH2, and CH3), and similarly the light chain comprise a VL followed by a CL. Depending on the heavy chain, immunoglobulins may be classed as IgG (e.g. IgG1, IgG2, IgG3, IgG4), IgA (e.g. IgA1, IgA2), IgD, IgE, or IgM. The light chain may be kappa (κ) or lambda (λ).
In some embodiments, the antigen-binding molecule described herein comprises, or consists of, an IgG (e.g. IgG1, IgG2, IgG3, IgG4), IgA (e.g. IgA1, IgA2), IgD, IgE, or IgM which binds to HER3.
In some embodiments, the antigen-binding molecule of the present invention is at least monovalent binding for HER3. Binding valency refers to the number of binding sites in an antigen-binding molecule for a given antigenic determinant. Accordingly, in some embodiments the antigen-binding molecule comprises at least one binding site for HER3.
In some embodiments the antigen-binding molecule comprises more than one binding site for HER3, e.g. 2, 3 or 4 binding sites. The binding sites may be the same or different. In some embodiments the antigen-binding molecule is e.g. bivalent, trivalent or tetravalent for HER3.
Aspects of the present invention relate to multispecific antigen-binding molecules. By “multispecific” it is meant that the antigen-binding molecule displays specific binding to more than one target. In some embodiments the antigen-binding molecule is a bispecific antigen-binding molecule. In some embodiments the antigen-binding molecule comprises at least two different antigen-binding domains (i.e. at least two antigen-binding domains, e.g. comprising non-identical VHs and VLs).
In some embodiments the antigen-binding molecule binds to HER3 and another target (e.g. an antigen other than HER3), and so is at least bispecific. The term “bispecific” means that the antigen-binding molecule is able to bind specifically to at least two distinct antigenic determinants.
It will be appreciated that an antigen-binding molecule according to the present invention (e.g. a multispecific antigen-binding molecule) may comprise antigen-binding molecules capable of binding to the targets for which the antigen-binding molecule is specific. For example, an antigen-binding molecule which is capable of binding to HER3 and an antigen other than HER3 may comprise: (i) an antigen-binding molecule which is capable of binding to HER3, and (ii) an antigen-binding molecule which is capable of binding to an antigen other than HER3.
It will also be appreciated that an antigen-binding molecule according to the present invention (e.g. a multispecific antigen-binding molecule) may comprise antigen-binding polypeptides or antigen-binding polypeptide complexes capable of binding to the targets for which the antigen-binding molecule is specific. For example, an antigen-binding molecule according to the invention may comprise e.g. (i) an antigen-binding polypeptide complex capable of binding to HER3, comprising a light chain polypeptide (comprising the structure VL-CL) and a heavy chain polypeptide (comprising the structure VH-CH1-CH2-CH3), and (ii) an antigen-binding polypeptide complex capable of binding to an antigen other than HER3, comprising a light chain polypeptide (comprising the structure VL-CL) and a heavy chain polypeptide (comprising the structure VH-CH1-CH2-CH3).
In some embodiments, a component antigen-binding molecule of a larger antigen-binding molecule (e.g. a multispecific antigen-biding molecule) may be referred to e.g. as an “antigen-binding domain” or “antigen-binding region” of the larger antigen-binding molecule.
In some embodiments the antigen-binding molecule comprises an antigen-binding molecule capable of binding to HER3, and an antigen-binding molecule capable of binding to an antigen other than HER3. In some embodiments, the antigen other than HER3 is an immune cell surface molecule. In some embodiments, the antigen other than HER3 is a cancer cell antigen. In some embodiments the antigen other than HER3 is a receptor molecule, e.g. a cell surface receptor. In some embodiments the antigen other than HER3 is a cell signalling molecule, e.g. a cytokine, chemokine, interferon, interleukin or lymphokine. In some embodiments the antigen other than HER3 is a growth factor or a hormone.
A cancer cell antigen is an antigen which is expressed or over-expressed by a cancer cell. A cancer cell antigen may be any peptide/polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. A cancer cell antigen's expression may be associated with a cancer. A cancer cell antigen may be abnormally expressed by a cancer cell (e.g. the cancer cell antigen may be expressed with abnormal localisation), or may be expressed with an abnormal structure by a cancer cell. A cancer cell antigen may be capable of eliciting an immune response. In some embodiments, the antigen is expressed at the cell surface of the cancer cell (i.e. the cancer cell antigen is a cancer cell surface antigen). In some embodiments, the part of the antigen which is bound by the antigen-binding molecule described herein is displayed on the external surface of the cancer cell (i.e. is extracellular). The cancer cell antigen may be a cancer-associated antigen. In some embodiments the cancer cell antigen is an antigen whose expression is associated with the development, progression or severity of symptoms of a cancer. The cancer-associated antigen may be associated with the cause or pathology of the cancer, or may be expressed abnormally as a consequence of the cancer. In some embodiments, the cancer cell antigen is an antigen whose expression is upregulated (e.g. at the RNA and/or protein level) by cells of a cancer, e.g. as compared to the level of expression by comparable non-cancerous cells (e.g. non-cancerous cells derived from the same tissue/cell type). In some embodiments, the cancer-associated antigen may be preferentially expressed by cancerous cells, and not expressed by comparable non-cancerous cells (e.g. non-cancerous cells derived from the same tissue/cell type). In some embodiments, the cancer-associated antigen may be the product of a mutated oncogene or mutated tumor suppressor gene. In some embodiments, the cancer-associated antigen may be the product of an overexpressed cellular protein, a cancer antigen produced by an oncogenic virus, an oncofetal antigen, or a cell surface glycolipid or glycoprotein.
In some embodiments the antigen other than HER3 is an antigen expressed by cells of a HER3-associated cancer. A HER3-associated cancer may be a cancer expressing HER3 (e.g. expressing HER3 protein at the cell surface); such cancers may be referred to as “HER3-positive” cancers. HER3-associated cancers include cancers for which HER3 gene/protein expression is a risk factor for, and/or is positively associated with, the onset, development, progression or severity of symptoms of the cancer, and/or metastasis. HER3-associated cancers include those described in Zhang et al., Acta Biochimica et Biophysica Sinica (2016) 48(1):39-48 and Sithanandam and Anderson Cancer Gene Ther (2008) 15(7):413-448, both of which are hereby incorporated by reference in their entirety. In some embodiments a HER3-associated cancer may be a lung cancer (e.g. NSCLC), melanoma, breast cancer, pancreatic cancer, prostate cancer, ovarian cancer, gastric cancer, colon cancer or oral cavity cancer.
An immune cell surface molecule may be any peptide/polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof expressed at or on the cell surface of an immune cell. In some embodiments, the part of the immune cell surface molecule which is bound by the antigen-binding molecule of the present invention is on the external surface of the immune cell (i.e. is extracellular). The immune cell surface molecule may be expressed at the cell surface of any immune cell. In some embodiments, the immune cell may be a cell of hematopoietic origin, e.g. a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. The lymphocyte may be e.g. a T cell, B cell, natural killer (NK) cell, NKT cell or innate lymphoid cell (ILC), or a precursor thereof (e.g. a thymocyte or pre-B cell). In some embodiments the immune cell surface molecule may be a costimulatory molecule (e.g. CD28, OX40, 4-1BB, ICOS or CD27) or a ligand thereof. In some embodiments the immune cell surface molecule may be a checkpoint molecule (e.g. PD-1, CTLA-4, LAG-3, TIM-3, VISTA, TIGIT or BTLA) or a ligand thereof.
Multispecific antigen-binding molecules according to the invention may be provided in any suitable format, such as those formats described in described in Brinkmann and Kontermann MAbs (2017) 9(2): 182-212, which is hereby incorporated by reference in its entirety. Suitable formats include those shown in
The skilled person is able to design and prepare bispecific antigen-binding molecules. Methods for producing bispecific antigen-binding molecules include chemically crosslinking of antigen-binding molecules or antibody fragments, e.g. with reducible disulphide or non-reducible thioether bonds, for example as described in Segal and Bast, 2001. Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14:IV:2.13:2.13.1-2.13.16, which is hereby incorporated by reference in its entirety. For example, N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used to chemically crosslink e.g. Fab fragments via hinge region SH— groups, to create disulfide-linked bispecific F(ab)2 heterodimers.
Other methods for producing bispecific antigen-binding molecules include fusing antibody-producing hybridomas e.g. with polyethylene glycol, to produce a quadroma cell capable of secreting bispecific antibody, for example as described in D. M. and Bast, B. J. 2001. Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14:IV:2.13:2.13.1-2.13.16.
Bispecific antigen-binding molecules according to the present invention can also be produced recombinantly, by expression from e.g. a nucleic acid construct encoding polypeptides for the antigen-binding molecules, for example as described in Antibody Engineering: Methods and Protocols, Second Edition (Humana Press, 2012), at Chapter 40: Production of Bispecific Antigen-binding molecules: Diabodies and Tandem scFv (Hornig and Farber-Schwarz), or French, How to make bispecific antigen-binding molecules, Methods Mol. Med. 2000; 40:333-339, the entire contents of both of which are hereby incorporated by reference. For example, a DNA construct encoding the light and heavy chain variable domains for the two antigen-binding fragments (i.e. the light and heavy chain variable domains for the antigen-binding fragment capable of binding HER3, and the light and heavy chain variable domains for the antigen-binding fragment capable of binding to another target protein), and including sequences encoding a suitable linker or dimerization domain between the antigen-binding fragments can be prepared by molecular cloning techniques. Recombinant bispecific antibody can thereafter be produced by expression (e.g. in vitro) of the construct in a suitable host cell (e.g. a mammalian host cell), and expressed recombinant bispecific antibody can then optionally be purified.
In some embodiments the antigen-binding molecules of the present invention comprise an Fc region.
In IgG, IgA and IgD isotypes an Fc region is composed of CH2 and CH3 regions from one polypeptide, and CH2 and CH3 regions from another polypeptide. The CH2 and CH3 regions from the two polypeptides together form the Fc region. In IgM and IgE isotypes the Fc regions contain three constant domains (CH2, CH3 and CH4), and CH2 to CH4 from the two polypeptides together form the Fc region.
In preferred embodiments in accordance with the various aspects of the present disclosure an Fc region comprises two polypeptides, each polypeptide comprising a CH2 region and a CH3 region.
In some embodiments, the antigen-binding molecule of the present invention comprises an Fc region comprising modification in one or more of the CH2 and CH3 regions promoting association of the Fc region. Recombinant co-expression of constituent polypeptides of an antigen-binding molecule and subsequent association leads to several possible combinations. To improve the yield of the desired combinations of polypeptides in antigen-binding molecules in recombinant production, it is advantageous to introduce in the Fc regions modification(s) promoting association of the desired combination of heavy chain polypeptides. Modifications may promote e.g. hydrophobic and/or electrostatic interaction between CH2 and/or CH3 regions of different polypeptide chains. Suitable modifications are described e.g. in Ha et al., Front. Immnol (2016) 7:394, which is hereby incorporated by reference in its entirety.
In some embodiments the antigen-binding molecule of the present invention comprises an Fc region comprising paired substitutions in the CH3 regions of the Fc region according to one of the following formats, as shown in Table 1 of Ha et al., Front. Immnol (2016) 7:394: KiH, KiHs-s, HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, SEED or A107.
In some embodiments, the Fc region comprises the “knob-into-hole” or “KiH” modification, e.g. as described e.g. in U.S. Pat. No. 7,695,936 and Carter, J Immunol Meth 248, 7-15 (2001). In such embodiments, one of the CH3 regions of the Fc region comprises a “knob” modification, and the other CH3 region comprises a “hole” modification. The “knob” and “hole” modifications are positioned within the respective CH3 regions so that the “knob” can be positioned in the “hole” in order to promote heterodimerisation (and inhibit homodimerisation) of the polypeptides and/or stabilise heterodimers. Knobs are constructed by substituting amino acids having small chains with those having larger side chains (e.g. tyrosine or tryptophan). Holes are created by substituting amino acids having large side chains with those having smaller side chains (e.g. alanine or threonine).
In some embodiments, one of the CH3 regions of the Fc region of the antigen-binding molecule of the present invention comprises the substitution (numbering of positions/substitutions in the Fc, CH2 and CH3 regions herein is according to the EU numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991) T366W, and the other CH3 region of the Fc region comprises the substitution Y407V. In some embodiments, one of the CH3 regions of the Fc region of the antigen-binding molecule comprises the substitution T366W, and the other CH3 region of the Fc region comprises the substitutions T366S and L368A. In some embodiments, one of the CH3 regions of the Fc region of the antigen-binding molecule comprises the substitution T366W, and the other CH3 region of the Fc region comprises the substitutions Y407V, T366S and L368A.
In some embodiments, the Fc region comprises the “DD-KK” modification as described e.g. in WO 2014/131694 A1. In some embodiments, one of the CH3 regions comprises the substitutions K392D and K409D, and the other CH3 region of the Fc region comprises the substitutions E356K and D399K. The modifications promote electrostatic interaction between the CH3 regions.
In some embodiments, the antigen-binding molecule of the present invention comprises an Fc region modified as described in Labrijn et al., Proc Natl Acad Sci USA. (2013) 110(13):5145-50, referred to as ‘Duobody’ format. In some embodiments one of the CH3 regions comprises the substitution K409R, and the other CH3 region of the Fc region comprises the substitution K405L.
In some embodiments, the antigen-binding molecule of the present invention comprises an Fc region comprising the “EEE-RRR” modification as described in Strop et al., J Mol Biol. (2012) 420(3):204-19. In some embodiments one of the CH3 regions comprises the substitutions D221E, P228E and L368E, and the other CH3 region of the Fc region comprises the substitutions D221R, P228R and K409R.
In some embodiments, the antigen-binding molecule comprises an Fc region comprising the “EW-RVT” modification described in Choi et al., Mol Cancer Ther (2013) 12(12):2748-59. In some embodiments one of the CH3 regions comprises the substitutions K360E and K409W, and the other CH3 region of the Fc region comprises the substitutions Q347R, D399V and F405T.
In some embodiments, one of the CH3 regions comprises the substitution S354C, and the other CH3 region of the Fc region comprises the substitution Y349C. Introduction of these cysteine residues results in formation of a disulphide bridge between the two CH3 regions of the Fc region, further stabilizing the heterodimer (Carter (2001), J Immunol Methods 248, 7-15).
In some embodiments, the Fc region comprises the “KiHs-s” modification. In some embodiments one of the CH3 regions comprises the substitutions T366W and S354C, and the other CH3 region of the Fc region comprises the substitutions T366S, L368A, Y407V and Y349C.
In some embodiments, the antigen-binding molecule of the present invention comprises an Fc region comprising the “SEED” modification as described in Davis et al., Protein Eng Des Sel (2010) 23(4):195-202, in which β-strand segments of human IgG1 CH3 and igA CH3 are exchanged.
In some embodiments, one of the CH3 regions comprises the substitutions S364H and F405A, and the other CH3 region of the Fc region comprises the substitutions Y349T and T394F (see e.g. Moore et al., MAbs (2011) 3(6):546-57).
In some embodiments, one of the CH3 regions comprises the substitutions T350V, L351Y, F405A and Y407V, and the other CH3 region of the Fc region comprises the substitutions T350V, T366L, K392L and T394W (see e.g. Von Kreudenstein et al., MAbs (2013) 5(5):646-54).
In some embodiments, one of the CH3 regions comprises the substitutions K360D, D399M and Y407A, and the other CH3 region of the Fc region comprises the substitutions E345R, Q347R, T366V and K409V (see e.g. Leaver-Fay et al., Structure (2016) 24(4):641-51).
In some embodiments, one of the CH3 regions comprises the substitutions K370E and K409W, and the other CH3 region of the Fc region comprises the substitutions E357N, D399V and F405T (see e.g. Choi et al., PLoS One (2015) 10(12):e0145349).
Fc-mediated functions include Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of the membrane attack complex (MAC), cell degranulation, cytokine and/or chemokine production, and antigen processing and presentation.
Modifications to antibody Fc regions that influence Fc-mediated functions are known in the art, such as those described e.g. in Wang et al., Protein Cell (2018) 9(1):63-73, which is hereby incorporated by reference in its entirety. Exemplary Fc region modifications known to influence antibody effector function are summarised in Table 1 of Wang et al., Protein Cell (2018) 9(1):63-73.
The combination of substitutions F243L/R292P/Y300L/V3051/P396L is described in Stavenhagen et al. Cancer Res. (2007) to increase binding to FcγRIIIa, and thereby enhance ADCC. The combination of substitutions S239D/1332E or S239D/1332E/A330L is described in Lazar et al., Proc Natl Acad Sci USA. (2006)103:4005-4010 to increase binding to FcγRIIIa, and thereby increase ADCC. The combination of substitutions S239D/1332E/A330L is also described to decrease binding to FcγRIIb, and thereby increase ADCC. The combination of substitutions S298A/E333A/K334A is described in Shields et al., J Biol Chem. (2001) 276:6591-6604 to increase binding to FcγRIIIa, and thereby increase ADCC. The combination of substitutions L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in one heavy chain, and the combination of substitutions D270E/K326D/A330M/K334E in the other heavy chain, is described in Mimoto et al., MAbs. (2013): 5:229-236 to increase binding to FcγRIIIa, and thereby increase ADCC. The combination of substitutions G236A/S239D/1332E is described in Richards et al., Mol Cancer Ther. (2008) 7:2517-2527 to increase binding to FcγRIIa and to increase binding to FcγRIIIa, and thereby increase ADCP.
The combination of substitutions K326W/E333S is described in Idusogie et al. J Immunol. (2001) 166(4):2571-5 to increase binding to C1q, and thereby increase CDC. The combination of substitutions S267E/H268F/S324T is described in Moore et al. MAbs. (2010) 2(2):181-9 to increase binding to C1q, and thereby increase CDC. The combination of substitutions described in Natsume et al., Cancer Res. (2008) 68(10):3863-72 is reported to increase binding to C1q, and thereby increase CDC. The combination of substitutions E345R/E430G/S440Y is described in Diebolder et al. Science (2014) 343(6176):1260-3 to increase hexamerisation, and thereby increase CDC.
The combination of substitutions M252Y/S254T/T256E is described in Dall'Acqua et al. J Immunol. (2002) 169:5171-5180 to increase binding to FcRn at pH 6.0, and thereby increase antigen-binding molecule half-life. The combination of substitutions M428L/N434S is described in Zalevsky et al. Nat Biotechnol. (2010) 28:157-159 to increase binding to FcRn at pH 6.0, and thereby increase antigen-binding molecule half-life.
Where a heavy chain constant region/Fc region/CH2-CH3 region/CH2 region/CH3 region is described herein as comprising position(s)/substitution(s) “corresponding to” reference position(s)/substitution(s), equivalent position(s)/substitution(s) in homologous heavy chain constant regions/Fc regions/CH2-CH3 regions/CH2 regions/CH3 regions are contemplated.
Where an Fc region is described as comprising specific position(s)/substitution(s), the position(s)/substitution(s) may be present in one or both of the polypeptide chains which together form the Fc region.
Unless otherwise specified, positions herein refer to positions of human immunoglobulin constant region amino acid sequences numbered according to the EU numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. By way of illustration, the substitutions L242C and K334C in human IgG1 correspond to L>C substitution at position 125, and K>C substitution at position 217 of the human IgG1 constant region numbered according to SEQ ID NO:171.
Homologous heavy chain constant regions are heavy chain constant regions comprising an amino acid sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the heavy chain constant region of Human IgG1 (i.e. the amino acid sequence shown in SEQ ID NO:171). Homologous Fc regions are Fc regions comprised of polypeptides comprising an amino acid sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to CH2-CH3 region of Human IgG1 (i.e. the amino acid sequences shown in SEQ ID NO:174 and 175). Homologous CH2 regions are CH2 regions comprising an amino acid sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to CH2 region of Human IgG1 (i.e. the amino acid sequence shown in SEQ ID NO:174). Homologous CH3 regions are CH3 regions comprising an amino acid sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to CH3 region of Human IgG1 (i.e. the amino acid sequence shown in SEQ ID NO:175).
Corresponding positions to those identified in human IgG1 can be identified by sequence alignment which can be performed e.g. using sequence alignment software such as ClustalOmega (Söding, J. 2005, Bioinformatics 21, 951-960).
In some embodiments the antigen-binding molecule of the present invention comprises an Fc region comprising modification to increase an Fc-mediated function. In some embodiments the Fc region comprises modification to increase ADCC. In some embodiments the Fc region comprises modification to increase ADCP. In some embodiments the Fc region comprises modification to increase CDC. An antigen-binding molecule comprising an Fc region comprising modification to increase an Fc-mediated function (e.g. ADCC, ADCP, CDC) induces an increased level of the relevant effector function as compared to an antigen-binding molecule comprising the corresponding unmodified Fc region.
In some embodiments, the antigen-binding molecule of the present invention comprises an Fc region comprising modification to increase affinity for one or more Fc receptors (e.g. FcγRIIa, FcγRIIIa). Modifications increasing affinity for Fc receptors can increase Fc-mediated effector function such as antibody-dependent cellular cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the antigen-binding molecule of the present invention comprises an Fc region comprising modification to reduce affinity for C1q; such modification reducing complement-dependent cytotoxicity (CDC), which can be desirable. In some embodiments, the antigen-binding molecule of the present invention comprises an Fc region comprising modification to increase hexamer formation. Modifications to the Fc region capable of increasing affinity for one or more Fc receptors, reducing affinity for C1q and/or increasing hexamer formation are described e.g. in Saxena and Wu Front Immunol. (2016) 7:580, which is hereby incorporated by reference in its entirety. In some embodiments the antigen-binding molecule of the present invention comprises an Fc region comprising CH2/CH3 comprising one or more of the substitutions shown in Table 1 of Saxena and Wu Front Immunol. (2016) 7:580.
In some embodiments the antigen-binding molecule of the present invention comprises an Fc comprising modification to increase binding to an Fc receptor. In some embodiments the Fc region comprises modification to increase binding to an Fcγ receptor. In some embodiments the Fc region comprises modification to increase binding to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa and FcγRIIIb. In some embodiments the Fc region comprises modification to increase binding to FcγRIIIa. In some embodiments the Fc region comprises modification to increase binding to FcγRIIa. In some embodiments the Fc region comprises modification to increase binding to FcγRIIb. In some embodiments the Fc region comprises modification to increase binding to FcRn. In some embodiments the Fc region comprises modification to increase binding to a complement protein. In some embodiments the Fc region comprises modification to increase or reduce binding to C1q. In some embodiments the Fc region comprises modification to promote hexamerisation of the antigen-binding molecule. In some embodiments the Fc region comprises modification to increase antigen-binding molecule half-life. In some embodiments the Fc region comprises modification to increase co-engagement.
In this specification an “Fcγ receptor” may be from any species, and includes isoforms, fragments, variants (including mutants) or homologues from any species. Similarly, “FcγRI”, “FcγRIIa”, “FcγRIIb”, “FcγRIIc”, “FcγRIIIa” and “FcγRIIIb” refer respectively to FcγRI/FcγRIIa/FcγRIIb/FcγRIIc/FcγRIIIa/FcγRIIIb from any species, and include isoforms, fragments, variants (including mutants) or homologues from any species. Humans have six different classes of Fc γ receptor (mouse orthologues are shown in brackets): FcγRI (mFcγRI), FcγRIIa (mFcγRIII), FcγRIIb (mFcγRIIb), FcγRIIc, FcγRIIIa (mFcγRIV) and FcγRIIIb. Variant Fc γ receptors include e.g. the 158V and 158F polymorphs of human FcγRIIIa, and the 167H and 167R polymorphs of human FcγRIIa.
In some embodiments the antigen-binding molecule of the present invention comprises an Fc region comprising (e.g. comprising one more polypeptides comprising a heavy chain constant region, or a CH2-CH3 region, comprising) one or more (e.g. 1, 2, 3, 4, 5, 6, 7 or 8) of the following: C at the position corresponding to position 242; C at the position corresponding to position 334; A at the position corresponding to position 236; D at the position corresponding to position 239; E at the position corresponding to position 332; L at the position corresponding to position 330; K at the position corresponding to position 345; and G at the position corresponding to position 430.
In some embodiments the antigen-binding molecule of the present invention comprises an Fc region comprising (e.g. comprising one more polypeptides comprising a heavy chain constant region, or a CH2-CH3 region, comprising) one or more (e.g. 1, 2, 3, 4, 5, 6, 7 or 8) of the following substitutions (or corresponding substitutions): L242C, K334C, G236A, S239D, 1332E, A330L, E345K, and E430G.
In some embodiments the antigen-binding molecule comprises an Fc region comprising (e.g. comprising one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) a C at the position corresponding to position 242. In some embodiments the Fc region comprises (e.g. comprises one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) a C at the position corresponding to position 334. In some embodiments the Fc region comprises (e.g. comprises one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) a C at the position corresponding to position 242 and a C at the position corresponding to position 334.
In some embodiments the antigen-binding molecule comprises an Fc region comprising (e.g. comprising one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) an A at the position corresponding to position 236. In some embodiments the Fc region comprises (e.g. comprises one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) a D at the position corresponding to position 239. In some embodiments the Fc region comprises (e.g. comprises one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) an A at the position corresponding to position 236, and a D at the position corresponding to position 239.
In some embodiments the antigen-binding molecule comprises an Fc region comprising (e.g. comprising one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) an E at the position corresponding to position 332. In some embodiments the Fc region comprises (e.g. comprises one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) an A at the position corresponding to position 236, a D at the position corresponding to position 239, and an E at the position corresponding to position 332.
In some embodiments the antigen-binding molecule comprises an Fc region comprising (e.g. comprising one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) an L at the position corresponding to position 330. In some embodiments the Fc region comprises (e.g. comprises one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) an A at the position corresponding to position 236, a D at the position corresponding to position 239, an E at the position corresponding to position 332, and an L at the position corresponding to position 330.
In some embodiments the antigen-binding molecule comprises an Fc region comprising (e.g. comprising one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH3 region, comprising) a K at the position corresponding to position 345. In some embodiments the Fc region comprises (e.g. comprises one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH3 region, comprising) a G at the position corresponding to position 430. In some embodiments the Fc region comprises (e.g. comprises one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) a K at the position corresponding to position 345, and a G at the position corresponding to position 430.
In some embodiments the antigen-binding molecule comprises an Fc region comprising (e.g. comprising one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) a C at the position corresponding to position 242, a C at the position corresponding to position 334, an A at the position corresponding to position 236, and a D at the position corresponding to position 239.
In some embodiments the antigen-binding molecule comprises an Fc region comprising (e.g. comprising one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) a C at the position corresponding to position 242, a C at the position corresponding to position 334, an A at the position corresponding to position 236, a D at the position corresponding to position 239, and an E at the position corresponding to position 332.
In some embodiments the antigen-binding molecule comprises an Fc region comprising (e.g. comprising one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) a C at the position corresponding to position 242, a C at the position corresponding to position 334, an A at the position corresponding to position 236, a D at the position corresponding to position 239, an E at the position corresponding to position 332, and an L at the position corresponding to position 330.
In some embodiments the antigen-binding molecule comprises an Fc region comprising (e.g. comprising one more polypeptides comprising a heavy chain constant region, or a CH2-CH3 region, comprising) a C at the position corresponding to position 242, a C at the position corresponding to position 334, a K at the position corresponding to position 345, and a G at the position corresponding to position 430.
In some embodiments the antigen-binding molecule comprises an Fc region comprising (e.g. comprising one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) the substitution L242C (or an equivalent substitution). In some embodiments the Fc region comprises (e.g. comprises one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) the substitution K334C (or an equivalent substitution). In some embodiments the Fc region comprises (e.g. comprises one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) the substitution L242C (or an equivalent substitution) and the substitution K334C (or an equivalent substitution).
In some embodiments the antigen-binding molecule comprises an Fc region comprising (e.g. comprising one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) the substitution G236A (or an equivalent substitution). In some embodiments the Fc region comprises (e.g. comprises one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) the substitution S239D (or an equivalent substitution). In some embodiments the Fc region comprises (e.g. comprises one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) the substitution G236A (or an equivalent substitution), and the substitution S239D (or an equivalent substitution).
In some embodiments the antigen-binding molecule comprises an Fc region comprising (e.g. comprising one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) the substitution 1332E (or an equivalent substitution). In some embodiments the Fc region comprises (e.g. comprises one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) the substitution G236A (or an equivalent substitution), the substitution S239D (or an equivalent substitution), and the substitution 1332E (or an equivalent substitution).
In some embodiments the antigen-binding molecule comprises an Fc region comprising (e.g. comprising one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) the substitution A330L (or an equivalent substitution). In some embodiments the Fc region comprises (e.g. comprises one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) the substitution G236A (or an equivalent substitution), the substitution S239D (or an equivalent substitution), the substitution 1332E (or an equivalent substitution), and the substitution A330L (or an equivalent substitution).
In some embodiments the antigen-binding molecule comprises an Fc region comprising (e.g. comprising one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH3 region, comprising) the substitution E345K (or an equivalent substitution). In some embodiments the Fc region comprises (e.g. comprises one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH3 region, comprising) the substitution E430G (or an equivalent substitution). In some embodiments the Fc region comprises (e.g. comprises one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) the substitution E345K (or an equivalent substitution), and the substitution E430G (or an equivalent substitution).
In some embodiments the antigen-binding molecule comprises an Fc region comprising (e.g. comprising one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) the substitution L242C (or an equivalent substitution), the substitution K334C (or an equivalent substitution), the substitution G236A (or an equivalent substitution), and the substitution S239D (or an equivalent substitution).
In some embodiments the antigen-binding molecule comprises an Fc region comprising (e.g. comprising one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) the substitution L242C (or an equivalent substitution), the substitution K334C (or an equivalent substitution), the substitution G236A (or an equivalent substitution), the substitution S239D (or an equivalent substitution), and the substitution 1332E (or an equivalent substitution).
In some embodiments the antigen-binding molecule comprises an Fc region comprising (e.g. comprising one more polypeptides comprising a heavy chain constant region, a CH2-CH3 region, or a CH2 region, comprising) the substitution L242C (or an equivalent substitution), the substitution K334C (or an equivalent substitution), the substitution G236A (or an equivalent substitution), the substitution S239D (or an equivalent substitution), the substitution 1332E (or an equivalent substitution), and the substitution A330L (or an equivalent substitution).
In some embodiments the antigen-binding molecule comprises an Fc region comprising (e.g. comprising one more polypeptides comprising a heavy chain constant region, or a CH2-CH3 region, comprising) the substitution L242C (or an equivalent substitution), the substitution K334C (or an equivalent substitution), the substitution E345K (or an equivalent substitution), and the substitution E430G (or an equivalent substitution).
In some embodiments the antigen-binding molecule comprises an Fc region comprising (e.g. comprising one more polypeptides comprising a heavy chain constant region, or a CH2-CH3 region, comprising) one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) of the following: L at the position corresponding to position 243, P at the position corresponding to position 292, L at the position corresponding to position 300, I at the position corresponding to position 305 and L at the position corresponding to position 396; D at the position corresponding to position 239 and E at the position corresponding to position 332; D at the position corresponding to position 239, E at the position corresponding to position 332 and L at the position corresponding to position 330; A at the position corresponding to position 298, A at the position corresponding to position 333 and A at the position corresponding to position 334; Y at the position corresponding to position 234, Q at the position corresponding to position 235, W at the position corresponding to position 236, M at the position corresponding to position 239, D at the position corresponding to position 268, E at the position corresponding to position 270 and A at the position corresponding to position 298; E at the position corresponding to position 270, D at the position corresponding to position 326, M at the position corresponding to position 330 and E at the position corresponding to position 334; A at the position corresponding to position 236, D at the position corresponding to position 239 and E at the position corresponding to position 332; W at the position corresponding to position 326 and S at the position corresponding to position 333; E at the position corresponding to position 267, F at the position corresponding to position 268 and T at the position corresponding to position 324; R at the position corresponding to position 345, G at the position corresponding to position 430 and Y at the position corresponding to position 440; Y at the position corresponding to position 252, T at the position corresponding to position 254 and E at the position corresponding to position 256; and L at the position corresponding to position 428 and S at the position corresponding to position 434.
In some embodiments the antigen-binding molecule comprises an Fc region comprising (e.g. comprising one more polypeptides comprising a heavy chain constant region, or a CH2-CH3 region, comprising) one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) of the following combinations of substitutions (or corresponding substitutions): F243L/R292P/Y300L/V3051/P396L; S239D/1332E; S239D/1332E/A330L; S298A/E333A/K334A; L234Y/L235Q/G236W/S239M/H268D/D270E/S298A; D270E/K326D/A330M/K334E; G236A/S239D/1332E; K326W/E333S; S267E/H268F/S324T; E345R/E430G/S440Y; M252Y/S254T/T256E; and M428L/N434S.
The present invention also provides polypeptide constituents of antigen-binding molecules. The polypeptides may be provided in isolated or substantially purified form.
The antigen-binding molecule of the present invention may be, or may comprise, a complex of polypeptides.
In the present specification where a polypeptide comprises more than one domain or region, it will be appreciated that the plural domains/regions are preferably present in the same polypeptide chain. That is, the polypeptide comprises more than one domain or region is a fusion polypeptide comprising the domains/regions.
In some embodiments a polypeptide according to the present invention comprises, or consists of, a VH as described herein. In some embodiments a polypeptide according to the present invention comprises, or consists of, a VL as described herein.
In some embodiments, the polypeptide additionally comprises one or more antibody heavy chain constant regions (CH). In some embodiments, the polypeptide additionally comprises one or more antibody light chain constant regions (CL).In some embodiments, the polypeptide comprises a CH1, CH2 region and/or a CH3 region of an immunoglobulin (Ig).
In some embodiments the polypeptide comprises one or more regions of an immunoglobulin heavy chain constant sequence. In some embodiments the polypeptide comprises a CH1 region as described herein. In some embodiments the polypeptide comprises a CH1-CH2 hinge region as described herein. In some embodiments the polypeptide comprises a CH2 region as described herein. In some embodiments the polypeptide comprises a CH3 region as described herein. In some embodiments the polypeptide comprises a CH2-CH3 region as described herein.
In some embodiments the polypeptide comprises a CH3 region comprising any one of the following amino acid substitutions/combinations of amino acid substitutions (shown e.g. in Table 1 of Ha et al., Front. Immnol (2016) 7:394, incorporated by reference hereinabove): T366W; T366S, L368A and Y407V T366W and S354C; T366S, L368A, Y407V and Y349C; S364H and F405A; Y349T and T394F; T350V, L351Y, F405A and Y407V; T350V, T366L, K392L and T394W; K360D, D399M and Y407A; E345R, Q347R, T366V and K409V; K409D and K392D; D399K and E356K; K360E and K409W; Q347R, D399V and F405T; K360E, K409W and Y349C; Q347R, D399V, F405T and S354C; K370E and K409W; and E357N, D399V and F405T.
In some embodiments the CH2 and/or CH3 regions of the polypeptide comprise one or more amino acid substitutions for promoting association of the polypeptide with another polypeptide comprising a CH2 and/or CH3 region.
In some embodiments the polypeptide comprises one or more regions of an immunoglobulin light chain constant sequence. In some embodiments the polypeptide comprises a CL region as described herein.
In some embodiments, the polypeptide according to the present invention comprises a structure from N to C-terminus according to one of the following:
Also provided by the present invention are antigen-binding molecules composed of the polypeptides of the present invention. In some embodiments, the antigen-binding molecule of the present invention comprises one of the following combinations of polypeptides:
In some embodiments the antigen-binding molecule comprises more than one of a polypeptide of the combinations shown in (A) to (1) above. By way of example, with reference to (D) above, in some embodiments the antigen-binding molecule comprises two polypeptides comprising the structure VH-CH1-CH2-CH3, and two polypeptides comprising the structure VL-CL.
In some embodiments, the antigen-binding molecule of the present invention comprises one of the following combinations of polypeptides:
Wherein: “VH(anti-HER3)” refers to the VH of an antigen-binding molecule capable of binding to HER3 as described herein, e.g. as defined in one of (1) to (61) above; “VL(anti-HER3)” refers to the VL of an antigen-binding molecule capable of binding to HER3 as described herein, e.g. as defined in one of (62) to (119) above.
In some embodiments the polypeptide comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of one of SEQ ID NOs:187 to 223.
In some embodiments the antigen-binding molecules and polypeptides of the present invention comprise a hinge region. In some embodiments a hinge region is provided between a CH1 region and a CH2 region. In some embodiments a hinge region is provided between a CL region and a CH2 region. In some embodiments the hinge region comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:173.
In some embodiments the antigen-binding molecules and polypeptides of the present invention comprise one or more linker sequences between amino acid sequences. A linker sequence may be provided at one or both ends of one or more of a VH, VL, CH1-CH2 hinge region, CH2 region and a CH3 region of the antigen-binding molecule/polypeptide.
Linker sequences are known to the skilled person, and are described, for example in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, which is hereby incorporated by reference in its entirety. In some embodiments, a linker sequence may be a flexible linker sequence. Flexible linker sequences allow for relative movement of the amino acid sequences which are linked by the linker sequence. Flexible linkers are known to the skilled person, and several are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences often comprise high proportions of glycine and/or serine residues.
In some embodiments, the linker sequence comprises at least one glycine residue and/or at least one serine residue. In some embodiments the linker sequence consists of glycine and serine residues. In some embodiments, the linker sequence has a length of 1-2, 1-3, 1-4, 1-5 or 1-10 amino acids.
The antigen-binding molecules and polypeptides of the present invention may additionally comprise further amino acids or sequences of amino acids. For example, the antigen-binding molecules and polypeptides may comprise amino acid sequence(s) to facilitate expression, folding, trafficking, processing, purification or detection of the antigen-binding molecule/polypeptide. For example, the antigen-binding molecule/polypeptide may comprise a sequence encoding a His, (e.g. 6XHis), Myc, GST, MBP, FLAG, HA, E, or Biotin tag, optionally at the N- or C-terminus of the antigen-binding molecule/polypeptide. In some embodiments the antigen-binding molecule/polypeptide comprises a detectable moiety, e.g. a fluorescent, lunminescent, immuno-detectable, radio, chemical, nucleic acid or enzymatic label.
The antigen-binding molecules and polypeptides of the present invention may additionally comprise a signal peptide (also known as a leader sequence or signal sequence). Signal peptides normally consist of a sequence of 5-30 hydrophobic amino acids, which form a single alpha helix. Secreted proteins and proteins expressed at the cell surface often comprise signal peptides.
The signal peptide may be present at the N-terminus of the antigen-binding molecule/polypeptide, and may be present in the newly synthesised antigen-binding molecule/polypeptide. The signal peptide provides for efficient trafficking and secretion of the antigen-binding molecule/polypeptide. Signal peptides are often removed by cleavage, and thus are not comprised in the mature antigen-binding molecule/polypeptide secreted from the cell expressing the antigen-binding molecule/polypeptide.
Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and/or can be identified/predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172-2176).
In some embodiments, the signal peptide of the antigen-binding molecule/polypeptide of the present invention comprises, or consists of, an amino acid sequence having at least 80%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of one of SEQ ID NOs:178 to 186.
In some embodiments the antigen-binding molecules of the present invention additionally comprise a detectable moiety.
In some embodiments the antigen-binding molecule comprises a detectable moiety, e.g. a fluorescent label, phosphorescent label, luminescent label, immuno-detectable label (e.g. an epitope tag), radiolabel, chemical, nucleic acid or enzymatic label. The antigen-binding molecule may be covalently or non-covalently labelled with the detectable moiety.
Fluorescent labels include e.g. fluorescein, rhodamine, allophycocyanin, eosine and NDB, green fluorescent protein (GFP) chelates of rare earths such as europium (Eu), terbium (Tb) and samarium (Sm), tetramethyl rhodamine, Texas Red, 4-methyl umbelliferone, 7-amino-4-methyl coumarin, Cy3, and Cy5. Radiolabels include radioisotopes such as Iodine123, Iodine125, Iodine126, Iodine131, Iodine133, Bromine77, Technetium99m, Indium111, Indium113m, Gallium67, Gallium68, Ruthenium95, Ruthenium97, Ruthenium103, Ruthenium105, Mercury207, Mercury203, Rhenium99m, Rhenium101, Rhenium105, Scandium47, Tellurium121m, Tellurium122m, Tellurium125m, Thulium165, Thuliuml167, Thulium168, Copper67, Fluorine18, Yttrium90, Palladium100, Bismuth217 and Antimony211. Luminescent labels include as radioluminescent, chemiluminescent (e.g. acridinium ester, luminol, isoluminol) and bioluminescent labels. Immuno-detectable labels include haptens, peptides/polypeptides, antibodies, receptors and ligands such as biotin, avidin, streptavidin or digoxigenin. Nucleic acid labels include aptamers. Enzymatic labels include e.g. peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase and luciferase.
In some embodiments the antigen-binding molecules of the present invention are conjugated to a chemical moiety. The chemical moiety may be a moiety for providing a therapeutic effect. Antibody-drug conjugates are reviewed e.g. in Parslow et al., Biomedicines. 2016 September; 4(3):14. In some embodiments, the chemical moiety may be a drug moiety (e.g. a cytotoxic agent). In some embodiments, the drug moiety may be a chemotherapeutic agent. In some embodiments, the drug moiety is selected from calicheamicin, DM1, DM4, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), SN-38, doxorubicin, duocarmycin, D6.5 and PBD.
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments the antigen-binding molecule comprises, or consists of:
In some embodiments, the antigen-binding molecule is produced by the cell line deposited 7 May 2021 as ATCC Patent Deposit Number PTA-127062, e.g. as described in GB 2108449.6, which is hereby incorporated by reference in its entirety.
The antigen-binding molecules described herein may be characterised by reference to certain functional properties. In some embodiments, the antigen-binding molecule described herein may possess one or more of the following properties:
The antigen-binding molecules described herein preferably display specific binding to HER3. As used herein, “specific binding” refers to binding which is selective for the antigen, and which can be discriminated from non-specific binding to non-target antigen. An antigen-binding molecule that specifically binds to a target molecule preferably binds the target with greater affinity, and/or with greater duration than it binds to other, non-target molecules.
The ability of a given polypeptide to bind specifically to a given molecule can be determined by analysis according to methods known in the art, such as by ELISA, Surface Plasmon Resonance (SPR; see e.g. Hearty et al., Methods Mol Biol (2012) 907:411-442), Bio-Layer Interferometry (see e.g. Lad et al., (2015) J Biomol Screen 20(4): 498-507), flow cytometry, or by a radiolabeled antigen-binding assay (RIA) enzyme-linked immunosorbent assay. Through such analysis binding to a given molecule can be measured and quantified. In some embodiments, the binding may be the response detected in a given assay.
In some embodiments, the extent of binding of the antigen-binding molecule to an non-target molecule is less than about 10% of the binding of the antibody to the target molecule as measured, e.g. by ELISA, SPR, Bio-Layer Interferometry or by RIA. Alternatively, binding specificity may be reflected in terms of binding affinity where the antigen-binding molecule binds with a dissociation constant (KD) that is at least 0.1 order of magnitude (i.e. 0.1×10n, where n is an integer representing the order of magnitude) greater than the KD of the antigen-binding molecule towards a non-target molecule. This may optionally be one of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0.
In some embodiments, the antigen-binding molecule displays binding to human HER3, mouse HER3, rat HER3 and/or cynomolgus macaque (Macaca fascicularis) HER3. That is, in some embodiments the antigen-binding molecule is cross-reactive for human HER3, mouse HER3, rat HER3 and/or cynomolgus macaque HER3. In some embodiments the antigen-binding molecule of the present invention displays cross-reactivity with HER3 of a non-human primate. Cross-reactivity to HER3 in model species allows in vivo exploration of efficacy in syngeneic models without relying on surrogate molecules.
In some embodiments the antigen-binding molecule binds to human HER3, mouse HER3, rat HER3 and/or cynomolgus macaque HER3; and does not bind to HER2 and/or EGFR (e.g. human HER2 and/or human EGFR).
In some embodiments, the antigen-binding molecule does not display specific binding to EGFR (e.g. human EGFR). In some embodiments, the antigen-binding molecule does not display specific binding to HER2 (e.g. human HER2). In some embodiments, the antigen-binding molecule does not display specific binding to (i.e. does not cross-react with) a member of the EGFR family of proteins other than HER3. In some embodiments, the antigen-binding molecule does not display specific binding to EGFR, HER2 and/or HER4.
In some embodiments, the antigen-binding molecule of the invention binds to HER3 (e.g. human HER3) with a KD of 10 μM or less, preferably one of ≤5 μM, ≤2 μM, ≤1 μM, ≤500 nM, ≤400 nM, ≤300 nM, ≤200 nM, ≤100 nM, ≤95 nM, ≤90 nM, ≤85 nM, ≤80 nM, ≤75 nM, ≤70 nM, ≤65 nM, ≤60 nM, ≤55 nM, ≤50 nM, ≤45 nM, ≤40 nM, ≤35 nM, ≤30 nM, ≤25 nM, ≤20 nM, ≤15 nM, ≤12.5 nM, ≤10 nM, ≤9 nM, ≤8 nM, ≤7 nM, ≤6 nM, ≤5 nM, ≤4 nM ≤3 nM, ≤2 nM, ≤1 nM, ≤900 μM, ≤800 μM, ≤700 μM, ≤600 μM, ≤500 μM, ≤400 μM, ≤300 μM, ≤200 μM, ≤100 μM, ≤90 μM, ≤80 μM, ≤70 μM, ≤60 μM, ≤50 μM, ≤40 μM, ≤30 μM, ≤20 μM, ≤10 μM, ≤9 μM, ≤8 μM, ≤7 μM, ≤6 μM, ≤5 μM, ≤4 μM, ≤3 μM, ≤2 μM, ≤1 μM.
The antigen-binding molecules of the present invention may bind to a particular region of interest of HER3. The antigen-binding region of an antigen-binding molecule according to the present invention may bind to a linear epitope of HER3, consisting of a contiguous sequence of amino acids (i.e. an amino acid primary sequence). In some embodiments, the antigen-binding molecule may bind to a conformational epitope of HER3, consisting of a discontinuous sequence of amino acids of the amino acid sequence.
In some embodiments, the antigen-binding molecule of the present invention binds to HER3. In some embodiments, the antigen-binding molecule binds to the extracellular region of HER3 (e.g. the region shown in SEQ ID NO:9). In some embodiments, the antigen-binding molecule binds to subdomain II of the extracellular region of HER3 (e.g. the region shown in SEQ ID NO:16).
In some embodiments, the antigen-binding molecule binds to the region of HER3 shown in SEQ ID NO:229. In some embodiments the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 shown in SEQ ID NO:229. In some embodiments, the antigen-binding molecule binds to the regions of HER3 shown in SEQ ID NOs:230 and 231. In some embodiments the antigen-binding molecule contacts one or more amino acid residues of the regions of HER3 shown in SEQ ID NOs:230 and 231. In some embodiments, the antigen-binding molecule binds to the region of HER3 shown in SEQ ID NO:230. In some embodiments the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 shown in SEQ ID NO:230. In some embodiments, the antigen-binding molecule binds to the region of HER3 shown in SEQ ID NO:231. In some embodiments the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 shown in SEQ ID NO:231. In some embodiments, the antigen-binding molecule binds to the region of HER3 shown in SEQ ID NO:23. In some embodiments the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 shown in SEQ ID NO:23. In some embodiments, the antigen-binding molecule binds to the region of HER3 shown in SEQ ID NO:21. In some embodiments the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 shown in SEQ ID NO:21. In some embodiments the antigen-binding molecule binds to the region of HER3 shown in SEQ ID NO:19. In some embodiments the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 shown in SEQ ID NO:19. In some embodiments, the antigen-binding molecule binds to the region of HER3 shown in SEQ ID NO:22. In some embodiments the antigen-binding molecule contacts one or more amino acid residues of the region of HER3 shown in SEQ ID NO:22.
In some embodiments, the antigen-binding molecule of the present invention is capable of binding to a polypeptide comprising, or consisting of, the amino acid sequence of one of SEQ ID NOs:1, 3, 4, 6 or 8. In some embodiments, the antigen-binding molecule is capable of binding to a polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:9. In some embodiments, the antigen-binding molecule is capable of binding to a polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:16. In some embodiments, the antigen-binding molecule is capable of binding to a peptide/polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:229. In some embodiments, the antigen-binding molecule is capable of binding to a peptide/polypeptide comprising, or consisting of, the amino acid sequences of SEQ ID NO:230 and 231. In some embodiments, the antigen-binding molecule is capable of binding to a peptide/polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:230. In some embodiments, the antigen-binding molecule is capable of binding to a peptide/polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:231. In some embodiments, the antigen-binding molecule is capable of binding to a peptide/polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:23. In some embodiments, the antigen-binding molecule is capable of binding to a peptide/polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:21. In some embodiments, the antigen-binding molecule is capable of binding to a peptide/polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:19. In some embodiments, the antigen-binding molecule is capable of binding to a peptide/polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:22.
In some embodiments, the antigen-binding molecule does not bind to the region of HER3 corresponding to positions 260 to 279 of SEQ ID NO:1. In some embodiments the antigen-binding molecule does not contact an amino acid residue of the region of HER3 corresponding to positions 260 to 279 of SEQ ID NO:1. In some embodiments, the antigen-binding molecule does not bind to the region of HER3 shown in SEQ ID NO:23. In some embodiments the antigen-binding molecule does not contact an amino acid residue of the region of HER3 shown in SEQ ID NO:23. In some embodiments, the antigen-binding molecule is not capable of binding to a peptide consisting of the amino acid sequence corresponding to positions 260 to 279 of SEQ ID NO:1. In some embodiments, the antigen-binding molecule is not capable of binding to a peptide consisting of the amino acid sequence of SEQ ID NO:23.
As used herein, a “peptide” refers to a chain of two or more amino acid monomers linked by peptide bonds. A peptide typically has a length in the region of about 2 to 50 amino acids. A “polypeptide” is a polymer chain of two or more peptides. Polypeptides typically have a length greater than about 50 amino acids.
The ability of an antigen-binding molecule to bind to a given peptide/polypeptide can be analysed by methods well known to the skilled person, including analysis by ELISA, immunoblot (e.g. western blot), immunoprecipitation, surface plasmon resonance and biolayer interferometry.
Ligand binding to HER3 promotes conformational changes that enables HER3 to homo- or heterodimerise, resulting in activation of downstream pathways. HER3 demonstrates ‘closed’ and ‘open’ conformations. By closed conformation it is meant that HER3 is in a tethered conformation and is unavailable for receptor homo- or heterodimerisation. By open conformation it is meant that HER3 is in an extended conformation and is available for receptor homo- or heterodimerisation.
In some embodiments the antigen-binding molecule is capable of binding to HER3 when HER3 is in the open conformation. In some embodiments the antigen-binding molecule is capable of binding to HER3 when HER3 is in the closed conformation. In some embodiments the antigen-binding molecule is capable of binding to HER3 when HER3 is in the open and/or closed conformation. In some embodiments the antigen-binding molecule is capable of binding to the HER3 ectodomain when HER3 is in the open and/or closed conformation. In some embodiments the antigen-binding molecule is capable of binding to the HER3 dimerisation arm when HER3 is in the open and/or closed conformation. Binding to the dimerisation arm enables an antigen-binding molecule to prevent interaction between HER3 and an interaction partner for HER3, e.g. as described herein.
In some embodiments the antigen-binding molecule is capable of binding to HER3 in the presence and/or absence of a ligand for HER3. In some embodiments the antigen-binding molecule is capable of binding to HER3 independently of a ligand for HER3. In some embodiments the ligand is NRG, NRG-1 and/or NRG-2. HER3 is activated by ligand binding to its extracellular domain which promotes conformational changes that enables HER3 to homo- or heterodimerise. Binding of an antigen-binding molecule to HER3 independently of ligand binding allows the antigen-binding molecule to inhibit the action of HER3 in both ligand-absent and ligand-present conformational states. In some embodiments the antigen-binding molecule does not compete with ligand binding to HER3. In some embodiments the antigen-binding molecule does not bind to HER3 at the ligand binding site.
In some embodiments, the antigen-binding molecule binds to HER3 similarly well in the presence or absence of ligand for HER3 (i.e. irrespective of whether HER3 is provided in the ligand-bound or unbound form).
In some embodiments, the antigen-binding molecule binds to HER3 in the presence of a ligand for HER3 with an affinity which is similar to the affinity of binding of the antigen-binding molecule to HER3 in the absence of ligand for HER3. Example 8.10 and
Herein, a binding affinity which is ‘similar’ to a reference binding affinity means a binding affinity which is within 50%, e.g. within one of 40%, 45%, 30%, 25%, 20% 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the reference binding affinity, as determined under comparable conditions.
In some embodiments, the antigen-binding molecule binds to HER3 in the presence of a ligand for HER3 (e.g. NRG1 or NRG2) with a KD which is within 50%, e.g. within one of 40%, 45%, 30%, 25%, 20% 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the KD Of the antigen-binding molecule for binding to HER3 in the absence of the ligand (as determined under comparable conditions).
In some embodiments, the antigen-binding molecule binds to HER3 in the presence of a ligand for HER3 (e.g. NRG1 or NRG2) with a Kon which is within 50%, e.g. within one of 40%, 45%, 30%, 25%, 20% 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the Kon of the antigen-binding molecule for binding to HER3 in the absence of the ligand (as determined under comparable conditions).
In some embodiments, the antigen-binding molecule binds to HER3 in the presence of a ligand for HER3 (e.g. NRG1 or NRG2) with a Koff which is within 50%, e.g. within one of 40%, 45%, 30%, 25%, 20% 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the Koff of the antigen-binding molecule for binding to HER3 in the absence of the ligand (as determined under comparable conditions).
In some embodiments the antigen-binding molecule is capable of binding the same region of HER3, or an overlapping region of HER3, to the region of HER3 which is bound by an antibody comprising the VH and VL sequences of one of clones 10D1, 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c89, 10D1_c90, 10D1_c91, 10D1_c92, 10D1_c93, 10A6, 4-35-B2 or 4-35-B4. In some embodiments the antigen-binding molecule is capable of binding the same region of HER3, or an overlapping region of HER3, to the region of HER3 which is bound by an antibody comprising the VH and VL sequences of one of clones 10D1_c89, 10D1_c90 or 10D1_c91. In some embodiments the antigen-binding molecule is capable of binding the same region of HER3, or an overlapping region of HER3, to the region of HER3 which is bound by an antibody comprising the VH and VL sequences of clone 10D1_c89.
The region of a peptide/polypeptide to which an antibody binds can be determined by the skilled person using various methods well known in the art, including X-ray co-crystallography analysis of antibody-antigen complexes, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange analysis by mass spectrometry, phage display, competition ELISA and proteolysis-based ‘protection’ methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21(3):145-156, which is hereby incorporated by reference in its entirety. Such methods can also be used to determine whether an antigen-binding molecule is capable of binding to proteins in different conformations.
In some embodiments the antigen-binding molecule of the present invention does not bind to HER3 in the same region of HER3, or an overlapping region of HER3, as an antibody comprising the VH and VL sequences of anti-HER3 antibody clone MM-121 (described e.g. in Schoeberl et al., Sci. Signal. (2009) 2(77): ra31) and/or LJM-716 (described e.g. Garner et al., Cancer Res (2013) 73: 6024-6035). In some embodiments the antigen-binding molecule of the present invention does not display competition with an antibody comprising the VH and VL sequences of anti-HER3 antibody clone MM-121 and/or LJM-716 for binding to HER3, e.g. as determined by SPR analysis.
In some embodiments the antigen-binding molecule of the present invention binds to HER3 in a region which is accessible to an antigen-binding molecule (i.e., an extracellular antigen-binding molecule) when HER3 is expressed at the cell surface (i.e. in or at the cell membrane). In some embodiments the antigen-binding molecule is capable of binding to HER3 expressed at the cell surface of a cell expressing HER3. In some embodiments the antigen-binding molecule is capable of binding to HER3-expressing cells (e.g. HER3+ cells, e.g. HER3+ cancer cells).
The ability of an antigen-binding molecule to bind to a given cell type can be analysed by contacting cells with the antigen-binding molecule, and detecting antigen-binding molecule bound to the cells, e.g. after a washing step to remove unbound antigen-binding molecule. The ability of an antigen-binding molecule to bind to immune cell surface molecule-expressing cells and/or cancer cell antigen-expressing cells can be analysed by methods such as flow cytometry and immunofluorescence microscopy.
The antigen-binding molecule of the present invention may be an antagonist of HER3. In some embodiments, the antigen-binding molecule is capable of inhibiting a function or process (e.g. interaction, signalling or other activity) mediated by HER3 and/or a binding partner for HER3 (e.g. HER3 (i.e. in the case of homodimerisation), HER2, EGFR, HER4, HGFR, IGF1R and/or cMet). Herein, ‘inhibition’ refers to a reduction, decrease or lessening relative to a control condition.
In some embodiments the antigen-binding molecule of the present invention is capable of inhibiting interaction between HER3 and an interaction partner for HER3. An interaction partner for HER3 may be expressed by the same cell as the HER3. An interaction partner or HER3 may be expressed at the cell surface (i.e. in or at the cell membrane). In some embodiments an interaction partner for HER3 may be a member of the EGFR family of proteins, e.g. HER3, HER2, EGFR, HER4, HGFR, IGF1R and/or cMet. In some embodiments an interaction partner for HER3 may be IGF1R and/or cMet. Interaction between HER3 and an interaction partner for HER3 may result in the formation of a polypeptide complex. Interaction between HER3 and an interaction partner for HER3 to form a polypeptide complex may be referred to as multimerisation. Where multimerisation is between polypeptide monomers multimerisation may be referred to as dimerisation.
In some embodiments the antigen-binding molecule is capable of inhibiting interaction between HER3 monomers. In some embodiments the antigen-binding molecule is capable of inhibiting interaction between HER3 and HER2. In some embodiments the antigen-binding molecule is capable of inhibiting interaction between HER3 and EGFR. In some embodiments the antigen-binding molecule is capable of inhibiting interaction between HER3 and HER4. In some embodiments the antigen-binding molecule is capable of inhibiting interaction between HER3 and HGFR. In some embodiments the antigen-binding molecule is capable of inhibiting interaction between HER3 and IGF1R. In some embodiments the antigen-binding molecule is capable of inhibiting interaction between HER3 and cMet.
Inhibition of interaction may be achieved by binding of the antigen-binding molecule to a region of HER3 required for interaction between HER3 and an interaction partner for HER3 (e.g. the dimerisation loop of HER3 shown in SEQ ID NO:19). In some embodiments the antigen-binding molecule contacts one or more residues of HER3 necessary for interaction between HER3 and an interaction partner for HER3; in this way the antigen-binding molecule makes the region unavailable, thereby inhibiting interaction. In some embodiments the antigen-binding molecule binds to HER3 in a manner which inhibits/prevents interaction between HER3 and an interaction partner for HER3. In some embodiments the antigen-binding molecule inhibits/prevents access of the interaction partner for HER3 to the region of HER3 required for interaction between HER3 and the interaction partner for HER3; this may be achieved in cases even where the antigen-binding molecule does not contact the region of HER3 required for interaction between HER3 and the interaction partner for HER3, e.g. through steric inhibition of access of the interaction partner for HER3 to the region of HER3 required for interaction between HER3 and the interaction partner.
In some embodiments the antigen-binding molecule is capable of inhibiting homodimerisation of HER3 monomers. In some embodiments the antigen-binding molecule is capable of inhibiting dimerisation between HER3 and HER2. In some embodiments the antigen-binding molecule is capable of inhibiting dimerisation between HER3 and EGFR. In some embodiments the antigen-binding molecule is capable of inhibiting dimerisation between HER3 and HER4. In some embodiments the antigen-binding molecule is capable of inhibiting dimerisation between HER3 and HGFR. In some embodiments the antigen-binding molecule is capable of inhibiting dimerisation between HER3 and IGF1R. In some embodiments the antigen-binding molecule is capable of inhibiting dimerisation between HER3 and cMet.
The ability of an antigen-binding molecule to inhibit interaction between two factors can be determined for example by analysis of interaction in the presence of, or following incubation of one or both of the interaction partners with, the antibody/fragment. Assays for determining whether a given antigen-binding molecule is capable of inhibiting interaction between two interaction partners include competition ELISA assays and analysis by SPR. In some embodiments the antigen-binding molecule is a competitive inhibitor of interaction between HER3 and an interaction partner for HER3.
In some embodiments, the antigen-binding molecule of the present invention is capable of inhibiting interaction between HER3 and an interaction partner for HER3 (e.g. HER3, HER2, EGFR, HER4, HGFR, IGF1R and/or cMet) to less than less than 1 times, e.g. ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level of interaction between HER3 and the interaction partner for HER3 in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule), in a suitable assay.
The ability of an antigen-binding molecule to inhibit interaction between interaction partners can also be determined by analysis of the downstream functional consequences of such interaction. For example, downstream functional consequences of interaction between HER3 and interaction partners for HER3 include P13K/AKT/mTOR and/or MAPK signalling. For example, the ability of an antigen-binding molecule to inhibit interaction of HER3 and an interaction partner for HER3 may be determined by analysis of P13K/AKT/mTOR and/or MAPK signalling following treatment with NRG in the presence of the antigen-binding molecule. P13K/AKT/mTOR and/or MAPK signalling can be detected and quantified e.g. using antibodies capable of detecting phosphorylated members of the signal transduction pathways.
The ability of an antigen-binding molecule to inhibit interaction of HER3 and an interaction partner for HER3 can also be determined by analysing proliferation of cells expressing HER3 following treatment with NRG in the presence of the antigen-binding molecule. Cell proliferation can be determined e.g. by detecting changes in number of cells over time, or by in vitro analysis of incorporation of 3H-thymidine or by CFSE dilution assay, e.g. as described in Fulcher and Wong, Immunol Cell Biol (1999) 77(6): 559-564, hereby incorporated by reference in entirety.
In some embodiments, the antigen-binding molecule of the present invention is capable of inhibiting proliferation of cells harbouring mutation to BRAF V600, e.g. cells comprising the BRAF V600E or V600K mutation (see Example 10).
In some embodiments the antigen-binding molecule inhibits HER3-mediated signalling. HER3-mediated signalling can be analysed e.g. using an assay of a correlate of HER3-mediated signalling, e.g. cell proliferation, and/or phosphorylation of one or more signal transduction molecules of the P13K/AKT/mTOR and/or MAPK signal transduction pathways.
In some embodiments, the antigen-binding molecule of the present invention is capable of inhibiting P13K/AKT/mTOR and/or MAPK signalling by HER3-expressing cells. The level of P13K/AKT/mTOR and/or MAPK signalling may be analysed by detection and quantification of the level of phosphorylation of one or more of the components of the P13K/AKT/mTOR and/or MAPK pathways, e.g. following stimulation with NRG (see Example 4.3).
In some embodiments, the antigen-binding molecule of the present invention is capable of inhibiting proliferation of HER3-expressing cells, e.g. in response to stimulation with NRG. In some embodiments, the antigen-binding molecule of the present invention is capable of inhibiting proliferation of HER3-expressing cells to less than less than 1 times, e.g. ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level of proliferation of HER3-expressing cells in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule), in a suitable assay. In some embodiments, the antigen-binding molecule of the present invention is capable of inhibiting P13K/AKT/mTOR and/or MAPK signalling by HER3-expressing cells to less than less than 1 times, e.g. ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level of signalling by HER3-expressing cells in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule), in a suitable assay.
HER3-mediated signalling can be investigated in vitro, e.g. as described in Example 8.9, or in vivo, e.g. as described in Example 11.
ADCC activity can be analysed e.g. according to the methods described in Yamashita et al., Scientific Reports (2016) 6:19772 (hereby incorporated by reference in its entirety), or by 51Cr release assay as described e.g. in Jedema et al., Blood (2004) 103: 2677-82 (hereby incorporated by reference in its entirety). ADCC activity can also be analysed using the Pierce LDH Cytotoxicity Assay Kit, in accordance with the manufacturer's instructions (as described in Example 5 herein).
ADCP can be analysed e.g. according to the method described in Kamen et al., J Immunol (2017) 198 (1 Supplement) 157.17 (hereby incorporated by reference in its entirety).
The ability to induce CDC can be analysed e.g. using a C1q binding assay, e.g. as described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466 (hereby incorporated by reference in its entirety).
Thermostability of antigen-binding molecules can be analysed by methods well known to the skilled person, including Differential Scanning Fuorimetry and Differential Scanning Calorimetry (DSC), which are described e.g. in He et al., J Pharm Sci. (2010) which is hereby incorporated by reference in its entirety. Thermostability may be reflected in terms of a melting temperature (Tm), unfolding temperature or disassembly temperature (expressed e.g. in ° C. or F°).
In some embodiments, an antigen-binding molecule comprising an Fc region as described herein binds to an activatory Fcγ receptor (e.g. hFcγRIIa (e.g. hFcγRIIa167H, hFcγRIIa167R), hFcγRIIIa (e.g. hFcγRIIIa158V, hFcγRIIIa158F), mFcγRIV, mFcγRIII) with an affinity of binding which is greater than 1 times, e.g. greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or greater than 20 times the affinity of binding to the activatory Fcγ receptor by an equivalent antigen-binding molecule having an Fc region comprised of CH2-CH3 having the amino acid sequence of SEQ ID NO:174-175. In some embodiments the KD of the antigen-binding molecule comprising an Fc region described herein for binding to the activatory Fcγ receptor is less than 1 times, e.g. less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06 or less than 0.05 times the KD of an equivalent antigen-binding molecule having an Fc region comprised of CH2-CH3 having the amino acid sequence of SEQ ID NO:174-175 for the activatory Fcγ receptor. In some embodiments, the antigen-binding molecule comprising an Fc region as described herein binds to an activatory Fcγ receptor (e.g. hFcγRIIa (e.g. hFcγRIIa167H, hFcγRIIa167R), hFcγRIIIa (e.g. hFcγRIIIa158V, hFcγRIIIa158F), mFcγRIV, mFcγRIII) with a KD of 1000 nM or less, preferably one of ≤500 nM, ≤100 nM, ≤75 nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, ≤15 nM, ≤12.5 nM, ≤10 nM, ≤9 nM, ≤8 nM, ≤7 nM, ≤6 nM, ≤5 nM, ≤4 nM ≤3 nM, ≤2 nM or ≤1 nM.
In some embodiments, an antigen-binding molecule comprising an Fc region as described herein binds to an FcRn (e.g. hFcRn, mFcRn) with an affinity of binding which is greater than 1 times, e.g. greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or greater than 20 times the affinity of binding to the FcRn by an equivalent antigen-binding molecule having an Fc region comprised of CH2-CH3 having the amino acid sequence of SEQ ID NO:174-175. In some embodiments the KD of the antigen-binding molecule comprising an Fc region described herein for binding to the FcRn is less than 1 times, e.g. less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06 or less than 0.05 times the KD of an equivalent antigen-binding molecule having an Fc region comprised of CH2-CH3 having the amino acid sequence of SEQ ID NO:174-175 for the FcRn.
In some embodiments, the antigen-binding molecule comprising an Fc region as described herein binds to an FcRn (e.g. hFcRn, mFcRn) with a KD of 1000 nM or less, preferably one of ≤500 nM, ≤100 nM, ≤75 nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, ≤15 nM, ≤12.5 nM, ≤10 nM, ≤9 nM, ≤8 nM, ≤7 nM, ≤6 nM, ≤5 nM, ≤4 nM ≤3 nM, ≤2 nM or ≤1 nM.
In some embodiments, an antigen-binding molecule comprising an Fc region as described herein binds to an inhibitory Fcγ receptor (e.g. hFcγRIIb mFcγRIIb) with an affinity of binding which is less than 1 times, e.g. less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or less than 0.1 times the affinity of binding to the inhibitory Fcγ receptor by an equivalent antigen-binding molecule having an Fc region comprised of CH2-CH3 having the amino acid sequence of SEQ ID NO:174-175. In some embodiments the KD of the antigen-binding molecule comprising an Fc region described herein for binding to the inhibitory Fcγ receptor is greater than 1 times, e.g. greater than 2, 3, 4, 5, 6, 7, 8, 9 or greater than 10 times the KD of an equivalent antigen-binding molecule having an Fc region comprised of CH2-CH3 having the amino acid sequence of SEQ ID NO:174-175 for the inhibitory Fcγ receptor.
In some embodiments, the antigen-binding molecule comprising an Fc region as described herein binds to an inhibitory Fcγ receptor (e.g. hFcγRIIb mFcγRIIb) with a KD 1 nM or greater, preferably one of ≥5 nM, ≥10 nM, ≥50 nM, ≥100 nM, ≥500 nM, ≥1000 nM, ≥2000 nM, ≥3000 nM, ≥4000 nM or ≥5000 nM.
In some embodiments the selectivity of binding for an activatory Fcγ receptor (e.g. hFcγRIIa) relative to an inhibitory Fcγ receptor (e.g. hFcγRIIb) for an antigen-binding molecule comprising an Fc region as described herein is greater than 1 times, e.g. greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or greater than 20 times selectivity of binding displayed by an equivalent antigen-binding molecule having an Fc region comprised of CH2-CH3 having the amino acid sequence of SEQ ID NO:174-175.
In some embodiments, an antigen-binding molecule comprising an Fc region as described herein displays ADCC which is greater than 1 times, e.g. greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or greater than 20 times the ADCC displayed by an equivalent antigen-binding molecule having an Fc region comprised of CH2-CH3 having the amino acid sequence of SEQ ID NO:174-175.
In some embodiments, the EC50 (ng/ml) determined for an antigen-binding molecule comprising an Fc region as described herein in an assay of ADCC activity less than 1 times, e.g. less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or less than 0.1 times the EC50 (ng/ml) determined for an equivalent antigen-binding molecule having an Fc region comprised of CH2-CH3 having the amino acid sequence of SEQ ID NO:174-175.
In some embodiments, the EC50 (ng/ml) for an antigen-binding molecule comprising an Fc region as described herein in an assay of ADCC activity is 500 ng/ml or less, preferably one of ≤400 ng/ml, ≤300 ng/ml, ≤200 ng/ml, ≤100 ng/ml, ≤90 ng/ml, ≤80 ng/ml, ≤70 ng/ml, ≤60 ng/ml, ≤50 ng/ml, ≤40 ng/ml, ≤30 ng/ml, ≤20 ng/ml, or ≤10 ng/ml.
In some embodiments, an antigen-binding molecule comprising an Fc region as described herein may have a melting temperature, unfolding temperature or disassembly temperature which is which is ≥0.75 times and ≤1.25 times, e.g. ≥0.8 times and ≤1.2 times, ≥0.85 times and ≤1.15 times, ≥0.9 times and ≤1.1 times, ≥0.91 times and ≤1.09 times, ≥0.92 times and ≤1.08 times, ≥0.93 times and ≤1.07 times, ≥0.94 times and ≤1.06 times, ≥0.95 times and ≤1.05 times, ≥0.96 times and ≤1.04 times, ≥0.97 times and ≤1.03 times, ≥0.98 times and ≤1.02 times, or ≥0.99 times and ≤1.01 times the melting temperature, unfolding temperature or disassembly temperature of an equivalent antigen-binding molecule having an Fc region comprised of CH2-CH3 having the amino acid sequence of SEQ ID NO:174-175.
In some embodiments, the antigen-binding molecule of the present invention is capable of increasing killing of HER3-expressing cells. Killing of HER3-expressing cells may be increased through an effector function of the antigen-binding molecule. In embodiments wherein antigen-binding molecule comprises an Fc region the antigen-binding molecule may increasing killing of HER3-expressing cells through one or more of complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP).
An antigen-binding molecule which is capable of increasing killing of HER3-expressing cells can be identified by observation of an increased level of killing of HER3-expressing cells in the presence of—or following incubation of the HER3-expressing cells with—the antigen-binding molecule, as compared to the level of cell killing detected in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule), in an appropriate assay. Assays of CDC, ADCC and ADCP are well known the skilled person. The level of killing of HER3-expressing cells can also be determined by measuring the number/proportion of viable and/or non-viable HER3-expressing cells following exposure to different treatment conditions.
In some embodiments, the antigen-binding molecule of the present invention is capable of increasing killing of HER3-expressing cells (e.g. HER3-expressing cancer cells) to more than 1 times, e.g. ≥1.01 times, ≥1.02 times, ≥1.03 times, ≥1.04 times, ≥1.05 times, ≥1.1 times, ≥1.2 times, ≥1.3 times, ≥1.4 times, ≥1.5 times, ≥1.6 times, ≥1.7 times, ≥1.8 times, ≥1.9 times, ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥6 times, ≥7 times, ≥8 times, ≥9 times or ≥10 times the level of killing observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule).
In some embodiments, the antigen-binding molecule of the present invention is capable of reducing the number of HER3-expressing cells (e.g. HER3-expressing cancer cells) to less than less than 1 times, e.g. ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the number of HER3-expressing cells (e.g. HER3-expressing cancer cells) detected following incubation in the absence of the antigen-binding molecule (or following incubation in the presence of an appropriate control antigen-binding molecule), in a comparable assay.
In some embodiments, the antigen-binding molecule of the present invention inhibits the development and/or progression of cancer in vivo.
In some embodiments the antigen-binding molecule causes an increase in the killing of cancer cells, e.g. by effector immune cells. In some embodiments the antigen-binding molecule causes a reduction in the number of cancer cells in vivo, e.g. as compared to an appropriate control condition. In some embodiments the antigen-binding molecule inhibits tumor growth, e.g. as determined by measuring tumor size/volume over time.
The antigen-binding molecule of the present invention may be analysed for the ability to inhibit development and/or progression of cancer in an appropriate in vivo model, e.g. cell line-derived xenograft model. The cell line-derived xenograft model may be derived from HER3-expressing cancer cells. In some embodiments the model is an N87 cell-derived model, a SNU16 cell-derived model, a FaDu cell-derived model, an OvCAR8 cell-derived model, a HCC95 cell-derived model, an A549 cell-derived model, an ACHN cell-derived model or a HT29 cell-derived model.
The cancer may be a HER3-associated cancer as described herein (i.e. cancers for which HER3 gene/protein expression is a risk factor for, and/or is positively associated with, the onset, development, progression or severity of symptoms of the cancer, and/or metastasis). The cancer may comprise HER3-expressing cells. In some embodiments the cancer comprises a HER3+ tumor.
In some embodiments, administration of an antigen-binding molecule according to the present invention may cause one or more of: inhibition of the development/progression of the cancer, a delay to/prevention of onset of the cancer, a reduction in/delay to/prevention of tumor growth, a reduction in/delay to/prevention of metastasis, a reduction in the severity of the symptoms of the cancer, a reduction in the number of cancer cells, a reduction in tumour size/volume, and/or an increase in survival (e.g. progression free survival), e.g. as determined in an appropriate HER3-expressing cancer cell line-derived xenograft model.
In some embodiments, the antigen-binding molecule of the present invention is capable of inhibiting tumor growth in a HER3-expressing cancer cell line-derived xenograft model to less than less than 1 times, e.g. ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the tumor growth observed in the absence of treatment with the antigen-binding molecule (or following treatment with an appropriate negative control antigen-binding molecule).
In some embodiments, treatment of a subject with an antigen-binding molecule or other article disclosed herein (e.g. composition, nucleic acid etc), e.g. wherein the antigen-binding molecule/article is administered to a subject at a dosage and/or in accordance with a dosage schedule described herein, may be associated with one or more of the following outcomes:
An adverse event (AE) is any untoward, undesired or unplanned medical occurrence in a patient administered an investigational medicinal product (IMP), a comparator product or an approved drug. An AE can be a sign, symptom, disease, and/or laboratory or physiological observation that may or may not be related to the IMP or comparator. An AE includes but is not limited to those in the following list.
A serious adverse event (SAE) is any AE, regardless of dose, causality or expectedness, that:
In some embodiments, response to treatment in accordance with the present disclosure can be characterised by reference to tumour/lesion responses. In some embodiments, tumour/lesion responses are evaluated in accordance with the response evaluation criteria in solid tumours (RECIST) criteria, e.g. the RECIST 1.1 criteria as described in Eisenhauer et al., Eur J Cancer. 2009 January;45(2):228-47, which is hereby incorporated by reference in its entirety. In some embodiments, tumour/lesion responses are evaluated in accordance with the Prostate Cancer Working Group 3 (PCWG3) criteria, e.g. as described in Scher et al., J Clin Oncol. 2016 Apr. 20; 34(12):1402-18, which is hereby incorporated by reference in its entirety.
In some embodiments treatment of a subject with an antigen-binding molecule or article described herein, e.g. wherein the antigen-binding molecule/article is administered to a subject at a dosage and/or in accordance with a dosage schedule described herein, may be associated with one or more of the following outcomes (as assessed in accordance with the RECIST 1.1 or PCWG3 criteria, as appropriate; see Example 16.10 for details and methods of assessment), e.g. at 12 and/or 24 months from the start of treatment:
Tumour responses can be evaluated using the appropriate imaging technique according to the tumour and its location, e.g. CT scan, MRI scan and FDG-PET. Appropriate techniques will be familiar to the skilled person and are described in Eisenhauer et al, supra, and/or in Example 16.10 herein.
The subject may be a subject defined herein, e.g. having, or determined to have, a cancer or solid tumour, e.g. according to the present disclosure.
The present invention also provides Chimeric Antigen Receptors (CARs) comprising the antigen-binding molecules or polypeptides of the present invention.
CARs are recombinant receptors that provide both antigen-binding and T cell activating functions. CAR structure and engineering is reviewed, for example, in Dotti et al., Immunol Rev (2014) 257(1), hereby incorporated by reference in its entirety. CARs comprise an antigen-binding region linked to a cell membrane anchor region and a signalling region. An optional hinge region may provide separation between the antigen-binding region and cell membrane anchor region, and may act as a flexible linker.
The CAR of the present invention comprises an antigen-binding region which comprises or consists of the antigen-binding molecule of the present invention, or which comprises or consists of a polypeptide according to the invention.
The cell membrane anchor region is provided between the antigen-binding region and the signalling region of the CAR and provides for anchoring the CAR to the cell membrane of a cell expressing a CAR, with the antigen-binding region in the extracellular space, and signalling region inside the cell. In some embodiments, the CAR comprises a cell membrane anchor region comprising or consisting of an amino acid sequence which comprises, consists of, or is derived from, the transmembrane region amino acid sequence for one of CD3-4, CD4, CD8 or CD28. As used herein, a region which is ‘derived from’ a reference amino acid sequence comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference sequence.
The signalling region of a CAR allows for activation of the T cell. The CAR signalling regions may comprise the amino acid sequence of the intracellular domain of CD3-4, which provides immunoreceptor tyrosine-based activation motifs (ITAMs) for phosphorylation and activation of the CAR-expressing T cell. Signalling regions comprising sequences of other ITAM-containing proteins such as FcγRI have also been employed in CARs (Haynes et al., 2001 J Immunol 166(1):182-187). Signalling regions of CARs may also comprise co-stimulatory sequences derived from the signalling region of co-stimulatory molecules, to facilitate activation of CAR-expressing T cells upon binding to the target protein. Suitable co-stimulatory molecules include CD28, OX40, 4-1BB, ICOS and CD27. In some cases CARs are engineered to provide for co-stimulation of different intracellular signalling pathways. For example, signalling associated with CD28 costimulation preferentially activates the phosphatidylinositol 3-kinase (P13K) pathway, whereas the 4-1BB-mediated signalling is through TNF receptor associated factor (TRAF) adaptor proteins. Signalling regions of CARs therefore sometimes contain co-stimulatory sequences derived from signalling regions of more than one co-stimulatory molecule. In some embodiments, the CAR of the present invention comprises one or more co-stimulatory sequences comprising or consisting of an amino acid sequence which comprises, consists of, or is derived from, the amino acid sequence of the intracellular domain of one or more of CD28, OX40, 4-1BB, ICOS and CD27.
An optional hinge region may provide separation between the antigen-binding domain and the transmembrane domain, and may act as a flexible linker. Hinge regions may be derived from IgG1. In some embodiments, the CAR of the present invention comprises a hinge region comprising or consisting of an amino acid sequence which comprises, consists of, or is derived from, the amino acid sequence of the hinge region of IgG1.
Also provided is a cell comprising a CAR according to the invention. The CAR according to the present invention may be used to generate CAR-expressing immune cells, e.g. CAR-T or CAR-NK cells. Engineering of CARs into immune cells may be performed during culture, in vitro.
The antigen-binding region of the CAR of the present invention may be provided with any suitable format, e.g. scFv, scFab, etc.
The present invention provides a nucleic acid, or a plurality of nucleic acids, encoding an antigen-binding molecule, polypeptide or CAR according to the present invention.
In some embodiments, the nucleic acid is purified or isolated, e.g. from other nucleic acid, or naturally-occurring biological material. In some embodiments the nucleic acid(s) comprise or consist of DNA and/or RNA.
The present invention also provides a vector, or plurality of vectors, comprising the nucleic acid or plurality of nucleic acids according to the present invention.
The nucleotide sequence may be contained in a vector, e.g. an expression vector. A “vector” as used herein is a nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell. The vector may be a vector for expression of the nucleic acid in the cell. Such vectors may include a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed. A vector may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express a peptide or polypeptide from a vector according to the invention.
The term “operably linked” may include the situation where a selected nucleic acid sequence and regulatory nucleic acid sequence (e.g. promoter and/or enhancer) are covalently linked in such a way as to place the expression of nucleic acid sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence. The resulting transcript(s) may then be translated into a desired peptide(s)/polypeptide(s).
Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g. yeast artificial chromosomes).
In some embodiments, the vector may be a eukaryotic vector, e.g. a vector comprising the elements necessary for expression of protein from the vector in a eukaryotic cell. In some embodiments, the vector may be a mammalian vector, e.g. comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.
Constituent polypeptides of an antigen-binding molecule according to the present invention may be encoded by different nucleic acids of the plurality of nucleic acids, or by different vectors of the plurality of vectors.
The present invention also provides a cell comprising or expressing an antigen-binding molecule, polypeptide or CAR according to the present invention. Also provided is a cell comprising or expressing a nucleic acid, a plurality of nucleic acids, a vector or a plurality of vectors according to the invention.
The cell may be a eukaryotic cell, e.g. a mammalian cell. The mammal may be a primate (rhesus, cynomolgous, non-human primate or human) or a non-human mammal (e.g. rabbit, guinea pig, rat, mouse or other rodent (including any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, e.g. dairy cows, or any animal in the order Bos), horse (including any animal in the order Equidae), donkey, and non-human primate).
The present invention also provides a method for producing a cell comprising a nucleic acid(s) or vector(s) according to the present invention, comprising introducing a nucleic acid, a plurality of nucleic acids, a vector or a plurality of vectors according to the present invention into a cell. In some embodiments, introducing an isolated nucleic acid(s) or vector(s) according to the invention into a cell comprises transformation, transfection, electroporation or transduction (e.g. retroviral transduction).
The present invention also provides a method for producing a cell expressing/comprising an antigen-binding molecule, polypeptide or CAR according to the present invention, comprising introducing a nucleic acid, a plurality of nucleic acids, a vector or a plurality of vectors according to the present invention in a cell. In some embodiments, the methods additionally comprise culturing the cell under conditions suitable for expression of the nucleic acid(s) or vector(s) by the cell. In some embodiments, the methods are performed in vitro.
The present invention also provides cells obtained or obtainable by the methods according to the present invention.
Antigen-binding molecules and polypeptides according to the invention may be prepared according to methods for the production of polypeptides known to the skilled person.
Polypeptides may be prepared by chemical synthesis, e.g. liquid or solid phase synthesis. For example, peptides/polypeptides can by synthesised using the methods described in, for example, Chandrudu et al., Molecules (2013), 18: 4373-4388, which is hereby incorporated by reference in its entirety.
Alternatively, antigen-binding molecules and polypeptides may be produced by recombinant expression. Molecular biology techniques suitable for recombinant production of polypeptides are well known in the art, such as those set out in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Press, 2012, and in Nat Methods. (2008); 5(2): 135-146 both of which are hereby incorporated by reference in their entirety. Methods for the recombinant production of antigen-binding molecules are also described in Frenzel et al., Front Immunol. (2013); 4: 217 and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100: 3451-3461, both of which are hereby incorporated by reference in their entirety.
In some cases the antigen-binding molecule of the present invention are comprised of more than one polypeptide chain. In such cases, production of the antigen-binding molecules may comprise transcription and translation of more than one polypeptide, and subsequent association of the polypeptide chains to form the antigen-binding molecule.
For recombinant production according to the invention, any cell suitable for the expression of polypeptides may be used. The cell may be a prokaryote or eukaryote. In some embodiments the cell is a prokaryotic cell, such as a cell of archaea or bacteria. In some embodiments the bacteria may be Gram-negative bacteria such as bacteria of the family Enterobacteriaceae, for example Escherichia coli. In some embodiments, the cell is a eukaryotic cell such as a yeast cell, a plant cell, insect cell or a mammalian cell, e.g. CHO, HEK (e.g. HEK293), HeLa or COS cells. In some embodiments, the cell is a CHO cell that transiently or stably expresses the polypeptides.
In some cases the cell is not a prokaryotic cell because some prokaryotic cells do not allow for the same folding or post-translational modifications as eukaryotic cells. In addition, very high expression levels are possible in eukaryotes and proteins can be easier to purify from eukaryotes using appropriate tags. Specific plasmids may also be utilised which enhance secretion of the protein into the media.
In some embodiments polypeptides may be prepared by cell-free-protein synthesis (CFPS), e.g. according using a system described in Zemella et al. Chembiochem (2015) 16(17): 2420-2431, which is hereby incorporated by reference in its entirety.
Production may involve culture or fermentation of a eukaryotic cell modified to express the polypeptide(s) of interest. The culture or fermentation may be performed in a bioreactor provided with an appropriate supply of nutrients, air/oxygen and/or growth factors. Secreted proteins can be collected by partitioning culture media/fermentation broth from the cells, extracting the protein content, and separating individual proteins to isolate secreted polypeptide(s). Culture, fermentation and separation techniques are well known to those of skill in the art, and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition; incorporated by reference herein above).
Bioreactors include one or more vessels in which cells may be cultured. Culture in the bioreactor may occur continuously, with a continuous flow of reactants into, and a continuous flow of cultured cells from, the reactor. Alternatively, the culture may occur in batches. The bioreactor monitors and controls environmental conditions such as pH, oxygen, flow rates into and out of, and agitation within the vessel such that optimum conditions are provided for the cells being cultured.
Following culturing the cells that express the antigen-binding molecule/polypeptide(s), the polypeptide(s) of interest may be isolated. Any suitable method for separating proteins from cells known in the art may be used. In order to isolate the polypeptide it may be necessary to separate the cells from nutrient medium. If the polypeptide(s) are secreted from the cells, the cells may be separated by centrifugation from the culture media that contains the secreted polypeptide(s) of interest. If the polypeptide(s) of interest collect within the cell, protein isolation may comprise centrifugation to separate cells from cell culture medium, treatment of the cell pellet with a lysis buffer, and cell disruption e.g. by sonification, rapid freeze-thaw or osmotic lysis.
It may then be desirable to isolate the polypeptide(s) of interest from the supernatant or culture medium, which may contain other protein and non-protein components. A common approach to separating protein components from a supernatant or culture medium is by precipitation. Proteins of different solubilities are precipitated at different concentrations of precipitating agent such as ammonium sulfate. For example, at low concentrations of precipitating agent, water soluble proteins are extracted. Thus, by adding different increasing concentrations of precipitating agent, proteins of different solubilities may be distinguished. Dialysis may be subsequently used to remove ammonium sulfate from the separated proteins.
Other methods for distinguishing different proteins are known in the art, for example ion exchange chromatography and size chromatography. These may be used as an alternative to precipitation, or may be performed subsequently to precipitation.
Once the polypeptide(s) of interest have been isolated from culture it may be desired or necessary to concentrate the polypeptide(s). A number of methods for concentrating proteins are known in the art, such as ultrafiltration or lyophilisation.
The present invention also provides compositions comprising the antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors and cells described herein.
The antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors and cells described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The composition may be formulated for topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral or transdermal routes of administration which may include injection or infusion.
Suitable formulations may comprise the antigen-binding molecule in a sterile or isotonic medium. Medicaments and pharmaceutical compositions may be formulated in fluid, including gel, form. Fluid formulations may be formulated for administration by injection or infusion (e.g. via catheter) to a selected region of the human or animal body.
In some embodiments the composition is formulated for injection or infusion, e.g. into a blood vessel or tumor.
In accordance with the invention described herein methods are also provided for the production of pharmaceutically useful compositions, such methods of production may comprise one or more steps selected from: producing an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein; isolating an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein; and/or mixing an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.
For example, a further aspect the invention described herein relates to a method of formulating or producing a medicament or pharmaceutical composition for use in the treatment of a disease/condition (e.g. a cancer), the method comprising formulating a pharmaceutical composition or medicament by mixing an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.
In aspects and embodiments of the present disclosure, the antigen-binding molecule may be provided in a composition comprising particular chemical constituents in specified concentrations/proportions.
In some embodiments, the antigen-binding molecule is provided in a buffer. As used herein, a “buffer” refers to a buffered solution that resists changes in pH by the action of its acid-base conjugate components. A buffer of the present disclosure preferably has a pH in the range from about 4.5 to about 7.0, preferably from about 5.0 to about 6.5. Examples of buffers that will control the pH in this range include acetate, histidine, histidine-arginine, histidine-methionine and other organic acid buffers.
In some embodiments, the composition comprising the antigen-binding molecule has a pH of 4.0 to 7.0, e.g. one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2. In some embodiments, the composition has a pH of ˜5.5. In some embodiments, the composition has a pH of ˜5.8. In some embodiments, the composition has a pH of ˜6.5.
In some embodiments, the antigen-binding molecule is provided in an acetate buffer, i.e. a buffer comprising acetate ions. In some embodiments, the antigen-binding molecule is provided in a composition comprising acetate at a final concentration of 2 mM to 200 mM acetate, e.g. one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM. In some embodiments, the composition may comprise ˜20 mM acetate.
In some embodiments, the antigen-binding molecule is provided in a histidine buffer, i.e. a buffer comprising histidine ions. In some embodiments, the antigen-binding molecule is provided in a composition comprising histidine at a final concentration of 2 mM to 200 mM histidine, e.g. one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM. In some embodiments, the composition may comprise ˜20 mM histidine.
In some embodiments, the antigen-binding molecule is provided in a sodium phosphate buffer, i.e. a buffer comprising sodium and phosphate ions. In some embodiments, the antigen-binding molecule is provided in a composition comprising sodium phosphate at a final concentration of 2 mM to 200 mM sodium phosphate, e.g. one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM. In some embodiments, the composition may comprise ˜20 mM sodium phosphate.
In some embodiments, the antigen-binding molecule is provided in a sodium acetate buffer, i.e. a buffer comprising sodium and acetate ions. In some embodiments, the antigen-binding molecule is provided in a composition comprising sodium acetate at a final concentration of 2 mM to 200 mM sodium acetate, e.g. one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM. In some embodiments, the composition may comprise ˜20 mM sodium acetate.
In some embodiments, the antigen-binding molecule is provided in an arginine buffer, i.e. a buffer comprising arginine ions. In some embodiments, the antigen-binding molecule is provided in a composition comprising arginine at a final concentration of 1 mM to 250 mM arginine, e.g. one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM. In some embodiments, the composition may comprise ˜20 mM arginine.
In some embodiments, the antigen-binding molecule is provided in a histidine-arginine buffer, i.e. a buffer comprising histidine and arginine ions. In some embodiments, the antigen-binding molecule is provided in a composition comprising histidine at a final concentration of 2 mM to 200 mM histidine, e.g. one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM, and arginine at a final concentration of 1 mM to 300 mM arginine, e.g. one of 10 mM to 250 mM, 50 mM to 200 mM, 75 mM to 200 mM, 100 mM to 180 mM, or 125 to 175 mM. In some embodiments, the composition may comprise ˜20 mM histidine and ˜150 mM arginine.
In some embodiments, the antigen-binding molecule is provided in a composition comprising sodium chloride. The sodium chloride component of the composition may be provided at a final concentration of 1 mM to 250 mM sodium chloride, e.g. one of 10 mM to 250 mM, 50 mM to 200 mM, 75 mM to 200 mM, 100 mM to 180 mM, or 125 to 175 mM. In some embodiments, the composition may comprise ˜150 mM sodium chloride.
In some embodiments, the antigen-binding molecule is provided in a composition comprising methionine. The methionine component of the composition may be provided at a final concentration of 1 mM to 250 mM methionine, e.g. one of 10 mM to 250 mM, 50 mM to 200 mM, 75 mM to 200 mM, 100 mM to 180 mM, or 125 to 175 mM. In some embodiments, the composition may comprise ˜150 mM methionine.
In some embodiments, the composition comprising the antigen-binding molecule comprises an isotonicity agent. Isotonicity agents may be used to provide isotonic formulations. Examples of isotonicity agents include e.g. salts (e.g. sodium chloride, potassium chloride) and sugars (e.g. sucrose, glucose, trehalose).
In some embodiments, the antigen-binding molecule is provided in a composition comprising sucrose. The sucrose component of the composition may be provided at a final concentration (in weight by volume) of 2% to 20%, e.g. one of 2% to 15%, 3% to 12%, or 4% to 10%. In some embodiments, the composition may comprise ˜2, ˜4, ˜6, or ˜8% (w/v) sucrose. The sucrose component of the composition may be provided at a final concentration of 200 to 300 nM, e.g. ˜240 mM.
In some embodiments, the composition comprising the antigen-binding molecule comprises a surfactant. As used herein, a “surfactant” refers to an agent which lowers interfacial tension. The surfactant is preferably a non-ionic surfactant. Examples of surfactants include polysorbate (polysorbate-20, polysorbate-80), poloxamer (poloxamer-188) and Triton X-100. The surfactant is preferably present in the composition in the range from about 0.001% (w/v) to about 0.5% (w/v).
In some embodiments, the antigen-binding molecule is provided in a composition comprising polysorbate-20. The polysorbate-20 component of the composition may be provided at a final concentration (in weight by volume) of 0.001% to 0.1%, e.g. one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%. In some embodiments, the composition may comprise ˜0.02% (w/v) polysorbate-20. In some embodiments, the composition may comprise ˜0.05% (w/v) polysorbate-20.
In some embodiments, the antigen-binding molecule is provided in a composition comprising polysorbate-80. The polysorbate-80 component of the composition may be provided at a final concentration (in weight by volume) of 0.001% to 0.1%, e.g. one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%. In some embodiments, the composition may comprise ˜0.01% (w/v) polysorbate-80. In some embodiments, the composition may comprise ˜0.02% (w/v) polysorbate-80.
In some embodiments, the antigen-binding molecule is provided in a composition comprising:
In some embodiments, the antigen-binding molecule is provided in a composition comprising:
In some embodiments, the antigen-binding molecule is provided in a composition comprising:
In some embodiments, the antigen-binding molecule is provided in a composition comprising:
In some embodiments, the antigen-binding molecule is provided in a composition comprising:
In some embodiments, the antigen-binding molecule is provided in a composition comprising:
In some embodiments, the antigen-binding molecule is provided in a composition comprising:
In some embodiments, the antigen-binding molecule is provided in a composition comprising:
In some embodiments, the antigen-binding molecule is provided in a composition comprising:
In some embodiments, the antigen-binding molecule is provided in a composition comprising:
In some embodiments, the antigen-binding molecule is provided in a composition comprising:
In some embodiments, the antigen-binding molecule is provided in a composition comprising:
In some embodiments, the antigen-binding molecule is provided in a composition comprising:
In some embodiments, the antigen-binding molecule is provided in a composition comprising:
In some embodiments, the antigen-binding molecule is provided in a composition comprising:
In some embodiments, the antigen-binding molecule is provided in a composition comprising:
The composition may comprise about 0.5 mg/mL to about 100 mg/mL antigen-binding molecule. The composition may comprise about 0.5 mg/mL to about 80 mg/mL antigen-binding molecule. The composition may comprise about 0.75 mg/mL to about 70 mg/mL antigen-binding molecule. The composition may comprise about 1 mg/mL to about 60 mg/mL antigen-binding molecule. The composition may comprise about 1.2 mg/mL to about 50 mg/mL antigen-binding molecule.
The antigen-binding molecule may be formulated at a concentration of about 30 mg/mL, about 35 mg/mL, about 40 mg/mL, about 45 mg/mL, about 50 mg/mL, about 55 mg/mL, about 60 mg/mL, about 65 mg/mL, or about 70 mg/mL, or any ranges therein, e.g. in a composition according to the present disclosure. The antigen-binding molecule may be formulated at a concentration of about 50 mg/mL, e.g. in a composition according to the present disclosure.
The antigen-binding molecule may be formulated at a concentration of about 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL, 1.0 mg/mL, 1.1 mg/mL, 1.2 mg/mL, 1.3 mg/mL, 1.4 mg/mL, 1.5 mg/mL, 1.6 mg/mL, 1.7 mg/mL, 1.8 mg/mL, 1.9 mg/mL, or 2.0 mg/mL, e.g. in a composition according to the present disclosure. The antigen-binding molecule may be formulated at a concentration of about 1.2 mg/mL, e.g. in a composition according to the present disclosure.
The 50 mg/mL antigen-binding molecule solution may be diluted before administration using any suitable excipient. In some embodiments, the 50 mg/mL antigen-binding molecule solution is diluted for administration in 0.9% sodium chloride (NaCl). In some embodiments, the 50 mg/mL antigen-binding molecule solution is diluted for administration to a concentration of at least 1.2 mg/mL, e.g. in a volume of 100 to 250 mL. In some embodiments, the diluted antigen-binding molecule solution is then administered to a subject, e.g. via intravenous administration, e.g. using a dose/dosing regime according to the present disclosure, e.g. to treat a disease/condition according to the present disclosure. In some embodiments, the diluted antigen-binding molecule solution is administered to the subject within 48 hours from the initial dilution step.
The antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors, cells and compositions described herein find use in therapeutic and prophylactic methods.
The present invention provides an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein for use in a method of medical treatment or prophylaxis. Also provided is the use of an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein in the manufacture of a medicament for treating or preventing a disease or condition. Also provided is a method of treating or preventing a disease or condition, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein.
The methods may be effective to reduce the development or progression of a disease/condition, alleviation of the symptoms of a disease/condition or reduction in the pathology of a disease/condition. The methods may be effective to prevent progression of the disease/condition, e.g. to prevent worsening of, or to slow the rate of development of, the disease/condition. In some embodiments the methods may lead to an improvement in the disease/condition, e.g. a reduction in the symptoms of the disease/condition or reduction in some other correlate of the severity/activity of the disease/condition. In some embodiments the methods may prevent development of the disease/condition a later stage (e.g. a chronic stage or metastasis).
It will be appreciated that the articles of the present invention may be used for the treatment/prevention of any disease/condition that would derive therapeutic or prophylactic benefit from a reduction in the number and/or activity of cells expressing HER3. For example, the disease/condition may be a disease/condition in which cells expressing HER3 are pathologically implicated, e.g. a disease/condition in which an increased number/proportion of cells expressing HER3 is positively associated with the onset, development or progression of the disease/condition, and/or severity of one or more symptoms of the disease/condition, or for which an increased number/proportion of cells expressing HER3, is a risk factor for the onset, development or progression of the disease/condition.
In some embodiments, the disease/condition to be treated/prevented in accordance with the present invention is a disease/condition characterised by an increase in the number/proportion/activity of cells expressing HER3, e.g. as compared to the number/proportion/activity of cells expressing HER3 in the absence of the disease/condition.
In some embodiments the disease/condition to be treated/prevented is a cancer.
The cancer may be any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation), neoplasm or tumor. The cancer may be benign or malignant and may be primary or secondary (metastatic). A neoplasm or tumor may be any abnormal growth or proliferation of cells and may be located in any tissue. The cancer may be of tissues/cells derived from e.g. the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain) cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g. renal epithelia), gallbladder, oesophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissues, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, and/or white blood cells.
Tumors to be treated may be nervous or non-nervous system tumors. Nervous system tumors may originate either in the central or peripheral nervous system, e.g. glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, Schwannoma, neurofibrosarcoma, astrocytoma and oligodendroglioma. Non-nervous system cancers/tumors may originate in any other non-nervous tissue, examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, Non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma, prostate carcinoma, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic carcinoma, NSCLC, hematologic cancer and sarcoma.
HER3 and its association with and role in cancer is reviewed e.g. in Karachaliou et al., BioDrugs. (2017) 31(1):63-73 and Zhang et al., Acta Biochimica et Biophysica Sinica (2016) 48(1): 39-48, both of which are hereby incorporated by reference in their entirety.
In some embodiments, a cancer is selected from: a cancer comprising cells expressing HER3, a solid tumor, breast cancer, breast carcinoma, ductal carcinoma, gastric cancer, gastric carcinoma, gastric adenocarcinoma, colorectal cancer, colorectal carcinoma, colorectal adenocarcinoma, head and neck cancer, squamous cell carcinoma of the head and neck (SCCHN), lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, ovarian cancer, ovarian carcinoma, ovarian serous adenocarcinoma, kidney cancer, renal cell carcinoma, renal clear cell carcinoma, renal cell adenocarcinoma, renal papillary cell carcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, cervical cancer, cervical squamous cell carcinoma, skin cancer, melanoma, esophageal cancer, esophageal adenocarcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, uterine cancer, uterine corpus endometrial carcinoma, thyroid cancer, thyroid carcinoma, pheochromocytoma, paraganglioma, bladder cancer, bladder urothelial carcinoma, prostate cancer, prostate adenocarcinoma, sarcoma and thymoma.
In some embodiments the cancer to be treated in accordance with the present invention is selected from: a HER3-expressing cancer, gastric cancer (e.g. gastric carcinoma, gastric adenocarcinoma, gastrointestinal adenocarcinoma), head and neck cancer (e.g. head and neck squamous cell carcinoma), breast cancer (e.g. triple negative breast cancer), ovarian cancer (e.g. ovarian carcinoma), lung cancer (e.g. NSCLC, lung adenocarcinoma, squamous lung cell carcinoma), melanoma, prostate cancer (e.g. castration resistant prostate cancer), oral cavity cancer (e.g. oropharyngeal cancer), renal cancer (e.g. renal cell carcinoma) or colorectal cancer (e.g. colorectal carcinoma; RAS wild type colorectal cancer), oesophageal cancer, pancreatic cancer, a solid cancer and/or a liquid cancer.
In some embodiments the cancer to be treated in accordance with the present invention is a solid cancer expressing/overexpressing HER3 selected from: gastric cancer (e.g. gastric carcinoma, gastric adenocarcinoma, gastrointestinal adenocarcinoma), head and neck cancer (e.g. head and neck squamous cell carcinoma), breast cancer (e.g. triple negative breast cancer), ovarian cancer (e.g. ovarian carcinoma), lung cancer (e.g. NSCLC, lung adenocarcinoma, squamous lung cell carcinoma, invasive mucinous adenocarcinoma), melanoma, prostate cancer (e.g. castration resistant prostate cancer), oral cavity cancer (e.g. oropharyngeal cancer), colorectal cancer (e.g. colorectal carcinoma; RAS wild type colorectal cancer), oesophageal cancer, pancreatic cancer, bladder cancer, cervical cancer, endometrial cancer, or hepatocellular carcinoma (HCC). The cancer may be metastatic.
The treatment/prevention may be aimed at one or more of: delaying/preventing the onset/progression of symptoms of the cancer, reducing the severity of symptoms of the cancer, reducing the survival/growth/invasion/metastasis of cells of the cancer, reducing the number of cells of the cancer and/or increasing survival of the subject.
In some embodiments, the cancer to be treated/prevented comprises cells expressing an EGFR family member (e.g. HER3, EGFR, HER2 or HER4), and/or cells expressing a ligand for an EGFR family member. In some embodiments, the cancer to be treated/prevented is a cancer which is positive for an EGFR family member. In some embodiments, the cancer over-expresses an EGFR family member and/or a ligand for an EGFR family member. Overexpression of can be determined by detection of a level of expression which is greater than the level of expression by equivalent non-cancerous cells/non-tumor tissue.
In some embodiments, the cancer to be treated/prevented comprises cells expressing HER3 and another EGFR family member (e.g. EGFR, HER2 or HER4). In some embodiments, the cancer to be treated/prevented comprises cells overexpressing HER3 and overexpressing another EGFR family member (e.g. EGFR, HER2 or HER4). Overexpression of HER3/another EGFR family member can be determined by detection of a level of expression of HER3/another EGFR family member which is greater than the level of expression by equivalent non-cancerous cells/non-tumor tissue.
Expression may be determined by any suitable means. Expression may be gene expression (e.g. transcriptional upregulation) or protein expression. Gene expression can be determined e.g. by detection of mRNA encoding HER3, for example by quantitative real-time PCR (qRT-PCR). Protein expression can be determined e.g. by for example by antibody-based methods, for example by western blot, immunohistochemistry, immunocytochemistry, flow cytometry, or ELISA.
In some embodiments, the cancer to be treated/prevented comprises cells expressing HER3. In some embodiments, the cancer to be treated/prevented is a cancer which is positive for HER3. In some embodiments, the cancer over-expresses HER3. Overexpression of HER3 can be determined by detection of a level of expression of HER3 which is greater than the level of expression by equivalent non-cancerous cells/non-tumor tissue.
In some embodiments, a patient may be selected for treatment described herein based on the detection of a cancer expressing HER3, or overexpressing HER3, e.g. in a sample obtained from the patient.
In some embodiments, a patient may be selected for treatment described herein based on the detection of a cancer expressing HER3 and another EGFR family member (e.g. EGFR, HER2 or HER4), or overexpressing HER3 and another EGFR family member (e.g. EGFR, HER2 or HER4), e.g. in a sample obtained from the patient.
In some embodiments, the cancer to be treated/prevented comprises cells expressing a ligand for HER3 (e.g. NRG1 and/or NRG2). In some embodiments, the cancer to be treated/prevented comprises cells expressing a level of expression of NRG1 and/or NRG2 which is greater than the level of expression by equivalent non-cancerous cells/non-tumor tissue. The cancer may be described as comprising cells that overexpress NRG1 and/or NRG2.
HER3-binding antigen-binding molecules described herein are demonstrated to bind to HER3 with extremely high affinity when HER3 is bound by NRG (i.e. when HER3 is provided in the ‘open’ conformation), and also when HER3 is not bound by NRG (i.e. when HER3 is provided in the ‘closed’ conformation).
Thus the antigen-binding molecules of the present invention are particularly useful for the treatment/prevention of cancers characterised by HER3 ligand expression/overexpression, for example cancers/tumors comprising cells expressing/overexpressing a ligand for HER3.
In some embodiments, a patient may be selected for treatment described herein based on the detection of a cancer characterised by HER3 ligand expression/overexpression, such as a cancer comprising cells expressing/overexpressing NRG1 and/or NRG2, e.g. in a sample obtained from the subject. Selection based on detection of HER3 ligand expression/overexpression may be combined with selection based on detection of HER3 and/or another EGFR family member (e.g. EGFR, HER2 or HER4).
In some embodiments, the cancer to be treated in accordance with the present invention comprises cells harbouring a genetic variant (e.g. a mutation) which causes increased (gene and/or protein) expression of a ligand for HER3, relative to comparable cells harbouring a reference allele not comprising the genetic variant (e.g. a non-mutated, or ‘wildtype’ allele). The genetic variant may be or comprise insertion, deletion, substitution to, or larger-scale translocation/rearrangement of, the nucleotide sequence relative to the reference allele.
A mutation ‘resulting in’ increased expression of a ligand for HER3 may be known or predicted to cause, or may be associated with, increased gene/protein expression of a ligand for HER3. Mutations resulting in increased expression of a ligand for HER3 may be referred to as ‘activating’ mutations.
A mutation which causes increased expression of a ligand for HER3 may result in gene or protein expression of a ligand for HER3 which is not expressed by, and/or not encoded by genomic nucleic acid of, an equivalent cell not harbouring the mutation. That is, the ligand for HER3 may be a neoantigen arising as a result of the mutation, and thus ‘increased expression’ may be from no expression. By way of illustration, a cell comprising CD74-NRG1 gene fusion displays increased expression of the CD74-NRG1 fusion polypeptide encoded by the gene fusion relative to cells lacking the CD74-NRG1 gene fusion.
A mutation which causes increased expression of a ligand for HER3 may result in increased gene or protein expression of a ligand for HER3 which is expressed by, and/or which is encoded by genomic nucleic acid of, an equivalent cell not comprising the mutation. By way of illustration, a cell may comprise a mutation resulting in an increase in the level of transcription of nucleic acid encoding NRG1 relative to level of transcription of nucleic acid encoding NRG1 by an equivalent cell not comprising the mutation.
In some embodiments, a mutation which causes increased expression of a ligand for HER3 may cause an increase in gene expression of a ligand for HER3 relative to an equivalent cell not comprising the mutation. In some embodiments, a mutation which causes increased expression of a ligand for HER3 may cause an increase in protein expression of a ligand for HER3 relative to an equivalent cell not comprising the mutation.
In some embodiments, a mutation which causes increased expression of a ligand for HER3 may cause an increase in the level of a ligand for HER3 on or at the cell surface of a cell comprising the mutation, relative to an equivalent cell not comprising the mutation. In some embodiments, a mutation which causes increased expression of a ligand for HER3 may cause an increase in the level of a secretion of a ligand for HER3 from a cell comprising the mutation, relative to an equivalent cell not comprising the mutation.
Cells having increased expression of a ligand for HER3 relative to the level of expression of the ligand for HER3 by a reference cell (e.g. as a result of mutation) may be described as ‘overexpressing’ the ligand for HER3, or having ‘upregulated expression’ of the ligand for HER3. For example, a cancer comprising cells harbouring a mutation resulting in increased expression of a ligand for HER3 relative to equivalent cells lacking the mutation may be described as a cancer comprising cells displaying overexpression/upregulated expression of the ligand for HER3. In some embodiments, the reference cell lacking the mutation may be a non-cancerous cell (e.g. of equivalent cell type) or a cancerous cell (e.g. of equivalent cancer type).
Herein, a ‘ligand for HER3’ is generally intended to refer to molecule capable of binding to HER3 through the ligand binding region of HER3 formed by domains I and III of HER3. In some embodiments, a ligand for HER3 binds to HER3 via interaction with domains I and/or III of HER3. Exemplary ligands for HER3 include Neuregulins such as NRG1 and NRG2, which bind to HER3 via interaction between their EGF-like domains and the ligand binding region of HER3.
The HER3 ligand is preferably able to bind and trigger signalling through the HER3 receptor and/or receptor complexes comprising HER3. As will be clear from the present disclosure, receptor complexes comprising HER3 may further comprise an interaction partner for HER3 as described herein, e.g. HER3, HER2, EGFR, HER4, HGFR, IGF1R and/or cMet).
In some embodiments the ligand for HER3 is able to bind to HER3 receptor/receptor complex expressed by a cell other than the cell having increased expression of the HER3 ligand. For example, in some embodiments the ligand for HER3 is able to bind to a HER3-expressing cancer cell.
In some embodiments the ligand for HER3 is able to bind to HER3 receptor/receptor complex expressed by the cell having increased expression of the HER3 ligand.
In some embodiments the cancer to be treated comprises (i) cells expressing HER3, and (ii) cells expressing a ligand for HER3 (e.g. having increased expression of a ligand for HER3, e.g. as a consequence of mutation resulting in increased expression of a ligand for HER3).
In some embodiments the cancer to be treated comprises cells which (i) express HER3 and (ii) which also express a ligand for HER3 (e.g. which have increased expression of a ligand for HER3, e.g. as a consequence of mutation resulting in increased expression of a ligand for HER3).
In some embodiments, the ligand for HER3 comprises, or consists of, the amino acid sequence a HER3-binding region of a ligand for HER3, or an amino acid sequence derived from a HER3-binding region of a ligand for HER3. An amino acid sequence which is derived from a HER3-binding region of a ligand for HER3 may comprise at least 60% (e.g. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to the amino acid sequence from which it is derived.
In some embodiments, the ligand for HER3 comprises an EGF-like domain capable of binding to HER3, or a HER3-binding fragment thereof. In some embodiments, a HER3-binding EGF-like domain/fragment is, or is derived from, an EGF family member (e.g. heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor-α (TGF-α), amphiregulin (AR), epiregulin (EPR), epigen, betacellulin (BTC), NRG1, NRG2, NRG3 or NRG4).
EGF family members contain one or more repeats of the conserved amino acid sequence shown in SEQ ID NO:240, which contains six cysteine residues that form three intramolecular disulfide bonds, providing three structural loops required for high-affinity binding to their cognate receptors (see Harris et al. Experimental Cell Research (2003) 284(1): 2-13). In some embodiments, a ligand for HER3 comprises one or more copies of an amino acid sequence conforming to the consensus sequence shown in SEQ ID NO:240.
Exemplary ligands for HER3 include Neuregulins (NRGs). Neuregulins include NRG1, NRG2, NRG3 and NRG4. The amino acid sequence of human NRG1 (alpha isoform) is shown in SEQ ID NO:232. The alpha isoform and several other isoforms of human NRG1 (including alpha1a isoform (see UnitProt: Q02297-2), alpha2b isoform (see UnitProt: Q02297-3) and alpha3 isoform (see UnitProt: Q02297-4)) comprise the EGF-like domain shown in SEQ ID NO:233, through which they bind to HER3. The amino acid sequence of human NRG2 (isoform 1) is shown in SEQ ID NO:234. Isoform 1 and several other isoforms of human NRG2 (including isoform 3 (see UniProt:014511-3), isoform 5 (see UniProt:014511-5), isoform 6 (see UniProt:014511-6), isoform DON-1B (see UniProt:014511-7) and isoform DON-1R (see UniProt:014511-8)) comprise the EGF-like domain shown in SEQ ID NO:235, through which they bind to HER3. The amino acid sequence of human NRG3 is shown in SEQ ID NO:236, and the EGF-like domain of human NRG3 shown in SEQ ID NO:237. The amino acid sequence of human NRG4 is shown in SEQ ID NO:238, and the EGF-like domain of human NRG3 shown in SEQ ID NO:239. In some embodiments, an NRG is selected from NRG1, NRG2, NRG3 and NRG4. In some embodiments, an NRG is selected from NRG1 and NRG2.
In some embodiments an EGF-like domain/fragment comprises, or consists of, an amino acid sequence having at least 60% (e.g. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to the EGF-like domain of an NRG (NRG1, NRG2, NRG3 or NRG4). In some embodiments an EGF-like domain/fragment comprises, or consists of, an amino acid sequence having at least 60% (e.g. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to one of SEQ ID NOs:233, 235, 237 or 239.
In some embodiments, the ligand for HER3 is an NRG (e.g. NRG1, NRG2, NRG3 or NRG4; e.g. NRG1 or NRG2), or comprises an amino acid sequence derived from an amino acid sequence of an NRG (i.e. comprises an amino acid sequence having at least 60% (e.g. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to an amino acid sequence of an NRG.
In some embodiments, the ligand for HER3 comprises, or consists of, an amino acid sequence having at least 60% (e.g. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to the HER3-binding region of a ligand for HER3 (e.g. an NRG, e.g. NRG1, NRG2, NRG3 or NRG4; e.g. NRG1 or NRG2). In some embodiments, a ligand for HER3 comprises, or consists of, an amino acid sequence having at least 60% (e.g. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to the EGF-like domain of an NRG (e.g. NRG1, NRG2, NRG3 or NRG4; e.g. NRG1 or NRG2).
In some embodiments a ligand for HER3 is not an EGFR family protein (e.g. HER3, HER2, EGFR, HER4, HGFR, IGF1R, cMet).
In some embodiments, the mutation resulting in increased expression of a ligand for HER3 is an NRG gene fusion. In some embodiments, the ligand for HER3 is the product of (i.e. a polypeptide encoded by) an NRG gene fusion. In some embodiments the cancer comprises cells having an NRG gene fusion.
As used herein, an “NRG gene fusion” refers to a genetic variant encoding a polypeptide comprising (i) an amino acid sequence of an NRG protein (e.g. NRG1, NRG2, NRG3 or NRG4; e.g. NRG1 or NRG2), and (ii) an amino acid sequence of a protein other than the NRG protein.
It will be appreciated that an NRG gene fusion preferably encodes a HER3 ligand as described herein. In some embodiments, an NRG gene fusion encodes a polypeptide comprising a HER3-binding region of an NRG protein. In some embodiments, an NRG gene fusion encodes a polypeptide comprising the EGF-like domain of an NRG protein, or an amino acid sequence which is capable of binding to HER3 and having at least 60% (e.g. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to the EGF-like domain of an NRG protein.
In some embodiments, an NRG gene fusion encodes a fusion polypeptide comprising a transmembrane domain. In some embodiments, an NRG gene fusion encodes a fusion polypeptide comprising the transmembrane domain of a protein other than the NRG protein.
In some embodiments, an NRG gene fusion is an NRG1 gene fusion. In some embodiments, the NRG1 gene fusion encodes a polypeptide comprising the EGF-like domain of NRG1, or an amino acid sequence which is capable of binding to HER3 and having at least 60% (e.g. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to the EGF-like domain of NRG1.
NRG1 gene fusions are described e.g. in WO 2018/182422 A1, WO 2019/051155 A1, Dhanasekaran et al., Nat Commun. (2014) 5: 5893, Drilon et al., Cancer Discov. (2018) 8(6):686-695, Nagasaka et al., Journal of Thoracic Oncology (2019) 14(8):1354-1359 and Jonna et al., Clin Cancer Res. (2019) 25(16):4966-4972, all of which are hereby incorporated by reference in their entirety. The diversity of NRG1 gene fusions may result from NRG1 being located on chromosome 8, which is particularly susceptible to genomic translocation events (Adelaide et al., Genes Chromosomes Cancer. (2003) 37(4):333-45).
In some embodiments, an NRG1 gene fusion is selected from CLU-NRG1, CD74-NRG1, DOC4-NRG1, SLC3A2-NRG1, RBPMS-NRG1, WRN-NRG1, SDC4-NRG1, RAB21L1-NRG1, VAMP2-NRG1, KIF13B-NRG1, THAP7-NRG1, SMAD4-NRG1, MDK-NRG1, TNC-NRG1, DIP2B-NRG1, MRPL13-NRG1, PARP8-NRG1, ROCK1-NRG1, DPYSL2-NRG1, ATP1B1-NRG1, CDH6-NRG1, APP-NRG1, AKAP13-NRG1, THBS1-NRG1, FOXA1-NRG1, PDE7A-NRG1, RAB3IL1-NRG1, CDK1-NRG1, BMPRIB-NRG1, TNFRSF10B-NRG1, and MCPH1-NRG1. In some embodiments, an NRG1 gene fusion is CLU-NRG1.
CD74-NRG1 gene fusion is described e.g. in Fernandez-Cuesta et al. Cancer Discov. (2014) 4:415-22 and Nakaoku et al., Clin Cancer Res (2014) 20:3087-93. DOC4-NRG1 gene fusion is described e.g. in Liu et al., Oncogene. (1999) 18(50):7110-4 and Wang et al., Oncogene. (1999) 18(41):5718-21. SLC3A2-NRG1 gene fusion is described e.g. in Nakaoku et al., Clin Cancer Res (2014) 20:3087-93, Shin et al., Oncotarget (2016) 7:69450-65 and Shin et al., Mol Cancer Ther. (2018) 17(9):2024-2033. RBPMS-NRG1, WRN-NRG1, RAB2IL1-NRG1 and SDC4-NRG1 gene fusions are described e.g. in Dhanasekaran et al., Nat Commun. (2014) 5: 5893. VAMP2-NRG1 gene fusion is described e.g. in Jung et al., J Thorac Oncol. (2015) 10(7):1107-11 and Shim et al., J Thorac Oncol. (2015) 10(8):1156-62. KIF13B-NRG1 gene fusion is described e.g. in Xia et al., Int J Surg Pathol. (2017) 25(3):238-240. SMAD4-NRG1, AKAP13-NRG1, THBS1-NRG1, FOXA1-NRG1, PDE7A-NRG1, RAB3IL1-NRG1 and THAP7-NRG1 gene fusions are described e.g. in Drilon et al., Cancer Discov. (2018) 8(6):686-695. MDK-NRG1, TNC-NRG1, DIP2B-NRG1, MRPL13-NRG1, PARP8-NRG1, ROCK1-NRG1 and DPYSL2-NRG1 gene fusions are described e.g. in Jonna et al., Clin Cancer Res. (2019) 25(16):4966-4972. ATP1B1-NRG1 gene fusion is described e.g. in Drilon et al., Cancer Discov. (2018) 8(6):686-695 and Jones et al., Annals of Oncology (2017) 28:3092-3097. CLU-NRG1 gene fusion is described e.g. in Drilon et al., Cancer Discov. (2018) 8(6):686-695 and Nagasaka et al., Journal of Thoracic Oncology (2019) 14(8):1354-1359.
In some embodiments, an NRG gene fusion is an NRG2 gene fusion. In some embodiments, the NRG2 gene fusion encodes a polypeptide comprising the EGF-like domain of NRG2, or an amino acid sequence which is capable of binding to HER3 and having at least 60% (e.g. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to the EGF-like domain of NRG2.
NRG2 gene fusions include SLC12A2-NRG2 described e.g. in WO 2015/093557 A1, and ZNF208-NRG2 described in Dupain et al., Mol Ther. (2019) 27(1):200-218.
A cancer comprising cells having a mutation which results in increased expression of a ligand for HER3 (e.g. comprising cells having an NRG gene fusion, e.g. an NRG1 gene fusion or an NRG2 gene fusion) can be any cancer described herein. In some embodiments, such cancer may be of tissues/cells derived from the lung, breast, head, neck, kidney, ovary, pancreas, prostate, uterus, gallbladder, colon, rectum, bladder, soft tissue or nasopharynx.
In some embodiments, a cancer comprising cells having a mutation which results in increased expression of a ligand for HER3 (e.g. comprising cells having an NRG gene fusion, e.g. an NRG1 gene fusion or an NRG2 gene fusion) is selected from: lung cancer, non-small cell lung cancer, lung adenocarcinoma, invasive mucinous lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, breast carcinoma, breast invasive carcinoma, head and neck cancer, head and neck squamous cell carcinoma, renal cancer, renal clear cell carcinoma, ovarian cancer, ovarian serous cystadenocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, prostate cancer, prostate adenocarcinoma, endometrial cancer, uterine carcinosarcoma, gallbladder cancer, cholangiocarcinoma, colorectal cancer, bladder cancer, urothelial bladder cancer, sarcoma, soft tissue sarcoma, neuroendocrine tumor and neuroendocrine tumor of the nasopharynx.
In particular embodiments, the cancer to be treated in accordance with the present invention is lung cancer (e.g. non-small cell lung cancer, lung adenocarcinoma, invasive mucinous lung adenocarcinoma or lung squamous cell carcinoma) comprising cells having an NRG1 gene fusion.
It will be appreciated that in embodiments herein, cancers comprising cells having specified characteristics may be or comprise tumors comprising cells having those characteristics.
As is common in the art, a cancer/tumor comprising cells having specified characteristics may be referred to herein simply as a cancer/tumor having those characteristics. By way of illustration, a cancer/tumor comprising cells having an NRG1 gene fusion may be referred to simply as “a cancer/tumor comprising NRG1 gene fusion”, or “an NRG1 gene fusion cancer/tumor”.
Administration of the articles of the present invention is preferably in a “therapeutically effective” or “prophylactically effective” amount, this being sufficient to show therapeutic or prophylactic benefit to the subject. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease/condition and the particular article administered. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disease/disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.
Administration may be alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. The antigen-binding molecule or composition described herein and a therapeutic agent may be administered simultaneously or sequentially.
In some embodiments, the methods comprise additional therapeutic or prophylactic intervention, e.g. for the treatment/prevention of a cancer. In some embodiments, the therapeutic or prophylactic intervention is selected from chemotherapy, immunotherapy, radiotherapy, surgery, vaccination and/or hormone therapy. In some embodiments, the therapeutic or prophylactic intervention comprises leukapheresis. In some embodiments the therapeutic or prophylactic intervention comprises a stem cell transplant.
In some embodiments the antigen-binding molecule is administered in combination with an agent capable of inhibiting signalling mediated by an EGFR family member.
Accordingly, the invention provides compositions comprising an article according to the present invention (e.g. an antigen-binding molecule according to the invention) and another agent capable of inhibiting signalling mediated by an EGFR family member (e.g. EGFR, HER2, HER3 or HER4). Also provided is the use of such compositions in methods of medical treatment and prophylaxis of diseases/conditions described herein.
Also provided are methods for treating/preventing diseases/conditions described herein comprising administering articles of the present invention an article according to the present invention (e.g. an antigen-binding molecule according to the invention) and another agent capable of inhibiting signalling mediated by an EGFR family member.
Agents capable of inhibiting signalling mediated by EGFR family members are known in the art, and include e.g. small molecule inhibitors (e.g. tyrosine kinase inhibitors), monoclonal antibodies (and antigen-binding fragments thereof), peptide/polypeptide inhibitors (e.g. decoy ligands/receptors or peptide aptamers) and nucleic acids (e.g. antisense nucleic acid, splice-switching nucleic acids or nucleic acid aptamers). Inhibitors of signalling mediated by EGFR family members include agents that inhibit signalling through a direct effect on an EGFR family member, an interaction partner therefore, and/or a downstream factor involved in signalling mediated by the EGFR family member.
In some embodiments the antagonist of signalling mediated by an EGFR family member inhibits signalling mediated by one or more of EGFR, HER2, HER4 and HER3. Inhibitors of signalling mediated by EGFR family members are described e.g. in Yamaoka et al., Int. J. Mol. Sci. (2018), 19, 3491, which is hereby incorporated by reference in its entirety. In some embodiments the antagonist is a pan-ErbB inhibitor. In some embodiments the antagonist is an inhibitor of signalling mediated by EGFR (e.g. cetuximab, panitumumab, gefitinib, erlotinib, lapatinib, afatinib, brigatinib, icotinib, osimertinib, zalutumumab, vandetanib, necitumumab, nimotuzumab, dacomitinib, duligotuzumab or matuzumab). In some embodiments the antagonist is an inhibitor of signalling mediated by HER2 (e.g. trastuzumab, pertuzumab, lapatinib, neratinib, afatinib, dacomitinib, MM-111, MCLA-128 or margetuximab). In some embodiments the antagonist is an inhibitor of signalling mediated by HER3 (e.g. seribantumab, lumretuzumab, elgemtumab, KTN3379, AV-203, GSK2849330, REGN1400, MP-RM-1, EV20, duligotuzumab, MM-111, istiratumab, MCLA-128, patritumab, EZN-3920, RB200 or U3-1402). In some embodiments the antagonist is an inhibitor of signalling mediated by HER4 (e.g. lapatinib, ibrutinib, afatinib, dacomitinib or neratinib).
In some embodiments the antagonist of signalling mediated by an EGFR family member inhibits a downstream effector of signalling by an EGFR family member. Downstream effectors of signalling by an EGFR family members include e.g. P13K, AKT, KRAS, BRAF, MEK/ERK and mTOR. In some embodiments, the antagonist of signalling mediated by an EGFR family member is an inhibitor of the MAPK/ERK pathway. In some embodiments, the antagonist of signalling mediated by an EGFR family member is an inhibitor of the P13K/ATK/mTOR pathway. In some embodiments the antagonist is a P13K inhibitor (e.g. pictilisib, buparlisib, idelalisib, copanlisib or duvelisib). In some embodiments the antagonist is an AKT inhibitor (e.g. MK-2206, AZD5363, ipatasertib, VQD-002, perifosine or miltefosine). In some embodiments the antagonist is a BRAF inhibitor (e.g. vemurafenib, dabrafenib, SB590885, XL281, RAF265, encorafenib, GDC-0879, PLX-4720, sorafenib, or LGX818). In some embodiments the antagonist is a MEK/ERK inhibitor (e.g. trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, PD035901, or TAK-733). In some embodiments the antagonist is a mTOR inhibitor (e.g. rapamycin, deforolimus, temsirolimus, everolimus, ridaforolimus or sapanisertib).
In some embodiments, the cancer to be treated in accordance with an aspect of the present invention (including monotherapy or combination therapy) is a cancer which is resistant to treatment with an antagonist of signalling mediated by an EGFR family member (e.g. EGFR, HER2, HER4 and/or HER3), e.g. an antagonist as described in the preceding three paragraphs. In some embodiments the subject to be treated has a cancer which is resistant to treatment with an antagonist of signalling mediated by an EGFR family member. In some embodiments the subject to be treated has a cancer which has developed resistance to treatment with an antagonist of signalling mediated by an EGFR family member. In some embodiments the subject to be treated has a cancer which previously responded to treatment with an antagonist of signalling mediated by an EGFR family member, and which is now resistant to treatment with the antagonist. In some embodiments the subject to be treated has a cancer which has relapsed and/or progressed following treatment with an antagonist of signalling mediated by an EGFR family member. In some embodiments the subject to be treated has a cancer which initially responded to treatment with an antagonist of signalling mediated by an EGFR family member, but later progressed on said treatment.
In some embodiments a subject to be treated in accordance with the present invention may have been determined to have (i.e. may have been diagnosed as having) a cancer comprising cells having a mutation which causes increased expression of a ligand for HER3 (e.g. as described herein). In some embodiments the methods of the invention may comprise determining whether a subject has a cancer comprising cells having a mutation which causes increased expression of a ligand for HER3. In some embodiments, the methods comprise analysing nucleic acid from cells of a cancer. In some embodiments the methods comprise detecting a mutation which causes increased expression of a ligand for HER3.
The skilled person is readily able to identify cancers and subjects described herein. Such cancers and subjects may be identified e.g. through monitoring of the development/progression of the cancer (and/or correlates thereof) over time e.g. during the course of treatment with an antagonist of signalling mediated by an EGFR family member. In some embodiments, identification of such subjects/cancers may comprise analysis of a sample (e.g. a biopsy), e.g. in vitro. In some embodiments the cancer may be determined to comprise cells having a mutation which is associated with reduced susceptibility and/or resistance to treatment with the antagonist. In some embodiments the cancer may be determined to comprise cells having upregulated expression of an EGFR family member.
In particular embodiments, the cancer to be treated is a cancer which is resistant to treatment with an antagonist of signalling mediated by EGFR and/or HER2. In some embodiments the subject to be treated has a cancer which is resistant to treatment with an antagonist of signalling mediated by EGFR and/or HER2. In some embodiments the subject to be treated has a cancer which has developed resistance to treatment with an antagonist of signalling mediated by EGFR and/or HER2. In some embodiments the subject to be treated has a cancer which previously responded to treatment with an antagonist of signalling mediated by EGFR and/or HER2, and which is now resistant to treatment with the antagonist. In some embodiments the subject to be treated has a cancer which has relapsed and/or progressed following treatment with an antagonist of signalling mediated by EGFR and/or HER2. In some embodiments the subject to be treated has a cancer which initially responded to treatment with an antagonist of signalling mediated by EGFR and/or HER2, but later progressed on said treatment. In some embodiments the subject to be treated has a cancer associated with amplification of signalling of an EGFR family member, e.g. EGFR and/or HER2.
In particular embodiments, the cancer to be treated comprises mutation conferring resistance to treatment with an inhibitor of BRAF. In some embodiments, the mutation is mutation at BRAF V600. In some embodiments, the mutation is BRAF V600E or V600K. The cancer may be thyroid or colon cancer, e.g. RAS wild type colorectal cancer.
In particular embodiments, the cancer to be treated comprises mutation conferring resistance to treatment with an inhibitor of BRAF (e.g. mutation at BRAF V600), and the treatment comprises administration of vemurafenib or darafenib.
In some embodiments the antigen-binding molecule is administered in combination with an agent capable of inhibiting signalling mediated by an immune checkpoint molecule. In some embodiments the immune checkpoint molecule is e.g. PD-1, CTLA-4, LAG-3, VISTA, TIM-3, TIGIT or BTLA. In some embodiments the antigen-binding molecule is administered in combination with an agent capable of promoting signalling mediated by a costimulatory receptor. In some embodiments the costimulatory receptor is e.g. CD28, CD80, CD40L, CD86, OX40, 4-1BB, CD27 or ICOS.
Accordingly, the invention provides compositions comprising an article according to the present invention (e.g. an antigen-binding molecule according to the invention) and an agent capable of inhibiting signalling mediated by an immune checkpoint molecule. Also provided are compositions comprising the articles of the present invention and an agent capable of promoting signalling mediated by a costimulatory receptor. Also provided is the use of such compositions in methods of medical treatment and prophylaxis of diseases/conditions described herein.
Also provided are methods for treating/preventing diseases/conditions described herein comprising administering articles of the present invention an article according to the present invention (e.g. an antigen-binding molecule according to the invention) and an agent capable of inhibiting signalling mediated by an immune checkpoint molecule. Also provided are methods for treating/preventing diseases/conditions described herein comprising administering articles of the present invention an article according to the present invention (e.g. an antigen-binding molecule according to the invention) and an agent capable of promoting signalling mediated by a costimulatory receptor.
Agents capable of inhibiting signalling mediated by immune checkpoint molecules are known in the art, and include e.g. antibodies capable of binding to immune checkpoint molecules or their ligands, and inhibiting signalling mediated by the immune checkpoint molecule. Other agents capable of inhibiting signalling mediated by an immune checkpoint molecule include agents capable of reducing gene/protein expression of the immune checkpoint molecule or a ligand for the immune checkpoint molecule (e.g. through inhibiting transcription of the gene(s) encoding the immune checkpoint molecule/ligand, inhibiting post-transcriptional processing of RNA encoding the immune checkpoint molecule/ligand, reducing stability of RNA encoding the immune checkpoint molecule/ligand, promoting degradation of RNA encoding the immune checkpoint molecule/ligand, inhibiting post-translational processing of the immune checkpoint molecule/ligand, reducing stability the immune checkpoint molecule/ligand, or promoting degradation of the immune checkpoint molecule/ligand), and small molecule inhibitors.
Agents capable of promoting signalling mediated by costimulatory receptors are known in the art, and include e.g. agonist antibodies capable of binding to costimulatory receptors and triggering or increasing signalling mediated by the costimulatory receptor. Other agents capable of promoting signalling mediated by costimulatory receptors include agents capable of increasing gene/protein expression of the costimulatory receptor or a ligand for the costimulatory receptor (e.g. through promoting transcription of the gene(s) encoding the costimulatory receptor/ligand, promoting post-transcriptional processing of RNA encoding the costimulatory receptor/ligand, increasing stability of RNA encoding the costimulatory receptor/ligand, inhibiting degradation of RNA encoding the costimulatory receptor/ligand, promoting post-translational processing of the costimulatory receptor/ligand, increasing stability the costimulatory receptor/ligand, or inhibiting degradation of the costimulatory receptor/ligand), and small molecule agonists.
In particular embodiments the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signalling mediated by PD-1. The agent capable of inhibiting signalling mediated by PD-1 may be a PD-1- or PD-L1-targeted agent. The agent capable of inhibiting signalling mediated by PD-1 may e.g. be an antibody capable of binding to PD-1 or PD-L1 and inhibiting PD-1-mediated signalling.
In some embodiments, the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signalling mediated by CTLA-4. The agent capable of inhibiting signalling mediated by CTLA-4 may be a CTLA-4-targeted agent, or an agent targeted against a ligand for CTLA-4 such as CD80 or CD86. In some embodiments, the agent capable of inhibiting signalling mediated by CTLA-4 may e.g. be an antibody capable of binding to CTLA-4, CD80 or CD86 and inhibiting CTLA-4-mediated signalling.
In some embodiments, the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signalling mediated by LAG-3. The agent capable of inhibiting signalling mediated by LAG-3 may be a LAG-3-targeted agent, or an agent targeted against a ligand for LAG-3 such as MHC class 11. In some embodiments, the agent capable of inhibiting signalling mediated by LAG-3 may e.g. be an antibody capable of binding to LAG-3 or MHC class II and inhibiting LAG-3-mediated signalling.
In some embodiments, the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signalling mediated by VISTA. The agent capable of inhibiting signalling mediated by VISTA may be a VISTA-targeted agent, or an agent targeted against a ligand for VISTA such as VSIG-3 or VSIG-8. In some embodiments, the agent capable of inhibiting signalling mediated by VISTA may e.g. be an antibody capable of binding to VISTA, VSIG-3 or VSIG-8 and inhibiting VISTA-mediated signalling.
In some embodiments, the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signalling mediated by TIM-3. The agent capable of inhibiting signalling mediated by TIM-3 may be a TIM-3-targeted agent, or an agent targeted against a ligand for TIM-3 such as Galectin 9. In some embodiments, the agent capable of inhibiting signalling mediated by TIM-3 may e.g. be an antibody capable of binding to TIM-3 or Galectin 9 and inhibiting TIM-3-mediated signalling.
In some embodiments, the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signalling mediated by TIGIT. The agent capable of inhibiting signalling mediated by TIGIT may be a TIGIT-targeted agent, or an agent targeted against a ligand for TIGIT such as CD113, CD112 or CD155. In some embodiments, the agent capable of inhibiting signalling mediated by TIGIT may e.g. be an antibody capable of binding to TIGIT, CD113, CD112 or CD155 and inhibiting TIGIT-mediated signalling.
In some embodiments, the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signalling mediated by BTLA. The agent capable of inhibiting signalling mediated by BTLA may be a BTLA-targeted agent, or an agent targeted against a ligand for BTLA such as HVEM. In some embodiments, the agent capable of inhibiting signalling mediated by BTLA may e.g. be an antibody capable of binding to BTLA or HVEM and inhibiting BTLA-mediated signalling.
In some embodiments methods employing a combination of an antigen-binding molecule of the present invention and an agent capable of inhibiting signalling mediated by an immune checkpoint molecule (e.g. PD-1) provide an improved treatment effect as compared to the effect observed when either agent is used as a monotherapy. In some embodiments the combination of an antigen-binding molecule of the present invention and an agent capable of inhibiting signalling mediated by an immune checkpoint molecule (e.g. PD-1) provide a synergistic (i.e. super-additive) treatment effect.
Simultaneous administration refers to administration of the antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition and therapeutic agent together, for example as a pharmaceutical composition containing both agents (combined preparation), or immediately after each other and optionally via the same route of administration, e.g. to the same artery, vein or other blood vessel. Sequential administration refers to administration of one of the antigen-binding molecule/composition or therapeutic agent followed after a given time interval by separate administration of the other agent. It is not required that the two agents are administered by the same route, although this is the case in some embodiments. The time interval may be any time interval.
Chemotherapy and radiotherapy respectively refer to treatment of a cancer with a drug or with ionising radiation (e.g. radiotherapy using X-rays or γ-rays). The drug may be a chemical entity, e.g. small molecule pharmaceutical, antibiotic, DNA intercalator, protein inhibitor (e.g. kinase inhibitor), or a biological agent, e.g. antibody, antibody fragment, aptamer, nucleic acid (e.g. DNA, RNA), peptide, polypeptide, or protein. The drug may be formulated as a pharmaceutical composition or medicament.
The formulation may comprise one or more drugs (e.g. one or more active agents) together with one or more pharmaceutically acceptable diluents, excipients or carriers.
A treatment may involve administration of more than one drug. A drug may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. For example, the chemotherapy may be a co-therapy involving administration of two drugs, one or more of which may be intended to treat the cancer.
The chemotherapy may be administered by one or more routes of administration, e.g. parenteral, intravenous injection, oral, subcutaneous, intradermal or intratumoral.
The chemotherapy may be administered according to a treatment regime. The treatment regime may be a pre-determined timetable, plan, scheme or schedule of chemotherapy administration which may be prepared by a physician or medical practitioner and may be tailored to suit the patient requiring treatment. The treatment regime may indicate one or more of: the type of chemotherapy to administer to the patient; the dose of each drug or radiation; the time interval between administrations; the length of each treatment; the number and nature of any treatment holidays, if any etc. For a co-therapy a single treatment regime may be provided which indicates how each drug is to be administered.
Chemotherapeutic drugs may be selected from: Abemaciclib, Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, Acalabrutinib, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Afatinib Dimaleate, Afinitor (Everolimus), Akynzeo (Netupitant and Palonosetron Hydrochloride), Aldara (Imiquimod), Aldesleukin, Alecensa (Alectinib), Alectinib, Alemtuzumab, Alimta (Pemetrexed Disodium), Aliqopa (Copanlisib Hydrochloride), Alkeran for Injection (Melphalan Hydrochloride), Alkeran Tablets (Melphalan), Aloxi (Palonosetron Hydrochloride), Alunbrig (Brigatinib), Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil), Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, Aredia (Pamidronate Disodium), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arsenic Trioxide, Arzerra (Ofatumumab), Asparaginase Erwinia chrysanthemi, Atezolizumab, Avastin (Bevacizumab), Avelumab, Axicabtagene Ciloleucel, Axitinib, Azacitidine, Bavencio (Avelumab), BEACOPP, Becenum (Carmustine), Beleodaq (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, Besponsa (Inotuzumab Ozogamicin), Bevacizumab, Bexarotene, Bexxar (Tositumomab and Iodine I 131 Tositumomab), Bicalutamide, BiCNU (Carmustine), Bleomycin, Blinatumomab, Blincyto (Blinatumomab), Bortezomib, Bosulif (Bosutinib), Bosutinib, Brentuximab Vedotin, Brigatinib, BuMel, Busulfan, Busulfex (Busulfan), Cabazitaxel, Cabometyx (Cabozantinib-S-Malate), Cabozantinib-S-Malate, CAF, Calquence (Acalabrutinib), Campath (Alemtuzumab), Camptosar (Irinotecan Hydrochloride), Capecitabine, CAPOX, Carac (Fluorouracil-Topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, Carmubris (Carmustine), Carmustine, Carmustine implant, Casodex (Bicalutamide), CEM, Ceritinib, Cerubidine (Daunorubicin Hydrochloride), Cervarix (Recombinant HPV Bivalent Vaccine), Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Cladribine, Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Clolar (Clofarabine), CMF, Cobimetinib, Cometriq (Cabozantinib-S-Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (Dactinomycin), Cotellic (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cyramza (Ramucirumab), Cytarabine, Cytarabine Liposome, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin, Daratumumab, Darzalex (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Decitabine, Defibrotide Sodium, Defitelio (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DepoCyt (Cytarabine Liposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, Doxil (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, Dox-SL (Doxorubicin Hydrochloride Liposome), DTIC-Dome (Dacarbazine), Durvalumab, Efudex (Fluorouracil-Topical), Elitek (Rasburicase), Ellence (Epirubicin Hydrochloride), Elotuzumab, Eloxatin (Oxaliplatin), Eltrombopag Olamine, Emend (Aprepitant), Empliciti (Elotuzumab), Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride, EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Erivedge (Vismodegib), Erlotinib Hydrochloride, Erwinaze (Asparaginase Erwinia chrysanthemi), Ethyol (Amifostine), Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposome), Everolimus, Evista (Raloxifene Hydrochloride), Evomela (Melphalan Hydrochloride), Exemestane, 5-FU (Fluorouracil Injection), 5-FU (Fluorouracil-Topical), Fareston (Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Fluoroplex (Fluorouracil-Topical), Fluorouracil Injection, Fluorouracil-Topical, Flutamide, Folex (Methotrexate), Folex PFS (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), FU-LV, Fulvestrant, Gardasil (Recombinant HPV Quadrivalent Vaccine), Gardasil 9 (Recombinant HPV Nonavalent Vaccine), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, Gemtuzumab Ozogamicin, Gemzar (Gemcitabine Hydrochloride), Gilotrif (Afatinib Dimaleate), Gleevec (Imatinib Mesylate), Gliadel (Carmustine Implant), Gliadel wafer (Carmustine Implant), Glucarpidase, Goserelin Acetate, Halaven (Eribulin Mesylate), Hemangeol (Propranolol Hydrochloride), Herceptin (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, Hycamtin (Topotecan Hydrochloride), Hydrea (Hydroxyurea), Hydroxyurea, Hyper-CVAD, Ibrance (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig (Ponatinib Hydrochloride), Idamycin (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, Idhifa (Enasidenib Mesylate), Ifex (Ifosfamide), Ifosfamide, Ifosfamidum (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate, Imbruvica (Ibrutinib), Imfinzi (Durvalumab), Imiquimod, Imlygic (Talimogene Laherparepvec), Inlyta (Axitinib), Inotuzumab Ozogamicin, Interferon Alfa-2b, Recombinant, Interleukin-2 (Aldesleukin), Intron A (Recombinant Interferon Alfa-2b), Iodine I 131 Tositumomab and Tositumomab, Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposome, Istodax (Romidepsin), Ixabepilone, Ixazomib Citrate, Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate), JEB, Jevtana (Cabazitaxel), Kadcyla (Ado-Trastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance (Palifermin), Keytruda (Pembrolizumab), Kisqali (Ribociclib), Kymriah (Tisagenlecleucel), Kyprolis (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, Lartruvo (Olaratumab), Lenalidomide, Lenvatinib Mesylate, Lenvima (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, Leukeran (Chlorambucil), Leuprolide Acetate, Leustatin (Cladribine), Levulan (Aminolevulinic Acid), Linfolizin (Chlorambucil), LipoDox (Doxorubicin Hydrochloride Liposome), Lomustine, Lonsurf (Trifluridine and Tipiracil Hydrochloride), Lupron (Leuprolide Acetate), Lupron Depot (Leuprolide Acetate), Lupron Depot-Ped (Leuprolide Acetate), Lynparza (Olaparib), Marqibo (Vincristine Sulfate Liposome), Matulane (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, Mekinist (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, Mesnex (Mesna), Methazolastone (Temozolomide), Methotrexate, Methotrexate LPF (Methotrexate), Methylnaltrexone Bromide, Mexate (Methotrexate), Mexate-AQ (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, Mitozytrex (Mitomycin C), MOPP, Mozobil (Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (Paclitaxel Albumin-stabilized Nanoparticle Formulation), Navelbine (Vinorelbine Tartrate), Necitumumab, Nelarabine, Neosar (Cyclophosphamide), Neratinib Maleate, Nerlynx (Neratinib Maleate), Netupitant and Palonosetron Hydrochloride, Neulasta (Pegfilgrastim), Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate), Nilandron (Nilutamide), Nilotinib, Nilutamide, Ninlaro (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim), Obinutuzumab, Odomzo (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Ondansetron Hydrochloride, Onivyde (Irinotecan Hydrochloride Liposome), Ontak (Denileukin Diftitox), Opdivo (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-Intron (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor, Pomalidomide, Pomalyst (Pomalidomide), Ponatinib Hydrochloride, Portrazza (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride, Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopag Olamine), Propranolol Hydrochloride, Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Purixan (Mercaptopurine), [No Entries], Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, Relistor (Methylnaltrexone Bromide), R-EPOCH, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Ribociclib, R-ICE, Rituxan (Rituximab), Rituxan Hycela (Rituximab and Hyaluronidase Human), Rituximab, Rituximab and Hyaluronidase Human, Rolapitant Hydrochloride, Romidepsin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride), Rubraca (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib Phosphate, Rydapt (Midostaurin), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, Somatuline Depot (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, Sprycel (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Peginterferon Alfa-2b), Sylvant (Siltuximab), Synribo (Omacetaxine Mepesuccinate), Tabloid (Thioguanine), TAC, Tafinlar (Dabrafenib), Tagrisso (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, Tarabine PFS (Cytarabine), Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Tecentriq (Atezolizumab), Temodar (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, Thalomid (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, Tolak (Fluorouracil-Topical), Topotecan Hydrochloride, Toremifene, Torisel (Temsirolimus), Tositumomab and Iodine I 131 Tositumomab, Totect (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, Treanda (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, Trisenox (Arsenic Trioxide), Tykerb (Lapatinib Ditosylate), Unituxin (Dinutuximab), Uridine Triacetate, VAC, Valrubicin, Valstar (Valrubicin), Vandetanib, VAMP, Varubi (Rolapitant Hydrochloride), Vectibix (Panitumumab), VeIP, Velban (Vinblastine Sulfate), Velcade (Bortezomib), Velsar (Vinblastine Sulfate), Vemurafenib, Venclexta (Venetoclax), Venetoclax, Verzenio (Abemaciclib), Viadur (Leuprolide Acetate), Vidaza (Azacitidine), Vinblastine Sulfate, Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP, Vismodegib, Vistogard (Uridine Triacetate), Voraxaze (Glucarpidase), Vorinostat, Votrient (Pazopanib Hydrochloride), Vyxeos (Daunorubicin Hydrochloride and Cytarabine Liposome), Wellcovorin (Leucovorin Calcium), Xalkori (Crizotinib), Xeloda (Capecitabine), XELIRI, XELOX, Xgeva (Denosumab), Xofigo (Radium 223 Dichloride), Xtandi (Enzalutamide), Yervoy (Ipilimumab), Yescarta (Axicabtagene Ciloleucel), Yondelis (Trabectedin), Zaltrap (Ziv-Aflibercept), Zarxio (Filgrastim), Zejula (Niraparib Tosylate Monohydrate), Zelboraf (Vemurafenib), Zevalin (lbritumomab Tiuxetan), Zinecard (Dexrazoxane Hydrochloride), Ziv-Aflibercept, Zofran (Ondansetron Hydrochloride), Zoladex (Goserelin Acetate), Zoledronic Acid, Zolinza (Vorinostat), Zometa (Zoledronic Acid), Zydelig (Idelalisib), Zykadia (Ceritinib) and Zytiga (Abiraterone Acetate).
In some embodiments the antigen-binding molecule of the invention is administered in combination with one or more of: a HER2 inhibitor (e.g. an anti-HER2 antibody), an EGFR inhibitor (e.g. an anti-EGFR antibody), an alkylating agent, a pyrimidine analogue, a thymidylate synthase inhibitor (or precursor thereof), and/or an androgen receptor inhibitor.
In some embodiments the antigen-binding molecule of the invention is administered in combination with one or more of: trastuzumab, cetuximab, cisplatin, 5-FU or capecitabine. In some embodiments the antigen-binding molecule of the invention is administered in combination with trastuzumab and cisplatin, and 5-FU or capecitabine.
In some embodiments the antigen-binding molecule of the invention is administered in combination with an anti-EGFR antibody. In some embodiments the antigen-binding molecule of the invention is administered in combination with cetuximab. Administration in combination with cetuximab is contemplated in particular for the treatment of head and neck cancer (e.g. head and neck squamous cell carcinoma) or colorectal cancer (e.g. RAS wild type colorectal cancer).
In some embodiments the antigen-binding molecule of the invention is administered in combination with enzalutamide or another androgen receptor inhibitor (e.g. apalutamide, bicalutamide, flutamide, nilutamide, or darolutamide). Administration in combination with enzalutamide or other androgen receptor inhibitor is contemplated in particular for the treatment of prostate cancer (e.g. castration resistant prostate cancer).
In some embodiments the antigen-binding molecule of the invention is administered in combination with an anti-HER2 antibody. In some embodiments the antigen-binding molecule of the invention is administered in combination with trastuzumab. Administration in combination with trastuzumab is contemplated in particular for the treatment of breast cancer (e.g. triple negative breast cancer) or gastric cancer.
In some embodiments the antigen-binding molecule of the invention is administered in combination with another anti-HER3 antibody. In some embodiments the antigen-binding molecule of the invention is administered in combination with one or more of: MM121 (SAR256212/seribantumab; NCT04383210, NCT04790695), LJM-716 (elgemtumab; NCT02167854), AV203 (Sarantopoulos. J et al. 2014, Journal of Clinical Oncology, 32: 11113-13), CDX-3379/KTN3379 (NCT03254927, Duvvuri et al. Clin Cancer Res. 2019 Oct. 1; 25(19):5752-5758, NCT03254927), RG7116 (lumretuzumab; Meulendijks et al. 2016, Clin Cancer Res, 22: 877-85), RG7597 (duligotuzumab; Juric et al. 2015, Clin Cancer Res, 21: 2462-70), U3-1287 (patritumab/AMG 888; LoRusso et al. 2013, Clin Cancer Res, 19: 3078-87), GSK2849330 (NCT01966445), ISU-104 (Kim et al. 2019, Annals of Oncology, 30; NCT03552406), REGN-1400 (Papadopoulos et al. 2014, Journal of Clinical Oncology, 32: 2516-16), MCLA-128 (zenocutuzumab; Alsina et al. 2018 Annals of Oncology, 29; Calvo et al. AACR 107th Annual Meeting 2016; Apr. 16-20, 2016, NCT03321981, NCT02912949), MM-111, MM-141 (Calvo et al. 2016 supra, Kundranda et al. 2020, Ann Oncol, 31: 79-87), Varlitinib (NCT02992340, NCT03499626) or BDTX-189 (NCT04209465), with all references hereby incorporated by reference in their entirety.
Multiple doses of the antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition may be provided. References to “antigen-binding molecule” in the following paragraphs also encompass one or more other articles according to the disclosure (polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition), and vice versa. One or more, or each, of the doses may be accompanied by simultaneous or sequential administration of another therapeutic agent.
Multiple doses may be separated by a predetermined time interval, which may be selected to be one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or31 days, or 1, 2, 3, 4, 5, or 6 months. By way of example, doses may be given once every 7, 14, 21 or 28 days (plus or minus 3, 2, or 1 days: such as 4, 5, 6, 8, 9 or 10 days; 11, 12, 13, 15, 16 or 17 days; 18, 19, 20, 22, 23, or 24 days; or 25, 26, 27, 29, 30 or 31 days). That is, there may be one treatment event every 7 days, every 14 days, every 21 days, or every 28 days. There may be a treatment holiday between doses/administrations of about 7 days (plus or minus 3, 2, or 1 days), about 14 days (plus or minus 3, 2, or 1 days), about 21 days (plus or minus 3, 2, or 1 days), or about 28 days (plus or minus 3, 2, or 1 days), respectively.
In some aspects of the present disclosure, the antigen-binding molecule is administered once every week (e.g. once every 7 days plus or minus 3, 2, or 1 days), once every two weeks (e.g. once every 14 days plus or minus 3, 2, or 1 days), once every three weeks (e.g. once every 21 days plus or minus 3, 2, or 1 days) or once every four weeks (e.g. once every 28 days plus or minus 3, 2, or 1 days). That is, there may be one treatment event/administration every one week, every two weeks, every 3 weeks, or every 4 weeks.
In some embodiments there may be multiple administrations of the antigen-binding molecule per week, per two weeks, per three weeks or per four weeks. In some embodiments, the antigen-binding molecule is administered four times every 28 days/4 weeks, twice every 28 days/4 weeks, three times every 28 days/4 weeks, or 3 times every 21 days/3 weeks. Treatment may continue for 1, 2, 3, 4, 5, 6 or more months. Treatment may continue for 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or more weeks, or for 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 or more weeks.
In some embodiments the antigen-binding molecule is administered once every week, every four weeks (i.e. four doses in total per four-week period). That is, the antigen-binding molecule may be administered once every 7 days (plus/minus 3, 2, or 1 days), over a period of 28 days. This may be referred to as one administration ‘cycle’. One cycle may be 28 days (plus/minus 3, 2, or 1 days), or one month. There may be four doses per cycle. The antigen-binding molecule may be administered once every week for one, two, three, four, five, six or more cycles (that is, once every week for 1, 2, 3, 4, 5, 6 or more months, or periods of 28 days, or once every week for 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or more weeks).
In some embodiments the antigen-binding molecule is administered once every two weeks (e.g. biweekly) every four weeks (i.e. two doses in total per four-week period). That is, the antigen-binding molecule may be administered once every 14 days (plus/minus 3, 2, or 1 days), over a period of 28 days. This may be referred to as one administration ‘cycle’. One cycle may be 28 days (plus/minus 3, 2, or 1 days), or one month. There may be two doses per cycle. The antigen-binding molecule may be administered once every two weeks for one, two, three, four, five, six or more cycles (that is, once every 2 weeks over 1, 2, 3, 4, 5, 6 or more months, or periods of 28 days, or once every 2 weeks for 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or more weeks).
In some embodiments the antigen-binding molecule is administered once every three weeks. That is, the antigen-binding molecule may be administered once every 21 days (plus/minus 3, 2, or 1 days). This may be referred to as one administration ‘cycle’. One cycle may be 21 days (plus/minus 3, 2, or 1 days). There may be one dose per cycle. The antigen-binding molecule may be administered once every week for one, two, three, four, five, six or more cycles (that is, once every 1, 2, 3, 4, 5, 6 or more periods of 21 days, or once every three weeks for 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 or more weeks).
In some embodiments the antigen-binding molecule is administered once every four weeks. That is, the antigen-binding molecule may be administered once every 28 days (plus/minus 3, 2, or 1 days). This may be referred to as one administration ‘cycle’. One cycle may be 28 days (plus/minus 3, 2, or 1 days), or one month. There may be one dose per cycle. The antigen-binding molecule may be administered once every week for one, two, three, four, five, six or more cycles (that is, once every 1, 2, 3, 4, 5, 6 or more months, or periods of 28 days, or once every 4 weeks for 4, 8, 12, 16, 20, 24, 28, or more weeks).
In some embodiments, administration (e.g. in the schedules above, e.g. an administration once per 7, 14, 21 or 28 days, and/or e.g. total administration(s) over one or more periods of 21 or 28 days) comprises administering at least 150 mg of the antigen-binding molecule. In some embodiments, each administration comprises administering at least 175 mg, at least 200 mg, at least 225 mg, at least 250 mg, at least 275 mg, at least 300 mg, at least 325 mg, at least 350 mg, at least 375 mg, at least 400 mg, at least 425 mg, at least 450 mg, at least 475 mg, at least 500 mg, at least 525 mg, at least 550 mg, or at least 575 mg of the antigen-binding molecule. In some embodiments each administration comprises administering at least 600 mg of the antigen-binding molecule. In some embodiments each administration comprises administering no more than 3000 mg of the antigen-binding molecule.
In some embodiments, administration (e.g. in the schedules above, e.g. an administration once per 7, 14, 21 or 28 days, and/or e.g. total administration(s) over one or more periods of 21 or 28 days) comprises administering at least 600 mg of the antigen-binding molecule. In some embodiments, each administration comprises administering at least 625 mg, at least 650 mg, at least 675 mg, at least 700 mg, at least 725 mg, at least 750 mg, at least 775 mg, at least 800 mg, at least 825 mg, at least 850 mg, at least 875 mg, at least 900 mg, at least 925 mg, at least 950 mg, at least 975 mg, at least 1000 mg, at least 1050 mg, at least 1100 mg, at least 1150 mg, at least 1200 mg, at least 1250 mg, at least 1300 mg, at least 1350 mg, at least 1400 mg, at least 1450 mg, at least 1500 mg, at least 1550 mg, at least 1600 mg, at least 1650 mg, at least 1700 mg, at least 1750 mg, at least 1800 mg, at least 1850 mg, at least 1900 mg, at least 1950 mg, at least 2000 mg, at least 2050 mg, at least 2100 mg, at least 2150 mg, at least 2200 mg, at least 2250 mg, at least 2300 mg, at least 2350 mg, at least 2400 mg, at least 2450 mg, at least 2500 mg, at least 2550 mg, at least 2600 mg, at least 2650 mg, at least 2700 mg, at least 2750 mg, at least 2800 mg, at least 2850 mg, at least 2900 mg, or at least 2950 mg of the antigen-binding molecule.
In some embodiments each administration (e.g. in the schedules above, e.g. an administration once per 7, 14, 21 or 28 days) comprises administering 150-600 mg of the antigen-binding molecule. In some embodiments each administration comprises administering 600-3000 mg of the antigen-binding molecule. In some embodiments each administration comprises administering 900-3000 mg of the antigen-binding molecule. In some embodiments each administration comprises administering 900-2400 mg of the antigen-binding molecule. In some embodiments each administration comprises administering 1500-3000 mg of the antigen-binding molecule. In some embodiments each administration comprises administering 1500-2400 mg of the antigen-binding molecule. In some embodiments each administration comprises administering 1500-2100 mg of the antigen-binding molecule. In some embodiments each administration comprises administering about 1800 mg of the antigen-binding molecule. In some embodiments each administration comprises administering about 2100 mg of the antigen-binding molecule.
In some embodiments an administration comprises administering about 1800 mg of the antigen-binding molecule every 7 or 14 days (i.e. once every week or once every 2 weeks). In some embodiments an administration comprises administering about 2100 mg of the antigen-binding molecule every 7 or 14 days (i.e. once every week or once every 2 weeks).
Each administration cycle, comprising one or more administrations of antigen-binding molecule over a period of 21 or 28 days as described above, results in a total amount of administered antigen-binding molecule. In some embodiments each administration cycle (i.e. comprising one or more administrations of antigen-binding molecule over a period of 21 or 28 days, as above) comprises the administration of at least 150 mg of the antigen-binding molecule (i.e. via one administration, or in total after two or more administrations). In some embodiments each administration cycle comprises the administration of at least 300 mg of the antigen-binding molecule. In some embodiments each administration cycle comprises the administration of at least 600 mg of the antigen-binding molecule. In some embodiments each administration cycle comprises the administration of at least 900 mg of the antigen-binding molecule. In some embodiments each administration cycle comprises the administration of at least 1500 mg of the antigen-binding molecule. In some embodiments each administration cycle comprises the administration of at least 1800 mg of the antigen-binding molecule. In some embodiments each administration cycle comprises the administration of at least 2100 mg of the antigen-binding molecule. In some embodiments each administration cycle comprises the administration of at least 2400 mg of the antigen-binding molecule. In some embodiments each administration cycle comprises the administration of at least 2700 mg of the antigen-binding molecule.
In some embodiments each administration cycle comprises the administration of 150-600 mg of the antigen-binding molecule. In some embodiments each administration cycle comprises the administration of 600-3000 mg of the antigen-binding molecule. In some embodiments each administration cycle comprises the administration of 900-3000 mg of the antigen-binding molecule. In some embodiments each administration cycle comprises the administration of 900-2400 mg of the antigen-binding molecule. In some embodiments each administration cycle comprises the administration of 1500-3000 mg of the antigen-binding molecule. In some embodiments each administration cycle comprises the administration of 1500-2400 mg of the antigen-binding molecule. In some embodiments each administration cycle comprises the administration of 1500-2100 mg of the antigen-binding molecule.
In some embodiments each administration cycle comprises the administration of 3000-12000 mg of the antigen-binding molecule. In some embodiments each administration cycle comprises the administration of 5400-12000 mg of the antigen-binding molecule. In some embodiments each administration cycle comprises the administration of 3600-8400 mg of the antigen-binding molecule. In some embodiments each administration cycle comprises the administration of 4800-7200 mg of the antigen-binding molecule.
In some embodiments each administration cycle comprises the administration of about 4800 mg of the antigen-binding molecule. In some embodiments each administration cycle comprises the administration of about 7200 mg of the antigen-binding molecule. In some embodiments each administration cycle comprises the administration of about 8400 mg of the antigen-binding molecule. In some embodiments each administration cycle comprises the administration of about 12000 mg of the antigen-binding molecule. The antigen-binding molecule may be administered in multiple doses (e.g. once per week) to reach a total amount of antigen-binding molecule administered per administration cycle.
In some embodiments each administration cycle comprises the administration of no more than 2100 mg of the antigen-binding molecule. In some embodiments each administration cycle comprises the administration of no more than 2400 mg of the antigen-binding molecule. In some embodiments each administration cycle comprises the administration of no more than 3000 mg of the antigen-binding molecule. In some embodiments each administration cycle comprises the administration of no more than 5000 mg of the antigen-binding molecule.
An “administration cycle” is used herein to refer to a period of 21 or 28 days (e.g. 3 or 4 weeks). Treatment described herein may be administered according to one or more dosing schedules provided herein over 1, 2, 3, 4, 5, 6 or more administration cycles. Where an administration cycle is described as “comprising the administration of x mg antigen-binding molecule”, this may be written in the alternative as “x mg of the antigen-binding molecule is administered every 21 or 28 days” or “the antigen-binding molecule is administered at a dose of x mg every 21 or 28 days”.
The antigen-binding molecule may be administered in a composition, e.g. as described herein. Administration of the antigen-binding molecule or composition may be topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral or transdermal routes of administration which may include injection or infusion.
In some embodiments, administration of the antigen-binding molecule is intravenous injection or infusion. The antigen-binding molecule may be administered intravenously over 120 minutes. The antigen-binding molecule may be administered intravenously over 60 minutes. The antigen-binding molecule may be administered intravenously over 45-150 minutes, 50-135 minutes, or 60-120 minutes, or over any time period within those limits.
The invention also provides the articles of the present invention for use in methods for detecting, localizing or imaging HER3, or cells expressing HER3.
The antigen-binding molecules described herein may be used in methods that involve the antigen-binding molecule to HER3. Such methods may involve detection of the bound complex of the antigen-binding molecule and HER3.
As such, a method is provided, comprising contacting a sample containing, or suspected to contain, HER3, and detecting the formation of a complex of the antigen-binding molecule and HER3. Also provided is a method comprising contacting a sample containing, or suspected to contain, a cell expressing HER3, and detecting the formation of a complex of the antigen-binding molecule and a cell expressing HER3.
Suitable method formats are well known in the art, including immunoassays such as sandwich assays, e.g. ELISA. The methods may involve labelling the antigen-binding molecule, or target(s), or both, with a detectable moiety, e.g. a fluorescent label, phosphorescent label, luminescent label, immuno-detectable label, radiolabel, chemical, nucleic acid or enzymatic label as described herein. Detection techniques are well known to those of skill in the art and can be selected to correspond with the labelling agent.
Methods of this kind may provide the basis of methods for the diagnostic and/or prognostic evaluation of a disease or condition, e.g. a cancer. Such methods may be performed in vitro on a patient sample, or following processing of a patient sample. Once the sample is collected, the patient is not required to be present for the in vitro method to be performed, and therefore the method may be one which is not practised on the human or animal body. In some embodiments the method is performed in vivo.
Detection in a sample may be used for the purpose of diagnosis of a disease/condition (e.g. a cancer), predisposition to a disease/condition, or for providing a prognosis (prognosticating) for a disease/condition, e.g. a disease/condition described herein. The diagnosis or prognosis may relate to an existing (previously diagnosed) disease/condition.
Such methods may involve detecting or quantifying HER3 or cells expressing HER3, e.g. in a patient sample. Where the method comprises quantifying the relevant factor, the method may further comprise comparing the determined amount against a standard or reference value as part of the diagnostic or prognostic evaluation. Other diagnostic/prognostic tests may be used in conjunction with those described herein to enhance the accuracy of the diagnosis or prognosis or to confirm a result obtained by using the tests described herein.
A sample may be taken from any tissue or bodily fluid. The sample may comprise or may be derived from: a quantity of blood; a quantity of serum derived from the individual's blood which may comprise the fluid portion of the blood obtained after removal of the fibrin clot and blood cells; a tissue sample or biopsy; pleural fluid; cerebrospinal fluid (CSF); or cells isolated from said individual. In some embodiments, the sample may be obtained or derived from a tissue or tissues which are affected by the disease/condition (e.g. tissue or tissues in which symptoms of the disease manifest, or which are involved in the pathogenesis of the disease/condition).
The present invention also provides methods for selecting/stratifying a subject for treatment with a HER3-targeted agent. In some embodiments a subject is selected for treatment/prevention in accordance with the invention, or is identified as a subject which would benefit from such treatment/prevention, based on detection/quantification of HER3, or cells expressing HER3, e.g. in a sample obtained from the individual.
The subject in accordance with aspects the invention described herein may be any animal or human. The subject is preferably mammalian, more preferably human. The subject may be a non-human mammal, but is more preferably human. The subject may be male or female. The subject may be a patient. A subject may have been diagnosed with a disease or condition requiring treatment (e.g. a cancer), may be suspected of having such a disease/condition, or may be at risk of developing/contracting such a disease/condition.
In embodiments according to the present invention the subject is preferably a human subject. In some embodiments, the subject to be treated according to a therapeutic or prophylactic method of the invention herein is a subject having, or at risk of developing, a cancer. In embodiments according to the present invention, a subject may be selected for treatment according to the methods based on characterisation for certain markers of such disease/condition.
Subjects according to the present disclosure may comprise a cancer (e.g. cancer cells) expressing or overexpressing HER3, EGFR, HER2, HER4, NRG1, NRG2, and/or a ligand for HER3, e.g. as described herein. Subjects according to the present disclosure may comprise cells expressing an NRG gene fusion, e.g. as described herein. Subjects according to the present disclosure may comprise tumour cells that express or overexpress HER3, EGFR, HER2, HER4, NRG1, NRG2, and/or a ligand for HER3, and/or comprise an NRG gene fusion e.g. as described herein.
Subjects may comprise advanced or metastatic solid tumours, e.g. confirmed histologically (e.g via immunohistochemistry on tumour biopsy). The tumours may be resistant or refractory to conventional treatment, or no conventional therapy may exist. The subject may have a cancer that is known, or tested, to express HER3, e.g. bladder cancer, triple negative breast cancer, castration resistant prostate cancer, cervical cancer, RAS wild type colorectal cancer, endometrial cancer, gastric cancer, hepatocellular carcinoma (HCC), melanoma, non-small cell lung cancer (NSCLC), oesophageal cancer, ovarian cancer, pancreatic cancer, and/or squamous cell cancers of the head and neck (e.g. HNSCC).
Methods according to the present disclosure may comprise detecting a cancer/cancer cells expressing or overexpressing HER3, EGFR, HER2, HER4, NRG1, NRG2, a ligand for HER3 and/or an NRG gene fusion, e.g. as described herein, e.g. in a sample obtained from the subject. Methods according to the present disclosure may comprise selecting a subject for treatment based on the detection of said cancer/cancer cells/expression. For example, the presence of cancer/cancer cells expressing or overexpressing one or more of HER3, EGFR, HER2, HER4, NRG1, NRG2, a ligand for HER3, and/or an NRG gene fusion may indicate that a subject is suitable for treatment using an antigen-binding molecule disclosed herein.
Methods according to the present disclosure may comprise detecting other biomarkers, e.g. phosphorylated HER3 (pHER3), p70S6K activity, Ki67 expression, presence of cleaved caspase 3, circulating tumour markers including cell free DNA (cfDNA) alteration allele fraction/tumour fraction (e.g. tumour-derived cfDNA such as ctDNA), soluble HER3, soluble NRG1, P13/MAPK pathway activity and/or P13/MAPK pathway mutations. In some embodiments, expression/presence/activity of pHER3, p70S6K, Ki67, cleaved caspase 3, ctDNA, soluble HER3, soluble NRG1, P13/MAPK pathway activity and/or P13/MAPK pathway mutations in a subject (e.g. in a sample obtained from the subject) indicates that the subject is suitable for treatment using an antigen-binding molecule disclosed herein. In some embodiments, reduced expression/presence/activity of pHER3, p70S6K, Ki67, cleaved caspase 3, ctDNA, soluble HER3, soluble NRG1, P13/MAPK pathway activity and/or P13/MAPK pathway mutations in a subject (e.g. in a sample obtained from the subject; e.g. compared to a previous sample obtained from the subject) indicates a reduction/improvement in the development, progression or pathology of a cancer, or another positive outcome e.g. an outcome as described herein.
Expression of HER3, pHER3, another EGFR family member, a ligand for HER3 (e.g. NRG1), p70S6K, Ki67, and/or cleaved caspase 3 may be detected using e.g. standard immunohistochemistry techniques on tumour tissue obtained by biopsy. The presence of an NRG gene fusion in cells, the level of cfDNA/ctDNA in plasma, or P13/MAPK pathway activity and/or mutations in tumour tissue may be detected using e.g. next generation sequencing. Circulating soluble HER3 and/or soluble NRG1 may be detected using e.g. standard ELISA techniques in plasma/serum samples.
One or more tumour serum markers (or panels of markers) may be evaluated before, during and after the subject receives treatment, as appropriate for the subject's tumour type, including but not limited to CA-125, HE4 and OVA1 in ovarian cancer; CEA in colorectal cancer; CA19-9 in pancreatic and gastric cancer; PSA, PAP, PCA3, the Oncotype DX GPS signature and the Prolaris signature in prostate cancer; BTA, FGFR2 and/or FGFR3 gene mutations, NMP22, and chromosomes 3, 7, 17, and 9p21 in bladder cancer; ALK gene rearrangements and overexpression, EGFR gene mutation, NSE, PD-L1 and ROS1 gene rearrangement in NSCLC; BRCA1 and/or BRCA2 in ovarian and breast cancers; BRAF V600 (e.g. BRAF V600E/) and KRAS mutations in e.g. colorectal cancer and NSCLC; CA15-3/CA27.29, ER/PR, uPA, PAl-1 and the Mammaprint signature in breast cancer; cytokeratin fragment 21-1 in lung cancer; DCP in HCC, DPD mutation in breast, colorectal, gastric and pancreatic cancers; thyroglobulin in thyroid cancer; and NTRK gene fusion in any solid tumour.
Any biomarker described herein may be detected before, during and/or after treatment using the methods disclosed herein, e.g. to select a suitable subject for treatment and/or to monitor the course or success of the treatment. Detection of a biomarker after treatment may be compared with detection of that biomarker before treatment.
In some aspects of the invention described herein a kit of parts is provided. In some embodiments the kit may have at least one container having a predetermined quantity of an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein.
In some embodiments the kit comprises a 50 mg/mL solution of antigen-binding molecule, e.g. in a composition according to the present disclosure. In some embodiments the kit comprises 50 mg/mL antigen binding molecule in a composition comprising 20 mM histidine, 8% (w/v) sucrose, 0.02% (w/v) polysorbate 80 at pH 5.8.
The composition comprising 50 mg/mL antigen-binding molecule may be diluted before use. The kit may comprise instructions for dilution. The kit may comprise 0.9% sodium chloride (NaCl) for use in diluting the composition, e.g. to arrive at a composition suitable for intravenous administration. The kit may comprise instructions that the diluted composition must be administered to a patient within 48 hours of the time of initial dilution.
The kit may comprise a composition comprising antigen-binding molecule at a concentration of at least 1.2 mg/mL, e.g. provided as is, or after dilution with NaCl as above.
The antigen-binding molecule, or other article of the disclosure, may be lyophilised (i.e. the container may comprise lyophilised antigen-binding molecule or other article). The lyophilised agent may be reconstituted in a composition buffer, e.g. according to the present disclosure. The kit may comprise instructions for reconstituting the antigen-binding molecule/other article.
The container may be any suitable container, e.g. a glass vial.
In some embodiments, the kit may comprise materials for producing an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein.
The kit may provide the antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition together with instructions for administration to a patient in order to treat a specified disease/condition, e.g. using a dose/dosing regime as described herein and/or to treat a disease/condition as described herein.
In some embodiments the kit may further comprise at least one container having a predetermined quantity of another therapeutic agent (e.g. anti-infective agent or chemotherapy agent). In such embodiments, the kit may also comprise a second medicament or pharmaceutical composition such that the two medicaments or pharmaceutical compositions may be administered simultaneously or separately such that they provide a combined treatment for the specific disease or condition. The therapeutic agent may also be formulated so as to be suitable for injection or infusion to a tumor or to the blood. The therapeutic agent may be any such agent described herein, such as cetuximab, enzalutamide or another androgen receptor inhibitor, or trastuzumab.
As used herein, “sequence identity” refers to the percent of nucleotides/amino acid residues in a subject sequence that are identical to nucleotides/amino acid residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum percent sequence identity between the sequences. Pairwise and multiple sequence alignment for the purposes of determining percent sequence identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Söding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780 software. When using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used.
The following numbered paragraphs (paras) provide further statements of features and combinations of features which are contemplated in connection with the present invention:
1. An antigen-binding molecule, optionally isolated, which is capable of binding to HER3 in extracellular region subdomain II.
2. The antigen-binding molecule according to para 1, wherein the antigen-binding molecule inhibits interaction between HER3 and an interaction partner for HER3.
3. The antigen-binding molecule according to para 1 or para 2, wherein the antigen-binding molecule is capable of binding to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:16.
4. The antigen-binding molecule according to any one of paras 1 to 3, wherein the antigen-binding molecule is capable of binding to a polypeptide comprising the amino acid sequence of SEQ ID NO:23 or SEQ ID NO:229.
5. The antigen-binding molecule according to any one of paras 1 to 4, wherein the antigen-binding molecule is capable of binding to a polypeptide comprising the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:229.
6. The antigen-binding molecule according to any one of paras 1 to 5, wherein the antigen-binding molecule comprises:
7. The antigen-binding molecule according to any one of paras 1 to 6, wherein the antigen-binding molecule comprises:
8. The antigen-binding molecule according to any one of paras 1 to 6, wherein the antigen-binding molecule comprises:
9. The antigen-binding molecule according to any one of paras 1 to 6, wherein the antigen-binding molecule comprises:
10. The antigen-binding molecule according to any one of paras 1 to 6, wherein the antigen-binding molecule comprises:
11. The antigen-binding molecule according to any one of paras 1 to 6, wherein the antigen-binding molecule comprises:
12. The antigen-binding molecule according to any one of paras 1 to 6, wherein the antigen-binding molecule comprises:
13. The antigen-binding molecule according to any one of paras 1 to 6, wherein the antigen-binding molecule comprises:
14. The antigen-binding molecule according to any one of paras 1 to 6, wherein the antigen-binding molecule comprises:
15. The antigen-binding molecule according to any one of paras 1 to 6, wherein the antigen-binding molecule comprises:
16. The antigen-binding molecule according to any one of paras 1 to 6, wherein the antigen-binding molecule comprises:
17. The antigen-binding molecule according to any one of paras 1 to 5, wherein the antigen-binding molecule comprises:
18. The antigen-binding molecule according to any one of paras 1 to 4, wherein the antigen-binding molecule is capable of binding to a polypeptide comprising the amino acid sequence of SEQ ID NO:22.
19. The antigen-binding molecule according to any one of paras 1 to 4 or para 18, wherein the antigen-binding molecule comprises:
20. The antigen-binding molecule according to any one of paras 1 to 4 or para 18, wherein the antigen-binding molecule comprises:
21. The antigen-binding molecule according to any one of paras 1 to 4, wherein the antigen-binding molecule comprises:
22. The antigen-binding molecule according to any one of paras 1 to 21, wherein the antigen-binding molecule is capable of binding to human HER3 and one or more of mouse HER3, rat HER3 and cynomolgous macaque HER3.
23. An antigen-binding molecule, optionally isolated, comprising (i) an antigen-binding molecule according to any one of paras 1 to 22, and (ii) an antigen-binding molecule capable of binding to an antigen other than HER3.
24. The antigen-binding molecule according to any one of paras 1 to 23, wherein the antigen-binding molecule is capable of binding to cells expressing HER3 at the cell surface.
25. The antigen-binding molecule according to any one of paras 1 to 24, wherein the antigen-binding molecule is capable of inhibiting HER3-mediated signalling.
26. The antigen-binding molecule according to any one of paras 1 to 25, wherein the antigen-binding molecule comprises an Fc region, the Fc region comprising a polypeptide having: (i) C at the position corresponding to position 242, and C at the position corresponding to position 334, and (ii) one or more of: A at the position corresponding to position 236, D at the position corresponding to position 239, E at the position corresponding to position 332, L at the position corresponding to position 330, K at the position corresponding to position 345, and G at the position corresponding to position 430.
27. The antigen-binding molecule according to para 26 wherein the Fc region comprises a polypeptide having C at the position corresponding to position 242, C at the position corresponding to position 334, A at the position corresponding to position 236, D at the position corresponding to position 239, E at the position corresponding to position 332, and L at the position corresponding to position 330.
28. A chimeric antigen receptor (CAR) comprising an antigen-binding molecule according to any one of paras 1 to 27.
29. A nucleic acid, or a plurality of nucleic acids, optionally isolated, encoding an antigen-binding molecule according to any one of paras 1 to 27 or a CAR according to para 28.
30. An expression vector, or a plurality of expression vectors, comprising a nucleic acid or a plurality of nucleic acids according to para 29.
31. A cell comprising an antigen-binding molecule according to any one of paras 1 to 27, a CAR according to para 28, a nucleic acid or a plurality of nucleic acids according to para 29, or an expression vector or a plurality of expression vectors according to para 30.
32. A method comprising culturing a cell comprising a nucleic acid or a plurality of nucleic acids according to para 29, or an expression vector or a plurality of expression vectors according to para 30, under conditions suitable for expression of the antigen-binding molecule or CAR from the nucleic acid(s) or expression vector(s).
33. A composition comprising an antigen-binding molecule according to any one of paras 1 to 27, a CAR according to para 28, a nucleic acid or a plurality of nucleic acids according to para 29, an expression vector or a plurality of expression vectors according to para 30, or a cell according to para 31.
34. An antigen-binding molecule according to any one of paras 1 to 27, a CAR according to para 28, a nucleic acid or a plurality of nucleic acids according to para 29, an expression vector or a plurality of expression vectors according to para 30, a cell according to para 31, or a composition according to para 33 for use in a method of medical treatment or prophylaxis.
35. An antigen-binding molecule according to any one of paras 1 to 27, a CAR according to para 28, a nucleic acid or a plurality of nucleic acids according to para 29, an expression vector or a plurality of expression vectors according to para 30, a cell according to para 31, or a composition according to para 33, for use in a method of treatment or prevention of a cancer.
36. Use of an antigen-binding molecule according to any one of paras 1 to 27, a CAR according to para 28, a nucleic acid or a plurality of nucleic acids according to para 29, an expression vector or a plurality of expression vectors according to para 30, a cell according to para 31, or a composition according to para 33, in the manufacture of a medicament for use in a method of treatment or prevention of a cancer.
37. A method of treating or preventing a cancer, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen-binding molecule according to any one of paras 1 to 27, a CAR according to para 28, a nucleic acid or a plurality of nucleic acids according to para 29, an expression vector or a plurality of expression vectors according to para 30, a cell according to para 31, or a composition according to para 33.
38. The antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell or composition for use according to para 34 or para 35, the use according to para 36 or the method according to para 37, wherein the method additionally comprises administration of an inhibitor of signalling mediated by an EGFR family member, optionally wherein the inhibitor of signalling mediated by an EGFR family member is an inhibitor of signalling mediated by HER2 and/or EGFR.
39. The antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell or composition for use, the use or the method according to any one of paras 34 to para 38, wherein the cancer is selected from: a cancer comprising cells expressing an EGFR family member, a cancer comprising cells expressing HER3, a solid tumor, breast cancer, breast carcinoma, ductal carcinoma, gastric cancer, gastric carcinoma, gastric adenocarcinoma, colorectal cancer, colorectal carcinoma, colorectal adenocarcinoma, head and neck cancer, squamous cell carcinoma of the head and neck (SCCHN), lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, ovarian cancer, ovarian carcinoma, ovarian serous adenocarcinoma, kidney cancer, renal cell carcinoma, renal clear cell carcinoma, renal cell adenocarcinoma, renal papillary cell carcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, cervical cancer, cervical squamous cell carcinoma, skin cancer, melanoma, esophageal cancer, esophageal adenocarcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, uterine cancer, uterine corpus endometrial carcinoma, thyroid cancer, thyroid carcinoma, pheochromocytoma, paraganglioma, bladder cancer, bladder urothelial carcinoma, prostate cancer, prostate adenocarcinoma, sarcoma and thymoma.
40. A method of inhibiting HER3-mediated signalling, comprising contacting HER3-expressing cells with an antigen-binding molecule according to any one of paras 1 to 27.
41. A method of reducing the number or activity of HER3-expressing cells, the method comprising contacting HER3-expressing cells with an antigen-binding molecule according to any one of paras 1 to 27.
42. An in vitro complex, optionally isolated, comprising an antigen-binding molecule according to any one of paras 1 to 27 bound to HER3.
43. A method comprising contacting a sample containing, or suspected to contain, HER3 with an antigen-binding molecule according to any one of paras 1 to 27, and detecting the formation of a complex of the antigen-binding molecule with HER3.
44. A method of selecting or stratifying a subject for treatment with a HER3-targeted agent, the method comprising contacting, in vitro, a sample from the subject with an antigen-binding molecule according to any one of paras 1 to 27 and detecting the formation of a complex of the antigen-binding molecule with HER3.
45. Use of an antigen-binding molecule according to any one of paras 1 to 27 as an in vitro or in vivo diagnostic or prognostic agent.
46. Use of an antigen-binding molecule according to any one of paras 1 to 27 in a method for detecting, localizing or imaging a cancer, optionally wherein the cancer is selected from: a cancer comprising cells expressing an EGFR family member, a cancer comprising cells expressing HER3, a solid tumor, breast cancer, breast carcinoma, ductal carcinoma, gastric cancer, gastric carcinoma, gastric adenocarcinoma, colorectal cancer, colorectal carcinoma, colorectal adenocarcinoma, head and neck cancer, squamous cell carcinoma of the head and neck (SCCHN), lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, ovarian cancer, ovarian carcinoma, ovarian serous adenocarcinoma, kidney cancer, renal cell carcinoma, renal clear cell carcinoma, renal cell adenocarcinoma, renal papillary cell carcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, cervical cancer, cervical squamous cell carcinoma, skin cancer, melanoma, esophageal cancer, esophageal adenocarcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, uterine cancer, uterine corpus endometrial carcinoma, thyroid cancer, thyroid carcinoma, pheochromocytoma, paraganglioma, bladder cancer, bladder urothelial carcinoma, prostate cancer, prostate adenocarcinoma, sarcoma and thymoma.
47. An antigen-binding molecule, optionally isolated, which is capable of binding to HER3, wherein the antigen-binding molecule comprises:
48. The antigen-binding molecule according to para 47, wherein the antigen-binding molecule comprises:
49. The antigen-binding molecule according to para 47 or para 48, wherein the antigen-binding molecule comprises:
50. An antigen-binding molecule, optionally isolated, comprising (i) an antigen-binding molecule according to any one of paras 47 to 49, and (ii) an antigen-binding molecule capable of binding to an antigen other than HER3.
51. A chimeric antigen receptor (CAR) comprising an antigen-binding molecule according to any one of paras 47 to 50.
52. A nucleic acid, or a plurality of nucleic acids, optionally isolated, encoding an antigen-binding molecule according to any one of paras 47 to 50 or a CAR according to para 51.
53. An expression vector, or a plurality of expression vectors, comprising a nucleic acid or a plurality of nucleic acids according to para 52.
54. A cell comprising an antigen-binding molecule according to any one of paras 47 to 50, a CAR according to para 51, a nucleic acid or a plurality of nucleic acids according to para 52, or an expression vector or a plurality of expression vectors according to para 53.
55. A method comprising culturing a cell comprising a nucleic acid or a plurality of nucleic acids according to para 52, or an expression vector or a plurality of expression vectors according to para 53, under conditions suitable for expression of the antigen-binding molecule or CAR from the nucleic acid(s) or expression vector(s).
56. A composition comprising an antigen-binding molecule according to any one of paras 47 to 50, a CAR according to para 51, a nucleic acid or a plurality of nucleic acids according to para 52, an expression vector or a plurality of expression vectors according to para 53, or a cell according to para 54.
57. An antigen-binding molecule according to any one of paras 47 to 50, a CAR according to para 51, a nucleic acid or a plurality of nucleic acids according to para 52, an expression vector or a plurality of expression vectors according to para 53, a cell according to para 54, or a composition according to para 56 for use in a method of medical treatment or prophylaxis.
58. An antigen-binding molecule according to any one of paras 47 to 50, a CAR according to para 51, a nucleic acid or a plurality of nucleic acids according to para 52, an expression vector or a plurality of expression vectors according to para 53, a cell according to para 54, or a composition according to para 56, for use in a method of treatment or prevention of a cancer.
59. Use of an antigen-binding molecule according to any one of paras 47 to 50, a CAR according to para 51, a nucleic acid or a plurality of nucleic acids according to para 52, an expression vector or a plurality of expression vectors according to para 53, a cell according to para 54, or a composition according to para 56, in the manufacture of a medicament for use in a method of treatment or prevention of a cancer.
60. A method of treating or preventing a cancer, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen-binding molecule according to any one of paras 47 to 50, a CAR according to para 51, a nucleic acid or a plurality of nucleic acids according to para 52, an expression vector or a plurality of expression vectors according to para 53, a cell according to para 54, or a composition according to para 56.
61. The antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell or composition for use according to para 57 or para 58, the use according to para 59 or the method according to para 60, wherein the method additionally comprises administration of an inhibitor of signalling mediated by an EGFR family member, optionally wherein the inhibitor of signalling mediated by an EGFR family member is an inhibitor of signalling mediated by HER2 and/or EGFR.
62. The antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell or composition for use, the use or the method according to any one of paras 57 to para 61, wherein the cancer is selected from: a cancer comprising cells expressing an EGFR family member, a cancer comprising cells expressing HER3, a solid tumor, breast cancer, breast carcinoma, ductal carcinoma, gastric cancer, gastric carcinoma, gastric adenocarcinoma, colorectal cancer, colorectal carcinoma, colorectal adenocarcinoma, head and neck cancer, squamous cell carcinoma of the head and neck (SCCHN), lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, ovarian cancer, ovarian carcinoma, ovarian serous adenocarcinoma, kidney cancer, renal cell carcinoma, renal clear cell carcinoma, renal cell adenocarcinoma, renal papillary cell carcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, cervical cancer, cervical squamous cell carcinoma, skin cancer, melanoma, esophageal cancer, esophageal adenocarcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, uterine cancer, uterine corpus endometrial carcinoma, thyroid cancer, thyroid carcinoma, pheochromocytoma, paraganglioma, bladder cancer, bladder urothelial carcinoma, prostate cancer, prostate adenocarcinoma, sarcoma and thymoma.
63. A method of inhibiting HER3-mediated signalling, comprising contacting HER3-expressing cells with an antigen-binding molecule according to any one of paras 47 to 50.
64. A method of reducing the number or activity of HER3-expressing cells, the method comprising contacting HER3-expressing cells with an antigen-binding molecule according to any one of paras 47 to 50.
65. An in vitro complex, optionally isolated, comprising an antigen-binding molecule according to any one of paras 47 to 50 bound to HER3.
66. A method comprising contacting a sample containing, or suspected to contain, HER3 with an antigen-binding molecule according to any one of paras 47 to 50, and detecting the formation of a complex of the antigen-binding molecule with HER3.
67. A method of selecting or stratifying a subject for treatment with a HER3-targeted agent, the method comprising contacting, in vitro, a sample from the subject with an antigen-binding molecule according to any one of paras 47 to 50 and detecting the formation of a complex of the antigen-binding molecule with HER3.
68. Use of an antigen-binding molecule according to any one of paras 47 to 50 as an in vitro or in vivo diagnostic or prognostic agent. 69. Use of an antigen-binding molecule according to any one of paras 47 to 50 in a method for detecting, localizing or imaging a cancer, optionally wherein the cancer is selected from: a cancer comprising cells expressing an EGFR family member, a cancer comprising cells expressing HER3, a solid tumor, breast cancer, breast carcinoma, ductal carcinoma, gastric cancer, gastric carcinoma, gastric adenocarcinoma, colorectal cancer, colorectal carcinoma, colorectal adenocarcinoma, head and neck cancer, squamous cell carcinoma of the head and neck (SCCHN), lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, ovarian cancer, ovarian carcinoma, ovarian serous adenocarcinoma, kidney cancer, renal cell carcinoma, renal clear cell carcinoma, renal cell adenocarcinoma, renal papillary cell carcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, cervical cancer, cervical squamous cell carcinoma, skin cancer, melanoma, esophageal cancer, esophageal adenocarcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, uterine cancer, uterine corpus endometrial carcinoma, thyroid cancer, thyroid carcinoma, pheochromocytoma, paraganglioma, bladder cancer, bladder urothelial carcinoma, prostate cancer, prostate adenocarcinoma, sarcoma and thymoma.
The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
Aspects and embodiments of the present invention will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.
Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment.
Where a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated.
Methods described herein may preferably performed in vitro. The term “in vitro” is intended to encompass procedures performed with cells in culture whereas the term “in vivo” is intended to encompass procedures with/on intact multi-cellular organisms.
Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures.
In the following Examples, the inventors describe the generation of novel anti-HER3 antibody clones targeted to specific regions of interest in the HER3 molecule, and the biophysical and functional characterisation and therapeutic evaluation of these antigen-binding molecules.
The inventors selected two regions in the extracellular region of human HER3 (SEQ ID NO:9) for raising HER3-binding monoclonal antibodies.
Approximately 6 week old female BALB/c mice were obtained from InVivos (Singapore). Animals were housed under specific pathogen-free conditions and were treated in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines.
For hybridoma production, mice were immunized with proprietary mixtures of antigenic peptide, recombinant target protein or cells expressing the target protein.
Prior to harvesting the spleen for fusion, mice were either boosted with antigen mixture for three consecutive days or only for a single day. 24 h after the final boost total splenocytes were isolated and fused with the myeloma cell line P3X63.Ag8.653 (ATCC, USA), with PEG using ClonaCell-HY Hybridoma Cloning Kit, in accordance with the manufacturer's instructions (Stemcell Technologies, Canada).
Fused cells were cultured in ClonaCell-HY Medium C (Stemcell Technologies, Canada) overnight at 37° C. in a 5% CO2 incubator. The next day, fused cells were centrifuged and resuspended in 10 ml of ClonaCell-HY Medium C and then gently mixed with 90 ml of semisolid methylcellulose-based ClonaCell-HY Medium D (StemCell Technologies, Canada) containing HAT components, which combines the hybridoma selection and cloning into one step.
The fused cells were then plated into 96 well plates and allowed to grow at 37° C. in a 5% CO2 incubator. After 7-10 days, single hybridoma clones were isolated and antibody producing hybridomas were selected by screening the supernatants by Enzyme-linked immunosorbent assay (ELISA) and Fluorescence-activated cell sorting (FACs).
1.2 Antibody Variable Region Amplification and Sequencing Total RNA was extracted from hybridoma cells using TRIzol reagent (Life Technologies, Inc., USA) using manufacturer's protocol. Double-stranded cDNA was synthesized using SMARTer RACE 5/3′ Kit (Clontech™, USA) in accordance with the manufacturer's instructions. Briefly, 1 μg total RNA was used to generate full-length cDNA using 5′-RACE CDS primer (provided in the kit), and the 5′ adaptor (SMARTer II A primer) was then incorporated into each cDNA according to manufacturer's instructions. cDNA synthesis reactions contained: 5×First-Strand Buffer, DTT (20 mM), dNTP Mix (10 mM), RNase Inhibitor (40 U/μl) and SMARTScribe Reverse Transcriptase (100 U/μl).
The race-ready cDNAs were amplified using SeqAmp DNA Polymerase (Clontech™, USA). Amplification reactions contained SeqAmp DNA Polymerase, 2× Seq AMP buffer, 5′ universal primer provided in the 5′ SMARTer Race kit, that is complement to the adaptor sequence, and 3′ primers that anneal to respective heavy chain or light chain constant region primer. The 5′ constant region were designed based on previously reported primer mix either by Krebber et al. J. Immunol. Methods 1997; 201: 35-55, Wang et al. Journal of Immunological Methods 2000, 233; 167-177 or Tiller et al. Journal of Immunological Methods 2009; 350:183-193. The following thermal protocol was used: pre-denature cycle at 94° C. for 1 min; 35 cycles of 94° C., 30 s, 55° C., 30 s and 72° C., 45 s; final extension at 72° C. for 3 min.
The resulting VH and VL PCR products, approximately 550 bp, were cloned into pJET1.2/blunt vector using CloneJET PCR Cloning Kit (Thermo Scientific, USA) and used to transform highly competent E. coli DH5a. From the resulting transformants, plasmid DNA was prepared using Miniprep Kit (Qiagene, Germany) and sequenced. DNA sequencing was carried out by AITbiotech. These sequencing data were analyzed using the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue):D413-22) to characterize the individual CDRs and framework sequences. The signal peptide at 5′ end of the VH and VL was identified by SignalP (v 4.1; Nielsen, in Kihara, D (ed): Protein Function Prediction (Methods in Molecular Biology vol. 1611) 59-73, Springer 2017).
Four monoclonal anti-HER3 antibody clones were selected for further development: 10D1, 10A6, 4-35-B2, and 4-35-B4.
Humanised versions of 10D1 were designed in silico by grafting of complementarity determining regions (CDRs) into VH and VL comprising human antibody framework regions, and were further optimized for antigen binding by yeast display method.
For yeast display, humanized sequences were converted into single-chain fragment variable (scFv) format by polymerase chain reaction (PCR) and used as templates to generate mutant libraries by random mutagenesis. Mutant PCR libraries were then electroporated into yeast together with linearized pCTcon2 vector to generate yeast libraries. The libraries were stained with human HER3 antigen and sorted for top binders. After 4-5 rounds of sorting, individual yeast clones were sequenced to identify unique antibody sequences.
2.1 Cloning VH and VL into Expression Vectors:
DNA sequences encoding the heavy and light chain variable regions of the anti-HER3 antibody clones were subcloned into the pmAbDZ_IgG1_CH and pmAbDZ_IgG1_CL (InvivoGen, USA) eukaryotic expression vectors for construction of human-mouse chimeric antibodies.
Alternatively, DNA sequence encoding the heavy and light chain variable regions of the anti-HER3 antibody clones were subcloned into the pFUSE-CHIg-hG1 and pFUSE2ss-CLIg-hk (InvivoGen, USA) eukaryotic expression vectors for construction of human-mouse chimeric antibodies. Human IgG1 constant region encoded by pFUSE-CHIg-hG1 comprises the substitutions D356E, L358M (positions numbered according to EU numbering) in the CH3 region relative to Human IgG1 constant region (IGHG1; UniProt:P01857-1, v1; SEQ ID NO:176). pFUSE2ss-CLIg-hk encodes human IgG1 light chain kappa constant region (IGCK; UniProt: P01834-1, v2).
Variable regions along with the signal peptides were amplified from the cloning vector using SeqAmp enzyme (Clontech™, USA) following the manufacturer's protocol. Forward and reverse primers having 15-20 bp overlap with the appropriate regions within VH or VL plus 6 bp at 5′ end as restriction sites were used. The DNA insert and the vector were digested with restriction enzyme recommended by the manufacturer to ensure no frameshift was introduced and ligated into its respective plasmid using T4 ligase enzyme (Thermo Scientific, USA). The molar ratio of 3:1 of DNA insert to vector was used for ligation.
Antibodies were expressed using either 1) Expi293 Transient Expression System Kit (Life Technologies, USA), or 2) HEK293-6E Transient Expression System (CNRC-NRC, Canada) following the manufacturer's instructions.
HEK293F cells (Expi293F) were obtained from Life Technologies, Inc (USA). Cells were cultured in serum-free, protein-free, chemically defined medium (Expi293 Expression Medium, Thermo Fisher, USA), supplemented with 50 IU/ml penicillin and 50 μg/ml streptomycine (Gibco, USA) at 37° C., in 8% CO2 and 80% humidified incubators with shaking platform.
Expi293F cells were transfected with expression plasmids using ExpiFectamine 293 Reagent kit (Gibco, USA) according to its manufacturer's protocol. Briefly, cells at maintenance were subjected to a media exchange to remove antibiotics by spinning down the culture, cell pellets were re-suspended in fresh media without antibiotics at 1 day before transfection. On the day of transfection, 2.5×106/ml of viable cells were seeded in shaker flasks for each transfection. DNA-ExpiFectamine complexes were formed in serum-reduced medium, Opti-MEM (Gibco, USA), for 25 min at room temperature before being added to the cells. Enhancers were added to the transfected cells at 16-18 h post transfection. An equal amount of media was topped up to the transfectants at day 4 post-transfection to prevent cell aggregation. Transfectants were harvested at day 7 by centrifugation at 4000×g for 15 min, and filtered through 0.22 μm sterile filter units.
HEK293-6E cells were obtained from National Research Council Canada. Cells were cultured in serum-free, protein-free, chemically defined Freestyle F17 Medium (Invitrogen, USA), supplemented with 0.1% Kolliphor-P188 and 4 mM L-Glutamine (Gibco, USA) and 25 μg/ml G-418 at 37° C., in 5% CO2 and 80% humidified incubators with shaking platform.
HEK293-6E cells were transfected with expression plasmids using PEIpro™ (Polyplus, USA) according to its manufacturer's protocol. Briefly, cells at maintenance were subjected to a media exchange to remove antibiotics by centrifugation, cell pellets were re-suspended with fresh media without antibiotics at 1 day before transfection. On the day of transfection, 1.5-2×106 cells/ml of viable cells were seeded in shaker flasks for each transfection. DNA and PEIpro™ were mixed to a ratio of 1:1 and the complexes were allowed to form in F17 medium for 5 min at RT before adding to the cells. 0.5% (w/v) of Tryptone N1 was fed to transfectants at 24-48 h post transfection. Transfectants were harvested at day 6-7 by centrifugation at 4000×g for 15 min and the supernatant was filtered through 0.22 μm sterile filter units. Cells were transfected with vectors encoding the following combinations of polypeptides:
Antibodies secreted by the transfected cells into the culture supernatant were purified using liquid chromatography system AKTA Start (GE Healthcare, UK). Specifically, supernatants were loaded onto HiTrap Protein G column (GE Healthcare, UK) at a binding rate of 5 ml/min, followed by washing the column with 10 column volumes of washing buffer (20 mM sodium phosphate, pH 7.0). Bound mAbs were eluted with elution buffer (0.1 M glycine, pH 2.7) and the eluents were fractionated to collection tubes which contain appropriate amount of neutralization buffer (1 M Tris, pH 9). Neutralised elution buffer containing purified mAb were exchanged into PBS using 30K MWCO protein concentrators (Thermo Fisher, USA) or 3.5K MWCO dialysis cassettes (Thermo Fisher, USA). Monoclonal antibodies were sterilized by passing through 0.22 μm filter, aliquoted and snap-frozen in −80° C. for storage.
Antibody purity was analysed by size exclusion chromatography (SEC) using Superdex 200 10/30 GL columns (GE Healthcare, UK) in PBS running buffer, on a AKTA Explorer liquid chromatography system (GE Healthcare, UK). 150 μg of antibody in 500 μl PBS pH 7.2 was injected to the column at a flow rate of 0.75 ml/min at room temperature. Proteins were eluted according to their molecular weights.
The result for anti-HER3 antibody clone 10D1 ([1] of Example 2.2) is shown in
The results obtained for the different 10D1 variant clones are shown in
Sodium-Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE): Antibody purity was also analysed by SDS-PAGE under reducing and non-reducing conditions according to standard methods. Briefly, 4%-20% TGX protein gels (Bio-Rad, USA) were used to resolve proteins using a Mini-Protean Electrophoresis System (Bio-Rad, USA). For non-reducing condition, protein samples were denatured by mixing with 2× Laemmli sample buffer (Bio-Rad, USA) and boiled at 95° C. for 5-10 min before loading to the gel. For reducing conditions, 2× sample buffer containing 5% of β-mercaptoethanol (βME), or 40 mM DTT (dithiothreitol) was used. Electrophoresis was carried out at a constant voltage of 150V for 1 h in SDS running buffer (25 mM Tris, 192 mM glycine, 1% SDS, pH 8.3).
Protein samples (30 μg) were fractionated by SDS-PAGE as described above and transferred to nitrocellulose membranes. Membranes were then blocked and immunoblotted with antibodies overnight at 4° C. After washing three times in PBS-Tween the membranes were then incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies. The results were visualized via a chemiluminescent Pierce ECL Substrate Western blot detection system (Thermo Scientific, USA) and exposure to autoradiography film (Kodak XAR film).
The primary antibodies used for detection were goat anti-human IgG-HRP (GenScript Cat No. A00166) and goat anti-human kappa-HRP (SouterhnBiotech Cat No. 2060-05).
The result for anti-HER3 antibody clone 10D1 ([1] of Example 2.2) is shown in
Wildtype HEK293 cells (which do not express high levels of HER3) and HEK293 cells transfected with vector encoding human HER3 (i.e. HEK 293 HER O/E cells) were incubated with 20 μg/ml of anti-HER3 antibody or isotype control antibody at 4° C. for 1.5 hr. The anti-HER3 antibody clone LJM716 (described e.g. in Garner et al., Cancer Res (2013) 73: 6024-6035) was included in the analysis as a positive control.
The cells were washed thrice with FACS buffer (PBS with 5 mM EDTA and 0.5% BSA) and resuspended in FITC-conjugated anti-FC antibody (Invitrogen, USA) for 40 min at 2-8° C. Cells were washed again and resuspended in 200 μL of FACS flow buffer (PBS with 5 mM EDTA) for flow cytometric analysis using MACSQuant 10 (Miltenyi Biotec, Germany). After acquisition, all raw data were analyzed using Flowlogic software. Cells were gated using forward and side scatter profile percentage of positive cells was determined for native and overexpressing cell populations.
The results are shown in
ELISAs were used to determine the binding specificity of the antibodies. The antibodies were analysed for binding to human HER3 polypeptide, as well as respective mouse, rat and monkey homologues of HER3 (Sino Biological Inc., China). The antibodies were also analysed for their ability to bind to human EGFR and human HER2 (Sino Biological Inc., China).
ELISAs were carried out according to standard protocols. Briefly, 96-well plates (Nunc, Denmark) were coated with 0.1 μg/ml of target polypeptide in phosphate-buffered saline (PBS) for 16 h at 4° C. After blocking for 1 h with 1% BSA in Tris buffer saline (TBS) at room temperature, the anti-HER3 antibody was serially diluted with the highest concentration being 10 μg/ml, and added to the plate. Post 1 h incubation at room temperature, plates were washed three times with TBS containing 0.05% Tween 20 (TBS-T) and were then incubated with a HRP-conjugated anti-His antibody (Life Technologies, Inc., USA) for 1 h at room temperature. After washing, plates were developed with colorimetric detection substrate 3,3′,5,5′-tetramethylbenzidine (Turbo-TMB; Pierce, USA) for 10 min. The reaction was stopped with 2M H2SO4, and OD was measured at 450 nM.
The results of the ELISAs are shown in
Anti-HER3 antibody clone 10D1 was found not to bind to human HER2 or human EGFR even at high concentrations of the antibody (
Anti-HER3 antibody clone 4-35-B2 was found to bind to human HER2 and human EGFR (
Anti-HER3 antibody clone 4-35-B4 was found to bind to human HER2 and human EGFR (
All of the 10D1 variants were demonstrated to bind to human HER3 (
The anti-HER3 antibody clones in IgG1 format were analysed for binding affinity to human HER3. Bio-Layer Interferometry (BLI) experiments were performed using the Octet QK384 system (ForteBio). anti-Human IgG Capture (AHC) Octet sensor tips (Pall ForteBio, USA) were used to anti-HER3 antibodies (25 nM). All measurements were performed at 25° C. with agitation at 1000 rpm. Kinetic measurements for antigen binding were performed by loading His-tagged human HER3 antigens at different concentrations for 120 s, followed by a 120 s dissociation time by transferring the biosensors into assay buffer containing wells. Sensograms were referenced for buffer effects and then fitted using the Octet QK384 user software (Pall ForteBio, USA). Kinetic responses were subjected to a global fitting using a one site binding model to obtain values for association (Kon), dissociation (Koff) rate constants and the equilibrium dissociation constant (KD). Only curves that could be reliably fitted with the software (R2>0.90) were included in the analysis.
A representative sensorgram for the analysis of clone 10D1 is shown in
The humanised/optimized 10D1 variants bind to human HER3 with very high affinity. Representative sensorgrams are shown in
The affinities determined for 10D1 clone variants are shown below:
Clone 4-35-B2 was found to bind to human HER3 with an affinity of KD=80.9 nM (
The first derivative of the raw data obtained for Differential Scanning Fluorimetry analysis of the thermostability of antibody clone 10D1 is shown in
The analysis was also performed for the 10D1 variant clones and LJM716. The first derivative of the raw data and the determined Tms are shown in
Anti-HER3 antibody 10D1 was analysed to determine whether it competes with anti-HER3 antibodies MM-121 and/or LJM-716 for binding to HER3. The epitope for MM-121 has been mapped in domain I of HER3; it blocks the NRG ligand binding site. The epitope for LJM-716 has been mapped to conformational epitope distributed across domains II and IV, and it locks HER3 in an inactive conformation.
Bio-Layer Interferometry (BLI) experiments were performed using the Octet QK384 system (ForteBio). anti-Penta-HIS (HIS1K) coated biosensor tips (ForteBio, USA) were used to capture His-tagged human HER3 (75 nM; 300 s). Binding by saturating antibody (400 nm; 600 s) was detected, followed by a dissociation step (120s), followed by detection of binding with competing antibody (300 nM; 300 s), followed by a dissociation step (120s). The variable region of MM-121 antibody was cloned in the PDZ vector having human IgG2 and IgKappa Fc backbone. The variable region of LJM-716 antibody was cloned in the PDZ vector having human IgG1 and IgKappa Fc backbone.
The results of the analysis are shown in
10D1 was found to bind a distinct and topologically distant epitope of HER3 than MM-121 and/or LJM-716.
The epitope for 10D1 was mapped using overlapping 15-mer amino acids to cover the entire HER3 extracellular domain. Each unique 15-mer was elongated by a GS linker at C and N-terminals, conjugated to a unique well in 384 well plates, and the plates were incubated with 0.1, 1, 10 and 100 μg/ml of 10D1 antibody for 16 hrs at 4° C. The plates were washed and then incubated for 1 hr at 20° C. with POD-conjugated goat anti-human IgG. Finally POD substrate solution was added to the wells for 20 min. before binding was assessed by measurement of chemiluminescence at 425 nm using a LI-COR Odyssey Imaging System, and quantification and analysis was performed using the PepSlide Analyzer software package. The experiment was performed in duplicate.
The 10D1 epitope was found not to be directly located at a β-hairpin structure of the HER3 dimerisation arm located at domain II, but instead at a dimerisation interface N-terminal to the β-hairpin.
The site of HER3 to which 10D1 and 10D1-derived clones was determined to bind corresponds to positions 218 to 235 of the amino acid sequence of human HER3 (as shown e.g. in SEQ ID NO:1); the amino acid sequence for this region of HER3 is shown in SEQ ID NO:229. Within this region, two consensus binding site motifs were identified, and are shown in SEQ ID NOs:230 and 231.
Binding to this location of HER3 acts to impede HER family heterodimerisation and consequent downstream signalling pathways (see Example 4). Binding is ligand (NRG) independent. The 10D1 binding site is solvent accessible in both the open and closed HER3 conformations, is not conserved between HER3 and other HER family members, and is 100% conserved between human, mouse, rat and monkey HER3 orthologs.
The anti-HER3 antibodies were analysed for their ability to inhibit heterodimerisation of HER3 and HER2. Briefly, 96-well plates (Nunc, Denmark) were coated with 0.1 μg/ml His-tagged HER2 protein in PBS for 16 h at 4° C. After blocking for 1 h with 1% BSA in PBS at room temperature, recombinant biotinylated human HER3 protein was added in the presence of different concentrations of anti-HER3 antibody clone 10D1, and pates were incubated for 1 h at room temperature. Plates were subsequently washed three times, and then incubated with HRP-conjugated secondary antibody for 1 h at room temperature. After washing, plates were developed with colorimetric detection substrate 3,3′,5,5′-tetramethylbenzidine (Turbo-TMB; Pierce, USA) for 10 min. The reaction was stopped with 2M H2SO4, and OD was measured at 450 nM.
The results are shown in
In further experiments, inhibition of HER2:HER3 dimerisation was analysed
In further experiments, inhibition of HER2:HER3 dimerisation was evaluated using the PathHunter Pertuzumab Bioassay Kit (DiscoverX) according to the manufacturer's instructions.
Briefly, HER2 and HER3 overexpressing U2OS cells were thawed using 1 ml of pre-warmed CP5 media and 5,000 cells were seeded per well and cultured at 37° C. in 5% CO2 atmosphere for 4 hr. Cells were then treated with an 8-point serial dilution of 10D1F.FcA or Pertuzumab, starting from 25 μg/ml.
After 4 hr incubation, 30 ng/ml of heregulin-32 was added to each well and the cells were incubated for a further 16 hr. 10 μL of PathHunter bioassay detection reagent 1 was added to wells, and incubated for 15 min at room temperature in the dark. This was followed by addition of 40 μL PathHunter bioassay detection reagent 2, and incubation for 60 min at room temperature in the dark. Plates were then read using Synergy4 Biotek with 1 second delay.
The results are shown in
The inventors characterised expression of EGFR protein family members by cancer cell lines to identify appropriate cells to investigate inhibition of HER3.
Cell lines used in the experiments were purchased from ATCC and cultured as recommended. Briefly, cell lines maintained in the indicated cell culture medium, supplemented with 10% FBS and 1% Pen/Strep. Cells were cultured at 37° C., in 5% CO2 incubators. Cultured cells were plated at the appropriate seeding density in a 96 well plate: HT29, HCC95, FADU and OvCar8 cells were seeded at 2000 cells/well, NCI-N87 cells were seeded at 5000 cells/well, SNU-16, ACHN and cells were seeded at 1500 cells/well, and A549 cells were seeded at 1200 cells/well.
Anti-HER3 antibody 10D1 was analysed for its ability to inhibit HER-3 mediated signalling in vitro.
Briefly, N87 and FaDu cells were seeded in wells of a 6 well plate with 10% serum at 37° C., 5% CO2. After 16 hrs, cells were starved by culture overnight in 1% FBS cell culture medium (to reduce signalling elicited by growth factors in the serum). On the following day cells were treated with 50 μg/ml anti-HER3 antibody 10D1 for 4 hrs, followed by 15 min stimulation with NRG (100 ng/ml). Proteins were then extracted, quantified using standard Bradford protein assay, fractionated by SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were then blocked and immunoblotted with the following antibodies overnight at 4° C. anti-pHER3, anti-pAKT, pan anti-HER3, pan anti-AKT and anti-beta-actin. The blots were visualized via Bio-Rad Clarity Western ECL substrate, and bands were quantified using densiometric analysis; data were normalized to beta actin controls.
The results are shown in
In further experiments the inventors investigated the intracellular signalling pathways affected by anti-HER3 antibody-mediated inhibition of HER3.
FaDu cells were seeded in wells of a 6 well plate with 10% serum at 37° C., 5% CO2. After 16 hrs, cells were starved by culture overnight in 1% FBS cell culture medium. On the following day cells were treated with 50 μg/ml anti-HER3 antibody 10D1 for 4 hrs, followed by 15 min stimulation with NRG (100 ng/ml). Proteins were then extracted, quantified using standard Bradford protein assay, and incubated overnight with pre-blocked Phosphoprotein Antibody Array membrane (Ray Biotech) at 4° C. The membrane was then washed with washing buffer and incubated with detection antibody cocktail for 2 hrs at room temperature, followed by washing and incubation with HRP-Conjugated anti-IgG. After 2 hrs the membrane was washed and probed using the kit detection buffer. Images were captured with Syngene Gbox imaging system, the intensity of each dot/phosphoprotein was measured and percent inhibition was calculated by comparison with intensity measured for cells treated in the same way in the absence of the antibody.
The results are shown in
In further experiments the inventors investigated the effect of treatment with anti-HER3 antibody 10D1 on proliferation of HER3-expressing cells.
Briefly, N87 and FaDu cells were treated with serially diluted concentrations of anti-HER3 antibody 10D1, starting from 100 μg/ml with a 9-point half log dilution. Cell proliferation was measuring using the CCK-8 proliferation assay (Dojindo, Japan) after a period of 5 days, in accordance with the manufacturer's instructions. Briefly 1× CCK-8 solution was added to each well followed by incubation for 2 h at 37° C. The OD was then measured at 450 nm.
Anti-HER3 antibody 10D1 displayed dose-dependent inhibition of cell proliferation by N87 and FaDu cells.
Female NCr nude mice approximately 6-8 weeks old were housed under specific pathogen-free conditions and treated in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines. 500 μg anti-HER3 antibody was administered and blood was obtained from 3 mice by cardiac puncture at baseline (−2 hr), 0.5 hr, 6 hr, 24 hr, 96 hr, 168 hr and 336 hr after administration. Antibody in the serum was quantified by ELISA.
The results are shown in
Anti-HER3 antibody clone 10D1 was analysed in silico for safety and immunogenicity using IMGT DomainGapAlign (Ehrenmann et al., Nucleic Acids Res., 38, D301-307 (2010)) and IEDB deimmunization (Dhanda et al., Immunology. (2018) 153(1):118-132) tools.
Anti-HER3 antibody clone 10D1 had numbers of potential immunogenic peptides few enough to be considered low immunogenicity risk, and did not possess any other properties that could cause potential developability issues.
The Table of
Mice treated with anti-HER3 antibodies in the experiments described in Example 5.3 were monitored for changes in weight and gross necroscopy. No differences were detected in these mice as compared to mice treated with vehicle only.
Hemotoxicity was investigated in an experiment in which 6-8 week old female BALB/c mice (20-25 g) were injected intraperitoneally with a single dose of 1000 μg anti-HER3 10D1 antibody or an equal volume of PBS. Blood samples were obtained at 96 hours post injection and analysed for numbers of different types of white blood cells by flow cytometry and electrolyte indices for NA+, K+, and Cl−.
Mice were also analysed for correlates hepatotoxicity, nephrotoxicity and pancreatic toxicity at 96 hours post injection. The levels of alanine aminotransferase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), creatinine (CREA), alkaline phosphatase (ALP), glucose (GLU), calcium (CAL), total bilirubin (BIL), total protein (TPR) and albumin (ALB) detected following administration of a single dose of 1000 μg anti-HER3 antibody were found to be within the Charles River reference range and do not differ significantly from the levels of these markers in the PBS-treated group. These are shown in
Female NCr nude mice approximately 6-8 weeks old were purchased from InVivos (Singapore). Animals were housed under specific pathogen-free conditions and were treated in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines.
Cell lines used included N87 cells (gastric cancer), FaDu cells (head and neck cancer), OvCAR8 cells (ovarian cancer), SNU16 cells (gastric cancer), HT29 cells (colorectal cancer), A549 cells (lung cancer), HCC95 cells (lung cancer) and AHCN cells (kidney cancer).
Tumor volumes were measured 3 times a week using a digital caliper and calculated using the formula [L ×W2/2]. Study End point was considered to have been reaches once the tumors of the control arm measured >1.5 cm in length.
5.3.1 N87 model
10D1 was administered IP, biweekly at 500 μg per dose (for a total of 10 doses); a control treatment group received an equal volume of PBS.
Anti-HER3 antibody clone 10D1 was found to be highly potent in this model, and capable of inhibiting tumor growth by ˜76%.
10D1 was administered IP, biweekly at 500 μg per dose (for a total of 9 doses); a control treatment group received an equal volume of PBS.
Anti-HER3 antibody clone 10D1 was found to be highly potent in this model, and capable of inhibiting tumor growth by ˜68%.
10D1 was administered IP, weekly at 500 μg per dose (for a total of 4 doses). Control treatment groups received an equal volume of PBS, or the same dose of an isotype control antibody.
Anti-HER3 antibody clone 10D1 was found to be highly potent in this model, and capable of inhibiting tumor growth by ˜85%.
10D1 was administered IP, biweekly at 500 μg per dose (for a total of 8 doses); a control treatment group received an equal volume of PBS.
Anti-HER3 antibody clone 10D1 was found to be highly potent in this model, and capable of inhibiting tumor growth by ˜86%.
10D1 was administered IP, biweekly at 500 μg per dose (for a total of 9 doses); a control treatment group received an equal volume of PBS.
Anti-HER3 antibody clone 10D1 was found to be highly potent in this model, and capable of inhibiting tumor growth by ˜74%.
10D1 was administered IP, biweekly at 500 μg per dose (for a total of 4 doses); a control treatment group received an equal volume of PBS.
Anti-HER3 antibody clone 10D1 was found to be highly potent in this model, and capable of inhibiting tumor growth by ˜90%.
10D1 was administered IP, biweekly at 500 μg per dose (for a total of 10 doses); a control treatment group received an equal volume of PBS.
Anti-HER3 antibody clone 10D1 was found to be highly potent in this model, and capable of inhibiting tumor growth by ˜91%.
Anti-HER3 antibody clone 4-35-B2 was similarly found to be highly potent in this model, and capable of inhibiting tumor growth by ˜63%.
10D1 was administered IP, biweekly at 500 μg per dose (for a total of 7 doses); a control treatment group received an equal volume of PBS.
Anti-HER3 antibody clone 10D1 was found to be highly potent in this model, and capable of inhibiting tumor growth by ˜61%.
Patients with HER2+advanced gastric cancer who have failed or cannot receive trastuzumab are treated by intravenous injection of anti-HER3 antibody selected from: 10D1, 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c89, 10D1_c90, 10D1_c91, 10D1_c92 and 10D1_c93, at a dose calculated in accordance with safety-adjusted ‘Minimal Anticipated Biological Effect Level’ (MABEL) approach. Patients are monitored for 28 days post-administration.
The patients are then evaluated according to the Common Terminology Criteria for Adverse Events (CTCAE), to determine the safety and tolerability of the treatment, and to determine the pharmacokinetics of the molecules.
Treatment with the anti-HER3 antibodies is found to be safe and tolerable.
12-48 patients with HER2+ advanced gastric cancer who have failed or cannot receive trastuzumab are treated by intravenous injection of anti-HER3 antibody selected from: 10D1, 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c89, 10D1_c90, 10D1_c91, 10D1_c92 and 10D1_c93 (e.g. 10D1_c89, 10D1_c90 or 10D1_c91; e.g. 10D1_c89), in accordance with a 3+3 model based escalation with overdose control (EWOC) dose escalation.
The patients are then evaluated according to the Common Terminology Criteria for Adverse Events (CTCAE) to determine the safety and tolerability of the treatment, and the pharmacokinetics of the molecules and efficacy of the treatment is evaluated. The maximum tolerated dose (MTD) and maximum administered dose (MAD) are also determined.
Dose Escalation—Combination Therapy 9-18 patients with HER2+advanced gastric cancer who have failed or trastuzumab are treated by intravenous injection of anti-HER3 antibody selected from: 10D1, 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c89, 10D1_c90, 10D1_c91, 10D1_c92 and 10D1_c93 (e.g. 10D1_c89, 10D1_c90 or 10D1_c91; e.g. 10D1_c89) in combination with trastuzumab, in accordance with a 3+3 model based escalation with anti-PD-L1 antibody (3 mg/kg).
The patients are then evaluated according to the Common Terminology Criteria for Adverse Events (CTCAE) to determine the safety and tolerability of the treatment, and the pharmacokinetics of the molecules and efficacy of the treatment is evaluated.
Patients with HER2+ advanced gastric cancer who have recently failed trastuzumab, and whose tumours have been well-characterised genetically and histologically are treated with anti-HER3 antibody selected from: 10D1, 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c89, 10D1_c90, 10D1_c91, 10D1_c92, 10D1_c93 (e.g. 10D1_c89, 10D1_c90 or 10D1_c91; e.g. 10D1_c89) in combination with trastuzumab, cisplatin, and either 5-FU or capecitabine
The anti-HER3 antibodies are found to be safe and tolerable, to be able to reduce the number/proportion of cancer cells, reduce tumor cell marker expression, increase progression-free survival and increase overall survival.
Humanization of the variable regions of the parental mouse antibody 10D1P was done by CDR grafting. Human framework sequences for grafting were identified by blasting the parental amino acid sequence against the human V domain database and the genes with highest identity to the parental sequence were selected. Upon grafting the mouse CDRs into the selected human frameworks, residues in canonical positions of the framework were back mutated to the parental mouse sequence to preserve antigen binding. A total of 9 humanized variants of 10D1P were designed.
Affinity against human HER3 was increased by two rounds of affinity maturation using yeast display. In the first round, a mixed library of the 9 designed variants was constructed by random mutagenesis and screened by flow cytometry using biotinylated antigen. In the second round, one heavy chain and one light chain clones isolated in the first round were used as template to generate and screen a second library. A total of 10 humanized and affinity matured clones were isolated.
Potential liabilities (immunogenicity, glycosylation sites, exposed reactive residues, aggregation potential) in the variable regions of the designed and isolated humanized variants of 10D1P was assessed using in silico prediction tools. The sequences were deimmunised using IEDB deimmunisation tool. The final sequence of 10D1F was selected among the optimized variants based on its developability characteristics as well as in vitro physicochemical and functional properties.
Clone 10D1F comprises VH of SEQ ID NO:36 and VL of SEQ ID NO:83. 10D1F displays 89.9% homology with human heavy chain and 85.3% homology with human light chain.
The antigen-binding molecule comprising 10D1F variable regions and human IgG1 constant regions, and which is comprised of the polypeptides of SEQ ID NOs: 206 and 207, is designated 10D1F.FcA (also sometimes referred to herein as “10D1F.A” or “anti-HER3 clone 10D1_c89 IgG1”—see e.g. [16] of Example 2.2).
10D1 and 10D1 variants were engineered to comprise mutations in CH2 and/or CH3 regions to increase the potency of the antibodies, e.g. optimise Fc effector function, enhance antibody-dependent cellular cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP), and improve half-life. The Fc regions of clones 10D1 and 10D1F.FcA were modified to include modifications ‘GASDALIE’ (G236A, S239D, A330L, 1332E) and ‘LCKC’ (L242C, K334C) in the CH2 region. The GASDALIE substitutions were found to increase affinity for the FcγRIIa (GA) and FcγRIIIa (SDALIE) receptors and enhance ADCP and NK-mediated ADCC (see Example 8.8), whilst decreasing affinity for C1q (AL) and reducing CDC. The LCKC substitutions were found to increase thermal stability of the Fc region by creating a new intramolecular disulphide bridge.
The modified version of 10D1F.FcA heavy chain polypeptide comprising the GASDALIE and LCKC mutations is shown in SEQ ID NO:225. The antigen-binding molecule comprised of the polypeptides of SEQ ID NOs: 225 and 207 is designated 10D1F.FcB (also sometimes referred to herein as “10D1F.B”).
A modified version of 10D1 comprising the GASDALIE and LCKC substitutions in CH2 region was prepared and its ability to bind Fc receptor FcγRIIIa was analysed by Bio-Layer Interferometry. The sequence for 10D1 VH-CH1-CH2-CH3 comprising substitutions GASDALIE and LCKC corresponding to G236A, S239D, A330L, 1332E and L242C, K334C is shown in SEQ ID NO:227.
Briefly, anti-Penta-HIS (HIS1 K) coated biosensor tips (Pall ForteBio, USA) were used to capture His-tagged FcγRIIIa (V158) (270 nM) for 120 s. All measurements were performed at 25° C. with agitation at 1000 rpm. Association kinetic measurements for antigen binding were performed by incubating anti-HER3 antibodies at different concentrations (500 nM to 15.6 nM) for 60 s, followed by a 120 s dissociation time by transferring the biosensors into assay buffer (pH 7.2) containing wells. Sensograms were referenced for buffer effects and then fitted using the Octet QK384 user software (Pall ForteBio, USA). Kinetic responses were subjected to a global fitting using a one site binding model to obtain values for association (Kon), dissociation (Koff) rate constants and the equilibrium dissociation constant (KD). Only curves that could be reliably fitted with the software (R2>0.90) were included in the analysis.
The thermostability of the variant was also analysed by Differential Scanning Fluorimetry analysis as described in Example 3.4.
A construct for 10D1 comprising the GASD substitutions in CH2 region was also prepared; a sequence of 10D1 VH-CH1-CH2-CH3 comprising substitutions corresponding to G236A and S239D is shown in SEQ ID NO:228.
The affinity of anti-HER3 antibody clone 10D1 ([1] of Example 2.2) and the GASD variant thereof were analysed by Bio-Layer Interferometry for affinity of binding to FcγRIIIa. BLI was performed as described above.
The thermostability of the 10D1 GASD variant was also analysed by Differential Scanning Fluorimetry analysis as described in Example 3.4. The results are shown in
Another antibody variant was created comprising an N297Q substitution in the CH2 region. A representative sequence for 10D1F VH-CH1-CH2-CH3 comprising the N297Q substitution is shown in SEQ ID NO:226. This ‘silent form’ prevents both N-linked glycosylation of the Fc region and Fc binding to Fcγ receptors and is used as a negative control.
Wildtype (WT) HEK293 cells (which do not express high levels of HER3) and HEK293 cells transfected with vector encoding human HER3 (i.e. HEK293 HER O/E cells) were incubated with 10 μg/ml of humanised anti-HER3 antibody 10D1F.FcA (10D1F), anti-HER3 antibody 10D1 (10D1P) or isotype control antibody at 4° C. for 1.5 hr. The anti-HER3 antibody clone LJM716 (described e.g. in Garner et al., Cancer Res (2013) 73: 6024-6035, and Example 3.5) was included in the analysis as a positive control.
The cells were washed with buffer (PBS with 2 mM EDTA and 0.5% BSA) and resuspended in FITC-conjugated anti-FC antibody (Invitrogen, USA) at 10 μg/ml for 20 min at 4° C. Cells were washed again and resuspended in 200 μL of FACS flow buffer (PBS with 5 mM EDTA) for flow cytometric analysis using MACSQuant 10 (Miltenyi Biotec, Germany). Unstained WT and transfected HEK293 cells were included in the analysis as negative controls. After acquisition, all raw data were analyzed using Flowlogic software. Cells were gated using forward and side scatter profile percentage of positive cells was determined for native and overexpressing cell populations.
The results are shown in
ELISAs were used to confirm the binding specificity of the 10D1F.FcA antibody. The antibodies were analysed for their ability to bind to human HER3 polypeptide as well as human HER1 (EGFR) and human HER2 (Sino Biological Inc., China). Human IgG isotype and an irrelevant antigen were included as negative controls.
ELISAs were carried out according to standard protocols. Plates were coated with 0.1 μg/ml of target polypeptide in phosphate-buffered saline (PBS) for 16 h at 4° C. After blocking for 1 h with 1% BSA in Tris buffer saline (TBS) at room temperature, the anti-HER3 antibody was serially diluted with the highest concentration being 10 μg/ml, and added to the plate. Post 1 h incubation at room temperature, plates were washed three times with TBS containing 0.05% Tween 20 (TBS-T) and were then incubated with a HRP-conjugated anti-His antibody (Life Technologies, Inc., USA) for 1 h at room temperature. After washing, plates were developed with colorimetric detection substrate 3,3′,5,5′-tetramethylbenzidine (Turbo-TMB; Pierce, USA) for 10 min. The reaction was stopped with 2M H2SO4, and OD was measured at 450 nM.
The results are shown in
The ability of 10D1F.FcA to bind HER4 was analysed using flow cytometry. Wildtype (WT) HEK293 cells (which do not express high levels of HER4) and HEK293 cells transfected with vector encoding human HER4 (i.e. HEK293 HER O/E cells) were incubated with 10 μg/ml of anti-HER3 antibody 10D1F.FcA (10D1F) or isotype control antibody (negative control) at 4° C. for 1.5 hr. The anti-HER3 antibody clones LJM716 (described e.g. in Garner et al., Cancer Res (2013) 73: 6024-6035) and MM-121 (seribantumab), as described in Example 3.5, were included in the analysis as positive control. Also included was a commercial anti-HER4 antibody (Novus, Cat: FAB11311P). Unstained HEK293 cells were included in the analysis as negative controls.
HEK293 cells were incubated with 10 μg/ml of each antibody for 1 hour at 4° C. Flow cytometry was performed as described above. Cells were contacted with FITC-conjugated anti-FC antibody (Invitrogen, USA) at for 30 min at 4° C.
The results are shown in
In addition, antibody 10D1F.FcA was analysed for its ability to bind to HER3 polypeptide homologues from mouse, rat and monkey (Sino Biological Inc., China). M. musculus, R. norvegicus and M. cynomolgus HER3 homologues share 91.1, 91.0 and 98.9% sequence identity respectively with human HER3 and the HER3 signalling pathways are conserved between the four species.
ELISAs were performed as above.
The results are shown in
The anti-HER3 antibody clones 10D1F.FcA and 10D1F.FcB were analysed for binding affinity to human HER3.
Bio-Layer Interferometry (BLI) experiments were performed using the Octet QK384 system (ForteBio). Antibodies (25 nM) were coated onto anti-Human IgG Capture (AHC) Octet sensor tips (Pall ForteBio, USA). Binding was detected using titrated HIS-tagged human HER3 in steps of baseline (60 s), loading (120 s), baseline2 (60 s), association (120 s), dissociation (FcA 120 s, FcB 600 s) and regeneration (15 s). Antigen concentrations are shown in the table in
Representative sensorgrams for the analysis of clones 10D1F.FcA and 10D1F.FcB are shown in
Differential Scanning Fluorimetry was performed for antibodies 10D1F.FcA and 10D1F.FcB as described in Example 3.4.
The first derivative of the raw data obtained for Differential Scanning Fluorimetry analysis of the thermostability of antibody clone 10D1F.FcA is shown in
The first derivative of the raw data obtained for Differential Scanning Fluorimetry analysis of the thermostability of antibody clone 10D1F.FcB is shown in
The purity of antibodies 10D1F.FcA and 10D1F.FcB was analysed by size exclusion chromatography (SEC). 150 μg of 10D1F.FcA in 500 μl PBS pH 7.2 or 150 μg of 10D1F.FcB in 500 μl PBS pH 7.45 was injected on a Superdex 200 10/30 GL column in PBS running buffer at a flow rate of 0.75 min/ml or 0.5 min/ml, respectively, at room temperature and the A280 of flow through was recorded.
The results are shown in
Anti-HER3 antibody 10D1F.FcA was analysed to determine whether it competes with anti-HER3 antibodies M-05-74 or M-08-11 (Roche) for binding to HER3. Epitopes of M-05-74 and M-08-11 were both mapped to the β-hairpin structure of the HER3 dimerisation arm located at domain II. M-08-11 does not bind to HER4 whereas M-05-74 recognises the HER4 dimerisation arm. Binding of M-05-74 and M-08-11 to HER3 is ligand (NRG) independent.
BLI experiments were performed as described in Example 3.5 with one alteration: 400 nM of competing antibodies were used. The variable regions of M-05-74 and M-08-11 antibodies were cloned in the PDZ vector having human IgG1 and IgKappa Fc backbone.
The results of the analysis are shown in
Anti-HER3 antibody 10D1 F.FcA was analysed for its ability to inhibit heterodimerisation of HER3 and HER2.
Plate-based ELISA dimerisation assays were carried out according to standard protocols. Plates were coated with 1 μg/ml HER2-Fc protein. After blocking and washing, the plate was incubated with different concentrations of candidate antibodies 10D1F.FcA, MM-121, LJM716, Pertuzumab, Roche M05, Roche M08 or isotype control and constant HER3 His 2 μg/ml and NRG 0.1 μg/ml for 1 hour. Plates were then washed and incubated for 1 hour with secondary anti-HIS HRP antibody. Plates were washed, treated with TMB for 10 mins and the reaction was stopped using 2M H2SO4 stop solution. The absorbance was read at 450 nm.
The results are shown in
In another assay, inhibition of dimerization was detected using PathHunter® Pertuzumab Bioassay Kit (DiscoverX, San Francisco, USA). HER2 and HER3 overexpressing U20S cells were thawed using 1 ml of pre-warmed CP5 media and 5K cells were seeded per at 37° C., 5% CO2 for 4 hrs. Cells were treated with serially diluted concentrations of 10D1F.FcA, Seribantumab, or Pertuzumab starting from 25 μg/ml with an 8-point serial dilution. After 4 hrs incubation, 30 ng/ml of heregulin-β2 was added to each well and the plates were further incubated for 16 hrs. Following incubation, 10 μL PathHunter bioassay detection reagent 1 was added and incubated for 15 mins at room temperature in the dark, followed by addition of 40 μL PathHunter bioassay detection reagent 2 which was then incubated for 60 mins at room temperature in the dark. Plates were read using Synergy4 Biotek with 1 second delay. 10D1 F.FcA was found to have an EC50 value of 3.715e-11 for inhibition of HER2-HER3 heterodimerisation. In the same assay, the comparative EC50 value for Seribantumab/MM-121 was found to be 6.788e-10 and the comparative EC50 value for Pertuzumab was found to be 2.481e-10.
Anti-HER3 antibody 10D1F.FcA was analysed for its ability to inhibit heterodimerisation of EGFR and HER3.
Plate-based ELISA dimerisation assays were performed according to standard protocols. Plate was coated with 1 μg/ml human EGFR-His. After blocking and washing, the plate was incubated with different concentrations of candidate antibodies 10D1F.FcA, MM-121, LJM716, Pertuzumab, or isotype control with constant HER3-biotin 4 μg/ml and NRG 0.1 μg/ml for 1 hour. Plates were then washed and incubated for 1 hour with secondary anti-avidin HRP antibody. Plates were washed, treated with TMB for 10 mins and the reaction was stopped using 2M H2SO4 stop solution. The absorbance was read at 450 nm.
The results are shown in
Anti-HER3 antibody clones 10D1F.FcA and 10D1F.FcB were analysed for their ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC).
Target cells (HEK293 overexpressing HER3) were plated in U-bottom 96-well plates at a density of 20,000 cells/well. Cells were treated with a dilution series (50,000 ng/ml-0.18 ng/ml) of one of 10D1F.FcA, 10D1F.FcB, 10D1F.FcA_N297Q (silent form), LJM-716, Seribantumab (MM-121) or left untreated, and incubated at 37° C. and 5% CO2 for 30 min. Effector cells (Human Natural Killer Cell Line No-GFP-CD16.NK-92; 176V) were added to the plate containing target cells at a density of 60,000 cells/well.
The following controls were included: Target cell maximal LDH release (target cells only), spontaneous release (target cells and effector cells without antibody), background (culture media only). Plates were spun down and incubated at 37° C. and 5% CO2 for 21 hrs.
LDH release assay (Pierce LDH Cytotoxicity Assay Kit): before the assay, 10 μl of Lysis Buffer (10×) were added to target cell maximal LDH release controls and incubated at 37° C. and 5% CO2 for 20 min. After incubation, plates were spun down and 50 μL of the supernatant were transferred to clear flat-bottom 96-well plates. Reactions were started by addition of 50 μl of LDH assay mix containing substrate to the supernatants and incubated at 37° C. for 30 min. Reactions were stopped by addition of 50 μl of stop solution and absorbance was recorded at 490 nm and 680 nm with a BioTek Synergy HT microplate reader.
For data analysis, absorbance from test samples was corrected to background and spontaneous release from target cells and effector cells. Percent cytotoxicity of test samples was calculated relative to target cell maximal LDH release controls and plot as a function of antibody concentration.
The results are shown in
Anti-HER3 antibody 10D1F.FcA was analysed for its ability to inhibit HER-3 mediated signalling in vitro in cancer cell lines.
N87, FaDu or OvCAR8 cells were seeded in wells in a 6 well plate with 10% serum overnight at 37° C. with 5% CO2. Cells were starved with 0.2% FBS culture medium for 16 hrs, and were then treated for 0.5 hours with different antibodies at IC50 corresponding to the cell line. Antibodies tested were: 10D1F.FcA (10D1), Seribantumab (SBT), Elegemtumab (LJM), Pertuzumab (PTM), Cetuximab (CTX), and Trastuzumab (TZ).
Before harvesting, cells were stimulated with 100 ng/ml of NRG1. Protein extracted from cell lines were quantified using standard Bradford protein assay. Protein samples (50 μg) were fractionated by SDS-PAGE and transferred to nitrocellulose membrane. Membranes were then blocked and immunoblotted with the indicated antibodies. The results were visualized via Bio-Rad Clarity Western ECL substrate. The blots were quantified using densiometric analysis and data was normalised to beta actin.
The results are shown in
For the experiments using N87 cells, A549 cells, OvCar8 cells and FaDu cells total RNA was extracted at 16 hrs post antibody treatment was analysed to determine pathway activation based on the level of expression of key signal transduction pathway proteins by gene set enrichment analysis. The results of the analysis are shown in
In further experiments using A549 cells, in vitro phosphorylation assays were performed as above except that the cells were treated for 0.5 hours or 4 hours with the different antibodies. The results are shown in
In further experiments, anti-HER3 antibodies were analysed in order to determine the affinity of binding to human HER3 in the context of a human HER3:human NRG1 complex (that is, HER3 provided in the ligand-bound, ‘open’ conformation), as compared to the affinity of binding to human HER3 in the absence of NRG1 complex (that is, HER3 provided in the unbound, ‘closed’ conformation).
Binding of anti-HER3 antibodies to human HER3 was evaluated by BLI. Briefly, anti-Human IgG Capture (AHC) sensors (ForteBio) were loaded with anti-HER3 IgG antibodies (25 nM). Kinetic measurements were performed in absence or presence of NRG1. NRG1 was used at 1:1 molar ratio with HER3 wherein the complex was allowed to form at RT for 2 h. His-tagged human HER3 or HER3-NRG1 complexes were loaded to antibody coated AHC sensors at different concentrations for 120 s, followed by a 120 s dissociation time. All measurements were performed at 25° C. with agitation at 1000 rpm. Sensorgrams were referenced for buffer effects and then analyzed using the Octet QK384-software (ForteBio). Kinetic responses were globally fitted using a one-site binding model to obtain values for association (Kon), dissociation (Koff) rate constants and the equilibrium dissociation constant (KD).
The following anti-HER3 antibodies were analysed in the experiments: 10D1F.A ([16] of Example 2.2), MM-121 and LJM-716.
The results are shown in
Taken together, the results identify 10D1F as an anti-HER3 antibody which binds to an epitope of HER3 providing it with the unique combination of properties that (i) it competitively inhibits heterodimerisation of HER3 with EGFR or HER2 (see Example 8.7 and
The pharmacokinetics of anti-HER3 antibodies 10D1F.FcA or 10D1F.FcB administered intravenously were assessed in mouse and rat. 10D1F.FcA is also referred to herein as HMBD-001. The parameters for the pharmacokinetic analysis in rats and mice were derived from a non-compartmental model: maximum concentration (Cmax), AUC (0-336 hr), AUC (0-infinity), Half-life (t1/2), Clearance (CL), Volume of distribution at steady state (Vd). All animal studies have been performed in strict compliance with local ethics committee requirements, sector standards and applicable law.
500 μg anti-HER3 antibody 10D1F.FcA or 10D1F.FcB was administered and blood was obtained at baseline (−2 hr), 6 hr, 24 hr, 96 hr, 168 hr and 336 hr after administration. Antibody in the serum was quantified by ELISA.
The results are shown in
10D1 F variants were analysed to determine a single dose pharmacokinetic profile in female Sprague Dawley rats.
Antibody clones 10D1F.FcA and 10D1F.FcB were administered in a single dose of 4 mg (˜10 mg/kg), 10 mg (˜25 mg/kg), 40 mg (˜100 mg/kg) or 100 mg (˜250 mg/kg) via tail vein slow i.v. injection. Vehicle was administered as a negative control. Blood was obtained at baseline (−24 hr), 6 hr, 24 hr, 96 hr, 168 hr and 336 hr after administration. Antibody in the serum was quantified by ELISA.
The results are shown in
The toxicological effects of 10D1F.FcA and 10D1F.FcB were analysed.
BALB/c mice were injected intraperitoneally with a single dose of either 10D1F.FcA and 10D1F.FcB at one of doses: 200 ug (˜10 mg/kg), 500 ug (˜25 mg/kg), 2 mg (˜100 mg/kg), or 5 mg (˜250 mg/kg), or an equal volume of PBS. 3 mice were injected with each treatment, 4 mice were injected with PBS control. Blood samples were obtained at 96 hours post injection and analysed for RBC indices (total RBC count, haematocrit, haemoglobin, platelet count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration) and WBC indices (total WBC count, lymphocyte count, neutrophil count, monocyte count). Analysis was performed using a HM5 Hematology Analyser.
The results are shown in
Mice treated with 10D1F.FcA or 10D1F.FcB showed no abnormalities after 96 hours in weight, behaviour, skin condition, oral examination, stool and urine examination or eye examination. Enlarged spleen (splenomegaly) approximately 1.5 times the normal size was observed in mice treated with higher doses: 10D1F.FcA 250 mg/kg, 10D1F.FcB 100 mg/kg, 10D1F.FcB 250 mg/kg.
A further study was performed in the BALB/c mice to assess the toxicological effects of repeat doses of 500 μg (˜25 mg/kg) 10D1 F.FcA or 10D1F.FcB. Antibody was administered once a week for four weeks. Blood was obtained 28 days after the first administration. There was no effect observed on RBC, liver, kidney, pancreatic or electrolyte indices for either antibody, no signs of clinical abnormalities and no differences detected in gross necroscopy. Total WBC count, lymphocyte count and neutrophil count was observed to be decreased in mice treated with 10D1F.FcA or 10D1F.FcB but this was not considered to be adverse.
In another study, BALB/c mice were administered with a single dose of 10D1F.FcA or an equal volume of PBS (vehicle control), and analysed after 96 hours (vehicle) or 336 hours (10D1F.FcA). Representative results are shown in
Sprague Dawley rats were injected intraperitoneally with a single dose of either 10D1F.FcA or 10D1F.FcB antibody at one of doses: 4 mg (˜10 mg/kg), 10 mg (˜25 mg/kg), 40 mg (˜100 mg/kg), 100 mg (˜250 mg/kg). Blood was obtained at −24 hours, 6 hours, 24 hours, 96 hours, 168 hours and 336 hours. Up to 366 hours post injection there was no effect on RBC indices, no toxic effect on WBC indices, and no effect on liver, kidney, pancreatic or electrolyte indices. There were no signs of clinical abnormalities and no differences detected in gross necroscopy.
Representative results obtained from rats administered with 250 mg/kg 10D1F.FcA are shown in
The absence of toxicity signals in rodent toxicology models is a predictor of tolerability of these antibodies at clinical efficacious doses.
Further toxicokinetic studies are described in Example 16.4.
Anti-HER3 antibody 10D1F.FcA was analysed for its ability to inhibit tumour growth in vitro in a number of tumour models: N87 cells (gastric cancer), HCC95 cells (lung cancer), FaDu cells (head and neck cancer), SNU-16 cells (gastric cancer), A549 cells (lung cancer), OvCar8 cells (ovarian cancer), ACHN cells (kidney cancer) and HT29 cells (colorectal cancer). 10D1F.FcA efficacy was compared to other anti-HER3 antibodies seribantumab (MM-121) and LJM-716, and other EGFR family therapies cetuximab, trastuzumab and pertuzumab.
Cells were treated with serially diluted concentrations of therapeutic antibodies, starting from 1500 ug/ml with a 9-point dilution. Cell viability was measured using CCK-8 cell proliferation assay, 3-5 days post treatment. The percentage of cell inhibition shown is relative to cells treated with only buffer (PBS). Data points indicates average of three replicates.
The results are shown in
Anti-HER3 antibody clones 10D1F.FcA and 10D1F.FcB were assessed for their effect on tumour growth in in vivo cancer models.
Tumour cells were inserted subcutaneously into the right flanks of female NCr nude mice. Antibodies (25 mg/kg 10D1F.FcA, 10D1F.FcB, Cetuximab, LJM-716 or MM-121) or vehicle were administered biweekly for six weeks.
The results are shown in
Tumour cells were inserted with matrigel subcutaneously into the right flanks of female NCr nude mice. Antibodies (10 and 25 mg/kg 10D1F.FcA and 10D1F.FcB, or 25 mg/kg of Cetuximab, Trastuzumab, Pertuzumab, LJM-716 or MM-121) or vehicle were administered once a week for six weeks.
The results are shown in
Tumour cells were inserted with matrigel subcutaneously into the right flanks of female NCr nude mice. Antibodies (10 and 25 mg/kg 10D1F.FcA, or 25 mg/kg Cetuximab, LJM-716 or MM-121) or vehicle were administered once a week for six weeks.
The results are shown in
Tumour cells are inserted with matrigel subcutaneously into the right flanks of female NCr nude mice. Antibodies (25 mg/kg 10D1F.FcA, or 50 mg/kg of Trastuzumab, LJM-716 or MM-121) or vehicle were administered biweekly for six weeks.
The results are shown in
The following cell lines were investigated:
The cells were investigated for surface expression of EGFR family members by flow cytometry. Briefly, 300,000 cells were incubated with 20 μg/ml of 10D1F.FcA, cetuximab or trastuzumab for 1 hr at 4° C. Alexafluor 488-conjugated anti-human antibody was used at 10 μg/ml as a secondary antibody (40 min at 4° C.).
The results are shown in
The inventors investigated the ability of different HER3-binding antibodies to inhibit in vitro proliferation of different thyroid cancer cell lines harbouring the V600E BRAF mutation.
Briefly, cells of the different cell lines were seeded at a density of 1.5×105 cells/well, and treated the next day with a 10 point serial dilution starting from 1000 μg/ml of 10D1F.FcA, seribantumab, LJM-716, pertuzumab or isotype control antibody. After 3 days, proliferation was measured using a CCK-8 cell proliferation assay. Percent inhibition of proliferation was calculated relative to cells treated with an equal volume of PBS instead of antibodies.
The results are shown in
In further experiments, the ability of a combination of 10D1F.FcA and vemurafenib to inhibit in vitro proliferation of different thyroid cancer cell lines harbouring the V600E BRAF mutation was investigated.
Cells were seeded at a density of 1.5×105 cells/well, and treated the next day with a 10 point serial dilution starting from 1000 μg/ml of 10D1F.FcA or isotype control antibody, in the presence or absence of 200 nM vemurafenib. After 3 days, proliferation was measured using a CCK-8 cell proliferation assay. Percent inhibition of proliferation was calculated relative to cells treated with an equal volume of PBS instead of antibodies.
The results are shown in
The inventors investigated the ability of 10D1F.FcA to inhibit HER3-mediated signalling in vivo. 1×106 FaDu or OvCar8 cells were introduced subcutaneously into NCr nude mice, to establish ectopic xenograft tumors.
Once tumors had reached a volume of greater than 100 mm3, mice were with treated by biweekly intraperitoneal injection of 10D1F.FcA at a dose of 25 mg/kg, or an equal volume of vehicle (control). After 4 weeks, tumors were harvested. Protein extracts were prepared from the tumors and quantified via Bradford assay, 50 μg samples were fractionated by SDS-PAGE, and analysed by western blot using antibodies in order to determine in vivo phosphorylation of HER3 and AKT, as described in Example 4.3.
The results are shown in
The inventors investigated internalisation of anti-HER3 antibodies by HER3-expressing cells.
Briefly, 100,000 HEK293 cells engineered to express HER3, HCC95, N87 or OVCAR8 cells were seeded in wells of 96-well tissue culture plates and cultured overnight at 37° C. in 5% CO2. Cells were then treated with 120 nM of 10D1F.FcA, LJM-716, seribantumab or trastuzumab, and 360 nM of pHrodo iFL Green reagent, and incubated at 37° C. in 5% CO2. The cells in culture were imaged every 30 min for 24 hours, in 4 different fields of each well. The maximum signal intensity in the FITC channel of each field was quantified at 24 hours.
The results are shown in
No significant internalization of 10D1F.FcA was observed in HCC95, N87, or OvCar8 cells.
As expected, significant internalization of 10D1F.FcA, LJM-716, and seribantumab was observed in HEK293 cells overexpressing HER3.
In further experiments, antibody internalisation was investigated by flow cytometry.
N87 cells were seeded in wells of 96-well tissue culture plates at a density of 50,000 cells/well, and allowed to adhere overnight (37° C., 5% CO2). 10D1F.FcA or trastuzumab were mixed with labelling reagent, and the labelled complexes were added to cells. Samples were harvested at 0 min, 10 min, 30 min, 1 hour, 2 hour and 4.5 hour time points, by aspiration of cell culture medium, washing with PBS and treatment with accutase. Accutase activity was neutralised, and cells were resuspended in FACs buffer and analysed by flow cytometry.
The results are shown in
Anti-HER3 antibody 10D1F in mIgG2a format was evaluated for its ability to be used in immunohistochemistry for the detection of human HER3 protein.
Processing of sections was performed using Bond reagents (Leica Biosystems). Arrays of commercially-available frozen tissue sections were obtained. Slides were dried in a desiccator for 10 min and then subjected to the following treatments, with water washes and/or TBS-T rinses between steps: (i) fixation by treatment with 100% acetone for 10 min at room temperature; (ii) endogenous peroxidase blocking by treatment with 3% (v/v) H2O2 for 15 min at room temperature; (iii) blocking by treatment with 10% goat serum for 30 min at room temperature, (iv) incubation with 10D1 F-mIgG2a at 1:250 dilution of a 6.2 mg/ml solution overnight at 4° C., (v) incubation with HRP-polymer conjugated goat anti-mouse antibody for 30 min at room temperature, and (vi) development with Bond Mixed DAB Refine for 5 min at room temperature, followed by rinsing with deionised water and 1× Bond Wash to stop the reaction.
Slides were then dehydrated, mounted in synthetic mounting media and scanned with high resolution.
The results are shown in
In further experiments, an A549 xenograft tumor was harvested in cold PBS, embedded in OCT cryoembedding medium, frozen in dry-ice and stored at −80° C. 10 μm sections were obtained using a cryostat.
Slides were dried in a desiccator for 10 min and then subjected to the following treatments, with water washes and/or TBS-T rinses between steps: (i) fixation by treatment with 100% acetone for 10 min at room temperature; (ii) endogenous peroxidase blocking by treatment with 3% (v/v) H2O2 for 15 min at room temperature; (iii) blocking by treatment with 10% goat serum for 30 min at room temperature, (iv) incubation with 10D1F.FcA at 1:50 dilution of 8.8 mg/ml solution, or with 1:200 dilution of Sino Biological rabbit anti-HER3 (Cat. No. 10201-T24) overnight at 4° C., (v) incubation with Invitrogen F(ab′)2-Goat anti-Human IgG (H+L) HRP (A24470) (1:500), or HRP-polymer conjugated goat anti-rabbit antibody for 30 min at room temperature, and (vi) development with Bond Mixed DAB Refine for 5 min at room temperature, followed by rinsing with deionised water and 1× Bond Wash to stop the reaction.
Slides were then counterstained with haematoxylin, dehydrated, mounted in synthetic mounting media and scanned with high resolution.
The results are shown in
The inventors investigated the therapeutic efficacy of 10D1F in a patient-derived xenograft model of ovarian cancer comprising CLU-NRG1 fusion.
Female BALB/c nude mice approximately 5-7 weeks old were housed under specific pathogen-free conditions and were treated in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines.
A model of ovarian cancer comprising CLU-NRG1 fusion was established by inoculating mice subcutaneously at the right flank with four pellets of a patient-derived xenograft designated OV6308 (Crown Bioscience Inc.). OV6308 is an ovarian high-grade serous adenocarcinoma derived from a 51-year female patient, and comprises the CLU-NRG1 gene fusion.
Tumor volumes were measured 3 times a week using a digital caliper and calculated using the formula [L ×W2/2]. Study End point was considered to have been reached once the tumors of the control arm measured >1.5 cm in length.
Mice were administered biweekly by intraperitoneal injection with (i) 25 mg/kg of 10D1F.FcA (i.e. [16] of Example 2.2), or (ii) 25 mg/kg of hIgG1 isotype control antibody (n=10 for each treatment group).
The results are shown in
Formulation development was performed for an antigen-binding molecule comprising 10D1F variable regions and human IgG1 constant regions and which is comprised of the polypeptides of SEQ ID NOs: 206 and 207. The antigen-binding molecule is produced by a cell of the cell line deposited 7 May 2021 as ATCC Patent Deposit Number PTA-127062.
Nine liquid formulations comprising the antibody were generated as follows:
Formulation F10 comprised antibody and PBS. The formulations were evaluated for their stability under different conditions.
Stability of antibody in the different formulations was evaluated after storage at 40° C. for 1 month. All formulations comprised 8.4 mg/mL protein.
Stability of antibody in the different formulations was evaluated at high protein concentration.
Visual inspection for visible aggregates or particles showed that no visible particles were induced in any formulation at 10 mg/mL, 50 mg/mL, 100 mg/mL or 200 mg/mL (
Freeze-thaw cycles of −80° C. and room temperature were performed every 3+ days.
In conclusion, the antibody is more stable in formulations F1 to F7 compared to formulations F8 to F10.
Five further formulations were generated using buffer exchange as follows:
Formulation 5 comprises the same constituents as Formulation F2 in Example 15.1.
The five formulations were subjected to preliminary stress screening by freezing and thawing and mechanical stress by agitation and syringe aspiration.
Freeze-thawing: Three cycles of freezing at −70° C. and thawing at room temperature were performed. The vials were placed separately in the −70° C. freezer and were stored in the freezer for 90 hrs. The vials were thawed at room temperature, protected from light and placed separately. The vials were gently swirled two times per hour until fully thawed. After the three freeze/thaw cycles, the thawed vials were stored at 2-8° C. until analysis.
Syringe and needle aspiration: Samples were aspired and dispensed through a syringe and needle 10 times. The needle was as small as possible in order to induce aggregate and particle formation. A 5-mL syringe with a needle were used with manual aspiration and dispensing at a rate of approximately 1.5 mL/s.
Agitation: The samples were mounted on a shaking table and agitated 72 hrs at 2-8° C. A multi-rotator (Multi RS-60 BioSan) were used for end-over-end rotation including vibration.
The samples were analyzed for particle formation by appearance and microflow imaging (MFI), aggregation by SE-HPLC, purity and degradation by CE-SDS and protein content by A280. The results are shown below. No visible particles were detected in any formulation by visual inspection.
Protein content by A280. No significant change was seen in the protein concentration in the stressed samples compared to the reference samples.
Monomeric purity. No change in monomeric purity was observed after the stress tests in any of the formulations.
Number of subvisible particles/mL by Micro-Flow Imaging.
Each sample was analyzed with CE-SDS after stress tests. Results in
Formulations 1, 4 and 5 were advanced to a formulation stability study.
The stability of formulations 1, 4 and 5 was evaluated over 12 weeks at 50 mg/mL, at +5° C. and at +25° C. At 0, 4, 8 and 12 weeks, the samples were analysed for stability using the following techniques:
Each sample was analysed at 0 weeks and 12 weeks. No significant differences were seen in the protein concentrations at T0 compared to T12.
The samples were inspected for visible particles every 4 weeks. No visible particles were induced in any formulation at either temperature.
The samples were analysed by SE-HPLC every fourth week for 12 weeks.
The results are shown in
The charged distribution data from IE-HPLC is shown in
Subvisible particle count differed between samples and over time but was attributed to method variation rather than being an effect of buffer type or temperature. There was no clear trend in increase of subvisible particles in any formulation over time.
Data do not indicate any formation of low molecular weight species due to peptide bond disruption. After T4, a non-reducible heavy chain/light chain thioether bond could be detected which accounts for the missing percent in the summarized data for reduced conditions at T8 and T12.
15.3.7 pH
No change in pH was detected for formulations 1, 4 or 5 over 12 weeks incubation.
No change in osmolality was detected for formulation 1, 4 or 5 over 12 weeks incubation.
The antigen binding ELISA data is presented below. Differences are related to method variation and no significant change of binding capacity was observed. Formulation 1 (T0 and T12+5° C.) was used as internal control between ELISA plates.
1)Average of a duplicate, as specified sample was used as internal control in individual plates
Protein content measurement by A280 did not indicate any loss of drug product after 12 weeks incubation at either +5° C. or +25° C. Assessment by visual inspection did not indicate any formation of visible particles after 12 weeks incubation at either +5° C. or +25° C. Assessment by BMI did not indicate any significant formation of subvisible particles after 12 weeks incubation at either +5° C. or +25° C. CE-SDS results did not indicate any disruption of peptide bonds or disulfide bonds after 12 weeks incubation at either +5° C. or +25° C. Assessment of charged variants showed very small changes of the charged isoforms after 12 weeks incubation at either +5° C. or +25° C. Assessment of antigen-binding capacity did not indicate any change after 12 weeks incubation at either +5° C. or +25° C. Assessment of pH and osmolality did not indicate any change after 12 weeks incubation at either +5° C. or +25° C.
Assessment of the monomeric IgG purity by SE-HPLC indicated a drop in relative purity by 1.1%, for Formulations 1 and 4, and 1.8% for Formulation 5. The results for the respective formulations were plotted and extrapolated down to 95% monomeric IgG purity to estimate a longest shelf life of drug product in the different formulations. The predicted shelf life at +5° C. for all formulations was 28-29 weeks, with Formulation 5 having the steepest declining slope. At +25° C., Formulation 1 showed the shortest predicted shelf life of 14 weeks, and for Formulations 4 and 5 the predicted shelf life was 18 weeks and 21 weeks, respectively.
Formulation 5 (50 mg/mL HMBD-001, 20 mM histidine, 8% (w/v) sucrose (240 mM), 0.02% (w/v) polysorbate 80, final pH 5.8) was found to be the most suitable formulation and was chosen to be taken forward for clinical manufacture as a liquid fill drug product. Polysorbate 80 is added at a concentration of 0.02% (w/v) as a surfactant to prevent potential mechanical stress induced (agitation) instability of the protein and aggregation.
HMBD-001 formulated in 20 mM histidine, 8% (w/v) sucrose (240 mM), 0.02% (w/v) polysorbate 80, pH 5.8 was assessed further for:
pH and appearance of the formulation did not change after 7 or 15 days at 40° C. with 80% relative humidity. No soluble aggregation was detected after 15 days' incubation. Antibody integrity was not compromised after either length incubation.
HER3 binding and Fc reactivity were measured using Bio-Layer Interferometry. Bio-reactivity of the antibody was retained after incubation for 7 or 15 days, as below.
The antibody was concentrated to 200 mg/mL in 20 mM histidine, 8% (w/v) sucrose (240 mM), 0.02% (w/v) polysorbate 80, pH 5.8 and assessed for aggregation.
After 15 days' incubation at 40° C. with 80% relative humidity, the pH, appearance, solubility, protein integrity and bio-reactivity of the antibody are well retained in the formulation above. A concentration of 200 mg/ml was also achieved with no aggregation propensity.
The stability of HMBD-001 drug substance was assessed over 24 months at ≤−65° C. and 6 months at +5 (±3° C.) HMBD-001 drug product was assessed over 6 months at −20° C. and 6 months at +5 (±3° C.) 50 mg/mL HMBD-001 was formulated in 20 mM histidine, 8% (w/v) (240 mM) sucrose and 0.02% polysorbate 80, at pH 5.8 (batch HB0721-P5).
Evaluations included colour, visible particles, pH, osmolality, chemical instability (analytical HPLC-IEC and Iso Electric Focusing (IEF) for charge variation (deamidations and oxidations) and SDS-PAGE and HPLC-SEC for fragmentation), protein concentration (A280 (ε=1.64)2), aggregations (HPLC-SEC), loss of binding activity (ELISA), and product integrity/contaminants (endotoxin LAL and bioburden tests).
The results are shown in
Stability assessments are continued for up to 60 months, e.g. at 12, 18, 24, 36, 48 and 60 months.
As used herein, HMDB-001 refers to an IgG1 humanised monoclonal antigen-binding molecule comprising 10D1F variable regions and human IgG1 constant regions, and which is comprised of the polypeptides of SEQ ID NOs: 206 and 207.
HER3 is one of a family of receptors (‘the HER family’) which signal through the P13K/AKT/mTOR and the MAPK/ERK pathways to promote cell survival and proliferation. Aberrant activation or over-expression can therefore promote carcinogenesis and cancer progression. HER3 is unique among the family in that it lacks kinase activity, so must be activated by dimerising with a kinase-active partner (commonly epidermal growth factor receptor (EGFR) or HER2 which are other HER family members) for signal transduction to take place (Berger, Mendrola, and Lemmon 2004; Kim et al. 1998).
The HER3 extracellular domain exists in a reversible equilibrium between a “closed” inactive conformation and an “open” active conformation which can form dimers (Roskoski 2004; Cho and Leahy 2002). In the conventional model for activation, the equilibrium shifts to the open conformation as a result of ‘ligand dependent’ stabilisation (mediated by the HER3 ligand Neuregulin 1, [NRG1]). However, presence of any dimerisation partner at sufficient concentration can also shift the equilibrium, as the partner binds to and stabilizes HER3 that is transiently in the open conformation. This is known as ‘ligand-independent’ activation (Burgess et al. 2003; Lee-Hoeflich et al. 2008; Alimandi et al. 1995).
Overexpression of HER3 is frequently observed in multiple tumour types and is associated with a poorer clinical outcome. Enhanced expression of HER3 is found in colorectal carcinoma, head and neck squamous cell carcinoma (HNSCC), NSCLC, melanoma, and breast, gastric, ovarian, prostate, and bladder cancers. The impact of HER3 overexpression is greater in cancers where HER2 is also overexpressed e.g. breast, gastric and ovarian. HER3 is the preferred heterodimeric partner for EGFR in melanoma and pancreatic carcinoma.
HER3 represents a promising therapeutic target for the treatment of HER3 driven tumours and HER-therapy resistant tumours. Up-regulation of HER3 expression and activity is associated with resistance to multiple pathway inhibitors and associated with a poor prognosis. HER3 has a critical role in oncogenic EGFR family signalling. When HER3 heterodimerizes with EGFR or HER2 it triggers signalling through the P13K/AKT/mTOR pathway in addition to the MAPK pathway. HER3 activation has thus been implicated in acquired resistance to EGFR/HER2 therapies and other MAPK pathway therapies such as BRAF inhibitors, where the P13K pathway represents a common escape route for tumours. Although classically driven by ligand binding, HER3 heterodimerisation can also be driven by high levels of EGFR or HER2 binding partner.
A number of anti-HER3 therapies, including biological and small molecule agents, have been investigated clinically to date (Mishra et al. 2018). Apart from therapies engaging immune cells, all the HER3 therapies have the aim of inhibiting HER3 downstream signalling. HER3 signalling plays a major role in drug resistance to a variety of cancer therapies. In the case of resistance to EGFR or HER2 directed therapy, mechanisms include (i) transcriptional upregulation of HER3 (Abel et al. 2013; Wang et al. 2013), (ii) increased levels of NRG1 (Gwin and Spector 2014; Xia et al. 2013), and (iii) HER2 amplification (Vlacich and Coffey 2011; Yonesaka et al. 2011).
Oncogenic fusions of SLC3A2, CD74, or VAMP2 to NRG1 isoforms have been identified which promote secretion or extracellular expression of the EGF-like domain of NRG1 and thereby increase HER3 activation. NRG1 fusions were first identified and are most common in invasive mucinous adenocarcinoma (IMA) in which the fusion is present in 20-30% of cases but are also present in other types of solid tumours at lower frequency (<1%). Based on preclinical and limited clinical data, HER3-targeted therapy appears to be highly effective in NRG1 fusion driven IMA (Nakaoku et al. 2014). Efforts to target constitutively activating mutations in the downstream signalling cascades, such as BRAF V600E, which confer TKI resistance in thyroid and colon carcinomas may also fail through HER3 activation (Di Nicolantonio et al. 2008; Piscazzi et al. 2012; Frasca et al. 2013). Notably, activation of HER3 by NRG1 promotes resistance to the specific BRAF V600E inhibitor vemurafenib (Prasetyanti et al. 2015).
In support of the role of HER3 in drug-resistance, anti-HER3 antibodies restore sensitivity to vemurafenib in BRAF-V600E mutant colon cancer (Prasetyanti et al. 2015) and blockade of HER2:HER3 signalling with combination therapy overcomes trastuzumab resistance in HER2-positive breast cancer (Watanabe et al. 2019). HER3, therefore, represents a promising therapeutic target for the treatment of HER3 driven tumours and HER-therapy resistant tumours.
Due to the relationship of HER3 with sensitivity or resistance to HER-targeted therapy, with HER3 having to form heterodimer or heterotrimer complexes with other receptor tyrosine kinases in order to fully transduce signalling (Holbro et al. 2003; Lee-Hoeflich et al. 2008), the combination of HER3 and EGFR/HER2-targeted agents may be an efficient way to abrogate drug resistance and, thus, enhance anti-tumour activity in many solid tumours. For example, multiple preclinical models have shown that combination of anti-HER3 monoclonal antibodies with anti-EGFR therapies leads to enhanced anti-tumour activity (Gaborit, Lindzen, and Yarden 2016). The anti-HER3 antibody patritumab abrogates cetuximab resistance mediated by heregulin in colorectal cancer cells (Kawakami H. et al. 2014) and R3 expression has been shown to be upregulated in trastuzumab resistant tumour cells (Narayan M et al. 2009). Potential combinations include, but are not limited to, cetuximab, enzalutamide or other androgen receptor inhibitor, and trastuzumab.
A number of anti-HER3 therapies, including biological and small molecule agents, have been investigated clinically to date; approaches have been based on targeting HER3 by one of the following mechanisms: i) blocking ligand-binding sites; ii) locking HER3 in the tethered conformation; iii) trapping its primary ligand, NRG1; iv) triggering the internalisation of the HER3 receptor; or v) employing immune cells to kill cancer cells over expressing HER3 (Elenius et al. 1999).
Previous attempts to develop anti-HER3 antibodies have showed limited efficacy in clinical trials, possibly because they have been unable to completely block both ligand binding and heterodimerisation with EGFR or HER2. There remains considerable unmet need across tumour types where high HER3 expression is seen and where there is resistance to other EGFR/HER2 therapies. Additional non-cross resistant therapies are required.
Antibody HMBD-001 targets HER3 via a different mechanism. HMBD-001 is designed to directly block the heterodimerisation surface of HER3 and thus prevent both ligand dependent and independent heterodimerisation, thereby preventing cancer cells from becoming resistant to treatments such as cetuximab and trastuzumab. The most similar agents to HMBD-001 are LJM716 (elgemtumab) and CDX3379 (KTN3379) as these inhibit both ligand dependent and independent signalling, albeit by different mechanisms.
Furthermore, preclinical data support the exploration of an anti-HER3 antibody in the setting of resistance to anti-androgen therapies in prostate cancer. Zhang et al. 2020 have shown that cancer associated fibroblasts can promote anti-androgen resistance through increased levels of NRG1 in mouse models and in prostate organoid cultures, promoting resistance through activation of HER3. Furthermore, both anti-NRG1 and HER3 antibodies, in vitro and in vivo, in the setting of anti-androgen resistance have shown anti-tumour activity, providing a rationale for exploration of combining HMBD-001 with an androgen receptor inhibitor.
This is an open label, multi-centre, first-in-human (FIH), Phase I/Ila adaptive design trial with initial intra-patient dose escalation followed by inter-patient dose escalation to determine the recommended Phase II dose (RP2D) and schedule of HMBD-001 administration as a single agent.
Subsequently the combining of HMBD-001 with other anticancer combination agent(s) will be explored. For each novel combination, there will be a dose escalation arm and dose expansion cohort to further characterise the safety, pharmacokinetic (PK) and pharmacodynamic profiles of HMBD-001 and assess preliminary efficacy.
Part A: The first part of the study is a dose escalation stage conducted in 2 arms.
Part B: The second part of the study consists of two arms with HMBD-001 given as monotherapy and in combination in multiple combination cohorts.
HMBD-001 is an IgG1 humanised monoclonal antibody specifically targeting HER3, a receptor highly expressed on cancer cells in certain tumours.
HMBD-001 binds to an epitope on the domain II dimerisation interface of HER3. It exerts its pharmacological effect by directly inhibiting heterodimerisation with EGFR or HER2, thereby inhibiting the subsequent phosphorylation of HER3 and downstream signalling through the P13K/AKT/mTOR pathway, and inhibiting tumour cell proliferation. HMBD-001 is able to bind to its epitope whether HER3 is in the ‘open’ or ‘closed’ conformation, see e.g. Examples 3.5 and 8.10 above. This means it is pharmacologically active whether HER3 activation is driven in a ligand-dependent manner by NRG1 binding or in a ligand-independent manner (commonly by overexpression of HER2 or EGFR).
Preclinical pharmacology studies have demonstrated that HMBD-001 exerts its pharmacological effect by 1) binding to the receptor dimerisation interface of HER3 and blocking heterodimerisation with HER2 or EGFR; 2) inhibiting the subsequent phosphorylation of HER3 and downstream signalling through the P13K/AKT/mTOR pathway; 3) inhibiting tumour cell proliferation. See e.g. Examples 4.1, 4.3, 5.3, 8, 9 and 11 above).
HMBD-001 specifically binds with sub-nanomolar affinity to HER3, and inhibits dimerisation of HER3 with both HER2 and EGFR. It either fully or substantially inhibited phosphorylation of HER3 and AKT in both NRG1 driven and NRG1 independent cell line models. Inhibition of tumour cell proliferation by HMBD-001 was evident in a range of phenotypically distinct cell lines, and potency was superior to the other anti-HER3 comparator antibodies included, which were MM-121 (seribantumab) and LJM716 (elgemtumab). HMBD-001 was active in cells which were NRG1 driven, high HER2/EGFR driven, both NRG1 and HER2/EGFR driven, and in cells with the BRAF V600E mutation which confers tyrosine kinase inhibitor (TKI) resistance. HMBD-001 had negligible activity in natural killer (NK) cell mediated antibody-dependent cell-mediated cytotoxicity assays (ADCC) and complement dependent cytotoxicity (CDC) assays, suggesting these are not anticipated mechanisms of action.
Murine tumour efficacy studies were performed using cell-line derived xenograft (CDX) models in mice. Female NCr nude mice (5-8 weeks old, 21-29 g) were subcutaneously implanted with N87, A549, FaDu, OvCAR8 cells. Treatment was initiated when tumours reached approximately 100 to 200 mm3. Vehicle (PBS) or therapeutic antibodies (25 mg/kg) were administered intraperitoneally (IP) at indicated time points (weekly FaDu and OvCAR8, twice weekly N87 and A549). Tumours were measured twice per week using a calliper and tumour volume (mm3) was calculated. Each data point represents the mean tumour volume ±SEM (n=8 mice).
The results are shown in
HMBD-001 as monotherapy completely inhibited tumour growth at 25 mg/kg IP in high NRG1-driven A549 (lung carcinoma) and FaDu (hypopharyngeal carcinoma), and high NRG1- and HER2/EGFR-driven OvCAR8 (Ovarian) models. In the A549 and FaDu models, HMBD-001 showed superior efficacy to cetuximab and trastuzumab, respectively. HMBD-001 was either equivalent or superior to the comparator anti-HER3 antibodies in all models. In the N87 model (NRG1 independent), HMBD-001 showed significant anti-tumour efficacy (64% TGI), in contrast to the comparator anti-HER3 antibodies which showed no effect. HMBD-001 efficacy was also demonstrated in patient derived xenograft (PDX) models from non-small cell lung (NSCLC), oesophageal and ovarian tumours, including one with an NRG1 fusion.
Exploration of HMBD-001 dose response was performed in the A549 model. Antibody (10 mg/kg, 5 mg/kg or 2 mg/kg) or vehicle was administered biweekly. The results are shown in
HMBD-001 is supplied as a concentrate solution 50 mg/mL for dilution and IV infusion.
Formulation buffer: 20 mM histidine, 8% (w/v) sucrose, 0.02% (w/v) polysorbate 80 at pH 5.8.
HMBD-001 is stored frozen at −20° C. (±5° C.) and protected from light.
HMBD-001 concentrate solution 50 mg/mL for infusion is diluted in 0.9% sodium chloride (NaCl) to a concentration of not less than 1.2 mg/mL, to a minimum volume of 100 mL and a maximum volume of 250 mL. The prepared solution for infusion may be stored in a refrigerator between 2 and 8° C. for a maximum of 40 hours before administration and must be administered within 48 hours of initial dilution.
To determine the dosages of HMBD-001 to be given to human patients, the pharmacokinetic, pharmacology and toxicology data of HMBD-001 from studies in tumour bearing and non-tumour bearing mice and rats was used for scaling to human doses. To determine the expected biologically active dose, PK data from tumour efficacy studies where maximal anti-tumour effect was seen were used to define target trough levels of HMBD-001, see e.g.
In vivo studies for HMBD-001 have included a single dose IV toxicity study in mice and rats, and repeat dose IV toxicity studies in rats up to 28-day duration (weekly dosing) with 28 days recovery (see also Example 9.1). All animal studies have been performed in strict compliance with local ethics committee requirements, sector standards and applicable law.
In toxicokinetic studies, Wistar rats were administered four weekly doses of HMBD-001 at 25, 100 and 250 mg/kg via IV injection. Blood was obtained at a series of time points following Day 1 and either Day 22 or Day 29. Antibody in the serum was quantified by immunoassay.
The elimination half-life estimates at Day 1 ranged from 178 to 251 hours. Exposure, as assessed by HMBD-001 Cmax and AUC0-168, increased with the increase in HMBD-001 dose level from 25 to 250 mg/kg/dose, and overall was approximately dose proportional between 25 and 250 mg/kg/dose. The TK profiles for both studies showed HMBD-001 mean Cmax and AUC0-168 values were higher on Day 22 or 29 compared to Day 1, indicating accumulation of HMBD-001 after multiple doses of HMBD-001 in rats. There were no significant differences between male and female rats in any PK parameters.
A single dose PK study was performed in NCr Nude mice (25 mg/kg HMBD-001, IV). A single dose PK study including a range of dose levels (2, 5, 10 and 25 mg/kg HMBD-001, IP) was conducted in A549 tumour bearing mice. The pharmacokinetic parameters are included in Table 1.
In a GLP toxicity study using HMBD-001, the highest non-severely toxic dose (HNSTD) and no observed adverse effect level (NOAEL) in rats were 250 mg/kg/dose (the highest dose level tested). This dose corresponded with a Cmax value of 8490 μg/mL and an AUC0-168 value of 423,000 h*μg/mL on Day 29 of the main dosing phase. This is equivalent to a 66 mg/kg human equivalent dose (60 kg human) based on allometric scaling and analysis of exposure of other anti-HER3 antibodies which have been administered clinically.
The observed pharmacokinetic parameters for both rats and mice were in line with typical values for monoclonal antibodies in healthy animals, i.e. volume of distribution slightly over total plasma volume, biological half-life in the range of several days and clearance of several millilitres per day (Oitate et al. 2011; Liu 2018).
1Day of dosing to which the pharmacokinetic/toxicokinetic parameters are related.
2Denominator specifies ‘t’ (time).
3Male and female data were combined as there were no significant differences.
An in vitro cytokine release assay was carried out using PBMC from 10 healthy donors and HMBD-001 in a plate-immobilized format. The concentration of all tested analytes (TNF-α, IL-2, IFN-γ, IL-6 and IL-10) induced by immobilized HMBD-001 in the assay were equivalent to or lower than those induced by the negative controls.
In immunohistochemistry (IHC) studies, HMBD-001 weakly or weakly to moderately stained the membrane and cytoplasm of rare to occasional or occasional epithelial cells in a few of the human tissues examined, including appendix (mucosa), mammary gland (glands, ducts), pancreas (ducts), and uterus (surface, glands), consistent with the reported expression of HER3 at low levels in epithelial cells in normal tissues. No unexpected reactivity was observed with HMBD-001 in any of the human tissues examined. Specificity of HMBD-001 was also confirmed by screening on recombinant cells expressing >5000 human proteins.
Pharmacokinetic data from other HER3 targeting monoclonals where relevant information is available are presented in Table 3. For previous anti-HER3 antibodies where information was available, elimination half-life ranged from approximately 8 to 14 days once pharmacokinetics were in the linear phase, while shorter half-lives were observed at low doses, likely due to the contribution of target mediated drug disposition (TMDD). Dose normalised Cmax levels for other anti-HER3 agents varied by up to 2-fold (from 19-41 μg/ml per mg/kg dosed). Anti-HER3 antibodies, because of their blocking mechanism, tend to be escalated to target a pre-defined ‘trough’ level for pharmacological activity, which can be accompanied by direct analysis of target engagement using biomarkers, and/or by establishment of ‘saturating’ or ‘linear’ PK profiles as an indirect marker of target saturation.
HMBD-001 is administered to human subjects, e.g. using doses, dosage regimes, formulations and patient selection criteria as provided herein, and one or more of the following outcomes are observed:
HMBD-001 is administered as an intravenous once weekly, 2-weekly, 3-weekly, or 4-weekly infusion. Subjects have been administered HMBD-001 at 150 mg, 300 mg, 600 mg, 1200 mg and 1800 mg.
Each cycle of HMBD-001 consists of between 21 and 28 days depending on the dose frequency. The number of doses received per cycle will also be dependent on the dose frequency and patients may continue initially for up to 6 cycles. Refer to Table 4 for initial dosing schedules and cycle duration.
Treatment may continue for longer than 6 cycles where a patient is benefiting from treatment with HMBD-001 either as a monotherapy or in combination (i.e. has stable or responding disease as measured by RECIST 1.1 and the patient is not experiencing any Grade 3 or greater IMP-related adverse events).
HMBD-001 is administered as a slow intravenous infusion using an infusion pump over a fixed interval of 120 minutes for Cycle 1, starting with a weekly schedule in the early single patient cohorts. If the patient does not experience any infusion related reactions, the infusion rate may be reduced to 60 minutes after completion of Cycle 1.
A single patient, intra-patient dose escalation scheme is applied. During the intra-patient dose escalation phase, a Cycle is defined as 28 days and consists of 4 weekly doses of HMBD-001.
Assuming that the emerging pharmacokinetic properties of HMBD-001 are in line with expectations (see e.g. Example 17). Alternative, less frequent dosing schedules (i.e. every 2 or 3 weeks) will be considered. Any alternative dosing schedules will not be more frequent than once weekly dosing or be less frequent than once every 28 days.
Following the intra-patient dose escalation phase, a multi-patient cohort, inter-patient dose escalation scheme is being explored. The expected maximum dose to be administered in the single agent dose escalation phase is 2100 mg (e.g. per week).
The first patient in a cohort is observed for toxicity for 7 days from HMBD-001 administration on Cycle 1 Day 1 before the second and third patients can receive HMBD-001. Patients who appear to be benefiting from HMBD-001 (stable disease or complete or partial response) and who are not experiencing any severe adverse reactions, may continue to receive further cycles.
The anticipated maximum clinical dose is 60 mg/kg per 50 kg patient, which is equivalent to 3000 mg per patient.
The HMBD-001 starting dose for each combination dose escalation cohort is selected based upon the combination agent selected and the data from the monotherapy dose escalation phase.
On determining the RP2D and schedule of single agent HMBD-001 in Part A, Arm 1, patients are recruited to a monotherapy dose expansion cohort (Part B, Arm 1) in order to provide paired tumour biopsies for pharmacodynamic analysis. A 3-stage Bayesian adaptive design is used to assess anti-tumour activity, where a true response rate of 10% is considered undesirable and at least 25% as desirable. A minimum of 10 and a maximum of 25 evaluable patients are enrolled.
Patients are selected based upon high tumour cell HER3 expression status or confirmed NRG1 fusion rearrangement from the following tumour subtypes: RAS wild type colorectal, castration resistant prostate cancer, triple negative breast cancer and squamous cell carcinoma of the head and neck. Expression of phosphorylated HER3 and/or high NRG1 expression in fresh biopsies may also be evaluated as predictive biomarkers if indicated by the acquired data.
16.6.4 Part B Arm 2: Combination Agent(s) Dose Expansion Multiple combination cohorts using the RP2D for HMBD-001 that can be given with the selected combination agent(s) as determined in the combination agent dose escalation (Part A, Arm 2) in selected indications are enrolled. A 3-stage Bayesian adaptive design, is used to assess anti-tumour activity, allowing for early stopping if the intervention is futile.
Taking into account the type of compound (targeted biological), mechanism of action (competitive receptor blocking), and available preclinical pharmacology and toxicity data, the inventors determined that both anti-tumour activity and toxicity will be driven by area under the curve (AUC) and by maintenance of HMBD-001 plasma levels above a pharmacologically active level.
Since HMBD-001 is not an agonist and HER3 is not a high-risk target, the inventors chose to start at a level with some biological activity to minimise exposure of patients to sub-therapeutic dose levels. A pharmacologically active dose (PAD) approach was therefore used. Preclinical in vitro and in vivo pharmacology data was used to define target plasma levels for pharmacological activity and efficacy, and these have been used to inform the starting dose, and the expected efficacious level respectively.
The FIH clinical trial uses fixed dose levels rather than body weight based. This is based on studies including simulations and clinical pharmacology of administered antibodies, which have shown that interpatient variation in exposure is comparable after body weight and fixed dosing, and pharmacokinetic variability is moderate relative to the variability generally observed in pharmacodynamics, efficacy and safety (Bai et al. 2012; Hendrikx et al. 2017). Dose calculations/scaling between species have been carried out on a mg/kg basis and a body weight of 60 kg for human subsequently used for conversion.
Preclinical in vitro pharmacology data was used to define relevant target plasma levels for starting dose; the mean IC50 in proliferation assays in 6 cell lines was 108 μg/mL (range 19-168 μg/ml, n=3 per cell line). Allometric scaling of rat PK parameters was used to predict human PK parameters, and to model doses which would result in human plasma levels in the desired range for a limited period following the first dosing occasion (>19 μg/ml for less than 1 week). From this analysis, 2.5 mg/kg was selected as the appropriate dose level to achieve this profile.
Additionally, in setting the starting dose, the following factors were considered to ensure that the PAD-based approach had generated a starting dose which was within the range established as non-adverse by in vitro and in vivo toxicology studies and to compare to starting doses of other anti-HER3 antibodies in the clinic.
Toxicology and exposure data from rat studies was used to provide an upper limit for the starting dose. The NOAEL (no observed adverse effect level) was set at 250 mg/kg (dosed weekly) since this was the top dose included in the rat GLP repeat dose study. Modelling of the rat data from this dose level converged with a 2 compartmental model which generated human predictions for clearance and volume of distribution, and predicted that equivalent human exposure would be achieved at a dose of 66 mg/kg (based on 60 kg human), which is approximately 26 times higher than the proposed clinical starting dose and twice the proposed maximum dose in human. This supports the suitability of the 2.5 mg/kg proposed starting dose, considering that the affinity of HMBD-001 for HER3 and the expression and location of the target are broadly equivalent in human and rat.
In vitro cytokine release assays indicated that HMBD-001 did not elicit cytokine release above negative controls and other low risk mAb comparators. Although it is not possible to directly equate blood concentrations with the assay because of the immobilised antibody format, assumptions based on other anti-HER3 antibodies can be used to estimate that a Cmax of 50-70 μg/mL would result from the first administration of the 2.5 mg/kg starting dose, which is significantly lower than the top concentration of 100 μg/mL tested in vitro.
Alongside modelling of HMBD-001 preclinical data, starting dose calculations for HMBD-001 have taken into account pharmacokinetic data from other anti-HER3 antibodies (Table 3), including expected half-life and Cmax range, and the concept of target-mediated drug disposition driving faster clearance in early cohorts. For anti-HER3 antibodies already in the clinic, starting doses ranged from 18-360 mg (0.25-5.1 mg/kg) (all trials summarised by Mishra, 2018).
The starting doses of the anti-HER3 antibodies with the most similar mechanisms to HMBD-001 were KTN3379 (5 mg/kg) and LJM716 (3 mg/kg).
Based on a consideration of the factors above, a starting dose of 2.5 mg/kg was chosen. This dose would result in a predicted Cmax of 50-75 μg/mL on the first dosing occasion, based on normal human plasma volumes. This starting dose would be expected to have some pharmacological activity, but this would be limited to a duration of less than one week after each administration so would not be expected to be a therapeutic dose. Target mediated disposition would be expected in early cohorts, and would result in shorter exposure above the target level than predictions, but since this cannot be modelled accurately this starting dose level is considered to be appropriate.
The starting dose level of 2.5 mg/kg has been converted based on a 60 kg bodyweight to reach a starting dose of 150 mg fixed dose in Cohort 1.
Based on the in vivo data across the models used, 150 μg/mL is the plasma level where efficacy may be expected in human, although differences in tumour penetration may result in higher plasma levels being required in human to achieve equivalent intra-tumoural concentrations. Based on analysis of PK parameters, it is expected that for HMBD-001 this Cmin plasma level would be maintained with doses of between 10 and 30 mg/kg, with dosing intervals between weekly and three weekly depending on the elimination half-life. For this reason, the maximum human dose is expected to be a 2100 mg fixed dose, which is at the upper end of this range.
To identify the Maximum Tolerated Dose (MTD) of the HMBD-001 monotherapy, a one-stage Bayesian CRM based on a 5-dose empiric dose-toxicity model with a cohort size of 3, a starting dose at dose level 1 and prior guess of the MTD at dose level 3, is utilised. Using a Bayesian safety-monitoring framework with a beta-binomial conjugate analysis, the Bayesian CRM will allow stopping early if there is sufficient evidence that the lowest dose is too toxic, that is, if the posterior probability of the DLT rate at the lowest dose being >25% is greater than 85%,
A DLT is defined as a possibly, probably or highly probably HMBD-001-related AE occurring during the first 28 days of Cycle 1 Day 1 administration which fulfils one or more of the following criteria:
*Note: In the event of a Grade 4 neutropenia, a full blood count must be performed on Day 5 after the onset of the event to determine if a DLT has occurred. The investigator must continue to monitor the patient closely until resolution to Grade 1 or less.
The single agent maximum administered dose (MAD) will be defined as the highest dose level administered in the absence of defining the MTD.
Doses of prednisolone >10 mg daily (or equipotent doses of other corticosteroids) are not permitted whilst on the trial, or within 2 weeks of Cycle 1 Day 1, other than as pre-medication, where it has been approved by the CI and Sponsor as an alternative pre-medication, or if alternative steroids are required, or if clinically indicated to treat infusion related reactions.
Doses of prednisolone <10 mg daily (or equipotent doses of other corticosteroids) are permitted and inhaled corticosteroids are allowed.
Patients enrolled in the HMBD-001 monotherapy dose escalation arm will have tumour types known to overexpress HER3. High HER3 expression has been demonstrated in tumour histologies, including in gastric, colorectal, prostate, breast, lung, ovarian and head and neck cancers.
All patients recruited to the monotherapy expansion and combination dose escalation and combination dose expansion arms will be selected based on confirmed high HER3 expression status prior to trial enrolment. Patients with a confirmed NRG1 fusion rearrangement may also be enrolled.
Up to 135 evaluable patients will be recruited for the trial. The final number will depend on the number of dose escalations needed to reach the Maximum Tolerated Dose (MTD)/Maximum Administered Dose (MAD), the number of expansion phase cohorts explored and the number of patients that may need to be replaced.
In some tumour types the main clinical use will be in combination with other signalling pathway inhibitors.
In the monotherapy dose escalation phase, HER3 tumour expression will be measured retrospectively; patients with tumours expected to have a high expression of HER3 will be selected.
Patients with confirmed high HER3 expression will be selected for the monotherapy expansion, combination dose escalation and combination expansion cohorts and therefore this forms part of the eligibility criteria. High HER3 expression will be determined using a research assay under InVitro Diagnostic (IVD) exemption criteria for health institutions (commonly referred to as in-house manufacturing). Contingent on the acquired data, fresh biopsy phosphorylated HER3 expression and/or high NRG1 expression may also become mandated. Patients with a confirmed existing NRG1 fusion rearrangement will also be considered eligible. NRG1 fusion rearrangement will be assessed using a research assay performed within each local healthcare institute.
1. Histologically confirmed advanced or metastatic solid tumours resistant or refractory to conventional treatment, or for which no conventional therapy exists or is not considered appropriate by the Investigator or is declined by the patient.
Patients with Tumour Types Known to Overexpress HER3 Including:
Patients with castration resistant prostate cancer, RAS wild type colorectal cancer, triple negative breast cancer or squamous cell cancers of the head and neck with:
2. Life expectancy of at least 12 weeks.
3. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. (Appendix 2).
4. Haematological and biochemical indices within the ranges shown below. These measurements should be performed to confirm the patient's eligibility.
5. Patients with advanced prostate cancer must have castrate levels of testosterone and have received a next generation hormonal agent (at least one of abiraterone, enzalutamide, apalutamide or darolutamide).
6. Aged 16 years or over at the time consent is given.
For Part A Arm 2, Part B Arm 1 and 2: Fresh tumour biopsy is obtained for HER3 and NRG1 expression analysis by IHC and other pharmacodynamic analysis. This must be from a metastatic site. Confirmation of high HER3 expression must be obtained prior to trial enrolment.
Tumour serum markers are measured as appropriate for the patient's tumour type e.g. CA-125, CEA, CA19-9 or PSA.
1. Radiotherapy (except for palliative reasons), chemotherapy, endocrine therapy (with the exception of life-long hormone suppression such as luteinising hormone-releasing hormone (LHRH) agents in prostate cancer), immunotherapy or investigational medicinal products during the previous 4 weeks before trial Cycle 1 Day 1.
2. Patients with ongoing toxic manifestations of previous treatments greater than NCI-CTCAE Grade 1. Exceptions to this are alopecia of any grade, Grade 2 peripheral neuropathy and any other ongoing toxic manifestation which in the opinion of the Investigator and the Sponsor should not exclude the patient
3. Patients with symptomatic brain or leptomeningeal metastases should be excluded. Asymptomatic patients on a stable steroid dose ≤10 mg prednisolone or equipotent doses of other corticosteroids will be eligible for the trial.
4. Women of child-bearing potential (or are already pregnant or lactating). However, those patients who meet the following points are considered eligible:
5. Male patients with partners of child-bearing potential. However, those patients who meet the following points are considered eligible:
6. Major surgery from which the patient has not yet recovered.
7. At high medical risk because of non-malignant systemic disease including active uncontrolled infection. Patients with previous Hepatitis C exposure but no current infection are eligible to participate.
8. Known to be serologically positive for hepatitis B, hepatitis C or human immunodeficiency virus (HIV) infection. Patients with previous Hepatitis C exposure but no current infection are eligible to participate.
9. Known or suspected hypersensitivity reaction to previous biological therapy that in the opinion of the Investigator is a contraindication for their participation in this study.
10. Concurrent congestive heart failure, prior history of >class II cardiac disease (New York Heart Association [NYHA]-Appendix 3), history of clinically significant cardiac ischaemia or prior history of clinically significant cardiac arrhythmia. Patients with significant cardiovascular disease are excluded as defined by:
11. Patients with an active autoimmune disease including but not limited to myasthenia gravis, myositis, autoimmune hepatitis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, vascular thrombosis associated with antiphospholipid syndrome, Wegener's granulomatosis, Sjögren's syndrome, Guillain-Barre syndrome, multiple sclerosis, vasculitis or glomerulonephritis. Patients with controlled Type I diabetes mellitus on a stable dose of insulin and patients with hypothyroidism only requiring thyroid hormone replacement and on stable dose will be eligible. Patients with skin disorders (such as vitiligo, psoriasis or alopecia) not requiring systemic treatment, or conditions not expected to recur in the absence of an external trigger will be permitted to participate provided all the following criteria are met:
12. Patients receiving doses of prednisolone >10 mg daily (or equipotent doses of other corticosteroids) within 7 days prior to the first dose of study drug are not eligible unless administered as pre-medication.
13. Patients having received a live vaccination within 4 weeks prior to first dose of HMBD-001.
14. Is a participant or plans to participate in another interventional clinical trial, whilst taking part in this Phase/IIIa trial of HMBD-001. Participation in an observational trial or interventional clinical trial which does not involve administration of an IMP and which would not place an unacceptable burden on the patient in the opinion of the Investigator and Medical Advisor would be acceptable.
15. Any other condition which in the Investigator's opinion would not make the patient a good candidate for the clinical trial.
16. Current or prior malignancy which could affect safety or efficacy assessment of the IMP or compliance with the protocol or interpretation of results. Patients with curatively-treated non-melanoma skin cancer, non-muscle-invasive bladder cancer, or carcinomas-in-situ are generally eligible.
As monotherapy, HER3 monoclonal antibodies have been well tolerated at doses up to 40 mg/kg weekly IV and without reaching a maximum tolerated dose. Most adverse events have been NCI-CTCAE Grade 1-2. Based on preclinical experience with HMBD-001 and reported clinical data with other HER3 monoclonal antibodies, expected toxicities might include, but not be limited to, the following:
An adverse event (AE) is any untoward, undesired or unplanned medical occurrence in a patient administered an investigational medicinal product (IMP), a comparator product or an approved drug. An AE can be a sign, symptom, disease, and/or laboratory or physiological observation that may or may not be related to the IMP or comparator. An AE includes but is not limited to those in the following list.
A serious adverse event (SAE) is any AE, regardless of dose, causality or expectedness, that:
*A life-threatening event is defined as an event when the patient was at substantial risk of dying at the time of the adverse event occurring or with continued use of the device or other medical product which might have resulted in the death of the patient.
**A medically important event is defined as any event that may jeopardise the patient or may require intervention to prevent one of the outcomes listed above. Examples include allergic bronchospasm (a serious problem with breathing) requiring treatment in an emergency room, serious blood dyscrasias (blood disorders) or seizures/convulsions that do not result in hospitalisation. The development of drug dependence or drug abuse would also be examples of important medical events.
Infusion-related reactions (IRR) are often associated with the administration of monoclonal antibodies and other therapeutic agents.
Patients are assessed for expression of biomarkers, including:
HMBD-001 levels are measured in plasma by ELISA. The plasma/concentration/time data are analysed using non-compartmental methods. The pharmacokinetic (PK) parameters for HMBD-001 include the maximum observed plasma concentration (Cmax), time to reach Cmax (Tmax), area under the plasma concentration time curve (AUC), terminal elimination half-life (t1/2), steady/state volume of distribution (Vss) and total body clearance (CL).
In the single agent dose escalation phase (Part A, Arm 1), approximately 5 mL of blood is collected from patients at up to 20 time points during Cycles 1 to 3. The approximate total volume of blood withdrawn from each patient is a maximum of 120 mL. In the expansion phases (Part B, Arms 1 and 2) and combination agent(s) escalation phase (Part A, Arm 2) a 5 mL sample of blood is taken pre dose and 1 hour post end of infusion for Cycle 1 and 3 for each patient. The approximate total volume of blood withdrawn from each patient is a maximum of 45 mL.
To measure circulating Biomarkers (e.g. NRG1, soluble HER3), approximately 6 mL of blood is collected for analysis of potential surrogate markers of HMBD-001 activity at specific time points pre and post treatment during the dose escalation and expansion phases, according to agreed SOPs and validated methods to explore levels of soluble HER3 and ligand NRG1. The approximate total volume of blood withdrawn from each patient for these analyses is 96 mL.
Approximately 20 mL of blood is collected from patients at up to 6 time points pre, during treatment and at the Off-Study visit to obtain plasma and measure cfDNA. The cfDNA is measured in plasma from patients to detect changes in tumour fraction and genetic alterations. The cfDNA analysis is complemented by genomic and tumour DNA analysis to identify fragments originating from the tumour in contrast to fragments from other cell origin and demonstrate decrease in tumour fraction in serial cfDNA samples. The approximate total volume of blood withdrawn from each patient for this analysis is 120 mL.
Disease is measured according to the Response Evaluation Criteria in Solid Tumours (RECIST) v1.1 or Prostate Cancer Working Group 3 (PCWG3) Criteria for Clinical Trials as applicable.
Progression Free Survival (PFS) is defined as the time from date of administration of the first dose of HMBD-001 or first dose of combination treatment on this trial to disease progression or death from any cause. If a patient has not progressed and is still alive, the patient will be censored at the date of the last adequate disease assessment.
Overall Survival (OS) is defined as the time from date of administration of the first dose of HMBD-001 or first dose of the combination treatment on this trial to the date of death due to any cause. If a patient has not died, the patient will be censored at the most recent date known to be alive.
Overall response Rate (ORR) is defined as the proportion of patients who have achieved Complete Response (CR) or Partial Response (PR).
New response evaluation criteria in solid tumours (RECIST criteria): Revised RECIST guideline (Version 1.1). E. A. Eisenhauer et al. (2009). European Journal of Cancer 45: 228-247
At baseline, tumour lesions/lymph nodes will be categorised measurable or non-measurable as follows:
Tumour lesions: Must be accurately measured in at least one dimension (longest diameter in the plane of measurement is to be recorded) with a minimum size of:
Malignant lymph nodes: To be considered pathologically enlarged and measurable, a lymph node must be 15 mm in the short axis when assessed by CT scan (CT scan slice thickness recommended to be no greater than 5 mm). At baseline and in follow-up, only the short axis will be measured and followed.
All other lesions, including small lesions (longest diameter <10 mm or pathological lymph nodes with 10 to <15 mm short axis) as well as truly non-measurable lesions. Lesions considered truly non-measurable include: leptomeningeal disease, ascites, pleural or pericardial effusion, inflammatory breast disease, lymphangitic involvement of skin or lung, abdominal masses/abdominal organomegaly identified by physical exam that is not measurable by reproducible imaging techniques.
Bone lesions, cystic lesions, and lesions previously treated with local therapy require particular comment:
All measurements should be recorded in metric notation, using callipers if clinically assessed. All baseline evaluations should be performed as close as possible to the treatment start and never more than 4 weeks before the beginning of the treatment.
The same method of assessment and the same technique should be used to characterise each identified and reported lesion at baseline and during follow-up. Imaging based evaluation should always be done rather than clinical examination unless the lesion(s) being followed cannot be imaged but are assessable by clinical exam.
Clinical lesions will only be considered measurable when they are superficial and ≥10 mm diameter as assessed using callipers (e.g. skin nodules). For the case of skin lesions, documentation by colour photography including a ruler to estimate the size of the lesion is suggested. As noted above, when lesions can be evaluated by both clinical exam and imaging, imaging evaluation should be undertaken since it is more objective and may also be reviewed at the end of the trial.
Chest CT is preferred over chest X-ray, particularly when progression is an important endpoint, since CT is more sensitive than X-ray, particularly in identifying new lesions. However, lesions on chest X-ray may be considered measurable if they are clearly defined and surrounded by aerated lung.
CT is the best currently available and reproducible method to measure lesions selected for response assessment. This guideline has defined measurability of lesions on CT scan based on the assumption that CT slice thickness is 5 mm or less. When CT scans have slice thickness greater than 5 mm, the minimum size for a measurable lesion should be twice the slice thickness. MRI is also acceptable in certain situations (e.g. for body scans). More details concerning the use of both CT and MRI for assessment of objective tumour response evaluation are provided in the publication from Eisenhauer et al.
Ultrasound is not useful in assessment of lesion size and should not be used as a method of measurement. Ultrasound examinations cannot be reproduced in their entirety for independent review at a later date and, because they are operator dependent, it cannot be guaranteed that the same technique and measurements will be taken from one assessment to the next (described in greater detail in Appendix II). If new lesions are identified by ultrasound in the course of the trial, confirmation by CT or MRI is advised. If there is concern about radiation exposure at CT, MRI may be used instead of CT in selected instances.
The utilisation of these techniques for objective tumour evaluation is not advised. However, they can be useful to confirm complete pathological response when biopsies are obtained or to determine relapse in trials where recurrence following complete response or surgical resection is an endpoint.
Tumour markers alone cannot be used to assess objective tumour response. If markers are initially above the upper normal limit, however, they must normalise for a patient to be considered in complete response.
These techniques can be used to differentiate between PR and CR in rare cases if required by protocol (for example, residual lesions in tumour types such as germ cell tumours, where known residual benign tumours can remain). When effusions are known to be a potential adverse effect of treatment (e.g. with certain taxane compounds or angiogenesis inhibitors), the cytological confirmation of the neoplastic origin of any effusion that appears or worsens during treatment can be considered if the measurable tumour has met criteria for response or stable disease in order to differentiate between response (or stable disease) and progressive disease.
To assess objective response or future progression, it is necessary to estimate the overall tumour burden at baseline and use this as a comparator for subsequent measurements. Measurable disease is defined by the presence of at least one measurable lesion (as detailed above).
When more than one measurable lesion is present at baseline all lesions up to a maximum of five lesions total (and a maximum of two lesions per organ) representative of all involved organs should be identified as target lesions and will be recorded and measured at baseline (this means in instances where patients have only one or two organ sites involved a maximum of two and four lesions respectively will be recorded). For evidence to support the selection of only five target lesions, see analyses on a large prospective database in the article by Bogaerts et al. Target lesions should be selected on the basis of their size (lesions with the longest diameter), be representative of all involved organs, but in addition should be those that lend themselves to reproducible repeated measurements. It may be the case that, on occasion, the largest lesion does not lend itself to reproducible measurement in which circumstance the next largest lesion which can be measured reproducibly should be selected. An example in
Lymph nodes merit special mention since they are normal anatomical structures which may be visible by imaging even if not involved by tumour. Pathological nodes which are defined as measurable and may be identified as target lesions must meet the criterion of a short axis of ≥15 mm by CT scan. Only the short axis of these nodes will contribute to the baseline sum. The short axis of the node is the diameter normally used by radiologists to judge if a node is involved by solid tumour. Nodal size is normally reported as two dimensions in the plane in which the image is obtained (for CT scan this is almost always the axial plane; for MRI the plane of acquisition may be axial, sagital or coronal). The smaller of these measures is the short axis. For example, an abdominal node which is reported as being 20 mm×30 mm has a short axis of 20 mm and qualifies as a malignant, measurable node. In this example, 20 mm should be recorded as the node measurement. All other pathological nodes (those with short axis 10 mm but <15 mm) should be considered non-target lesions. Nodes that have a short axis ≥10 mm are considered non-pathological and should not be recorded or followed.
A sum of the diameters (longest for non-nodal lesions, short axis for nodal lesions) for all target lesions will be calculated and reported as the baseline sum diameters. If lymph nodes are to be included in the sum, then as noted above, only the short axis is added into the sum. The baseline sum diameters will be used as reference to further characterise any objective tumour regression in the measurable dimension of the disease.
All other lesions (or sites of disease) including pathological lymph nodes should be identified as non-target lesions and should also be recorded at baseline. Measurements are not required and these lesions should be followed as ‘present’, ‘absent’, or in rare cases ‘unequivocal progression’ (more details to follow). In addition, it is possible to record multiple non-target lesions involving the same organ as a single item on the case record form (e.g. ‘multiple enlarged pelvic lymph nodes’ or ‘multiple liver metastases’).
Complete Response (CR): Disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm.
Partial Response (PR): At least a 30% decrease in the sum of diameters of target lesions, taking as reference the baseline sum diameters.
Progressive Disease (PD): At least a 20% increase in the sum of diameters of target lesions, taking as reference the smallest sum on trial (this includes the baseline sum if that is the smallest on trial). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progression).
Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum diameters while on trial.
Lymph nodes identified as target lesions should always have the actual short axis measurement recorded (measured in the same anatomical plane as the baseline examination), even if the nodes regress to below 10 mm on trial. This means that when lymph nodes are included as target lesions, the ‘sum’ of lesions may not be zero even if complete response criteria are met, since a normal lymph node is defined as having a short axis of <10 mm. Case report forms or other data collection methods may therefore be designed to have target nodal lesions recorded in a separate section where, in order to qualify for CR, each node must achieve a short axis <10 mm. For PR, SD and PD, the actual short axis measurement of the nodes is to be included in the sum of target lesions.
Target Lesions that Become ‘too Small to Measure’:
While on trial, all lesions (nodal and non-nodal) recorded at baseline should have their actual measurements recorded at each subsequent evaluation, even when very small (e.g. 2 mm). However, sometimes lesions or lymph nodes which are recorded as target lesions at baseline become so faint on CT scan that the radiologist may not feel comfortable assigning an exact measure and may report them as being ‘too small to measure’. When this occurs it is important that a value be recorded on the case report form. If it is the opinion of the radiologist that the lesion has likely disappeared, the measurement should be recorded as 0 mm. If the lesion is believed to be present and is faintly seen but too small to measure, a default value of 5 mm should be assigned. (Note: It is less likely that this rule will be used for lymph nodes since they usually have a definable size when normal and are frequently surrounded by fat such as in the retroperitoneum; however, if a lymph node is believed to be present and is faintly seen but too small to measure, a default value of 5 mm should be assigned in this circumstance as well). This default value is derived from the 5 mm CT slice thickness (but should not be changed with varying CT slice thickness). The measurement of these lesions is potentially non-reproducible, therefore providing this default value will prevent false responses or progressions based upon measurement error. To reiterate, however, if the radiologist is able to provide an actual measure, that should be recorded, even if it is below 5 mm.
Lesions that Split or Coalesce on Treatment:
When non-nodal lesions ‘fragment’, the longest diameters of the fragmented portions should be added together to calculate the target lesion sum. Similarly, as lesions coalesce, a plane between them may be maintained that would aid in obtaining maximal diameter measurements of each individual lesion. If the lesions have truly coalesced such that they are no longer separable, the vector of the longest diameter in this instance should be the maximal longest diameter for the ‘coalesced lesion’.
While some non-target lesions may actually be measurable, they need not be measured and instead should be assessed only qualitatively at the time points specified in the protocol.
Complete Response (CR): Disappearance of all non-target lesions and normalisation of tumour marker level. All lymph nodes must be non-pathological in size (<10 mm short axis).
Non-CR/Non-PD: Persistence of one or more non-target lesion(s) and/or maintenance of tumour marker level above the normal limits.
Progressive Disease (PD): Unequivocal progression (see comments below) of existing non-target lesions. (Note: the appearance of one or more new lesions is also considered progression).
The concept of progression of non-target disease requires additional explanation as follows:
In this setting, to achieve ‘unequivocal progression’ on the basis of the non-target disease, there must be an overall level of substantial worsening in non-target disease such that, even in presence of SD or PR in target disease, the overall tumour burden has increased sufficiently to merit discontinuation of therapy. A modest ‘increase’ in the size of one or more non-target lesions is usually not sufficient to quality for unequivocal progression status. The designation of overall progression solely on the basis of change in non-target disease in the face of SD or PR of target disease will therefore be extremely rare.
This circumstance arises in some phase III trials when it is not a criterion of trial entry to have measurable disease. The same general concepts apply here as noted above, however, in this instance there is no measurable disease assessment to factor into the interpretation of an increase in non-measurable disease burden. Because worsening in non-target disease cannot be easily quantified (by definition: if all lesions are truly non-measurable) a useful test that can be applied when assessing patients for unequivocal progression is to consider if the increase in overall disease burden based on the change in non-measurable disease is comparable in magnitude to the increase that would be required to declare PD for measurable disease: i.e. an increase in tumour burden representing an additional 73% increase in ‘volume’ (which is equivalent to a 20% increase diameter in a measurable lesion). Examples include an increase in a pleural effusion from ‘trace’ to ‘large’, an increase in lymphangitic disease from localised to widespread, or may be described in protocols as ‘sufficient to require a change in therapy’. If ‘unequivocal progression’ is seen, the patient should be considered to have had overall PD at that point. While it would be ideal to have objective criteria to apply to non-measurable disease, the very nature of that disease makes it impossible to do so; therefore the increase must be substantial.
The appearance of new malignant lesions denotes disease progression; therefore, some comments on detection of new lesions are important. There are no specific criteria for the identification of new radiographic lesions; however, the finding of a new lesion should be unequivocal: i.e. not attributable to differences in scanning technique, change in imaging modality or findings thought to represent something other than tumour (for example, some ‘new’ bone lesions may be simply healing or flare of pre-existing lesions). This is particularly important when the patient's baseline lesions show partial or complete response. For example, necrosis of a liver lesion may be reported on a CT scan report as a ‘new’ cystic lesion, which it is not.
A lesion identified on a follow-up trial in an anatomical location that was not scanned at baseline is considered a new lesion and will indicate disease progression. An example of this is the patient who has visceral disease at baseline and while on trial has a CT or MRI brain ordered which reveals metastases. The patient's brain metastases are considered to be evidence of PD even if he/she did not have brain imaging at baseline.
If a new lesion is equivocal, for example because of its small size, continued therapy and follow-up evaluation will clarify if it represents truly new disease. If repeat scans confirm there is definitely a new lesion, then progression should be declared using the date of the initial scan.
While FDG-PET response assessments need additional study, it is sometimes reasonable to incorporate the use of FDG-PET scanning to complement CT scanning in assessment of progression (particularly possible ‘new’ disease). New lesions on the basis of FDG-PET imaging can be identified according to the following algorithm:
Prostate Cancer Working Group 3 (PCWG3) Criteria in Clinical Trials (Scher et al. 2016) Imaging-based tumour assessments of response will be conducted utilising CT scans of the thorax, abdomen and pelvis or MRI and bone scintigraphy according to the Prostate Cancer Working Group 3 (PCWG3) guidelines.
The PCWG3-includes modified RECIST 1.1 criteria are to be used for soft tissue lesion assessment and bone scans are to be used for assessment of bone disease. PCWG3 does advise following RECIST 1.1 for extra-skeletal disease but recommends the following modification: up to five lesions per site of metastatic spread (e.g. lung, liver, lymph nodes, adrenal and brain as separate sites) are to be recorded to address disease heterogeneity and to track patterns of metastatic progression.
In accord with RECIST 1.1, for measurable disease in lymph nodes, only report changes in lesions ≥1.5 cm in the short axis; for visceral measurable lesions, only report changes in lesions ≥1 cm in the longest dimension. Bone disease will not be considered as non-target lesions assessed by RECIST v1.1, but will be assessed for progressive disease by PCWG3. Bone lesions should be recorded separately using the bone scans.
All references described in Example 16 are hereby incorporated by reference in their entirety.
Human subjects were administered HMBD-001 as described in Example 16.
Eight subjects received HMBD-001 monotherapy as part of the dose escalation phase (Part A, Arm 1). Full PK profiles were obtained at Day 1 and 15 (1st and 3rd dosing occasions) and analyses of raw data using Phoenix WinNonLin were carried out. PK parameters were in line with those expected for a monoclonal antibody of this class (see Table 6). The mean concentration-time profiles show that mean concentrations of HMBD-001 generally increased with the increase in dose level. The increases in HMBD-001 Cmax and AUC0-168 values were generally dose proportional.
129115
102660-15570
194.5
159.1-229.9
Patients were observed for Treatment Emergent Adverse Events in accordance with the Common Terminology Criteria for Adverse Events (NCI-CTCAE). No AEs were observed at grade 4 or 5, and no DLTs were observed (as set out in Example 16.6.6). Over 85% of observed AEs were graded as mild or moderate (grades 1-2).
Human subjects were administered HMBD-001 as described in Example 16.
Eight subjects received HMBD-001 monotherapy as part of the dose escalation phase (Part A, Arm 1). Full PK profiles were obtained at Day 1 and 15 (1st and 3rd dosing occasions) and analyses of raw data using Phoenix WinNonLin were carried out. PK parameters were in line with those expected for a monoclonal antibody of this class (see Table 6). The mean concentration-time profiles show that mean concentrations of HMBD-001 generally increased with the increase in dose level. The increases in HMBD-001 Cmax and AUC0-168 values were generally dose proportional.
129115
102660-15570
194.5
159.1-229.9
Patients were observed for Treatment Emergent Adverse Events in accordance with the Common Terminology Criteria for Adverse Events (NCI-CTCAE). No AEs were observed at grade 4 or 5, and no DLTs were observed (as set out in Example 16.6.6). Over 85% of observed AEs were graded as mild or moderate (grades 1-2).
| Number | Date | Country | Kind |
|---|---|---|---|
| 10202109667X | Sep 2021 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/074520 | 9/2/2022 | WO |
