HK1208911B

HK1208911B - Identification of patients in need of pd-l1 inhibitor cotherapy

Info

Publication number: HK1208911B
Application number: HK15109664.9A
Authority: HK
Inventors: Anton Belousov; Giampaolo BIANCHINI; Luca Gianni; Marlene Thomas
Original assignee: 霍夫曼-拉罗奇有限公司
Priority date: 2012-11-30
Filing date: 2013-11-29
Publication date: 2018-07-06

Description

Identification of patients in need of PD-L1 inhibitor combination therapy

The present invention relates to means and methods for determining whether a patient is in need of PD-L1 inhibitor cotherapy (cotherapy). A patient is determined to be in need of PD-L1 inhibitor cotherapy if low or absent ER expression levels and elevated expression levels of programmed death ligand 1(PD-L1) compared to a control are measured in vitro in a sample from the patient. The patient is undergoing therapy comprising a modulator of HER2/neu (ErbB2) signaling pathway, such as trastuzumab (trastuzumab), and a chemotherapeutic agent, such as docetaxel (dodetaxel), or such therapy is contemplated for the patient. Also provided herein are means and methods for treating cancer in a cancer patient for whom therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway (such as trastuzumab) and a chemotherapeutic agent (such as docetaxel) is contemplated, wherein the patient is to receive PD-L1 inhibitor combination therapy.

The HER family of receptor tyrosine kinases is an important mediator of cell growth, differentiation and survival. The receptor family includes four distinct members, including epidermal growth factor receptor (EGFR, ErbB1, or HER1), HER2(ErbB2 or p 185)^neu) HER3(ErbB3) and HER4(ErbB4 or tyro 2).

EGFR encoded by the erbB1 gene has been causally linked to human malignancy. In particular, increased EGFR expression has been observed in breast, bladder, lung, head, neck and stomach cancers and glioblastomas. Elevated EGFR receptor expression is often associated with increased production of EGFR ligands (i.e., transforming growth factor alpha (TGF- α)) by the same tumor cells, leading to receptor activation by an autocrine stimulatory pathway. Baselga and Mendelsohn, Pharmac. Ther.64: 127-. Monoclonal antibodies against EGFR, or its ligands TGF-alpha and EGF, have been evaluated as therapeutic agents in the treatment of such malignancies. See, e.g., Baselga and Mendelsohn, supra; masui et al, Cancer Research 44:1002-1007 (1984); and Wu et al, J.Clin.invest.95:1897-1905 (1995).

Second member of the HER family, p185^neuOriginally identified as the product of a transformed gene from a neuroblastoma from chemically treated rats. The activated form of the neu proto-oncogene is derived from a point mutation (valine to glutamic acid) in the transmembrane region of the encoded protein. Amplification of the human homolog of neu was observed in breast and ovarian cancers and correlated with poor prognosis (Slamon et al, Science 235: 177-. To date, no point mutation similar to that in the neu protooncogene has been reported for human tumors. HER2 overexpression (often but not uniformly due to gene amplification) has also been observed in other carcinomas including gastric, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic and bladder cancers. See King et al, Science 229:974 (1985); yokota et al, Lancet 1:765- > 767 (1986); fukushige et al, Mol Cell biol.6:955-958 (1986); guerin et al, Oncogene Res.3:21-31 (1988); cohen et al, Oncogene 4:81-88 (1989); yonemura et al, Cancer Res.51:1034 (1991); borst et al, Gynecol.Oncol.38:364 (1990); weiner et al, Cancer Res.50: 421-; kern et al, Cancer Res.50:5184 (1990); park et al, cancer Res.49:6605 (1989); zhau et al, mol.Carcinog.3: 254-; aasland et al, Br.J. cancer57:358-363 (1988); williams et al, Pathiology 59:46-52 (1991); and McCann et al, Cancer 65:88-92 (1990); and so on. HER2 can be overexpressed in prostate Cancer (Gu et al, Cancer Lett.99:185-9 (1996); Ross et al, hum. Pathol.28:827-33 (1997); Ross et al, Cancer 79:2162-70 (1997); and Sadasivan et al, J.Urol.150:126-31 (1993)).

P185 has been described for rat^neuAnd the human HER2 protein product. Drebin and colleagues have generated the product p185 against rat neu gene^neuThe antibody of (1). See, e.g., Drebin et al, Cell 41: 695-one 706 (1985); myers et al, meth.enzym.198:277-290 (1991); and WO 94/22478. Drebin et al, Oncogene 2:273-^neuThe mixture of antibodies reactive with the two distinct regions of (a) results in a synergistic anti-tumor effect on neu-transformed NIH-3T3 cells implanted in nude mice. See also U.S. patent No.5,824,311 issued at 10/20 of 1998.

Hudziak et al, mol.cell.biol.9(3):1165-1172(1989) describe the generation of a panel of HER2 antibodies characterized using the human breast tumor cell line SK-BR-3. The relative cell proliferation of SK-BR-3 cells after exposure to antibody was determined by crystal violet staining of monolayers after 72 hours. Using this assay, the maximum inhibition was obtained with an antibody designated 4D5, which inhibits cell proliferation by 56%. Other antibodies in this panel reduced cell proliferation to a lesser extent in this assay. It was further found that antibody 4D5 sensitizes HER2 overexpressing breast tumor cell line to the cytotoxic effects of TNF- α. U.S. Pat. No.5,677,171, issued 10, 14, 1997, can also be seen. HER2 antibodies discussed in Hudziak et al, Fendly et al, Cancer Research 50: 1550-; kotts et al, In Vitro 26(3):59A (1990); sarup et al, Growth Regulation 1:72-82 (1991); shepard et al, J.Clin.Immunol.11(3): 117. ang. 127 (1991); kumar et al, mol.cell.biol.11(2): 979-; lewis et al, cancer Immunol.Immunother.37:255-263 (1993); pietras et al, Oncogene 9:1829-1838 (1994); vitetta et al, Cancer Research 54: 5301-; sliwkowski et al, J.biol.chem.269(20):14661-14665 (1994); scott et al, J.biol.chem.266:14300-5 (1991); d' souza et al, Proc. Natl. Acad. Sci.91:7202-7206 (1994); lewis et al, Cancer Research 56: 1457-; and Schaefer et al, Oncogene 15:1385-1394 (1997).

A recombinant humanized form of murine HER2 antibody 4D5 (huMAb4D5-8, rhuMAb HER2, trastuzumab or herceptin)^TM(Herceptin); U.S. Pat. No.5,821,337) has clinical activity in patients with HER2 overexpressing metastatic breast cancer who have received extensive prior anti-cancer therapies (Baselga et al, J.Clin. Oncol.14:737-744 (1996)). Trastuzumab received marketing approval from the food and drug administration at 9, 25 of 1998 for the treatment of patients with metastatic breast cancer whose tumors overexpress HER2 protein.

Humanized anti-ErbB 2 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8As described in table 3 of U.S. patent 5,821,337 (which is expressly incorporated herein by reference); humanized 520C9(WO 93/21319) and humanized 2C4 antibodies as described in WO 01/000245 (which is expressly incorporated herein by reference).

Pertuzumab (Pertuzumab) (see, e.g., WO 01/000245) binds to HER2 at the dimerization domain of HER2, thereby inhibiting its ability to form an active dimeric receptor complex, and thus blocks the downstream signaling cascade that ultimately leads to Cell growth and division (see, Franklin, m.c., Cancer Cell 5:317-328 (2004)). Pertuzumab is a fully humanized recombinant monoclonal antibody directed against the extracellular domain of HER 2. binding of Pertuzumab to HER2 on human epithelial cells prevents HER2 from forming complexes with other members of the HER family (including EGFR, HER3, HER4) and possibly also HER2 homodimerization. by blocking the complexes, Pertuzumab prevents the formation of ligands by HER1, HER3 and HER4 (e.g., TGF α, amphiregulin, survival and heregulin) and the stimulation of Cell growth and the effect of Cell growth by blocking the two signal residues of EGF 36449, c.2, heavy chain of Pertuzumab (see, 685) is based on the two sequences of EGF, Pertuzumab, and Pertuzumab (c.In contrast, pertuzumab has a12 amino acid difference in the light chain and a 29 amino acid difference in the IgG1 heavy chain.

Other HER2 antibodies with various properties have been described in Tagliabue et al, int.J. cancer 47:933-937 (1991); McKenzie et al, Oncogene 4: 543-; maier et al, Cancer Res.51: 5361-; bacillus et al, Molecular Carcinogenesis 3: 350-; stancovski et al, PNAS (USA)88: 8691-; bacus et al, Cancer Research 52: 2580-; xu et al, int.J.cancer 53:401-408 (1993); WO 94/00136; kasprzyk et al, Cancer Research 52: 2771-; hancock et al, Cancer Res.51: 4575-; shawver et al, Cancer Res.54: 1367-; aretag et al, Cancer Res.54:3758-3765 (1994); harwerth et al, J.biol.chem.267:15160-15167 (1992); U.S. Pat. Nos. 5,783,186; and Klaper et al, Oncogene14:2099-2109 (1997).

Homology screening has led to the identification of two other HER receptor family members; HER3 (U.S. Pat. Nos. 5,183,884 and 5,480,968 and Kraus et al, PNAS (USA)86: 9193-. Both of these receptors exhibit elevated expression on at least some breast cancer cell lines.

HER receptors are generally found in various combinations in cells, and heterodimerization is thought to increase the diversity of cellular responses to various HER ligands (Earp et al, Breast Cancer Research and Treatment 35:115-132 (1995)). EGFR is bound by 6 different ligands; epidermal Growth Factor (EGF), transforming Growth factor alpha (TGF-. alpha.), amphiregulin, heparin-binding epidermal Growth factor (HB-EGF), betacellulin (betacellulin), and epiregulin (Epiregin) (Groenen et al, Growth Factors 11:235-257 (1994)). One family of alternatively spliced heregulin proteins derived from a single gene are the ligands of HER3 and HER 4. The heregulin family includes the alpha, beta and gamma heregulins (Holmes et al, Science256:1205-1210 (1992); U.S. Pat. No.5,641,869; and Schaefer et al, Oncogene 15:1385-1394 (1997)); neu Differentiation Factor (NDF), Glial Growth Factor (GGF); acetylcholine Receptor Inducing Activity (ARIA); and sensory and motor neuron derived factor (SMDF). For a review, see Groenen et al, Growth Factors 11:235-257 (1994); lemke, G., Molec. & cell. Neurosci.7: 247-. Recently, 3 additional HER ligands were identified; reported as neuregulin-2 (NRG-2) binding to HER3 or HER4 (Chang et al, Nature 387509-; HER 4-binding neuregulin-3 (Zhang et al, PNAS (USA)94(18):9562-7 (1997)); and neuregulin-4 which binds HER 4(Harari et al, Oncogene 18:2681-89 (1999)). HB-EGF, betacellulin and epiregulin also bind HER 4.

Although EGF and TGF α do not bind HER2, EGF stimulates EGFR and HER2 to form a heterodimer, which activates EGFR and causes transphosphorylation of HER2 in the heterodimer. Dimerization and/or transphosphorylation appear to activate HER2 tyrosine kinase. See Earp et al, supra. Similarly, when HER3 is co-expressed with HER2, an active signaling complex is formed and antibodies to HER2 are able to disrupt this complex (Sliwkowski et al, J.biol.chem.269(20):14661-14665 (1994)). In addition, HER3 affinity for regulatory proteins (HRG) was elevated to a higher affinity state when co-expressed with HER 2. For the HER2-HER3 protein complex, Levi et al, Journal of Neuroscience 15:1329-1340 (1995); morrissey et al, Proc. Natl. Acad. Sci. USA 92:1431-1435 (1995); and Lewis et al, Cancer Res.56: 1457-. Like HER3, HER4 forms an active signaling complex with HER2 (Carraway and Cantley, Cell 78:5-8 (1994)).

Antibody variant compositions are also described in the art. U.S. patent No.6,339,142 describes a HER2 antibody composition comprising a mixture of an anti-HER 2 antibody and one or more acidic variants thereof, wherein the amount of the acidic variants is less than about 25%. Trastuzumab is an exemplary HER2 antibody. The Reid et al poster "Effects of Cell culture Processes Change on human Antibody Processes diagnostics" presented at the Well Characterized biotech therapeutics conference (1 month 2003) describes an unnamed, Humanized IgG1 Antibody composition having N-terminal heterogeneity due to the combination of VHS signal peptide, N-terminal glutamine, and pyroglutamic acid on its heavy chain. Harris et al, lecture "The Ideal Chromatographic Antibody Characterisation Method" presented at The IBC Antibody Productivity conference (month 2 2002), reported a VHS extension on The heavy chain of E25 (a humanized anti-IgE Antibody). The Rouse et al poster "GlycoproteinCharacterisation by High Resolution Mass Spectrometry and Its Application to Biopharmacological Development" presented on WCBP (6-9.1.2004) describes a monoclonal antibody composition with N-terminal heterogeneity derived from AHS or HS signal peptide residues in Its light chain. In the introduction "structural Use of complexity students and Assays for Well CharacterizedBiologicals" at the IBC conference (9.2000), Jill Porter discusses a late eluting form of ZENAPAAXTM with 3 additional amino acid residues in its heavy chain. US 2006/0018899 describes a composition comprising the main species pertuzumab antibody and amino-terminal leader extension variants as well as other variant forms of pertuzumab antibody.

Patent publications relating to HER antibodies include: US5,677,171, US5,720,937, US5,720,954, US5,725,856, US5,770,195, US5,772,997, US6,165,464, US6,387,371, US6,399,063, US 2002/0192211 a 2002/0192211, US 4,968,603, US5,821,337, US6,054,297, US 2002/0192211 a 2002/0192211, US5,648,237, US6,267,958, US 2002/0192211, WO 2002/0192211, US 127,526, US 2002/0192211, WO 2002/0192211, US 2002/0192211 a 2002/0192211, US 2002/0192211B 2002/0192211, US 2002/0192211 a 2002/0192211, US 2002/0192211B 2002/0192211, US 2002/0192211 a 2002/0192211, US 2002/0192211B 2002/0192211, US 2002/0192211B, US6,632,979B 1, WO 01/00244, US 2002/0090662A 1, WO 01/89566, US 2002/0064785, US 2003/0134344, WO 04/24866, US 2004/0082047, US 2003/0175845A 1, WO 03/087131, US 2003/0228663, WO 2004/008099A2, US 2004/0106161, WO 2004/048525, US 2004/0258685A 1, US5,985,553, US5,747,261, US 4,935,341, US5,401,638, US5,604,107, WO 87/07646, WO 89/10412, WO 91/05264, EP 412,116B 1, EP 494,135B 1, US5,824,311, EP 444,181B 1, EP1,006,194A 2, US 2002/0155527A 1, WO 91/02062, US5,571,894, US5,939,531, EP 502,812B 1, WO 93/03741, EP 554,441B 1, EP 656,367A 1, US5,288,477, US5,514,554, US5,587,458, WO 93/12220, WO 93/16185, US5,877,305, WO 93/21319, WO 93/21232, US5,856,089, WO 94/22478, US5,910,486, US6,028,059, WO 96/07321, US5,804,396, US5,846,749, EP 711,565, WO 96/16673, US5,783,404, US5,977,322, US6,512,097, WO 97/00271, US6,270,765, US6,395,272, US5,837,243, WO 96/40789, US5,783,186, US6,458,356, WO 97/20858, WO 97/38731, US6,214,388, US5,925,519, WO 98/02463, US5,922,845, WO 98/18489, WO 98/33914, US5,994,071, WO 98/45479, US6,358,682B 1, US2003/0059790, WO 99/55367, WO 01/20033, US 2002/0076695A 1, WO 00/78347, WO 01/09187, WO 01/21192, WO 01/32155, WO 01/53354, WO 01/56604, WO 01/76630, WO 02/05791, WO 02/11677, US6,582,919, US 2002/0192652A 1, US 2003/0211530A 1, WO 02/44413, US 2002/0142328, US6,602,670B 2, WO 02/45653, WO 02/055106, US 2003/0152572, US 2003/0165840, WO 02/087619, WO 03/006509, WO 03/012072, WO 03/028638, US 2003/0068318, WO 03/041736, EP1,357,132, US 2003/0202973, US 2004/0138160, US5,705,157, US6,123,939, EP 616,812B 1, US 2003/0103973, US 2003/0108545, US6,403,630B 1, WO 00/61145, WO 00/61185, US6,333,348B 1, WO 01/05425, WO 01/64246, WO 6,582,919, US2003/0022918, US 2002/0051785A 1, US6,767,541, WO 01/76586, US 2003/0144252, WO01/87336, US 2002/0031515A 1, WO 01/87334, WO 02/05791, WO 02/09754, US 2003/0157097, US 2002/0076408, WO 02/055106, WO 02/070008, WO 02/089842 and WO 03/86467.

Selection of trastuzumab/herceptin with HER2 antibody based on HER2 protein overexpression/gene amplification^TMA patient being treated for treatment; see, e.g., WO 99/31140(Paton et al), US 2003/0170234A1(Hellmann, S.), and US2003/0147884(Paton et al); and WO 01/89566, US 2002/0064785, and US 2003/0134344(Mass et al). For Immunohistochemistry (IHC) and Fluorescence In Situ Hybridization (FISH) for detection of overexpression and amplification of HER2, see also US 2003/0152987(Cohen et al). WO 2004/053497 and US 2004/024815A 1(Bacus et al), and US2003/0190689(Crosby and Smith) mention determining or predicting response to trastuzumab therapy. US 2004/013297a1(Bacus et al) is concerned with determining or predicting response to ABX0303 EGFR antibody therapy. WO 2004/000094(Bacus et al) is concerned with determining the response to GW572016, a small molecule EGFR-HER2 tyrosine kinase inhibitor. WO 2004/063709(Amler et al) mentions biomarkers and methods for determining sensitivity to the EGFR inhibitor erlotinib hydrochloride (erlotinib HCl). US 2004/0209290(Cobleigh et al) is concerned with gene expression markers for the prognosis of breast cancer.

Patients to be treated with a HER2 dimerization inhibitor (e.g., pertuzumab, as described in more detail above) may be selected for treatment based on HER activation or dimerization. Patent publications on pertuzumab and the selection of patients for treatment therewith include: WO 01/00245(Adams et al); US 2003/0086924(Sliwkowski, M.); US 2004/0013667a 1(Sliwkowski, M.); and WO 2004/008099A2, and US 2004/0106161(Bossenmai et al).

Indicating herceptin in the art^TMTrastuzumab for the treatment of metastatic breast cancer patients whose tumors overexpress the HER2 protein or have HER2 gene amplification:

a) as a monotherapy for the treatment of those patients who have received at least two chemotherapy regimens for their metastatic disease. Prior chemotherapy must already include at least anthracyclines and taxanes unless the patient is not suitable for these treatments. Hormone receptor positive patients must also have received hormone therapy, unless the patient is not suitable for such treatment,

b) in combination with paclitaxel (paclitaxel) for the treatment of patients who have not received chemotherapy for their metastatic disease and for whom an anthracycline is not suitable, and

c) in combination with docetaxel (docetaxel) for the treatment of patients who have not received chemotherapy for their metastatic disease.

Herceptin may also be used^TMTrastuzumab as an adjunct treatment in early breast cancer. Herceptin^TMTrastuzumab is also approved for the treatment of HER2 positive early stage breast cancer patients after surgery, chemotherapy (neoadjuvant (i.e. pre-surgery) or adjuvant), and radiation therapy (if applicable). In addition, the combination of herceptin with capecitabine (capecitabine) or 5-fluorouracil and cisplatin is indicated for the treatment of HER2 positive locally advanced or metastatic gastric or gastroesophageal junction adenocarcinoma patients who have not received prior anti-cancer therapy for their metastatic disease. The efficacy and safety of neoadjuvant pertuzumab and trastuzumab therapy has been evaluated in phase 2 trials (neoshere); gianni (2012) Lancet Oncol 13, 25-32.

In the art, for example, treatment of breast cancer patients with herceptin TM/trastuzumab is recommended and routine for patients with HER2 positive cancer. A HER2 positive cancer is present if a high HER2 (protein) expression level as detected by immunohistochemical methods (e.g. HER2(+++)) or HER2 gene amplification as detected by in situ hybridization (e.g. ISH positive, such as HER2 gene copy number higher than 4 copies of HER2 gene per tumor cell or the ratio of HER2 gene copy number to CEP17 signal number ≧ 2.0) or both, is found in a sample obtained from the patient, such as in breast tissue biopsy or breast tissue resection or tissue derived from metastatic sites.

WO 2011/109789, WO 2011/066342, WO 2009/089149 and WO2006/133396 disclose therapeutic uses of PD-L1 inhibitors. Furthermore, WO2010/077634 discloses anti-PD-L1 antibodies and therapeutic uses thereof.

The present invention relates to a method of determining the need for a PD-L1 inhibitor combination therapy in a cancer patient, (i) wherein a therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for said patient or (ii) wherein said patient is undergoing a therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent, said method comprising the steps of:

a) measuring in vitro the expression levels of Estrogen Receptor (ER) and programmed death ligand 1(PD-L1) in a sample from said patient,

b) determining that the patient is in need of PD-L1 inhibitor cotherapy if a low or absent ER expression level and an elevated expression level of programmed death ligand 1(PD-L1) compared to the control is measured in step (a).

Thus, the present invention provides a method for determining the need for a cancer patient for a PD-L1 inhibitor cotherapy in combination with a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising the steps of:

-testing a tumor sample of a patient for whom a therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated or which patient is undergoing said therapy;

-determining the expression level of Estrogen Receptor (ER) and programmed death ligand 1(PD-L1) in said tumor sample,

wherein a low or absent ER expression level and an elevated expression level of programmed death ligand 1(PD-L1) as compared to a control is indicative of successful use of PD-L1 modulator combination therapy in said patient.

As demonstrated in the accompanying examples, it has been surprisingly found in the present invention that if the expression level of programmed death ligand 1(PD-L1) is increased in a sample of an ER negative (ER (-)) cancer patient compared to a control, then the treatment with a modulator of the HER2/neu (ErbB2) signaling pathway (such as herceptin TM/trastuzumab) and a chemotherapeutic agent (such as docetaxel @) is being undertaken(Taxotere)) shows a significantly worse pathological complete response (pCR) to the therapy in Estrogen Receptor (ER) negative (ER (-) cancer patients (cancer patients with low or even absent ER expression levels) compared to Estrogen Receptor (ER) positive (ER (+)) cancer patients. The terms "programmed death ligand 1", "CD 274", and "PD-L1" are used interchangeably herein. Thus, ER negatives (ER (-)) with elevated expression levels of programmed death ligand 1(PD-L1) compared to controls) Cancer patients would benefit from additional combination therapy with PD-L1 inhibitors. It is expected that these patients will be on the market with a modulator of the HER2/neu (ErbB2) signaling pathway (e.g., herceptin TM/trastuzumab) and a chemotherapeutic agent (e.g., docetaxel @) The therapy of (a) is in addition to the combination therapy with the PD-L1 inhibitor, the pathological complete response rate (pCR) in this patient group is increased. In other words, if the expression level of programmed death ligand 1(PD-L1) in a sample from a patient is increased compared to a control, ER negative (ER (-)) cancer patients will have a modulator of the HER2/neu (ErbB2) signaling pathway (e.g., trastuzumab) and a chemotherapeutic agent (e.g., docetaxel @) in the patient) Is externally connected with a programmed death ligand 1(PD-L1) inhibitor. In the following, ER-negative cancer patients or (biological/tumor) samples derived from ER-negative cancer patients are denoted herein as "ER (-)". Likewise, ER-positive cancer patients or (biological/tumor) samples derived from ER-positive cancer patients are denoted herein as "ER (+)".

In accordance with the above, the present invention relates to a method of treating cancer in a cancer patient for whom a therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an elevated expression level of programmed death ligand 1(PD-L1) as compared to a control, and administering to the patient an effective amount of a modulator of the HER2/neu (ErbB2) signaling pathway, a chemotherapeutic agent and an inhibitor of programmed death ligand 1 (PD-L1). Likewise, the present invention relates to a method of treating cancer in a cancer patient undergoing therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent, comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an elevated expression level of programmed death ligand 1(PD-L1) as compared to a control, and administering to said patient an effective amount of an inhibitor of programmed death ligand 1 (PD-L1). Thus, encompassed herein are pharmaceutical compositions for use in treating cancer comprising a modulator of the HER2/neu (ErbB2) signaling pathway and an inhibitor of programmed death ligand 1(PD-L1), wherein the cancer is determined to have a low or absent ER expression level and to have an elevated expression level of programmed death ligand 1(PD-L1) as compared to a control.

In accordance with the above, the methods provided herein for determining the need for a PD-L1 inhibitor combination therapy in a cancer patient may comprise an additional step prior to step a), wherein said step is or comprises obtaining a sample from said cancer patient. Thus, the present invention provides a method of determining the need for a PD-L1 inhibitor combination therapy in a cancer patient, (i) wherein a therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for said patient or (ii) wherein said patient is undergoing a therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent, said method comprising the step of obtaining a sample from said cancer patient, said method further comprising the following steps

a) Measuring in vitro in a sample from the patient the expression levels of Estrogen Receptor (ER) and programmed death ligand 1 (PD-L1);

Furthermore, it has been found herein and demonstrated in the accompanying examples that the need for PD-L1 inhibitor cotherapy in a patient can be determined even more reliably if the expression level of interferon-gamma (IFN γ) in a sample of the patient is measured in addition to the expression level of programmed death ligand 1 (PD-L1). It is shown herein that patients with low or absent ER expression have a significantly worse pathologically complete response to therapy with a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent if the expression level of programmed death ligand 1(PD-L1) is elevated and the expression level of several interferon-gamma (IFN γ) is reduced.

Thus, preferably, the methods provided herein further comprise measuring the expression level of interferon-gamma (IFN γ) in a sample from the patient, wherein if the expression level of interferon-gamma (IFN γ) is decreased as compared to a control, then the patient is determined to be in need of PD-L1 inhibitor cotherapy. In accordance with the above, in a preferred aspect, the present invention relates to a method of determining the need for a PD-L1 inhibitor combination therapy in a cancer patient, (i) wherein a therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for said patient or (ii) wherein said patient is undergoing a therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent, said method comprising the following steps

a) Measuring in vitro in a sample from the patient the expression level of Estrogen Receptor (ER), the expression level of programmed death ligand 1(PD-L1), and the expression level of interferon- γ (IFN γ);

b) determining that the patient is in need of PD-L1 inhibitor cotherapy if low or absent ER expression levels, an elevated expression level of programmed death ligand 1(PD-L1) compared to a control, and a reduced expression level of interferon-gamma (IFN gamma) compared to a control are measured in step (a).

Thus, a decreased level of interferon-gamma (IFN γ) expression as compared to a control is indicative of successful use of PD-L1 inhibitor cotherapy in the patient. In accordance with the above, the pharmaceutical composition provided herein is for use in treating a cancer, wherein the cancer is determined to have a low or absent ER expression level, the cancer is determined to have an elevated expression level of programmed death ligand 1(PD-L1) as compared to a control, and the cancer is determined to have a reduced expression level of interferon-gamma (IFN γ) as compared to a control. Thus, provided herein is a pharmaceutical composition for use in treating cancer comprising a modulator of the HER2/neu (ErbB2) signaling pathway and an inhibitor of programmed death ligand 1(PD-L1), wherein the cancer is determined to have a low or absent ER expression level and to have an elevated expression level of programmed death ligand 1(PD-L1) compared to a control and to have a reduced expression level of interferon- γ (IFN γ) compared to a control.

As used herein, the term "cancer patient" refers to a patient suspected of having cancer, or being predisposed to having cancer. The cancer to be treated according to the present invention may be a solid cancer, such as breast cancer or gastric cancer. In addition, the cancer may be ovarian cancer or colorectal cancer. Preferably, the cancer is a "HER 2 positive" cancer.

Preferably, the cancer is breast cancer, such as early breast cancer. The breast cancer may be early stage breast cancer or metastatic breast cancer. Thus, a cancer patient (to be treated) is suspected of having, suffering from, or susceptible to developing a solid cancer, wherein the solid cancer may be breast cancer or gastric cancer. Preferably, the cancer is breast cancer, such as early stage breast cancer. Preferably, the patient is a human.

As mentioned above, the expression levels of Estrogen Receptor (ER) and programmed death ligand (PD-L1), and optionally interferon-gamma (IFN- γ), can be measured in vitro in a sample from a patient. Preferably, the methods provided herein comprise measuring interferon-gamma (IFN- γ) in vitro in a sample from a patient. Preferably, the sample to be evaluated/analyzed herein is a tumor tissue sample. Determining that the patient (or group of patients) is in need of PD-L1 inhibitor cotherapy if low or absent ER expression levels and elevated expression levels of programmed death ligand 1(PD-L1) and optionally reduced expression levels of interferon-gamma (IFN-gamma) compared to a control are measured in vitro in said sample.

The term "ER" is an abbreviation for "estrogen receptor". Likewise, the terms "PD-L1" and "IFN- γ" are abbreviations for the terms "programmed death ligand" and "interferon- γ", respectively. Thus, the term "ER" is used interchangeably herein with "estrogen receptor". Likewise, the terms "PD-L1" and "IFN- γ" are used interchangeably herein with the terms "programmed death ligand" and "interferon- γ", respectively.

Preferably, the (tumor/biological) sample of the patient and/or cancer to be treated is characterized by or associated with a low or absent Estrogen Receptor (ER) expression level. Preferably, the sample of the patient is a tumor sample. ER expression levels can be ER negative (ER (-). The term "ER (-)" is used interchangeably herein with the term "ER negative".

The "ER negative" expression level can be determined by conventional and standard procedures, as described, for example, in Guideline one Receptor Testing in Breast cancer.S.nofech-Mozes, E.Vella, S.Dhesy-Thind, and W.Hanna (evaluation Initiative of the Program in evaluation-Based Care (PEBC), Cancer Care Ontario (CCO); Report Date: April 8,2011). This guide (and references cited therein) is incorporated herein by reference in its entirety. These guidelines are on the world wide web on the cancer. https:// www.cancercare.on.ca/toolbox/qualitygeidelines/clin-program/pathlabebs/available.

Conventional and standard procedures for determining "ER negative" expression levels are described in these guidelines and also in the following references: Nofech-Mozes S, Vella ET, Dhesy-Thind S, Hagerty KL, Mangu PB, Tenin S, etc., Systematic review on hormone receiver testing in noise cancer. applied Immunohistochem Mol Morphol.2012May; 20(3) 214-63.doi:10.1097/PAI.0b013e318234aa12.Epub 2011Nov 11.

Nofech-Mozes S,Vella ET,Dhesy-Thind S,Hanna WM.Cancer Care Ontarioguideline recommendations for hormone receptor testing in breast cancer.ClinOncol(R Coll Radiol).Epub 2012May 17。

"ER negative" expression can be determined by IHC (immunohistochemistry) if, for example, ER expression levels are low or absent and/or if Progesterone Receptor (PR) expression levels are low or absent. The abbreviation "PR" is used herein interchangeably with the term "progesterone receptor". Samples or patients may be assessed herein as "ER negative" according to the following staining pattern (by IHC):

only nuclear (not cytoplasmic) staining should be scored.

There are three categories of staining:

positive: more than or equal to 10% staining in ER or PR

Low positive: 1% to 9% staining in ER or PR

Negative: < 1% staining in ER and PR

Thus, if according to IHC, the sample shows the following staining pattern: (iii) staining at < 1% in ER and PR, then in particular, the sample or patient may be assessed herein as "ER negative".

If according to IHC the sample shows a "positive" staining: samples or patients may be assessed herein as "ER positive" if ≧ 1% staining (i.e., more than 1% of the examined/assessed cells have/show staining for estrogen receptor according to IHC (immunohistochemistry)).

Preferably, if according to IHC, the sample shows the following staining pattern: samples or patients are evaluated herein as "ER negative" if < 1% staining in ER (i.e. less than 1% of the examined/evaluated cells have/show staining in estrogen receptor according to IHC (immunohistochemistry)). Most preferably, a sample or patient is assessed as "ER negative" if the nuclei in a tumor tissue sample show < 1% staining in terms of ER staining according to IHC. Thus, from the three categories provided above, the assessment of "ER negative" is based on < 1% staining in ER according to IHC.

Likewise, "ER negative" expression can be determined by other methods routinely employed in the art. For example, "ER negative" may be determined if mRNA/RNA expression levels are low or absent. Conventional methods to be used include, but are not limited to: allred score, IRS, Remmele score or any other suitable biochemical detection method. The skilled person knows that the cut-off of such a method must match the cut-off as defined above via IHC.

The nucleic acid sequences and amino acid sequences of Progesterone Receptor (PR), Estrogen Receptor (ER), programmed death ligand 1(PD-L1), and/or interferon-gamma (IFN γ) to be used herein are well known and can be obtained from databases such as NCBI. Exemplary sequences are provided herein (see, e.g., SEQ ID NOS: 38-51).

The methods and sample types used to establish the cut-off values for markers such as programmed death ligand 1(PD-L1) and/or interferon-gamma (IFN- γ) and to measure the sample obtained from the individual or patient to be analyzed are matched to each other or are the same. A cut-off value can be obtained in the control group, i.e. a value above which overexpression, e.g. increased expression of programmed death ligand 1(PD-L1) compared to the control, is acknowledged. A cut-off value, i.e. a value below which a reduced expression, e.g. a reduced expression of interferon-gamma (IFN- γ) compared to a control, is acknowledged, may be obtained in the control group.

The control group on which the cut-off value is based is selected to match the investigated individual/patient group, in other words, if the method of the invention is used to determine the need for PD-L1 combination therapy in patients with breast or gastric cancer, respectively, the control group is also a patient with breast or gastric cancer, respectively. The control group used to establish the cut-off values for PD-L1 and/or IFN- γ comprised at least 40, or at least 50, or at least 100 individuals/patient. Expression levels above the cut-off or corresponding values are considered to represent overexpression, and values at or below the cut-off are considered to be reduced expression.

In one embodiment, the expression level of "IFN- γ" in a tumor tissue sample from an individual/patient is compared to a cut-off value. Values above the cut-off are considered to represent overexpression of IFN- γ, and values at or below the cut-off are considered to be reduced IFN- γ expression. In one embodiment, decreased expression is admitted if the IFN- γ expression level is at or below the highest pentad, quartile or tertile value, respectively, as established in a control group. In one embodiment, the cutoff for IFN- γ is the highest quartile. In one embodiment, the cut-off value is a value between the 70 th and 80 th percentiles. In one embodiment, the cutoff value for IFN- γ is the 73 th percentile, i.e., values above this cutoff are considered representative of overexpression of IFN- γ, and values at or below the 73 th percentile are considered reduced IFN- γ expression. In one embodiment, if IFN- γ expression in a sample (e.g., a tumor tissue sample) is reduced (i.e., below or at an IFN- γ cutoff), then the individual/patient is determined to be in need of PD-L1 combination therapy. In one embodiment, if IFN- γ is overexpressed (i.e., above the IFN- γ cutoff, as described above), then the individual/patient is determined not to require PD-L1 combination therapy.

In one embodiment, the expression level of PD-L1 in a tumor tissue sample from an individual/patient is compared to a cut-off value. Values above the cut-off are considered to represent overexpression of PD-L1, and values at or below the cut-off are considered to be reduced PD-L1 expression. In one embodiment, overexpression of PD-L1 is admitted if the PD-L1 expression level is higher than the cut-off value between the 50 th percentile and the 75 th percentile, as established in the control group. In one embodiment, overexpression of PD-L1 is admitted if the PD-L1 expression level is higher than the cut-off value between the 50 th percentile and the 70 th percentile of the control group. In one embodiment, the individual/patient is determined to be in need of PD-L1 combination therapy if PD-L1 is overexpressed (i.e., the measured expression level of PD-L1 is higher than the PD-L1 cutoff).

In a particular embodiment, overexpression of PD-L1 is established in an individual/patient subgroup with a reduced level of IFN- γ expression in a tumor tissue sample. In one embodiment, overexpression of PD-L1 is admitted if the PD-L1 expression level is above the cut-off value between the 40 th percentile and the 65 th percentile, as established in this subgroup. In one embodiment, overexpression of PD-L1 is admitted if the PD-L1 expression level is above the cut-off value between the 50 th percentile and the 60 th percentile, as established in this subgroup. In one embodiment, if the expression level of PD-L1 in the subgroup with reduced IFN- γ expression is higher than the 54 th percentile, the individual/patient is determined to be in need of PD-L1 combination therapy.

In one embodiment, if IFN- γ expression in the tumor tissue sample is reduced (i.e., below or at an IFN- γ cutoff) and PD-L1 is overexpressed (i.e., above a PD-L1 cutoff), then the individual/patient is determined to be in need of PD-L1 combination therapy.

The term "elevated expression level of programmed death ligand 1(PD-L1) compared to a control" may be used herein interchangeably with "expression level of programmed death ligand 1(PD-L1) above the PD-L1 cutoff value", as defined and explained above.

The term "reduced expression level of interferon-gamma (IFN γ) compared to a control" may be used herein interchangeably with "expression level of interferon-gamma (IFN γ) below or at an IFN γ cutoff value".

The present invention relates to the following aspects.

The present invention relates to a method of determining the need for a PD-L1 inhibitor combination therapy for a cancer patient, (i) wherein a therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for said patient or (ii) wherein said patient is undergoing a therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent, said method comprising the steps of:

a) measuring in vitro in a sample from the patient the expression levels of Estrogen Receptor (ER), programmed death ligand 1(PD-L1), and interferon- γ (IFN γ);

b) determining that the patient is in need of PD-L1 inhibitor cotherapy if low or absent ER expression levels (e.g., ER (-)/ER negative), programmed death ligand 1(PD-L1) expression levels above the PD-L1 cutoff, and interferon-gamma (IFN γ) expression levels below or at the IFN γ cutoff are measured in step (a).

The present invention relates to a method of treating cancer in a cancer patient for whom therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated, the method comprising selecting a cancer patient whose cancer is determined to be having a low or absent ER expression level (such as ER (-)/ER negative) and having an expression level of programmed death ligand 1(PD-L1) above the PD-L1 cutoff and having an expression level of interferon- γ (IFN γ) below or at the IFN γ cutoff, and administering to the patient an effective amount of a modulator of the HER2/neu (ErbB2) signaling pathway, a chemotherapeutic agent and an inhibitor of programmed death ligand 1 (PD-L1).

The present invention relates to a method of treating cancer in a cancer patient undergoing therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising selecting a cancer patient whose cancer is determined to be having a low or absent ER expression level (such as ER (-)/ER negative) and having an expression level of programmed death ligand 1(PD-L1) above the PD-L1 cutoff and having an expression level of interferon-gamma (IFN γ) below or at the IFN γ cutoff, and administering to the patient an effective amount of an inhibitor of programmed death ligand 1 (PD-L1).

The present invention relates to a pharmaceutical composition comprising a modulator of the HER2/neu (ErbB2) signaling pathway and an inhibitor of programmed death ligand 1(PD-L1) for use in the treatment of cancer, wherein the cancer is determined to have a low or absent ER expression level (such as ER (-)/ER negative) and to have an expression level of programmed death ligand 1(PD-L1) above the PD-L1 cutoff and to have an expression level of interferon-gamma (IFN γ) below or at the IFN γ cutoff.

All explanations and definitions given herein for "PD-L1 inhibitor", "PD-L1 inhibitor combination therapy", "cancer patient", "HER 2/neu (ErbB2) signaling pathway modulator", "chemotherapeutic agent", "sample", "expression level" and the like apply mutatis mutandis to the above-described aspects of the invention.

The expression levels of Estrogen Receptor (ER), programmed death ligand 1(PD-L1), and interferon-gamma (IFN γ) in a sample from a patient can be measured simultaneously or sequentially in any combination in vitro. For example, the expression levels of Estrogen Receptor (ER), programmed death ligand 1(PD-L1), and interferon- γ (IFN γ) can be measured simultaneously. Expression levels of Estrogen Receptor (ER) can be measured first, followed by measurement of programmed death ligand 1(PD-L1) and interferon-gamma (IFN γ). The expression level of programmed death ligand 1(PD-L1) may be measured first, followed by (simultaneous or sequential) measurement of Estrogen Receptor (ER) and interferon-gamma (IFN γ). The expression level of interferon-gamma (IFN γ) may be measured first, followed by (simultaneously or sequentially) measurement of Estrogen Receptor (ER) and programmed death ligand 1 (PD-L1). Any order/combination of measurement of expression levels of Estrogen Receptor (ER), programmed death ligand 1(PD-L1), and interferon-gamma (IFN γ) in a sample from a patient is contemplated and included herein.

It is contemplated herein that a patient is determined to be in need of PD-L1 inhibitor cotherapy if a low or absent ER expression level is measured in the first step (1) (e.g., ER (-)/ER negative), and if an interferon-gamma (IFN γ) expression level below or at an IFN γ cutoff is measured in the second step (2), and if a programmed death ligand 1(PD-L1) expression level above the PD-L1 cutoff is measured in the third step (3).

The present invention relates to the following aspects:

b) determining that the patient is in need of PD-L1 inhibitor cotherapy if a low or absent level of ER expression is measured in the first step (1) (e.g., ER (-)/ER negative), and if a level of interferon-gamma (IFN γ) expression is measured in the second step (2) that is below or at the IFN γ cutoff, and if a level of programmed death ligand 1(PD-L1) expression is measured in the third step (3) that is above the PD-L1 cutoff.

The present invention relates to a method of treating cancer in a cancer patient for whom a therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level in a first step (1) (such as ER (-)/ER negative) and in a second step (2) an interferon-gamma (IFN γ) expression level below or at an IFN γ cutoff and in a third step (3) an expression level of programmed death ligand 1(PD-L1) above a PD-L1 cutoff, and administering to the patient effective amounts of a modulator of the HER2/neu (ErbB2) signaling pathway, a chemotherapeutic agent and an inhibitor of death ligand 1 (PD-L1).

The present invention relates to a method of treating cancer in a cancer patient undergoing therapy comprising a modulator of the HER2/neu (ErbB2) signalling pathway and a chemotherapeutic agent, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level in step (1) (such as ER (-)/ER negative) and an interferon-gamma (IFN γ) expression level in step (2) that is below or at the IFN γ cutoff and a programmed death ligand 1(PD-L1) expression level in step (3) that is above the PD-L1 cutoff, and administering to the patient an effective amount of an inhibitor of programmed death ligand 1 (PD-L1).

The present invention relates to a pharmaceutical composition comprising a modulator of the HER2/neu (ErbB2) signaling pathway and an inhibitor of programmed death ligand 1(PD-L1) for use in the treatment of cancer, wherein the cancer is determined to have a low or absent ER expression level (such as ER (-)/ER negative), to have an interferon-gamma (IFN γ) expression level below or at an IFN γ cutoff, to have an expression level of programmed death ligand 1(PD-L1) above a PD-L1 cutoff.

The following relates to an exemplary cut-off value that allows for determining that a patient is in need of PD-L1 inhibitor combination therapy in accordance with the present invention. Whether the expression level of PD-L1 and/or IFN- γ in a sample from a patient is below or above such a cut-off value can be readily determined by conventional techniques such as Affymetrix.

If the gene expression analysis gave results for IFN- γ expression higher than or equal to 4.8, no combination therapy (HER2 targeting and PDL1 targeting) was recommended and further PDL1 assessment was not necessary. If gene expression analysis gives IFN- γ results below 4.8, then parallel assessment of PD-L1 is necessary. If the PD-L1 gene expression analysis then gave results higher than or equal to 5.3, a combination therapy (HER2 targeting and PDL1 targeting) is recommended. This exemplary scheme is shown in fig. 19.

In this context, Affymetrix may be implemented as follows: total RNA from tumor cells was extracted from FFPE tumor sections using the Light Cycler pertuzumab FFPET RNA kit (Roche Diagnostics). RNA was processed for hybridization using WT-ovationFPE System V2(Nugen), and hybridized with AffymetrixHuman genome U133Plus 2.0 array hybridization. The hybridized arrays were washed and stained on Affymetrix Fluidics Station (Fluidics Station)450 and washed with AffymetrixThe scanner 30007G scans.

As mentioned, the expression level of PD-L1 and/or IFN- γ in a sample from a patient may be determined by conventional techniques (such as Affymetrix). The following relates to an exemplary protocol for such assays (also referred to herein as gene expression profiling):

tumor biopsies can be profiled for gene expression on the AFFYMETRIX HG-U133Plus 2 whole human genome microarray platform. Roche HighPure RNA extraction, NuGen amplification and standard AFFYMETRIX hybridization and scanning protocols can be used. These schemes and the like are incorporated herein by reference. All array scans were typically passed through standard AFFYMETRIX QC.

A Robust multi-array algorithm (RMA) may be used to preprocess the original signal (irigary et al, 2003. world wide web in. ncbi. nlm. nih. gov/pubmed/12925520; incorporated herein by reference). All sets of probes available for the gene of interest can be acquired as reported below. For gene CD274, where several probe sets are available to represent the gene, the probe set with the highest mean expression value (defined as the arithmetic mean of the expression of a given probe set) was selected to represent the gene:

CD274(PDL1)

223834_ at, for PDL1 selection

227458_at

The probe set selected corresponded to the last exon/3' UTR of the gene and captured all known RefSEq mrnas (see fig. 6).

IFNG

210354_at

This probe set also represents the last exon/3' UTR of the gene and captures all known refseqmrnas (see fig. 7).

In accordance with the above, the expression level of interferon-gamma may be measured prior to the expression level of Estrogen Receptor (ER) and/or prior to the expression level of programmed death ligand 1 (PD-L1). The step of measuring the expression levels of Estrogen Receptor (ER) and programmed death ligand 1(PD-L1) may even be absent.

For example, as shown in the appended examples, the expression level of several interferon-gammas (IFN γ) is higher than or equal to (about) 4.8, as determined by conventional methods (e.g., Affymetrix), then PD-L1 combination therapy may not be recommended.

Thus, the present invention provides a method of determining the need for a PD-L1 inhibitor combination therapy in a cancer patient, wherein a therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for said patient or wherein said patient is undergoing a therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent, said method comprising the steps of:

(a) measuring in vitro the expression level of interferon-gamma (IFN γ) in a sample from said patient;

(b) if the expression level of interferon-gamma (IFN γ) is higher than or equal to (about) 4.8, as determined by conventional methods (e.g., Affymetrix) in step (a), then the patient is determined not to require PD-L1 inhibitor cotherapy.

If the expression level of interferon-gamma (IFN γ) is below (about) 4.8, as determined by conventional methods, such as Affymetrix, the expression level of programmed death ligand 1(PD-L1) and optionally Estrogen Receptor (ER) may be measured in vitro in a sample from the patient.

(a) measuring in vitro the expression levels of interferon-gamma (IFN γ), of Estrogen Receptor (ER) and of programmed death ligand 1(PD-L1) in a sample from said patient,

(b) determining that the patient is in need of PD-L1 inhibitor cotherapy if the expression level of interferon-gamma (IFN γ) is below (about) 4.8, as determined by conventional methods such as Affymetrix, and if a low or absent ER expression level and optionally an elevated expression level of programmed death ligand 1(PD-L1) compared to a control is measured in step (a).

If the measured expression level of programmed death ligand 1(PD-L1) in a sample from the patient is elevated compared to a control, then the patient may be determined to be in need of PD-L1 inhibitor cotherapy in accordance with the present invention. For example, the expression level of programmed death ligand 1(PD-L1) can be greater than or equal to (about) 5.3 as determined by conventional methods (e.g., Affymetrix).

All explanations and definitions given herein for "PD-L1 inhibitor", "PD-L1 inhibitor combination therapy", "cancer patient", "HER 2/neu (ErbB2) signaling pathway modulator", "chemotherapeutic agent", "sample", "expression level" etc., as administered herein, apply in this context mutatis mutandis.

Thus, the present invention relates to a method of treating cancer in a cancer patient for whom a therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an elevated expression level of programmed death ligand 1(PD-L1) compared to a control and an expression level of interferon-gamma (IFN γ) determined according to conventional methods (such as Affymetrix) to be below (about) 4.8, and administering to the patient an effective amount of a modulator of the HER2/neu (ErbB2) signaling pathway, a chemotherapeutic agent and an inhibitor of programmed death ligand 1 (PD-L1).

Furthermore, the present invention relates to a method of treating cancer in a cancer patient undergoing therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an elevated expression level of programmed death ligand 1(PD-L1) compared to a control, and to have an expression level of interferon-gamma (IFN γ) below (about) 4.8 as determined according to conventional methods such as Affymetrix, and administering to said patient an effective amount of an inhibitor of programmed death ligand 1 (PD-L1).

Pharmaceutical compositions comprising a modulator of the HER2/neu (ErbB2) signaling pathway and an inhibitor of programmed death ligand 1(PD-L1) for use in the treatment of cancer are provided, wherein the cancer is determined to have a low or absent ER expression level and to have an elevated expression level of programmed death ligand 1(PD-L1) compared to a control and an expression level of interferon-gamma (IFN γ) of less than (about) 4.8 as determined according to conventional methods such as Affymetrix.

The pharmaceutical composition for use in treating cancer may further comprise a chemotherapeutic agent.

In accordance with the above, the methods provided herein can include the steps of measuring the expression level of interferon-gamma (IFN γ) in the sample, and determining that the patient is in need of PD-L1 inhibitor cotherapy if a reduced expression level of interferon-gamma (IFN γ) is measured as compared to a control. For example, a "reduced expression level" of interferon-gamma (IFN γ) may be an expression level of less than (about) 4.8, as determined by conventional methods such as Affymetrix. Thus, a cancer determined to have a reduced level of interferon-gamma (IFN γ) expression compared to a control may be determined to have a level of interferon-gamma (IFN γ) expression of less than (about) 4.8, as determined by conventional methods such as Affymetrix.

It is contemplated herein that expression levels may be reflected in the activity of the gene product/protein. Thus, the activity of ER, PD-L1 and/or IFN- γ can also be measured and assessed in accordance with the invention in addition to or instead of expression levels. Those skilled in the art are aware of corresponding means and methods for detecting and assessing ER, PD-L1 and IFN- γ expression levels and/or activity. Exemplary methods to be used include, but are not limited to, molecular evaluation such as Western blot, Northern blot, real-time PCR, and the like. Such methods are described in detail herein.

The expression level of ER, PD-L1, and/or IFN- γ can be the mRNA expression level of ER, PD-L1, and/or IFN- γ. If the gene product is RNA, particularly mRNA (e.g., unspliced, partially spliced, or spliced mRNA), the determination can be carried out by using Northern blot techniques, in situ hybridization, hybridization on a DNA chip or microarray equipped with one or more probes or probe sets specific for mRNA transcripts, or PCR techniques, such as quantitative PCR techniques, such as real-time PCR. These and other methods suitable for binding (specific) mRNA are well known in the art and are described, for example, in Sambrook and Russell (2001, supra). The skilled person is able to determine the amount of a component, in particular the gene product, by using a correlation, preferably a linear correlation, between the intensity of the detection signal and the amount of the gene product to be determined.

The expression level may be the protein expression level of ER, PD-L1 and/or IFN- γ. Quantification of protein expression levels can be performed by using well-known techniques such as Western blot techniques, immunoassays, gel or blot based methods, IHC, mass spectrometry, flow cytometry, FACS, and the like. Generally, the person skilled in the art knows methods for the quantification of polypeptides/proteins. The amount of purified polypeptide in solution can be determined by physical means (e.g., photometry). The method of quantifying a particular polypeptide in a mixture can depend, for example, on the specific binding of an antibody. Specific detection and quantification methods that exploit antibody specificity include, for example, immunohistochemistry (in situ). Western blot combinations separate protein mixtures by electrophoresis and specific detection with antibodies. The electrophoresis may be multi-dimensional, such as 2D electrophoresis. Typically, polypeptides are separated in 2D electrophoresis according to their apparent molecular weight along one dimension and their isoelectric point along the other direction. Alternatively, the protein quantification method may involve, but is not limited to, mass spectrometry or enzyme-linked immunosorbent assay methods.

The use of High Throughput Screening (HTS) is also contemplated in the context of the present invention. Suitable HTS methods are known in the art. Those skilled in the art will be readily able to adapt such protocols or known HTS methods to practice the methods of the present invention. Such assays are typically carried out in a liquid phase, where at least one reaction batch is completed for each cell/tissue/cell culture to be tested. A typical vessel to be used is a microtiter plate (i.e. a plurality of "prime" 96 reaction vessels) having, for example, 384, 1536, or 3456 wells. Robotics, data processing and control software, and sensitivity instrumentation are further commonly used components of HTS devices. Often, robotic systems are used to transport the microtiter plate from station to add and mix samples and reagents, incubate reagents and finally read out (detection). In general, HTS can be used in the simultaneous preparation, incubation and analysis of many plates. The assay may be carried out in a single reaction (which is generally preferred), however washing and/or transfer steps may also be included. Detection can be carried out using radioactivity, luminescence, or fluorescence, such as Fluorescence Resonance Energy Transfer (FRET), Fluorescence Polarization (FP), and the like. The biological samples described herein may also be used in such contexts. In particular, cellular and in vivo assays may be employed in HTS. Cellular assays may also include cell extracts, i.e., extracts from cells, tissues, etc. However, it is preferred herein to use cells or tissues as biological samples (in particular samples obtained from patients/subjects suffering from or susceptible to cancer), whereas in vivo assays are particularly useful for validating modulators/inhibitors/chemotherapeutic agents to be used herein. Depending on the results of the first assay, subsequent assays can be performed by rerunning the experiment to collect further data on the narrowed set (e.g., samples found to be "positive" in the first assay), which confirms and refines the observations.

As used in the context of the methods of the present invention, preferably non-limiting examples of "controls" are controls from patients who do not require PD-L1 inhibitor cotherapy, such as samples/cells/tissues obtained from one or more healthy subjects or one or more patients who have cancer/tumor and are known not to require PD-L1 inhibitor cotherapy treatment. For example, such controls (samples) may be from patients who do not benefit from additional PD-L1 inhibitor combination therapy. Another non-limiting example of a "control" is an "internal standard," such as a mixture of purified or synthetically produced proteins and/or peptides or RNAs, wherein the amount of each protein/peptide/RNA is quantified by using the controls described above.

Yet another non-limiting example of a "control" may be a "healthy" control, such as a sample/cell/tissue obtained from a healthy subject or a patient not suffering from cancer/tumor or cells obtained from such a subject. In accordance with the above, the reference or control expression level of ER, PD-L1 and/or IFN- γ is determined in (a sample of) the corresponding healthy control subject/patient, i.e. it is the "normal" state of ER, PD-L1 and/or IFN- γ. The control may also be a sample/cell/tissue obtained from an individual or patient suspected of having cancer, as long as the sample/cell/tissue does not contain tumor or cancer cells. In yet another alternative, a "control" may be a sample/cell/tissue obtained from an individual or patient having cancer that has been obtained prior to the development or diagnosis of the cancer described below.

A sample to be evaluated according to the methods provided herein can include non-diseased cells and/or diseased cells, i.e., non-cancerous cells and/or cancerous cells. However, the content of cancerous cells in non-cancerous cells should be higher than e.g. 50%. The sample may also (or even only) comprise cancer/tumor cells, such as breast cancer/tumor cells. The term "sample" shall generally mean any biological sample obtained from a tumor of a patient. The sample may be a tissue resection or tissue biopsy. The sample may also be a metastatic lesion or a section of a metastatic lesion or a blood sample known or suspected to contain circulating tumor cells. According to the above, the biological sample may comprise cancer cells and to some extent, i.e. less than e.g. 50% non-cancer cells (other cells). A skilled pathologist is able to distinguish cancer cells from normal tissue cells. Methods for obtaining tissue biopsies, tissue resections, bodily fluids, and the like from mammals, such as humans, are well known in the art.

As explained above, a cancer patient determined to be in need of a PD-L1 inhibitor combination therapy according to the present invention is undergoing therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent or such therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for patients. Therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent is indicated for patients having a "HER 2 positive cancer", such as patients suspected of having a HER2 positive cancer, having a HER2 positive cancer or being predisposed to having a HER2 positive cancer. Preferably, the cancer to be treated is according to the invention a "HER 2 positive cancer", in particular a "HER 2 positive breast cancer". The "HER 2 positive cancer" can be "HER 2 positive breast cancer" or "HER 2 positive gastric cancer". Furthermore, the HER2 positive cancer may be ovarian cancer, lung cancer, colorectal cancer, kidney cancer, bone marrow cancer, bladder cancer, skin cancer, prostate cancer, esophageal cancer, salivary gland cancer, pancreatic cancer, liver cancer, head and neck cancer, CNS (especially brain) cancer, cervical cancer, cartilage cancer, colon cancer, genitourinary cancer, gastrointestinal cancer, pancreatic cancer, synovial cancer, testicular cancer, thymus cancer, thyroid cancer and uterine cancer.

As used herein, the term "HER 2 positive cancer" refers to cancerous/neoplastic tissue or the like comprising cancer cells having higher than normal HER2 levels. For the purposes of the present invention, a "HER 2 positive cancer" has an Immunohistochemistry (IHC) score of at least 2+ and/or an In Situ Hybridization (ISH) amplification rate ≧ 2.0 (i.e., ISH positive). Thus, if high HER2 (protein) expression levels, e.g. as detected by immunohistochemical methods and/or HER2 gene amplification as detected by in situ hybridization (ISH positivity, e.g. a ratio of HER2 gene copy number higher than 4 copies of HER2 gene per tumor cell or HER2 gene copy number to CEP17 signal number ≧ 2.0) are found in a sample obtained from the patient, such as breast tissue biopsy or breast tissue resection or tissue derived from a metastatic site, then a HER2 positive cancer is present. In one embodiment, a "HER 2 positive cancer" has an Immunohistochemical (IHC) score of HER2(3+) and/or is ISH positive.

The expression level of HER2 can be detected by immunohistochemical methods and the HER2 gene amplification status can be measured by in situ hybridization methods, such as Fluorescence In Situ Hybridization (FISH) techniques. Corresponding assays and kits for protein expression assays and for detecting gene amplification are well known in the art. Alternatively, other methods (such as qRT-PCR) may be used to detect HER2 gene expression levels.

HER2 expression levels can be detected by immunohistochemical methods and the like. Such methods are well known in the art and corresponding commercial kits are available. An exemplary kit that may be used in accordance with the present invention is HerceptTest manufactured and sold by Dako corporation^TMOr as Ventana Pathway^TMTest (2)And the like. Herceptest can be used^TMThe reagents provided and followed Herceptest^TMTo assess HER2 protein expression levels. The skilled person will know additional means and methods for determining the expression level of HER2 by immunohistochemical methods; see, for example, WO 2005/117553. Thus, the expression level of HER2 can be easily and reproducibly determined by a person skilled in the art without undue burden. However, to ensure accurate and reproducible results, the tests must be conducted in specialized laboratories that ensure the validity of the test protocols.

HER2 expression levels can be classified among low, moderate and high expression levels. It is preferred in the context of the present invention that HER2 positive disease is defined as a strong expression level of HER2 (e.g. HER2(3+) according to IHC), e.g. as determined in a sample of a cancer patient.

The scoring system recommended for assessing IHC staining patterns reflecting HER2 expression levels assigned herein to HER2(0), HER2(+), HER2(++) and HER2(+++) is as follows:

the IHC staining pattern described above is routinely used to determine HER2 positive breast cancer. The terms HER2(+), HER2(+ +) and HER2(+++), as used herein, are equivalent to the terms HER2(1+), HER2(2+) and HER2(3 +). As used in the context of the present invention, a "low protein expression level" corresponds to a score of 0 or 1+ (according to the "negative evaluation" of the table shown herein above), a "weak to medium protein expression level" corresponds to a score of 2+ (weak to medium overexpression, see table above), and a "high protein expression level" corresponds to a score of 3+ (strong overexpression, see table above). As described in detail herein above, the assessment of protein expression levels (i.e. the scoring system shown in the table) is based on results obtained by immunohistochemical methods. Thus, as a standard or routinely, two commercially available FDA approved kits (i.e., Dako Herceptin) are used^TMAnd Ventana Pathway^TM) One performed HER2 status by immunohistochemistry. These are semi-quantitative assays that stratify expression levels into 0 (less than 20,000 receptors per cell, no visible expression by IHC staining), 1+ (about 100,000 receptors per cell, partial membrane staining, less than 10% of cells overexpressing HER2), 2+ (about 500,000 receptors per cell, weak to moderate complete membrane staining, greater than 10% of cells overexpressing HER2), and 3+ (about 2,000,000 receptors per cell, strong complete membrane staining, greater than 10% of cells overexpressing HER 2).

Alternatively, other methods may be used to assess HER2 protein expression levels, such as Western blots, ELISA-based detection systems, and the like.

HER2 positive cancer can also be diagnosed by assessing the gene amplification status of HER 2. Thus, if this assessment according to ISH is positive, HER2 positive cancer is diagnosed. According to this assessment, HER2 positive cancers may specifically involve an average HER2 gene copy number per tumor cell of greater than 4 copies of the HER2 gene (for those test systems without internal centromere control probes) or a HER2/CEP17 ratio of > -2.0 (for those test systems using internal chromosome 17 centromere control probes). In other words, HER2 positive cancers may specifically involve a HER2 gene copy number of greater than 4. The level of amplification of the HER2 gene can be readily identified by In Situ Hybridization (ISH), such as Fluorescence In Situ Hybridization (FISH), Chromogenic In Situ Hybridization (CISH) and Silver In Situ Hybridization (SISH). These methods are known to the skilled worker. The principles of these methods can be deduced from standard textbooks. Commercial kits for determining the amplification status of the HER2 gene by in situ hybridization are available.

The following IHC staining pattern was recommended for the determination of HER2 positive gastric cancer (see Dako herceptin package insert).

In Hercep Test^TMIn a stained biopsy, at least 5 clusters of stained tumor cells are recommended. A cluster of at least 5 stained tumor cells consists of 5 linked HER2 stained tumor cells.

Table 9: interpretation and scoring of HER2 immunohistochemical staining

Based on the criteria of Hofmann et al (40).

A more refined IHC staining pattern for determining HER2 positive gastric cancer is as follows:

as indicated above, the HER2 positive cancer to be treated according to the invention may be breast cancer, such as early stage breast cancer. As used herein, the term "early stage breast cancer" refers to breast cancer that has not spread out of the breast or axillary lymph nodes. In general, such cancers can be treated with neoadjuvant or adjuvant therapy. As used herein, the term "neoadjuvant therapy" refers to systemic therapy given preoperatively. The term "adjuvant therapy" refers to systemic therapy administered post-operatively. In accordance with the above, the treatment may be neoadjuvant or adjuvant therapy of early stage breast cancer.

In accordance with the above, the sample to be assessed may be from (obtained from) a patient having a HER2 positive cancer as defined above. For example, the sample may be obtained from a tumor tissue, a tumor, and thus a tumor cell or tumor tissue suspected of being a HER2 positive tumor, such as a breast tumor, and the like. The skilled person is able to identify such tumors and/or individuals/patients suffering from the respective cancers using standard techniques known in the art and the methods disclosed herein. In general, the tumor cells or cancer cells can be obtained from any biological source/organism, particularly any biological source/organism that is afflicted with the cancer described above. Particularly useful cells in the context of the present invention are preferably human cells. These cells may be obtained from, for example, a biopsy or biological sample. Tumor/cancer/tumor cell/cancer cell is a solid tumor/cancer/tumor cell/cancer cell. In accordance with the above, the cancer/tumor cells may be breast cancer/tumor cells, or the sample comprises cancer/tumor cells, such as breast cancer/tumor cells. In accordance with the above, the tumor/cancer may be a breast tumor/cancer.

The modulator of the HER2/neu (ErbB2) signaling pathway may be a HER2 inhibitor, such as a HER dimerization/signaling inhibitor. The HER dimerization inhibitor may be a HER2 dimerization inhibitor. The HER dimerization inhibitor may inhibit HER heterodimerization or HER homodimerization. The HER dimerization inhibitor may be an anti-HER antibody. The term "antibody" is used herein in the broadest sense and specifically encompasses intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. Also included are human and humanized and CDR grafted antibodies.

The HER antibody may bind to a HER receptor selected from the group consisting of: EGFR, HER2, and HER 3. Preferably, the antibody binds HER 2. The anti-HER 2 antibody may bind to domain II of HER2 extracellular domain. The antibody may bind to the junction between domains I, II and III of the extracellular domain of HER 2. The anti-HER 2 antibody may be pertuzumab.

For purposes herein, "pertuzumab" and "rhuMAb 2C 4" (which are used interchangeably) refer to antibodies comprising a variable light domain and a variable heavy domain (the amino acid sequences of which are shown in SEQ ID nos. 5 and 6, respectively, as depicted in fig. 2). Also shown in figure 2 are the variable light and variable heavy domains of variant 574/pertuzumab (the amino acid sequences are shown in SEQ ID nos. 7 and 8, respectively, as depicted in figure 2). Where pertuzumab is an intact antibody, preferably, it comprises an IgG1 antibody; in one embodiment comprising a light chain amino acid sequence, preferably, it comprises light and heavy chain amino acid sequences as shown in figures 3A/3B and 5A/5B, respectively (figures 5A/5B show the light and heavy chain amino acid sequences of the variant pertuzumab). Optionally, the heavy chain amino acid sequence of pertuzumab as shown in fig. 3B may include an additional amino acid "K" at position 449 of the C-terminus. Optionally, the antibody is produced by recombinant Chinese Hamster Ovary (CHO) cells. The terms "pertuzumab" and "rhuMAb 2C 4" are encompassed herein with names Adopted in the United States (United States addressed Name, usa n) or International non-proprietary Name (INN): biologically similar forms of the drug of pertuzumab. Again, the corresponding sequences are shown in fig. 2 to 5.

The modulator of the HER2/neu (ErbB2) signaling pathway may be a HER shedding inhibitor, such as a HER2 shedding inhibitor. HER shedding inhibitors may inhibit HER heterodimerization or HER homodimerization. The inhibitor of HER shedding may be an anti-HER antibody.

The term "antibody" is used herein in the broadest sense and specifically encompasses intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. Also included are human and humanized and CDR grafted antibodies.

The anti-HER antibody may bind to a HER receptor selected from the group consisting of: EGFR, HER2, and HER 3. Preferably, the antibody binds HER 2. The HER2 antibody may bind to subdomain IV of HER2 extracellular domain. Preferably, the HER2 antibody is herceptin^TMTrastuzumab.

For purposes herein, "herceptin^TM"/" trastuzumab "and" rhuMAb4D5-8 "(which are used interchangeably) refer to antibodies comprising a variable light domain and a variable heavy domain (the amino acid sequences of which are shown in FIG. 4, respectively; the domains are indicated by arrows). In case trastuzumab is an intact antibody, preferably it comprises the IgG1 antibody; in one embodiment, it comprises light and heavy chain amino acid sequences as shown in figure 4. Optionally, by Chinese hamster ovary (C)HO) cells produce antibodies. The terms "trastuzumab" and "rhuMAb 4D 5-8" are encompassed herein with either a U.S. adopted name (USAN) or an international non-proprietary name (INN): biologically similar forms of trastuzumab.

The programmed death ligand 1(PD-L1) inhibitor may be an antibody that specifically binds to PD-L1 (anti-PD-L1 antibody). Again, the term "antibody" is used in the broadest sense and specifically encompasses intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. Also included are human and humanized and CDR grafted antibodies.

Exemplary anti-PD-L1 antibodies are disclosed in WO2010/077634, which is incorporated herein in its entirety. The following describes a corresponding exemplary anti-PD-L1 antibody to be used according to the invention.

The anti-PD-L1 antibody may comprise a heavy chain variable region polypeptide comprising HVR-H1, HVR-H2, and HVR-H3 sequences, wherein:

(a) the HVR-H1 sequence is GFTFSX1SWIH (SEQ ID NO: 1);

(b) the HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG (SEQ ID NO: 2);

(c) the HVR-H3 sequence is RHWPGGFDY (SEQ ID NO: 3); further, wherein: x1 is D or G; x2 is S or L; x3 is T or S. X1 can be D; x2 can be S and X3 can be T.

The polypeptide may further comprise a variable region heavy chain framework sequence juxtaposed between HVRs according to the following formula: (HC-FR1) - (HVR-H1) - (HC-FR2) - (HVR-H2) - (HC-FR3) - (HVR-H3) - (HC-FR 4). The framework sequence may be derived from a human consensus framework sequence. The framework sequence may be a VH subgroup III consensus framework. One or more framework sequences may be as follows:

HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:4)

HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO:5)

HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:6)

HC-FR4 is WGQGTLVTVSA (SEQ ID NO: 7).

The heavy chain polypeptide may be combined with a variable region light chain comprising HVR-L1, HVR-L2, and HVR-L3, wherein:

(a) the HVR-L1 sequence is RASQX4X5X6TX7X8A (SEQ ID NO: 8);

(b) the HVR-L2 sequence is SASX9LX10S, and (SEQ ID NO: 9);

(c) the HVR-L3 sequence is QQX11X12X13X14PX15T (SEQ ID NO: 10); further, wherein: x4 is D or V; x5 is V or I; x6 is S or N; x7 is A or F; x8 is V or L; x9 is F or T; x10 is Y or A; x11 is Y, G, F, or S; x12 is L, Y, F or W; x13 is Y, N, A, T, G, F or I; x14 is H, V, P, T or I; x15 is A, W, R, P or T.

X4 can be D; x5 may be V; x6 can be S; x7 may be a; x8 may be V; x9 may be F; x10 may be Y; x11 may be Y; x12 can be L; x13 may be Y; x14 can be H; x15 may be a.

The polypeptide may further comprise a variable region light chain framework sequence juxtaposed between HVRs according to the following formula: (LC-FR1) - (HVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR 4). The framework sequence may be derived from a human consensus framework sequence. The framework sequence may be a VL κ I consensus framework. One or more framework sequences may be as follows:

LC-FR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 11);

LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO: 12);

LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 13);

LC-FR4 is FGQGTKVEIKR (SEQ ID NO: 14).

The anti-PD-L1 antibody (or antigen-binding fragment thereof) may comprise heavy and light chain variable region sequences, wherein:

(a) the heavy chain comprises HVR-H1, HVR-H2, and HVR-H3, wherein further:

(i) the HVR-H1 sequence is GFTFSX1SWIH (SEQ ID NO: 1);

(ii) the HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG (SEQ ID NO: 2);

(iii) the HVR-H3 sequence is RHWPGGFDY, and (SEQ ID NO: 3); (b) the light chain comprises HVR-L1, HVR-L2, and HVR-L3, wherein further:

(iv) the HVR-L1 sequence is RASQX4X5X6TX7X8A (SEQ ID NO: 8);

(v) the HVR-L2 sequence is SASX9LX10S (SEQ ID NO: 9);

(vi) the HVR-L3 sequence is QQX11X12X13X14PX15T (SEQ ID NO: 10); wherein: x1 is D or G; x2 is S or L; x3 is T or S; x4 can be D or V; x5 can be V or I; x6 can be S or N; x7 can be a or F; x8 can be V or L; x9 can be F or T; x10 can be Y or A; x11 can be Y, G, F, or S; x12 can be L, Y, F or W; x13 can be Y, N, A, T, G, F or I; x14 can be H, V, P, T or I; x15 can be A, W, R, P or T.

X1 can be D; x2 can be S and X3 can be T. Further, the positions may be as follows: x4 ═ D, X5 ═ V, X6 ═ S, X7 ═ a and X8 ═ V, X9 ═ F, and X10 ═ Y, X11 ═ Y, X12 ═ L, X13 ═ Y, X14 ═ H, and/or X15 ═ a. Further, the positions may be as follows: x1 ═ D, X2 ═ S and X3 ═ T, X4 ═ D, X5 ═ V, X6 ═ S, X7 ═ a and X8 ═ V, X9 ═ F, and X10 ═ Y, X11 ═ Y, X12 ═ L, X13 ═ Y, X14 ═ H and X15 ═ a.

The antibody (or antigen-binding fragment thereof) may further comprise

(a) A variable region heavy chain framework sequence juxtaposed between HVRs according to the formula: (HC-FR1) - (HVR-H1) - (HC-FR2) - (HVR-H2) - (HC-FR3) - (HVR-H3) - (HC-FR4), and

(b) a variable region light chain framework sequence juxtaposed between HVRs according to the formula: (LC-FR1) - (HVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR 4). The framework sequence may be derived from a human consensus framework sequence.

The variable region heavy chain framework sequence may be a VH subgroup III consensus framework. One or more framework sequences may be as follows:

HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 4);

HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO: 5);

HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 6);

HC-FR4 is WGQGTLVTVSA (SEQ ID NO: 7).

The variable region light chain framework sequence may be a VL kappa I consensus framework. One or more framework sequences may be as follows:

LC-FR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 11);

LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO: 12);

LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC and (SEQ ID NO: 13);

LC-FR4 is FGQGTKVEIKR (SEQ ID NO: 14).

The antibody (or antigen binding fragment thereof) may be or may comprise

(a) The variable heavy chain framework sequence is as follows:

(i) HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 4);

(ii) HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO: 5);

(iii) HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 6);

(iv) HC-FR4 is WGQGTLVTVSA; and (SEQ ID NO: 7);

(b) the variable light chain framework sequences are as follows:

(i) LC-FR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 11);

(ii) LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO: 12);

(iii) LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 13);

(iv) LC-FR4 is FGQGTKVEIKR (SEQ ID NO: 14).

The antibody (or fragment thereof) may further comprise a human constant region. The constant region may be selected from the group consisting of: IgG1, IgG2, IgG3, and IgG 4. The constant region may be IgG 1. The antibody (or fragment thereof) may further comprise a murine constant region. The constant region may be selected from the group consisting of: IgG1, IgG2A, IgG2B, and IgG 3. The constant region may be IgG 2A.

The antibody (or fragment thereof) may have reduced or minimal effector function. Minimal effector function may result from effector-lowering Fc mutations. The effector reducing Fc mutation may be N297A. The effector-reducing Fc mutation may be D265A/N297A. Minimal effector function may result from aglycosylation.

The antibody (or fragment thereof) may comprise heavy and light chain variable region sequences, wherein:

(a) the heavy chain comprises HVR-H1, HVR-H2, and HVR-H3, which have at least 85% overall sequence identity to GFTFSDSWIH (SEQ ID NO:15), AWISPYGGSTYYADSVKG (SEQ ID NO:16), and RHWPGGFDY (SEQ ID NO:3), respectively, and

(b) the light chain comprises HVR-L1, HVR-L2, and HVR-L3, which have at least 85% overall sequence identity to RASQDVSTAVA (SEQ ID NO:17), SASFLYS (SEQ ID NO:18), and QQYLYHPAT (SEQ ID NO:19), respectively.

The sequence identity may be at least 90%.

The antibody (or fragment thereof) may further comprise:

(a) variable region heavy chain (VH) framework sequences juxtaposed between HVRs according to the following formula: (HC-FR1) - (HVR-H1) - (HC-FR2) - (HVR-H2) - (HC-FR3) - (HVR-H3) - (HC-FR4), and

(b) a variable region light chain (VL) framework sequence juxtaposed between HVRs according to the formula: (LC-FR1) - (HVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR 4).

The antibody (or fragment thereof) may further comprise VH and VL framework regions derived from human consensus sequences. The VH framework sequences may be derived from Kabat subgroup I, II, or III sequences. The VH framework sequence may be a Kabat subgroup III consensus framework sequence. The VH framework sequences may be as follows:

HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 4);

HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO: 5);

HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 6);

HC-FR4 is WGQGTLVTVSA (SEQ ID NO: 7).

VL framework sequences may be derived from Kabat kappa I, II, III or IV subgroup sequences. The VL framework sequence may be a Kabat kappa I consensus framework sequence.

The VL framework sequence may be as follows:

LC-FR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 11);

LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO: 12);

LC-FR3 is GVPSRFSGSGSGTDFTITISSLQPEDFATYYC (SEQ ID NO: 13);

LC-FR4 is FGQGTKVEIKR (SEQ ID NO: 14).

(a) the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPG

KGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC

ARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO:20), and

(b) the light chain sequence has at least 85% sequence identity to the following light chain sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGK

APKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(SEQ ID NO：21)。

the sequence identity may be at least 90%.

(a) the heavy chain comprises the sequence: EVQLVESGGGLVQPGGSLRLS

CAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO:20), and

(b) the light chain comprises the sequence: DIQMTQSPSSLSASVGDRVTITC

RASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(SEQ ID NO：21)。

Furthermore, the anti-PD-L1 antibody may be encoded by a nucleic acid. Thus, isolated nucleic acids encoding the above polypeptides/antibodies (or fragments thereof) are described herein.

Provided herein are isolated nucleic acids encoding a light chain or heavy chain variable sequence of an anti-PD-L1 antibody or antigen-binding fragment, wherein:

(a) the heavy chain further comprises HVR-H1, HVR-H2, and HVR-H3 sequences having at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO:15), AWISPYGGSTYYADSVKG (SEQ ID NO:16), and RHWPGGFDY (SEQ ID NO:3), respectively, or

(b) The light chain further comprises HVR-L1, HVR-L2, and HVR-L3 sequences that have at least 85% sequence identity to RASQDVSTAVA (SEQ ID NO:17), SASFLYS (SEQ ID NO:18), and QQYLYHPAT (SEQ ID NO:19), respectively.

The sequence identity may be 90%. The anti-PD-L1 antibody may further comprise VL and VH framework regions derived from a human consensus sequence. The VH sequences may be derived from Kabat subgroup I, II, or III sequences. The VL sequence may be derived from a Kabat kappa I, II, III or IV subgroup sequence. The anti-PD-L1 antibody may comprise a constant region derived from a murine antibody. The anti-PD-L1 antibody can comprise constant regions derived from a human antibody. The constant region may be IgG 1. Antibodies encoded by nucleic acids may have reduced or minimal effector function. Minimal effector function may result from effector-lowering Fc mutations. The effector reducing Fc mutation may be N297A.

Further provided herein are vectors comprising the nucleic acids, host cells comprising the vectors. The host cell may be eukaryotic. The host cell may be mammalian. The host cell may be a Chinese Hamster Ovary (CHO) cell. The host cell may be prokaryotic. The host cell may be E.coli. Also provided herein are methods for producing an anti-PD-L1 antibody, comprising culturing the above-described host cell under conditions suitable for expression of a vector encoding an anti-PD-L1 antibody or antigen-binding fragment, and recovering the antibody or fragment.

The means and methods provided herein for treating cancer and/or cancer patients are described in more detail below.

Thus, encompassed herein are pharmaceutical compositions for use in treating cancer comprising a modulator of the HER2/neu (ErbB2) signaling pathway, such as trastuzumab, and an inhibitor of programmed death ligand 1(PD-L1) such as the anti-PD-L1 antibody described herein, wherein the cancer is determined to have a low or absent ER expression level and to have an elevated expression level of programmed death ligand 1(PD-L1) as compared to a control. The cancer may be determined to have a reduced level of interferon-gamma (IFN γ) expression compared to a control. The pharmaceutical composition may further comprise a chemotherapeutic agent (e.g. taxol or taxol derivatives, such as docetaxel)

In accordance with the above, the present invention provides a method for treating cancer comprising administering to a subject in need thereof an effective amount of a modulator of the HER2/neu (ErbB2) signaling pathway, a chemotherapeutic agent, and an inhibitor of programmed death ligand 1 (PD-L1). The cancer may be determined to have a reduced level of interferon-gamma (IFN γ) expression compared to a control.

Provided herein are modulators of the HER2/neu (ErbB2) signaling pathway and inhibitors of programmed death ligand 1(PD-L1) for use in the treatment of cancer, wherein the cancer is determined to have a low or absent ER expression level and to have an elevated expression level of programmed death ligand 1(PD-L1) as compared to a control. Further, provided herein are modulators of the HER2/neu (ErbB2) signaling pathway, programmed death ligand 1(PD-L1) inhibitors, and chemotherapeutic agents (e.g., taxol or taxol derivatives, such as docetaxel) For use in treating cancer, wherein the cancer is determined to have a low or absent expression level of ER and to have an elevated expression level of programmed death ligand 1(PD-L1) as compared to a control. The cancer may be determined to have a reduced level of interferon-gamma (IFN γ) expression compared to a control.

As discussed above, the present invention provides a method of treating cancer in a cancer patient for whom therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an elevated expression level of programmed death ligand 1(PD-L1) as compared to a control, and administering to the patient effective amounts of a modulator of the HER2/neu (ErbB2) signaling pathway, a chemotherapeutic agent and an inhibitor of programmed death ligand 1 (PD-L1). Likewise, the present invention provides a method of treating cancer in a cancer patient undergoing therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an elevated expression level of programmed death ligand 1(PD-L1) as compared to a control, and administering to the patient an effective amount of an inhibitor of programmed death ligand 1 (PD-L1).

The explanations and definitions given above in terms of "cancer", "cancer patient", "PD-L1 inhibitor", "PD-L1 inhibitor therapy", "modulator of the HER2/neu (ErbB2) signaling pathway", "chemotherapeutic agent", "low or absent ER expression level", "elevated expression level of programmed death ligand 1 (PD-L1)", "reduced expression level of interferon-gamma (IFN-gamma)", etc. apply in the context herein after making the necessary corrections.

The terms "treatment" and the like are used herein generally to mean obtaining a desired pharmacological and/or physiological effect. The effect may be preventative in terms of complete or partial prevention of the disease or symptoms thereof and/or therapeutic in terms of partial or complete cure of the disease and/or adverse effects due to the disease. As used herein, the term "treatment" encompasses any treatment of a disease in a patient and includes: (a) preventing the associated disease in a patient who may be predisposed to the disease; (b) inhibiting the disease, i.e. preventing its formation; or (c) relieving the disease, i.e., causing regression of the disease.

For purposes of the present invention, "patient" includes both humans and other animals (particularly mammals), and other organisms. As such, the methods are applicable to both human therapy and veterinary applications. Preferably, the patient is a human.

The following explanation relates in more detail to the treatment/therapy of these patients/this patient group according to the invention.

The formulation and administration of the pharmaceutical composition will be carried out in a manner consistent with good medical practice, taking into account the clinical condition of the individual patient, the site of delivery of the pharmaceutical composition, the method of administration, the schedule of administration, and other factors known to practitioners. For purposes herein, an "effective amount" of a pharmaceutical composition may thus be determined in light of such considerations.

The skilled person will appreciate that the effective amount of one of the PD-L1 inhibitor, modulator of the HER2/neu (ErbB2) signaling pathway, and chemotherapeutic agent described herein in a pharmaceutical composition administered to an individual will depend, inter alia, on the nature of the compound. For example, if the compound is a (poly) peptide or protein, the total pharmaceutically effective amount of the pharmaceutical composition administered parenterally per dose will be in the range of about 1 μ g protein/kg/day to 10mg protein/kg/day of patient body weight, although as noted above, this will be determined by therapeutic judgment. More preferably, this dose is at least 0.01mg protein/kg/day, and most preferably, for humans, between about 0.01 and 1mg protein/kg/day.

For trastuzumab, the following administration may be employed:

dosimetry and method of administration

The HER2 test was mandatory before initiating therapy. Herceptin treatment should only be initiated by a physician who is experienced in administering cytotoxic chemotherapy.

MBC

Daily schedule every three weeks

The recommended initial loading dose is 8mg/kg body weight. The recommended maintenance dose for the three week interval was 6mg/kg body weight, starting 3 weeks after the loading dose.

Daily schedule for each week

The recommended initial loading dose of herceptin is 4mg/kg body weight. The recommended weekly maintenance dose of herceptin is 2mg/kg body weight, starting 1 week after the loading dose.

Administration in combination with paclitaxel or docetaxel

In the pivot test (H0648g, M77001), paclitaxel or docetaxel was administered the day after the first dose of herceptin (for the dose, see summary of herceptin or docetaxel product characteristics) and immediately after the subsequent dose of herceptin (if the prior dose of herceptin was well tolerated).

Administered in combination with an aromatase inhibitor

In the pivot test (BO16216), herceptin and anastrozole (anastrozole) were administered from day 1 onward. There is no limit to the relative timing of the herceptin and anastrozole at the time of administration (for dose see summary of product characteristics for anastrozole or other aromatase inhibitors).

EBC

Daily schedule for every three weeks and every week

As a three week regimen, the recommended initial loading dose of herceptin is 8mg/kg body weight. The recommended maintenance dose for the three week interval of herceptin was 6mg/kg body weight, starting 3 weeks after the loading dose.

As a weekly regimen (initial loading dose of 4mg/kg, followed by 2mg/kg weekly), paclitaxel following chemotherapy with doxorubicin (doxorubicin) and cyclophosphamide (cyclophosphamide) was accompanied.

(for chemotherapy combination administration, see section 5.1).

MGC

Daily schedule every three weeks

Breast cancer (MBC and EBC) and gastric cancer (MGC)

Duration of treatment

Patients with MBC or MGC should be treated with herceptin until the disease progresses. Patients with EBC should be treated with herceptin for 1 year or until disease recurrence, whichever occurs first.

Dose reduction

No reduction of herceptin dose was performed during the clinical trial. Patients may continue therapy during the reversible chemotherapy-induced myelosuppression period, but should be carefully monitored for complications of neutropenia during this time. For information on dose reduction or delay, refer to the summary of product characteristics of paclitaxel, docetaxel or aromatase inhibitors.

Missed dose

If a patient misses a dose of herceptin for one week or less, the usual maintenance dose should be given as early as possible (weekly schedule: 2 mg/kg; every three weeks schedule: 6 mg/kg). Not waiting until the next planning cycle. The subsequent maintenance dose should then be given according to the previous schedule (weekly schedule: 2 mg/kg; every three weeks schedule: 6mg/kg, respectively).

If the patient misses a dose of herceptin for more than one week, a reload dose of herceptin should be given in about 90 minutes (weekly regimen: 4 mg/kg; every three weeks regimen: 8 mg/kg). Then, from this point on, the subsequent maintenance doses of herceptin (weekly protocol: weekly; every three weeks protocol: every 3 weeks, respectively: 2mg/kg for the weekly protocol; 6mg/kg for the three weeks protocol) should be administered.

Special patient population

Clinical data showed that the distribution of herceptin did not change with age or serum creatinine. In clinical trials, elderly patients did not receive reduced doses of herceptin. No special pharmacokinetic studies have been conducted in the elderly and in subjects with renal or hepatic injury. However, in population pharmacokinetic analysis, it was shown that age and kidney injury did not affect trastuzumab distribution.

Application method

The herceptin loading dose should be administered as a 90 minute intravenous infusion. It is not administered as an intravenous bolus or as a bolus. The herceptin intravenous infusion should be administered by a healthcare provider who is ready to manage allergies, and first aid kits should be available. Patients should be observed for symptoms (such as fever and chills or other infusion-related symptoms) at least 6 hours after the start of the first infusion and 2 hours after the start of the subsequent infusions (see sections 4.4 and 4.8). Interrupting or slowing the rate of infusion may help control such symptoms. Infusion may be continued after the symptoms have been alleviated.

If the initial loading dose is well tolerated, subsequent doses can be administered as a 30 minute infusion. The pharmaceutical compositions of the present invention may be administered parenterally.

Preferably, the pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier. By "pharmaceutically acceptable carrier" is meant any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary. As used herein, the term "parenteral" refers to modes of administration, which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intraarticular injection and infusion. Administration of the compositions provided herein can specifically include twice daily, every other day, every three days, every four days, every five days, once a week, once every two weeks, once every three weeks, once monthly, and the like.

Pharmaceutical compositions are also suitably administered by sustained release systems. Suitable examples of sustained-release compositions include semipermeable polymeric matrices in the form of shaped articles, for example films, or microcapsules. Sustained release matrices include polylactide (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid (Sidman, U.S. et al, Biopolymers 22:547- & 556(1983)), poly (2-hydroxyethylmethacrylate) (R.Langer et al, J.biomed.Mater.Res.15:167- & 277(1981), and R.Langer, chem.Tech.12:98-105(1982)), ethylene-vinyl acetate (R.Langer et al, supra), or poly-D- (-3-hydroxybutyric acid (EP 133,988). Sustained release pharmaceutical compositions also include liposome-entrapped compounds. The liposomes containing the pharmaceutical composition are prepared by methods known per se: DE 3,218,121; epstein et al, Proc.Natl.Acad.Sci. (USA)82: 3688-; hwang et al, Proc.Natl.Acad.Sci. (USA)77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP143,949; EP 142,641; japanese patent application 83-118008; U.S. patent nos. 4,485,045 and 4,544,545; and EP102,324. Typically, liposomes are of the small (about 200-800 angstroms) monolayer type, wherein the lipid content is greater than about 30mol.

For parenteral administration, the pharmaceutical compositions are generally formulated by mixing them in a unit dose injectable form (solution, suspension, or emulsion) in the desired purity with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to the recipient at the dosages and concentrations employed and is compatible with the other ingredients of the formulation.

In general, formulations are prepared such that the components of the pharmaceutical composition are in uniform and intimate contact with a liquid carrier or a finely divided solid carrier, or both. The product is then shaped, if necessary, into the desired formulation. Preferably, the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as are liposomes. Suitably, the carrier contains minor amounts of additives such as substances which enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or salts thereof; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) (poly) peptides, such as polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions, such as sodium; and/or a non-ionic surfactant, such as a polysorbate, poloxamer (poloxamer), or PEG.

The components of the pharmaceutical composition to be used for therapeutic administration must be sterile. Sterility is readily achieved by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Typically, the therapeutic component of the pharmaceutical composition is placed into a container having a sterile access port (e.g., an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle).

Typically, the components of the pharmaceutical composition will be stored in unit or multi-dose containers (e.g., sealed ampoules or vials), either as aqueous solutions or as lyophilized formulations for reconstitution. As an example of a lyophilized formulation, a 10ml vial was filled with 5ml of sterile filtered 1% (w/v) aqueous solution and the resulting mixture was lyophilized. Infusion solutions were prepared by reconstituting the lyophilized compound with bacteriostatic water for injection.

Comprising a modulator of the HER2/neu (ErbB2) signalling pathway, an inhibitor of programmed death ligand 1(PD-L1) and a chemotherapeutic agent (such as taxol or a taxol derivative, such as docetaxel) may be effected via simultaneous, sequential or separate administration of the individual components of the treatment) The cancer treatment provided herein. For example, one or more modulators of the HER2/neu (ErbB2) signaling pathway as defined herein (e.g., trastuzumab) may be administered concurrently with one or more inhibitors of the programmed death ligand 1(PD-L1) as defined herein (e.g., anti-PD-L1 antibodies as provided and described herein). It is contemplated herein that the sequential administration of a modulator of the HER2/neu (ErbB2) signaling pathway as defined herein, such as trastuzumab, may also be performed concurrently with one or more of the programmed death ligand 1(PD-L1) inhibitors defined herein, such as the anti-PD-L1 antibodies provided and described herein, to be used in accordance with the present invention. A modulator of the HER2/neu (ErbB2) signaling pathway as defined herein (e.g., trastuzumab) and one or more inhibitor of programmed death ligand 1(PD-L1) as defined herein (e.g., an anti-PD-L1 antibody as provided and described herein) may also be administered separately as defined herein. For example, one or more modulators of the HER2/neu (ErbB2) signaling pathway as defined herein (such as trastuzumab) may be administered in a first step, followed by administration of one or more programmed death ligand 1(PD-L1) inhibitors (such as the anti-PD-L1 antibodies provided and described herein) in a second step, and vice versa. Also, can be the same asThe chemotherapeutic agent is administered either concurrently, sequentially or separately. Included herein are modulators of the HER2/neu (ErbB2) signaling pathway, inhibitors of programmed death ligand 1(PD-L1), and chemotherapeutic agents (e.g., taxol or taxol derivatives, such as docetaxel) Simultaneously, sequentially or separately.

The cancer treatments provided herein, comprising a modulator of the HER2/neu (ErbB2) signaling pathway, a programmed death ligand 1(PD-L1) inhibitor, and a chemotherapeutic agent (e.g., taxol or a taxol derivative, such as docetaxel), can be applied as a monotherapy). However, it may also be applied together with (i.e. in further combination therapy with) one or more additional therapies (e.g. conventional therapies such as surgery, radiotherapy and/or one or more additional chemotherapeutic agents).

Surgery may comprise the step of partial or complete tumor resection prior to, during or after administration of a cancer treatment provided herein comprising a modulator of the HER2/neu (ErbB2) signaling pathway, a programmed death ligand 1(PD-L1) inhibitor and a chemotherapeutic agent (such as taxol or a taxol derivative, such as docetaxel)). Modulators of the HER2/neu (ErbB2) signaling pathway, programmed death ligand 1(PD-L1) inhibitors and chemotherapeutic agents (e.g., taxol or taxol derivatives, such as docetaxel) provided herein may be administered in a neoadjuvant or adjuvant setting, particularly neoadjuvant or adjuvant cancer therapy)。

Modulators of the HER2/neu (ErbB2) signaling pathway, chemotherapeutic agents, and inhibitors of programmed death ligand 1(PD-L1) may be administered in a neoadjuvant setting. Modulators of the HER2/neu (ErbB2) signaling pathway, chemotherapeutic agents, and programmed death ligand 1(PD-L1) inhibitors may be administered in an adjuvant setting or in a metastatic setting.

Thus, modulators of the HER2/neu (ErbB2) signaling pathway, programmed death ligand 1(PD-L1) inhibitors and chemotherapeutic agents (such as taxol or taxol derivatives, such as docetaxel) provided herein, may be administered to patients in need of such treatment during or after surgical intervention/resection of cancerous tissue). Thus, the present invention can be used for neoadjuvant therapy, i.e., treatment in which a patient/patient group in need thereof is administered the therapy provided herein prior to surgery. It can also be used for adjuvant therapy (i.e. after surgery).

Preferably, the chemotherapeutic agent to be used herein is a taxane (the term "taxol" is used interchangeably herein with "taxane") or a taxane derivative (taxol derivative), such as docetaxelOr a paclitaxel. Docetaxel is particularly preferred hereinThe use of (1).

The (further) chemotherapeutic agent may be one or more of the following exemplary non-limiting drugs or agents:

cisplatin, vinorelbine (Vinorelbin), Carboplatin (Carboplatin), paclitaxel, gemcitabine (Gemcitabin), docetaxel, Bevacizumab (Bevacizumab), Pemetrexed (Pemetrexed), etoposide (Etoposid), Irinotecan (Irinotecan), ifosfamide (Ifosfamid), Topotecan (Topotecan),

anti-angiogenic agents such as VEGF blockers (such as bevacizumab/Avastin or sutent (sunitinib malate) -SU-11248)), linoamine (linomide), integrin α v β 3 function inhibitors, angiostatin (angiostatin), propylenimine (razaxin), thalidomide (thalidomide), and including vascular targeting agents (such as combretastatin phosphate or N-acetyl colchicol-O-phosphate (N-acetyl colchinol-O-phosphate));

cytostatic agents, such as antiestrogens (e.g. tamoxifen (tamoxifen), toremifene (toremifene), raloxifene (raloxifene), droloxifene (droloxifene), indoxifene (iodoxyfene)), progestogens (progestens) (e.g. megestrol acetate (megestrol acetate)), aromatase inhibitors (e.g. anastrozole, letrozole (letrozole), vorozole (vorazole), exemestane (exemestane)), antiprogestins, antiandrogens (e.g. flutamide), nilutamide (nilutamide), bicalutamide (bicalutamide), cyproterone acetate (prosterone acetate)), rh agonists and antagonists (e.g. goserelin acetate (goserelinacetate), leuprolide (leuprolide)), testosterone 5 α -dihydrotestosterone inhibitors (e.g. renin-derived androgen acetate (e.g. inhibitors of the function of the growth factor (e.g. amaurokinase), and inhibitors of such as inhibitors of the growth factor (e.g. amaurokinase (e.g. inhibitors of the growth factor (e) including the growth factor (e.g. amaurokinase (amaurokinase), inhibitors of this kind of growth factor (e) and inhibitors such as well as inhibitors of growth factor (e, e.g. amaurokinase (amaurokinase), and inhibitors of growth factor (e Growth factors and hepatocyte growth factors, such inhibitors including growth factor antibodies, growth factor receptor antibodies, tyrosine kinase inhibitors, and serine/threonine kinase inhibitors);

biological response modifiers (e.g., interferons); antimetabolite agents (e.g., gemcitabine); anti-hormonal compounds such as anti-estrogens; antibodies (e.g., edrecolomab); adjuvant (anti) hormone therapy (i.e., therapy with an adjuvant (anti) hormone drug such as tamoxifen); gene therapy methods (e.g., antisense therapy); and/or immunotherapy methods.

Chemotherapy may also (additionally) include the use of one or more antiproliferative/anti-neobiological drugs and combinations thereof, as used in medical oncology, such as tyrosine kinase inhibitors, raf inhibitors, ras inhibitors, dual tyrosine kinase inhibitors, taxol, taxanes (such as paclitaxel or docetaxel), anthracyclines, such as doxorubicin (doxorubicin) or epirubicin (epirubicin), aromatase inhibitors (such as anastrozole or letrozole), and/or vinorelbine (vinorelbine); cyclophosphamide (cyclophosphamide), methotrexate (methotrexate) or fluorouracil (also known as 5-FU) can be used in such combination therapy individually or in the form of a combination therapy ("CMF therapy") comprising these three drugs, optionally in combination with any other therapy provided herein. Specific examples of chemotherapeutic agents for use with the combination therapies of the invention are pemetrexed (pemetrexed), raltitrexed (raltitrexed), etoposide (etoposide), vinorelbine, paclitaxel, docetaxel, cisplatin, oxaliplatin (oxaliplatin), carboplatin, gemcitabine, irinotecan (irinotecan) (CPT-11), 5-fluorouracil (5-FU (including capecitabine)), doxorubicin, cyclophosphamide, temozolomide, hydroxyurea, (iii) antiproliferative/anti-neobiological agents such as those used in medical oncology, and combinations thereof, such as antimetabolites (e.g., antifolates such as methotrexate, fluoropyrimidines (fluoropyrnidines) such as 5-fluorouracil, adenine and adenosine analogs, cytosine arabinoside); anti-tumor antibiotics (e.g., anthracyclines such as doxorubicin, daunorubicin, epirubicin and idarubicin, mitomycin-C, dactinomycin, mithramycin); platinum derivatives (e.g., cisplatin, carboplatin); alkylating agents (e.g., nitrogen mustards, melphalan (melphalan), chlorambucil (chlorambucil), busulfan (busulfan), cyclophosphamide (cyclophosphamide), ifosfamide (ifosfamide), nitrosoureas (nitrosureas), thiotepa (thiotepa)); antimitotic agents (e.g., vinca alkaloids such as vincristine and taxanes such as taxol, taxotere); topoisomerase inhibitors (e.g. epipodophyllotoxins such as etoposide and teniposide, amsacrine, topotecan, and also irinotecan); also enzymes (e.g., asparaginase); and thymidylate synthase inhibitors (e.g., raltitrexed); and other types of chemotherapeutic agents.

The inhibitor/modulator/chemotherapeutic agent used in accordance with the present invention is described herein and generally refers to known and/or commercially available inhibitor/modulator/chemotherapeutic agents. However, the use of known compounds for which inhibitors yet to be produced or for which inhibitory activity is to be tested is also covered in the context of the present invention.

In yet another aspect, the invention relates to the use of a nucleic acid or antibody capable of detecting expression levels of ER, PD-L1 and optionally IFN γ for determining the need of a patient for a combination therapy with a PD-L1 inhibitor in combination with a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent. A corresponding explanation of the terms has been given above and is suitable for the purposes of this document, mutatis mutandis.

Preferably, the nucleic acid (e.g., oligonucleotide) is about 15 to 100 nucleotides in length. Those skilled in the art are readily able to identify and/or prepare oligo-or polynucleotides capable of detecting expression levels of ER, PD-L1, and optionally IFN γ based on his general knowledge and the teachings provided herein. In particular, these nucleic acids (e.g., oligo or polynucleotides) can be used as probes in the methods described herein, e.g., in the measurement of expression levels. The skilled person will be aware of, for example, computer programs that can be used to identify the corresponding probes to be used herein. For example, a nucleic acid (or portion of a nucleic acid) encoding an estrogen receptor (e.g., SEQ ID NO:38), a nucleic acid (or portion of a nucleic acid) encoding PD-L1 (e.g., SEQ ID NO:42), and optionally a nucleic acid (or portion of a nucleic acid) encoding IFN γ (e.g., SEQ ID NO:44) can be used in this context to identify specific probes for detecting the expression levels of ER, PD-L1, and IFN γ, respectively. Exemplary nucleic acid sequences encoding ER, PD-L1 and IFN γ are available on corresponding databases (such as the NCBI database, world wide web in NCBI.

Furthermore, provided herein are compositions that are diagnostic compositions further optionally comprising means for detecting/determining/assessing ER, PD-L1 and IFN γ expression levels. Such means for detection are, for example, the nucleotides and/or antibodies described above. Thus, the present invention relates to such means (e.g. such nucleotides and/or antibodies) for the preparation of a diagnostic composition for the determination of patients in need of a PD-L1 inhibitor cotherapy.

In an alternative aspect, the present invention relates to such means for detecting (e.g. the nucleic acids and/or antibodies described above and/or the "binding molecules" described below in the context of a kit to be used according to the present invention) for determining a patient in need of a PD-L1 inhibitor cotherapy. Preferably, the invention relates to antibodies for use in identifying patients in need of PD-L1 inhibitor combination therapy.

Furthermore, the present invention also relates to a kit for carrying out the methods provided herein, the kit comprising a nucleic acid or antibody capable of detecting the expression level of ER, PD-L1, and optionally IFN γ. Also encompassed herein is the use of the kits described herein for carrying out the methods provided herein. The kits useful for practicing the methods and uses described herein may comprise oligonucleotides or polynucleotides capable of determining the expression level of ER, PD-L1, and optionally IFN γ. For example, the kit may comprise one or more compounds required for the specific measurement of ER, PD-L1 and optionally IFN γ expression levels. Furthermore, the present invention relates to the use of one or more compounds required for the specific measurement of ER, PD-L1 and optionally IFN γ expression levels for the preparation of a kit for carrying out the method or use of the invention. Based on the teachings of the present invention, the skilled person knows which compound/compounds are required for specific measurement of ER, PD-L1 and optionally IFN γ expression levels. For example, such compounds may be "binding molecules". In particular, such compounds may be one or more (nucleotide) probes, primers (pairs), antibodies and/or aptamers specific to the (gene) product of the ER gene/coding sequence, the PD-L1 gene/coding sequence and optionally the IFN γ/coding sequence. The kit of the invention (to be prepared in the context) may be a diagnostic kit.

The kit of the invention (to be prepared in the context) or the method and use of the invention may further comprise or be provided with a manual of useful instructions. For example, the instruction manual may instruct the skilled person (how) to determine the (reference/control) expression levels of ER, PD-L1 and optionally IFN γ or (how) to determine the patient's need for PD-L1 inhibitor therapy. In particular, the instruction manual may comprise instructions for using or applying the methods or uses provided herein. The kit of the invention (to be prepared in the context) may further comprise substances/chemicals and/or equipment suitable/required for carrying out the method and use of the invention. For example, such substances/chemicals and/or devices are solvents, diluents and/or buffers for stabilizing and/or storing the compounds required for specific measurement of ER, PD-L1 and optionally IFN γ expression levels.

As used herein, the terms "comprises" and "comprising," or grammatical variations thereof, are to be understood as specifying the recited features, integers, steps, or components, but do not preclude the addition of one or more individual features, integers, steps, components, or groups thereof. This term encompasses the terms "consisting of … …" and "consisting essentially of … …". Thus, the terms "comprising"/"including"/"having" mean that any additional component (or, similarly, feature, integer, step, etc.) may be present.

The term "consisting of … …" means that no other component (or, likewise, feature, integer, step, etc.) is present.

The term "consisting essentially of … …" or grammatical variations thereof as used herein is to be understood as specifying the recited features, integers, steps or components but does not preclude the addition of one or more individual features, integers, steps, components or groups thereof but only if the additional features, integers, steps, components or groups thereof do not materially alter the basic and novel characteristics of the claimed compositions, devices or methods. Thus, the term "consisting essentially of … …" means that certain other components (or, similarly, features, integers, steps, etc.) may be present, i.e., those that do not materially affect the essential characteristics of the composition, device, or method. In other words, the term "consisting essentially of … …" (which may be used interchangeably herein with the term "consisting essentially of") allows for the presence of additional components in a composition, device, or method other than the mandatory components (or likewise, features, integers, steps, etc.) so long as the essential features of the device or method are not substantially affected by the presence of the additional components.

The term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, biological and biophysical arts.

As used herein, the term "isolated" refers to a composition that has been removed from its in vivo location (e.g., an aquatic organism or moss). Preferably, the isolated composition of the invention is substantially free of other materials (e.g., other proteins that do not comprise an anti-adhesion effect) present in its in vivo location (i.e., purified or semi-purified).

As used herein, the term "about" means ± 10%.

The invention also relates to the following:

1. a method of determining the need for PD-L1 inhibitor combination therapy in a cancer patient,

(i) wherein a therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for the patient or (ii) wherein the patient is undergoing a therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising the steps of:

2. A method of treating cancer in a cancer patient for whom a therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an elevated expression level of programmed death ligand 1(PD-L1) as compared to a control, and administering to the patient an effective amount of a modulator of the HER2/neu (ErbB2) signaling pathway, a chemotherapeutic agent, and an inhibitor of programmed death ligand 1 (PD-L1).

3. A method of treating cancer in a cancer patient undergoing therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an elevated expression level of programmed death ligand 1(PD-L1) as compared to a control, and administering to the patient an effective amount of an inhibitor of programmed death ligand 1 (PD-L1).

4. A pharmaceutical composition for use in treating cancer comprising a modulator of the HER2/neu (ErbB2) signaling pathway and an inhibitor of programmed death ligand 1(PD-L1), wherein the cancer is determined to have a low or absent ER expression level and to have an elevated expression level of programmed death ligand 1(PD-L1) as compared to a control.

5. The pharmaceutical composition of item 4 for use in treating cancer, further comprising a chemotherapeutic agent.

6. The method of any one of items 1 to 3, further comprising measuring in vitro the expression level of interferon-gamma (IFN γ) in a sample from the patient, and determining that the patient is in need of PD-L1 inhibitor cotherapy if a reduced expression level of interferon-gamma (IFN γ) is measured as compared to a control.

7. The method of any one of items 1, 2,3 and 6; or items 4 and 5, wherein the ER expression level is ER (-).

8. The method of any one of items 1, 2,3, 6, and 7; or the pharmaceutical composition of any one of items 4,5 and 7, wherein the modulator of the HER2/neu (ErbB2) signaling pathway is the HER2 antibody herceptin/trastuzumab.

9. The method of any one of items 1, 2,3, 6,7 and 8; or the pharmaceutical composition of any one of items 5,7 and 8, wherein the chemotherapeutic agent is taxol or a taxol derivative.

10. The method of any one of items 1, 2,3, 6,7, and 8 to 9; or any one of items 4,5, and 7 to 9, wherein the programmed death ligand 1(PD-L1) inhibitor is an antibody that specifically binds to PD-L1 (anti-PD-L1 antibody).

11. The method of any one of items 1, 2,3, and 6 to 10; or any one of items 4,5, and 7 to 10, wherein the cancer is a solid cancer.

12. The method of item 11; the pharmaceutical composition of item 11 or item 11, wherein the solid tumor is breast cancer or gastric cancer.

13. The method of any one of items 1, 2,3, and 6 to 12; or any one of items 4,5, and 7 to 12, wherein the PD-L1 expression level is an mRNA expression level.

14. Use of a nucleic acid or antibody capable of detecting expression levels of ER, PD-L1 and optionally IFN γ for determining the need of a patient for combination therapy with a PD-L1 inhibitor in combination with a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent.

15. The method of any one of items 1, 2,3, and 6 to 14; or any one of items 4,5 and 7 to 14, wherein the modulator of the HER2/neu (ErbB2) signaling pathway, the chemotherapeutic agent, and the inhibitor of programmed death ligand 1(PD-L1) are to be administered in a neo-adjuvant setting.

The invention is further described by reference to the following non-limiting figures and examples. Unless otherwise indicated, established methods using recombinant genetic techniques, as described, for example, in Sambrook, Russell, "Molecular Cloning, Laboratory Manual," Cold Spring Harbor Laboratory, N.Y. (2001)), are incorporated herein by reference in their entirety.

Brief Description of Drawings

Fig. 1.

Figure 1 provides a schematic representation of the structure of HER2 protein, and the amino acid sequences of domains I-IV of its extracellular domain.

Fig. 2.

FIGS. 2A and 2B depict variable lightness (V) of murine monoclonal antibody 2C4_L) (FIG. 2A) and variable weight (V)_H) (FIG. 2B) domains (SEQ ID Nos. 5 and 6, respectively); variant 574/V of pertuzumab_LAnd V_HDomains (SEQ ID Nos. 7 and 8, respectively), and human V_LAnd V_HAlignment of the amino acid sequences of the consensus framework (hum κ 1, light κ subgroup I; humIII, heavy subgroup III) (SEQ ID Nos. 9 and 10, respectively). Asterisks identify differences between the variable domains of pertuzumab and murine monoclonal antibody 2C4 or between the variable domains of pertuzumab and the human framework. Complementarity Determining Regions (CDRs) are in parentheses.

Fig. 3.

Fig. 3A and 3B show the amino acid sequences of the pertuzumab light chain (fig. 3A) and heavy chain (fig. 3B). CDRs are shown in bold. The calculated molecular weights of the light and heavy chains were 23,526.22Da and 49,216.56Da (cysteine in reduced form). The carbohydrate module is attached to Asn 299 of the heavy chain.

Fig. 4.

Fig. 4A and 4B show the amino acid sequences of the trastuzumab light chain (fig. 4A) and heavy chain (fig. 4B), respectively. The boundaries of the variable light and variable heavy fields are indicated by arrows.

Fig. 5.

Fig. 5A and 5B depict a variant pertuzumab light chain sequence (fig. 5A) and a variant pertuzumab heavy chain sequence (fig. 5B), respectively.

Fig. 6.

The known mRNA transcript of gene CD274 and the location of the target region for AFFYMETRIX probe set. Exons are shown as gray bold rectangles and the junction regions are indicated by thin horizontal lines. The probe set is shown as a black bold rectangle, and their sequences are located against the mRNA sequence. The coordinates provided are genomic coordinates on chromosome 9.

Fig. 7.

Known mRNA transcripts of the gene IFNG and the location of the target region with respect to AFFYMETRIX probe set. Exons are shown as gray bold rectangles and the junction regions are indicated by thin horizontal lines. The probe set is shown as a black bold rectangle, and their sequences are located against the mRNA sequence. The coordinates provided are genomic coordinates on chromosome 12.

Fig. 8.

Distribution of expression of genes IFNG and CD274 in samples of ER-and ER + populations. The symbol type corresponds to the final pCR state (solid: pCR achieved, open: pCR not achieved).

Fig. 9.

A) Block diagram of expression of gene CD274 for ER-responders (pCR yes) and non-responders (pCR no). The right provides a bar chart of the representation of these two categories.

B) Distribution of t-test statistics (H0 assumed no difference). Vertical marks indicate the actual values found in the samples involved. The area of the shaded area corresponds to the alpha level.

Fig. 10.

A) Block diagram of the expression of the gene IFNG in ER-responders (pCR yes) and non-responders (pCR no). The right provides a bar chart of the representation of these two categories.

Fig. 11.

A) Block diagram of expression of gene CD274 for ER + responders (pCR yes) and non-responders (pCR no). The right provides a bar chart of the representation of these two categories.

Fig. 12.

A) Block diagram of the expression of the gene IFNG in ER + responders (pCR yes) and non-responders (pCR no). The right provides a bar chart of the representation of these two categories.

FIG. 13.

Recipient operating characteristics of the final logistic regression model of the ER-population. A positive level is considered a positive response status (pCR — yes).

Fig. 14.

LIFT curves of the final logistic regression model of the ER population. A positive level is considered a positive response status (pCR — yes). The Y-axis shows how much the fraction of the population is compared to the ratio of the response level as a whole in the selected response level (the upper curve corresponds to pCR ═ yes).

Fig. 15.

One example of a predicted clinical response status of an ER-population. Shown is a predicted response profile, governed by patient age, cancer type, pN status, and expression of the two genes involved. The actual predicted pCR probability (which equals 0.443) for N0LABC patients around 60 years old and with expression of both genes around the median level is given.

Fig. 16.

Recipient operational characteristics of the final logistic regression model of the ER + population. A positive level is considered a positive response status (pCR — yes).

Fig. 17.

ER-the age distribution of patients.

Fig. 18.

Age distribution of ER + patients.

Fig. 19.

Decision tree view of IFNG and CD274 gene expression in ER-patients predicting clinical response. The first two splits required to explain pCR are for IFNG and CD 274.

Examples

Example 1: if the expression level of ER is low or absent (ER-negative) and if the expression level of PD-L1 is elevated Cancer patients undergoing HER2 targeted therapy and chemotherapy benefit from a PD-L1 inhibitor combination therapy

The assessment of gene expression was carried out with the aid of the R Bioconductor package "affy", R version 2.15.0. All heuristic analysis and prediction models were generated using SASJMP version 10.0.

48 HER2+, ER + and 39 HER2+, ER-breast cancer biopsies were obtained from a NeoSphere clinical trial. Samples have been taken at diagnosis from patients who were later treated with docetaxel and trastuzumab in a neoadjuvant setting. The distribution of the main clinical covariates at baseline and clinical responses in the implicated population (as assessed at surgery) was as follows:

ER negative samples:

patient age (see FIG. 17)

Quantile

Cancer type

pT (pathological staining of tumor)

pN (pathological staining of knots)

G (grade)

ER positive samples:

patient age (see fig. 18)

Quantile

Cancer type

pT

pN

G

Tandem analysis of the complete response to pathology (pCR) by Estrogen receptor status (ER)

Gene expression profiling

Tumor biopsies were profiled for gene expression on the AFFYMETRIX HG-U133Plus 2 whole human genome microarray platform. Roche HighPure RNA extraction, NuGen amplification and standard AFFYMETRIX hybridization and scanning protocols were used. All array scans passed standard AFFYMETRIX QC.

The original signal was preprocessed using a robust multi-array algorithm (RMA) (Irizarry et al, 2003.http:// www.ncbi.nlm.nih.gov/pubmed/12925520). All probe sets available for the gene of interest were taken as reported below. For gene CD274, the probe set with the highest mean expression value (defined as the arithmetic mean of the expression of a given probe set) was selected to represent the gene when several probe sets were available to represent the gene:

CD274 (PDL1)

223834_ at, selected for PD-L1

227458_at

IFNG

210354_at

FIG. 8 shows the combined distribution of the expression of the above genes in samples of both the ER-and ER + populations. The symbol type corresponds to the final pCR state (solid: pCR achieved, open: pCR not achieved).

For more details on the distribution of CD274 and IFNG expression between ER and pCR layers, see appendix I.

For each ER subpopulation, a logistic regression model was constructed that correlated the expression of selected genes with the clinical response modulated for patient age, cancer type, and node status:

response-patient age + cancer type + pN + CD274+ IFNG

ER-populations.

The summarized model output is given below. The Odds Ratio (OR) per unit change of biomarker values is given. Given expression values on the log2 scale, a1 unit change would correspond to 2-fold overexpression. See the appendix for details.

The final model used to predict the probability of a particular patient responding to treatment includes CD274 and IFNG expression and looks like:

p(pCR)＝-3.737+1.607*CD274-1.069*IFNG

ER + population.

The effect of PDL1 expression was evident in the ER-subpopulation of HER2+ breast cancer patients undergoing combined treatment with trastuzumab and chemotherapy in a neoadjuvant setting. That is, overexpression of PDL1 at diagnosis corresponds to a lower response rate to neoadjuvant therapy (i.e., a lower response rate to the combination therapy of trastuzumab and chemotherapy). This is true regardless of the patient's age, type of cancer, or lymph node status. Baseline assessment of gene expression of either of the two biomarkers PDL1 and INFG allowed identification of whether a patient is likely to experience greater benefit if PD-L1 targeted therapy was added to trastuzumab and chemotherapy.

The following relates to cut-off values that allow a patient to be determined in accordance with the present invention as being in need of a PD-L1 inhibitor combination therapy.

If the gene expression analysis gave IFNG expression results higher than or equal to 4.8, no combination therapy (HER2 targeting and PD-L1 targeting) was recommended and further PD-L1 assessment would not be necessary. If gene expression analysis gives an IFNG result below 4.8, then parallel assessment of PD-L1 is necessary. If the PD-L1 gene expression analysis then gave results higher than or equal to 5.3, a combination therapy (HER2 targeting and PDL1 targeting) was recommended (see fig. 19).

Appendix I

ER-subgroup

Single factor analysis of CD274 expression by pCR ER ═ ER negativity (see FIG. 9A)

t test

Yes-no

Assuming unequal variances

The results are also shown in fig. 9B.

Single factor analysis of IFNG expression by pCR ER ═ ER negative

The results are shown in fig. 10A.

t test

Yes-no

Assuming unequal variances

The results are shown in fig. 10B.

ER + subgroup

Single factor analysis of CD274 expression by pCR ER ═ ER positive

The results are shown in fig. 11A.

t test

Yes-no

Assuming unequal variances

The results are shown in fig. 11B.

Single factor analysis of IFNG expression by pCR ER ═ ER positive

The results are shown in fig. 12A.

t test

Yes-no

Assuming unequal variances

The results are shown in fig. 12B.

Appendix II

Nominal logical fit of pCR ER ═ ER negative

Gradient convergence, 5 repeats

Full model inspection

Parameter estimation

Logarithmic yield for no/yes

Effect likelihood ratio test

Yield ratio

pCR yield for No vs. Yes

The check and confidence intervals for the yield ratio are based on likelihood ratios.

Specific yield ratio

Per unit change of regression

Odds ratio of cancer types

Yield ratio of pN

Recipient operating feature

(see FIG. 13)

Positive levels were determined using pCR ═ YES-

AUC

0.79718

Confusion matrix

In fact

Predicted

Whether to practice yes or not

NO 225

Is 615

Curve of lift force

(see FIG. 14)

pCR

-no

-is

Prediction order analyzer (Prediction Profiler)

(see FIG. 15)

Nominal logical fit of pCR ER ═ ER positive

Gradient convergence, 19 repeats

Full model inspection

Lack of fit

Parameter estimation

Logarithmic yield for no/yes

Effect likelihood ratio test

Yield ratio

pCR yield for No vs. Yes

Specific yield ratio

Per unit change of regression

Odds ratio of cancer types

Yield ratio of pN

Recipient operating feature

(see FIG. 16)

Positive levels were determined using pCR ═ YES-

AUC

0.77273

Confusion matrix

In fact

Predicted

Whether to practice yes or not

NO 321

Is 60

The present invention relates to the following nucleotide and amino acid sequences:

the sequences provided herein are available in the NCBI database in particular and disclosed in WO2010/077634, and can be obtained from the world wide web in NCBI. db ═ gene; these sequences also relate to annotated and modified sequences. The invention also provides techniques and methods in which homologous sequences and variants of the conciseness sequences provided herein are used.

1-21 define an anti-PD-L1 antibody to be used according to the invention. SEQ ID NOS: 1-21 are shown in the sequence listing.

SEQ ID Nos. 22 to 37 show the sequence of the amino acid sequence of domains I-IV of the HER2 protein (SEQ ID Nos. 22 to 25, see also FIG. 1) and the sequence of an anti-HER 2 antibody (SEQ ID Nos. 26 to 37, see also FIGS. 2 to 5).

SEQ ID No.26：

Variable lightness (V) of murine monoclonal antibody 2C4 (SEQ ID Nos. 5 and 6, respectively)_L) (FIG. 2A) the amino acid sequence of the domain, as shown in FIG. 2.

SEQ ID No.27：

Variable weight (V) of murine monoclonal antibody 2C4_H) (FIG. 2B) the amino acid sequence of the domain, as shown in FIG. 2. SEQ ID No. 28:

variant 574/variable lightness of pertuzumab (V)_L) (FIG. 2A) the amino acid sequence of the domain, as shown in FIG. 2. SEQ ID No. 29:

variant 574/variable weight (V) of pertuzumab_H) (FIG. 2B) the amino acid sequence of the domain, as shown in FIG. 2. SEQ ID No. 30:

human V_LConsensus framework (hum κ 1, light κ subgroup I; humIII, heavy subgroup III), as shown in FIG. 2. SEQ ID No. 31:

human V_HConsensus framework (hum κ 1, light κ subgroup I; humIII, heavy subgroup III), as shown in FIG. 2. SEQ ID No. 32:

the amino acid sequence of pertuzumab light chain, as shown in fig. 3A.

SEQ ID No.33：

The amino acid sequence of pertuzumab heavy chain, as shown in fig. 3B.

SEQ ID No.34：

The amino acid sequence of the trastuzumab light chain domain as shown in fig. 4A. The boundaries of the variable light field are indicated by arrows.

SEQ ID No.35：

The amino acid sequence of trastuzumab heavy chain as shown in fig. 4B. The boundaries of the variable weight domain are indicated by arrows. SEQ ID No. 36:

amino acid sequence of variant pertuzumab light chain sequence (fig. 5A).

SEQ ID No.37：

Amino acid sequence of variant pertuzumab heavy chain sequence (fig. 5B).

SEQ ID NO.38

Nucleotide sequence encoding human Progesterone Receptor (PR)

NCBI reference sequence: NC _000011.9

C101000544-100900355 human chromosome 11, grch37.p10 primary assembly SEQ id No. 39:

amino acid sequence of human Progesterone Receptor (PR)

PRGR _ HUMAN length: type 15:10, 12/h/07/h in 9332012: checking by P: 6067. SEQ ID No.40:

nucleotide sequence encoding human Estrogen Receptor (ER)

(NM_000125.3)

SEQ ID NO.41：

Nucleotide sequence encoding human Estrogen Receptor (ER)

NCBI reference sequence: NC _000006.11

(> gi |224589818:152011631-

SEQ ID No.42：

Amino acid sequence of human Estrogen Receptor (ER)

>ENST00000206249_6

SEQ ID No.43：

Nucleotide sequence encoding human programmed death ligand 1(PD-L1)

NCBI reference sequence: NC _000009.11

(> gi 224589821:5450503-5470567 human chromosome 9, GRCh37.p10 initial assembly

SEQ ID NO.44：

Nucleotide sequence encoding human programmed death ligand 1(PD-L1) (CD274), transcript variant 1, mRNA

NCBI reference sequence: NM _014143.3

(> gi |292658763| ref | NM — 014143.3| human CD274 molecule (CD274), transcript variant 1, mRNA

SEQ ID No.45：

Amino acid sequence of human programmed death ligand 1(PD-L1) (programmed cell death 1 ligand 1 isoform a precursor [ human ])

NCBI reference sequence: NP-054862.1

(> gi 7661534| ref | NP-054862.1 | programmed cell death 1 ligand 1 isoform a precursor [ human ]

SEQ ID No.46：

Nucleotide sequence encoding human programmed death ligand 1(PD-L1) (CD274), transcript variant 2, mRNA

NCBI reference sequence: NM _001267706.1

(> gi |390979638| ref | NM — 001267706.1| human CD274 molecule (CD274), transcript variant 2, mRNA

SEQ ID No.47:

Amino acid sequence of human programmed death ligand 1(PD-L1) (programmed cell death 1 ligand 1 isoform b precursor [ human ])

NCBI reference sequence: NP-001254635.1

(> gi |390979639| ref | NP _001254635.1| pro-b isoform b precursor of programmed cell death 1 ligand 1 [ human ] SEQ ID No.48:

nucleotide sequence encoding human programmed death ligand 1(PD-L1) (human CD274 molecule (CD274), transcript variant 3, non-coding RNA)

NCBI reference sequence: NR _052005.1

(> gi |390979640| ref | NR _052005.1| human CD274 molecule (CD274), transcript variant 3, non-coding RNA

SEQ ID No.49:

Nucleotide sequence encoding human interferon gamma (human chromosome 12, GRCh37.p10 primary assembly)

NCBI reference sequence: NC _000012.11

(> gi 224589803: c68553521-68548550 human chromosome 12, GRCh37.p10 initial assembly

SEQ ID No.50:

Nucleotide sequence for coding human interferon gamma, mRNA

NCBI reference sequence: NM _000619.2

(> gi |56786137| ref | NM-000619.2 | human interferon, γ (IFNG), mRNA

SEQ ID No.51：

Amino acid sequence of human interferon gamma, precursor of interferon gamma [ human ]

NCBI reference sequence: NP-000610.2

(> gi |56786138| ref | NP-000610.2 | Interferon gamma precursor [ human ]

All publications cited herein are incorporated by reference in their entirety. Having now fully described this invention, it will be appreciated by those skilled in the art that the invention can be carried out within a wide and equivalent range of conditions, parameters, and the like, without affecting the spirit or scope of the invention or any embodiment thereof.

Claims

1. Use of a nucleic acid or antibody capable of detecting expression levels of Estrogen Receptor (ER) and programmed death ligand 1(PD-L1) for the preparation of a reagent or kit for use in a method of determining the need for PD-L1 inhibitor combination therapy in a cancer patient, (i) wherein therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for the patient or (ii) wherein the patient is undergoing therapy comprising a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising the steps of:

2. The use of claim 1, wherein the method further comprises measuring in vitro the expression level of interferon-gamma (IFN γ) in a sample from the patient, and determining that the patient is in need of PD-L1 inhibitor cotherapy if a reduced expression level of interferon-gamma (IFN γ) is measured as compared to a control.

3. Use according to claim 1 or 2, wherein the ER expression level is ER (-).

4. Use according to any one of claims 1 to 3, wherein the modulator of the HER2/neu (ErbB2) signaling pathway is a HER shedding inhibitor.

5. The use of claim 4 wherein the HER shedding inhibitor is a HER2 shedding inhibitor.

6. The use of claim 4 or 5 wherein the inhibitor of HER shedding inhibits HER heterodimerization or HER homodimerization.

7. The use of any one of claims 4 to 6 wherein the inhibitor of HER shedding is an anti-HER antibody.

8. The use of claim 7 wherein the anti-HER antibody binds to a HER receptor selected from the group consisting of: EGFR, HER2, and HER 3.

9. The use of claim 8 wherein the anti-HER antibody is an anti-HER 2 antibody that binds HER 2.

10. The use of claim 9, wherein the anti-HER 2 antibody binds to subdomain IV of HER2 extracellular domain.

11. The use of claim 9 or 10, wherein the anti-HER 2 antibody is Herceptin (Herceptin)/Trastuzumab (Trastuzumab).

12. The use of any one of claims 1 to 3, wherein the modulator of the HER2/neu (ErbB2) signaling pathway is a HER signaling inhibitor.

13. The use of any one of claims 1 to 3, wherein the modulator of the HER2/neu (ErbB2) signaling pathway is a HER dimerization inhibitor.

14. The use of claim 13 wherein the HER dimerization inhibitor is a HER2 dimerization inhibitor.

15. The use of claim 13 or 14 wherein the HER dimerization inhibitor inhibits HER heterodimerization or HER homodimerization.

16. The use of any one of claims 13 to 15 wherein the HER dimerization inhibitor is an anti-HER antibody.

17. The use of claim 16 wherein the anti-HER antibody binds to a HER receptor selected from the group consisting of: EGFR, HER2, and HER 3.

18. The use of claim 17 wherein the anti-HER antibody is an anti-HER 2 antibody that binds HER 2.

19. The use of claim 18, wherein the anti-HER 2 antibody binds to domain II of HER2 extracellular domain.

20. The use of claim 19, wherein the anti-HER 2 antibody binds to the junction between domains I, II and III of the HER2 extracellular domain.

21. The use of claim 18 or 20, wherein the anti-HER 2 antibody is Pertuzumab (Pertuzumab).

22. The use of any one of claims 1 to 21, wherein the chemotherapeutic agent is taxol or a taxol derivative.

23. The use of claim 22, wherein the taxol derivative is docetaxel (dodetaxel).

24. The use of any one of claims 1 to 23, wherein the programmed death ligand 1(PD-L1) inhibitor is an anti-PD-L1 antibody that specifically binds PD-L1.

25. The use of claim 24, wherein the anti-PD-L1 antibody comprises a heavy chain variable region polypeptide comprising HVR-H1, HVR-H2, and HVR-H3 sequences, wherein:

(a) the HVR-H1 sequence is GFTFSX1SWIH (SEQ ID NO: 1);

(b) the HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG (SEQ ID NO: 2);

(c) the HVR-H3 sequence is RHWPGGFDY (SEQ ID NO: 3);

further, wherein: x1 is D or G; x2 is S or L; x3 is T or S.

26. The use of claim 25, wherein X1 is D; x2 is S and X3 is T.

27. The use of claim 25, wherein the heavy chain variable region polypeptide further comprises a heavy chain variable region framework sequence juxtaposed between HVRs according to the formula: (HC-FR1) - (HVR-H1) - (HC-FR2) - (HVR-H2) - (HC-FR3) - (HVR-H3) - (HC-FR 4).

28. The use of claim 27, wherein the framework sequence is derived from a human consensus framework sequence.

29. The use of claim 28, wherein the framework sequence is a VH subgroup III consensus framework.

30. The use of claim 29, wherein one or more of the framework sequences is as follows:

HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:4)

HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO:5)

HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:6)

HC-FR4 is WGQGTLVTVSA (SEQ ID NO: 7).

31. The use of claim 25, wherein the heavy chain variable region polypeptide is in combination with a light chain variable region polypeptide comprising HVR-L1, HVR-L2 and HVR-L3, wherein:

(a) the HVR-L1 sequence is RASQX4X5X6TX7X8A (SEQ ID NO: 8);

(b) the HVR-L2 sequence is SASX9LX10S, and (SEQ ID NO: 9);

(c) the HVR-L3 sequence is QQX11X12X13X14PX15T (SEQ ID NO: 10);

further, wherein: x4 is D or V; x5 is V or I; x6 is S or N; x7 is A or F; x8 is V or L; x9 is F or T; x10 is Y or A; x11 is Y, G, F, or S; x12 is L, Y, F or W; x13 is Y, N, A, T, G, F or I; x14 is H, V, P, T or I; x15 is A, W, R, P or T.

32. The use of claim 31, wherein X4 is D; x5 is V; x6 is S; x7 is A; x8 is V; x9 is F; x10 is Y; x11 is Y; x12 is L; x13 is Y; x14 is H; x15 is A.

33. The use of claim 31, wherein the light chain variable region polypeptide further comprises a light chain variable region framework sequence juxtaposed between HVRs according to the formula: (LC-FR1) - (HVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR 4).

34. The use of claim 33, wherein the framework sequence is derived from a human consensus framework sequence.

35. The use of claim 33, wherein the framework sequence is a VL kappa I consensus framework.

36. The use of claim 35, wherein one or more of the framework sequences is as follows:

LC-FR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 11);

LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO: 12);

LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 13);

LC-FR4 is FGQGTKVEIKR (SEQ ID NO: 14).

37. The use of claim 25, wherein the anti-PD-L1 antibody comprises a heavy chain variable region sequence and a light chain variable region sequence, wherein:

(a) the heavy chain variable region sequence comprises HVR-H1, HVR-H2, and HVR-H3, wherein further:

(i) the HVR-H1 sequence is GFTFSX1SWIH (SEQ ID NO: 1);

(ii) the HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG (SEQ ID NO: 2);

(iii) the HVR-H3 sequence is RHWPGGFDY, and (SEQ ID NO: 3);

(b) the light chain variable region sequence comprises HVR-L1, HVR-L2 and HVR-L3, wherein further:

(iv) the HVR-L1 sequence is RASQX4X5X6TX7X8A (SEQ ID NO: 8);

(v) the HVR-L2 sequence is SASX9LX10S (SEQ ID NO: 9);

(vi) the HVR-L3 sequence is QQX11X12X13X14PX15T (SEQ ID NO: 10);

wherein: x1 is D or G; x2 is S or L; x3 is T or S; x4 is D or V; x5 is V or I; x6 is S or N; x7 is A or F; x8 is V or L; x9 is F or T; x10 is Y or A; x11 is Y, G, F, or S; x12 is L, Y, F or W; x13 is Y, N, A, T, G, F or I; x14 is H, V, P, T or I; x15 is A, W, R, P or T.

38. The use of claim 37, wherein X1 is D; x2 is S and X3 is T.

39. Use according to claim 37, wherein X4-D, X5-V, X6-S, X7-a and X8-V, X9-F, and X10-Y, X11-Y, X12-L, X13-Y, X14-H and X15-a.

40. Use according to claim 37, wherein X1 ═ D, X2 ═ S and X3 ═ T, X4 ═ D, X5 ═ V, X6 ═ S, X7 ═ a and X8 ═ V, X9 ═ F, and X10 ═ Y, X11 ═ Y, X12 ═ L, X13 ═ Y, X14 ═ H and X15 ═ a.

41. The use of any one of claims 37 to 40, wherein the anti-PD-L1 antibody further comprises:

(a) a heavy chain variable region framework sequence juxtaposed between HVRs according to the formula: (HC-FR1) - (HVR-H1) - (HC-FR2) - (HVR-H2) - (HC-FR3) - (HVR-H3) - (HC-FR4), and

(b) a light chain variable region framework sequence juxtaposed between HVRs according to the formula: (LC-FR1) - (HVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR 4).

42. The use of claim 41, wherein the heavy chain variable region framework sequence and the light chain variable region framework sequence are derived from a human consensus framework sequence.

43. The use of claim 42, wherein the heavy chain variable region framework sequence is a VH subgroup III consensus framework.

44. The use of claim 43, wherein one or more of the framework sequences is as follows:

HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 4);

HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO: 5);

HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 6);

HC-FR4 is WGQGTLVTVSA (SEQ ID NO: 7).

45. The use of claim 42, wherein the light chain variable region framework sequence is a VL kappa I consensus framework.

46. The use of claim 45, wherein one or more of the framework sequences is as follows:

LC-FR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 11);

LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO: 12);

LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC, and (SEQ ID NO: 13);

LC-FR4 is FGQGTKVEIKR (SEQ ID NO: 14).

47. The use of claim 42, wherein:

(a) the heavy chain variable region framework sequence is as follows:

(i) HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 4);

(ii) HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO: 5);

(iii) HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 6);

(iv) HC-FR4 is WGQGTLVTVSA; and (SEQ ID NO: 7);

(b) the light chain variable region framework sequence is as follows:

(i) LC-FR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 11);

(ii) LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO: 12);

(iii) LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 13);

(iv) LC-FR4 is FGQGTKVEIKR (SEQ ID NO: 14).

48. The use of claim 47, wherein the anti-PD-L1 antibody further comprises a human constant region.

49. The use of claim 48, wherein the constant region is selected from the group consisting of: IgG1, IgG2, IgG3, and IgG 4.

50. The use of claim 49, wherein the constant region is IgG 1.

51. The use of claim 47, wherein the anti-PD-L1 antibody further comprises a murine constant region.

52. The use of claim 51, wherein the constant region is selected from the group consisting of: IgG1, IgG2A, IgG2B, and IgG 3.

53. The use of claim 52, wherein the constant region is IgG 2A.

54. The use of claim 49 or 52, wherein the anti-PD-L1 antibody has reduced or minimal effector function.

55. The use of claim 54, wherein said minimal effector function results from effector-reducing Fc mutations.

56. The use of claim 55, wherein the effector-reducing Fc mutation is N297A.

57. The use of claim 55, wherein the effector reducing Fc mutation is D265A and/or N297A.

58. The use of claim 54, wherein the minimal effector function is derived from aglycosylation (aglycosylation).

59. The use of claim 25, wherein the antibody comprises a heavy chain variable region sequence and a light chain variable region sequence, wherein:

(a) the heavy chain variable region sequence comprises HVR-H1, HVR-H2, and HVR-H3, which have at least 85% overall sequence identity to GFTFSDSWIH (SEQ ID NO:15), AWISPYGGSTYYADSVKG (SEQ ID NO:16), and RHWPGGFDY (SEQ ID NO:3), respectively, and

(b) the light chain variable region sequence comprises HVR-L1, HVR-L2, and HVR-L3, which have at least 85% overall sequence identity to RASQDVSTAVA (SEQ ID NO:17), SASFLYS (SEQ ID NO:18), and QQYLYHPAT (SEQ ID NO:19), respectively.

60. The use of claim 59, wherein said sequence identity is at least 90%.

61. The use of claim 60, wherein the anti-PD-L1 antibody further comprises:

(a) a heavy chain variable region (VH) framework sequence juxtaposed between HVRs according to the formula: (HC-FR1) - (HVR-H1) - (HC-FR2) - (HVR-H2) - (HC-FR3) - (HVR-H3) - (HC-FR4), and

(b) a light chain variable region (VL) framework sequence juxtaposed between HVRs according to the formula: (LC-FR1) - (HVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR 4).

62. The use of claim 61, wherein the anti-PD-L1 antibody further comprises VH and VL framework sequences derived from a human consensus sequence.

63. The use of claim 62, wherein the VH framework sequence is derived from Kabat subgroup I, II, or a III sequence.

64. The use of claim 63, wherein the VH framework sequence is Kabat subgroup III consensus framework sequence.

65. The use of claim 64, wherein the VH framework sequence is as follows:

HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 4);

HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO: 5);

HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 6);

HC-FR4 is WGQGTLVTVSA (SEQ ID NO: 7).

66. The use of claim 62, wherein the VL framework sequence is derived from a Kabat kappa I, II, III or IV subgroup sequence.

67. The use of claim 66, wherein the VL framework sequence is a Kabat kappa I consensus framework sequence.

68. The use of claim 67, wherein the VL framework sequence is as follows:

LC-FR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 11);

LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO: 12);

LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 13);

LC-FR4 is FGQGTKVEIKR (SEQ ID NO: 14).

69. The use of claim 25, wherein the anti-PD-L1 antibody comprises a heavy chain variable region sequence and a light chain variable region sequence, wherein:

(a) the heavy chain variable region sequence has at least 85% sequence identity to a heavy chain variable region sequence selected from the group consisting of:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO:20), and

(b) the light chain variable region sequence has at least 85% sequence identity to the following light chain variable region sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(SEQ ID NO:21)。

70. the use of claim 69, wherein the sequence identity is at least 90%.

71. The use of claim 25, wherein the anti-PD-L1 antibody comprises a heavy chain variable region sequence and a light chain variable region sequence, wherein:

(a) the heavy chain variable region sequence comprises the sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO:20), and

(b) the light chain variable region sequence comprises the sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 21).

72. The use of any one of claims 1 to 71, wherein the cancer is a solid cancer.

73. The use of claim 72, wherein the solid cancer is breast cancer or gastric cancer.

74. The use of claim 72, wherein the solid cancer is breast cancer.

75. The use of any one of claims 1 to 74, wherein the expression level of PD-L1, as determined by conventional methods, is greater than or equal to 5.3.

76. The use of claim 75, wherein the level of PD-L1 expression is determined by Affymetrix.

77. The use of any one of claims 1 to 76, wherein the PD-L1 expression level is an mRNA expression level.

78. The use of claim 77, wherein the mRNA expression level of PD-L1 is assessed by in situ hybridization, microarray, or real-time PCR.

79. The use of any one of claims 1 to 75, wherein the PD-L1 expression level is a protein expression level.

80. The use of claim 79, wherein the protein expression level of PD-L1 is assessed by an immunoassay.

81. The use of claim 79, wherein said protein expression level of PD-L1 is assessed by a gel or blot-based method.

82. The use of claim 79, wherein the protein expression level of PD-L1 is assessed by IHC.

83. The use of claim 79, wherein the protein expression level of PD-L1 is assessed by mass spectrometry.

84. The use of claim 79, wherein the protein expression level of PD-L1 is assessed by flow cytometry.

85. The use of claim 79, wherein the protein expression level of PD-L1 is assessed by FACS.

86. The use of any one of claims 1 to 85, wherein the patient to be treated is a human.

87. Use of a nucleic acid or antibody capable of detecting the expression levels of ER, PD-L1 and optionally IFN γ for the preparation of a reagent or kit for determining the need of a patient for a PD-L1 inhibitor cotherapy in combination with a modulator of the HER2/neu (ErbB2) signaling pathway and a chemotherapeutic agent.

88. A kit useful for performing the method of any one of claims 1 to 86, comprising a nucleic acid or antibody capable of detecting the expression level of ER and PD-L1.

89. The kit of claim 88, further comprising a nucleic acid or antibody capable of detecting the expression level of IFN γ.

90. The use of any one of claims 1 to 86, wherein said modulator of the HER2/neu (ErbB2) signaling pathway, said chemotherapeutic agent and said inhibitor of programmed death ligand 1(PD-L1) are to be administered in a neoadjuvant setting or an adjuvant setting or a metastatic setting.