The present invention relates to the field of medicine. More particularly, the present invention relates to bispecific antibodies that bind human programmed cell death 1 ligand 1 (PD-L1) and T-cell immunoglobulin- and mucin-domain-containing protein-3 (TIM-3), and may be useful for treating solid and hematological tumors alone and in combination with chemotherapy and other cancer therapeutics.

Tumor cells escape detection and elimination by the immune system through multiple mechanisms. Immune checkpoint pathways are used in maintenance of self-tolerance and control of T cell activation, but cancer cells can use the pathways to suppress the anti-tumor response and prevent their destruction.

The PD-L1/human programmed cell death 1 (PD-1) pathway is one such immune checkpoint. Human PD-1 is found on T cells and PD-L1 is aberrantly expressed by a variety of tumor types; binding of PD-L1 to PD-1 inhibits T cell proliferation and cytokine production. The PD-1/PD-L1 inhibitory axis has been subjugated by tumors as part of the natural selective process that shapes tumor evolution in the context of an anti-tumor immune response.

TIM-3 is a checkpoint receptor identified to play a key role in the functional suppression of exhausted T-cells. T cells recognizing tumor antigens can be isolated from patients and mouse models, but such cells can exhibit an exhausted phenotype characterized by impairment in cytotoxic functions, effector cytokine production, and proliferation. These T cells can express high levels of the checkpoint regulator TIM-3.

While therapeutic targeting of the PD-1/PD-L1 pathway is clinically validated, there is a variable objective response in cancer patients of between 9-45% in PD-L1-negative and PD-L2-high-positive tumors, and many patients do not achieve durable response. Combined blockade of two or more checkpoint receptors may increase the frequency of objective responses and achieve efficacy in cancer patients who may be refractory to monotherapy with a PD-1 or PD-L1 blocking antibody.

In vitro treatment of tumor infiltrating lymphocytes harvested from CT26 tumor bearing mice with two antibodies, an anti-mouse TIM-3 antibody that can interact with T cells and an anti-mouse PD-L1 antibody that can separately interact with the mouse tumor cells, resulted in more effective control of mouse tumor growth than targeting either pathway alone (Sakuishi et. al. JEM 2010, 207: 2187 and WO 2015/048312).

An oral, small molecule that antagonizes PD-L1 and TIM-3 has been reported (Sasikumar et al., AACR; Cancer Res 2016; 76(14 Suppl):Abstract 4861). This small molecule compound was reported to demonstrate efficacy in syngenic mouse tumor models.

There remains a need to provide compounds that can bind and inhibit both human PD-L1 and human TIM-3, but also have the target specificity and pharmacokinetics of an antibody. In particular, there remains a need to provide heterodimeric bispecific antibodies that bind both human PD-L1 and human TIM-3 where the heterodimeric bispecific antibody enables pairing of two different heavy chains and two different light chains into a single IgG-like antibody. Further, there is a need for the bispecific antibody to resemble native antibody structure as measured by modeling, mass spectrometry, and stability analyses for purposes such as low immunogenicity, stable in vivo pharmacokinetics, and adequate stability. Additionally, there remains a need to provide anti-human PD-L1 and anti-human TIM-3 bispecific antibodies that possess one or more of the following features: simultaneously bind and bridge PD-L1 expressing tumor cells and TIM-3 expressing T-cells, suppress PD-1 and TIM-3 checkpoint inhibitor pathways, exhibit better anti-tumor activity as measured in a tumor model than a combination of an anti-PD-L1 antibody and an anti-TIM-3 antibody, and lack antibody immune effector functions.

Accordingly, the present invention provides an antibody that binds human PD-L1 (SEQ ID NO: 1) and human TIM-3 (SEQ ID NO: 2) comprising:a) a first heavy chain (HC) comprising a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO: 3,b) a first light chain (LC) comprising a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO: 4,c) a second HC comprising a HCVR having the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7,d) a second LC comprising a LCVR having the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10;
and wherein, the first LC forms an inter-chain disulfide bond with the first HC, the second LC forms an inter-chain disulfide bond with the second HC, and the first HC forms two inter-chain disulfide bonds with the second HC, and the first HC and second HC are human IgG1 isotype.

The present invention further provides an antibody wherein the second HC comprises a HCVR having the amino acid sequence of SEQ ID NO: 5, and the second LC comprises a LCVR having the amino acid sequence of SEQ ID NO: 8. The present invention further provides an antibody wherein the second HC comprises a HCVR having the amino acid sequence of SEQ ID NO: 6, and the second LC comprises a LCVR having the amino acid sequence of SEQ ID NO: 9. The present invention further provides an antibody, wherein the second HC comprises a HCVR having the amino acid sequence of SEQ ID NO: 7, and the second LC comprises a LCVR having the amino acid sequence of SEQ ID NO: 10.

The present invention further provides an antibody, wherein:a) the first HC comprises the amino acid sequences of SEQ ID NO: 19 and SEQ ID NO: 22,b) the second HC comprises the amino acid sequences of SEQ ID NO: 23 and SEQ ID NO: 26,c) the first LC comprises the amino acid sequence of SEQ ID NO: 27, andd) the second LC comprises the amino acid sequence of SEQ ID NO: 28.

The present invention provides an antibody that binds human PD-L1 (SEQ ID NO: 1) and human TIM-3 (SEQ ID NO: 2) comprising:a) a first HC comprising the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 19 and SEQ ID NO: 22,b) a first LC comprising the amino acid sequences of SEQ ID NO: 4, and SEQ ID NO: 27,c) a second HC comprising the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 23 and SEQ ID NO: 26,d) a second LC comprising the amino acid sequences of SEQ ID NO: 8, and SEQ ID NO: 28;
and wherein, the first LC forms an inter-chain disulfide bond with the first HC, the second LC forms an inter-chain disulfide bond with the second HC, and the first HC forms two inter-chain disulfide bonds with the second HC, and the first HC and second HC are human IgG1 isotype.

The present invention provides an antibody that binds human PD-L1 (SEQ ID NO: 1) and human TIM-3 (SEQ ID NO: 2) comprising:a) a first HC comprising the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 19 and SEQ ID NO: 22,b) a first LC comprising the amino acid sequences of SEQ ID NO: 4, and SEQ ID NO: 27,c) a second HC comprising the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 23 and SEQ ID NO: 26,d) a second LC comprising the amino acid sequences of SEQ ID NO: 9, and SEQ ID NO: 28;
and wherein, the first LC forms an inter-chain disulfide bond with the first HC, the second LC forms an inter-chain disulfide bond with the second HC, and the first HC forms two inter-chain disulfide bonds with the second HC, and the first HC and second HC are human IgG1 isotype.

The present invention provides an antibody that binds human PD-L1 (SEQ ID NO: 1) and human TIM-3 (SEQ ID NO: 2) comprising:a) a first HC comprising the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 19 and SEQ ID NO: 22,b) a first LC comprising the amino acid sequences of SEQ ID NO: 4, and SEQ ID NO: 27,c) a second HC comprising the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 23 and SEQ ID NO: 26,d) a second LC comprising the amino acid sequences of SEQ ID NO: 10, and SEQ ID NO: 28;
and wherein, the first LC forms an inter-chain disulfide bond with the first HC, the second LC forms an inter-chain disulfide bond with the second HC, and the first HC forms two inter-chain disulfide bonds with the second HC, and the first HC and second HC are human IgG1 isotype.

The present invention further provides an antibody that binds human PD-L1 (SEQ ID NO: 1) and human TIM-3 (SEQ ID NO: 2), wherein:a) the first HC comprises the amino acid sequences of SEQ ID NO: 20 and SEQ ID NO: 21, andb) the second HC comprises the amino acid sequences of SEQ ID NO: 24 and SEQ ID NO: 25.

For SEQ ID NOs: 19-28, Table 1 maps where the sequences correlate on the antibody. For SEQ ID NOs: 3-10, Table 2 maps where the sequences correlate on the antibody.

The present invention provides an antibody that binds human PD-L1 (SEQ ID NO: 1) and human TIM-3 (SEQ ID NO: 2) comprising two light chains (LC) and two heavy chains (HC), wherein:a) the first LC has the amino acid sequence of SEQ ID NO: 12,b) the first HC has the amino acid sequence of SEQ ID NO: 11,c) the second LC has the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18, andd) the second HC has the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

The present invention provides an antibody comprising two light chains (LC) and two heavy chains (HC), wherein:a) the first LC has the amino acid sequence of SEQ ID NO: 12,b) the first HC has the amino acid sequence of SEQ ID NO: 11,c) the second LC has the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18, andd) the second HC has the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

The present invention provides an antibody that binds human PD-L1 (SEQ ID NO: 1) and human TIM-3 (SEQ ID NO: 2) comprising two light chains (LC) and two heavy chains (HC), wherein:a) the first LC has the amino acid sequence of SEQ ID NO: 12, and the first HC has the amino acid sequence of SEQ ID NO: 11, andb) the second LC and the second HC have the amino acid sequences of SEQ ID NO: 16 and SEQ ID NO: 13, SEQ ID NO: 17 and SEQ ID NO: 14, or SEQ ID NO: 18 and SEQ ID NO: 15, respectively.

The present invention further provides an antibody, wherein the second LC has the amino acid sequence of SEQ ID NO: 16, and the second HC has the amino acid sequence of SEQ ID NO: 13. The present invention further provides an antibody, wherein the second LC has the amino acid sequence of SEQ ID NO: 17, and the second HC has the amino acid sequence of SEQ ID NO: 14. The present invention further provides an antibody, wherein the second LC has the amino acid sequence of SEQ ID NO: 18, and the second HC has the amino acid sequence of SEQ ID NO: 15.

The present invention provides a half-antibody that binds human PD-L1 (SEQ ID NO: 1) comprising a LC and a HC, wherein the LC has the amino acid sequence of SEQ ID NO: 12, and the HC has the amino acid sequence of SEQ ID NO: 11.

The present invention provides a half-antibody that binds human TIM-3 (SEQ ID NO: 2) comprising a LC and a HC, wherein the LC and the HC have the amino acid sequences of SEQ ID NO: 16 and SEQ ID NO: 13, SEQ ID NO: 17 and SEQ ID NO: 14, or SEQ ID NO: 18 and SEQ ID NO: 15, respectively.

TABLE 1Sequences mapped to region of IgG1 heavy chain or light chainSEQIDRe-Amino acid positions of Antibody C that correlates withNOgionsequence19CH1128-190 of PD-L1 antibody heavy chain (SEQ ID NO: 11)20CH2237-271 of PD-L1 antibody heavy chain (SEQ ID NO: 11)21CH2330-340 of PD-L1 antibody heavy chain (SEQ ID NO: 11)22CH3350-410 of PD-L1 antibody heavy chain (SEQ ID NO: 11)23CH1128-190 of TIM-3 antibody heavy chain (SEQ ID NO: 15)24CH2231-265 of TIM-3 antibody heavy chain (SEQ ID NO: 15)25CH2330-340 of TIM-3 antibody heavy chain (SEQ ID NO: 15)26CH3350-410 of TIM-3 antibody heavy chain (SEQ ID NO: 15)27CL131-181 of PD-L1 antibody light chain (SEQ ID NO: 12)28CL131-181 of TIM-3 antibody light chain (SEQ ID NO: 18)

The present invention provides a mammalian cell comprising a DNA molecule comprising a polynucleotide sequence encoding polypeptides having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 12, and SEQ ID NO: 18, wherein the cell is capable of expressing an antibody of the present invention.

The present invention provides a mammalian cell comprising a first DNA molecule and a second DNA molecule, wherein the first DNA molecule comprises a polynucleotide sequence encoding polypeptides having the amino acid sequence of SEQ ID NO: 11 and SEQ ID NO: 12, and wherein the second DNA molecule comprises a polynucleotide sequence encoding polypeptides having the amino acid sequence of SEQ ID NO: 15 and SEQ ID NO: 18, wherein the cell is capable of expressing an antibody of the present invention.

The present invention provides a mammalian cell comprising a first DNA molecule, a second DNA molecule, a third DNA molecule, and a fourth DNA molecule, wherein the first DNA molecule comprises a polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 11, the second DNA molecule comprises a polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 12, the third DNA molecule comprises a polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 15, and the fourth DNA molecule comprises a polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 18, wherein the cell is capable of expressing an antibody of the present invention.

The present invention provides a process for producing an antibody of the present invention comprising cultivating a mammalian cell of the present invention under conditions such that the antibody is expressed, and recovering the expressed antibody.

The present invention provides an antibody produced by a process of the present invention.

The present invention provides an antibody produced by cultivating a mammalian cell comprising a DNA molecule comprising a polynucleotide sequence encoding polypeptides having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 12, and SEQ ID NO: 18 under conditions such that the antibody is expressed, and recovering the expressed antibody.

The present invention provides an antibody produced by cultivating a mammalian cell comprising a first DNA molecule and a second DNA molecule, wherein the first DNA molecule comprises a polynucleotide sequence encoding polypeptides having the amino acid sequence of SEQ ID NO: 11 and SEQ ID NO: 12, and wherein the second DNA molecule comprises a polynucleotide sequence encoding polypeptides having the amino acid sequence of SEQ ID NO: 15 and SEQ ID NO: 18 under conditions such that the antibody is expressed, and recovering the expressed antibody.

The present invention provides a pharmaceutical composition, comprising an antibody of the present invention and an acceptable carrier, diluent, or excipient.

The present invention provides a method of treating cancer, comprising administering to a patient in need thereof, an effective amount of an antibody of the present invention. The present invention further provides a method of treating cancer, comprising administering to a patient in need thereof, an effective amount of an antibody of the present invention, wherein the cancer is melanoma, lung cancer, head and neck cancer, liver cancer, colorectal cancer, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, endometrial cancer, esophageal cancer, soft tissue sarcoma, cholangiocarcinoma, or hepatocellular carcinoma.

The present invention provides a method of treating cancer, wherein the cancer is melanoma. The present invention further provides a method of treating cancer, wherein the cancer is lung cancer. The present invention further provides a method of treating cancer, wherein the lung cancer is non-small cell lung cancer, small cell lung cancer, or mesothelioma. The present invention further provides a method of treating cancer, wherein the cancer is head and neck cancer. The present invention further provides a method of treating cancer, wherein the cancer is liver cancer. The present invention further provides a method of treating cancer, wherein the cancer is colorectal cancer. The present invention further provides a method of treating cancer, wherein the cancer is pancreatic cancer. The present invention further provides a method of treating cancer, wherein the cancer is gastric cancer. The present invention further provides a method of treating cancer, wherein the cancer is kidney cancer. The present invention further provides a method of treating cancer, wherein the cancer is bladder cancer. The present invention further provides a method of treating cancer, wherein the cancer is prostate cancer. The present invention further provides a method of treating cancer, wherein the cancer is breast cancer. The present invention further provides a method of treating cancer, wherein the cancer is ovarian cancer. The present invention further provides a method of treating cancer, wherein the cancer is endometrial cancer. The present invention further provides a method of treating cancer, wherein the cancer is esophageal cancer. The present invention further provides a method of treating cancer, wherein the cancer is soft tissue sarcoma. The present invention further provides a method of treating cancer, wherein the cancer is cholangiocarcinoma. The present invention further provides a method of treating cancer, wherein the cancer is hepatocellular carcinoma.

The present invention further provides methods comprising the administration of an effective amount of the antibody of the present invention in simultaneous, separate, or sequential combination with one or more standard of care agents selected from the group consisting of cisplatin, carboplatin, dacarbazine, liposomal doxorubicin, docetaxel, cyclophosphamide and doxorubicin, navelbine, eribulin, paclitaxel, paclitaxel protein-bound particles for injectable suspension, ixabepilone, capecitabine, FOLFOX (leucovorin, fluorouracil, and oxaliplatin), FOLFIRI (leucovorin, fluorouracil, and irinotecan), gemcitabine, topotecan, liposomal irinotecan, pemetrexed, and cetuximab.

The present invention further provides methods comprising the administration of an effective amount of the antibody of the present invention in simultaneous, separate, or sequential combination with one or more antitumor agents selected from the group consisting of nivolumab, ipilimumab, pidilizumab, pembrolizumab, tremelimumab, urelumab, lirilumab, atezolizumab, epacadostat, and durvalumab.

The present invention further provides methods comprising the administration of an effective amount of the antibody of the present invention comprising simultaneous, separate, or sequential combination with ionizing radiation.

The present invention provides an antibody of the present invention, for use in therapy. The present invention provides an antibody of the present invention, for use in the treatment of cancer. The present invention provides an antibody of the present invention, for use in the treatment of cancer, wherein the cancer is melanoma, lung cancer, head and neck cancer, liver cancer, colorectal cancer, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, endometrial cancer, esophageal cancer, soft tissue sarcoma, cholangiocarcinoma, or hepatocellular carcinoma.

The present invention provides an antibody of the present invention, for use in the treatment of melanoma. The present invention provides an antibody of the present invention, for use in the treatment of lung cancer. The present invention further provides an antibody of the present invention, wherein the lung cancer is non-small cell lung cancer, small cell lung cancer, or mesothelioma. The present invention provides an antibody of the present invention, for use in the treatment of head and neck cancer. The present invention provides an antibody of the present invention, for use in the treatment of liver cancer. The present invention provides an antibody of the present invention, for use in the treatment of colorectal cancer. The present invention provides an antibody of the present invention, for use in the treatment of pancreatic cancer. The present invention provides an antibody of the present invention, for use in the treatment of gastric cancer. The present invention provides an antibody of the present invention, for use in the treatment of kidney cancer. The present invention provides an antibody of the present invention, for use in the treatment of bladder cancer. The present invention provides an antibody of the present invention, for use in the treatment of prostate cancer. The present invention provides an antibody of the present invention, for use in the treatment of breast cancer. The present invention provides an antibody of the present invention, for use in the treatment of ovarian cancer. The present invention provides an antibody of the present invention, for use in the treatment of endometrial cancer. The present invention provides an antibody of the present invention, for use in the treatment of esophageal cancer. The present invention provides an antibody of the present invention, for use in the treatment of soft tissue sarcoma. The present invention provides an antibody of the present invention, for use in the treatment of cholangiocarcinoma. The present invention provides an antibody of the present invention, for use in the treatment of hepatocellular carcinoma.

The present invention provides the antibody of the present invention for use in simultaneous, separate, or sequential combination with one or more standard of care agents selected from the group consisting of cisplatin, carboplatin, dacarbazine, liposomal doxorubicin, docetaxel, cyclophosphamide and doxorubicin, navelbine, eribulin, paclitaxel, paclitaxel protein-bound particles for injectable suspension, ixabepilone, capecitabine, FOLFOX (leucovorin, fluorouracil, and oxaliplatin), FOLFIRI (leucovorin, fluorouracil, and irinotecan), gemcitabine, topotecan, liposomal irinotecan, pemetrexed, and cetuximab, in the treatment of cancer.

The present invention provides the antibody of the present invention for use in simultaneous, separate, or sequential combination with one or more antitumor agents selected from the group consisting of nivolumab, ipilimumab, pidilizumab, pembrolizumab, tremelimumab, urelumab, lirilumab, atezolizumab, epacadostat, and durvalumab, in the treatment of cancer.

The present invention provides the antibody of the present invention for use in simultaneous, separate, or sequential combination with ionizing radiation, in the treatment of cancer.

The present invention provides a pharmaceutical composition for use in treating cancer, comprising an effective amount of an antibody of the present invention. The present invention further provides a pharmaceutical composition for use in treating cancer, comprising an effective amount of an antibody of the present invention, wherein the cancer is melanoma, lung cancer, head and neck cancer, liver cancer, colorectal cancer, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, endometrial cancer, esophageal cancer, soft tissue sarcoma, cholangiocarcinoma, or hepatocellular carcinoma. The present invention further provides a pharmaceutical composition for use in treating cancer, comprising an effective amount of an antibody of the present invention, wherein the lung cancer is non-small cell lung cancer, small cell lung cancer, or mesothelioma.

The present invention provides a pharmaceutical composition for use in treating melanoma, comprising an effective amount of an antibody of the present invention. The present invention provides a pharmaceutical composition for use in treating lung cancer, comprising an effective amount of an antibody of the present invention. The present invention provides a pharmaceutical composition for use in treating head and neck cancer, comprising an effective amount of an antibody of the present invention. The present invention provides a pharmaceutical composition for use in treating liver cancer, comprising an effective amount of an antibody of the present invention. The present invention provides a pharmaceutical composition for use in treating colorectal cancer, comprising an effective amount of an antibody of the present invention. The present invention provides a pharmaceutical composition for use in treating pancreatic cancer, comprising an effective amount of an antibody of the present invention. The present invention provides a pharmaceutical composition for use in treating gastric cancer, comprising an effective amount of an antibody of the present invention. The present invention provides a pharmaceutical composition for use in treating kidney cancer, comprising an effective amount of an antibody of the present invention. The present invention provides a pharmaceutical composition for use in treating bladder cancer, comprising an effective amount of an antibody of the present invention. The present invention provides a pharmaceutical composition for use in treating prostate cancer, comprising an effective amount of an antibody of the present invention. The present invention provides a pharmaceutical composition for use in treating breast cancer, comprising an effective amount of an antibody of the present invention. The present invention provides a pharmaceutical composition for use in treating ovarian cancer, comprising an effective amount of an antibody of the present invention. The present invention provides a pharmaceutical composition for use in treating endometrial cancer, comprising an effective amount of an antibody of the present invention. The present invention provides a pharmaceutical composition for use in treating esophageal cancer, comprising an effective amount of an antibody of the present invention. The present invention provides a pharmaceutical composition for use in treating soft tissue sarcoma, comprising an effective amount of an antibody of the present invention. The present invention provides a pharmaceutical composition for use in treating cholangiocarcinoma, comprising an effective amount of an antibody of the present invention. The present invention provides a pharmaceutical composition for use in treating hepatocellular carcinoma, comprising an effective amount of an antibody of the present invention.

The present invention further provides a pharmaceutical composition for use in treating cancer, wherein said pharmaceutical composition is administered in simultaneous, separate, or sequential combination with one or more standard of care agents selected from the group consisting of cisplatin, carboplatin, dacarbazine, liposomal doxorubicin, docetaxel, cyclophosphamide and doxorubicin, navelbine, eribulin, paclitaxel, paclitaxel protein-bound particles for injectable suspension, ixabepilone, capecitabine, FOLFOX (leucovorin, fluorouracil, and oxaliplatin), FOLFIRI (leucovorin, fluorouracil, and irinotecan), gemcitabine, topotecan, liposomal irinotecan, pemetrexed, and cetuximab.

The present invention further provides a pharmaceutical composition for use in treating cancer, wherein said pharmaceutical composition is administered in simultaneous, separate, or sequential combination with one or more antitumor agents selected from the group consisting of nivolumab, ipilimumab, pidilizumab, pembrolizumab, tremelimumab, urelumab, lirilumab, atezolizumab, epacadostat, and durvalumab.

The present invention further provides a pharmaceutical composition for use in treating cancer, wherein said pharmaceutical composition is administered in simultaneous, separate, or sequential combination with ionizing radiation.

The present invention provides the use of an antibody of the present invention for the manufacture of a medicament for the treatment of cancer. The present invention further provides the use of an antibody of the present invention for the manufacture of a medicament for the treatment of cancer, wherein the cancer is melanoma, lung cancer, head and neck cancer, liver cancer, colorectal cancer, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, endometrial cancer, esophageal cancer, soft tissue sarcoma, cholangiocarcinoma, or hepatocellular carcinoma. The present invention further provides the use of an antibody of the present invention for the manufacture of a medicament for the treatment of cancer, wherein the lung cancer is non-small cell lung cancer, small cell lung cancer, or mesothelioma.

The present invention further provides the use of an antibody of the present invention in the manufacture of a medicament for the treatment of cancer wherein said medicament is to be administered simultaneously, separately, or sequentially with one or more standard of care agents selected from the group consisting of cisplatin, carboplatin, dacarbazine, liposomal doxorubicin, docetaxel, cyclophosphamide and doxorubicin, navelbine, eribulin, paclitaxel, paclitaxel protein-bound particles for injectable suspension, ixabepilone, capecitabine, FOLFOX (leucovorin, fluorouracil, and oxaliplatin), FOLFIRI (leucovorin, fluorouracil, and irinotecan), gemcitabine, topotecan, liposomal irinotecan, pemetrexed, and cetuximab.

The present invention further provides the use of an antibody of the present invention in the manufacture of a medicament for the treatment of cancer wherein said medicament is to be administered simultaneously, separately, or sequentially with one or more antitumor agents selected from the group consisting of nivolumab, ipilimumab, pidilizumab, pembrolizumab, tremelimumab, urelumab, lirilumab, atezolizumab, epacadostat, and durvalumab.

The present invention provides the use of an antibody of the present invention in the manufacture of a medicament for the treatment of cancer wherein said medicament is to be administered simultaneously, separately, or sequentially with ionizing radiation.

An antibody of the present invention is an engineered, non-naturally occurring polypeptide complex. A DNA molecule of the present invention is a non-naturally occurring DNA molecule that comprises a polynucleotide sequence encoding a polypeptide having the amino acid sequence of one of the polypeptides in an antibody of the present invention.

The antibody of the present invention is an IgG type antibody and has “heavy” chains and “light” chains that are cross-linked via intra- and inter-chain disulfide bonds. Each heavy chain is comprised of an N-terminal HCVR and a heavy chain constant region (“HCCR”). Each light chain is comprised of a LCVR and a light chain constant region (“LCCR”). Light chains each form disulfide bonds with a heavy chain, and the two heavy chains form two disulfide bonds between each other.

The constant region of the heavy chains contains CH1, CH2, and CH3 domains. CH1 comes after the HCVR; the CH1 and HCVR form the heavy chain portion of a Fab. CH2 comes after the hinge region and before CH3. CH3 comes after CH2 and is at the carboxy-terminal end of the heavy chain.

The constant region of the light chains contains one domain, CL. CL comes after the LCVR; the CL and LCVR form the light chain portion of a Fab.

Antibodies of the present invention are heterodimeric in that each arm of the antibody exhibits selective monovalent binding to its cognate antigen due to two different heavy chains and two different light chains forming the antibody. In the present invention one arm of the antibody binds human PD-L1 (SEQ ID NO: 1), and the other arm binds human TIM-3 (SEQ ID NO: 2). In order to ensure proper assembly of these bispecific antibodies, mutations are incorporated into the sequence of the heavy chains within the CH1 and CH3 region and into the sequence of the light chains within the light chain constant region. The CH1 and LC mutations are made to favor native pairing of the requisite light chain and heavy chain pairs and disfavor light chain mispairing. The CH3 mutations are made to favor heterodimeric pairing of the two distinct heavy chains and disfavor formation of homodimers. Mutations in the CH3 region of the anti-PD-L1 portion of the antibody preferably includes positions 356, 372, 398, and 400 as numbered by absolute position in SEQ ID NO: 11 (positions 350, 366, 392, and 394 if using EU numbering). Mutations in the CH3 region of the anti-TIM-3 portion of the antibody preferably includes positions 350, 351, 356, 405, and 407 as numbered by absolute position in SEQ ID NO: 15 (positions 350, 351, 405, and 407 if using EU numbering). Mutations in the CH1 region of the anti-PD-L1 portion of the antibody preferably includes positions 189 as numbered by absolute position in SEQ ID NO: 11 (position 183 if using EU numbering). Mutations in the CH1 region of the anti-TIM-3 portion of the antibody preferably includes positions 128, 147, 175, and 183 as numbered by absolute position in SEQ ID NO: 15 (positions 128, 147, 175, and 183 if using EU numbering).

Mutations in the CL region of the anti-PD-L1 portion of the antibody preferably includes positions 179 and 181 as numbered by absolute position in SEQ ID NO: 11 (positions 176 and 178 if using EU numbering). Mutations in the CL region of the anti-TIM-3 portion of the antibody preferably includes positions 131, 133, 176, and 178 as numbered by absolute position in SEQ ID NO: 15 (positions 131, 133, 176, and 178 if using EU numbering).

In certain antibodies of the present invention, heavy chain heterodimeric pairing mutations yield a CH3 thermal stability greater than 80° C., which is comparable to native antibodies (Table 1 and Von Kreudenstein, et al., 2014).

When expressed in certain biological systems, antibodies having Fc sequences are glycosylated in the Fc region. Typically, glycosylation occurs in the Fc region of the antibody at a highly conserved N-glycosylation site. N-glycans typically attach to asparagine. Antibodies may be glycosylated at other positions as well.

Optionally, certain antibodies of the present invention contain an Fc portion which is derived from human IgG1. IgG1 is well known to bind to the proteins of the Fc-gamma receptor family (FcγR) as well as C1q. Interaction with these receptors can induce antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Therefore, certain amino acid substitutions are introduced into IgG1 Fc for certain antibodies of the present invention, including Antibody A, B, and C, to ablate immune effector function. Mutations in the CH2 region of the anti-PD-L1 portion of the antibody may include positions 240, 241, and 271 as numbered by absolute position in SEQ ID NO: 11 (positions 234, 235, and 265 if using EU numbering). Mutations in the CH2 region of the anti-TIM-3 portion of the antibody may include positions 234, 235, and 265 as numbered by absolute position in SEQ ID NO: 15 (positions 234, 235, and 265 if using EU numbering).

An isolated DNA encoding a HCVR region can be converted to a full-length heavy chain gene by operably linking the HCVR-encoding DNA to another DNA molecule encoding heavy chain constant regions. The sequences of human, as well as other mammalian, heavy chain constant region genes are known in the art. DNA fragments encompassing these regions can be obtained e.g., by standard PCR amplification.

An isolated DNA encoding a LCVR region may be converted to a full-length light chain gene by operably linking the LCVR-encoding DNA to another DNA molecule encoding a light chain constant region. The sequences of human, as well as other mammalian, light chain constant region genes are known in the art. DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region. Preferably for antibodies of the present invention, the light chain constant region of the anti-PD-L1 portion of the antibody is a lambda light chain and the light chain constant region of the anti-TIM-3 portion of the antibody is a kappa light chain.

The polynucleotides of the present invention will be expressed in a host cell after the sequences have been operably linked to an expression control sequence. The expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors will contain selection markers, e.g., tetracycline, neomycin, and dihydrofolate reductase, to permit detection of those cells transformed with the desired DNA sequences.

The antibody of the present invention may readily be produced in mammalian cells such as CHO, NS0, HEK293 or COS cells. The host cells are cultured using techniques well known in the art.

The vectors containing the polynucleotide sequences of interest (e.g., the polynucleotides encoding the polypeptides of the antibody and expression control sequences) can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host.

Various methods of protein purification may be employed and such methods are known in the art and described, for example, in Deutscher,Methods in Enzymology182: 83-89 (1990) and Scopes,Protein Purification: Principles and Practice,3rd Edition, Springer, NY (1994).

In another embodiment of the present invention, the antibody, or the nucleic acids encoding the same, is provided in isolated form. As used herein, the term “isolated” refers to a protein, peptide, or nucleic acid which is free or substantially free from any other macromolecular species found in a cellular environment. “Substantially free” as used herein means the protein, peptide, or nucleic acid of interest comprises more than 80% (on a molar basis) of the macromolecular species present, preferably more than 90%, and more preferably more than 95%.

The antibody of the present invention, or pharmaceutical compositions comprising the same, may be administered by parenteral routes (e.g., subcutaneous and intravenous). An antibody of the present invention may be administered to a patient alone with pharmaceutically acceptable carriers, diluents, or excipients in single or multiple doses. Pharmaceutical compositions of the present invention can be prepared by methods well known in the art (e.g.,Remington: The Science and Practice of Pharmacy,22nded. (2012), A. Loyd et al., Pharmaceutical Press) and comprise an antibody, as disclosed herein, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

The term “treating” (or “treat” or “treatment”) refers to slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

“Binds” as used herein in reference to the affinity of an antibody for human PD-L1 (SEQ ID NO: 1), human TIM-3 (SEQ ID NO: 2), or both is intended to mean, unless indicated otherwise, a KDof less than about 1×10−6M, preferably, less than about 1×10−9M as determined by common methods known in the art, including by use of a surface plasmon resonance (SPR) biosensor at 37° C. essentially as described herein.

“Effective amount” means the amount of an antibody of the present invention or pharmaceutical composition comprising an antibody of the present invention that will elicit the biological or medical response of or desired therapeutic effect on a tissue, system, animal, mammal or human that is being sought by the researcher, medical doctor, or other clinician. An effective amount of the antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effect of the antibody is outweighed by the therapeutically beneficial effects.

This invention is further illustrated by the following non-limiting example.

