Combination therapy for treating cancer

Abstract

Provided herein are methods and compositions for the treatment of cancer, such methods comprising the combined use of a ROR1 antagonist and a checkpoint inhibitor antagonist.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 476,333, filed December 20, 2022, which is incorporated herein by reference in its entirety and for all purposes.

[0003] INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING

[0004] This application is submitted with a sequence listing in electronic format. The sequence listing is provided as a file named 51956-724_601_SL.xml, created on December 18, 2023, and having a size of 25,134 bytes. The information in the electronic sequence listing is incorporated by reference in its entirety. Background Art

[0005] Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an evolutionarily conserved type I membrane protein that belongs to the ROR subfamily and has an extracellular domain containing immunoglobulin (Ig)-like, Frizzled, and Kringle domains. Existing data suggest that ROR1 can play a role in non-canonical WNT signaling to promote the survival of malignant cells. Checkpoint inhibitors work by blocking the binding of checkpoint proteins to partner proteins, allowing T cells to kill cancer cells. Checkpoint inhibitor drugs include those targeting PD-1, PD-L1, and PD-L2. Summary of the Invention

[0006] Described herein are methods of treating cancer that combine the use of a ROR1 antagonist and a checkpoint inhibitor antagonist. Surprisingly and unexpectedly, the combination of a ROR1 antagonist and a checkpoint inhibitor was observed to provide a synergistic effect in treating cancer in subjects in need thereof, which is superior to the effect of using either the ROR1 antagonist or the checkpoint inhibitor alone.

[0007] In certain aspects, described herein is a method for treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a tyrosine kinase-like orphan receptor 1 (ROR1) antagonist and administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor comprises a PD-1 inhibitor or a PD-L1 inhibitor. In certain embodiments, the immune checkpoint inhibitor is a small molecule drug. In certain embodiments, the immune checkpoint inhibitor comprises an antibody, a peptide, or a protein. In certain embodiments, the immune checkpoint inhibitor comprises a PD-1 inhibitor. In certain embodiments, the PD-1 inhibitor is a PD-1 inhibitor antibody. In certain embodiments, the PD-1 inhibitor antibody comprises nivolumab, pembrolizumab, cemiplimab, dorstarlimab, or a combination thereof. In certain embodiments, the PD-1 inhibitor antibody comprises pembrolizumab. In certain embodiments, the PD-1 inhibitor antibody includes JTX-4014, spartalizumab (PDR001), camrelizumab (SHR1210), sintilmab (IBI308), tislelizumab (BGB-A317), toripalimab (JS 001), Retifanlimab (INCMGA00012) (MGA012), AMP-224, AMP-514 or a combination thereof. In certain embodiments, the immune checkpoint inhibitor includes a PD-L1 inhibitor. In certain embodiments, the PD-L1 inhibitor is a PD-L1 inhibitor antibody. In certain embodiments, the PD-L1 inhibitor antibody includes atezolizumab, avelumab, durvalumab or a combination thereof. In certain embodiments, the PD-L1 inhibitor antibody includes Envafolimab (KN035), Cosibelimab (CK-301), AUNP12, CA-170, BMS-986189, or a combination thereof. In certain embodiments, the ROR1 antagonist is an antibody or a small molecule. In certain embodiments, the antibody includes Fab, F(ab')2, Fv, or scFv. In certain embodiments, the ROR1 antagonist is an anti-ROR1 antibody. In certain embodiments, the antibody is zilovertamab.In certain embodiments, the antibody binds to a ROR1 amino acid sequence as set forth in SEQ ID NO: 30 or SEQ ID NO: 31. In certain embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are administered in a synergistic amount in combination. In certain embodiments, the method further comprises reducing growth of a tumor. In certain embodiments, the method reduces growth of a tumor by about 20% or more. In certain embodiments, the method reduces growth of a tumor by about 25% to about 45% when compared to administration of the immune checkpoint inhibitor and the ROR1 antagonist, respectively. In certain embodiments, the method further comprises reducing mass or volume of a tumor. In certain embodiments, the antibody comprises a humanized heavy chain variable region and a humanized light chain variable region, wherein the humanized heavy chain variable region comprises the sequence set forth in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3; and wherein the humanized light chain variable region comprises the sequence set forth in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7; and wherein the light chain variable region comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 8.

[0008] The method of any one of the preceding claims, wherein the immune cell infiltrate of the cancer expresses PD-1. In certain embodiments, the cancer expresses PD-L1. In certain embodiments, the cancer overexpresses PD-L1. In certain embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are administered substantially simultaneously. In certain embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are administered separately. In certain embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are administered sequentially. In certain embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are co-formulated for administration by a single formulation. In certain embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are administered in separate compositions. In certain embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are mixed prior to administration. In certain embodiments, the immune checkpoint inhibitor is administered by intravenous infusion. In certain embodiments, the immune checkpoint inhibitor is administered once every two weeks. In certain embodiments, the immune checkpoint inhibitor is administered once every three weeks. In certain embodiments, the immune checkpoint inhibitor is administered once every four weeks. In certain embodiments, the immune checkpoint inhibitor is administered at a dose of about 200 milligrams to about 2000 milligrams. In certain embodiments, the immune checkpoint inhibitor is administered at a dose of about 200 milligrams to about 800 milligrams. In certain embodiments, the immune checkpoint inhibitor is administered at a dose of about 300 milligrams to about 600 milligrams. In certain embodiments, the immune checkpoint inhibitor is administered at a dose of about 300 milligrams. In certain embodiments, the immune checkpoint inhibitor is administered at a dose of about 600 milligrams. In certain embodiments, the immune checkpoint inhibitor is administered in an amount of 200 mg every six weeks, 300 mg every six weeks, or 400 mg every six weeks. In certain embodiments, the immune checkpoint inhibitor is administered in an amount of 100 mg every three weeks or 300 mg every six weeks. In certain embodiments, the immune checkpoint inhibitor is administered in an amount of 2 mg / kg or 1 mg / kg every 3 weeks. In certain embodiments, the immune checkpoint inhibitor is administered in an amount of 50 mg every three weeks or 100 mg every six weeks. In certain embodiments, the immune checkpoint inhibitor is administered in an amount of about 50 mg to about 400 mg every three weeks. In certain embodiments, the immune checkpoint inhibitor is administered as a 25 mg / mL solution. In certain embodiments, the immune checkpoint inhibitor is administered by injection or intravenous infusion.In certain embodiments, the immune checkpoint inhibitor is administered at a dose of about 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.0, 2.25, 2.50, 2.75, 3.00, 3.5, 4, 4.5, 5, 5.5, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mg / kg. In certain embodiments, the ROR1 antagonist is administered at a dose of about 200 milligrams to about 2000 milligrams. In certain embodiments, the ROR1 antagonist is administered at a dose of about 200 milligrams to about 800 milligrams. In certain embodiments, the ROR1 antagonist is administered at a dose of about 300 milligrams to about 600 milligrams. In certain embodiments, the ROR1 antagonist is administered at a dose of about 300 milligrams. In certain embodiments, the ROR1 antagonist is administered at a dose of about 600 milligrams. In certain embodiments, the ROR1 antagonist is administered at a dose of about 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.0, 2.25, 2.50, 2.75, 3.00, 3.5, 4, 4.5, 5, 5.5, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mg / kg. In certain embodiments, the ROR1 antagonist is administered at 25 mg / mL solution. In certain embodiments, the ROR1 antagonist is administered once every two weeks. In certain embodiments, the ROR1 antagonist is administered once every three weeks. In certain embodiments, the ROR1 antagonist is administered once every four weeks. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the cancer is lymphoma, leukemia, myeloma, renal cell carcinoma, colon cancer, colorectal cancer, breast cancer, epidermoid carcinoma, melanoma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, uterine cancer, adenocarcinoma, sarcoma, or adrenal cancer. In certain embodiments, the cancer is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, mantle cell lymphoma (MCL), marginal zone B-cell lymphoma, Burkitt's Lymphoma, myeloma, AML, B-ALL, T-ALL, or B-cell leukemia.

[0009] In another aspect, described herein is a pharmaceutical composition comprising a therapeutically effective amount of a Receptor Tyrosine Kinase-Like Orphan Receptor 1 (ROR1) antagonist and a therapeutically effective amount of an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor comprises a PD-1 inhibitor or a PD-L1 inhibitor. In certain embodiments, the immune checkpoint inhibitor is a small molecule drug. In certain embodiments, the immune checkpoint inhibitor is an antibody. In certain embodiments, the immune checkpoint inhibitor comprises a PD-1 inhibitor. In certain embodiments, the PD-1 inhibitor is a PD-1 inhibitor antibody. In certain embodiments, the PD-1 inhibitor antibody comprises Nivolumab, Pembrolizumab, Cemiplimab, Dostarlimab or a combination thereof. In certain embodiments, the PD-1 inhibitor antibody comprises Pembrolizumab. In certain embodiments, the PD-1 inhibitor antibody comprises JTX-4014, Spartalizumab (PDR001), Camrelizumab (SHR1210), Cisplatin (IBI308), Tislelizumab (BGB-A317), Toripalimab (JS 001), Refluzimab (INCMGA00012) (MGA012), AMP-224, AMP-514 or a combination thereof. In certain embodiments, the immune checkpoint inhibitor comprises a PD-L1 inhibitor. In certain embodiments, the PD-L1 inhibitor is a PD-L1 inhibitor antibody. In certain embodiments, the PD-L1 inhibitor antibody comprises Atezolizumab, Avelumab, Durvalumab or a combination thereof. In certain embodiments, the PD-L1 inhibitor antibody comprises Enfortumab (KN035), Coxadylizumab (CK-301), AUNP12, CA-170, BMS-986189 or a combination thereof. In certain embodiments, the ROR1 antagonist is an antibody or a small molecule. In certain embodiments, the antibody comprises a Fab, a F(ab’)2, a Fv or a scFv. In certain embodiments, the ROR1 antagonist is an anti-ROR1 antibody. In certain embodiments, the antibody is Zanolimumab. In certain embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are administered in a synergistic amount in combination. In certain embodiments, the method further comprises reducing the growth of a tumor. In certain embodiments, the pharmaceutical composition reduces the growth of a tumor by about 20% or more. In certain embodiments, the pharmaceutical composition reduces the growth of a tumor by about 25% to about 45% when compared to the administration of the immune checkpoint inhibitor and the ROR1 antagonist, respectively. In certain embodiments, the pharmaceutical composition reduces the mass or volume of a tumor.In certain embodiments, the antibody comprises a humanized heavy chain variable region and a humanized light chain variable region, wherein the humanized heavy chain variable region comprises the sequence set forth in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3; and wherein the humanized light chain variable region comprises the sequence set forth in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7; and wherein the light chain variable region comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the immune checkpoint inhibitor is included in an amount of about 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.0, 2.25, 2.50, 2.75, 3.00, 3.5, 4, 4.5, 5, 5.5, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mg / kg. In certain embodiments, the composition comprises the ROR1 antagonist in an amount of about 200 milligrams to about 2000 milligrams. In certain embodiments, the composition comprises the ROR1 antagonist in an amount of about 200 milligrams to about 800 milligrams. In certain embodiments, the composition comprises the ROR1 antagonist in an amount of about 300 milligrams to about 600 milligrams. In certain embodiments, the composition comprises the ROR1 antagonist in an amount of about 300 milligrams to about 600 milligrams. In certain embodiments, the composition comprises the ROR1 antagonist in an amount of about 300 milligrams. In certain embodiments, the composition comprises the ROR1 antagonist in an amount of about 600 milligrams. In certain embodiments, the composition comprises the ROR1 antagonist in an amount of about 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.0, 2.25, 2.50, 2.75, 3.00, 3.5, 4, 4.5, 5, 5.5, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mg / kg.In certain embodiments, the composition comprises the ROR1 antagonist in an amount of about 10, 15, 20, 25, or 30 mg / mL. In certain embodiments, the composition comprises the immune checkpoint inhibitor in an amount of about 200 milligrams to about 800 milligrams. In certain embodiments, the composition comprises the immune checkpoint inhibitor in an amount of about 300 milligrams to about 600 milligrams. In certain embodiments, the composition comprises the immune checkpoint inhibitor in an amount of about 300 milligrams to about 600 milligrams. In certain embodiments, the composition comprises the immune checkpoint inhibitor in an amount of about 300 milligrams. In certain embodiments, the composition comprises the immune checkpoint inhibitor in an amount of about 600 milligrams. In certain embodiments, the composition comprises the immune checkpoint inhibitor in an amount of about 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.0, 2.25, 2.50, 2.75, 3.00, 3.5, 4, 4.5, 5, 5.5, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mg / kg. In certain embodiments, the composition comprises the immune checkpoint inhibitor in an amount of about 10, 15, 20, 25, or 30 mg / mL. In certain embodiments, the composition comprises a synergistic amount of the ROR1 antagonist and the immune checkpoint inhibitor in combination. In certain embodiments, the composition comprises a synergistic amount of the ROR1 antagonist and the immune checkpoint inhibitor in combination effective for treating a cancer in a subject. In certain embodiments, the cancer comprises a mutated PD-L1 gene. In certain embodiments, the cancer expresses PD-L1. In certain embodiments, the cancer overexpresses PD-L1. In certain embodiments, the cancer is a lymphoma, a leukemia, a myeloma, a renal cell carcinoma, a colon cancer, a colorectal cancer, a breast cancer, an epidermoid carcinoma, a melanoma, a gastric cancer, a brain cancer, a lung cancer, a pancreatic cancer, a cervical cancer, an ovarian cancer, a liver cancer, a bladder cancer, a prostate cancer, a testicular cancer, a thyroid cancer, a head and neck cancer, a uterine cancer, an adenocarcinoma, a sarcoma, or an adrenal cancer. In certain embodiments, the cancer is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, mantle cell lymphoma (MCL), marginal zone B-cell lymphoma, Burkitt lymphoma, myeloma, AML, B-ALL, T-ALL, or B-cell leukemia.

[0010] Also described herein is the use of a therapeutically effective amount of a tyrosine kinase-like orphan receptor 1 (ROR1) antagonist and a therapeutically effective amount of an immune checkpoint inhibitor in the manufacture of a medicament for treating a cancer in a subject in need thereof.

[0011] Aspects disclosed herein include the use of a therapeutically effective amount of a tyrosine kinase-like orphan receptor 1 (ROR1) antagonist and a therapeutically effective amount of an immune checkpoint inhibitor in a method of treating cancer in a subject in need thereof comprising administering to the subject the pharmaceutical composition. Aspects disclosed herein include the use of a therapeutically effective amount of a tyrosine kinase-like orphan receptor 1 (ROR1) antagonist and a therapeutically effective amount of an immune checkpoint inhibitor for the manufacture of a medicament for treating cancer in a subject in need thereof. In some embodiments, the immune checkpoint inhibitor comprises a PD-1 inhibitor or a PD-L1 inhibitor. In some embodiments, the immune checkpoint inhibitor is a small molecule drug. In some embodiments, the immune checkpoint inhibitor comprises an antibody, a peptide, or a protein. In some embodiments, the immune checkpoint inhibitor comprises a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor antibody. In some embodiments, the PD-1 inhibitor antibody comprises nivolumab, pembrolizumab, cemiplimab, dostarlimab, or a combination thereof. In some embodiments, the PD-1 inhibitor antibody comprises pembrolizumab. In some embodiments, the PD-1 inhibitor antibody comprises JTX-4014, spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), toripalimab (JS 001), refroalimab (INCMGA00012) (MGA012), AMP-224, AMP-514, or a combination thereof. In some embodiments, the immune checkpoint inhibitor comprises a PD-L1 inhibitor. In some embodiments, the PD-L1 inhibitor is a PD-L1 inhibitor antibody. In some embodiments, the PD-L1 inhibitor antibody comprises atezolizumab, avelumab, durvalumab, or a combination thereof. In some embodiments, the PD-L1 inhibitor antibody comprises enflurigimab (KN035), corimatrix (CK-301), AUNP12, CA-170, BMS-986189, or a combination thereof. In some embodiments, the ROR1 antagonist is an antibody or a small molecule. In some embodiments, the antibody comprises a Fab, a F(ab’)2, a Fv, or a scFv. In some embodiments, the ROR1 antagonist is an anti-ROR1 antibody. In some embodiments, the antibody is zanolimumab. In some embodiments, the antibody binds to a ROR1 amino acid sequence as set forth in SEQ ID NO: 30 or SEQ ID NO: 31. In some embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are administered in a combined synergistic amount. In some embodiments, the use comprises reducing the growth of a tumor. In some embodiments, the use reduces the growth of a tumor by about 20% or more.In some embodiments, the use reduces tumor growth by about 25% to about 45% when compared to separate administration of the immune checkpoint inhibitor and the ROR1 antagonist. In some embodiments, the use comprises reducing the mass or volume of a tumor. In some embodiments, the antibody comprises a humanized heavy chain variable region and a humanized light chain variable region, wherein the humanized heavy chain variable region comprises the sequence set forth in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3; and wherein the humanized light chain variable region comprises the sequence set forth in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7; and wherein the light chain variable region comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, immune cell infiltrates of the cancer express PD-1. In some embodiments, the cancer expresses PD-L1. In some embodiments, the cancer overexpresses PD-L1. In some embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are administered substantially simultaneously. In some embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are administered separately. In some embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are administered sequentially. In some embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are co-formulated for administration by a single formulation. In some embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are administered in separate compositions. In some embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are mixed prior to administration. In some embodiments, the immune checkpoint inhibitor is administered by intravenous infusion. In some embodiments, the immune checkpoint inhibitor is administered once every two weeks. In some embodiments, the immune checkpoint inhibitor is administered once every three weeks. In some embodiments, the immune checkpoint inhibitor is administered once every four weeks. In some embodiments, the immune checkpoint inhibitor is administered at a dose of about 200 milligrams to about 2000 milligrams. In some embodiments, the immune checkpoint inhibitor is administered at a dose of about 200 milligrams to about 800 milligrams. In some embodiments, the immune checkpoint inhibitor is administered at a dose of about 300 milligrams to about 600 milligrams. In some embodiments, the immune checkpoint inhibitor is administered at a dose of about 300 milligrams. In some embodiments, the immune checkpoint inhibitor is administered at a dose of about 600 milligrams.In some embodiments, the immune checkpoint inhibitor is administered in an amount of 200 mg every six weeks, 300 mg every six weeks, or 400 mg every six weeks. In some embodiments, the immune checkpoint inhibitor is administered in an amount of 100 mg every three weeks or 300 mg every six weeks. In some embodiments, the immune checkpoint inhibitor is administered in an amount of 2 mg / kg or 1 mg / kg every 3 weeks. In some embodiments, the immune checkpoint inhibitor is administered in an amount of 50 mg every three weeks or 100 mg every six weeks. In some embodiments, the immune checkpoint inhibitor is administered in an amount of about 50 mg to about 400 mg every three weeks. In some embodiments, the immune checkpoint inhibitor is administered as a 25 mg / mL solution. In some embodiments, the immune checkpoint inhibitor is administered by injection or intravenous infusion. In some embodiments, the immune checkpoint inhibitor is administered at a dose of about 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.0, 2.25, 2.50, 2.75, 3.00, 3.5, 4, 4.5, 5, 5.5, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mg / kg. In some embodiments, the ROR1 antagonist is administered at a dose of about 200 milligrams to about 2000 milligrams. In some embodiments, the ROR1 antagonist is administered at a dose of about 200 milligrams to about 800 milligrams. In some embodiments, the ROR1 antagonist is administered at a dose of about 300 milligrams to about 600 milligrams. In some embodiments, the ROR1 antagonist is administered at a dose of about 300 milligrams. In some embodiments, the ROR1 antagonist is administered at a dose of about 600 milligrams. In some embodiments, the ROR1 antagonist is administered at a dose of about 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.0, 2.25, 2.50, 2.75, 3.00, 3.5, 4, 4.5, 5, 5.5, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mg / kg. In some embodiments, the ROR1 antagonist is administered as a 25 mg / mL solution. In some embodiments, the ROR1 antagonist is administered once every two weeks. In some embodiments, the ROR1 antagonist is administered once every three weeks. In some embodiments, the ROR1 antagonist is administered once every four weeks. In some embodiments, the subject is a mammal.In some embodiments, the subject is a human. In some embodiments, the cancer is lymphoma, leukemia, myeloma, renal cell carcinoma, colon cancer, colorectal cancer, breast cancer, squamous cell carcinoma of the epithelium, melanoma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, uterine cancer, adenocarcinoma, sarcoma, or adrenal cancer. In some embodiments, the cancer is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, mantle cell lymphoma (MCL), marginal zone B-cell lymphoma, Burkitt's lymphoma, myeloma, AML, B-ALL, T-ALL, or B-cell leukemia.

[0012] INCORPORATION BY REFERENCE

[0013] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF DRAWINGS

[0014] The features and advantages of the present application will be better understood with reference to the following detailed description and accompanying drawings, of which:

[0015] FIG. 1 Depicts the percentage of Jeko-1 cell toxicity in a mixture of donor cells and Jeko-1 cells.

[0016] FIG. 2A Depicts the change in tumor volume over time for donor 451 cells treated with anti-RORl antibody, pembrolizumab, or a combination.

[0017] FIG. 2B Depicts the change in tumor volume over time for donor 244 cells treated with anti-RORl antibody, pembrolizumab, or a combination. DETAILED DESCRIPTION

[0018] Provided herein are methods of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a tyrosine kinase-like orphan receptor 1 (ROR1) antagonist and a therapeutically effective amount of an immune checkpoint inhibitor. Surprisingly and unexpectedly, it was observed that the combination of a ROR1 antagonist and a checkpoint inhibitor provided a synergistic effect in the treatment of cancer in a subject in need thereof, which was superior to the effect of using either a ROR1 antagonist or a checkpoint inhibitor alone.

[0019] Immune checkpoint inhibitors

[0020] In some embodiments, an immune checkpoint inhibitor is disclosed herein. In some embodiments, the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-l 1 inhibitor, a PD-L2 inhibitor, or a combination thereof. In some embodiments, the immune checkpoint inhibitor is a small molecule inhibitor. In some embodiments, the immune checkpoint inhibitor is an antibody, a peptide, or a protein.

[0021] In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor antibody. In some embodiments, the PD-1 inhibitor antibody comprises nivolumab, pembrolizumab, cemiplimab, dostarlimab, JTX-4014, spartalizumab (PDR001), camrelizumab (SHR1210), sendirnab (IBI308), tislelizumab (BGB-A317), toripalimab (JS 001), relatrnmab (INCMGA00012) (MGA012), AMP-224, AMP-514, or a combination thereof. In some embodiments, the PD-1 inhibitor antibody comprises nivolumab, pembrolizumab, cemiplimab, dostarlimab, or a combination thereof. In some embodiments, the PD-1 inhibitor antibody comprises JTX-4014, spartalizumab (PDR001), camrelizumab (SHR1210), sendirnab (IBI308), tislelizumab (BGB-A317), toripalimab (JS 001), relatrnmab (INCMGA00012) (MGA012), AMP-224, AMP-514, or a combination thereof. In some embodiments, the PD-1 inhibitor antibody comprises nivolumab. In some embodiments, the PD-1 inhibitor antibody comprises pembrolizumab. In some embodiments, the PD-1 inhibitor antibody comprises cemiplimab. In some embodiments, the PD-1 inhibitor antibody comprises dostarlimab. In some embodiments, the PD-1 inhibitor antibody comprises JTX-4014. In some embodiments, the PD-1 inhibitor antibody comprises spartalizumab (PDR001). In some embodiments, the PD-1 inhibitor antibody comprises camrelizumab (SHR1210). In some embodiments, the PD-1 inhibitor antibody comprises sendirnab (IBI308). In some embodiments, the PD-1 inhibitor antibody comprises tislelizumab (BGB-A317). In some embodiments, the PD-1 inhibitor antibody comprises toripalimab (JS 001). In some embodiments, the PD-1 inhibitor antibody comprises relatrnmab (INCMGA00012) (MGA012). In some embodiments, the PD-1 inhibitor antibody comprises AMP-224. In some embodiments, the PD-1 inhibitor antibody comprises AMP-514.

[0022] In some embodiments, the immune checkpoint inhibitor comprises a PD-L1 inhibitor. In some embodiments, the PD-L1 inhibitor comprises a PD-L1 inhibitor antibody. In some embodiments, the PD-L1 inhibitor is a small molecule. In some embodiments, the PD-L1 inhibitor comprises atezolizumab, avelumab, durvalumab, envafolimab (KN035), cosibelimab (CK-301), AUNP12, CA-170, BMS-986189, or a combination thereof. In some embodiments, the PD-L1 inhibitor comprises atezolizumab, avelumab, durvalumab, or a combination thereof. In some embodiments, the PD-L1 inhibitor comprises envafolimab (KN035), cosibelimab (CK-301), AUNP12, CA-170, BMS-986189, or a combination thereof. In some embodiments, the PD-L1 inhibitor comprises atezolizumab. In some embodiments, the PD-L1 inhibitor comprises avelumab. In some embodiments, the PD-L1 inhibitor comprises durvalumab. In some embodiments, the PD-L1 inhibitor comprises KN035. In some embodiments, the PD-L1 inhibitor comprises CK-301. In some embodiments, the PD-L1 inhibitor comprises AUNP12. In some embodiments, the PD-L1 inhibitor comprises CA-170. In some embodiments, the PD-L1 inhibitor comprises BMS-986189.

[0023] In some embodiments, the immune checkpoint inhibitor comprises a PD-L2 inhibitor. In some embodiments, the immune checkpoint inhibitor comprises CA-170, rHIgM12B, or a combination thereof. In some embodiments, the immune checkpoint inhibitor comprises CA-170. In some embodiments, the immune checkpoint inhibitor comprises rHIgM12B.

[0024] ROR1 antagonists

[0025] In certain aspects, a ROR1 antagonist is used in the methods described herein. In some embodiments, the ROR1 antagonist is a small molecule. In some embodiments, the ROR1 antagonist is a ROR1 binding molecule or binding fragment thereof. In some embodiments, the RoR1 binding molecule or ROR1 binding fragment thereof is an anti-ROR1 antibody as described herein. In some embodiments, the ROR1 binding molecule or ROR1 binding fragment thereof is an IgG antibody.

[0026] The provided anti-ROR1 antibodies include monoclonal anti-ROR1 antibodies and anti-ROR1 antibody fragments. Anti-ROR1 antibodies include anti-ROR1 antibody conjugates and molecules comprising anti-ROR1 antibodies, such as chimeric molecules. Thus, anti-ROR1 antibodies include, but are not limited to, full-length and natural anti-ROR1 antibodies, as well as fragments and portions thereof that retain its binding specificity, such as any specific binding portion thereof, including those of any number of immunoglobulin classes and / or isotypes (e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM); and biologically relevant (antigen binding) fragments or specific binding portions thereof, including but not limited to Fab, F(ab')2, Fv, and scFv (single chain or related entities). A monoclonal antibody is typically one member of a composition of substantially homogeneous antibodies; thus, each individual antibody comprised in a monoclonal antibody composition is identical except for possible naturally-occurring mutations that can be present in minor amounts. Monoclonal antibodies can comprise a human IgGl constant region. Monoclonal antibodies can comprise a human IgG4 constant region.

[0027] The term "antibody" herein is used in the broadest sense and includes monoclonal antibodies, and includes intact antibodies and functional (antigen binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (sFv or scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific (e.g., bispecific) antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise specified, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and subtypes thereof, IgM, IgE, IgA, and IgD. Antibodies can comprise a human IgGl constant region. Antibodies can comprise a human IgG4 constant region.

[0028] The terms "complementarity determining region" and "CDR," synonymous with "hypervariable region" or "HVR," are known in the art to refer to non-contiguous sequences of amino acids within the variable region of an antibody, which confer antigen specificity and / or binding affinity. Generally, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). "Framework region" and "FR" are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. Generally, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4).The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5thEd. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 Jan;27(l):55-77 (“IMGT” numbering scheme); Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309(3):657-70, (“Aho” numbering scheme); and Whitelegg NR and Rees AR, “WAM: an improved algorithm for modelling antibodies on the WEB,” Protein Eng. 2000 Dec;13(12):819-24 (“AbM” numbering scheme). In certain embodiments, the CDRs of the antibodies described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or a combination thereof.

[0029] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. The numbering of the Kabat and Chothia schemes is based on the most common antibody region sequence lengths, where insertions are accommodated by inserted letters, for example, "30a", and deletions occur in some antibodies. The two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact scheme is based on analysis of complex crystal structures and is similar to the Chothia numbering scheme in many respects.

[0030] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a natural antibody (V H and V L ) generally have a similar structure, wherein each domain comprises four conserved framework regions (FRs) and three CDRs (see, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). Single V H or V L The V domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen may use V H or V L The domains are separated from antibodies that bind to the antigen to screen for complementary V H or V L Libraries of domains (see, eg, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0031] The specific binding of the antibody molecules described herein refers to the binding mediated by one or more CDR portions of the antibody. It may not be necessary for all CDRs to be specifically bound. Specific binding can be demonstrated by an ELISA for a specific target or antigen, where the binding is significantly increased compared to an isotype control antibody.

[0032] In some embodiments, the anti-ROR1 antibody or antigen-binding fragment thereof comprises cirmtuzumab. In certain embodiments, the anti-ROR1 antibody or antigen-binding fragment thereof is cirmtuzumab. In some embodiments, the anti-ROR1 antibody comprises the variable heavy chain (VH) of cirmtuzumab. In some embodiments, the anti-ROR1 antibody comprises the VH CDR1, VH CDR2, and VH CDR3 derived from cirmtuzumab. In some embodiments, the anti-ROR1 antibody comprises the variable light chain (VL) of cirmtuzumab. In some embodiments, the anti-ROR1 antibody comprises the VL CDR1, VL CDR2, and VL CDR3 derived from cirmtuzumab.

[0033] In some embodiments, the ROR1 binding molecule or ROR1 binding fragment thereof comprises: a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 1; b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 2; c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 3; d) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 4; e) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 5; and / or f) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the ROR1 binding molecule or ROR1 binding fragment thereof comprises: a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 11; b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 12; c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13; d) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 14; e) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 15; and / or f) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the ROR1 binding molecule or ROR1 binding fragment thereof comprises: a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 21; b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 22; c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 23; d) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 24; e) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 25; and / or f) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0034] In some embodiments, the ROR1 binding molecule or ROR1 binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 7; and / or b) an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 8. In some embodiments, the ROR1 binding molecule or ROR1 binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7; and / or b) an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the ROR1 binding molecule or ROR1 binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 17; and / or b) an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 18. In some embodiments, the ROR1 binding molecule or ROR1 binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 17; and / or b) an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, the ROR1 binding molecule or ROR1 binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 27; and / or b) an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 28. In some embodiments, the ROR1 binding molecule or ROR1 binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 27; and / or b) an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 28.

[0035] In some embodiments, the ROR1 binding molecule or ROR1 binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9; and / or b) an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10.

[0036] The provided anti-RORl antibodies include anti-RORl antibody fragments. An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of anti-RORl antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv or sFv); and multispecific antibodies formed from antibody fragments. In particular embodiments, an anti-RORl antibody is a single-chain antibody fragment comprising a variable heavy chain region and / or a variable light chain region, such as a scFv.

[0037] Anti-RORl antibody fragments can be made by a variety of techniques, including but not limited to proteolytic digestion of an intact anti-RORl antibody and production by recombinant host cells. In some embodiments, an anti-RORl antibody is a recombinantly produced fragment, such as a fragment comprising a non-naturally occurring arrangement, such as those with two or more antibody regions or chains joined by a synthetic linker (e.g., a polypeptide linker), and / or those not produced by enzymatic digestion of a naturally occurring intact antibody. In some aspects, an anti-RORl antibody fragment is a scFv.

[0038] A "humanized" antibody refers to an antibody in which all or substantially all CDR amino acid residues are derived from a non-human CDR and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody can optionally include at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody, refers to a variant of a non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody, for example, to preserve or improve antibody specificity or affinity.

[0039] The provided anti-RORl antibodies include anti-RORl human antibodies. A "human antibody" is an antibody that has an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from an animal that has been raised using a human antibody repertoire or other human antibody encoding sequences, including a human antibody library. The term does not include humanized forms of non-human antibodies, such as those in which all or substantially all CDRs are non-human.

[0040] Human antibodies can be prepared by administering the immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to an antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin loci are typically inactivated. Human antibodies can also be derived from human antibody libraries, including phage display and cell-free libraries, containing antibody-encoding sequences derived from a human repertoire.

[0041] The terms "polypeptide" and "protein" are used interchangeably and refer to polymers of amino acid residues, and are not limited to a minimum length. Polypeptides, including the provided antibodies and antibody chains, as well as other peptides (e.g., linkers and binding peptides), can comprise amino acid residues, including natural and / or non-natural amino acid residues. The term also encompasses post-expression modifications of the polypeptide, e.g., glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, a polypeptide can contain modifications relative to the natural or native sequence, so long as the protein maintains a desired activity. These modifications can be intentional, as by site-directed mutagenesis, or can be accidental, such as by mutation of a host producing the protein or by errors due to PCR amplification. In some embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. Variants will typically differ by one or more substitutions, deletions, additions and / or insertions in one or more of the polypeptides specifically disclosed herein. Such variants can be naturally occurring or can be synthetically generated, e.g., by modifying one or more of the above polypeptide sequences of the application, and assessing one or more biological activities of the polypeptide as described herein and / or using any of a variety of known techniques. For example, it can be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and / or additions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding.

[0042] Amino acid sequence insertions and deletions include amino- and / or carboxyl-terminal fusions including fusions to polypeptides having more than 100 or more amino acids as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include an antibody with an N-terminal or C-terminal fusion to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody. Examples of intrasequence insertional variants include an antibody with an insert of an additional 3 amino acids in the light chain.

[0043] In some embodiments, the antibody is altered to increase or decrease its glycosylation (e.g., by altering the amino acid sequence such that one or more glycosylation sites are created or eliminated). The carbohydrate attached to the Fc region of the antibody can be altered. Native antibodies from mammalian cells typically comprise biantennary complex oligosaccharides attached by N-linkages to Asn297 of the CH2 domain of the Fc region (see, e.g., Wright et al. TIBTECH 15:26-32 (1997)). The oligosaccharide can be various carbohydrates, e.g., mannose, N-acetylglucosamine (GlcNAc), galactose, sialic acid, a fucose attached to a GlcNAc in the stem of the bi-antennary oligosaccharide structure. For example, modifications can be made to the oligosaccharide in an antibody to create antibody variants with certain improved properties. Antibody glycosylation variants can have improved ADCC and / or CDC function. In some embodiments, provided antibody variants have carbohydrate structures that lack (directly or indirectly) fucose attached to the Fc region. For example, the amount of fucose in such antibodies can be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the total sum of all sugar structures attached to Asn297 (see, e.g., WO 08 / 077546). Asn297 refers to the asparagine residue located at about position 297 of the Fc region (EU numbering of Fc region residues; see, e.g., Edelman et al. Proc Natl Acad Sci U S A. 1969 May;63(l):78-85). However, due to minor sequence variations in antibodies, Asn297 can occur at a different position. 297 297 297 297 297 ​​​​Also located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants can have improved ADCC function (see, e.g., Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); and Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004)). Cell lines, e.g., knockout cell lines, and methods of using the same, can be used to produce defucosylated antibodies, e.g., Lec13 CHO cells deficient in protein fucosylation and CHO cells knocked out for the alpha-1,6-fucosyltransferase gene (FUT8) (see, e.g., Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006)). Other antibody glycosylation variants are also included (see, e.g., U.S. Patent No. 6,602,684).

[0044] In some embodiments, an anti-ROR1 antibody provided herein has a dissociation constant (K - 8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M) for an antibody target. In some embodiments, an anti-ROR1 antibody provided herein has a dissociation constant (K D ) of about 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, or 0.001 nM or less, e.g., 10 -8 M or less, e.g., 10 -8 M to 10 - 13 M, e.g., 10 -9 M to 10 -13 M) for an antibody target. In some embodiments, an anti-ROR1 antibody provided herein has a dissociation constant (K D ) of about 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, or 0.001 nM or less, e.g., 10 DThe KD can be measured by any suitable assay. In certain embodiments, the KD can be measured using a surface plasmon resonance assay (e.g., using a Biacore® or Octet).

[0045] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an anti-ROR1 antibody provided herein, thereby generating an Fc region variant. An Fc region herein is the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of the constant region. Fc regions include native sequence Fc regions and variant Fc regions. Fc region variants can comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0046] In some cases, the Fc region of an immunoglobulin is important for many important antibody functions (e.g., effector functions), such as antigen-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADCP), although by different mechanisms, leading to the killing of target cells. Thus, in some embodiments, the antibodies described herein comprise a variable domain of the application in combination with a constant domain comprising a different Fc region, selected based on the biological activity of the antibody for the intended use. In certain cases, for example, human IgG can be classified into four subclasses, IgGl, IgG2, IgG3, and IgG4, and each of these comprises an Fc region with unique characteristics for binding to one or more of the following: Fcy receptors (activating receptors FcyRI (CD64), FcyRIIA, FcyRIIC (CD32); FcyRIIIA and FcyRIIIB (CD16) and inhibitory receptor FcyRIIB), and for the complement first component (Clq). Human IgGl and IgG3 bind to all Fcy receptors; IgG2 binds to FcyRIIA H131 , and binds to FcyRIIA R131 FcyRIIIA V158 with lower affinity; IgG4 binds to FcyRI, FcyRIIA, FcyRIIB, FcyRIIC and FcyRIIIA V158 ; and the inhibitory receptor FcyRIIB has lower affinity for IgGl, IgG2 and IgG3 than all other Fcy receptors. Studies have shown that FcyRI does not bind to IgG2, and FcyRIIIB does not bind to IgG2 and IgG4. Id. Generally, with respect to ADCC activity, human IgGl > IgG3 » IgG4 > IgG2.

[0047] ​In some embodiments, the anti-ROR1 antibodies of the present disclosure are variants with reduced effector function, which make them desirable candidates in applications where certain effector functions, such as complement fixation and ADCC, are unnecessary or absent. Such anti-ROR1 antibodies can have reduced complement dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the anti-ROR1 antibodies of the present disclosure are variants with increased effector function for use in applications where increased immunogenicity would be beneficial. Such anti-ROR1 antibodies can have increased CDC, ADCC, or ADCP, or a combination thereof. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent Nos. 5,500,362 and 5,821,337. Alternatively, non-radioactive assay methods (e.g., ACTI TM and CytoTox Non-radioactive Cytotoxicity Assay). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs), monocytes, macrophages, and natural killer (NK) cells.

[0048] Anti-ROR1 antibodies can have increased half-lives and improved binding to the neonatal Fc receptor (FcRn) (see, e.g., US 2005 / 0014934). Such anti-ROR1 antibodies can include an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn and include those with substitutions at one or more of the following Fc region residues according to the EU numbering system: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434 (see, e.g., U.S. Patent No. 7,371,826). Other examples of Fc region variants are contemplated (see, e.g., Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent Nos. 5,648,260 and 5,624,821; and WO 94 / 29351).

[0049] In some embodiments, it can be desirable to create cysteine engineered anti-RORl antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with cysteine residues. In some embodiments, the residue(s) substituted are in a position of the antibody that is accessible. The reactive thiol can be positioned for conjugation to other moieties, such as drug moieties or linker drug moieties, to create an immunoconjugate. In some embodiments, any one or more of the following residues can be substituted with cysteine: V205 (Kabat numbering) of the light chain; Al l 8 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region.

[0050] In some embodiments, the anti-RORl antibodies provided herein can be further modified to contain additional non-protein moieties that are known and available. Moieties suitable for derivatization of the antibody include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), co-polymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-l,3-dioxolane, poly-l,3,6-trioxane, ethylene / maleic acid copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly(nvinylpyrrolidinone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde can have advantages in manufacturing due to its stability in water. The polymers can have any suitable molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if two or more polymers are attached, they can be the same or different molecules.

[0051] The present disclosure provides immunoconjugates comprising the anti-RORl antibodies described herein. Immunoconjugates are antibodies conjugated to one or more heterologous molecules. For example, an immunoconjugate can comprise an anti-RORl antibody conjugated to one or more cytotoxic agents such as a chemotherapeutic agent or drug, a growth inhibitory agent, a proteinaceous toxin, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope. In some embodiments, an immunoconjugate can comprise an anti-RORl antibody or fragment thereof (e.g., scFv).

[0052] The anti-ROR1 antibodies described herein can be encoded by a nucleic acid. Nucleic acids are a class of polynucleotides comprising two or more nucleotide bases. In certain embodiments, a nucleic acid is a component of a vector that can be used to transfer a polynucleotide encoding a polypeptide into a cell. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a genomic integrating vector, or “integrating vector,” which can become integrated into the chromosomal DNA of the host cell. Another type of vector is an “episomal” vector, i.e., a nucleic acid capable of extra-chromosomal replication. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors.” Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, and the like. In an expression vector, regulatory elements such as promoters, enhancers, polyadenylation signals, and the like for controlling transcription can be derived from mammalian, microbial, viral, or insect genes. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene can additionally be introduced to facilitate recognition of transformants. Viral-derived vectors can be employed, such as lentivirus, retrovirus, adenovirus, adeno-associated virus, and the like. Plasmid vectors can be linearized for integration into the genome.

[0053] As used herein, the term “homologous,” “homology,” or “percent homology” when used herein to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, modified in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such formula is incorporated into the Basic Local Alignment Search Tool (BLAST) program of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). Percent homology of sequences can be determined using the latest version of BLAST as of the filing date of the present application.

[0054] The percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity (and not considering any conservative substitutions as part of the sequence identity). Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been filed with the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. ALIGN-2 is publicly available from Genentech, Inc., South San Francisco, Calif, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0055] In the case of amino acid sequence comparisons with ALIGN-2, the percent amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A having or comprising a certain percent amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in a program-aligned comparison of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where amino acid sequence A has a length not equal to that of amino acid sequence B, the percent amino acid sequence identity of A to B will not equal that of B to A. Unless otherwise specified, all percent amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0056] Nucleic acids encoding the anti-RORl antibodies described herein can be used to infect, transfect, transform, or otherwise transgenically render suitable cells with the nucleic acids, enabling production of the antibodies for commercial or therapeutic use. Standard cell lines and methods for producing antibodies from large-scale cell cultures are known in the art. See, e.g., Li et al., "Cell culture processes for monoclonal antibody production." Mabs. 2010 Sep-Oct; 2(5): 466-477. In certain embodiments, the cells are eukaryotic cells. In certain embodiments, the eukaryotic cells are mammalian cells. In certain embodiments, the mammalian cells are cell lines useful for producing antibodies, are Chinese hamster ovary (CHO) cells, NS0 murine myeloma cells, or PER.C6® human cells. In certain embodiments, the nucleic acids encoding the antibodies are integrated into a genomic locus of the cells useful for producing the antibodies. In certain embodiments, described herein is a method of making an anti-RORl antibody comprising culturing a cell comprising a nucleic acid encoding the antibody under in vitro conditions sufficient for the antibody to be produced and secreted.

[0057] In certain embodiments, described herein is a master cell bank comprising: (a) a mammalian cell line comprising a nucleic acid encoding an antibody described herein integrated at a genomic location; and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol or DMSO. In certain embodiments, the master cell bank comprises: (a) a CHO cell line comprising a nucleic acid encoding an antibody having (i) a heavy chain amino acid sequence set forth in any one of SEQ ID NOs: 9, 19, or 29; and (ii) a light chain amino acid sequence set forth in any one of SEQ ID NOs: 10, 20, or 30 integrated at a genomic location; and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol or DMSO. In certain embodiments, the master cell bank is housed in a suitable vial or container capable of withstanding liquid nitrogen freezing.

[0058] Also described herein are methods of making the anti-RORl antibodies described herein. Such methods comprise incubating a cell or cell line comprising a nucleic acid encoding an anti-RORl antibody in a cell culture medium under conditions sufficient to allow expression and secretion of the antibody, and further harvesting the anti-RORl antibody from the cell culture medium. Harvesting can further comprise one or more purification steps to remove live cells, cell debris, non-antibody proteins or polypeptides, undesired salts, buffers, and media components. In certain embodiments, the additional purification steps comprise centrifugation, ultracentrifugation, protein A, protein G, protein A / G, or protein L purification, and / or ion exchange chromatography.

[0059] Methods of administration and uses of pharmaceutical compositions

[0060] In some embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are administered at the same time. In some embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are administered simultaneously. In some embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are administered substantially simultaneously. In some embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are administered sequentially. In some embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are administered separately.

[0061] In some embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are administered in a synergistic amount in combination. In some embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are co-formulated for administration by a single formulation. In some embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are mixed prior to administration. In some embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are administered in separate compositions.

[0062] The immune checkpoint inhibitors and ROR1 antagonists described herein can be administered by a variety of routes, including, but not limited to, subcutaneously, intraperitoneally, intravenously, intramuscularly, intratumorally, intracerebrally, or orally. In some embodiments, the immune checkpoint inhibitor is administered by intravenous infusion. In some embodiments, the immune checkpoint inhibitor is administered orally. In some embodiments, the ROR1 antagonist is administered by intravenous infusion.

[0063] In certain aspects, the methods described herein comprise reducing the growth of a tumor. In some embodiments, the method reduces the growth of a tumor by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 70% or more, about 80% or more, or about 90% or more. In some embodiments, the method reduces the growth of a tumor by about 10% or more. In some embodiments, the method reduces the growth of a tumor by about 20% or more. In some embodiments, the method reduces the growth of a tumor by about 30% or more. In some embodiments, the method reduces the growth of a tumor by about 40% or more. In some embodiments, the method reduces the growth of a tumor by about 50% or more. In certain embodiments, the growth of a tumor is reduced when compared to the separate administration of an immune checkpoint inhibitor and a ROR1 antagonist. In certain embodiments, the growth of a tumor is reduced when compared to the administration of an immune checkpoint inhibitor or a ROR1 antagonist alone.

[0064] In certain embodiments, the method reduces tumor growth by about 25% to about 45%. In certain embodiments, tumor growth is reduced by about 15% to about 55%. In certain embodiments, tumor growth is reduced by about 5% to about 65%. In certain embodiments, tumor growth is reduced by about 5% to about 25%. In certain embodiments, tumor growth is reduced by about 15% to about 35%. In certain embodiments, tumor growth is reduced by about 35% to about 55%. In certain embodiments, tumor growth is reduced by about 45% to about 65%.

[0065] In certain embodiments, the method includes reducing the mass or volume of the tumor. In certain embodiments, the method includes reducing the mass of the tumor by at least about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 70% or more, about 80% or more or about 90% or more. In certain embodiments, the method includes reducing the volume of the tumor by at least about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 70% or more, about 80% or more or about 90% or more. In certain embodiments, when compared to administering an immune checkpoint inhibitor and a ROR1 antagonist respectively, the mass or volume of the tumor is reduced. In certain embodiments, the mass or volume of the tumor is reduced when compared to administration of the immune checkpoint inhibitor or ROR1 antagonist alone.

[0066] In certain embodiments, the checkpoint inhibitor is administered on a suitable dosage schedule, for example, weekly, twice a week, monthly, twice a month, once every two weeks, once every three weeks, or once a month, etc. In some embodiments, the checkpoint inhibitor is administered once over a period of at least two weeks. In some embodiments, the checkpoint inhibitor is administered once over a period of at least three weeks. In some embodiments, the checkpoint inhibitor is administered once over a period of at least four weeks. The antibody can be administered in any therapeutically effective amount. A therapeutically effective amount includes an amount sufficient to improve one or more symptoms associated with the disease or affliction to be treated. In some embodiments, the checkpoint inhibitor is administered in a therapeutically acceptable amount.

[0067] In some embodiments, the checkpoint inhibitor is administered in an amount of at least about 100 mg, about 200 mg, about 30 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, or about 2000 mg. In some embodiments, the checkpoint inhibitor is administered in an amount of no more than about 100 mg, about 200 mg, about 30 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, or about 2000 mg.In some embodiments, the checkpoint inhibitor is administered in an amount of about 100 mg to about 1000 mg, about 200 mg to about 1000 mg, about 300 mg to about 1000 mg, about 400 mg to about 1000 mg, about 500 mg to about 1000 mg, about 600 mg to about 1000 mg, about 700 mg to about 1000 mg, about 800 mg to about 1000 mg, about 900 mg to about 1000 mg, about 100 mg to about 900 mg, about 200 mg to about 900 mg, about 300 mg to about 900 mg, about 400 mg to about 900 mg, about 500 mg to about 900 mg, about 600 mg to about 900 mg, about 700 mg to about 900 mg, about 800 mg to about 900 mg, about 100 mg to about 800 mg, about 200 mg to about 800 mg, about 300 mg to about 800 mg, about 400 mg to about 800 mg, about 500 mg to about 800 mg, about 600 mg to about 800 mg, about 700 mg to about 800 mg, about 100 mg to about 700 mg, about 200 mg to about 700 mg, about 300 mg to about 700 mg, about 400 mg to about 700 mg, about 500 mg to about 700 mg, about 600 mg to about 700 mg, about 100 mg to about 600 mg, about 200 mg to about 600 mg, about 300 mg to about 600 mg, about 400 mg to about 600 mg, about 500 mg to about 600 mg, about 100 mg to about 500 mg, about 200 mg to about 500 mg, about 300 mg to about 500 mg, about 400 mg to about 500 mg, about 100 mg to about 400 mg, about 200 mg to about 400 mg, about 300 mg to about 400 mg, about 100 mg to about 300 mg, about 200 mg to about 300 mg, or about 100 mg to about 200 mg. In some embodiments, the checkpoint inhibitor is administered in an amount of about 200 mg to about 800 mg.

[0068] In some embodiments, the checkpoint inhibitor is administered in an amount of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, or about 2000 mg. In some embodiments, the checkpoint inhibitor is administered in an amount of about 600 mg.

[0069] The checkpoint inhibitor can be administered at a dose based on the weight or mass of the individual to whom the checkpoint inhibitor is administered. In certain embodiments, the checkpoint inhibitor is administered at between about 0.1 mg / kg to about 50 mg / kg. In certain embodiments, the checkpoint inhibitor is administered at between about 1 mg / kg to about 40 mg / kg. In certain embodiments, the checkpoint inhibitor is administered at between about 5 mg / kg to about 30 mg / kg. In certain embodiments, the checkpoint inhibitor is administered at between about 2 mg / kg to about 20 mg / kg. In certain embodiments, the checkpoint inhibitor is administered at between about 2 mg / kg to about 10 mg / kg. In certain embodiments, the checkpoint inhibitor is administered at between about 2 mg / kg to about 5 mg / kg. In certain embodiments, the checkpoint inhibitor is administered at between about 3 mg / kg to about 10 mg / kg. In certain embodiments, the checkpoint inhibitor is administered at between about 3 mg / kg to about 5 mg / kg. In certain embodiments, the checkpoint inhibitor is administered at between about 4 mg / kg to about 10 mg / kg. In certain embodiments, the checkpoint inhibitor is administered at between about 5 mg / kg to about 10 mg / kg. In certain embodiments, the checkpoint inhibitor is administered at least about 0.5 mg / kg, about 0.75 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 1.75 mg / kg, about 2 mg / kg, about 2.25 mg / kg, about 2.5 mg / kg, about 2.75 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about 5.5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg, about 7.5 mg / kg, about 8 mg / kg, about 8.5 mg / kg, about 9 mg / kg, about 9.5 mg / kg, about 10 mg / kg, about 10.5 mg / kg, about 11 mg / kg, about 11.5 mg / kg, about 12 mg / kg, about 12.5 mg / kg, about 13 mg / kg, about 13.5 mg / kg, about 14 mg / kg, about 14.5 mg / kg, about 15 mg / kg, about 15.5 mg / kg, about 16 mg / kg, about 16.5 mg / kg, about 17 mg / kg, about 17.5 mg / kg, about 18 mg / kg, about 18.5 mg / kg, about 19 mg / kg, about 19.5 mg / kg, about 20 mg / kg, about 21 mg / kg, about 22 mg / kg, about 23 mg / kg, about 24 mg / kg, about 25 mg / kg, about 30 mg / kg, about 40 mg / kg, or about 50 mg / kg.

[0070] In certain embodiments, the checkpoint inhibitor is administered in a 5 mg / ml solution, a 10 mg / ml solution, a 15 mg / ml solution, a 20 mg / ml solution, a 25 mg / ml solution, a 30 mg / ml solution, a 35 mg / ml solution, a 40 mg / ml solution, a 45 mg / ml solution, or a 50 mg / ml solution. In certain embodiments, the checkpoint inhibitor is administered in a 15 mg / ml solution.

[0071] In certain embodiments, the antibody is administered on a suitable dosage schedule, e.g., weekly, twice a week, monthly, twice a month, every two weeks, every three weeks, or monthly, etc. In some embodiments, the ROR1 binding molecule or ROR1 binding fragment is administered once over a period of at least two weeks. In some embodiments, the ROR1 binding molecule or ROR1 binding fragment is administered once over a period of at least three weeks. In some embodiments, the ROR1 binding molecule or ROR1 binding fragment is administered once over a period of at least four weeks. The antibody can be administered in any therapeutically effective amount. A therapeutically effective amount includes an amount sufficient to improve one or more symptoms associated with the disease or affliction to be treated. In some embodiments, the ROR1 binding molecule or ROR1 binding fragment is administered in a therapeutically acceptable amount.

[0072] In some embodiments, the ROR1 binding molecule or ROR1 binding fragment is administered in an amount of at least about 100 milligrams, about 200 milligrams, about 30 milligrams, about 400 milligrams, about 500 milligrams, about 600 milligrams, about 700 milligrams, about 800 milligrams, about 900 milligrams, about 1000 milligrams, about 1100 milligrams, about 1200 milligrams, about 1300 milligrams, about 1400 milligrams, about 1500 milligrams, about 1600 milligrams, about 1700 milligrams, about 1800 milligrams, about 1900 milligrams, or about 2000 milligrams. In some embodiments, the ROR1 binding molecule or ROR1 binding fragment is administered in an amount of no more than about 100 milligrams, about 200 milligrams, about 30 milligrams, about 400 milligrams, about 500 milligrams, about 600 milligrams, about 700 milligrams, about 800 milligrams, about 900 milligrams, about 1000 milligrams, about 1100 milligrams, about 1200 milligrams, about 1300 milligrams, about 1400 milligrams, about 1500 milligrams, about 1600 milligrams, about 1700 milligrams, about 1800 milligrams, about 1900 milligrams, or about 2000 milligrams.In some embodiments, the ROR1 binding molecule or ROR1 binding fragment is administered at a dosage of about 100 mg to about 1000 mg, about 200 mg to about 1000 mg, about 300 mg to about 1000 mg, about 400 mg to about 1000 mg, about 500 mg to about 1000 mg, about 600 mg to about 1000 mg, about 700 mg to about 1000 mg, about 800 mg to about 1000 mg, about 900 mg to about 1000 mg, about 100 mg g to about 900 mg, about 200 mg to about 900 mg, about 300 mg to about 900 mg, about 400 mg to about 900 mg, about 500 mg to about 900 mg, about 600 mg to about 900 mg, about 700 mg to about 900 mg, about 800 mg to about 900 mg, about 100 mg to about 800 mg, about 200 mg to about 800 mg, about 300 mg to about 800 mg, about 400 mg to about 800 mg, about 500 mg g to about 800 mg, about 600 mg to about 800 mg, about 700 mg to about 800 mg, about 100 mg to about 700 mg, about 200 mg to about 700 mg, about 300 mg to about 700 mg, about 400 mg to about 700 mg, about 500 mg to about 700 mg, about 600 mg to about 700 mg, about 100 mg to about 600 mg, about 200 mg to about 600 mg, about 300 mg to about 600 mg, about 400 mg to about 700 mg, about 500 mg to about 700 mg, about 600 mg to about 700 mg, about 100 mg to about 600 mg, about 200 mg to about 600 mg, about 300 mg to about 600 mg, about 400 In some embodiments, the ROR1 binding molecule or ROR1 binding fragment is administered in an amount of about 200 mg to about 800 mg.

[0073] In some embodiments, the ROR1 binding molecule or ROR1 binding fragment is administered in an amount of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, or about 2000 mg. In some embodiments, the ROR1 binding molecule or ROR1 binding fragment is administered in an amount of about 600 mg.

[0074] The ROR1 binding molecule or ROR1 binding fragment can be administered at a dose based on the body weight or mass of the individual to whom the ROR1 binding molecule or ROR1 binding fragment is administered. In certain embodiments, the ROR1 binding molecule or ROR1 binding fragment is administered at between about 0.1 mg / kg to about 50 mg / kg. In certain embodiments, the ROR1 binding molecule or ROR1 binding fragment is administered at between about 1 mg / kg to about 40 mg / kg. In certain embodiments, the ROR1 binding molecule or ROR1 binding fragment is administered at between about 5 mg / kg to about 30 mg / kg. In certain embodiments, the ROR1 binding molecule or ROR1 binding fragment is administered at between about 2 mg / kg to about 20 mg / kg. In certain embodiments, the ROR1 binding molecule or ROR1 binding fragment is administered at at least about 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, 1.5 mg / kg, 1.75 mg / kg, 2 mg / kg, 2.25 mg / kg, 2.5 mg / kg, 2.75 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 7.5 mg / kg, 8 mg / kg, 8.5 mg / kg, 9 mg / kg, 9.5 mg / kg, 10 mg / kg, 10.5 mg / kg, 11 mg / kg, 11.5 mg / kg, 12 mg / kg, 12.5 mg / kg, 13 mg / kg, 13.5 mg / kg, 14 mg / kg, 14.5 mg / kg, 15 mg / kg, 15.5 mg / kg, 16 mg / kg, 16.5 mg / kg, 17 mg / kg, 17.5 mg / kg, 18 mg / kg, 18.5 mg / kg, 19 mg / kg, 19.5 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, or about 50 mg / kg.

[0075] In some embodiments, the ROR1 antagonist is administered as a 5 mg / mL solution, a 10 mg / mL solution, a 15 mg / mL solution, a 20 mg / mL solution, a 25 mg / mL solution, a 30 mg / mL solution, a 35 mg / mL solution, a 40 mg / mL solution, a 45 mg / mL solution, or a 50 mg / mL solution. In some embodiments, the ROR1 antagonist is administered as a 25 mg / mL solution.

[0076] Aspects disclosed herein include use of a therapeutically effective amount of a tyrosine kinase-like orphan receptor 1 (ROR1) antagonist and a therapeutically effective amount of an immune checkpoint inhibitor in a method of treating cancer in a subject in need thereof comprising administering to the subject a pharmaceutical composition. Aspects disclosed herein include use of a therapeutically effective amount of a tyrosine kinase-like orphan receptor 1 (ROR1) antagonist and a therapeutically effective amount of an immune checkpoint inhibitor for the manufacture of a medicament for treating cancer in a subject in need thereof. In some embodiments, the immune checkpoint inhibitor comprises a PD-1 inhibitor or a PD-L1 inhibitor. In some embodiments, the immune checkpoint inhibitor is a small molecule drug. In some embodiments, the immune checkpoint inhibitor comprises an antibody, a peptide, or a protein. In some embodiments, the immune checkpoint inhibitor comprises a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor antibody. In some embodiments, the PD-1 inhibitor antibody comprises nivolumab, pembrolizumab, cemiplimab, dostarlimab, or a combination thereof. In some embodiments, the PD-1 inhibitor antibody comprises pembrolizumab. In some embodiments, the PD-1 inhibitor antibody comprises JTX-4014, spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), toripalimab (JS001), refravlimab (INCMGA00012) (MGA012), AMP-224, AMP-514, or a combination thereof. In some embodiments, the immune checkpoint inhibitor comprises a PD-L1 inhibitor. In some embodiments, the PD-L1 inhibitor is a PD-L1 inhibitor antibody. In some embodiments, the PD-L1 inhibitor antibody comprises atezolizumab, avelumab, durvalumab, or a combination thereof. In some embodiments, the PD-L1 inhibitor antibody comprises enflurigimab (KN035), corimatrix (CK-301), AUNP12, CA-170, BMS-986189, or a combination thereof. In some embodiments, the ROR1 antagonist is an antibody or a small molecule. In some embodiments, the antibody comprises a Fab, a F(ab’)2, a Fv, or a scFv. In some embodiments, the ROR1 antagonist is an anti-ROR1 antibody. In some embodiments, the antibody is zanolimumab. In some embodiments, the antibody binds to a ROR1 amino acid sequence as set forth in SEQ ID NO: 30 or SEQ ID NO: 31. In some embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are administered in a combined synergistic amount. In some embodiments, the use comprises reducing growth of a tumor. In some embodiments, the use reduces growth of a tumor by about 20% or more.In some embodiments, the use reduces tumor growth by about 25% to about 45% when compared to separate administration of the immune checkpoint inhibitor and the ROR1 antagonist. In some embodiments, the use comprises reducing the mass or volume of a tumor. In some embodiments, the antibody comprises a humanized heavy chain variable region and a humanized light chain variable region, wherein the humanized heavy chain variable region comprises the sequence set forth in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3; and wherein the humanized light chain variable region comprises the sequence set forth in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7; and wherein the light chain variable region comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the immune cell infiltrate of the cancer expresses PD-1. In some embodiments, the cancer expresses PD-L1. In some embodiments, the cancer overexpresses PD-L1. In some embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are administered substantially simultaneously. In some embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are administered separately. In some embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are administered sequentially. In some embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are co-formulated for administration by a single formulation. In some embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are administered in separate compositions. In some embodiments, the immune checkpoint inhibitor and the ROR1 antagonist are mixed prior to administration. In some embodiments, the immune checkpoint inhibitor is administered by intravenous infusion. In some embodiments, the immune checkpoint inhibitor is administered once every two weeks. In some embodiments, the immune checkpoint inhibitor is administered once every three weeks. In some embodiments, the immune checkpoint inhibitor is administered once every four weeks. In some embodiments, the immune checkpoint inhibitor is administered at a dose of about 200 milligrams to about 2000 milligrams. In some embodiments, the immune checkpoint inhibitor is administered at a dose of about 200 milligrams to about 800 milligrams. In some embodiments, the immune checkpoint inhibitor is administered at a dose of about 300 milligrams to about 600 milligrams. In some embodiments, the immune checkpoint inhibitor is administered at a dose of about 300 milligrams. In some embodiments, the immune checkpoint inhibitor is administered at a dose of about 600 milligrams. In some embodiments, the immune checkpoint inhibitor is administered in an amount of 200 mg every six weeks, 300 mg every six weeks, or 400 mg every six weeks.In some embodiments, the immune checkpoint inhibitor is administered in an amount of 100 mg every three weeks or 300 mg every six weeks. In some embodiments, the immune checkpoint inhibitor is administered in an amount of 2 mg / kg or 1 mg / kg every 3 weeks. In some embodiments, the immune checkpoint inhibitor is administered in an amount of 50 mg every three weeks or 100 mg every six weeks. In some embodiments, the immune checkpoint inhibitor is administered in an amount of about 50 mg to about 400 mg every three weeks. In some embodiments, the immune checkpoint inhibitor is administered as a 25 mg / mL solution. In some embodiments, the immune checkpoint inhibitor is administered by injection or intravenous infusion. In some embodiments, the immune checkpoint inhibitor is administered at a dose of about 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.0, 2.25, 2.50, 2.75, 3.00, 3.5, 4, 4.5, 5, 5.5, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mg / kg. In some embodiments, the ROR1 antagonist is administered at a dose of about 200 milligrams to about 2000 milligrams. In some embodiments, the ROR1 antagonist is administered at a dose of about 200 milligrams to about 800 milligrams. In some embodiments, the ROR1 antagonist is administered at a dose of about 300 milligrams to about 600 milligrams. In some embodiments, the ROR1 antagonist is administered at a dose of about 300 milligrams. In some embodiments, the ROR1 antagonist is administered at a dose of about 600 milligrams. In some embodiments, the ROR1 antagonist is administered at a dose of about 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.0, 2.25, 2.50, 2.75, 3.00, 3.5, 4, 4.5, 5, 5.5, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mg / kg. In some embodiments, the ROR1 antagonist is administered as a 25 mg / mL solution. In some embodiments, the ROR1 antagonist is administered once every two weeks. In some embodiments, the ROR1 antagonist is administered once every three weeks. In some embodiments, the ROR1 antagonist is administered once every four weeks. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.In some embodiments, the cancer is a lymphoma, leukemia, myeloma, renal cell carcinoma, colon cancer, colorectal cancer, breast cancer, squamous cell carcinoma of the epithelium, melanoma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, uterine cancer, adenocarcinoma, sarcoma, or adrenal cancer. In some embodiments, the cancer is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, mantle cell lymphoma (MCL), marginal zone B-cell lymphoma, Burkitt's lymphoma, myeloma, AML, B-ALL, T-ALL, or B-cell leukemia.

[0077] Cancer

[0078] As used herein and unless otherwise specified, the term "tumor" or "cancer" refers to neoplastic cell growth and includes pre-cancerous and cancerous cells and tissues. Tumors typically exist as lesions or masses. As used herein, "treating" a tumor means reducing, ameliorating, inhibiting, placing in or maintaining in a state of remission one or more symptoms of the disease, such as the tumor itself, vascularization of the tumor, or other parameters characteristic of the disease. "Treating" a tumor also means that one or more markers of the tumor can be eliminated, reduced, or prevented by the treatment. Non-limiting examples of such markers include uncontrolled degradation of the basement membrane and proximal extracellular matrix, migration, division, and organization of endothelial cells into new functional capillaries, and persistence of such functional capillaries.

[0079] Cancers, pre-cancerous diseases, tumors, and / or neoplastic diseases treated with the methods and compositions of the present disclosure are not limited to any particular cell or tumor type or specific cancer, and include any such disease (e.g., cancer) present in a cell, such as a cancer cell. In certain aspects, the peptide component of the antigen / MCH / co-stimulatory molecule / nanoparticle complex is an antigen or antigenic epitope expressed or present in a tumor cell, cancer cell, pre-cancerous cell, or neoplastic cell, or is derived or designed from a mimetic thereof. Various such proteins or epitopes have been identified for various cancers.

[0080] In some embodiments, the cancer is a lymphoma, leukemia, myeloma, renal cell carcinoma, colon cancer, colorectal cancer, breast cancer, squamous cell carcinoma of the epithelium, melanoma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, uterine cancer, adenocarcinoma, sarcoma, or adrenal cancer. In some embodiments, the cancer is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, mantle cell lymphoma (MCL), marginal zone B-cell lymphoma, Burkitt's lymphoma, myeloma, AML, B-ALL, T-ALL, or B-cell leukemia. In some embodiments, the cancer is mantle cell lymphoma.

[0081] In some embodiments, the cancer comprises a mutated PD-L1 gene. In some embodiments, the cancer expresses PD-L1. In some embodiments, the cancer overexpresses PD-L1. Pharmaceutically acceptable excipients, carriers, and diluents

[0082] In certain embodiments, the anti-ROR1 antibody and / or checkpoint inhibitor of the present disclosure is comprised in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. In certain embodiments, the anti-ROR1 antibody and / or checkpoint inhibitor of the present disclosure is administered suspended in a sterile solution. In certain embodiments, the solution comprises about 0.9% NaCl. In certain embodiments, the solution comprises about 5.0% dextrose. In certain embodiments, the solution further comprises one or more of: a buffering agent, e.g., acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylamino methane (Tris); a surfactant, e.g., polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; a polyol / di- / polysaccharide, e.g., glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; an amino acid, e.g., glycine or arginine; an antioxidant, e.g., ascorbic acid, methionine; or a chelating agent, e.g., EDTA or EGTA.

[0083] In certain embodiments, the anti-ROR1 antibody and / or checkpoint inhibitor of the present disclosure can be shipped / stored lyophilized and reconstituted prior to administration. In certain embodiments, the lyophilized anti-ROR1 antibody and / or checkpoint inhibitor formulation comprises a bulking agent, such as mannitol, sorbitol, sucrose, trehalose, dextran 40, or a combination thereof. The lyophilized formulation can be housed in a vial made of glass or other suitable non-reactive material. When formulated, whether reconstituted or not, the anti-ROR1 antibody and / or checkpoint inhibitor can be buffered at a certain pH, typically less than 7.0. In certain embodiments, the pH can be between 4.5 and 7.0, 4.5 and 6.5, 4.5 and 6.0, 4.5 and 5.5, 4.5 and 5.0, or 5.0 and 6.0.

[0084] Also described herein are kits comprising one or more of the anti-ROR1 antibody and / or checkpoint inhibitor described herein in a suitable container, and one or more additional components selected from the group consisting of: instructions for use, diluents, excipients, carriers, and devices for administration.

[0085] In certain embodiments, described herein is a method of preparing a plasma cell neoplasm therapeutic comprising mixing one or more pharmaceutically acceptable excipients, carriers, or diluents with an anti-ROR1 antibody and / or checkpoint inhibitor of the present disclosure. In certain embodiments, described herein is a method of preparing a cancer therapeutic for storage or transport comprising lyophilizing one or more anti-ROR1 antibodies and / or checkpoint inhibitors of the present disclosure.

[0086] Definitions

[0087] Unless otherwise defined, all technical and scientific terms, symbols and other technical and scientific terms or nomenclature used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0088] Throughout this application, various embodiments can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed the end values and individual numerical values within that range, and any combination of the range with any other disclosed numerical values. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and individual numbers within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0089] As used in the specification and claims, the singular form “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof.

[0090] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein, and refer to forms of measurement. The terms include determining whether an element is present or not (e.g., detecting). The terms can include quantitative, qualitative, or both quantitative and qualitative determinations. Assessing can be relative or absolute. Depending on the context, “detecting the presence of” can include detecting the amount of something present, as well as detecting whether it is present or not.

[0091] As used herein, the term "ROR1" relates to human ROR1 (synonyms: tyrosine-protein kinase transmembrane receptor ROR1, EC = 2.7.10.1, neurotrophic tyrosine kinase, receptor-related 1, UniProtKB Q01973), which is a tyrosine-protein kinase receptor. The extracellular domain of ROR1 consists of amino acids 30-406. As used herein, the term "antibody against ROR1, anti-ROR1 antibody or ROR1 Mab" relates to an antibody which specifically binds to human ROR1. The antibody specifically binds to the extracellular domain of ROR1. The antibody specifically binds to a fragment of the extracellular domain which is an Ig-like C2-type domain, a frizzled domain or a kringle domain. These fragments are mentioned in WO2005100605. It is further disclosed that the antibody can specifically bind to the extracellular domain fragment WNISSELNKDSYLTL of ROR1. This fragment is disclosed in Daneshmanesh A H et al., Int. J. Cancer, 123 (2008) 1190-1195.

[0092] As used herein, the term "about" refers to an amount that is near the stated amount by 10% or less.

[0093] As used herein, the term "individual," "patient," or "subject" refers to an individual diagnosed with, suspected of having, or at risk of developing at least one disease for which the described compositions and methods are useful in treating. In certain embodiments, the individual is a mammal. In certain embodiments, the individual is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, vicuna, alpaca, or yak. In certain embodiments, the individual is a human.

[0094] As used herein, the term "treat" or "treatment" refers to the intended intervention in the physiological state of a disease, resulting in a decrease in severity of the disease or condition; a decrease in duration of the condition; an improvement or elimination of one or more symptoms associated with the disease or condition; or providing a beneficial effect to the subject with the disease or condition. Treatment does not require curing the underlying disease or condition.

[0095] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes the regression of a disease, as evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The ability of a therapeutic agent to promote the regression of a disease can be assessed using a variety of methods known to the skilled artisan, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0096] As used herein, reference to "carrier," "excipient," or "diluent," "pharmaceutically acceptable" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. In some aspects, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, intraspinal, or transdermal use (e.g., by injection or infusion). Depending on the route of administration, the active compound (i.e., antibody) can be coated in a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound.

[0097] The pharmaceutical compounds described herein can comprise one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salt" refers to salts that retain the desired biological activity of the parent compound and do not impart any undesired toxicological effects (see, e.g., Berge, S.M., et al. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous, and the like, as well as those derived from nontoxic organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and

[0098] As described herein, an "epitope" refers to the minimum amount of binding determinants of an antibody or fragment described herein necessary for specific binding of the antibody or fragment to a target antigen. When the target antigen is a polypeptide, the epitope will be a contiguous or discontinuous epitope. A contiguous epitope is formed from one region of the target antigen, while a discontinuous epitope can be formed from two or more separate regions. For example, a discontinuous epitope can be formed when the target antigen adopts a tertiary structure that brings together two amino acid sequences and forms a three-dimensional structure that is bound by the antibody. When the target antigen is a polypeptide, the epitope will typically be a plurality of amino acids that are linked together in a polypeptide chain. A contiguous epitope can comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids. While an epitope can comprise a contiguous polymer of amino acids, not every amino acid of the polymer can be in contact with an amino acid residue of the antibody. Such non-contacting amino acids will still form part of the epitope because they can be important to the structure and linkage of the contacting amino acids. One of skill in the art can determine whether a given antibody binds to the epitope of a reference antibody, e.g., by cross-blocking experiments with the reference antibody. In certain embodiments, antibodies are described herein that bind to the same epitope of the described antibodies. In certain embodiments, antibodies are described herein that are competitively blocked by the described antibodies. In certain embodiments, antibodies are described herein that compete for binding with the described antibodies.

[0099] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0100] EMBODIMENTS

[0101] The following examples are included solely for the purpose of illustration and are not intended to limit the scope of the present application.

[0102] Example 1: Combination treatment of cancer with pembrolizumab and zanolimumab

[0103] Human donor cells were screened in vitro. Donor cells from two different donors were combined with Jeko-1 cells and stimulated with anti-CD3 / CD28 (5 pg / mL each), pembrolizumab (1 pg / mL), zanolimumab (20 pg / mL), or a combination of pembrolizumab (1 pg / mL) and zanolimumab (20 pg / mL) and incubated at the indicated effector to target ratios. As shown in FIG. 1 Zanolimumab modulates T cell activity in vitro and synergizes with pembrolizumab, with the synergy being more apparent at lower effector to target cell concentrations. Donor lines 451 and 244 were not highly allogeneic to Jeko-1 cells and were thus selected for in vivo studies.

[0104] The efficacy of anti-RORl, anti-PDl, and the combination of anti-RORl and anti-PDl was evaluated in the Jeko-1 MiXeno model in NSG mice. Tumor volume was measured over 25 days. Mice were treated with vehicle, zanolimomab (10 mg / kg), pembrolizumab (10 mg / kg), or the combination of zanolimomab and pembrolizumab (10 mg / kg each). The results are shown in Figure 1 and Table 1. Tumor growth inhibition (TGI) is expressed as a mean percentage of inhibition of delta and calculated using the following formula: [(C-C0)-(T-T0) / (C-C0)]*100%, where C refers to the tumor volume on a particular day of dosing with vehicle, C0 refers to the tumor volume on the first day of dosing with vehicle, T refers to the tumor volume on a particular day of treatment, and T0 refers to the tumor volume on the first day of treatment in the treatment group. The combination treatment resulted in greater tumor growth inhibition in both donor cells. The combination of the RORl antagonist and the checkpoint inhibitor was observed to provide a synergistic effect in the treatment of cancer in a subject in need thereof, which was superior to the effect of the RORl antagonist or the checkpoint inhibitor alone. FIGS. 2A-2B and Table 1. Tumor growth inhibition (TGI) is expressed as a mean percentage of inhibition of delta and calculated using the following formula: [(C-C0)-(T-T0) / (C-C0)]*100%, where C refers to the tumor volume on a particular day of dosing with vehicle, C0 refers to the tumor volume on the first day of dosing with vehicle, T refers to the tumor volume on a particular day of treatment, and T0 refers to the tumor volume on the first day of treatment in the treatment group. The combination treatment resulted in greater tumor growth inhibition in both donor cells. The combination of the RORl antagonist and the checkpoint inhibitor was observed to provide a synergistic effect in the treatment of cancer in a subject in need thereof, which was superior to the effect of the RORl antagonist or the checkpoint inhibitor alone.

[0105] Table 1: Tumor growth inhibition

[0106]

[0107] Delta is the difference between combination therapy and monotherapy

[0108] Example 2: Clinical trial of combination treatment of lymphoma with pembrolizumab and zanolimumab

[0109] This study aims to determine the efficacy of the combination of pembrolizumab and zanolimomab in the treatment of lymphoma. In addition, this study will also generate safety, tolerability, and pharmacokinetic data for the combination therapy in subjects with mantle cell lymphoma. Subjects will be administered placebo or the combination therapy and monitored. The following primary and secondary outcome measures will be assessed:

[0110] Primary outcome measure

[0111] The primary outcome measure includes tumor growth reduction.

[0112] Secondary outcome measures

[0113] The secondary outcome measures include tumor mass reduction, tumor volume reduction, and survival extension over time.

[0114] Inclusion criteria

[0115] Subjects with mantle cell lymphoma

[0116] Regimen

[0117] Subjects will be administered either placebo or a combination of pembrolizumab and zoliflutabine. At the end of the trial, subjects will be evaluated according to the criteria described above.

[0118] While preferred embodiments of the application have been shown and described herein, it is to be understood that the application is not limited to these embodiments. It will be apparent to those skilled in the art that various modifications, changes, and substitutions can be made thereto without departing from the application. It is the intention, therefore, to limit the application by the scope of the following claims and by the equivalents thereof.

[0119] Sequence

[0120]

[0121]

Claims

1. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a tyrosine kinase-like orphan receptor 1 (ROR1) antagonist and administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor.

2. The method according to any one of the preceding claims, wherein the immune checkpoint inhibitor comprises a PD-1 inhibitor or a PD-L1 inhibitor.

3. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor is a small molecule drug.

4. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor comprises an antibody, a peptide, or a protein.

5. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor comprises a PD-1 inhibitor. The method of claim 5 , wherein the PD-1 inhibitor is a PD-1 inhibitor antibody.

7. The method of claim 6, wherein the PD-1 inhibitor antibody comprises nivolumab, pembrolizumab, cemiplizumab, dotalimumab, or a combination thereof.

8. The method of claim 7, wherein the PD-1 inhibitor antibody comprises pembrolizumab.

9. The method of claim 6, wherein the PD-1 inhibitor antibody comprises JTX-4014, spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), toripalimab (JS 001), rivalizumab (INCMGA00012) (MGA012), AMP-224, AMP-514, or a combination thereof.

10. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor comprises a PD-L1 inhibitor. The method of claim 10 , wherein the PD-L1 inhibitor is a PD-L1 inhibitor antibody.

12. The method of claim 11, wherein the PD-L1 inhibitor antibody comprises atezolizumab, avelumab, durvalumab, or a combination thereof.

13. The method of claim 11, wherein the PD-L1 inhibitor antibody comprises enfalizumab (KN035), coxilimab (CK-301), AUNP12, CA-170, BMS-986189, or a combination thereof.

14. The method of any one of the preceding claims, wherein the ROR1 antagonist is an antibody or a small molecule.

15. The method of any one of the preceding claims, wherein the antibody comprises a Fab, a F(ab')2, a Fv, or a scFv.

16. The method of any one of the preceding claims, wherein the ROR1 antagonist is an anti-ROR1 antibody.

17. The method of any one of the preceding claims, wherein the antibody is zanetuzumab.

18. The method of any one of the preceding claims, wherein the antibody binds to the ROR1 amino acid sequence as shown in SEQ ID NO: 30 or SEQ ID NO:

31.

19. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor and the ROR1 antagonist are administered in a combined synergistic amount.

20. The method of any preceding claim, further comprising reducing the growth of a tumor.

21. The method of any one of the preceding claims, wherein the method reduces tumor growth by about 20% or more.

22. The method of any one of the preceding claims, wherein the method reduces tumor growth by about 25% to about 45% when compared to administering the immune checkpoint inhibitor and the ROR1 antagonist separately.

23. The method of any one of the preceding claims, further comprising reducing the mass or volume of a tumor.

24. The method of any one of the preceding claims, wherein the antibody comprises a humanized heavy chain variable region and a humanized light chain variable region, wherein the humanized heavy chain variable region comprises the sequence shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3; and wherein the humanized light chain variable region comprises the sequence shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.

6.

25. The method of any of the preceding claims, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:7; and wherein the light chain variable region comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

8.

26. The method of any one of the preceding claims, wherein the immune cell infiltrate of the cancer expresses PD-1.

27. The method of any one of the preceding claims, wherein the cancer expresses PD-L1.

28. The method of any one of the preceding claims, wherein the cancer overexpresses PD-L1.

29. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor and the ROR1 antagonist are administered substantially simultaneously.

30. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor and the ROR1 antagonist are administered separately.

31. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor and the ROR1 antagonist are administered sequentially.

32. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor and the ROR1 antagonist are co-formulated for administration via a single formulation.

33. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor and the ROR1 antagonist are administered as separate compositions.

34. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor and the ROR1 antagonist are mixed prior to administration.

35. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered by intravenous infusion.

36. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered once every two weeks.

37. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered once every three weeks.

38. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered once every four weeks.

39. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered at a dose of about 200 mg to about 2000 mg.

40. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered at a dose of about 200 mg to about 800 mg.

41. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered at a dose of about 300 mg to about 600 mg.

42. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered at a dose of about 300 mg.

43. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered at a dose of about 600 mg.

44. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered in an amount of 200 mg every six weeks, 300 mg every six weeks, or 400 mg every six weeks.

45. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered in an amount of 100 mg every three weeks or 300 mg every six weeks.

46. ​​The method of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered in an amount of 2 mg / kg or 1 mg / kg every 3 weeks.

47. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered in an amount of 50 mg every three weeks or 100 mg every six weeks.

48. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered in an amount of about 50 mg to about 400 mg every three weeks.

49. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered as a 25 mg / mL solution.

50. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered by injection or intravenous infusion.

51. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered at a dose of about 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.0, 2.25, 2.50, 2.75, 3.00, 3.5, 4, 4.5, 5, 5.5, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mg / kg.

52. The method of any one of the preceding claims, wherein the ROR1 antagonist is administered at a dose of about 200 mg to about 2000 mg.

53. The method of any one of the preceding claims, wherein the ROR1 antagonist is administered at a dose of about 200 mg to about 800 mg.

54. The method of any one of the preceding claims, wherein the ROR1 antagonist is administered at a dose of about 300 mg to about 600 mg.

55. The method of any one of the preceding claims, wherein the ROR1 antagonist is administered at a dose of about 300 mg.

56. The method of any one of the preceding claims, wherein the ROR1 antagonist is administered at a dose of about 600 mg.

57. The method of any one of the preceding claims, wherein the RORl antagonist is administered at a dose of about 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.0, 2.25, 2.50, 2.75, 3.00, 3.5, 4, 4.5, 5, 5.5, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mg / kg.

58. The method of any preceding claim, wherein the ROR1 antagonist is administered as a 25 mg / mL solution.

59. The method of any one of the preceding claims, wherein the ROR1 antagonist is administered biweekly.

60. The method of any preceding claim, wherein the ROR1 antagonist is administered once every three weeks.

61. The method of any preceding claim, wherein the ROR1 antagonist is administered once every four weeks.

62. The method of any preceding claim, wherein the subject is a mammal.

63. The method of any of the preceding claims, wherein the subject is a human.

64. The method of any one of the preceding claims, wherein the cancer is lymphoma, leukemia, myeloma, renal cell carcinoma, colon cancer, colorectal cancer, breast cancer, epithelial squamous cell carcinoma, melanoma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, uterine cancer, adenocarcinoma, sarcoma, or adrenal cancer.

65. The method of any one of the preceding claims, wherein the cancer is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, mantle cell lymphoma (MCL), marginal zone B-cell lymphoma, Burkitt lymphoma, myeloma, AML, B-ALL, T-ALL, or B-cell leukemia.

66. A pharmaceutical composition comprising a therapeutically effective amount of a tyrosine kinase-like orphan receptor 1 (ROR1) antagonist and a therapeutically effective amount of an immune checkpoint inhibitor.

67. The pharmaceutical composition of any one of the preceding claims, wherein the immune checkpoint inhibitor comprises a PD-1 inhibitor or a PD-L1 inhibitor.

68. A pharmaceutical composition according to any one of the preceding claims, wherein the immune checkpoint inhibitor is a small molecule drug.

69. A pharmaceutical composition according to any one of the preceding claims, wherein the immune checkpoint inhibitor is an antibody.

70. The pharmaceutical composition of any one of the preceding claims, wherein the immune checkpoint inhibitor comprises a PD-1 inhibitor.

71. The pharmaceutical composition of claim 70, wherein the PD-1 inhibitor is a PD-1 inhibitor antibody.

72. The pharmaceutical composition of claim 71, wherein the PD-1 inhibitor antibody comprises nivolumab, pembrolizumab, cemiplizumab, dotarizumab, or a combination thereof.

73. The pharmaceutical composition of claim 72, wherein the PD-1 inhibitor antibody comprises pembrolizumab.

74. The pharmaceutical composition of claim 71, wherein the PD-1 inhibitor antibody comprises JTX-4014, spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), toripalimab (JS 001), rivalizumab (INCMGA00012) (MGA012), AMP-224, AMP-514, or a combination thereof.

75. The pharmaceutical composition of any one of the preceding claims, wherein the immune checkpoint inhibitor comprises a PD-L1 inhibitor.

76. The pharmaceutical composition of claim 75, wherein the PD-L1 inhibitor is a PD-L1 inhibitor antibody.

77. The pharmaceutical composition of claim 76, wherein the PD-L1 inhibitor antibody comprises atezolizumab, avelumab, durvalumab, or a combination thereof.

78. The pharmaceutical composition of claim 75, wherein the PD-L1 inhibitor antibody comprises enfalizumab (KN035), coxilimab (CK-301), AUNP12, CA-170, BMS-986189, or a combination thereof.

79. The pharmaceutical composition of any preceding claim, wherein the ROR1 antagonist is an antibody or a small molecule.

80. The pharmaceutical composition of any one of the preceding claims, wherein the antibody comprises a Fab, a F(ab')2, a Fv, or a scFv.

81. The pharmaceutical composition of any preceding claim, wherein the ROR1 antagonist is an anti-ROR1 antibody.

82. The pharmaceutical composition of any one of the preceding claims, wherein the antibody is zalotuzumab.

83. The pharmaceutical composition of any one of the preceding claims, wherein the immune checkpoint inhibitor and the ROR1 antagonist are administered in a combined synergistic amount.

84. The pharmaceutical composition of any one of the preceding claims, wherein when administered to a subject, the pharmaceutical composition reduces the growth of a tumor.

85. The pharmaceutical composition of any one of the preceding claims, wherein when administered to a subject, the pharmaceutical composition reduces the growth of a tumor by about 20% or more.

86. The pharmaceutical composition of any of the preceding claims, wherein when administered to a subject, the pharmaceutical composition reduces the growth of a tumor by about 25% to about 45% when compared to administering the immune checkpoint inhibitor and the ROR1 antagonist separately.

87. The pharmaceutical composition of any one of the preceding claims, wherein when administered to a subject, the pharmaceutical composition reduces the mass or volume of a tumor.

88. A pharmaceutical composition according to any of the preceding claims, wherein the antibody comprises a humanized heavy chain variable region and a humanized light chain variable region, wherein the humanized heavy chain variable region comprises the sequence shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3; and wherein the humanized light chain variable region comprises the sequence shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.

6.

89. A pharmaceutical composition according to any of the preceding claims, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:7; and wherein the light chain variable region comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

8.

90. A pharmaceutical composition according to any of the preceding claims, wherein the immune checkpoint inhibitor is included in an amount of about 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.0, 2.25, 2.50, 2.75, 3.00, 3.5, 4, 4.5, 5, 5.5, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5 or 20 mg / kg.

91. The pharmaceutical composition of any of the preceding claims, wherein the composition comprises the ROR1 antagonist in an amount of about 200 mg to about 2000 mg.

92. The pharmaceutical composition of any of the preceding claims, wherein the composition comprises the ROR1 antagonist in an amount of about 200 mg to about 800 mg.

93. The pharmaceutical composition of any of the preceding claims, wherein the composition comprises the ROR1 antagonist in an amount of about 300 mg to about 600 mg.

94. The pharmaceutical composition of any of the preceding claims, wherein the composition comprises the ROR1 antagonist in an amount of about 300 mg to about 600 mg.

95. The pharmaceutical composition of any one of the preceding claims, wherein the composition comprises the ROR1 antagonist in an amount of about 300 mg.

96. The pharmaceutical composition of any of the preceding claims, wherein the composition comprises the ROR1 antagonist in an amount of about 600 mg.

97. The pharmaceutical composition of any of the preceding claims, wherein the composition comprises the ROR1 antagonist in an amount of about 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.0, 2.25, 2.50, 2.75, 3.00, 3.5, 4, 4.5, 5, 5.5, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mg / kg.

98. The pharmaceutical composition of any of the preceding claims, wherein the composition comprises the ROR1 antagonist in an amount of about 10, 15, 20, 25, or 30 mg / mL.

99. The pharmaceutical composition of any one of the preceding claims, wherein the composition comprises the immune checkpoint inhibitor in an amount of about 200 mg to about 800 mg.

100. The pharmaceutical composition of any one of the preceding claims, wherein the composition comprises the immune checkpoint inhibitor in an amount of about 300 mg to about 600 mg.

101. The pharmaceutical composition of any one of the preceding claims, wherein the composition comprises the immune checkpoint inhibitor in an amount of about 300 mg to about 600 mg.

102. A pharmaceutical composition according to any of the preceding claims, wherein the composition comprises the immune checkpoint inhibitor in an amount of about 300 mg.

103. A pharmaceutical composition according to any of the preceding claims, wherein the composition comprises the immune checkpoint inhibitor in an amount of about 600 mg.

104. The pharmaceutical composition of any of the preceding claims, wherein the composition comprises the immune checkpoint inhibitor in an amount of about 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.0, 2.25, 2.50, 2.75, 3.00, 3.5, 4, 4.5, 5, 5.5, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mg / kg.

105. The pharmaceutical composition of any of the preceding claims, wherein the composition comprises the immune checkpoint inhibitor in an amount of about 10, 15, 20, 25, or 30 mg / mL.

106. The pharmaceutical composition of any one of the preceding claims, wherein the composition comprises a combined synergistic amount of the ROR1 antagonist and the immune checkpoint inhibitor.

107. The pharmaceutical composition of any one of the preceding claims, wherein the composition comprises a combined synergistic amount of the ROR1 antagonist and the immune checkpoint inhibitor effective for treating cancer in a subject.

108. The pharmaceutical composition of claim 107, wherein the cancer comprises a mutated PD-L1 gene.

109. The pharmaceutical composition of claim 107, wherein the cancer expresses PD-L1.

110. The pharmaceutical composition of claim 107, wherein the cancer overexpresses PD-L1.

111. The pharmaceutical composition of claim 107, wherein the cancer is lymphoma, leukemia, myeloma, renal cell carcinoma, colon cancer, colorectal cancer, breast cancer, epithelial squamous cell carcinoma, melanoma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, uterine cancer, adenocarcinoma, sarcoma, or adrenal cancer.

112. The pharmaceutical composition of claim 107, wherein the cancer is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, mantle cell lymphoma (MCL), marginal zone B-cell lymphoma, Burkitt lymphoma, myeloma, AML, B-ALL, T-ALL, or B-cell leukemia.

113. The pharmaceutical composition of any one of the preceding claims, wherein the ROR1 antagonist and the immune checkpoint inhibitor are in separate compositions.

114. Use of the pharmaceutical composition of any one of claims 66-113 in a method of treating cancer in a subject in need thereof, comprising administering the pharmaceutical composition to the subject.

115. Use of a therapeutically effective amount of a tyrosine kinase-like orphan receptor 1 (ROR1) antagonist and a therapeutically effective amount of an immune checkpoint inhibitor in a method of treating cancer in a subject in need thereof, comprising administering the pharmaceutical composition to the subject.

116. Use of a therapeutically effective amount of a tyrosine kinase-like orphan receptor 1 (ROR1) antagonist and a therapeutically effective amount of an immune checkpoint inhibitor for the preparation of a medicament for treating cancer in a subject in need thereof.

117. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor comprises a PD-1 inhibitor or a PD-L1 inhibitor.

118. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor is a small molecule drug.

119. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor comprises an antibody, a peptide or a protein.

120. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor comprises a PD-1 inhibitor.

121. The use according to claim 120, wherein the PD-1 inhibitor is a PD-1 inhibitor antibody.

122. The use according to claim 121, wherein the PD-1 inhibitor antibody comprises nivolumab, pembrolizumab, cemiplizumab, dotalizumab, or a combination thereof.

123. The use of claim 122, wherein the PD-1 inhibitor antibody comprises pembrolizumab.

124. The use according to claim 121, wherein the PD-1 inhibitor antibody comprises JTX-4014, spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), toripalimab (JS 001), rivalizumab (INCMGA00012) (MGA012), AMP-224, AMP-514 or a combination thereof.

125. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor comprises a PD-L1 inhibitor.

126. The use according to claim 125, wherein the PD-L1 inhibitor is a PD-L1 inhibitor antibody.

127. The use according to claim 126, wherein the PD-L1 inhibitor antibody comprises atezolizumab, avelumab, durvalumab, or a combination thereof.

128. The use according to claim 126, wherein the PD-L1 inhibitor antibody comprises Enfalizumab (KN035), Coxilimab (CK-301), AUNP12, CA-170, BMS-986189 or a combination thereof.

129. The use of any preceding claim, wherein the ROR1 antagonist is an antibody or a small molecule.

130. The use of any preceding claim, wherein the antibody comprises a Fab, a F(ab')2, a Fv, or a scFv.

131. The use of any preceding claim, wherein the ROR1 antagonist is an anti-ROR1 antibody.

132. The use of any preceding claim, wherein the antibody is zelotuzumab.

133. The use of any preceding claim, wherein the antibody binds to the ROR1 amino acid sequence shown in SEQ ID NO: 30 or SEQ ID NO:

31.

134. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor and the ROR1 antagonist are administered in a combined synergistic amount.

135. The use of any one of the preceding claims, further comprising reducing the growth of a tumor.

136. The use of any one of the preceding claims, wherein the use reduces the growth of a tumor by about 20% or more.

137. The use of any one of the preceding claims, wherein the use reduces the growth of a tumor by about 25% to about 45% when compared to administering the immune checkpoint inhibitor and the ROR1 antagonist separately.

138. The use of any preceding claim, further comprising reducing the mass or volume of a tumor.

139. The use of any of the preceding claims, wherein the antibody comprises a humanized heavy chain variable region and a humanized light chain variable region, wherein the humanized heavy chain variable region comprises the sequence shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3; and wherein the humanized light chain variable region comprises the sequence shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.

6.

140. The use of any of the preceding claims, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:7; and wherein the light chain variable region comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

8.

141. The use of any preceding claim, wherein the immune cell infiltrate of the cancer expresses PD-1.

142. The use of any preceding claim, wherein the cancer expresses PD-L1.

143. The use of any preceding claim, wherein the cancer overexpresses PD-L1.

144. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor and the ROR1 antagonist are administered substantially simultaneously.

145. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor and the ROR1 antagonist are administered separately.

146. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor and the ROR1 antagonist are administered sequentially.

147. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor and the ROR1 antagonist are co-formulated for administration via a single formulation.

148. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor and the ROR1 antagonist are administered as separate compositions.

149. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor and the ROR1 antagonist are mixed prior to administration.

150. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered by intravenous infusion.

151. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered once every two weeks.

152. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered once every three weeks.

153. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered once every four weeks.

154. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered at a dose of about 200 mg to about 2000 mg.

155. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered at a dose of about 200 mg to about 800 mg.

156. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered at a dose of about 300 mg to about 600 mg.

157. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered at a dose of about 300 mg.

158. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered at a dose of about 600 mg.

159. The use of any of the preceding claims, wherein the immune checkpoint inhibitor is administered in an amount of 200 mg every six weeks, 300 mg every six weeks, or 400 mg every six weeks.

160. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered in an amount of 100 mg every three weeks or 300 mg every six weeks.

161. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered in an amount of 2 mg / kg or 1 mg / kg every 3 weeks.

162. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered in an amount of 50 mg every three weeks or 100 mg every six weeks.

163. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered in an amount of about 50 mg to about 400 mg every three weeks.

164. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered as a 25 mg / mL solution.

165. The use of any one of the preceding claims, wherein the immune checkpoint inhibitor is administered by injection or intravenous infusion.

166. The use of any of the preceding claims, wherein the immune checkpoint inhibitor is administered at a dose of about 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.0, 2.25, 2.50, 2.75, 3.00, 3.5, 4, 4.5, 5, 5.5, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mg / kg.

167. The use of any preceding claim, wherein the ROR1 antagonist is administered at a dose of about 200 mg to about 2000 mg.

168. The use of any preceding claim, wherein the ROR1 antagonist is administered at a dose of about 200 mg to about 800 mg.

169. The use of any preceding claim, wherein the ROR1 antagonist is administered at a dose of about 300 mg to about 600 mg.

170. The use of any preceding claim, wherein the ROR1 antagonist is administered at a dose of about 300 mg.

171. The use of any preceding claim, wherein the ROR1 antagonist is administered at a dose of about 600 mg.

172. The use of any of the preceding claims, wherein the ROR1 antagonist is administered at a dose of about 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.0, 2.25, 2.50, 2.75, 3.00, 3.5, 4, 4.5, 5, 5.5, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5 or 20 mg / kg.

173. The use of any preceding claim, wherein the ROR1 antagonist is administered as a 25 mg / mL solution.

174. The use of any preceding claim, wherein the ROR1 antagonist is administered once every two weeks.

175. The use of any preceding claim, wherein the ROR1 antagonist is administered once every three weeks.

176. The use of any preceding claim, wherein the ROR1 antagonist is administered once every four weeks.

177. The use of any preceding claim, wherein the subject is a mammal.

178. The use of any preceding claim, wherein the subject is a human.

179. The use according to any of the preceding claims, wherein the cancer is lymphoma, leukemia, myeloma, renal cell carcinoma, colon cancer, colorectal cancer, breast cancer, epithelial squamous cell carcinoma, melanoma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, uterine cancer, adenocarcinoma, sarcoma or adrenal cancer.

180. The use of any one of the preceding claims, wherein the cancer is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, mantle cell lymphoma (MCL), marginal zone B-cell lymphoma, Burkitt's lymphoma, myeloma, AML, B-ALL, T-ALL, or B-cell leukemia.

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