Crystals of anti-human PD-1 monoclonal antibodies
Crystals of pembrolizumab and structurally similar anti-PD-1 monoclonal antibodies are provided, as well as methods of producing such crystals, and uses of compositions comprising such antibody crystals, e.g. in treatment of cancers. The present invention satisfies these needs and more by providing pembrolizumab crystals and a method producing pembrolizumab crystals. One embodiment of the method of the invention produces crystals suitable for X-ray diffraction, and the inventors herein used such crystals to solve the three-dimensional structure of pembrolizumab to 2.3 A resolution.
1. A crystal of an anti-PD-1 monoclonal antibody (mAb), wherein the anti-PD-1 mAb is pembrolizumab and the crystal is characterized by unit cell dimensions of a=63.5 to 78.9 Å, b=110.2 to 112.2 Å, c=262.5 to 306 Å, α=90, β=90, γ =90° and a space group of P2 1 2 1 2 1 .
2. The crystal of claim 1 , wherein the crystal is characterized by having a length in a range of: 1 to 200 microns.
3. The crystal of claim 1 , which is capable of diffracting X-rays to a resolution selected from the group consisting of 2.3 Å to 3.5 Å, 2.3 Å to 3.0 Å, 2.3 Å to 2.75 Å, 2.3 Å to 2.5 Å and 2.3 Å.
4. A method for producing crystals of an anti-PD-1 monoclonal antibody (mAb), wherein the anti-PD-1 mAb is pembrolizumab and the crystal is characterized by unit cell dimensions of a=63.5 to 78.9 Å, b=110.2 to 112.2 Å, c=262.5 to 306 Å, α=90, β=90, γ =90° and a space group of P2 1 2 1 2 1 and the method comprises exposing a solution comprising the anti-PD-1 mAb to a precipitant solution at a temperature that is at least 25° C. and is no greater than 50° C. for a time sufficient for crystal formation, wherein the precipitant solution has a pH of 4.0 to 5.0 and comprises 1.0 M to 2.5 M ammonium dihydrogen phosphate.
5. The method of claim 4 , wherein the exposing step comprises mixing the antibody solution and the precipitant solution to form a crystallization mixture and applying a crystallization process to the mixture, wherein the crystallization process is selected from the group consisting of hanging drop vapor diffusion, sitting drop vapor diffusion and batch.
6. The method of claim 5 , wherein the crystallization process is a batch process and the method further comprises seeding the crystallization mixture with crystals of the anti-PD-1 mAb.
7. The method of claim 4 , wherein the antibody solution comprises the anti-PD-1 mAb at a concentration of 2 to 200 mg/ml, 3 to 100 mg/ml, 10 to 90 mg/ml, 20 to 80 mg/ml, 30 to 70 mg/ml, 40 to 60 mg/ml or about 50 mg/ml and the precipitant solution has a pH selected from the group consisting of 4.2 to 4.8, 4.4 to 4.6 and 4.5.
8. The method of claim 7 wherein the precipitant solution comprises (a) 1.5 M to 2.0 M ammonium dihydrogen phosphate and 100 to 120 mM Tris-HCl or (b) 1.9 M ammonium dihydrogen phosphate and 0.09 M ammonium hydrogen phosphate.
9. The method of claim 8 , wherein the exposing step is performed for at least 3, 4 or 5 days at a temperature of about 30° C.
10. A method for crystallizing an anti-PD-1 monoclonal antibody (mAb) from a solution comprising the anti-PD-1 mAb, wherein the antibody is pembrolizumab and the method comprises: (a) combining the anti-PD-1 mAb solution with a precipitant solution and seed crystals of the anti-PD-1 mAb to produce a seeded crystallization mixture; (b) incubating the seeded crystallization mixture at a temperature of at least 20° C. and no greater than about 40° C.; and (c) harvesting the crystals, wherein the seed crystals are from a seed stock of crystals of the anti-PD-1 mAb that were produced by a method of claim 4 .
11. A pharmaceutical composition comprising (a) crystals of an anti-PD-1 monoclonal antibody (mAb), wherein the antibody is pembrolizumab and the crystal is characterized by unit cell dimensions of a=63.5 to 78.9 Å, b=110.2 to 112.2 Å, c=262.5 to 306 Å, α=90, β=90, γ=90° and a space group of P2 1 2 1 2 1 and (b) at least one pharmaceutically acceptable excipient.
12. The composition of claim 11 , herein the anti-PD-1 mAb crystals are suspended in a liquid and the anti-PD-1 mAb concentration in the composition is at least 50 mg/ml, at least 100 mg/ml, at least 200 mg/ml or at least 250 mg/ml.
13. The composition of claim 11 , which is a solid.
14. A method of treating a human subject for a cancer, which comprises administering to the patient a therapeutically effective amount of a pharmaceutical composition of claim 11 .
15. The method of claim 14 , wherein the cancer is bladder cancer, breast cancer, clear cell kidney cancer, head/neck squamous cell carcinoma, lung squamous cell carcinoma, malignant melanoma, non-small-cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer, small-cell lung cancer (SCLC), triple negative breast cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CIVIL), diffuse large B-cell lymphoma (DLBCL), EBV-positive DLBCL, primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich large B-cell lymphoma, follicular lymphoma, Hodgkin's lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid cell leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), or small lymphocytic lymphoma (SLL).
16. The method of claim 15 , wherein the pharmaceutical composition comprises at least 200 mg/ml of the mAb and is administered subcutaneously.
17. The method of claim 15 , wherein the cancer is a solid tumor and a tissue section of the cancer removed from the subject prior to a first administration of the pharmaceutical composition tested positive for expression of one or both of PD-L1 and PD-L2.
18. A method for producing crystals of an anti-PD-1 monoclonal antibody (mAb), wherein the anti-PD1 mAb is pembrolizumab and the method comprises exposing a solution comprising the anti-PD-1 mAb to a precipitant solution at a temperature that is at least 25° C. and is no greater than 50° C. for a time sufficient for crystal formation, wherein the precipitant solution has a pH of 4.0 to 5.0 and comprises 1.0 M to 2.5 M ammonium dihydrogen phosphate.