Anti-human PD-1 antibody crystals and methods of use thereof

Abstract

The present invention provides a method for producing a crystalline anti-PD-1 monoclonal antibody (mAb) wherein the mAb is pembumab or a pembumab variant, the method comprising (1) mixing a solution comprising (a) mAb, (b) polyethylene glycol (PEG), and (c) an additive to form a crystalline solution, the additive is selected from caffeine, theophylline, 2 '-deoxyguanosine-5'-monophosphate, bioactive gibberellin and pharmaceutically acceptable salts of the bioactive gibberellin; (2) incubating the crystallization solution for a time sufficient for crystal formation; and (3) optionally harvesting the crystalline anti-PD-1mAb from the solution. In a particular embodiment, PEG is PEG 3350, and the additive is caffeine. The present invention also relates to novel anti-human PD-1mAb crystals produced by the methods described herein. Characterization of the redissolved crystal suspension using several biochemical methods shows that the biophysical properties of the redissolved mAb crystals coincide with the intact antibody starting sample. The crystals and methods of the present invention are suitable for a variety of pharmaceutical applications, such as purification, storage, formulation and drug delivery.

Description

[0001] This application is a divisional application of patent application No. 201980071745.8, filed on October 28, 2019, entitled "Anti-human PD-1 antibody crystal and method of use thereof". Technical Field

[0002] This invention relates to a method for producing a crystal suspension of an anti-PD-1 monoclonal antibody. The invention further relates to antibody crystals produced by the methods described herein, pharmaceutical compositions comprising the crystals, and methods of using the same.

[0003] Cross-references to related applications

[0004] This application claims the benefit of USSN 62 / 753,615, filed October 31, 2018, which is incorporated herein by reference in its entirety.

[0005] Reference to the electronically submitted sequence list

[0006] The sequence listing of this application was submitted electronically via EFS-Web as an ASCII format sequence listing, with the file name "24638WOPCT-SEQLIST-17OCT2019.TXT", creation date of October 9, 2019, and size of 10Kb. This sequence listing submitted via EFS-Web is part of this specification and is incorporated herein by reference in its entirety. Background Technology

[0007] Therapeutic and diagnostic antibodies have become the fastest-growing sector in the biopharmaceutical industry. A key aspect of the success of antibodies as therapeutic agents is the development of improved methods for expressing, purifying, and identifying these proteins. Generally, antibody therapeutics are large (typically greater than 150 kDa) and complex in nature, thus requiring administration at stoichiometric rather than catalytic levels. Consequently, production and purification have reached previously unattainable scales. Stable formulation and delivery strategies still need to be developed for such large quantities of complex molecules.

[0008] Developing stable formulations containing high concentrations of active agents, such as antibodies or antigen-binding fragments, is particularly important for biological agents intended for subcutaneous administration to patients, as the volume of solution delivered to the patient is significantly reduced. Subcutaneous administration is the preferred method of administration for many antibodies, partly because it allows for self-administration or makes administration easier for healthcare professionals such as pharmacists, doctors, or nurses. Therapeutic antibodies are traditionally prepared in lyophilized form or in solution. Lyophilized forms exhibit enhanced long-term stability but require reconstitution before use, making them less suitable for self-administration. On the other hand, the development of stable liquid formulations is more challenging and often requires refrigeration before use.

[0009] Immune checkpoint therapy targeting the programmed death receptor-1 (PD-1) axis has made groundbreaking progress in clinical response to various human cancers (Brahmer et al., N Engl J Med 2012, 366:2455-65; Garon et al. N Engl J Med 2015, 372:2018-28; Hamid et al., N Engl J Med 2013, 369:134-44; Robert et al., Lancet 2014, 384:1109-17; Robert et al., N Engl J Med 2015, 372:2521-32; Robert et al., N Engl J Med 2015, 372:320-30; Topalian et al., N Engl J Med 2012, 366:2443-54; Topalian et al., J Clin Oncol). 2014, 32:1020-30; Wolchok et al., N Engl J Med 2013, 369:122-33). The interaction between the PD-1 receptor on T cells and its ligands PD-L1 and PD-L2 on tumor and immune-infiltrating cells regulates T cell-mediated immune responses and may play a role in immune evasion in human tumors (Pardoll DM. Nat Rev Cancer 2012, 12:252-64). Binding of PD-1 to either of its ligands results in the delivery of inhibitory stimuli to T cells. Immunotherapy targeting the PD-1 axis includes monoclonal antibodies targeting the PD-1 receptor (KEYTRUDA). TM (pembrolizumab), Merck and Co., Inc., Kenilworth, NJ and OPDIVO TM (nivolumab), Bristol-Myers Squibb, Princeton, NJ) and monoclonal antibodies that bind to PD-L1 ligands (MPDL3280A; TECENTRIQ) TM (Atezolizumab), Genentech, San Francisco, CA). Both treatment approaches have demonstrated anti-tumor efficacy in multiple cancer types.

[0010] There is a need for stable formulations of improved anti-PD-1 antibodies for the treatment of, for example, cancer patients. Preferably, these antibody formulations do not require reconstitution prior to administration. Furthermore, these formulations enable the administration of higher antibody concentrations than are easily achievable with typical solution formulations, and preferably support high concentrations with low viscosity sufficient for convenient subcutaneous delivery. Summary of the Invention

[0011] In one aspect, the present invention relates to a method for producing crystalline anti-PD-1 monoclonal antibody (mAb), comprising: (a) mixing: (i) an aqueous buffer solution containing about 5 mg / mL to about 80 mg / mL of mAb, wherein the anti-PD-1 monoclonal antibody is pembrolizumab or a pembrolizumab variant, (ii) polyethylene glycol (PEG), and (iii) an additive selected from caffeine, theophylline, 2'-deoxyguanosine-5'-monophosphate, bioactive gibberellins such as gibberellin A3, and pharmaceutically acceptable salts of gibberellins, to form a crystallization solution having a pH of about 6.0 to about 8.8 and containing about 2% to about 40% by weight / volume (w / v) of PEG and about 0.1% to about 0.30% by w / v of additives; (b) incubating the crystallization solution for a period of time sufficient to form crystals; and (c) optionally harvesting the crystalline anti-PD-1 mAb from the solution.

[0012] In some embodiments, the mAb is pembrolizumab. In further embodiments, the mAb is a pembrolizumab variant that retains both the ability to bind to PD-1 and the ability to bind to additives.

[0013] In a particular implementation, the additive is caffeine.

[0014] In some embodiments, the crystallization solution further comprises about 1% to about 10% sodium dextran sulfate.

[0015] In one aspect, the present invention relates to isolated anti-PD-1 crystals prepared by the method of the present invention.

[0016] In another aspect, the present invention relates to a separated crystal comprising pembrolizumab in combination with caffeine, wherein the crystal is characterized by space group P2221. α = β = γ = 90°.

[0017] On the other hand, the present invention relates to crystalline pembrolizumab comprising pembrolizumab complexed with caffeine, characterized by solid-state NMR showing peaks at approximately 182.16, 181.54, 179.99, 109.36, 108.23, 103.58, 76.88, and 76.04 ppm. 13 C spectrum.

[0018] On the other hand, the present invention relates to crystalline pembrolizumab comprising pembrolizumab complexed with caffeine, characterized by solid-state NMR showing peaks at approximately 183.07, 182.16, 181.54, 180.55, 179.99, 110.70, 110.15, 109.36, 108.23, 103.58, 101.49, 99.75, 98.56, 76.88, 76.04, 74.97, 74.41, 73.52, 72.69, 13.85, 13.27, 12.26, and 11.13 ppm. 13 C spectrum.

[0019] This document also provides compositions comprising the anti-PD-1 mAb crystals of the present invention and a pharmaceutically acceptable carrier.

[0020] In one aspect, the present invention provides a method for treating cancer and / or infectious diseases by administering the crystals or compositions of the invention to a patient in need. In a particular embodiment, the composition is administered to the patient via intravenous infusion. In an alternative embodiment, the crystals are administered to the patient via subcutaneous injection. Attached Figure Description

[0021] The patent or application document contains at least one color drawing. A color copy of the patent or patent application disclosure with color drawings is provided by the Patent Office upon request and payment of the necessary fees.

[0022] Figure 1A-1C Micrographs of crystals in a pembrolizumab crystal suspension are shown, obtained by vapor diffusion at 30°C using a precipitant solution of Silver Bullet Bio Crystallization Reagent A2 and 12.5% ​​w / v PEG 3350, 0.05 M HEPES buffer, pH 6.8. See Example 1. Micrographs at 200x magnification were obtained using SONICC. TM The imaging system acquired the data after 30 days. Figure 1A Visible light images of the crystal taken at 200x magnification are provided. Figure 1B and 1C They respectively provide the use of SONICC TM Images generated by the UV-TPEF and SHG modes of the imaging system. Positive images from SHG and UV-TPEF indicate protein crystals.

[0023] Figure 2A-2C Visible light micrographs of crystals within a pembrolizumab crystal suspension generated using droplet vapor diffusion are provided at 200x magnification, as described in Example 2. Figure 2ACrystals formed from 0.20% caffeine, 12% PEG 3350, 50 mM HEPES, and pH 6.8 are shown. Figure 2B Crystals formed from 0.2% theophylline + 0.2% ethanolamine + 10% PEG 3350 are shown. Figure 2C Crystals formed using 0.2% theophylline + 0.2% 2'-deoxyguanosine 5-monophosphate sodium salt hydrate + 16% PEG 3350 are shown.

[0024] Figure 3 Micrographs of pembrolizumab crystals at 200x magnification are provided, which were produced by a crystallization method (10 mL scale) involving incubation of pembrolizumab with 9.8% PEG 3350, 45 mM HEPES, pH 7.7, and 0.23% caffeine at 30°C for 18 hours.

[0025] Figures 4A-4F Images are provided of a crystal suspension prepared as described in Example 6 using a 10.18% PEG 3350, 50 mM HEPES, pH 7.2 solution. The images show crystals formed after incubation of the crystallization solution at 2°C and 50°C, respectively, using SONICC. TM Visible light mode of imaging system ( Figure 4A and 4D ), UV-TPEF mode ( Figure 4B and 4E ) and SHG mode ( Figure 4C and 4F ) is characterized.

[0026] Figure 5 Micrographs of pembrolizumab crystals prepared using the procedure described in Example 10 are shown. The crystals selected for full structural characterization are also shown.

[0027] Figure 6A A graphical representation of the pembrolizumab / caffeine complex in the low-salt PEG / caffeine crystalline form described in Example 10 is shown. The protein backbone is shown as bands; the glycosyl groups linked to the protein and the ordered caffeine molecules bound to the protein are depicted as bars. Figure 6A In the color version, the protein backbone is shown as the following colored bands: orange VL, magenta CL, green VH, cyan CH1, yellow CH2, and gray CH3. Figure 6B This image shows a close-up view of caffeine molecules that have been found to be ordered and mediate crystal contacts. The protein backbone is represented as bands with side chains surrounding the caffeine molecule, which is depicted as a rod. In the color version, the color rules are consistent with... Figure 6A same

[0028] Figures 7A-7CThis demonstrates the use of batch crystallization (175 mL scale) and the conditions described in Example 11, utilizing SONICC. TM Visible light in imaging systems ( Figure 7A ), UV-TPEF Figure 7B ) and SHG ( Figure 7C Crystal images generated by the ) mode.

[0029] Figure 8A The viscosity (cP) of a 200 mg / mL pembrolizumab crystal suspension is shown compared to the shear rate (s) of BD Hypak 1 mL PFS with 27 G RW and 29 G TW x” needles. -1 See Example 12. Figure 8B The syringe injection force (N) is shown as the displacement (mm) of 200 (triangular), 175 (square), and 150 (rhomboid) mg / mL pembrolizumab crystal suspensions produced as described in Example 11.

[0030] Figure 9 The required injection force (N) for producing a 200 mg / mL crystal pembrolizumab suspension in a variety of 1 mL plastic and glass syringes over distance (mm) is provided. See Example 12. The crystal suspension was produced under the conditions described in Example 11.

[0031] Figure 10A The solid state of the pembrolizumab crystal suspension prepared as described in Example 11 is depicted. 13 C NMR CP MAS. Figure 10B Depicts Figure 10A The magnified spectral region of the spectrum.

[0032] Figure 11A and Figure 11B The image depicts a pembrolizumab-caffeine crystal suspension (solid line) and caffeine-only crystals (dashed line). 13 C( Figure 11A )and 15 N( Figure 11B CP MAS spectrum. 2- 13 C and 1,3- 15 N-isotope-labeled caffeine was used in these spectra. Detailed Implementation

[0033] This invention provides pembrolizumab antibodies and variants thereof in crystalline form, suspensions of these crystals, and pharmaceutical formulations of these suspensions. Highly purified pembrolizumab monoclonal antibodies are used in high-throughput (HT) vapor diffusion sparse matrix screening experiments. Novel crystalline suspensions are obtained using various additives at 30°C and room temperature. This invention also provides a method for preparing said novel monoclonal antibody (mAb) crystalline suspensions, for example, using bulk crystallization (batch and dialysis) to prepare them in high yield, wherein the mAb is pembrolizumab or a variant thereof.

[0034] In one aspect, the present invention relates to a method for producing crystalline anti-PD-1 mAb, comprising: (a) mixing: (i) an aqueous buffer solution containing about 5 mg / mL to about 80 mg / mL mAb, (ii) polyethylene glycol (PEG), and (iii) an additive selected from caffeine, theophylline, 2'-deoxyguanosine-5'-monophosphate, bioactive gibberellin, and pharmaceutically acceptable salts of bioactive gibberellin; to form a crystallization solution having a pH of about 6.0 to about 8.8 and containing about 5% to about 40% by weight / volume (w / v) PEG and about 0.10% to about 0.30% by w / v of the additive; (b) incubating the crystallization solution for a period of time sufficient to form crystals; and (c) optionally harvesting the crystalline anti-PD-1 mAb from the solution. The resulting crystal suspension contains anti-PD-1 mAb crystals, such as pembrolizumab crystals, having a particle size of 0.5-200 micrometers after harvesting. In a particular embodiment, the method further includes the step of homogenizing the crystals formed in step (b). In a further embodiment, the crystal anti-PD-1 mAb is harvested from a crystallization solution, or at least partially purified from a crystallization solution, and then the harvested or purified crystals are homogenized. The resulting anti-PD-1 mAb crystals, such as pembrolizumab crystals, have a particle size of about 0.5 to about 50 micrometers after homogenization.

[0035] This invention further provides various methods for preparing the crystalline pembrolizumab antibody of the present invention, as described in more detail in Examples 1-18. Examples 1 and 2 provide a vapor diffusion-based method that can be used for screening to determine crystallization conditions. Such methods are also suitable for producing large crystals for X-ray diffraction studies, for example, to determine the three-dimensional structure of anti-PD-1 antibodies. In some embodiments, sodium dextran sulfate is added to the crystallization solution to allow for better control of nucleation; thereby allowing the growth of larger crystals.

[0036] Examples 5, 11, and 15-17 provide crystallization methods suitable for large-scale production, such as batch crystallization and body dialysis crystallization, which can be used for commercial-scale production of crystalline pembrolizumab or pembrolizumab variants for therapeutic purposes. For example, examples 11, 14, and 15 provide a method for harvesting the crystals of the present invention using centrifugation; however, filtration methods known in the art, such as hollow fiber tangential flow filtration, can also be used to harvest crystals, for example, on a commercial scale.

[0037] Although the disclosed specific embodiments employ a 1:1 and / or 1:3 mixture of antibody solution and precipitant solution, any modification to the disclosed method that produces substantially the same concentration of solution components in the final crystallization solution (from which crystals are generated) is equivalent. For example, if a precipitant solution (containing PEG and additives, as defined herein) is used, comprising less than or greater than 50% of the final volume of the crystallization solution, the concentration of components in the precipitant solution can be increased or decreased proportionally.

[0038] The crystallization method of the present invention also provides a method for purifying pembrolizumab or pembrolizumab variant antibodies, even if such crystals are redissolved before use. In one embodiment, the pembrolizumab antibody is generated and at least partially purified by methods described herein and known in the art. The antibody is then crystallized, for example by batch crystallization or dialysis. The crystallized antibody is then recovered and washed, for example as described in Example 5 (or by filtration), and redissolved in a buffer, such as 10 mM histidine buffer pH 5.4, or any suitable buffer for the intended use of the purified antibody. For therapeutic use, suitable pharmaceutically acceptable buffers and excipients are used.

[0039] The crystallization method of this invention also provides a method for storing purified pembrolizumab antibodies, even if such crystals are redissolved before use. In one embodiment, pembrolizumab or a pembrolizumab variant antibody is generated and at least partially purified by methods described herein and known in the art. The antibody is then crystallized, for example by batch crystallization or dialysis. The resulting concentrated pembrolizumab crystal suspension is stored as a stable concentrated formulation suitable for transport and reconstitution at global formulation sites.

[0040] The crystal pembrolizumab antibody of the present invention has several advantageous properties for therapeutic use, including the ability to be formulated at high concentrations and low viscosity. This high concentration allows for more effective administration to the subject, for example, via subcutaneous injection. The crystal suspension of the present invention can be used to prepare pharmaceutical formulations up to 300-400 mg / mL, thereby enabling higher dosing with lower injection volumes and thus reducing discomfort. The crystal suspension of the present invention can be delivered via subcutaneous injection using a small-bore needle (e.g., a 27G insulin injector). The reduced volume, lower viscosity, and use of a smaller needle all contribute to reducing patient discomfort during subcutaneous administration.

[0041] The crystalline pembrolizumab antibody of the present invention also possesses other advantageous properties. The suspension of the crystalline pembrolizumab antibody exhibits stability comparable to the starting solution formulation and allows for a longer shelf life. Furthermore, the ability to store the suspension of the crystals of the present invention at room temperature provides significant advantages in pharmaceutical product handling and supply chain management.

[0042] Previous pembrolizumab crystal suspensions were prepared using a high-salt process. See WO2016 / 137850. The novel pembrolizumab crystals of this invention do not require high salt levels, which is advantageous for drug preparation processes because high salt levels are unsuitable for drug formulations intended for subcutaneous administration.

[0043] I. Definitions and abbreviations

[0044] Where used throughout the specification and appended claims, the following abbreviations shall apply:

[0045] CDR Complementary Determining Area

[0046] CHO Chinese hamster ovary

[0047] CP cross-polarization

[0048] CPS combined positive score

[0049] DFS disease-free survival

[0050] ELISA (Enzyme-Linked Immunosorbent Assay)

[0051] FR Frame Area

[0052] GRAS is generally considered safe

[0053] HEPES Hydroxyethyl-piperazine ethane-sulfonic acid buffer

[0054] HT High Throughput

[0055] IEX Ion Exchange

[0056] IHC immunohistochemistry or immunohistochemical IPTG isopropyl β-d-1-thiogalactopyranoside IV intravenous mAb monoclonal antibody MAS magic-angle spin NCI National Cancer Institute NMR PBS phosphate-buffered saline PD progressive disease PD-1 programmed cell death 1 PD-L1 programmed cell death 1 ligand 1

[0057] PD-L2 programmed cell death 1 ligand 2

[0058] PEG (Polyethylene Glycol) PFS (Progression-Free Survival PK) Pharmacokinetics PR (Partial Response OR) Overall Response OS (Overall Survival Q2W) One dose every two weeks Q3W One dose every three weeks QD (One dose daily) RECIST Response Evaluation Criteria for Solid Tumors RPLC (Reversed-Phase Liquid Chromatography) RPM (Revolutions per Minute SC) Subcutaneous SD (Stable Disease or Standard Deviation, Context-Specific) SHG (Second Harmonic Generation SONICC) Second-Order Nonlinear Imaging of Chiral Crystals T / C (Treatment / Control Tumor Volume Ratio TPS) Tumor Proportion Score

[0059] UV-TPEF ultraviolet two-photon excitation fluorescence

[0060] VH Immunoglobulin Heavy Chain Variable Region

[0061] VK Immunoglobulin κ Light Chain Variable Region

[0062] w / v weight / volume

[0063] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined elsewhere herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0064] The singular forms “a,” “an,” and “the” used throughout the specification and appended claims include plural references unless the context clearly specifies otherwise.

[0065] The use of "or" indicates one or both possibilities, unless the context explicitly specifies one of the indicated possibilities. In some cases, "and / or" is used to emphasize one or both possibilities.

[0066] "Treatment" or "treatment" refers to the application of the compositions of the present invention to a patient to induce a positive therapeutic effect. This term does not necessarily imply the complete elimination of all symptoms of disease or disorder. "Treatment" of cancer or immune disorders refers to the application of the crystal suspension or composition of the present invention to a patient suffering from an immune disorder or cancerous condition, or diagnosed as having or being susceptible to cancer or pathogen infection (e.g., virus, bacteria, fungus), to achieve at least one positive therapeutic effect, such as a reduction in the number of cancer cells, a reduction in tumor size, a reduction in the rate of cancer cell invasion into peripheral organs, or a reduction in the rate of tumor metastasis or tumor growth. "Treatment" may include one or more of the following: inducing / increasing an anti-tumor immune response; stimulating an immune response to pathogens, toxins, and / or self-antigens; stimulating an immune response to viral infection; reducing the number of one or more tumor markers; inhibiting the growth or survival of tumor cells; eliminating or reducing the size of one or more cancerous lesions or tumors; reducing the level of one or more tumor markers; improving or reducing the severity or duration of cancer; and prolonging the survival of a patient relative to the expected survival of a similarly untreated patient.

[0067] "Immune disorders" or "immune dysfunctions" include, for example, pathological inflammation, inflammatory disorders, and autoimmune disorders or diseases. "Immune disorders" also refer to infections, persistent infections, and proliferative disorders such as cancer, tumors, and angiogenesis, including infections, tumors, and cancers that resist elimination by the immune system. "Cancer disorders" include, for example, cancer cells, tumors, angiogenesis, and precancerous conditions such as developmental abnormalities.

[0068] "Inflammatory disorders" refer to disorders or pathological conditions in which the pathology is wholly or partially caused by changes in the number, migration rate, or activation of immune system cells. Immune system cells include, for example, T cells, B cells, monocytes or macrophages, antigen-presenting cells (APCs), dendritic cells, microglia, NK cells, NKT cells, neutrophils, eosinophils, mast cells, or any other cells particularly associated with immunity, such as endothelial cells or epithelial cells that produce cytokines.

[0069] The effectiveness of active treatment for cancer can be measured in several ways (see WA Weber, J. Nucl. Med. 50: 1S-10S (2009)). For example, regarding tumor growth inhibition, according to NCI criteria, a T / C ≤ 42% is the minimum level of antitumor activity. A T / C < 10% is considered a high level of antitumor activity, where T / C (%) = median tumor volume treated / median tumor volume in the control group × 100. In some embodiments, the treatment achieved by administering the formulation of the present invention is any one of progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS). PFS, also known as “time to tumor progression,” refers to the length of time during and after treatment when cancer does not grow, and includes the amount of time a patient experiences a complete or partial response, as well as the amount of time a patient experiences stable disease. DFS refers to the length of time a patient remains disease-free during and after treatment. OS refers to the extension of life expectancy compared to an original or untreated individual or patient. While embodiments of the formulations, treatments, and uses of the present invention may not be effective in achieving a positive therapeutic effect in every patient, they should achieve a positive therapeutic effect in a number of subjects who are statistically significant as determined by any statistical test known in the art (e.g., Student's t-test, chi2 test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test).

[0070] The term "patient" (or "subject" or "individual" herein) refers to a mammal (e.g., rat, mouse, dog, cat, rabbit) capable of being treated with the formulations of the present invention, preferably a human. The term "patient" may also include non-human animals, including livestock and domestic animals, including but not limited to cattle, horses, sheep, pigs, goats, rabbits, cats, dogs, and other mammals requiring treatment. In some embodiments, the patient is an adult patient. In other embodiments, the patient is a child patient. A patient "requiring treatment" is an individual diagnosed with, suspected of having, or susceptible to a disease or disorder that the crystal suspension or composition of the present invention is intended to treat, or a patient requiring prevention of a disorder.

[0071] The term "antibody" refers to any form of antibody that exhibits the desired biological activity. Therefore, it is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, humanized, fully human antibodies, and chimeric antibodies. "Parental antibody" refers to an antibody obtained by exposing the immune system to an antigen prior to antibody modification for its intended use (e.g., humanization of an antibody for use as a human therapeutic antibody).

[0072] Generally, the basic antibody structural unit comprises a tetramer. Each tetramer consists of two identical polypeptide chain pairs, each pair having a "light" chain (approximately 25 kDa) and a "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The variable region of each light / heavy chain pair forms the antibody binding site. Therefore, generally, a complete antibody has two binding sites. The carboxyl-terminal portion of the heavy chain can define a constant region primarily responsible for effector function. Typically, human light chains are classified as κ and λ light chains. Furthermore, human heavy chains are typically classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both the light and heavy chains, the variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also includes a "D" region of approximately 10 or more amino acids. See also General. Fundamental Immunology Ch.7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)).

[0073] Typically, the variable domains of heavy and light chains contain three hypervariable regions, also known as complementarity-determining regions (CDRs), located within relatively conserved framework regions (FRs). CDRs are usually aligned with the framework regions, enabling the binding of specific epitopes. Generally, from the N-terminus to the C-terminus, the variable domains of light and heavy chains include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Generally, amino acids are assigned to each domain according to… Sequences of Proteins of Immunological Interest , Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32: 1-75; al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

[0074] An antibody that "specifically binds" to a specific target protein is an antibody that preferentially binds to that target compared to other proteins, but this specificity does not require absolute binding specificity. An antibody is considered "specific" to its intended target if its binding confirms the presence of the target protein in a sample, for example, without producing an undesirable result such as a false positive. Antibodies or their binding fragments used in this invention bind to the target protein, i.e., human PD-1, with an affinity at least two times, preferably at least ten times, more preferably at least 20 times, and most preferably at least 100 times higher than that for non-target proteins. As used herein, if an antibody binds to a polypeptide containing a given amino acid sequence (e.g., the amino acid sequence of the mature human PD-1 molecule) but not to a protein lacking that sequence, it is said to specifically bind to the polypeptide containing that sequence.

[0075] The term "effective amount of medicine" or "therapeutic effective amount" refers to an amount sufficient to introduce a therapeutic composition or formulation into a patient to treat a disease or condition. Those skilled in the art will recognize that this level can vary depending on the characteristics of the patient (e.g., age, weight, etc.). When used in the crystal suspensions or compositions of the present invention, the term "effective amount" refers to an amount of suspension or composition sufficient to treat the pathological condition (e.g., cancerous condition or inflammatory disorder) to be treated. The "effective amount" of the crystals or compositions of the present invention refers to an amount sufficient to elicit the sought response in cells, tissues, systems, animals, or humans. In one embodiment, the effective amount is a "therapeutic effective amount" for alleviating the symptoms of the treated disease or condition. When the active compound (i.e., the active ingredient) is administered as a salt, the amount of the active ingredient refers to the free acid or free base form of the compound.

[0076] When referring to the amount of a modified substance or composition (e.g., mM or M), the percentage of a formulation component (v / v or w / v), the pH of the solution / formulation, or the parameter value of a step in a characterization method, the term "about" refers to a variation in a numerical quantity that may occur, for example, through typical measurement, operation, and sampling procedures involved in the preparation, characterization, and / or use of the substance or composition; through random errors in these procedures; through differences in the manufacture, origin, or purity of the ingredients used to make or use the composition or to perform the procedure; etc. In some embodiments, "about" may refer to a variation of ±0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 3.0, 4.0, or 5.0 in appropriate units. In some embodiments, "about" may refer to a variation of ±0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%. In some embodiments, for solid-state NMR purposes, the term "about" refers to ±0.1 ppm.

[0077] The terms “cancer,” “cancerous,” or “malignant” refer to or describe a physiological condition in mammals that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinomas, lymphomas, leukemias, blastomas, and sarcomas. More specific examples of such cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, gastrointestinal (intestinal) cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, and head and neck cancer.

[0078] When used to refer to the crystal antibody suspension of the present invention, "concentration" refers to the amount of antibody (e.g., pembrolizumab) present in a given macroscopic unit volume of solution. Although suspensions have inherent heterogeneity compared to conventional solutions, the term concentration is used in its conventional meaning. The antibody concentration in a crystal suspension is equal to the concentration of an equivalent sample in which the antibody is not in a crystalline form.

[0079] "Anti-PD-1 mAb crystals" or "crystal anti-PD-1 mAb," as used herein, refers to crystals containing antibodies arranged in a three-dimensional, periodically repeating lattice structure. In contrast, the solid, amorphous forms of monoclonal antibodies, such as those produced by lyophilizing mAbs dissolved in solution, do not exhibit the typical optical properties of crystalline antibody forms, such as refractive index and birefringence.

[0080] "Antibody solution" refers to a solution of anti-human PD-1 antibody (e.g., pembrolizumab) used to produce the crystallized antibody of the present invention. "Precipitant solution" refers to a second solution mixed with the antibody solution, typically in a 1:1 volume ratio (i.e., equal volumes of the two solutions mixed), to produce a "crystallization solution" from which the antibody grows. For convenience, the concentrations of the antibody and precipitant solutions in a 1:1 mixture are provided herein; however, those skilled in the art will recognize that the volume ratio used to prepare the mixture can be varied, and therefore the concentrations of the solutions constituting the mixture can also be varied. If such modifications produce the same crystallization conditions (i.e., the same crystallization solution) as the mixture described herein, then such modifications fall within the scope of the present invention.

[0081] Regarding the dialysis-based crystallization method, "dialysis solution" refers to the solution of pembrolizumab ("antibody solution") that is dialyzed to drive the formation of the crystal antibodies of the present invention. "Residue" refers to the antibody solution after dialysis, which may include the harvested antibody crystals. The antibody solution / residue is located on one side of the dialysis membrane, while the dialysis solution is located on the opposite side.

[0082] The term "homogenization" refers to the mechanical reduction of crystal grain size, resulting in smaller, more uniformly distributed grains. Homogenization can be performed by any known method, such as by using a homogenizer, or by forcing crystal grains through a smaller aperture (Venturi effect), such as a syringe, to break the grains into smaller sizes.

[0083] The terms “micrometer” and “one millionth of a meter” are used interchangeably in this article, both referring to 1 / 1,000,000 of a meter.

[0084] "PD-L1" or "PD-L2" expression refers to any detectable level of expression of a specified PD-L protein on the cell surface or a specified PD-L mRNA within cells or tissues. PD-L protein expression can be detected in immunohistochemical (IHC) analysis of tumor tissue sections using diagnostic PD-L antibodies or by flow cytometry. Alternatively, PD-L protein expression in tumor cells can be detected by PET imaging using a binding agent (e.g., antibody fragment, affibody, etc.) that specifically binds to the desired PD-L target (e.g., PD-L1 or PD-L2). Techniques for detecting and measuring PD-L mRNA expression include RT-PCR and real-time quantitative RT-PCR.

[0085] Several methods for quantifying PD-L1 protein expression in IHC analysis of tumor tissue sections have been described. See, for example, RH, et al., Proc. Natl. Acad. Sci USA 101(49):17174-17179 (2004); Thompson, RH et al., Cancer Res. 66:3381-3385 ​​(2006); Gadiot, J., et al., Cancer 117:2192-2201 (2011); Taube, J Metal., Sci Transl Med 4:127ra37 (2012); and Toplian, S Letal., New Eng. J Med. 366(26):2443-2454 (2012).

[0086] One approach employs a simple binary endpoint of PD-L1 expression positivity or negativity, with a positive result defined as the percentage of tumor cells showing histological evidence of cell surface membrane staining. Tumor tissue sections are counted as PD-L1 positive, representing at least 1%, preferably 5%, of the total tumor cells.

[0087] In another approach, PD-L1 expression in tumor tissue sections is quantified in tumor cells and in infiltrating immune cells that predominantly contain lymphocytes. The percentages of membrane-stained tumor cells and infiltrating immune cells are quantified as <5%, 5% to 9%, and then increased in 10% increments up to 100%. In some embodiments, PD-L1 expression in tumor cells is counted as negative if the score is below 5%, and as positive if the score is ≥5%. PD-L1 expression in immune infiltrates is reported as a semi-quantitative measure called the Adjusted Inflammation Score (AIS) (which is determined by multiplying the percentage of membrane-stained cells by the infiltration intensity, graded as none (0), mild (score 1, rare lymphocytes), moderate (score 2, tumor locally infiltrated by lymphohistiocytic aggregates), or severe (score 3, diffuse infiltration)). If the AIS ≥ 5, the tumor tissue section is counted as positive for PD-L1 expression in the immune infiltrates.

[0088] Tissue sections from tumors stained with diagnostic PD-L1 antibodies via IHC can also be used to score PD-L1 protein expression by evaluating PD-L1 expression in tumor cells and infiltrating immune cells within the tissue sections using a scoring process. See WO 2014 / 165422. One PD-L1 scoring process involves examining each tumor nest in the stained tissue section and assigning one or both of a modified H score (MHS) and a modified proportional score (MPS) to the tissue section. For MHS assignment, four separate percentages are estimated for all viable tumor cells and stained mononuclear inflammatory cells in all examined tumor nests: (a) unstained cells (intensity = 0), (b) weakly stained cells (intensity = 1+), (c) moderately stained cells (intensity = 2+), and (d) strongly stained cells (intensity = 3+). Cells must have at least partial membrane staining to be included in the weak, moderate, or strong staining percentages. The estimated percentages (summing up to 100%) are then entered into the formula 1x (percentage of weakly stained cells) + 2x (percentage of moderately stained cells) + 3x (percentage of strongly stained cells), and the result is assigned to the tissue section as the MHS. The MPS is assigned by estimating the percentage of cells with at least partial membrane staining of any intensity among all viable tumor cells and stained mononuclear inflammatory cells in all examined tumor nests, and the resulting percentage is assigned to the tissue section as the MPS. In some embodiments, if the MHS or MPS is positive, the tumor is designated as PD-L1 expressing positive.

[0089] "CPS" or "Comprehensive Positive Score" refers to an algorithm used to determine PD-L1 expression scores from a patient's tumor sample. CPS can be used to select patients for treatment with specific regimens, including those administering anti-PD-1 antibodies, where PD-L1 expression is associated with a higher response rate in a specific patient population relative to the same patient population that does not express PD-L1. CPS is determined by identifying the number of surviving PD-L1-positive tumor cells, the number of surviving PD-L1-negative tumor cells, and the number of surviving PD-L1-positive monocytes (MICs) in the tumor tissue of a patient with tumors and calculating CPS using the following formula:

[0090]

[0091] TPS, or "tumor proportion score," refers to the percentage of tumor cells that express PD-L1 on their cell membrane. TPS typically includes the percentage of tumor cells expressing PD-L1 at any intensity (weak, intermediate, or strong), which can be determined as described above using immunohistochemical analysis with diagnostic anti-human PD-L1 mAbs (e.g., antibodies 20C3 and 22C3). Cells are considered to express PD-L1 if membrane staining is present, including cells with partial membrane staining.

[0092] The level of PD-L mRNA expression can be compared with the mRNA expression level of one or more reference genes (such as ubiquitin C) commonly used in quantitative RT-PCR.

[0093] In some embodiments, the PD-L1 expression levels (protein and / or mRNA) of malignant cells and / or infiltrating immune cells within a tumor are determined to be “overexpressed” or “elevated” based on a comparison with appropriate controls of PD-L1 expression levels (protein and / or mRNA). For example, the control PD-L1 protein or mRNA expression level may be the level quantified in the same type of non-malignant cells or in sections from matched normal tissue. In some preferred embodiments, elevated PD-L1 expression in a tumor sample is determined if the PD-L1 protein (and / or PD-L1 mRNA) in the tumor sample is at least 10%, 20%, 30%, 40%, or 50% higher than in a control sample.

[0094] Pembrolizumab is an IgG4 monoclonal antibody, the structure of which is described in WHO Drug Information, Vol. 27, No. 2, pp. 161-162 (2013) (Merck Sharp & Dohme Corp., Whitehouse Station, NJ). Each light chain of pembrolizumab contains a light chain complementarity-determining region (CDR) containing the amino acid sequences shown in SEQ ID NO: 1, 2, and 3, and a heavy chain CDR containing the amino acid sequences shown in SEQ ID NO: 4, 5, and 6. Pembrolizumab's variable light chain (V... L ) and heavy chain (V H The product contains the amino acid sequences shown in SEQ ID NO:7 and SEQ ID NO:8, respectively, and the full-length light chain and heavy chain contain or are composed of the amino acid sequences shown in SEQ ID NO:9 and SEQ ID NO:10, respectively. Pembrolizumab is approved by the US FDA for the treatment of patients with unresectable or metastatic melanoma, as adjuvant therapy for patients with melanoma that has been completely resected and has metastases involving lymph nodes, and for the treatment of certain patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC), classic Hodgkin's lymphoma (cHL), urothelial carcinoma, gastric cancer, cervical cancer, primary mediastinal large B-cell lymphoma, microsatellite instability-high (MSI-H) cancer, esophageal cancer, hepatocellular carcinoma, Merkel cell carcinoma, renal cell carcinoma, endometrial cancer, small cell lung cancer, and non-small cell lung cancer, as described in Prescribing Information for KEYTRUDA. TM As described in (Merck & Co., Inc., Whitehouse Station, NJ USA; Initial US Approval 2014, Updated September 2019).

[0095] As used herein, “pembrolizumab variant” refers to a derivative of a pembrolizumab antibody that (1) substantially retains its biological activity of binding to and inhibiting the activity of the antigen (i.e., human PD-1) (e.g., blocking the binding of PD-1 to PD-L1 and / or PD-L2), and (2) retains the ability of the antibody to bind to an additive used in the crystallization solution in the method of the present invention, wherein the additive is caffeine, theophylline, 2'-deoxyguanosine-5'-monophosphate, a bioactive gibberellin such as gibberellin A3 or a pharmaceutically acceptable salt thereof. In embodiments of the invention, the pembrolizumab variant comprises the same sequences as the light and heavy chain sequences (SEQ ID NO: 9 and 10, respectively) in pembrolizumab, except that it has up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions located outside the light chain CDR and heavy chain CDR, for example, the variant position is located in the frame region or constant region. In a further embodiment, the pembrolizumab variant has up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions located outside the pembrolizumab light and heavy chain CDRs and further outside the caffeine-binding pembrolizumab residues, specifically outside TYR 436 and ASN 434 (positions 434 and 436 of SEQ ID NO: 10) of the pembrolizumab heavy chain. In other words, pembrolizumab and pembrolizumab variants contain the same CDR sequence but are different from each other due to conserved amino acid substitutions at no more than ten other positions in their full-length light and heavy chain sequences, respectively. The pembrolizumab variants are substantially identical to pembrolizumab in the following properties: binding affinity to PD-1, ability to block the binding of PD-L1 and PD-L2 to PD-1 respectively, and ability to bind to additives selected from: caffeine, theophylline, 2'-deoxyguanosine-5'-monophosphate, bioactive gibberellins (such as gibberellin A3), and pharmaceutically acceptable salts of said bioactive gibberellins.

[0096] A "precipitant" is a compound that reduces the solubility of a polypeptide (such as an antibody) in a concentrated solution. In batch crystallization methods, the precipitant may be included in a "precipitant solution," while in in vivo dialysis methods, the precipitant may be included in a "dialysis solution." The precipitant induces crystallization by forming an energy-disadvantaged precipitant depletion layer around the polypeptide molecules. To minimize the relative amount of this depletion layer, the polypeptide associates and eventually forms crystals. This process is explained in Weber (1991) Advances in Protein Chemistry 41:1. Various precipitants are known in the art. In the method of this invention, the precipitant is polyethylene glycol (e.g., PEG 3350).

[0097] In addition to the precipitant, one or more crystallization-promoting additives, selected from caffeine, theophylline, 2'-deoxyguanosine-5'-monophosphate, bioactive gibberellin, and pharmaceutically acceptable salts of bioactive gibberellin, are added to the polypeptide precipitant solution or crystallization solution. Two additives (caffeine and theophylline) suitable for use in the method of this invention have been found to share the following structural similarities:

[0098]

[0099] This paper also demonstrates that gibberellin A3 (or GA3 or gibberellic acid) is a useful reagent in the crystallization method of the present invention. Gibberellins (also known as GAs) are a class of hormones found in plants that share a common diterpene acid structure and regulate various developmental processes. “Bioactive gibberellins” involve different aspects of plant germination and share the following structural characteristics: 1) a hydroxyl group at C-3β, 2) a carboxyl group at C-6, and 3) a lactone between C-4 and C-10 (see below). Based on the similar structure and function of “bioactive gibberellins” (which include gibberellin A1 (GA1), gibberellin A3 (GA3), gibberellin A4 (GA4), and gibberellin A7 (GA7)) or their pharmaceutically acceptable salts, it is contemplated that any bioactive gibberellin or its pharmaceutically acceptable salt can be used in the method of the present invention.

[0100]

[0101]

[0102] In addition to precipitants, one or more other excipients may be added to the peptide precipitant solution or crystallization solution. Excipients include buffers (such as Tris or HEPES) to adjust the pH of the solution (thereby adjusting the surface charge on the peptide) and salts (such as sodium chloride, lithium chloride, and sodium citrate) to reduce the solubility of the peptide.

[0103] A "tissue slice" refers to a single part or small piece of a tissue sample, such as a thin slice of tissue cut from a sample of normal tissue or a tumor.

[0104] The term “Tris” (2-amino-2-hydroxymethyl-propane-1,3-diol) used in this article is synonymous with TRIS, Tris base, Trizma, Trisamine, THAM, aminobutanetriol, tromethamine, tris(hydroxymethyl)aminomethane, and thromboxane.

[0105] When used in a subject diagnosed with or suspected of having cancer, the term "tumor" refers to a malignant or potentially malignant tumor or mass of tissue of any size, including primary tumors and secondary growths. A solid tumor is an abnormal growth or mass of tissue that typically does not contain cysts or fluid-filled areas. Different types of solid tumors are named according to the type of cells that form them. Examples of solid tumors include sarcomas, carcinomas, and lymphomas. Leukemia (cancer of the blood) typically does not form solid tumors (National Cancer Institute, Dictionary of Cancer Terms).

[0106] "Tumor burden," also known as "tumor load," refers to the total amount of tumor material distributed throughout the body. Tumor burden refers to the total number of cancer cells or the total size of the tumor throughout the body (including lymph nodes and bone marrow). Tumor burden can be determined by a variety of methods known in the art, for example, by measuring the size of the tumor upon removal from the subject, for example, using a caliper, or by measuring the size of the tumor while it is in the body using imaging techniques (e.g., ultrasound, bone scan, computed tomography (CT), or magnetic resonance imaging (MRI) scans).

[0107] The term "tumor size" refers to the total size of a tumor, which can be measured as the length and width of the tumor. Tumor size can be determined by a variety of methods known in the art, such as by measuring the tumor size when it is removed from the subject, for example, using a caliper, or by measuring the tumor size while it is inside the body using imaging techniques such as bone scans, ultrasound, CT, or MRI scans.

[0108] "Humanized antibodies" refer to antibody forms that contain sequences derived from non-human (e.g., mouse) antibodies as well as human antibodies. These antibodies contain minimal sequences derived from non-human immunoglobulins. Generally, humanized antibodies contain at least one, and typically substantially all, of two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulins, and all or substantially all of the FR regions are sequences of human immunoglobulins. Humanized antibodies also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically of human immunoglobulins. Humanized forms of rodent antibodies typically contain the same CDR sequence as the parent rodent antibody, although certain amino acid substitutions may be included to improve the affinity of the humanized antibody, enhance stability, or for other reasons.

[0109] Antibodies that can be used in the compositions of the present invention also include antibodies having modified (or blocked) Fc regions to provide altered effector functions. See, for example, U.S. Patent Nos. 5,624,821; WO2003 / 086310; WO2005 / 120571; WO2006 / 0057702; Presta (2006) Adv. Drug Delivery Rev. 58:640-656. Such modifications can be used to enhance or suppress various immune system responses, potentially having beneficial effects in diagnosis and treatment. Changes to the Fc region include amino acid alterations (substitution, deletion, and insertion), glycosylation or deglycosylation, and the addition of multiple Fc regions. Changes in Fc can also alter the half-life of antibodies in therapeutic antibodies, and a longer half-life will result in a lower dosing frequency while increasing convenience and reducing material usage. See Presta (2005) J. Allergy Clin. Immunol. 116:731,734-35.

[0110] The "hypervariant region" refers to the antibody amino acid residues responsible for antigen binding and which vary between different antibody sequences. The hypervariable region contains amino acid residues from the "complementarity-determining region" or "CDR" (e.g., residues 24-34 (CDRL1), 50-56 (CDRL2), and 89-97 (CDRL3) in the light chain variable domain, and residues 31-35 (CDRH1), 50-65 (CDRH2), and 95-102 (CDRH3) in the heavy chain variable domain, as indicated by the Kabat numbering system (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health). The residues determined by the National Institutes of Health, Bethesda, Md., and / or those from the “hypervariant ring” (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) of the light chain variable domain and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) of the heavy chain variable domain (Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917). As used herein, the term “frame” or “FR” residues refer to those variable domain residues other than the hypervariant residues defined herein as CDR residues. CDR and FR residues are determined according to Kabat’s standard sequence definition. Kabat et al. (1987) Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda Md.

[0111] "Conservatively modified variants" or "conservative substitutions" refer to amino acid substitutions known to those skilled in the art, and can generally be performed without altering the biological activity of the resulting molecule, even in essential regions of the peptide. These exemplary substitutions are preferably performed according to those listed in Table 1 below:

[0112] Table 1. Exemplary Conservative Amino Acid Substitutions

[0113]

[0114]

[0115] Furthermore, those skilled in the art will recognize that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not significantly alter its biological activity. See, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th edition).

[0116] The phrase “consistently composed of…” or variations thereof, such as “consistently composed of…” or “consistently made of…”, used throughout the specification and claims, indicates the inclusion of any of the stated elements or groups of elements, and optionally other elements that are similar to or different in nature from the stated elements, without substantially altering the fundamental or novel properties of the specified dosing regimen, method, or composition. As a non-limiting example, a binding compound consisting substantially of the stated amino acid sequence may also include one or more amino acids that do not substantially affect the properties of the binding compound, including substitutions of one or more amino acid residues.

[0117] The word “comprising” or variations such as “including,” “containing,” or “comprising” are used throughout the specification and claims in an inclusive sense, that is, to specify the presence of the stated and featured features, but not to exclude the presence or addition of further features that may substantially enhance the operation or use of any embodiment of the invention, unless the context requires otherwise due to explicit language or necessary implied meaning.

[0118] "Isolated antibody" and "isolated antibody fragment" refer to a purified state and, in the context, to the molecule being referred to as being substantially free of other biomolecules such as nucleic acids, proteins, lipids, carbohydrates, or other materials (such as cell debris and growth media). Generally, the term "isolated" is not intended to mean the complete absence of these materials or the absence of water, buffers, or salts, unless their presence in amounts would significantly interfere with the experimental or therapeutic use of the binding compound described herein.

[0119] "Monoclonal antibody," or "mAb," or "Mab," as used herein, refers to a substantially homogeneous population of antibodies, meaning that the antibody molecules constituting this population are identical in amino acid sequence, except for the possible presence of small amounts of naturally occurring mutations. In contrast, conventional (polyclonal) antibody formulations typically comprise a large number of distinct antibodies with different amino acid sequences in their variable domains (especially CDRs), which are generally specific to different epitopes. The modifier "monoclonal" indicates that the antibody is derived from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody used according to the invention can be prepared by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or by a recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, those described in Clackson et al. (1991) Nature 352:624-628 and Marks et al. (1991) J. Mol. Biol. 222:581-597. See also Presta (2005) J. Allergy Clin. Immunol. 116:731.

[0120] The term "buffer" encompasses those reagents that maintain the solution pH of the formulations of the present invention within an acceptable range, or, for the lyophilized formulations of the present invention, provide an acceptable solution pH prior to lyophilization.

[0121] The term "pharmaceutical formulation" refers to a preparation whose form allows the active ingredient to be effective and does not contain any additional components that would be toxic to the subject to which the formulation is administered.

[0122] "Pharmaceutical acceptable" means an active ingredient that is reasonably administered to a subject to provide an effective dose and is "generally considered safe," such as excipients (mediators, additives) and compositions that, when administered to humans, are physiologically tolerable and generally do not produce allergic reactions or similar adverse reactions (e.g., stomach upset). In another embodiment, the term refers to molecular entities and compositions approved by federal or state regulatory agencies or listed in the United States Pharmacopeia or other recognized pharmacopoeias for use in animals, particularly humans.

[0123] As used in this article, “room temperature” or “RT” refers to a temperature ranging from about 18°C ​​to about 25°C (about 64 to about 77 degrees Fahrenheit).

[0124] A “stable” formulation is one in which the protein substantially retains its physical and / or chemical stability and / or biological activity during storage. Various analytical techniques for measuring protein stability are available in the art and have been reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at a selected temperature over a selected time period. For example, in one embodiment, a stable formulation is one in which no significant changes have been observed for at least 12 months at refrigerated temperatures (2-8°C). In another embodiment, a stable formulation is one in which no significant changes have been observed for at least 18 months at refrigerated temperatures (2-8°C). In yet another embodiment, a stable formulation is one in which no significant changes have been observed for at least 3 months at room temperature (23-27°C). In another embodiment, a stable formulation is one in which no significant changes are observed at room temperature (23-27°C) for at least 6 months. In another embodiment, a stable formulation is one in which no significant changes are observed at room temperature (23-27°C) for at least 12 months. In yet another embodiment, a stable formulation is one in which no significant changes are observed at room temperature (23-27°C) for at least 18 months.

[0125] As used herein, “substantially pure” appropriately means at least about 60% by weight, typically at least about 70% by weight, preferably at least about 80% by weight, more preferably at least about 90% by weight (e.g., about 90% by weight to about 99% by weight), even more preferably at least about 95% by weight (e.g., about 95% by weight to about 99% by weight, or about 98% by weight to 100% by weight), and most preferably at least about 99% by weight (e.g., 100% by weight) of a product comprising a crystallized anti-PD-1 antibody (e.g., crystallized pembrolizumab or a variant thereof) or its salt (e.g., a product separated from a reaction mixture providing a crystallized anti-PD-1 antibody or salt) composed of a crystallized anti-PD-1 antibody or salt. The purity level of the crystallized anti-PD-1 antibody and salt can be determined using standard analytical methods, such as thin-layer chromatography, gel electrophoresis, high-performance liquid chromatography, and / or mass spectrometry. If more than one analytical method is used and these methods provide experimentally significant differences in the determined purity levels, the method providing the highest purity level shall prevail. 100% pure crystal anti-PD-1 antibody or salt is crystal anti-PD-1 antibody or salt that is free from detectable impurities as determined by standard analytical methods.

[0126] II. Anti-PD-1 antibody used in the method of the present invention

[0127] In the method for producing anti-PD-1 mAb crystals and in the method of use / treatment of this invention, the anti-human PD-1 antibody is pembrolizumab or a variant of pembrolizumab. The amino acid sequence of pembrolizumab is provided in Table 2.

[0128] Table 2. Pembrolizumab antibody sequences

[0129]

[0130]

[0131] The crystal anti-PD-1 mAb of the present invention comprises three light chain CDRs (CDRL1, CDRL2, and CDRL3) and three heavy chain CDRs (CDRH1, CDRH2, and CDRH3). In one embodiment, the three light chain CDRs are SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and the three heavy chain CDRs are SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0132] In some embodiments, the present invention provides a light chain variable region (V L ) and heavy chain variable region (V H The pembrolizumab contains a crystal anti-PD-1 mAb, wherein the light chain variable region comprises SEQ ID NO:7 or a variant of SEQ ID NO:7, and the heavy chain variable region comprises SEQ ID NO:8 or a variant of SEQ ID NO:8. In some embodiments, the variable region sequence of the variant light chain or heavy chain is identical to the reference sequence, except having one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. In a particular embodiment, the amino acid substitutions are conserved amino acid substitutions. The substitutions in the pembrolizumab variants are located in the framework region (i.e., outside the CDR) or the constant region, and are located outside any residues that inhibit the binding of the pembrolizumab variant to the additives used in the methods herein, thereby inhibiting crystallization.

[0133] In one embodiment of the present invention, the crystalline anti-human PD-1 antibody comprises a light chain variable region (V1). L ) and heavy chain variable region (V H The light chain variable region includes or is composed of SEQ ID NO:7, and the heavy chain variable region includes or is composed of SEQ ID NO:8.

[0134] In another embodiment, the crystal anti-PD-1 mAb of the present invention comprises the same as the above-mentioned V L Domain or V H The V domain has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% sequence homology. L Domain and / or VH The domain exhibits specific binding to PD-1. In another embodiment, the crystalline anti-PD-1 mAb comprises a V domain having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid substitutions. L and V H The domain showed specific binding to PD-1.

[0135] In any of the above embodiments, the anti-PD-1 crystal of the present invention may comprise a full-length anti-PD-1 antibody (e.g., pembrolizumab), or may comprise a truncated antigen-binding fragment that (1) comprises the light chain CDR of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 and the heavy chain CDR of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, (2) specifically binds to human PD-1, and (3) specifically binds to the additive used in the method of the present invention. In some embodiments, the anti-PD-1 antibody is a full-length anti-PD-1 antibody selected from any class of immunoglobulins (including IgM, IgG, IgD, IgA, and IgE). Preferably, the antibody is an IgG antibody. Any isotype of IgG, including IgG1, IgG2, IgG3, and IgG4, may be used. Different constant domains may be attached to the V provided herein. L and V H Regions. For example, if the specific intended use of the antibody (or fragment) of the present invention requires altered effector functions, a heavy chain constant domain other than IgG1 can be used. Although IgG1 antibodies provide a longer half-life and effector functions such as complement activation and antibody-dependent cytotoxicity, these activities may not be required for all uses of the antibody. In these cases, for example, an IgG4 constant domain can be used.

[0136] In embodiments of the present invention, the crystal anti-PD-1 mAb is an anti-PD-1 antibody comprising a light chain containing or consisting of a sequence of amino acid residues shown in SEQ ID NO: 9 and a heavy chain containing or consisting of a sequence of amino acid residues shown in SEQ ID NO: 10. In some embodiments of the present invention, the crystal anti-PD-1 mAb of the present invention is a crystal pembrolizumab or a pembrolizumab biosimilar.

[0137] In a further embodiment, the crystal anti-PD-1 mAb is a pembrolizumab variant having up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions located outside the light and heavy chain CDRs of the pembrolizumab, and further outside the caffeine-binding pembrolizumab residues (i.e., outside pembrolizumab heavy chain TYR 436 and ASN 434 (434 and 436 of SEQ ID NO:10)).

[0138] Typically, the amino acid sequence variants of the crystalline pembrolizumab variants of the present invention have amino acid sequences that are identical to those of the reference antibody (e.g., heavy chain, light chain, V). H or V L The sequence has at least 90% amino acid sequence identity, more preferably at least 95%, 98%, or 99%. Identity or homology relative to a given sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical to anti-PD-1 residues after aligning the sequence and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of sequence identity. N-terminal, C-terminal, or internal extensions, deletions, or insertions of the antibody sequence are not interpreted as affecting sequence identity or homology.

[0139] Sequence identity refers to the degree to which two polypeptides have identical amino acids at equivalent positions when two sequences are optimally aligned. Sequence identity can be determined using the BLAST algorithm, where the algorithm's parameters are selected to yield the maximum match between corresponding sequences across the entire length of the respective reference sequence. The following references relate to the BLAST algorithm commonly used in sequence analysis: BLAST ALGORITHMS: Altschul, SF, et al., (1990) J. Mol. Biol. 215: 403-410; Gish, W., et al., (1993) Nature Genet. 3: 266-272; Madden, TL, et al., (1996) Meth. Enzymol. 266: 131-141; Altschul, SF, et al., (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J., et al., (1997) Genome Res. 7: 649-656; Wootton, JC, et al., (1993) Comput. Chem. 17: 149-163; Hancock, JMet al., (1994) Comput.Appl.Biosci.10:67-70; ALIGNMENT SCORINGSYSTEMS: Dayhoff, MO, et al., "A model of evolutionary change in proteins." Atlas ofProtein Sequence and Structure,(1978)vol.5,suppl.3.MODayhoff(ed.),pp.345-352,Natl.Biomed.Res.Found.,Washington,DC;Schwartz,RM,et al.,"Matrices for detecting distant relationships."Atlas of Protein Sequence andStructure,(1978)vol.5,suppl.3."MODayhoff(ed.),pp.353-358,Natl.Biomed.Res.Found.,Washington,DC;Altschul,SF,(1991)J.Mol.Biol.219:555-565;States,DJ,et al.,(1991)Methods 3:66-70; Henikoff,S.,et al.,(1992)Proc.Natl.Acad.Sci.USA 89:10915-10919; Altschul, SF, et al., (1993) J. Mol. Evol. 36: 290-300; ALIGNMENT STATISTICS: Karlin, S., et al., (1990) Proc.Natl.Acad.Sci.USA 87:2264-2268; Karlin, S., et al., (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877; Dembo, A., et al., (1994) Ann. Prob. 22: 2022-2039; and Altschul, SF "Evaluating the statistical significance of multipledistinct local alignments."Theoretical and Computational Methods inGenome Research (S. Suhai, ed.), (1997) pp.1-14, Plenum, New York. .

[0140] III. Methods for producing crystal antibody suspensions

[0141] In one aspect, the present invention relates to a method for producing crystalline anti-PD-1 monoclonal antibody (mAb), comprising: (a) mixing: (i) an aqueous buffer solution containing about 5 mg / mL to about 80 mg / mL mAb, wherein the anti-PD-1 mAb is pembrolizumab or a pembrolizumab variant, (ii) polyethylene glycol (PEG), and (iii) an additive selected from caffeine, theophylline, 2'-deoxyguanosine-5'-monophosphate, bioactive gibberellin, and pharmaceutically acceptable salts of gibberellin; to form a crystallization solution having a pH of about 6.0 to about 8.8 and containing about 2% to about 40% by weight (w / v) PEG and about 0.1% to about 0.30% by w / v of the additive; (b) incubating the crystallization solution for a period of time sufficient to form crystals; and (c) optionally harvesting the crystalline anti-PD-1 mAb from the solution.

[0142] In a particular embodiment of the invention, the method includes the step of harvesting crystals of anti-PD-1 mAb from the solution. Methods for harvesting crystals are known to those skilled in the art and include centrifugation, decantation, lyophilization, and filtration, such as hollow fiber tangential flow filtration.

[0143] In some embodiments, the method further includes a step of homogenizing the anti-PD-1 mAb crystals after harvesting them from the crystallization solution. The homogenization step provides anti-PD-1 mAb crystals with a smaller particle size (e.g., 0.5 to 50 micrometers). These smaller crystal particles can be used, for example, in high-concentration pharmaceutical formulations.

[0144] In some embodiments, the method further includes the step of homogenizing the anti-PD-1 mAb crystals without first harvesting the crystals from the crystallization solution. In this method, the crystallization solution may be homogenized after incubation for a time sufficient for crystal formation, for example, by forced passage through a syringe without first harvesting. Smaller-sized anti-PD-1 mAb crystals may optionally be harvested after homogenization.

[0145] In a specific embodiment of the invention, the PEG and additives are mixed together to form a precipitant solution before being mixed with an aqueous buffer solution containing mAb. The precipitant solution and the aqueous buffer solution containing mAb are then mixed together to form a crystallization solution.

[0146] In an alternative embodiment of the invention, PEG is mixed into an aqueous buffer solution containing mAb to form a PEG-mAb solution. An additive, either a solid or a solution, is then added to the PEG-mAb solution to form a crystalline solution.

[0147] In other embodiments, an aqueous buffer solution containing mAb is mixed with an additive to form an aqueous buffer solution containing both mAb and the additive. This solution is then mixed with PEG, either as a solid or as a solution.

[0148] In any of the above embodiments, the additive is caffeine, theophylline, 2'-deoxyguanosine-5'-monophosphate, bioactive gibberellin, or a pharmaceutically acceptable salt of gibberellin.

[0149] In one implementation, the additive is caffeine.

[0150] In another implementation, the additive is theophylline.

[0151] In yet another implementation, the additive is 2'-deoxyguanosine-5'-monophosphate.

[0152] In a further embodiment, the additive is a bioactive gibberellin or a pharmaceutically acceptable salt thereof. In a specific embodiment, the bioactive gibberellin is gibberellin A1, a pharmaceutically acceptable salt of gibberellin A1, gibberellin A3, a pharmaceutically acceptable salt of gibberellin A3, gibberellin A4, a pharmaceutically acceptable salt of gibberellin A4, gibberellin A7, or a pharmaceutically acceptable salt of gibberellin A7.

[0153] In certain embodiments, the additive is gibberellin A3 or a pharmaceutically acceptable salt thereof. In some embodiments, the additive is gibberellin A3. In other embodiments, the additive is a sodium salt of gibberellin A3. In other embodiments, the additive is a potassium salt of gibberellin A3. In other embodiments, the additive is an ammonium salt of gibberellin A3.

[0154] The amount of additive in the final crystallization solution is about 0.10% to about 0.30% w / v. In other embodiments, the amount of additive is about 0.15% to about 0.30% w / v, about 0.175% to about 0.30% w / v, about 0.20% to about 0.30% w / v, about 0.225% to about 0.30% w / v, about 0.25% to about 0.30% w / v, about 0.10% to about 0.25% w / v, about 0.10% to about 0.275% w / v, about 0.10% to about 0.25% w / v, about 0.10% to about 0.225% w / v, or about 0.10% to about 0.20% w / v. In a further embodiment, the amount of additive is about 0.10% w / v, about 0.125% w / v, about 0.15% w / v, about 0.175% w / v, about 0.20% w / v, about 0.225% w / v, about 0.25% w / v, about 0.275% w / v, or about 0.30% w / v.

[0155] In one embodiment, the additive is caffeine, which is present in the final crystallization solution at an amount of about 0.15% w / v to about 0.30% w / v.

[0156] In another embodiment, the additive is theophylline, which is present in the final crystallization solution at an amount of about 0.25% w / v to about 0.30% w / v.

[0157] In any of the above embodiments, the crystallization solution may further contain about 1% to about 10% w / v of sodium dextran sulfate, which reduces the nucleation rate and allows for the growth of larger crystals. In some cases, it may be desirable to form larger crystals, for example, for characterization studies such as X-ray crystallography. In further embodiments, the crystallization solution contains about 1%, about 1.5% w / v, about 2% w / v, about 2.5% w / v, about 3% w / v, about 3.5% w / v, about 4% w / v, about 4.5% w / v, about 5% w / v, about 5.5% w / v, about 6% w / v, about 6.5% w / v, about 7% w / v, about 7.5% w / v, about 8% w / v, about 8.5% w / v, about 9% w / v, about 9.5% w / v, or about 10% w / v of sodium dextran sulfate. In alternative embodiments, the crystallization solution comprises about 1% to about 9% w / v, about 1% to about 8% w / v, about 1% to about 7% w / v, about 1% to about 6% w / v, about 1% to about 5% w / v, about 1% to about 4% w / v, about 1% to about 3% w / v, about 1% to about 2% w / v, about 2% to about 10% w / v, about 2% to about 9% w / v, about 2% to about 8% w / v, about 2% to about 7% w / v, about 2% to about 6% w / v, about 2% to about 5% w / v, about 2% to about 4%, about 2% to about 3%, about 3% to about 10%, about 3% to about 9%, about 3% to about 8% w / v, about 3% to about 7% w / v, about 3% to about 6% w / v, about 3% to about 5% w / v. Sodium dextran sulfate, about 3% to about 4% w / v, about 4% to about 10% w / v, about 4% to about 9% w / v, about 4% to about 8% w / v, about 4% to about 7% w / v, about 4% to about 6% w / v, about 4% to about 5% w / v, about 5% to about 10% w / v, about 5% to about 9% w / v, about 5% to about 8% w / v, about 5% to about 7% w / v, about 5% to about 6% w / v, about 6% to about 10% w / v, about 6% to about 8% w / v, about 6% to about 7% w / v, about 7% to about 10% w / v, about 7% to about 9% w / v, about 7% to about 8% w / v, about 8% to about 10% w / v, about 8% to about 8% w / v, or about 9% to about 10% w / v.

[0158] In any of the above embodiments of the invention, the crystallization solution contains about 2% to about 40% w / v of PEG. The average molecular weight of the PEG is about 2,500 to about 35,000. In a particular embodiment, the PEG is PEG 3350. In alternative embodiments, PEG is PEG 2,500 (i.e., having an average molecular weight of 2,500), PEG 3,000, PEG 4,000, PEG 5,000, PEG 6,000, PEG 7,000, PEG 8,000, PEG 9,000, PEG 10,000, PEG 12,000, PEG 14,000, PEG 15,000, PEG 1600, PEG 1800, PEG 20,000, PEG 22,000, PEG 24,000, PEG 25,000, PEG 26,000, PEG 28,000, PEG 30,000, PEG 32,000, PEG 34,000, or PEG 35,000.

[0159] The amount of PEG in the crystallization solution is from about 2% to about 40% w / v; however, those skilled in the art will recognize that the method of the present invention uses PEG of different molecular weights to vary the amount of PEG. In some embodiments, PEG is present in the crystallization solution in an amount from about 5% to about 15% w / v. In alternative embodiments, PEG is present in the crystallization solution in an amount from about 10% to about 30% w / v. In further embodiments, PEG is present in amounts from about 5% to about 35% w / v, from about 5% to about 30% w / v, from about 5% to about 25% w / v, from about 5% to about 10% w / v, from about 10% to about 40% w / v, from about 5% to about 35% w / v, from about 10% to about 30% w / v, from about 10% to about 25% w / v, from about 10% to about 20% w / v, from about 10% to about 15% w / v, from about 15% to about 40% w / v, from about 15% to about 35% w / v. An amount of about 15% to about 30% w / v, about 15% to about 25% w / v, about 15% to about 20% w / v, about 20% to about 40% w / v, about 20% to about 35% w / v, about 20% to about 30% w / v, about 20% to about 25% w / v, about 25% to about 40% w / v, about 25% to about 35% w / v, about 25% to about 30% w / v, about 30% to about 40% w / v, or about 30% to about 35% w / v is present in the crystallization solution.

[0160] In the method of the present invention, the crystallization solution is prepared by combining the following: (1) an aqueous buffer solution containing anti-PD-1 mAb (i.e., pembrolizumab or a pembrolizumab variant), (2) PEG, and (3) an additive, as described herein; wherein the components of the crystallization solution may be added in any order. In an embodiment of the present invention, the aqueous buffer solution containing anti-PD-1 mAb has a pH of about 6.0 to about 8.8. In a further embodiment, the pH is about 6.0, about 6.2, about 6.4, about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, about 8.0, about 8.2, about 8.4, about 8.6, or about 8.8. In a further embodiment, the pH of the aqueous buffer solution containing anti-PD-1 mAb is about 5.0 to about 6.0. In another embodiment, the pH is about 6.8 to about 8.4.

[0161] In a further embodiment, the pH of the aqueous buffer solution containing anti-PD-1 mAb is about 6.2 to about 8.8, about 6.2 to about 8.6, about 6.2 to about 8.4, about 6.2 to about 8.2, about 6.2 to about 8.0, about 6.2 to about 7.8, about 6.2 to about 7.6, about 6.2 to about 7.4, about 6.2 to about 7.2, about 6.2 to about 7.0, about 6.2 to about 6.8, about 6.2 to about 6.6, about 6.2 to about 6.4, about 6.4 to about 8.8, about 6.4 to about 8.6, about 6.4 to about 8.4, about 6.4 to about 8.2, about 6.4 to about 8. 0, about 6.4 to about 7.8, about 6.4 to about 7.6, about 6.4 to about 7.4, about 6.4 to about 7.2, about 6.4 to about 7.0, about 6.4 to about 6.8, about 6.4 to about 6.6, about 6.6 to about 8.8, about 6.6 to about 8.6, about 6.6 to about 8.4, about 6.6 to about 8.2, about 6.6 to about 8.0, about 6.6 to about 7.8, about 6.6 to about 7.6, about 6.6 to about 7.4, about 6.6 to about 7.2, about 6.6 to about 7.0, about 6.6 to about 6.8, about 6.8 to about 8.8, about 6.8 to about 8.6, about 6.8 to about 8.6. 4. Approximately 6.8 to approximately 8.2, approximately 6.8 to approximately 8.0, approximately 6.8 to approximately 7.8, approximately 6.8 to approximately 7.6, approximately 6.8 to approximately 7.4, approximately 6.8 to approximately 7.2, approximately 6.8 to approximately 7.0, approximately 7.0 to approximately 8.8, approximately 7.0 to approximately 8.6, approximately 7.0 to approximately 8.4, approximately 7.0 to approximately 8.0, approximately 7.0 to approximately 7.8, approximately 7.0 to approximately 7.6, approximately 7.0 to approximately 7.4, approximately 7.0 to approximately 7.2, approximately 7.2 to approximately 8.8, approximately 7.2 to approximately 8.6, approximately 7.2 to approximately 8.4, approximately 7.2 to approximately 8.2, approximately 7.2 to approximately 8.0 Approximately 7.2 to approximately 7.8, approximately 7.2 to approximately 7.6, approximately 7.2 to approximately 7.4, approximately 7.4 to approximately 8.8, approximately 7.4 to approximately 8.6, approximately 7.4 to approximately 8.4, approximately 7.4 to approximately 8.2, approximately 7.4 to approximately 8.0, approximately 7.4 to approximately 7.8, approximately 7.4 to approximately 7.6, approximately 7.6 to approximately 8.8, approximately 7.6 to approximately 8.6, approximately 7.6 to approximately 8.4, approximately 7.6 to approximately 8.2, approximately 7.6 to approximately 8.0, approximately 7.6 to approximately 7.8, approximately 7.8 to approximately 8.8, approximately 7.8 to approximately 8.6, approximately 7.8 to approximately 8.4, approximately 7.8 to approximately 8.2 or approximately 7.8 to approximately 8.0.

[0162] In a particular embodiment of any method described herein, the aqueous buffer solution containing mAb further comprises a histidine buffer with a pH of about 5.0 to about 6.0. In a particular embodiment, the aqueous buffer solution containing mAb further comprises a 20 mM histidine buffer with a pH of 5.4.

[0163] In a specific embodiment of the method of the present invention, the pH of the crystallization solution and the amount of PEG present in the solution are selected from:

[0164] a) The pH of the crystallization solution is approximately 6.0, and the amount of PEG is approximately 2%–4% w / v.

[0165] b) The pH of the crystallization solution is approximately 6.4, and the amount of PEG is approximately 2%–6% w / v.

[0166] c) The pH of the crystallization solution is approximately 6.8 to 8.4, and the amount of PEG is approximately 6% to approximately 12% w / v.

[0167] d) The pH of the crystallization solution is about 8.8, and the amount of PEG is about 10% to 12% w / v.

[0168] In some implementations of the above method, PEG is PEG 3350.

[0169] In embodiments of the method of the present invention, the concentration of the anti-PD-1 mAb in the crystallization solution is from about 5 mg / mL to about 50 mg / mL. In further embodiments, the concentration of the anti-PD-1 mAb in the crystallization solution is from about 5 mg / mL to about 45 mg / mL, from about 5 mg / mL to about 40 mg / mL, from about 5 mg / mL to about 35 mg / mL, from about 5 mg / mL to about 30 mg / mL, from about 5 mg / mL to about 25 mg / mL, from about 5 mg / mL to about 20 mg / mL, from about 5 mg / mL to about 15 mg / mL, from about 5 mg / mL to about 10 mg / mL, from about 10 mg / mL to about 50 mg / mL, from about 10 mg / mL to about 45 mg / mL. mL, about 10 mg / mL to about 40 mg / mL, about 10 mg / mL to about 35 mg / mL, about 10 mg / mL to about 30 mg / mL, about 10 mg / mL to about 25 mg / mL, about 10 mg / mL to about 20 mg / mL, about 10 mg / mL to about 15 mg / mL, about 15 mg / mL to about 50 mg / mL, about 15 mg / mL to about 45 mg / mL, about 15 mg / mL to about 40 mg / mL, about 15 mg / mL to about 35 mg / mL, about 15 mg / mL to about 30 mg / mL L, about 15 mg / mL to about 25 mg / mL, about 15 mg / mL to about 20 mg / mL, about 20 mg / mL to about 50 mg / mL, about 20 mg / mL to about 45 mg / mL, about 20 mg / mL to about 40 mg / mL, about 20 mg / mL to about 35 mg / mL, about 20 mg / mL to about 30 mg / mL, about 20 mg / mL to about 25 mg / mL, about 25 mg / mL to about 50 mg / mL, about 25 mg / mL to about 45 mg / mL, about 25 mg / mL to about 40 mg / mL Approximately 25 mg / mL to approximately 35 mg / mL, approximately 25 mg / mL to approximately 30 mg / mL, approximately 30 mg / mL to approximately 50 mg / mL, approximately 30 mg / mL to approximately 45 mg / mL, approximately 30 mg / mL to approximately 40 mg / mL, approximately 30 mg / mL to approximately 35 mg / mL, approximately 35 mg / mL to approximately 50 mg / mL, approximately 35 mg / mL to approximately 45 mg / mL, approximately 35 mg / mL to approximately 40 mg / mL, approximately 40 mg / mL to approximately 50 mg / mL, or approximately 40 mg / mL to approximately 45 mg / mL.

[0170] In a particular embodiment of any method of the present invention, the crystallization solution further comprises about 25 mM to about 250 mM HEPES buffer. In some embodiments, the crystallization solution further comprises about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 110 mM, about 120 mM, about 125 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 175 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 225 mM, about 230 mM, about 240 mM, about 245 mM, or about 250 mM HEPES buffer.

[0171] In other embodiments of the method of the present invention, the crystallization solution further comprises any amount of Tris buffer (i.e., instead of HEPES buffer) specified above. In alternative embodiments, the crystallization solution further comprises PIPES, MOPS, TES, DIPSO, MOBS, or TAPSO buffer.

[0172] After mixing (1) an aqueous buffer solution containing anti-PD-1 mAb, (2) PEG, and (3) additives, the crystallization solution is incubated at a temperature of about 2°C to about 37°C for a sufficient time to form crystals. In some embodiments, the incubation temperature of the crystallization solution is about 18°C ​​to about 25°C. In other embodiments, the incubation temperature of the crystallization solution is about 2°C to about 35°C, about 2°C to about 30°C, about 2°C to about 25°C, about 2°C to about 20°C, about 2°C to about 15°C, about 2°C to about 10°C, about 5°C to about 37°C, about 5°C to about 35°C, about 5°C to about 30°C, about 5°C to about 25°C, about 5°C to about 20°C, about 5°C to about 15°C, about 5°C to about 10°C, about 10°C to about 37°C, about 10°C to about 35°C, about 10°C to about 30°C, about 10°C to about 25°C, about 10°C to about 20°C, about 10°C to about 15°C, about 15°C to about 37°C, about 15°C to about 35°C, about 15°C to about 30°C, about 15°C to about 25°C, about 15°C to about 20°C, about 20°C to about 37°C, about 20°C to about 35°C, about 20°C to about 30°C, about 20°C to about 25°C, about 25°C to about 37°C, about 25°C to about 35°C, about 25°C to about 30°C, about 30°C to about 37°C, or about 30°C to about 35°C.

[0173] In a further embodiment, the crystallization solution is heated to about 50°C (at which it remains in solution) and then cooled, wherein the crystallization solution crystallizes only when cooled to about 37°C or lower.

[0174] In a further embodiment, the crystallization solution is heated to about 50°C and then cooled to a temperature of about 18°C ​​to about 25°C or to about 25°C or lower.

[0175] In another embodiment, the crystallization solution is heated to about 50°C and then cooled to about 4°C.

[0176] In a specific embodiment of the method of the present invention, the incubation temperature is increased from about 4°C to about 10-40°C.

[0177] In any of the methods described herein, the crystallization solution is incubated for a sufficient period of time to form crystals. This can be achieved, for example, by visual inspection or SONIC. TM Imaging detection of crystal formation. In certain embodiments, the crystallization solution is incubated for about 15 minutes or longer. In some embodiments, the crystallization solution is incubated for about 2 hours or longer. In some embodiments, the crystallization solution is incubated overnight. In some embodiments, the crystallization solution is incubated for 18 hours or longer. In certain embodiments, the crystallization solution is incubated for about 30 minutes or longer, about 1 hour or longer, about 3 hours or longer, about 4 hours or longer, about 5 hours or longer, about 6 hours or longer, about 7 hours or longer, about 8 hours or longer, about 9 hours or longer, about 10 hours or longer, about 11 hours or longer, about 12 hours or longer, about 13 hours or longer, about 14 hours or longer, about 15 hours or longer, about 16 hours or longer, about 17 hours or longer, about 20 hours or longer, or about 24 hours or longer. In another embodiment, the crystallization solution is incubated for approximately 2, 3, 4, 5 days, 1 week, 10 days, 2 weeks, 15 days, 3 weeks, or more than 3 weeks.

[0178] In a particular embodiment of any of the methods described herein, the crystallization solution is rotated or stirred during incubation.

[0179] Various methods for protein crystallization are known. Giege et al. (1994) Acta Crystallogr. D50:339; McPherson (1990); Eur. J. Biochem. 189:1. These techniques include hanging drop vapor diffusion (McPherson (1976) J. Biol. Chem. 251:6300), sitting drop vapor diffusion, micro-batch, and dialysis.

[0180] Both hanging drop and sitting drop vapor diffusion require equilibrating a droplet containing purified protein, buffer, and precipitant with a larger reservoir containing a similar buffer and a higher concentration of precipitant. Initially, the droplet of protein solution contains insufficient concentration of precipitant for crystallization, but as water evaporates from the droplet and transfers to the reservoir, the precipitant concentration increases to the optimal level for crystallization. These optimal conditions are maintained until crystallization is complete because the system is in equilibrium. The difference between hanging drop and sitting drop lies in the vertical orientation of the protein solution droplets within the system.

[0181] In the micro-batch method, the peptide is mixed with a precipitant to achieve supersaturation, and the container is sealed and left to stand until crystals appear.

[0182] In dialysis, peptides remain on one side of the dialysis membrane that comes into contact with a solution containing a precipitant. The transmembrane equilibrium increases the precipitant concentration, leading to a supersaturation level of the peptide.

[0183] Some of these techniques are used to prepare the pembrolizumab crystals of the present invention, as described in more detail in the examples.

[0184] In any particular embodiment of any of the methods described herein, the crystallization solution is produced by vapor diffusion or batch crystallization.

[0185] In a particular embodiment of any method described herein for producing crystallized anti-PD-1 monoclonal antibodies, the method further includes a step of inoculating a crystallization solution with crystals of anti-PD-1 mAb prior to or during an incubation step.

[0186] Anti-PD-1 mAb crystals can be analyzed using various methods to examine or characterize their physical properties, such as crystal size, shape, surface morphology, total surface area, and porosity. These analytical techniques include, for example, electron diffraction and solid-state nuclear magnetic resonance (ssNMR), optical microscopy, transmission electron microscopy, scanning electron microscopy, atomic force microscopy, and various light scattering techniques. Furthermore, the biological activity and / or biophysical properties of the anti-PD-1 mAb in the crystals of this invention can be analyzed by “redissolving” the antibody crystals or by dissolving them in a buffer suitable for the desired analytical technique. For example, the dissolved anti-PD-1 mAb can be analyzed by one or more of ELISA, size exclusion chromatography, SDS-PAGE, and dynamic light scattering.

[0187] IV. Anti-PD-1 crystal antibody suspension and composition

[0188] In one aspect, the present invention provides isolated crystals formed by any method of the present invention, namely any method described herein for producing anti-PD-1mAb crystals.

[0189] The present invention also relates to a separated crystal comprising pembrolizumab in combination with caffeine, wherein the crystal is characterized by space group P2221. α = β = γ = 90°.

[0190] In one embodiment, the present invention provides a pembrolizumab crystal comprising a polypeptide, wherein the polypeptide is characterized by having structural coordinates of conserved residue backbone atoms with a root mean square deviation (RMSD) of less than about 2.0 angstroms when superimposed on backbone atoms described by structural coordinates in Table 7.

[0191] In some embodiments, the pembrolizumab crystals or pembrolizumab variant crystals of the present invention have a particle size of about 0.5 to 200 micrometers after harvesting. In a particular embodiment, the anti-PD-1 mAb crystals (e.g., pembrolizumab crystals) are homogenized after crystallization, resulting in a homogenized particle size of about 0.5 to about 50 micrometers.

[0192] In one embodiment, the present invention relates to crystalline pembrolizumab comprising pembrolizumab complexed with caffeine, characterized by solid-state NMR showing peaks at approximately 182.16, 181.54, 179.99, 109.36, 108.23, 103.58, 76.88, and 76.04 ppm. 13 C-spectrum. In another embodiment, a crystalline pembrolizumab complexed with caffeine is provided, characterized by solid-state NMR showing peaks at approximately 183.07, 182.16, 181.54, 180.55, 179.99, 110.70, 110.15, 109.36, 108.23, 103.58, 101.49, 99.75, 98.56, 76.88, 76.04, 74.97, 74.41, 73.52, 72.69, 13.85, 13.27, 12.26, and 11.13 ppm. 13 C-spectrum. In another embodiment, the crystalline pembrolizumab is characterized by, as Figure 10A Solid-state NMR as shown 13 C spectrum.

[0193] In another aspect, the present invention relates to pharmaceutical compositions comprising the novel anti-PD-1 crystal of the present invention (i.e., novel pembrolizumab crystal or pembrolizumab variant crystal) and a pharmaceutically acceptable carrier. To prepare the pharmaceutical composition, the anti-PD-1 mAb crystal of the present invention, or anti-PD-1 mAb dissolved from such crystal, is mixed with at least one pharmaceutically acceptable carrier or excipient. See, for example, Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984). The anti-PD-1 mAb crystal used in the pharmaceutical compositions of the present invention need not have any specific diffraction properties, as long as the biological activity and stability of the antibody remain within the desired range.

[0194] In some embodiments, the excipient is added directly to the crystallization solution during or after crystallization. In other embodiments, crystals are first harvested from the crystallization solution, washed by suspension in a stable solution, harvested from the stable solution, and then suspended in a liquid solution containing the excipient. The composition of the liquid can be any pharmaceutically acceptable medium and can include, for example, aqueous solutions and water-in-oil mixtures.

[0195] Pharmaceutical compositions in solid form can be prepared by drying a liquid suspension containing crystals and desired excipients, for example by passing nitrogen, air, or an inert gas through the crystals, by air drying, vacuum drying, or freeze drying. The moisture content in the final product is typically less than 10%, 7%, 5%, or 3% (by weight).

[0196] A pharmaceutical composition comprising pembrolizumab crystals dissolved from a liquid suspension or dried solid can be prepared by adding the desired amount of crystals to a pharmaceutically acceptable dissolution buffer and incubating at 4°C until the crystals dissolve. In one embodiment, the dissolution buffer comprises 10 mM histidine, pH 5.6, 0.02% polysorbate 80 w / v, and up to 4% sucrose w / v. In one embodiment, any particles in the resulting composition are removed prior to administration, for example, by centrifugation or filtration.

[0197] In a particular embodiment, the pharmaceutical composition is a crystal suspension, and the concentration of the anti-PD-1 mAb is about 5-400 mg / mL. In other embodiments, the concentration of the anti-PD-1 mAb is ≥75 mg / mL, ≥100 mg / mL, ≥125 mg / mL, ≥150 mg / mL, ≥175 mg / mL, ≥200 mg / mL, ≥225 mg / mL, ≥250 mg / mL, ≥275 mg / mL, ≥300 mg / mL, ≥325 mg / mL, or ≥350 mg / mL.

[0198] In certain embodiments, the pharmaceutical composition of the present invention further comprises a buffer of about 5 mM to about 50 mM. In some embodiments, the amount of buffer is about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM.

[0199] In specific embodiments, the pharmaceutical composition of the present invention further comprises about 0.01% to about 0.10% w / v of a nonionic surfactant. In some embodiments, the amount of nonionic surfactant is about 0.01% to about 0.05% w / v, about 0.01% to about 0.04% w / v, 0.02% to about 0.05% w / v, or 0.02% to about 0.04% w / v. In further embodiments, the pharmaceutical composition of the present invention does not contain any surfactant.

[0200] V. How to use

[0201] In one aspect, the present invention relates to a method for treating cancer in a patient requiring treatment, the method comprising administering to a subject an effective amount of (1) the anti-PD-1 mAb crystal of the present invention; i.e., a crystal of pembrolizumab or a crystal of a variant of pembrolizumab prepared by the methods described herein, or (2) administering to the patient a composition comprising the anti-PD-1 mAb crystal of the present invention and a pharmaceutically acceptable carrier. In some embodiments of the invention, the pembrolizumab crystal is dissolved in a solution (e.g., formulated as an aqueous preparation) prior to administration to the patient. In specific embodiments of the method, the composition is administered to the subject by intravenous administration. In other embodiments, the composition is administered to the subject by subcutaneous administration.

[0202] In some embodiments of the treatment method of the present invention, the dose of anti-PD-1 mAb is 200 mg, which is administered to the patient approximately every 3 weeks. In an alternative embodiment, the dose of the crystal monoclonal antibody is 400 mg, which is administered to the patient approximately every 6 weeks.

[0203] In some embodiments of the invention, a patient is given pembrolizumab crystal, a pembrolizumab variant crystal, or a composition containing pembrolizumab crystal or a pembrolizumab variant crystal every three weeks for 12 weeks or longer. In other embodiments, the crystals or compositions of the present invention are administered to the patient once every three weeks for a period of 15 weeks or longer, 18 weeks or longer, 21 weeks or longer, 24 weeks or longer, 27 weeks or longer, 30 weeks or longer, 33 weeks or longer, 36 weeks or longer, 39 weeks or longer, 42 weeks or longer, 45 weeks or longer, 48 weeks or longer, 51 weeks or longer, 54 weeks or longer, 57 weeks or longer, 60 weeks or longer, 63 weeks or longer, 66 weeks or longer, 69 weeks or longer, 72 weeks or longer, 75 weeks or longer, 78 weeks or longer, 81 weeks or longer, 84 weeks or longer, 87 weeks or longer, or 90 weeks or longer.

[0204] In other embodiments of the invention, a pembrolizumab crystal, a pembrolizumab variant crystal, or a composition comprising a pembrolizumab crystal or a pembrolizumab variant crystal is administered to a patient every six weeks for 12 weeks or longer. In other embodiments, the crystal or composition of the invention is administered to a patient every six weeks for 18 weeks or longer, 24 weeks or longer, 30 weeks or longer, 36 weeks or longer, 42 weeks or longer, 48 weeks or longer, 54 weeks or longer, 60 weeks or longer, 66 weeks or longer, 72 weeks or longer, 78 weeks or longer, 84 weeks or longer, 90 weeks or longer, 96 weeks or longer, 102 weeks or longer, 108 weeks or longer, 114 weeks or longer, 120 weeks or longer, 126 weeks or longer, or 132 weeks or longer.

[0205] In a first embodiment (E1), the present invention includes a method of treating cancer in a human patient, comprising administering an effective amount of the pembrolizumab crystal of the present invention to the patient.

[0206] In a second embodiment (E2), the present invention includes a method of treating melanoma in a human patient, comprising administering an effective amount of the pembrolizumab crystal of the present invention to the patient.

[0207] In a sub-implementation of implementation scheme E2, the melanoma is unresectable or metastatic.

[0208] In a further sub-implementation of implementation scheme E2, the melanoma is an adjuvant melanoma. In a specific implementation, the melanoma is a resected stage III melanoma.

[0209] In a third embodiment (E3), the present invention includes a method of treating a human patient with metastatic non-small cell lung cancer (NSCLC) by administering an effective amount of the pembrolizumab crystal of the present invention to the patient.

[0210] In a sub-implementation of Example E3, the NSCLC is scaly. In an alternative implementation, the NSCLC is non-scaly.

[0211] In a sub-implementation of implementation scheme E3, the method further includes administering carboplatin-paclitaxel or nab-paclitaxel to the patient.

[0212] In the sub-implementation plan of implementation plan E3 (implementation plan E3-A), the patient has a tumor with high PD-L1 expression [(tumor proportion score (TPS) ≥ 50%)] and has not previously received platinum-based chemotherapy.

[0213] In another sub-implementation of implementation plan E3 (implementation plan E3-B), the patient has a tumor with PD-L1 expression (TPS ≥ 1%) and has previously received platinum-based chemotherapy. In a specific implementation plan of implementation plan E3-B, the patient experiences disease progression during or after receiving platinum-based chemotherapy.

[0214] In some implementation schemes of scheme E3, the patient has a tumor with PD-L1 expression (TPS≥1%) and has not previously received platinum-based chemotherapy.

[0215] In certain embodiments of Example E3 (including embodiments E3-A and E3-B), PD-L1 TPS are determined by FDA-approved testing.

[0216] In certain implementation schemes of scheme E3 (including schemes E3-A and E3-B), the patient’s tumor does not have EGFR or ALK genomic abnormalities.

[0217] In certain implementation schemes of scheme E3 (including schemes E3-A and E3-B), the patient’s tumor had an EGFR or ALK genomic abnormality prior to receiving an anti-PD-1 antibody or its antigen-binding fragment, and the disease progressed during or after treatment for the EGFR or ALK abnormality.

[0218] In a fourth embodiment (E4), the present invention includes a method for treating a human patient with metastatic non-small cell lung cancer (NSCLC), comprising: (1) administering an effective amount of the pembrolizumab crystal of the present invention to the patient, and (2) administering pemetrexed and carboplatin to the patient. In a sub-implementation of E4, the patient has not previously received anticancer therapy prior to initiating a combination therapy regimen of the pembrolizumab crystal of the present invention with pemetrexed and carboplatin.

[0219] In specific embodiments (including sub-implementations) of Examples E3 and E4, the patient has non-squamous non-small cell lung cancer.

[0220] In the sub-implementation of implementation plan E4, pemetrexed is administered at 500 mg / m². 2 The dosage is administered to the patient.

[0221] In a sub-implementation of implementation plan E4, pemetrexed is administered to the patient every 21 days via intravenous infusion. In a specific implementation plan, the infusion time is approximately 10 minutes.

[0222] In a sub-implementation of implementation scheme E4 (implementation scheme E4-A), the invention further includes administering approximately 400 μg to approximately 1000 μg of folic acid once daily to the patient, starting approximately 7 days before administering pemetrexed and continuing until approximately 21 days after the patient has received the last dose of pemetrexed. In some embodiments, the folic acid is administered orally.

[0223] In a sub-implementation of implementation schemes E4 and E4-A (implementation scheme E4-B), the present invention further includes administering approximately 1 mg of vitamin B to the patient approximately one week before the first administration of pemetrexed and approximately every three cycles of pemetrexed administration (i.e., approximately every nine weeks). 12 In some implementations, vitamin B... 12 Intramuscular application.

[0224] In a sub-implementation of implementation schemes E4, E4-A, and E4-B (implementation scheme E4-C), the invention further includes administering approximately 4 mg of dexamethasone twice daily to the patient one day before, on, and one day after pemetrexed administration. In some embodiments, dexamethasone is administered orally.

[0225] In a fifth embodiment (E5), the present invention includes a method of treating recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) in a human patient, comprising administering an effective amount of the pembrolizumab crystal of the present invention to the patient.

[0226] In certain sub-implementations of Example E5, the patient had not previously received platinum-based chemotherapy and the patient's tumor expressed PD-L1 (Comprehensive Positive Score (CPS) ≥20).

[0227] In some sub-implementations of Example E5, the patient has recurrent or metastatic HNSCC.

[0228] In a sub-implementation of implementation plan E5, the patient had previously received platinum-based chemotherapy. In some implementation plans, the patient experienced disease progression during or after platinum-based chemotherapy.

[0229] In a sixth embodiment (E6), the present invention includes a method for treating refractory classical Hodgkin's lymphoma (cHL) in a human patient, comprising administering an effective amount of the pembrolizumab crystal of the present invention to the patient.

[0230] In the seventh embodiment (E7), the present invention includes a method for treating classical Hodgkin's lymphoma (cHL) in a human patient, comprising administering an effective amount of the pembrolizumab crystal of the present invention to the patient, wherein the patient has relapsed after 3 or more lines of treatment for cHL.

[0231] In the sub-implementations of implementation schemes E6 and E7, the patient is an adult patient.

[0232] In the alternative sub-implementations of implementation schemes E6 and E7, the patients are pediatric patients.

[0233] In the eighth embodiment (E8), the present invention includes a method for treating locally advanced or metastatic urothelial carcinoma in a human patient, comprising administering an effective amount of the pembrolizumab crystal of the present invention to the patient.

[0234] In the sub-implementation of implementation plan E8, the patient is not suitable for chemotherapy containing cisplatin.

[0235] In a sub-implementation of implementation scheme E8, the patient has a tumor expressing PD-L1. In some implementation schemes, the PD-L1 expression level is characterized by CPS ≥ 10.

[0236] In the sub-implementation of implementation plan E8, the patient experiences disease progression during or after platinum-based chemotherapy or within 12 months after neoadjuvant or adjuvant treatment with platinum-based chemotherapy.

[0237] In the ninth embodiment (E9), the present invention includes a method for treating unresectable or metastatic solid tumors with high microsatellite instability (MSI-H) or mismatch repair deficiency in human patients, comprising administering an effective amount of the pembrolizumab crystal of the present invention to the patient.

[0238] In a sub-implementation of implementation plan E9, the patient experiences disease progression following prior anticancer treatment.

[0239] In the tenth embodiment (E10), the present invention includes a method for treating unresectable or metastatic colorectal cancer with high microsatellite instability (MSI-H) or mismatch repair deficiency in human patients, comprising administering an effective amount of the pembrolizumab crystal of the present invention to the patient.

[0240] In a sub-implementation of implementation plan E10, the patient experienced disease progression following prior treatment with fluoropyrimidine, oxaliplatin, and irinotecan.

[0241] In the eleventh embodiment (E11), the present invention includes a method for treating recurrent locally advanced or metastatic gastric cancer in a human patient, comprising administering to the patient an effective amount of the pembrolizumab crystal of the present invention.

[0242] In the twelfth embodiment (E12), the present invention includes a method for treating recurrent locally advanced or metastatic adenocarcinoma of the gastroesophageal junction in a human patient, comprising administering to the patient an effective amount of the pembrolizumab crystal of the present invention.

[0243] In the sub-implementation schemes of implementation schemes E11 and E12, the patient's tumor expresses PD-L1 [Comprehensive Positive Score (CPS) ≥1].

[0244] In the sub-implementations of implementation schemes E11 and E12, the patient experiences disease progression during or after two or more prior line treatments, including fluoropyrimidine and platinum-based chemotherapy.

[0245] In the sub-implementations of implementation schemes E11 and E12, the patient experiences disease progression during or after two or more prior line treatments, including HER2 / neu targeted therapy.

[0246] In the thirteenth embodiment (E13), the present invention includes a method for treating cervical cancer in a human patient, comprising administering an effective amount of the pembrolizumab crystal of the present invention to the patient.

[0247] In a sub-implementation of implementation plan E13, the patient has recurrent or metastatic cervical cancer.

[0248] In a further sub-implementation of implementation plan E13, the patient experiences disease progression during or after chemotherapy.

[0249] In another sub-implementation of implementation scheme E13, the patient has a tumor that expresses PD-L1 [CPS≥1].

[0250] In the fourteenth embodiment (E14), the present invention includes a method of treating cancer in a human patient, comprising administering to the patient an effective amount of the pembrolizumab crystal of the present invention, wherein the patient has cancer selected from the following: melanoma, lung cancer, head and neck cancer, bladder cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, lymphoma, renal cancer, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, Merkel cell carcinoma, and salivary gland cancer.

[0251] In the fifteenth embodiment (E15), the present invention includes a method of treating cancer in a human patient, comprising administering an effective amount of the pembrolizumab crystal of the present invention to the patient, wherein the patient has small cell lung cancer.

[0252] In a sub-implementation of implementation plan E15, the patient has metastatic SCLC. In some sub-implementations, the patient previously received platinum-based chemotherapy (during or after platinum-based chemotherapy) and at least one other previous line of treatment. In some sub-implementations, the patient experienced disease progression during or after platinum-based chemotherapy and at least one other previous line of treatment.

[0253] In the sixteenth embodiment (E16), the present invention includes a method of treating non-Hodgkin's lymphoma in a human patient, comprising administering an effective amount of the pembrolizumab crystal of the present invention to the patient.

[0254] In a sub-implementation of implementation plan E16, non-Hodgkin's lymphoma is mediastinal large B-cell lymphoma. In some implementation plans, non-Hodgkin's lymphoma is refractory primary mediastinal large B-cell lymphoma (PMBCL). In other implementation plans, the patient has PMBCL and has relapsed after two or more prior lines of therapy.

[0255] In the seventeenth embodiment (E17), the present invention includes a method for treating breast cancer in a human patient, comprising administering an effective amount of the pembrolizumab crystal of the present invention to the patient.

[0256] In the sub-implementation of implementation plan E17, breast cancer is triple-negative breast cancer.

[0257] In the sub-implementation of implementation scheme E17, breast cancer is ER+ / HER2- breast cancer.

[0258] In the eighteenth embodiment (E18), the present invention includes a method for treating nasopharyngeal carcinoma in a human patient, comprising administering an effective amount of the pembrolizumab crystal of the present invention to the patient.

[0259] In the nineteenth embodiment (E19), the present invention includes a method of treating thyroid cancer in a human patient, comprising administering an effective amount of the pembrolizumab crystal of the present invention to the patient.

[0260] In the twentieth embodiment (E20), the present invention includes a method for treating salivary gland cancer in a human patient, comprising administering an effective amount of the pembrolizumab crystal of the present invention to the patient.

[0261] In the twenty-first embodiment (E21), the present invention includes a method of treating Merkel cell carcinoma (MCC) in a human patient, comprising administering an effective amount of the pembrolizumab crystal of the present invention to the patient. In this sub-implementation, the MCC is recurrent locally advanced or metastatic.

[0262] In the twenty-second embodiment (E22), the present invention includes a method for treating cancer in a human patient, the method comprising administering to the patient an effective amount of the pembrolizumab crystal of the present invention, wherein the cancer is selected from: melanoma, non-small cell lung cancer, relapsed or refractory classical Hodgkin's lymphoma, head and neck squamous cell carcinoma, cervical cancer, urothelial carcinoma, esophageal cancer, gastric cancer, primary mediastinal large B-cell lymphoma, and hepatocellular carcinoma.

[0263] In the twenty-third embodiment (E23), the present invention includes a method of treating cancer in a human patient, comprising administering to the patient an effective amount of the pembrolizumab crystal of the present invention, wherein the cancer is a heme malignancy.

[0264] In the sub-implementation of implementation plan E23, heme malignancies are selected from: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), EBV-positive DLBCL, primary mediastinal large B-cell lymphoma, T-cell / histiocytic large B-cell lymphoma, follicular lymphoma, Hodgkin's lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid leukemia-1 protein (MCL-1), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), and small lymphocytic lymphoma (SLL).

[0265] In the twenty-fourth embodiment (E24), the present invention includes a method of treating cancer in a human patient, comprising administering to the patient an effective amount of the pembrolizumab crystal of the present invention, wherein the patient has a tumor with a high mutation burden.

[0266] In a twenty-sixth embodiment (E26), the present invention includes a method of treating hepatocellular carcinoma in a human patient, comprising administering to the patient an effective amount of the pembrolizumab crystal of the present invention. In a sub-implementation of E26, the patient has previously received sorafenib treatment.

[0267] In a twenty-seventh embodiment (E27), the present invention includes a method of treating renal cell carcinoma in a human patient, comprising administering an effective amount of the pembrolizumab crystal of the present invention to the patient. In a sub-implementation of E27, the renal cell carcinoma is clear cell renal cell carcinoma.

[0268] In a twenty-eighth embodiment (E28), the present invention includes a method of treating esophageal cancer in a human patient, comprising administering an effective amount of the pembrolizumab crystal of the present invention to the patient. In a sub-implementation of E28, the esophageal cancer is a recurrent locally advanced or metastatic squamous cell carcinoma of the esophagus. In a further sub-implementation, the patient has experienced disease progression after one or more lines of systemic therapy. In a further sub-implementation, the patient's tumor expresses PD-L1 [Comprehensive Positive Score (CPS) ≥10].

[0269] In the twenty-ninth embodiment (E29), the present invention includes a method for treating ovarian cancer in a human patient, comprising administering to the patient an effective amount of the pembrolizumab crystal of the present invention.

[0270] In the thirtieth embodiment (E30), the present invention includes a method for treating colorectal cancer in a human patient, comprising administering to the patient an effective amount of the pembrolizumab crystal of the present invention.

[0271] In the thirty-first embodiment (E31), the present invention includes a method of treating cancer in a human patient, the method comprising administering to the patient an effective amount of the pembrolizumab crystal of the present invention, wherein the cancer is selected from: melanoma, lung cancer, head and neck cancer, bladder cancer, breast cancer, gastrointestinal cancer, multiple myeloma, lymphoma, renal cancer, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, salivary gland cancer, prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), pancreatic cancer, colon cancer, esophageal cancer, liver cancer, thyroid cancer, endometrial cancer, hepatocellular carcinoma, Merkel cell carcinoma, glioblastoma, glioma, and other neoplastic malignancies.

[0272] In any of the methods of the invention described herein, “pembrolizumab crystal of the invention” or “anti-PD-1 crystal mAb of the invention” can be any pembrolizumab crystal or pembrolizumab variant crystal of the invention (i.e., crystals described herein or prepared by the methods described herein), or a composition comprising the pembrolizumab crystal or pembrolizumab variant crystal of the invention, as detailed in Section 2 of this invention (titled “…”). Anti-PD-1 antibody used in the method of the present invention As described in or as in Section 4 (titled "") Anti-PD-1 antibody crystals, antibody suspensions and compositions As stated in ").

[0273] Malignancies exhibiting disease-free and overall survival improvements associated with the presence of tumor-infiltrating lymphocytes in biopsies or surgical materials, such as melanoma, colorectal cancer, liver cancer, kidney cancer, gastric / esophageal cancer, breast cancer, pancreatic cancer, and ovarian cancer, are covered by the methods and treatments described herein. These cancer subtypes are known to be susceptible to T-lymphocyte immune control. Furthermore, refractory or recurrent malignancies whose growth can be suppressed using the antibodies described herein are also included.

[0274] In some embodiments, the compositions of the present invention are administered to subjects suffering from cancers characterized by elevated expression of PD-L1 and / or PD-L2 in the tested tissue sample, including ovarian cancer, renal cancer, colorectal cancer, pancreatic cancer, breast cancer, liver cancer, gastric cancer, esophageal cancer, and melanoma. Other cancers that may benefit from treatment with the compositions of the present invention include those associated with persistent viral infections, such as human immunodeficiency virus (HIV), hepatitis A, B, and C viruses, Epstein-Barr virus (EBV), and human papillomavirus (HPV) known to be causally associated with, for example, Kaposi's sarcoma, liver cancer, nasopharyngeal carcinoma, lymphoma, cervical cancer, vulvar cancer, anal cancer, penile cancer, and oral cancer.

[0275] Another aspect includes methods for treating patients who have, are suspected of having, or are at risk of having an infection or infectious disease using the anti-PD-1 mAb crystals or pharmaceutical compositions of the present invention. Therefore, the present invention provides a method for treating chronic infections in mammalian subjects, comprising administering to the subject an effective amount of the anti-PD-1 crystal mAb of the present invention or a composition comprising the anti-PD-1 crystal mAb of the present invention. In some specific embodiments of the method, the composition is administered to the subject intravenously. In other embodiments, the composition is administered to the subject subcutaneously.

[0276] In this respect, the compositions of the present invention can be used alone or in combination with vaccines to stimulate an immune response against pathogens, toxins, and self-antigens. The compositions of the present invention can be used to stimulate an immune response against infectious viruses in humans, including but not limited to: human immunodeficiency virus, hepatitis A, B, and C viruses, Epstein-Barr virus (EBV), human cytomegalovirus, human papillomavirus (HPV), and herpesvirus. The compositions of the present invention comprising antagonistic anti-PD-1 antibodies or antibody fragments can be used to stimulate an immune response against infections caused by bacteria, fungi, parasites, and other pathogens. Hepatitis B and C infections and HIV infections are particularly considered chronic viral infections.

[0277] The anti-PD-1 mAb crystals and compositions of the present invention can be administered to patients in combination with one or more "additional therapeutic agents". These additional therapeutic agents can be biological therapeutic agents (including, but not limited to, antibodies against VEGF, EGFR, Her2 / neu, VEGF receptors, other growth factor receptors, CD20, CD40, CD-40L, OX-40, 4-1BB, and ICOS), growth inhibitors, immunogenic agents (e.g., attenuated cancer cells, tumor antigens, antigen-presenting cells (e.g., dendritic cells stimulated with tumor-derived antigens or nucleic acids), immunostimulatory cytokines (e.g., IL-2, IFNα2, GM-CSF), and cells transfected with genes encoding immunostimulatory cytokines (e.g., but not limited to GM-CSF).

[0278] As described above, in some embodiments of the method of the present invention, the method further includes administering additional therapeutic agents. In specific embodiments, the additional therapeutic agent is an anti-LAG3 antibody or its antigen-binding fragment, an anti-GITR antibody or its antigen-binding fragment, an anti-TIGIT antibody or its antigen-binding fragment, or an anti-CD27 antibody or its antigen-binding fragment. In one embodiment, the additional therapeutic agent is a Newcastle disease virus vector expressing IL-12. In a further embodiment, the additional therapeutic agent is dinaciclib. In a still further embodiment, the additional therapeutic agent is a STING agonist. In a still further embodiment, the additional therapeutic agent is a PARP inhibitor. In a still further embodiment, the additional therapeutic agent is a multityrosine kinase inhibitor. In another embodiment, the additional therapeutic agent is a MEK inhibitor. In another embodiment, the additional therapeutic agent is a CXCR2 antagonist. In another embodiment, the additional therapeutic agent is navarixin. In another embodiment, the additional therapeutic agent is olarparib. In another embodiment, the additional therapeutic agent is selumetinib. In another embodiment, the additional therapeutic agent is axitinib.

[0279] Other appropriate routes of administration for therapeutic agents may include, for example, parenteral delivery, including intramuscular, subcutaneous, intrathecal, direct intraventricular, intravenous, and intraperitoneal delivery. Drugs can be administered via a variety of conventional methods, such as intraperitoneal, parenteral, intra-arterial, or intravenous injection.

[0280] The choice of dosage for additional therapeutic agents depends on several factors, including serum or tissue turnover of the entity, symptom level, immunogenicity of the entity, and accessibility of target cells, tissues, or organs in the individual being treated. The dosage of additional therapeutic agents should be an amount that provides an acceptable level of side effects. Therefore, the dosage and frequency of administration for each additional therapeutic agent (e.g., a biologic or chemotherapeutic agent) will depend in part on the specific agent, the severity of the cancer being treated, and patient characteristics. We provide guidelines for selecting appropriate dosages of antibodies, cytokines, and small molecules. See, for example, Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al. (2003) New Engl. J. Med. 348: 601-608; Milgrom et al. (1999) New Engl. J. Med. 341: 1966-1973; Slamon et al. al.(2000)New Engl.J.Med.342:613-619; Ghosh et al.(2003)New Engl.J.Med.348:24-32; Lipsky et al.(2000)New Engl.J.Med.343:1594-1602; Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed); Medical Economics Company; ISBN:1563634457; 57th edition (November 2002). Clinicians may determine an appropriate dosing regimen, for example, using parameters or factors known or suspected to affect or expected to affect treatment in the field, and will depend on, for example, the patient's clinical history (e.g., previous treatment), the type and stage of the cancer to be treated, and biomarkers of response to one or more therapeutic agents in combination therapy.

[0281] Various literature references are available to facilitate the selection of pharmaceutically acceptable carriers or excipients for alternative therapeutic agents. See, for example, Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984); Hardman et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parental Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms:Disperse Systems,Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY.

[0282] In some implementations, additional therapeutic agents are administered via continuous infusion or at intervals such as daily, 1-7 times per week, weekly, bi-weekly, bi-weekly, monthly, bi-monthly, etc. Preferred dosing regimens are those involving the maximum dose or frequency that avoids serious adverse side effects. The total weekly dose is typically at least 0.05 μg / kg, 0.2 μg / kg, 0.5 μg / kg, 1 μg / kg, 10 μg / kg, 100 μg / kg, 0.2 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 10 mg / kg, 25 mg / kg, 50 mg / kg body weight or more. See, for example, Yang et al. (2003) New Engl. J. Med. 349:427-434; Herold et al. (2002) New Engl. J. Med. 346:1692-1698; Liu et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456; Portielji et al. (2003) Cancer Immunol. Immunother. 52:133-144. The required doses of small molecule therapeutics (e.g., peptide mimics, natural products, or organic chemicals) are roughly the same as the required doses (in moles per kilogram) of antibodies or peptides.

[0283] In some embodiments, dosing includes administering additional therapeutic agents to the subject in escalating doses of 1.0, 3.0, and 10 mg / kg throughout the course of treatment. The formulation may be a reconstituted liquid formulation, or it may be a previously undylated liquid formulation. The timeline may vary and may continue as long as the desired effect is achieved. In some embodiments, dose escalation will continue up to a dose of approximately 10 mg / kg. In some embodiments, the subject has a histological or cytological diagnosis of melanoma or other forms of solid tumor, and in some cases, the subject may have an unmeasurable disease. In some embodiments, the subject has received treatment with other chemotherapy agents, while in other embodiments, the subject has not received treatment.

[0284] In some embodiments, the dosing regimen includes administering doses from about 0.005 mg / kg to about 10 mg / kg, with dose escalation within the patient. In some embodiments, doses of 5 mg / kg or 10 mg / kg are administered at intervals of 3 weeks or 2 weeks. In yet another embodiment, for patients with melanoma or other solid tumors, a dose of 3 mg / kg is administered at three-week intervals. In these embodiments, the patient should have an unresectable disease; however, the patient may have previously undergone surgery.

[0285] In some embodiments, the subject is administered a 30-minute IV infusion of any of the pharmaceutical formulations described herein. In some embodiments of escalating dosing, the dosing interval between the first and second doses is approximately 28 days (±1 day). In some embodiments, the interval between the second and third doses is approximately 14 days (±2 days). In some embodiments, the dosing interval for doses following the second dose is approximately 14 days (±2 days).

[0286] Subcutaneous administration can be achieved by injection using a syringe or by using other injection devices (e.g. Devices; injection pens; or needle-free devices (e.g., MediJector and...) It is administered via injection.

[0287] Embodiments of the present invention also include one or more anti-PD-1 mAb crystals of the present invention (e.g., crystal pembrolizumab or pembrolizumab variants) or formulations comprising crystals prepared by the methods described herein or by the methods described herein, which (i) are used for the following purposes, (ii) are used as a medicament or composition for the following purposes, or (iii) are used to prepare a medicament for the following purposes: (a) (e.g., human) treatment; (b) pharmaceutical; (c) inducing or increasing an antitumor immune response; (d) reducing the amount of one or more tumor markers in a patient; (e) stopping or delaying the growth of a tumor or blood cancer; (f) stopping or delaying PD-1-related (g) Disease progression; (h) Stopping or delaying cancer progression; (i) Stabilizing PD-1-related disease; (j) Inhibiting tumor cell growth or survival; (k) Eliminating or shrinking the size of one or more cancerous lesions or tumors; (l) Reducing the progression, onset, or severity of PD-1-related disease; (m) Reducing the severity or duration of clinical symptoms of PD-1-related disease (e.g., cancer); (n) Prolonging patient survival relative to the expected survival of similar untreated patients; (o) Inducing complete or partial remission of cancerous symptoms or other PD-1-related diseases; (p) Treatment of cancer or infectious diseases.

[0288] For the purpose of describing and disclosing the methods and materials that can be used in conjunction with the present invention, all publications mentioned herein are incorporated herein by reference.

[0289] Different embodiments of the invention have been described herein with reference to the accompanying drawings, but it should be understood that the invention is not limited to these precise embodiments, and that various changes and modifications can be made therein without departing from the scope or spirit of the invention as defined in the appended claims.

[0290] Example 1

[0291] High-throughput crystallization screening of pembrolizumab

[0292] The ability of various small molecule reagents to promote pembrolizumab crystallization was screened, including amino acids, peptides, organic salts and acids, as well as bioactive small molecules (from Hampton Research Silver Bullet Bio Screening (catalog #HR2-088)). Solutions containing pembrolizumab (44 mg / mL) at 10 mM histidine and pH 5.6 were screened in 1536 unique crystallization plates (microbatch-under-oil) using 0.2 μl of pembrolizumab and 0.2 μl of screening solution (Luft et al., Journal of Structural Biology 142:170–179 (2003)). The screening solutions could be broken down into three main categories: (1) salts and buffers (combinations of 36 salts and eight buffers at three concentrations); (2) PEGs, salts, and buffers (combinations of eight PEGs at two concentrations with 36 salts and eight buffers); and (3) PEGs and Silver Bullet Bio reagents. The Silver Bullets Bio Screening consisted of 96 solutions in high-throughput form using a single deep-well module (Greiner780261). Each reagent was a mixture of small or large molecule digests in 0.02 M HEPES sodium pH 6.8 buffer. Each solution contained 2 to 20 small molecules. The Silver Bullets Bio Screening was diluted 1:10 in 15% PEG 3350, 0.02 M HEPES, pH 6.8 as a precipitation reagent. Experiments were performed at 4 °C, 20–22 °C, and 30 °C. Crystallization formation in the wells over time was monitored microscopically.

[0293] One month later, several molecules that induce pembrolizumab crystallization were identified. Crystallization was performed using SONICC. TM Visualization using an imaging system (Formulatrix, Bedford, MA). Second-order nonlinear imaging of chiral crystals (SONICC) is an imaging technique for visualizing protein crystals, enabling their discovery and identification. Two techniques—second harmonic generation (SHG) to detect crystallinity and ultraviolet two-photon excitation fluorescence (UV-TPEF) specific to protein samples—are combined to actively identify protein crystals. Crystals appear white against a completely black background, allowing for identification even in low-light environments. SONICC can also detect extremely small crystals, or microcrystals defined as having at least one dimension less than 1 μm.

[0294] One of the molecules identified at 30°C, ammonium dihydrogen phosphate, was also identified as a crystallizing agent compatible with previously developed high-salt processes. See WO 2016 / 137850. Furthermore, novel crystallizing agents, different from those used in previous high-salt processes, were identified as mixtures. Table 3 below provides the mixtures that can be used to produce crystals and the temperatures at which crystallization screening is positive.

[0295] Table 3. Crystallization Screening Results

[0296]

[0297] Four molecules were identified for further investigation: caffeine, theophylline, 2'-deoxyguanosine-5'-monophosphate, and gibberellin A3. No crystals were observed for any of the molecules tested in this screening at 4°C. Figure 1 shows an image of crystals formed using Silver BullerBio A2 as a crystallizing additive.

[0298] Example 2

[0299] Confirmation of crystallizing agent using droplet vapor diffusion

[0300] A seated-drop vapor diffusion experiment (96-well Swissci plate, 3 drops) was performed to confirm the crystallizing agent identified in Example 1. An antibody solution containing 44 mg / mL pembrolizumab was prepared in 10 mM histidine, pH 5.6. Several different mixtures were prepared containing 50 mM HEPES per mixture, pH 6.8, 12-15% w / v PEG 3350, and an additive (total volume 0.6 μL). The drop ratios were varied as follows: drop 1: 0.4 μL mixture + 0.2 μL pembrolizumab, drop 2: 0.3 μL mixture + 0.3 μL pembrolizumab, and drop 3: 0.2 μL mixture + 0.4 μL pembrolizumab. The experiment was performed at 23 °C. Crystal formation in the plate wells was monitored microscopically over time.

[0301] The results confirmed that 0.1-0.18% caffeine alone facilitated the crystallization of pembrolizumab in the presence of 12-15% w / v PEG 3350 and 50 mM HEPES (pH 6.8). 0.15% w / v caffeine and 0.15% w / v gibberellin A3, whether mixed together or independently, also effectively produced pembrolizumab crystals in the presence of 12-15% w / v PEG 3350, 50 mM HEPES, and pH 6.8. Theophylline did not produce crystals at 0.15% w / v, but at higher concentrations of 0.25% and 0.30% w / v, it effectively crystallized pembrolizumab in the presence of 12-15% w / v PEG 3350, 50 mM HEPES, and pH 6.8. 0.15% w / v theophylline, when paired with other Silver Bullet reagents, does indeed produce crystals when mixed with 0.2% w / v 2'-deoxyguanosine 5'-monophosphate sodium hydrate, 0.2% ethanolamine, 0.2% IPTG, 0.2% thiamine pyrophosphate, and 0.2% choline base solution. See Figure 2. 50 mM HEPES buffer and 12–15% w / v PEG 3350 alone do not produce any crystals.

[0302] Example 3

[0303] Batch crystallization of pembrolizumab

[0304] The experiment was designed to determine the optimal differential batch crystallization conditions for producing a pembrolizumab crystal suspension with a uniform particle size distribution of 10–50 micrometers.

[0305] The pembrolizumab stock solution in 10 mM histidine buffer (pH 5.6) was concentrated in a concentrator to achieve a final protein concentration of 44 mg / mL. The concentrated solution was then diluted to 20 mg / mL pembrolizumab in histidine buffer. A 2.5% w / v caffeine solution was prepared by adding 1.25 g of caffeine (Sigma catalog number C7731-250G) to 50 mL of 20 mM histidine (pH 5.4) and heating the resulting mixture to 40°C until the caffeine dissolved and formed a solution.

[0306] 0.2% caffeine in 10 mM tris (pH 8.0) was mixed with 16% PEG 3350 and 50 mM HEPES in 20 mg / mL pembrolizumab solution (50 mM histidine buffer, pH 5.4), with the pH changed at 0.1 intervals from 6.8 to 7.4. Batch crystallization was performed in 1.5 mL Eppendorf tubes with a total volume of 200 μL at pembrolizumab:mixture ratios of 1:1, 1:2, and 1:3. The tubes were placed on a rotating platform or stirring plate. All experiments were performed at room temperature, except for early batch crystallization at 4°C (which produced mostly precipitate instead of crystals).

[0307] For experiments conducted at room temperature, crystals formed on the first day and continued to form for more than 18 hours. A small amount of the crystallized solution was extracted for imaging on a batch plate. The optimal conditions for obtaining single-needle crystals of 10–50 micrometers were 15 mg / mL pembrolizumab, 0.20% caffeine, 14% PEG 3350, 50 mM HEPES, and pH 7.3 at room temperature for 18 hours.

[0308] Example 4

[0309] Batch crystallization scale-up experiment (1 mL scale) — Comparison of static method and rotation method

[0310] Two 1 mL batch crystallization experiments were conducted by mixing 333 μL of 19.4 mg / mL pembrolizumab, 0.175% caffeine, and 50 mM histidine at pH 5.5 (part A) with 666 μL of 50 mM HEPES at pH 7.7 and 10.18% PEG 3350 (part B) in a 1.5 mL Eppendorf tube. Part A solution preparation: The 44 mg / mL pembrolizumab solution was diluted to 20 mg / mL with 50 mM histidine at pH 5.5. 112 μL of 2.5% caffeine at 10 mM histidine at pH 5.5 was added to 1.4 mL of the diluted solution. The final composition of Part A was 19.4 mg / mL pembrolizumab, 7% caffeine, 50 mM histidine, and pH 5.5. Preparation of Part B solution: A 50 mM HEPES solution with pH 7 and 10.18% PEG 3350 was prepared using an Optimatrix maker liquid handling system.

[0311] One test tube was incubated under static conditions, while the other was placed on a Labnet MiniLabRoller H5500 at 30°C for 18 hours. Experiments under 200x microscopic examination revealed crystal clusters under static conditions, while a uniform suspension of needle-like crystals (10–30 μm) was observed in the rotated sample, indicating that rotation is the preferred procedure compared to static incubation.

[0312] Example 5

[0313] Pembrolizumab fractional crystallization using caffeine / PEG 3350 process (10 mL scale batches)

[0314] A 20 mg / mL pembrolizumab solution was prepared by diluting a 44 mg / mL pembrolizumab stock solution to a total volume of 3.33 mL with 20 mM histidine buffer, pH 5.4. 6.66 mL of 13% PEG 3350, 50 mM HEPES, pH 7.7, and 1.0 mL of 2.5% caffeine in 20 mM histidine buffer, pH 5.4 were added to this solution. The final composition of the resulting solution was 6.7 mg / mL pembrolizumab, 9.8% PEG 3350, 45 mM HEPES, pH 7.7, 6.6 mM histidine, and 0.23% caffeine. The solution was incubated at 30°C on a 24 RPM Labnet Mini LabRoller H5500 rotisserie. The solution was initially clear but became turbid after 18 hours. The turbid suspension was examined under a microscope, and microneedle formation was confirmed by 200x magnification. See the micrograph of the derived crystal. Figure 3 .

[0315] The derived crystal suspension was further processed to remove non-crystalline pembrolizumab and excess caffeine from the suspension, and the crystallization yield was measured.

[0316] Centrifuge 1 mL aliquots of the crystal suspension at 3000 RPM for 3 minutes in a microcentrifuge. Resuspend the resulting precipitate in 1 mL of 13% PEG 3350, 50 mM HEPES, pH 7.7, and label the supernatant as Wash Buffer 1. Centrifuge the suspension at 3000 RPM for 3 minutes in a microcentrifuge. Resuspend the resulting precipitate in 1 mL of 13% PEG 3350, 50 mM HEPES, pH 7.7, and label the supernatant as Wash Buffer 2. Centrifuge the suspension at 3000 RPM for 3 minutes in a microcentrifuge. Redissolve the resulting precipitate in 1 mL of cold 20 mM histidine buffer, pH 5.4. The precipitate dissolves within 5 minutes.

[0317] Protein concentration was determined using a nano-drop spectrophotometer with an extinction coefficient of 1.4. The mother liquor protein concentration was 78 mg / mL (due to caffeine distortion), wash buffer 1: 24 mg / mL (due to caffeine distortion), wash buffer 2: 3.87 mg / mL (due to caffeine distortion), and the final redissolved crystals: 6.2 mg / mL (280:260 nm ratio of 0.52, the same as the starting pembrolizumab solution). Based on protein determination, the overall yield was 94%.

[0318] Example 6

[0319] Crystallization screening in temperature range of 0-50℃

[0320] A solution of 44 mg / mL pembrolizumab in 20 mM histidine buffer, pH 5.4 was prepared using sterile, pyrogen-free aqueous solution (0.2 μm filtration).

[0321] A 2.5% caffeine, 20mM histidine, pH 5.4 solution was prepared by adding 1.25g of caffeine (Sigma; lot number SLBK4804V) to 50mL of 20mM histidine (Sigma; H-8000), pH 5.4. The mixture was heated to 60°C until the caffeine was incorporated into the solution. The resulting solution was cooled to room temperature before use.

[0322] A solution of 10.18% PEG 3350, 50mM HEPES, pH 7.4 was prepared by adding 2.5 mL of 1M HEPES (1M solution, pH 7.4; Hampton Research HR2-941-27), pH 7.4, and 10.2 mL of 50% PEG 3350 to 37.3 mL of sterile water for injection. The resulting solution was filtered through a 0.2-micron filter.

[0323] At room temperature, 66 μl of a solution of 10.18% PEG 3350, 50 mM HEPES, pH 7.4 was added to 33 μl of pembrolizumab solution (44 mg / mL) in 20 mM histidine buffer, pH 5.4. Then, 10 μl of 2.5% caffeine, 20 mM histidine buffer, pH 5.4 was added to the resulting solution. A mixture of 1.45 mg pembrolizumab, 6 mM histidine, pH 5.4, 6.1% PEG 3350, 30 mM HEPES, and 0.23% caffeine (measured at pH 7.2) was incubated (in solution) at 2 °C (on wet ice) or 50 °C (in a water bath) for 18 hours. Crystals were observed under a microscope in samples at 2 °C. Samples at 50 °C were clarified for 18 hours and then cooled to room temperature for 1 hour before crystallization.

[0324] In the attached SONICC TM The analysis showed that SONICC was performed on two samples diluted 1 / 16 in 10.18% PEG 3350, 50mM HEPES, pH 7.4 solution. TM Analysis. Both experiments showed positive UV and SHG imaging, consistent with chiral protein crystals. See Figure 4.

[0325] Example 7

[0326] pH range crystallinity study

[0327] This study was designed to investigate the pH of the solution to determine which pH range is effective for crystal formation.

[0328] Using Formulatrix Formulator TM Using a liquid handling system, the pH 6.0 to 8.8 grid was dispensed into 96-well microbatch plates (Hampton HR267) with 50 mM HEPES buffer per row and 1–12% PEG 3350 per column, resulting in a final volume of 66 μl per well. At room temperature, 33 μl of a solution containing pembrolizumab (44 mg / mL) in 20 M histidine buffer (pH 5.4) was added to each well, followed by 10 μl of 2.5% caffeine in 20 mM histidine buffer (pH 5.4). The 1.45 mg pembrolizumab, 6 mM histidine, pH 5.4, and 0.23% caffeine plate components were mixed using a 7X aspiration and dispensing step. The mixture (in solution) was incubated at 22 °C for 18 hours using SONICC. TM Analysis confirmed crystal formation.

[0329] Crystals were observed across the entire pH range from 6.0 to 8.8. In the lower pH range of 6.0 to 6.4, fewer crystals were observed than at higher pH levels, and a mixture of crystals and precipitate was observed. Optimal crystals based on size and mass were observed at pH 6.7–8.0. Crystals were observed above pH 8.0, but only when using a higher percentage of PEG. Crystallinity was confirmed using SONICC imaging. See Table 4.

[0330] Table 4. Results of the pH range study

[0331]

[0332] Example 8

[0333] Crystallization screening using PEGs of various molecular weights

[0334] A 44 mg / mL pembrolizumab solution was prepared in 20 mM histidine buffer, pH 5.4 using sterile, pyrogen-free aqueous solution (0.2 μm filter).

[0335] A 2.5% caffeine, 20mM histidine, pH 5.4 solution was prepared by adding 1.25g caffeine (Sigma; lot number SLBK4804V) to 50mL of 20mM histidine (Sigma; H-8000), pH 5.4, and heating to 60°C until dissolved. The solution was cooled to room temperature before use.

[0336] A solution of 10.18% PEG 3350, 50mM HEPES, pH 7.4 was prepared by adding 2.5 mL of 1M HEPES (1M solution, pH 7.4; Hampton Research HR2-941-27), pH 7.4, and 10.2 mL of 50% PEG 3350 to 37.3 mL of sterile water for injection. The resulting solution was filtered through a 0.2-micron filter.

[0337] Using Formulatrix Formulator TM Liquids were dispensed into 96-well microbatch plates (Hampton HR267) using a 1-12% linear gradient of PEG 200, 400, 3000, 3350, 8000, 10,000, and 20,000, along with 50 mM HEPES, pH 7.2, varying in each column. 33 μl of pembrolizumab (44 mg / mL) in 20 mM histidine buffer, pH 5.4 was added at room temperature, followed by 10 μl of 2.5% caffeine in 20 mM histidine buffer, pH 5.4. The plate components were mixed using a 7X aspiration and dispensing procedure. The mixture (in solution) was incubated at 22°C for 18 hours.

[0338] Except for the PEG 200 and PEG 400 rows, crystals were observed under a microscope in all rows. SONICC TM Analysis was performed using aliquots in a Whatman Fast Frame 4 slide wellplate. All wells containing PEG molecules with molecular weights ranging from 3,000 to 20,000 showed positive UV and SHG imaging consistent with chiral protein crystals. See Table 5.

[0339] Table 5. Crystallization screening of PEG molecules of various molecular weights

[0340]

[0341] Example 9

[0342] Monoclonal antibody crystallization screening

[0343] This study was conducted to determine whether the aforementioned PEG / caffeine conditions (which can be used to crystallize pembrolizumab) also effectively crystallize other monoclonal antibodies.

[0344] Several human recombinant monoclonal antibodies (10–40 mg / mL) were screened in 1536 unique crystallization plates using the suboil microbatch method described in Luft et al. (Journal of Structural Biology 142 (2003) 170–179) with 0.2 μl of monoclonal antibody (10–40 mg / mL) and 0.2 μl of precipitation solution (commercially available screening, including Silver Bullets Bio screening). Crystallization screening was performed at 4 °C, room temperature, and 30 °C. After 1 month, none of the screened monoclonal antibodies, except pembrolizumab, crystallized at any test temperature under the conditions of 0.16–0.2% caffeine, 12–15% PEG 3350, 0.05 M HEPES, pH 6.8, including the anti-PD-1 antibody nivolumab. A list of mAb targets and IgG types is provided in Table 6.

[0345] Table 6. Crystallization screening using different antibodies

[0346] mAb target IgG type Crystallization with PEG / caffeine IL23 1 - PD-1 (pembrolizumab) 4 + PD-1 (nivolumab) 4 - GITR 4 - GITR 1 - LAG3 4 - IGF-1R 4 - cCAM 4 - RSV 1 - FXIa 4 - CTLA4 1 - FXIa 4 -

[0347] Example 10

[0348] Preparation of pembrolizumab crystals suitable for X-ray diffraction analysis

[0349] Hampton Research Additive Screening (HR2-138), consisting of 96 unique additives, was used in a sitz-drop vapor diffusion plate with a baseline setting of 12% PEG 3350, 0.1M HEPES, pH 6.8, and 0.2% caffeine (72 μL). 10% of the additive screening (8 μL) was added to the reservoir. The reservoir was mixed using a Crystal Gryphon (Art Robbins Instruments, LLC, Sunnyvale, CA) and dispensed into 1-3 to 3-well Intelli-96 plates with single droplets of 0.4, 0.3, and 0.2 μL. Pembrolizumab (20 mg / mL) was added to 3-droplet single droplets in 3-well single droplets, respectively, to produce reservoir-to-pembrolizumab droplet ratios of 2:1, 1:1, and 1:2 for each replenishment reservoir. The plates were incubated at 14°C. One day later, crystals appeared in many pores, and the pores containing sodium dextran sulfate as an additive (Hampton additive screening condition E3) produced thicker needle-like crystals than those with other additives.

[0350] Prior to data collection, the crystals were harvested at room temperature and transferred to a cryoprotectant solution made from a precipitant mixture enhanced with 20% ethylene glycol. After immersion in this cryoprotectant solution for approximately 20 seconds, the crystals were fished out using a cryoloop and frozen in liquid nitrogen. The frozen crystals were then loaded onto a goniometer equipped with a nitrogen-cooled flow SER-CAT beamline at the Advanced Photon Source (APS) of Argonne National Laboratory (Argonne, IL, USA). X-ray diffraction was collected using a Rayonix MX300 HS detector. Pembrolizumab crystals prepared under the following conditions were fully characterized: 12% PEG 3350, 0.1M HEPES pH 6.8, 0.2% caffeine, 3% sodium dextran sulfate, 20 mg / mL pembrolizumab, 1:1 ratio (0.3 μl pembrolizumab / 0.3 μl 12% PEG 3350, 0.1M HEPES pH 6.8, 0.2% caffeine, 3% sodium dextran sulfate). Data were integrated and calibrated using the autoPROC program (Global Phasing), configured to use XDS for integration, POINTLESS for space group confirmation, AIMLESS for calibration, and STARANISO for anisotropy analysis and amplitude conversion. Micrographs of the crystals are shown below. Figure 5 .

[0351] The characteristics and data collection statistics of PEG / caffeine crystals are provided below:

[0352]

[0353]

[0354] Packaging analysis using the MATTHEWS program revealed that the asymmetric unit contained half of the antibody, with the other half generated by applying second-order axisymmetry to the crystal. The crystal structure was resolved using the molecular substitution package MOLREP with PDB entry 5DK3 as the search model. The search was conducted by sequentially searching for each rigid portion, maintaining the antibody portion already positioned at fixed coordinates. This portion was located in the following order: VL and VH, CL and CH1, CH2, CH3. Optimization was performed using the autoBUSTER program, which is part of the Global Phasing package. An image of the antibody is shown below. Figure 6A . Figure 6B A magnified view showing the interaction between caffeine and its environment within a crystal is provided.

[0355] Complete structural information and characterization of pembrolizumab crystals are provided in Table 7.

[0356]

[0357] Table 7. Three-dimensional crystal coordinates of the caffeine-pembrolizumab complex.

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508]

[0509]

[0510]

[0511]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524] Compared to pembrolizumab crystals prepared using the aforementioned high-salt process, this crystal exhibits a distinct quaternary structure. See WO 2016 / 137850. The caffeine binding site discovered in this pembrolizumab crystal is novel compared to the pembrolizumab antibody structure determined using crystals grown using the previous high-salt process.

[0525] Example 11

[0526] Batch crystallization method (175 mL scale)

[0527] A 42.7 mg / mL pembrolizumab solution in 20 mM histidine buffer, pH 5.4 was prepared using sterile, pyrogen-free aqueous solution (0.2 μm filtration). A solution of 10.18% PEG 3350 (50% solution RigakuItem#108058), 50 mM HEPES (1 M solution, pH 7.4, Hampton Research HR2-941-27), pH 7.0 was prepared by adding 20 mL of 1 M HEPES (pH 7.4) and 81.6 mL of 50% PEG 3350 to 298.4 mL of sterile water for injection (Hospira RI-4469) to 400 mL of PEG 3350. The resulting solutions were filtered through a 0.2 μm filter and stored at room temperature.

[0528] A solution of 2.5% caffeine (Sigma batch number SLBK4804V), 20mM histidine (Sigma H-8000), and pH 5.4 was prepared by adding 1.25g of caffeine to 50mL of 20mM histidine. The solution was heated to 60°C until the caffeine was infused into the solution. The solution was cooled to room temperature before use.

[0529] In 50 mL polypropylene centrifuge tubes (Fisherbrand) TM In the Sterile catalog #05-539-8, add 13.32 mL of pembrolizumab (42.7 mg / mL) in 20 mM histidine buffer (pH 5.4) at room temperature, and 26.4 mL of a solution containing 10.18% PEG 3350, 50 mM HEPES, pH 7.0. Then add 4 mL of a solution containing 2.5% caffeine, 20 mM histidine buffer, pH 5.4. Repeat this process for a total of four 50 mL centrifuge tubes.

[0530] The tubes (in solution) were placed on a Labnet rotisserie (catalog #H5600) and rotated at room temperature. Visible turbidity was observed after 15 minutes. Rotation of the batch continued for 2 hours at room temperature. Formulatrix SONIC was performed on each tube analyzed at a 1:10 dilution in 10% PEG 3350, 50 mM HEPES, pH 7.0 solution in Whatman Fast Frame 4 slide plates. TM Crystallinity was analyzed and verified. Figure 7 shows the results using SONICC. TM Representative analysis of imaging systems.

[0531] Centrifuge 50 mL tubes at 2300 RPM for 10 minutes at room temperature using a Beckman Coulter Allegra X-15R centrifuge. Decant the resulting mother liquor. Resuspend the precipitate in each 50 mL conical tube with 40 mL of 10% PEG 3350, 50 mM HEPES, and pH 7.0 buffer. Repeat this process. (via SONICC) TM The crystallinity of the resulting precipitate was analyzed. The concentration of pembrolizumab was measured by dissolving it in cold phosphate-buffered saline (PBS); weight:volume, 100 mg suspension: 1 mL PBS. Using an ultramicro UV spectrophotometer, the measured A280 reading for 19.5 mg in 10 mL PBS solution was 195 mg / mL (9.4 mL), representing an overall yield of 84%. Further dilutions were performed to prepare suspensions of 175 and 150 mg / mL using 10% PEG3350, 50 mM HEPES, and pH 7.0 buffer.

[0532] The experiment demonstrated that the crystallization process is scalable and reproducible, yielding a crystal suspension in high yield within 2 hours at room temperature. The results also showed that the pembrolizumab crystal suspension could be concentrated to high concentrations.

[0533] Example 12

[0534] Characterization of pembrolizumab crystal suspension

[0535] The pembrolizumab crystal suspension prepared in Example 11 was characterized by measuring particle size, dynamic viscosity and injectability, as described below.

[0536] Particle size analysis

[0537] The Horiba LA-960 was used to measure average particle size. The Horiba LA-960 combines modern sizing techniques with optimizations that allow measurement of suspension samples from 10 nm to 5 mm. The core theory of laser diffraction is that particles scatter light at angles determined by their size. Larger particles scatter at smaller angles, while smaller particles scatter at wider angles. The aggregate of particles produces a scattered light pattern defined by intensity and angle, which can be translated into particle size distribution results. Samples were diluted 1:10 with 10% PEG 3350, 50 mM HEPES, pH 7.0 buffer. The average particle size was 4.4 μm.

[0538] Dynamic viscosity measurement

[0539] The Rheosense m-VROC instrument uses the Hagen-Poiseuille formula to derive viscosity from pressure drop. Shear scans are performed in the range of 1,500–95,000 (1 / s) to measure dynamic viscosity. Viscosities of 200 mg / mL formulations are measured using a BD Hypak 1 ml pre-filled syringe with a 27-gauge standard wall (RW) or 29-gauge thin wall (TW) needle and plotted against different shear rates. Figure 8A Viscosity and shear rate data are provided. For the 200 mg / mL crystal suspension sample, the viscosity at room temperature was 26 cP at a shear rate of approximately 2000 sec⁻¹. As the shear rate was further increased to 80000 sec⁻¹ and 180000 sec⁻¹, an accompanying decrease in viscosity was observed. The viscosity decreased from 18 cP to 12 cP, which is within acceptable limits for high-concentration injectable products such as monoclonal antibodies. This unexpected shear-thinning behavior in the crystal suspension formulation could be used to facilitate the injection of pharmaceutical products from devices such as syringes or autoinjectors.

[0540] Injectability measurement

[0541] Preliminary injection force feasibility tests were conducted on a 200 mg / mL pembrolizumab suspension using various 1 mL plastic and glass syringes and needles. See Table 8.

[0542] Table 8. Injection force of 200 mg / mL pembrolizumab crystal suspension

[0543]

[0544]

[0545] Syringe injection force is the force required to dispense the contents of a syringe at a fixed rate. This force is typically measured using a tensile / compression tester (i.e., INSTRON). The tensile / compression tester (INSTRON, Norwood, MA) is used to measure pembrolizumab crystal suspensions filled with 200, 175, and 150 mg / mL in 1-mL polycarbonate syringes at injection rates of 120 mm / min (175 mg / mL and 200 mg / mL) or 225 mm / min (150 mg / mL). The injection force of the three different concentrations of pembrolizumab suspensions is provided by... Figure 8B In the middle, and the injection force of the sample filled in the glass syringe is shown in Figure 9 middle.

[0546] The results showed that for the 200 mg / mL suspension, the break-loose force and sliding force were relatively high (7.08 N and 4.52–5.12 N, respectively), while for lower concentrations (175 and 150 mg / mL), the break-loose force and sliding force were relatively low (3.96 N and 3.57–3.97 N, respectively). See also Figure 8B The injection force varies depending on the syringe barrel material (plastic or glass), injection rate, needle size (27 or 29G), and needle thickness (thin-walled or standard-walled). Figure 9 The injection force of plastic syringes (<8.5N) is lower compared to glass syringes (>12N). See also Figure 8B .

[0547] For a 200 mg / mL suspension, a higher injection rate requires a higher injection force. See Table 8 and... Figure 9 For example, in a BD Hypak 1mL glass syringe, the injection force was 12.1N at an injection rate of 133.86 mm / min, compared to 16.2N at an injection rate of 300 mm / min. The observed injection forces (6.36–18.41N) are within acceptable limits for subcutaneous injection of 200 mg / mL pembrolizumab crystalloid suspension. Overall, these data suggest that for injection of 200 mg / mL pembrolizumab crystalloid suspension, thin or conventional-walled 27 or 29G stainless steel needles made of polycarbonate plastic or glass can be used at injection rates from 133.86 to 300 mm / min, employing injection forces acceptable for subcutaneous injection.

[0548] Example 13

[0549] High-performance ion exchange chromatography analysis

[0550] Material

[0551] A 44 mg / mL pembrolizumab solution was prepared in 20 mM histidine buffer, pH 5.4 using sterile, pyrogen-free aqueous solution (0.2 μm filter).

[0552] A solution of 10.18% PEG 3350, 50mM HEPES, pH 7.4 was prepared by adding 2.5 ml of 1M HEPES (Hampton Research HR2-941-27), pH 7.4, and 10.2 ml of 50% PEG 3350 (50% solution; Rigaku Item#108058) to 37.3 mL of sterile water for injection (Hospira RI-4469). The resulting solution was filtered through a 0.2-micron filter.

[0553] A 2.5% caffeine, 20mM histidine, pH 5.4 solution was prepared by adding 1.25g of caffeine (Sigma; batch number SLBK4804V) to 50mL of 20mM histidine (Sigma; H-8000), pH 5.4. The solution was heated to 60°C until the caffeine was infused into the solution. The solution was cooled to room temperature before use.

[0554] Batch crystallization method (1 mL)

[0555] At room temperature, 333 μl of pembrolizumab (44 mg / mL) was added to 666 μl of 10.18% PEG 3350, 50 mM HEPES, pH 7.2 in 20 M histidine buffer, pH 5.4. 100 μl of 2.5% caffeine, 20 mM histidine buffer, pH 5.4 was added to the resulting solution. The mixture (in solution) was placed on a Labnet rotisserie at room temperature. Visible turbidity was observed after 15 minutes. The batch was continued to rotate at room temperature for 2 hours. Crystals were observed based on microscopic examination.

[0556] At room temperature, the crystal suspension was centrifuged at 3000 RPM for 3 minutes in a microcentrifuge. The mother liquor was removed. The precipitate was resuspended in 1 mL of 10.18% PEG 3350, 50 mM HEPES, and pH 7.2, and centrifuged at 3000 RPM for 3 minutes in a microcentrifuge at room temperature. The washings were removed. The precipitate was redissolved in 1 mL of PBS at 4°C for 8 minutes, and then centrifuged at 3000 RPM for 3 minutes in a microcentrifuge at 4°C.

[0557] HP-IEX method

[0558] Charge distributions of crystalline pembrolizumab and amorphous materials were evaluated using high-performance ion-exchange chromatography (HP-IEX). Ion-exchange HPLC was performed using a Dionex ProPac WCX-10 column and a 280 nm UV detector. Samples were diluted in purified water, and 80 g was injected for analysis. Different charge variants were eluted using gradients of the following mobile phases (Mobile Phase A: 24 mM MES, pH 6, 4% acetonitrile (v / v); Mobile Phase B: 20 ​​mM phosphate, 95 mM NaCl, pH 8, 4% acetonitrile (v / v)). The major peaks (representing non-degraded pembrolizumab) and the percentage areas of different charge variants in the pembrolizumab starting material and dissolved pembrolizumab crystals are provided in Table 9. The results indicate that the percentage charge variants of pembrolizumab in the crystalline suspension were similar to those in the starting material in the aqueous solution.

[0559] Table 9. IEX analysis of pembrolizumab crystal suspension and amorphous starting material

[0560]

[0561] Example 14

[0562] Pembrolizumab competitively binds to ELISA

[0563] Preparation of crystal suspensions for bioassay analysis

[0564] At room temperature, 333 μl of pembrolizumab (44 mg / mL) was added to 20 M histidine buffer, pH 5.4, along with 666 μl of 10.18% PEG 3350, 50 mM HEPES, pH 7.2. Then, 100 μl of 2.5% caffeine, 20 mM histidine buffer, pH 5.4 was added to the resulting solution. The mixture (in solution) was incubated at 30 °C for 1 month. Crystals were observed under a microscope.

[0565] The crystal suspension was centrifuged at 3000 RPM for 3 minutes in a microcentrifuge at room temperature. The mother liquor was removed. The precipitate was resuspended in 1 mL of 10.18% PEG 3350, 50 mM HEPES, pH 7.2, and centrifuged at 3000 RPM for 3 minutes in a microcentrifuge at room temperature. The washings were removed. The precipitate was redissolved in 1 mL of PBS at 4°C for 8 minutes, and then centrifuged at 3000 RPM for 3 minutes in a microcentrifuge at 4°C. The protein concentration was determined to be 6.061 mg / mL based on the Nanodrop 280 nm reading; the total volume was 1 mL. The sample was used for bioassay analysis as described below.

[0566] Competition combined with ELISA

[0567] Pembrolizumab competitive binding ELISA was used to assess the ability of pembrolizumab to competitively bind to PD-1 / Fc immobilized on an ELISA plate against PD-L1 (PD-1 ligand). A sample of amorphous pembrolizumab (“Reference”) was used as a reference material to test the potency of a crystalline pembrolizumab suspension (“Test Sample”) prepared by the above method. 4.5 μg / mL of both the Reference and Test Samples were serially diluted 2-fold in PBS pH 6.5, 1% BSA, and mixed with an equal volume of 600 ng / mL rhPD-L1 / Fc chimera (“PD-L1”, Bio-techne, R&D Systems (catalog number 156-B7), Minneapolis, MN) before being transferred to an ELISA plate. The final concentrations of the analytes were 2.25 μg / mL (Reference and Test Samples) and 300 ng / mL (PD-L1). The level of PD-L1 binding to PD-1 / Fc was detected by biotinylated anti-PD-L1 (Bio-techne, R&D Systems (catalog number BAF156)), followed by peroxidase-conjugated streptavidin and chemiluminescent substrate. Luminescence was measured using a microplate reader, and the resulting inhibition response curves were analyzed using 4-PL curve fitting software (SoftMax Pro).

[0568] The IC50 value derived from this study is a measure of the ability of pembrolizumab to inhibit PD-L1 binding to PD-1 / Fc. The biopotency of the crystalline samples is expressed as a percentage of relative potency of the pembrolizumab reference material. The geometric mean of the relative potency, along with the geometric standard deviation (%GSD) and 95% confidence interval, for multiple replicates (N=3) of the same samples are reported. The results indicate that the dissolved crystalline samples possess 95% of the relative potency compared to the reference (non-crystalline) pembrolizumab. See Table 10.

[0569] Table 10. Relative potency of pembrolizumab crystals

[0570]

[0571] Example 15

[0572] Laboratory batch crystallization method—non-cleanroom conditions

[0573] A 42.7 mg / mL solution of pembrolizumab (batch number W15-MK3475P-081) in 20 mM histidine buffer at pH 5.4 was prepared using sterile, pyrogen-free aqueous solution (0.2 μm filter).

[0574] A solution of 10.18% PEG 3350 (50% solution RigakuItem#10805850) mM HEPES, pH 7.0 (400 mL) was prepared by adding 20 mL of 1M HEPES (Hampton Research HR2-941-27), pH 7.4, and 81.6 mL of 50% PEG 3350 to 298.4 mL of sterile water. The resulting solution was filtered through a 0.2-micron filter and stored at room temperature.

[0575] A 2.5% caffeine, 20mM histidine, pH 5.4 solution was prepared by adding 1.25g of caffeine (Sigma batch number SLBK4804V) to 50mL of 20mM histidine (Sigma H-8000), pH 5.4. The resulting solution was heated to 60°C until the caffeine was incorporated. The solution was cooled to room temperature before use.

[0576] Prepare four 43.72 mL crystallization solutions. Add 26.4 mL of 10.18% PEG 3350, 50 mM HEPES, pH 7.0 to 13.32 mL of pembrolizumab (42.7 mg / mL) in 20 M histidine buffer, pH 5.4 at room temperature. Add 4 mL of 2.5% caffeine, 20 mM histidine buffer, pH 5.4 to the resulting solution in a 50 mL tube. Place the mixture (in solution) on a Labnet rotisserie at room temperature. Visible turbidity was observed after 15 minutes. Rotate the batch for another 2 hours at room temperature. SONICC analysis was performed to confirm crystallinity.

[0577] Centrifuge 50 mL conical tubes at 2600 RPM for 10 minutes at room temperature in a Beckman Coulter Allegra X-15R centrifuge. Decant the supernatant. Resuspend the precipitate in each 50 mL conical tube in 40 mL of 10% PEG 3350, 50 mM HEPES, pH 7.0. Repeat the centrifugation, decanting, and resuspension process. For the final centrifugation step, centrifuge the flasks at 3500 RPM for 20 minutes at room temperature in a Beckman Coulter Allegra X-15R centrifuge. Discard the supernatant. The protein concentration, measured by weight:volume, 1:10, A280 reading, was 216 mg / mL. Final volume: 9.7 mL (92% yield). Particle size analysis using a Horiba particle size analyzer showed an average particle size of 1.3 μm.

[0578] Example 16

[0579] Batch crystallization (43.72 mL scale) — Cleanroom conditions

[0580] A 2.5% caffeine, 20mM histidine, pH 5.4 solution was prepared by adding 1.25g of caffeine (Sigma batch number SLBK4804V) to 50mL of 20mM histidine (Sigma H-8000), pH 5.4. The mixture was heated to 60°C until the caffeine was infused into the solution. The solution was then cooled to room temperature and aseptically filtered before use.

[0581] A solution of 10.18% PEG 3350, 50mM HEPES, pH 7.0 was prepared by adding 20mL of 1M HEPES (Hampton Research HR2-941-27), pH 7.4, and 81.6mL of 50% PEG 3350 (RigakuItem#108058) to 298.4mL of sterile water. The resulting solution was filtered through a 0.2-micron filter and stored at room temperature.

[0582] A 42.7 mg / mL pembrolizumab solution was prepared using sterile, pyrogen-free water in 20 mM histidine buffer, pH 5.4 (filtered through a 0.2 μm filter).

[0583] To a sterile, filtered 13.32 mL solution of pembrolizumab (42.7 mg / mL) in 20 M histidine buffer (4 × 50 mL tubes), pH 5.4, add 26.4 mL of 10.18% PEG 3350, 50 mM HEPES, pH 7.0. In a sterile 50 mL conical tube in a cleanroom, add 4 mL of 2.5% caffeine, 20 mM histidine buffer, pH 5.4. After mixing, the crystallization solution in the 4 × 50 mL conical tubes has the following concentrations: 6 mM histidine, 6% PEG 3350, 30 mM HEPES, 0.23% caffeine, pH 6.8 containing 2.28 g of pembrolizumab.

[0584] The mixture (in solution) was placed on a Labnet rotisserie at room temperature. Visible turbidity was observed after 15 minutes. Four 50 mL sterile conical tubes were then rotated at room temperature for 2 hours. SONICC was performed after 2 hours. TM Analyze and confirm the degree of crystallinity.

[0585] Four 50 mL sterile conical tubes were centrifuged at 2600 RPM for 10 minutes each in a Beckman Coulter Allegra X-15R centrifuge at room temperature. The supernatant was decanted. The precipitate was resuspended; each 50 mL conical tube was centrifuged in 40 mL of 10% PEG 3350, 50 mM HEPES, pH 7.0. This process was repeated. For the final centrifugation step, the conical tubes were centrifuged at 3500 RPM for 20 minutes in a Beckman Coulter Allegra X-15R centrifuge at room temperature. The supernatant was decanted. The protein concentration, measured by a weight:volume ratio of 1:10 A280, was 231 mg / mL. The sample was diluted to 0.8 mL of 10% PEG 3350, 50 mM HEPES, pH 7.0. The final protein concentration, measured by a weight:volume ratio of 1:10 A280, was 200.3 mg / mL. The final volume was 8.4 mL (74% yield). The protein concentration, measured by RPLC, was 192.5 mg / mL (1.2 mM), and the caffeine concentration was 0.5 mg / mL (2.5 mM). The average particle size, measured using a Horiba particle size analyzer, was 1.3 μm.

[0586] Example 17

[0587] Batch dialysis crystallization method

[0588] 400 μl of 44 mg / mL pembrolizuab (batch number: W12123475P-17C) stock solution was placed in a Spectra / Por™ CE (cellulose ester) irradiated DispoDialyzer, MW cutoff: 10000, diameter 5 mm, sample volume 500 μl. The bag was placed in a 15 mL flat-bottomed tube containing 10 mL of 50 mM HEPES, pH 6.8, 10% PEG 3350, and 100 mM caffeine, and stirred at room temperature with a magnetic stir bar.

[0589] Slight turbidity was observed after 3 hours, and the turbidity increased significantly after 18 hours. Aliquots were characterized and crystal formation was confirmed by SONICC analysis. The resulting suspension was centrifuged at 3000 RPM for 3 minutes in a microcentrifuge. The precipitate was washed with 1 mL of 50 mM HEPES, pH 6.8, and 10% PEG 3350, and then centrifuged again at 3000 RPM for 3 minutes in a microcentrifuge. The precipitate was dissolved in 10 mL of normal PBS (at room temperature for 5 minutes). The final A280 reading was 1.6 mg / mL (16 mg total protein). The overall yield was 91% (17.6 mg of initial total mAb content). This experiment demonstrates that a dialysis-based crystallization method can produce crystalline pembrolizumab suspensions in high yields within 18 hours at room temperature.

[0590] Example 18

[0591] Pharmacokinetic studies of pembrolizumab crystal formulation

[0592] A pharmacokinetic comparability study was conducted in male Wistar Hen rats using pembrolizumab crystal formulations. All groups received pembrolizumab at a dose of 50 mg / kg. Pembrolizumab at a dose of 20 mg / mL in both the liquid IV formulation (7% sucrose, 0.02% polysorbate 80, 10 mM histidine, pH 5.5 (Group 1)) and the liquid SC formulation (7% sucrose, 0.02% polysorbate 80, 10 mM histidine, pH 5.5, 10 mM methionine (Group 2)) served as the baseline for bioavailability controls (N=3 for both Groups 1 and 2).

[0593] Pembrolizumab crystal suspensions were prepared as described in Example 16 and used for subcutaneous administration of pembrolizumab crystal formulations containing 20 mg / mL (Group 3, N=4), 40 mg / mL (Group 4, N=4), and 100 mg / mL (Group 5, N=4) pembrolizumab at concentrations listed in Table 11, along with 50 mM HEPES buffer, pH 7.0, and 10.18% PEG 3350. To ensure accurate dosing for each group, BD Hypak 2.25 mL pre-filled syringes for each group listed in Table 11 were precisely filled using a weight / density measurement (1 g / mL).

[0594] Table 11. Crystallization groups tested in rat studies

[0595] Group dose Concentration (mg / mL) Syringe filling*(g) 3 50mpk 20 1.1 4 50mpk 40 0.7 5 50mpk 100 0.5

[0596] *Due to reverse filling, there is a dead space of 0.1g in each syringe.

[0597] For each group, add the prescribed weight of crystalloid suspension to a tattered BDHypak™ 2.25 mL pre-filled glass syringe using a sterile 10 mL volumetric pipette. Use an evacuation tool to bring the plunger cap to the surface of the suspension in each filled syringe. Prepare a total of six syringes per group.

[0598] To establish a control against potential caffeine effects, the study included a caffeine-free pembrolizumab formulation (50 mM HEPES, pH 6.8, 10% PEG 3350, Group 6) and a PEG-free pembrolizumab formulation (50 mM HEPES, pH 6.8, caffeine, Group 7).

[0599] Blood was collected at 0.5, 3, 6, 24, 48, 72, 96, 168, 216, 336, 408, and 504 hours post-administration, and serum was prepared from 0.3 mL of post-administration whole blood. Pembrolizumab levels in serum were measured using an MSD (Meso Scale Discovery) immunoassay. PK parameters were calculated using Phoenix PK software 64.6.3. Bioavailability (F) was calculated based on the AUC of the liquid formulation IV group (F = AUC of SC / AUC of IV * 100%). Injection site monitoring was conducted throughout the study.

[0600] The results showed that the tested pembrolizumab SC liquid formulation at 20 mg / mL had similar bioavailability to the 20 mg / mL crystal formulation. See Table 12. The results also showed that the highest concentration (C... max Exponent exposure (area under the curve, AUC) and bioavailability (F) increased in a concentration-dependent manner: the higher the concentration, the higher the C. max The higher the concentrations, the higher the AUC and F. The shorter the time (T0) for the highest concentration of the crystal formulation to reach peak serum concentration compared to lower concentrations. max A shorter Ka indicates a faster absorption rate (Ka) for the highest concentration of the crystal formulation.

[0601] Table 12. Bioavailability of liquid and crystal formulations

[0602]

[0603] Example 19

[0604] Solid-state NMR characterization of pembrolizumab crystal suspension

[0605] Solid-state NMR spectra were obtained on a Bruker Avance III HD 400MHz spectrometer equipped with a 4.0 mm H / F / X magic angle spin (MAS) probe and a Bruker Avance III 500MHz spectrometer equipped with a 4.0 mm H / C / N MAS probe. For the 400MHz spectrometer... 13 For the C (carbon-13) experiment, the probe was tuned to double resonance C / H; for the 500MHz spectrometer 13 C (carbon-13) and 15 For the N(Nitrogen-15) experiment, the probe was tuned to triple resonance C / N / H. The MAS frequency was 12 kHz for all experiments. Sample temperature was controlled at 10 °C on the 400 MHz spectrometer and at 21 °C on the 500 MHz spectrometer. On the 400 MHz spectrometer, data was collected at 90.9 kHz with 1H dipole decoupling during acquisition. 13 C-cross polarization (CP) MAS spectra were acquired with a CP contact time of 1 ms and a recycle delay of 2 s. The data was collected at 71.4 kHz with 1H dipole decoupling on a 500 MHz spectrometer. 13 C CPMAS spectra were obtained with a CP contact time of 1 ms and a recovery delay of 2 seconds. Data was collected at 71.4 kHz with 1H dipole decoupling on a 500 MHz spectrometer. 15 The N CP MAS spectrum shows a CP contact time of 2.5 milliseconds and a recovery delay of 2 seconds. 13 C chemical shift reference: carbonyl carbon of glycine (α-form) at 176.45 ppm 13 C signal. For solid-state NMR purposes, the term "about" refers to ±0.1 ppm.

[0606] Using the above solid 13 Pembrolizumab crystals were measured using a 400MHz NMR instrument and procedure. Specifically, pembrolizumab crystals were prepared using the method described in Example 11. Pembrolizumab-caffeine crystals were obtained. 13 C(carbon-13)CPMAS NMR spectrum. Figure 10A and Figure 10B The complete spectrum and some magnified regions are shown. Characteristic peaks of pembrolizumab-caffeine crystals were observed at approximately 183.07, 182.16, 181.54, 180.55, 179.99, 110.70, 110.15, 109.36, 108.23, 103.58, 101.49, 99.75, 98.56, 76.88, 76.04, 74.97, 74.41, 73.52, 72.69, 13.85, 13.27, 12.26, and 11.13 ppm.

[0607] Using the above solid 13 C and 15 N 500MHz NMR equipment and procedures, measured using 2- 13 C and 1,3- 15 Pembrolizumab-caffeine crystals were prepared using N isotope-labeled caffeine. Pembrolizumab-caffeine crystals were obtained. 13 C (carbon-13) and 15 N(Nitrogen-15)CP MAS NMR spectrum. Figure 11A and Figure 11B The following are examples of the displayed parsing: 13 C and 15 A magnified spectral region of the N-caffeine peak. Peaks of approximately 1.69 ppm and 0.92 ppm were observed between pembro-caffeine crystals and caffeine-only crystals, respectively. 13 C and 15 Characteristic chemical shift differences in the N-caffeine peak.

Claims

1. A method for producing crystallized anti-PD-1 monoclonal antibody (mAb), comprising: a) Mixing: i. An aqueous buffer solution containing approximately 5 mg / mL to approximately 80 mg / mL of the mAb, wherein the anti-PD-1 mAb is pembrolizumab or a pembrolizumab variant. ii. Polyethylene glycol (PEG), and iii. Additives selected from the following: caffeine, theophylline, 2'-deoxyguanosine-5'-monophosphate, bioactive gibberellin and pharmaceutically acceptable salts of said bioactive gibberellin; To form a crystallization solution having a pH of about 6.0 to about 8.8 and containing about 2% to about 40% by weight / volume (w / v) of PEG and about 0.1% to about 0.30% by weight (w / v) of additives; b) Incubating the crystallization solution for a period of time sufficient for crystal formation; and c) Optionally, the crystal anti-PD-1 mAb is harvested from the solution.

2. The method of claim 1, wherein the aqueous buffer solution comprising the mAb further comprises a histidine buffer with a pH of about 5.0 to about 6.

0.

3. The method according to claim 1 or 2, wherein the PEG and the additive are mixed together to form a precipitant solution before being mixed with the aqueous buffer solution containing the mAb.

4. The method according to claim 1 or 2, wherein the aqueous buffer solution comprising the mAb is mixed with the PEG prior to mixing with the additive.

5. The method according to claim 1 or 2, wherein the aqueous buffer solution comprising the mAb is mixed with the additive prior to mixing with the PEG.

6. The method according to claim 1, wherein the additive is gibberellin A3 or a pharmaceutically acceptable salt thereof.

7. The method of claim 1, wherein the additive is about 0.15% to about 0.30% w / v of caffeine or about 0.25% to about 0.30% w / v of theophylline.

8. The method of claim 1, wherein the additive is caffeine, and the crystallizing solution further comprises about 1% to about 10% w / v sodium dextran sulfate.

9. The method according to claim 8, wherein the amount of sodium dextran sulfate is about 5% w / v.

10. The method according to any one of claims 1-9, wherein the PEG is present in the crystallization solution in an amount of about 5% to about 15% w / v.

11. The method according to any one of claims 1-9, wherein the PEG is present in the crystallization solution in an amount of about 10% to about 30% w / v.

12. The method according to any of the preceding claims, wherein the molecular weight of the PEG is from about 2,500 to about 35,000.

13. The method of claim 12, wherein the PEG is PEG 3350.

14. The method of claim 13, wherein the pH of the crystallization solution and the amount of PEG present in the crystallization solution are selected from the group consisting of: a) The pH of the crystallization solution is approximately 6.0, and the amount of PEG is 2-4% w / v. b) The pH of the crystallization solution is approximately 6.4, and the amount of PEG is 2-6% w / v. c) The pH of the crystallization solution is approximately 6.8-8.4, and the amount of PEG is 6-12% w / v. d) The pH of the crystallization solution is about 8.8, and the amount of PEG is 10-12% w / v.

15. The method according to any of the preceding claims, wherein the crystallization solution is incubated at an incubation temperature of about 2°C to about 37°C.

16. The method according to any of the preceding claims, wherein the crystallization solution is incubated at an incubation temperature of about 18°C ​​to about 25°C.

17. The method according to any one of claims 1-14, wherein the crystallization solution is heated to about 50°C and then cooled to about 37°C or lower.

18. The method of claim 17, wherein the crystallization solution is cooled to a temperature of about 18°C ​​to about 25°C.

19. The method of claim 17, wherein the crystallization solution is cooled to a temperature of about 4°C.

20. The method of claim 17, wherein the incubation temperature is increased from about 4°C to about 10-40°C.

21. The method according to any of the preceding claims, wherein the crystallization solution is incubated for about 15 minutes or longer.

22. The method of claim 21, wherein the crystallization solution is incubated for about 2 hours or longer.

23. The method according to any of the preceding claims, wherein the crystallization solution is rotated or stirred during incubation.

24. The method according to any of the preceding claims, wherein the concentration of the anti-PD-1 mAb in the crystallization solution is from about 5 mg / mL to about 50 mg / mL.

25. The method according to any of the preceding claims, wherein the crystallization solution is produced by vapor diffusion, batch crystallization, or dialysis.

26. The method according to any of the preceding claims, wherein the crystallization solution further comprises about 25 mM to about 250 mM HEPES buffer.

27. The method of claim 26, wherein the crystallization solution comprises about 50 mM HEPES buffer.

28. The method according to any of the preceding claims, wherein the mAb is pembrolizumab.

29. The method according to any of the preceding claims further comprises the step of seeding the crystallization solution with the crystals of the anti-PD-1mAb.

30. The method according to any of the preceding claims, further comprising the step of homogenizing the crystal anti-PD-1mAb.

31. A separated crystal formed by any of the preceding claims.

32. A crystal comprising a separated pembrolizumab complexed with caffeine, wherein the crystal is characterized by space group P2221. α = β = γ = 90°.

33. The crystal of claim 32, comprising a polypeptide, wherein the polypeptide is characterized in that, when superimposed on the backbone atoms described by the structural coordinates in Table 7, it comprises structural coordinates of conserved residue backbone atoms with a root mean square deviation (RMSD) of less than about 2.0 angstroms.

34. The crystal according to any one of claims 31-33, wherein the particle size of the crystal is about 0.5 to 50 micrometers.

35. A crystal pembrolizumab comprising pembrolizumab complexed with caffeine, characterized in that... Solid-state NMR showing peaks at approximately 182.16, 181.54, 179.99, 109.36, 108.23, 103.58, 76.88, and 76.04 ppm 13 C spectrum.

36. The crystalline pembrolizumab according to claim 35, further showing peaks at approximately 183.07, 180.55, 110.70, 110.15, 101.49, 99.75, 98.56, 74.97, 74.41, 73.52, 72.69, 13.85, 13.27, 12.26 and 11.13 ppm.

37. The crystalline pembrolizumab according to any one of claims 35-36, characterized in that... Solid-state NMR as shown in Figure 10A 13 C spectrum.

38. A composition comprising a crystal of any one of claims 31-34 or a crystal of any one of claims 35-37, and a pharmaceutically acceptable carrier.

39. The composition according to claim 38, wherein the composition is a crystal suspension and the concentration of the anti-PD-1 mAb is 5-400 mg / mL.

40. The composition of claim 38, wherein the concentration of the anti-PD-1 mAb is ≥75 mg / mL.

41. The composition according to any one of claims 35-40, further comprising about 5 mM to about 20 mM of a buffer.

42. The composition according to any one of claims 33-39, further comprising about 0.01% to about 0.10% w / v of a nonionic surfactant.

43. The composition according to any one of claims 33-40, further comprising a second active pharmaceutical ingredient (API).

44. The composition of claim 43, wherein the second API is a small molecule or a biological agent.

45. A method of treating cancer in a human patient, comprising administering a crystal of any one of claims 31-34, a crystal of pembrolizumab of any one of claims 35-37, or a composition of any one of claims 38-44 to a human patient in need.

46. ​​The method of claim 45, wherein the crystal or the composition is administered intravenously or subcutaneously to the patient.

47. The method according to claim 45 or 46, wherein the cancer is selected from: melanoma, non-small cell lung cancer, small cell lung cancer, Hodgkin's lymphoma, head and neck cancer, primary mediastinal large B-cell lymphoma, urothelial carcinoma, gastric cancer, esophageal cancer, kidney cancer, endometrial cancer, hepatocellular carcinoma, Merkel cell carcinoma, and cervical cancer.

48. The method according to claim 45 or 46, wherein the cancer is a solid tumor or colorectal cancer with high microsatellite instability or mismatch repair deficiency.

49. The method of claim 45 or 46, wherein the patient’s tumor has a high mutational burden.

50. The method according to any one of claims 45 to 48, wherein the dose of anti-PD-1 mAb is 200 mg, which is administered to the patient approximately every 3 weeks.

51. The method according to any one of claims 45 to 48, wherein the dose of the crystal mAb is 400 mg, which is administered to the patient approximately every 6 weeks.

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