EA046787B1 - AQUEOUS PHARMACEUTICAL COMPOSITION OF ANTI-PD-1 ANTIBODY PROLGOLIMAB AND ITS APPLICATION - Google Patents

Description

Field of technology

The invention relates to new aqueous compositions for anti-PD-1 antibodies, and, in particular, to new aqueous compositions for the anti-PD-1 antibody prolgolimab, which can be used as a drug for the treatment of malignant neoplasms.

State of the art

Programmed death protein 1 (PD-1) is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed by activated B cells, T cells and myeloid cells (Agata et al., supra; Okazaki et al. (2002) Curr. Opin. Immunol. 14: 391779-82; Bennet et al. (2003) J Immunol 170 :711-8). The original members of this family, CD28 and ICOS, were found to function in increasing T cell proliferation following the addition of monoclonal antibodies (Hutloff et al. (1999) Nature 397:263-266; Hansen et al. (1980) Immunogenics 10:247 -260). PD-1 was discovered by screening for differential expression in apoptotic cells (Ishida et al. (1992) EMBO J 11:3887-95). Other members of this family, CTLA-4 and BTLA, were detected by screening for differential expression in cytotoxic T lymphocytes and TH1 cells, respectively. CD28, ICOS and CTLA-4 all have an unpaired cysteine residue allowing homodimerization. In contrast, PD-1 is predicted to exist as a monomer, lacking the unpaired cysteine residue characteristic of other CD28 family members.

PD-1 is a 55 kDa type I transmembrane protein that is part of the Ig gene superfamily (Agata et al. (1996) Int Immunol 8:765-72). PD-1 contains a membrane-proximal immunoreceptor tyrosine-based inhibitory motif (ITIM) and a membrane-distal tyrosine-based switch motif (ITSM) (Thomas, M. L. (1995) J Exp Med 181:1953-6; Vivier, E. and Daeron, M. ( 1997) Immunol Today 18:286-91). PD-1, although structurally similar to CTLA-4, lacks the MYPPPY motif, which is critical for the binding of B7-1 and B7-2. Two ligands for PD-1, PD-L1 and PD-L2, have been identified and have been shown to negatively regulate T cell activation upon binding to PD-1 (Freeman et al. (2000) J Exp Med 192:1027-34; Latchman et al. (2001) Nat Immunol 2:261-8; Carter et al. (2002) Eur J Immunol 32:634-43). Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1 but not to other CD28 family members.

One ligand for PD-1, PD-L1, is abundant in various types of human cancer (Dong et al. (2002) Nat. Med. 8:787-9). The interaction between PD-1 and PD-L1 leads to a decrease in the number of tumor-infiltrating lymphocytes, a decrease in T-cell receptor-mediated proliferation, and immune evasion of cancer cells (Dong et al. (2003) J. Mol. Med. 81:281-7 ; Blank et al. (2005) Cancer Immunol. 54:307-314; Immunosuppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1, and this effect is additive when blocking the interaction of PD-1 with PD-L2 (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99 :12293-7; Brown et al. (2003) J Immunol 170:1257-66).

PD-1 is an inhibitory member of the CD28 family expressed on activated B cells, T cells and myeloid cells (Agata et al., supra; Okazaki et al. (2002) Curr Opin Immunol 14: 391779-82; Bennett et al. (2003) ) J Immunol 170:711-8). PD-1-deficient animals develop various autoimmune phenotypes, including autoimmune cardiopathy and a lupus-like syndrome with arthritis and nephritis (Nishimura et al. (1999) Immunity 11:141-51; Nishimura et al. (2001) Science 291:31922). Additionally, PD-1 has been found to play a role in autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease (GVHD), type I diabetes, and rheumatoid arthritis (Salama et al. (2003) J Exp Med 198:71-78 ; Prokunina and Alarcon-Riquelme (2004) Hum Mol Genet 13:R143; Nielsen et al. (2004) Lupus 13:510). In the murine tumor B cell line ITSM, PD-1 has been shown to be essential for blocking BCR-mediated Ca2+ influx and tyrosine phosphorylation of downstream effector molecules (Okazaki et al. (2001) PNAS 98:13866-71) .

Currently, a number of antibodies against PD-1 are known, for example, nivolumab (BMS), pembrolizumab (Merck), which are a human monoconal antibody of the IgG4 isotype.

Also known is a new anti-PD-1 antibody, prolgolimab (also known as BCD-100), which is a human monoclonal antibody of the IgG1 isotype with effectorless mutations L234A, L235A. Prolgolimab showed increased affinity for PD-1 and increased aggregation stability compared to antibodies of the IgG4 isotype. In addition, prolgolimab is currently in clinical trials for various types of malignancies, including melanoma, including unresectable or metastatic melanoma, early stage melanoma before and after curative treatment; lung cancer, non-small cell lung cancer (NSCLC), including unresectable or metastatic non-small cell lung cancer.

Thus, the development of new and improved stable aqueous pharmaceutical compositions for the anti-PD-1 antibody prolgolimab is currently relevant.

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Brief description of drawings

The invention will become better understood from the following detailed description of embodiments of the present invention with reference to the accompanying drawings, in which:

fig. 1, 2 are graphs illustrating the dynamics of BCD-100 concentrations in the blood serum of patients over 6 administrations of the drug (taking into account the coefficient) in μg/ml. (Study BCD-100-1);

fig. 3 is a graph illustrating the design of the BCD-1002/MIRACULUM study;

fig. 4 is a graph illustrating a flowchart of the study;

fig. 5 is a graph illustrating the overall survival of patients in group 1 (BCD-100, 1 mg/kg every 2 weeks) from the BCD-100-2/MIRACULUM study;

fig. 6 is a graph illustrating the overall survival of patients in group 2 (BCD-100, 3 mg/kg every 3 weeks) from the BCD-100-2/MIRACULUM study;

fig. 7 is a graph illustrating the progression-free survival of patients in the BCD-100, 1 mg/kg group (according to irRECIST criteria) from the BCD100-2/MIRACULUM study;

fig. 8 is a graph illustrating the progression-free survival of patients in the BCD-100, 3 mg/kg group (according to irRECIST criteria) from the BCD100-2/MIRACULUM study;

fig. 9 is a graph illustrating the concentration of BCD-100 in patients receiving 1 mg/kg every 2 weeks after a single dose of the drug (BCD100-2/MIRACULUM study results);

fig. 10 is a graph illustrating the concentration of BCD-100 in patients receiving 3 mg/kg every 3 weeks (results from the BCD-100-2/MIRACULUM study);

fig. 11 is a graph illustrating the proportion of Th9 in the total T helper cell population in subgroups of patients treated with BCD-100 at a dose of 1 mg/kg Q2W with different types of response to therapy (results from the BCD-100-2/MIRACULUM study);

fig. 12 is a graph illustrating the proportion of Th9 in the total T helper cell population in subgroups of patients treated with BCD-100 at a dose of 3 mg/kg Q3W with different types of response to therapy (results from the BCD-100-2/MIRACULUM study).

Description of the invention

Definitions

Terms used herein generally have their common art-specific meanings within the context of the invention and in the particular context in which each term is used. Below, or elsewhere in the description, certain terms that are used to describe the invention are defined to provide the practitioner with additional guidance regarding the description of the invention. Synonyms of some terms are given. The presentation of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere herein, including examples of any terms described herein, is for illustrative purposes only and does not in any way limit the scope or meaning of the invention or any subject matter illustrated. The invention is not limited to the various embodiments described in the present description.

Monoclonal antibody, as used herein, refers to a humanized antibody or a fully human antibody, unless otherwise specified in this application. Monoclonal antibodies of the invention can be produced using, for example, recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies or other technologies well known in the art.

A monoclonal antibody population, as used herein, refers to a homogeneous or substantially homogeneous population of antibodies (i.e., at least about 96%, but more preferably at least about 97 or 98%, or even more preferably at least 99 % of antibodies in the population will compete in an ELISA for the same antigen or epitope, or more preferably the antibodies are identical in amino acid sequence).

A full-length antibody found in nature is an immunoglobulin molecule that consists of four polypeptide chains (two heavy (H) chains (approximately 50-70 kDa full length) and two light (L) chains (approximately 25 kDa full length) ), linked by disulfide bridges. The amino-terminal portion of each chain includes a variable domain of approximately 100-110 or more amino acids that is responsible for antigen binding. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Light chains are classified as kappa or lambda and are characterized by a specific constant region. Each light chain consists of an N-terminal light chain variable region (VL or VK in this application) and a light chain constant region consisting of a single domain

- 2 046787 (CL or CK). Heavy chains are classified as γ (gamma), δ (delta), alpha (α), mu (μ) or epsilon (ε), and they define the isotype of the antibody, such as IgG, IgM, IgA, IgD and IgE, respectively, and several of them can be further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Each type of heavy chain is characterized by a specific Fc constant region. Each heavy chain consists of an N-terminal heavy chain variable region (VH in this application) and a CH constant region (heavy chain). The heavy chain constant region consists of three domains (CH1, CH2 and CH3) for IgG, IgD and IgA and 4 domains (CH1, CH2, CH3 and CH4) for IgM and IgE. The VH and VL variable domains can be further divided into hypervariable regions (hypervariable regions, CDRs) interspersed with more conserved framework regions (FRs). Each variable domain consists of three CDRs and four FRs, arranged in the following order from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

The variable regions of each light/heavy chain pair form the antigen-binding sites of the antibody. Thus, an intact IgG antibody has two binding sites. With the exception of bifunctional or bispecific antibodies, the two binding sites are the same. As used herein, an antigen-binding moiety, or antigen-binding region, or antigen-binding domain refers, interchangeably, to that part of an antibody molecule that contains amino acid residues that interact with the antigen and determine the specificity and affinity of the antibody for the antigen. This part of the antibody includes framework amino acid residues necessary to maintain the proper conformation of antigen-binding residues.

The antibody fragment may be an antibody fragment or an antibody fragment having the activity of a full-length antibody. Said antibody fragment may be F(ab') ₂ , F(ab) ₂ , Fab', Fab Fv and scFv.

The term inhibit or neutralize, as used herein, with respect to the functional activity of an antibody of the invention, means the ability to substantially inhibit, prevent, limit, slow, stop, reduce or reverse, for example, the development or severity of that which is inhibited, including , but not limited to the foregoing, a biological activity (eg, PD-1 activity) or property, disease or condition. The inhibition or neutralization of PD-1 activity resulting from binding of an antibody of the invention to PD-1 is preferably at least about 20, 30, 40, 50, 60, 70, 80, 90, 95% or greater.

The term isolated or isolated when used with respect to a nucleic acid or protein preparation (eg, an antibody) refers to a nucleic acid molecule or protein molecule that is identified and separated from at least one contaminant with which it is typically associated in a natural source. Preferably, the isolated antibody is an antibody that is substantially free of other antibodies having distinctive antigen specificity (e.g., the pharmaceutical compositions of the present invention contain an isolated antibody that specifically binds PD-1 and is substantially free of antibodies that specifically bind antigens other than PD-1).

A polynucleotide is operably linked if it has functional links to another polynucleotide. For example, a promoter or enhancer is operably linked to a coding sequence if it affects transcription of the sequence. A polypeptide is operably linked to another polypeptide if the polynucleotides encoding them are operably linked, preferably if they are in the same open reading frame.

The term Specific binding between an antibody and an antigen target (antigen) refers to immunological specificity. An antibody can specifically bind to a target antigen if it binds an epitope on the antigen to a greater extent than other epitopes on the antigen. Specific binding does not exclude cross-reactivity with other antigens bearing similar antigen epitopes.

The VL domains in antibodies according to the invention may be of the VL lambda type or the VL kappa type. The term VL domain refers to both VK lambda and VL kappa isotypes that contain one or more amino acid substitutions, insertions or deletions.

The term pharmaceutical composition refers to a composition and/or composition containing an antibody according to the invention in a therapeutically effective amount and excipients or excipients (carriers, diluents, fillers, solvents and other excipients).

As used herein, the term buffer or buffer solution refers to an aqueous solution containing a mixture of an acid (usually a weak acid such as, for example, acetic acid, citric acid) and its conjugate base (such as, for example, an acetate or citrate salt, e.g. sodium acetate, sodium citrate, as well as hydrates of these salts, for example sodium acetate trihydrate) or alternatively a mixture of a base (usually a weak base, for example histidine) and its conjugate acid (for example histidine hydrochloride). The pH value of a buffer solution changes little when a small amount of a strong base or strong acid is added to it, or when diluted or concentrated, due to the buffering effect provided by the buffering agent.

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In the present application, the buffer system contains one or more buffer agents and/or their conjugate(s) with an acid or base, and more suitably contains one or more buffer agents and their conjugate(s) with an acid or base, and most suitably contains only one buffering agent and its acid/base conjugate. Unless otherwise indicated, any concentrations specified in the present invention in relation to the buffer system (buffer concentration) may refer to the combined concentration of the buffer(s) agent(s) and/or its conjugate(s) with the acid or base. In other words, the concentrations reported herein with respect to a buffer system may refer to the combined concentration of the relevant buffer species (ie species in dynamic equilibrium with each other, eg citrate/citric acid). The total pH value of a composition containing a relevant buffer system is a reflection of the equilibrium concentration of each of the relevant buffer species (ie, the balance of the buffer(s) agent(s) with its acid or base conjugate(s).

As used herein, the term buffering agent refers to the acidic or alkaline component (usually a weak acid or weak base) of a buffer or buffer solution. A buffering agent helps maintain the pH of a given solution at or near a predetermined value, and buffering agents are typically selected to complement the predetermined value. The buffering agent may be a single compound that produces the desired buffering effect, particularly if said buffering agent is mixed with (and suitably proton exchangeable with) a suitable amount (depending on the predetermined desired value) of its corresponding acid/base conjugate .

The term solubilizer as used herein means a pharmaceutically acceptable non-ionic surfactant. A single solubilizer as well as combinations of solubilizers can be used. Examples of solubilizers include, but are not limited to, polysorbate 20 or polysorbate 80, poloxamer 184 or poloxamer 188, or PLURONIC®.

The terms osmotic agent or tonicity adjusting agent and osmolytic as used herein refer to an excipient that can provide the desired osmotic pressure to a liquid antibody solution. In some embodiments, the tonicity adjusting agent can adjust the osmotic pressure of the liquid antibody preparation to isotonicity such that the antibody preparation is physiologically compatible with the tissue cells of the subject. In yet another embodiment, the tonicity adjusting agent may help increase the stability of the antibodies. An isotonic drug is a drug that has an osmotic pressure equivalent to human blood. Isotonic drugs typically have an osmotic pressure of approximately 250 to 350 mOsm/kg. The term hypotonic describes a drug with an osmotic pressure less than that of human blood. Accordingly, the term hypertonic is used to describe a drug with an osmotic pressure greater than that of human blood. Isotonicity can be measured using, for example, a steam or cryoscopic osmometer. The tonicity adjusting agent may be in enantiomeric (eg, L- or D-enantiomer) or racemic form; in the form of isomers such as alpha or beta, including alpha, alpha; or beta, beta; or alpha, beta; or beta, alpha; in free acid or free base form; in the form of salt; in hydrated form (eg monohydrate) or anhydrous form. Examples of osmotic agents include, but are not limited to, sugars (trehalose dihydrate, sucrose, glucose), polyols (mannitol, sorbitol), amino acids (proline, arginine, glycine), or salts (sodium chloride, potassium chloride, magnesium chloride).

The term long-term storage or long-term stability should be understood to mean that the pharmaceutical composition can be stored for three months or more, for six months or more, and preferably for one year or more, most preferably with a minimum shelf life in a stable state at least two years. In general, the terms long-term storage and long-term stability further include steady-state storage durations that are at least comparable to or better than the steady-state shelf life typically required for currently available commercial formulations of the anti-PD-1 antibody prolgolimab without losses in stability that may render the formulation unsuitable for its intended pharmaceutical use.

The term parenteral administration refers to modes of administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intratracheal, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and epigastric injection or infusion.

The term use refers to the possibility of using an antibody according to the invention, or a pharmaceutical composition containing it, for the treatment, alleviation of diseases, to accelerate remission, reduce the frequency of relapses due to diseases or disorders mediated by receptors to which the antibody according to the invention can bind. Examples of diseases include, but are not limited to, malignant neoplasms, including melanoma, including

- 4 046787 inoperable or metastatic melanoma, early stages of melanoma before and after radical treatment; lung cancer, non-small cell lung cancer (NSCLC), including unresectable or metastatic non-small cell lung cancer; non-squamous non-small cell lung cancer, squamous cell lung cancer; small cell lung cancer, including unresectable or metastatic small cell lung cancer; early stage lung cancer before and after radical treatment; cervical cancer, including metastatic cervical cancer, early stages of cervical cancer before and after radical treatment; head and neck tumors, including squamous cell carcinoma of the head and neck; Hodgkin's lymphoma; tumors of the stomach and intestines, metastatic squamous cell carcinoma of the esophagus; bladder cancer, including metastatic urothelial carcinoma, kidney cancer; endometrial cancer, including metastatic endometrial cancer, early stages of endometrial cancer before and after radical treatment; breast cancer, including metastatic breast cancer, early stages of endometrial cancer before and after radical treatment; liver cancer, including metastatic or inoperable liver cancer, early stages of liver cancer before and after radical treatment; unresectable or metastatic solid tumor, including unresectable or metastatic solid tumor with signs of microsatellite instability.

The term treatment method refers to the possibility of using an antibody according to the invention or a pharmaceutical composition containing it, for the treatment, alleviation of diseases, to accelerate remission, reduce the frequency of relapses due to diseases or disorders associated with PD1 activity. Treating or treating a disease, disorder or condition may include preventing or delaying the onset of clinical symptoms of the disease, disorder or condition developing in a person, inhibiting the disease, disorder or condition, that is, stopping, reducing or slowing the progression of the disease or its relapse (in the case of maintenance therapy ) or at least one clinical or subclinical symptom thereof, or alleviation or attenuation of a disease, that is, causing regression of a disease, disorder or condition. Examples of diseases include, but are not limited to, malignancies, including melanoma, including unresectable or metastatic melanoma, early stages of melanoma before and after radical surgical treatment; lung cancer, non-small cell lung cancer (NSCLC), including unresectable or metastatic non-small cell lung cancer.

The term aqueous composition as used herein refers to a water-based composition; the water can be water, water for injection, saline solution (0.9-1.0% aqueous sodium chloride solution).

In one embodiment of the invention, the subject of treatment or patient is a mammal, preferably a human subject. The above subject may be male or female of any age.

In the present specification and in the following claims, unless the context otherwise requires, the words have, include and contain, or variations thereof such as has, having, includes, including, contains or containing, are to be understood to include the specified whole or group of wholes, but not to the exclusion of any other whole or group of wholes.

The essence of the invention

The present invention discloses aqueous pharmaceutical compositions of the anti-PD-1 antibody prolgolimab, which has improved aggregation stability and increased affinity compared to known anti-PD-1 antibodies that are based on the human IgG4 isotype antibody.

As described in international patent application publication WO/2018/013017, incorporated herein by reference, the anti-PD-1 antibody prolgolimab, which is a human monoclonal antibody of the IgG1 isotype with the effectorless mutations L234A, L235A, (referred to as herein, the antibody of the invention) has improved aggregation stability, increased affinity and improved pharmacokinetic parameters, such as 11/2β (hour) or Cmax (μg/ml), compared to known antibodies to PD-1, which are based on a human antibody IgG4 isotype, such as nivolumab. Prolgolimab has a weight average molecular mass of approximately 146 kDa and is specific for human PD-1. Prolgolimab has a heavy chain of 459 amino acids (SEQ ID NO: 1) and a human lung of 214 amino acids (SEQ ID NO: 2), the constant portion (Fc) of prolgolimab contains mutations L234A, L235A.

In this regard, it would be useful to administer an aqueous antibody composition of the invention to a patient with cancer.

A broad aspect of the present invention is an aqueous pharmaceutical composition suitable for administration to a subject to inhibit the activity of the PD-1 protein. The aqueous pharmaceutical composition contains a pharmaceutically effective amount of the anti-PD-1 antibody prolgolimab, an effective amount of trehalose dihydrate, a buffering agent based on acetate or histidine.

In accordance with one broad aspect of the present invention, there is provided an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 15 to 40 mg/ml as an antibody;

- 5 046787 (b) trehalose dihydrate at a concentration of 80 to 110 mg/ml;

(c) sodium acetate trihydrate at a concentration of 0.2 to 2.5 mg/ml and (d) acetic acid to a pH of 4.5 to 5.5.

In some embodiments, said prolgolimab may be at a concentration of 15 to 25 mg/ml.

In some embodiments, said prolgolimab may be at a concentration of 20 mg/ml.

In some embodiments, said trehalose dihydrate may be present at a concentration of 95 to 105 mg/ml.

In some embodiments, said trehalose dihydrate may be present at a concentration of 100 mg/ml.

In some embodiments, said sodium acetate trihydrate may be present at a concentration of 1.6 to 1.9 mg/ml.

In some embodiments, said sodium acetate trihydrate may be present at a concentration of 1.7 to 1.8 mg/ml.

In some embodiments, said sodium acetate trihydrate may be present at a concentration of 1.742 mg/ml.

In some embodiments, said acetic acid may be added to pH 5.0.

In some embodiments, said acetic acid may be present at a concentration of from 0.04 to 0.77 mg/ml.

In some embodiments, said acetic acid may be present at a concentration of 0.40 to 0.50 mg/ml.

In some embodiments, said acetic acid may be present at a concentration of 0.43 mg/ml.

In accordance with one broad aspect of the present invention there is provided an aHTu-PD-1 antibody aqueous pharmaceutical composition comprising:

(a) prolgolimab at a concentration of 90 to 150 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 50 to 110 mg/ml;

(c) sodium acetate trihydrate at a concentration of 0.2 to 2.5 mg/ml and (d) acetic acid to a pH of 4.5 to 5.5.

In some embodiments, said prolgolimab may be at a concentration of 90 to 110 mg/ml.

In some embodiments, said prolgolimab may be at a concentration of 100 mg/ml.

In some embodiments, said trehalose dihydrate may be present at a concentration of 75 to 85 mg/ml.

In some embodiments, said trehalose dihydrate may be present at a concentration of 80 mg/ml.

In some embodiments, said sodium acetate trihydrate may be present at a concentration of 1.6 to 1.9 mg/ml.

In some embodiments, said sodium acetate trihydrate may be present at a concentration of 1.7 to 1.8 mg/ml.

In some embodiments, said sodium acetate trihydrate may be present at a concentration of 1.742 mg/ml.

In some embodiments, said acetic acid may be added to a pH of 5.0 to 5.5.

In some embodiments, said acetic acid may be added to pH 5.0.

In some embodiments, said acetic acid may be present at a concentration of 0.045 to 0.77 mg/ml.

In some embodiments, said acetic acid may be present at a concentration of 0.40 to 0.50 mg/ml.

In some embodiments, said acetic acid may be present at a concentration of 0.43 mg/ml.

In accordance with one broad aspect of the present invention there is provided an aqueous pharmaceutical composition of an anti-PD-i antibody comprising:

(a) prolgolimab at a concentration of 5 mg/ml to 150 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 70 to 110 mg/ml;

(c) L-histidine at a concentration of 0.2 to 2.5 mg/ml; and (d) L-histidine hydrochloride at a concentration of 0.2 to 3.5 mg/ml.

In some embodiments, said prolgolimab may be in

- 6 046787 concentrations from 15 to 40 mg/ml.

In some embodiments, said prolgolimab may be at a concentration of 15 to 25 mg/ml.

In some embodiments, said prolgolimab may be at a concentration of 20 mg/ml.

In some embodiments, said trehalose dihydrate may be present at a concentration of 95 to 105 mg/ml.

In some embodiments, said trehalose dihydrate may be present at a concentration of 100 mg/ml.

In some embodiments, said L-histidine may be present at a concentration of 0.7 to 1.0 mg/ml.

In some embodiments, said L-histidine may be present at a concentration of 0.92 mg/ml.

In some embodiments, said L-histidine hydrochloride may be present at a concentration of 2.8 to 3.3 mg/ml.

In some embodiments, said L-histidine hydrochloride may be present at a concentration of 2.96 mg/ml.

In some embodiments of the invention, the composition may have a pH of from 5.5 to 6.5.

In some embodiments, the composition may have a pH of 5.5.

In some embodiments, said prolgolimab may be at a concentration of 90 to 110 mg/ml.

In some embodiments, said prolgolimab may be at a concentration of 100 mg/ml.

In some embodiments, said trehalose dihydrate may be present at a concentration of 75 to 85 mg/ml.

In some embodiments, said trehalose dihydrate may be present at a concentration of 80 mg/ml.

In some embodiments, said L-histidine may be present at a concentration of 0.7 to 1.0 mg/ml.

In some embodiments, said L-histidine may be present at a concentration of 0.92 mg/ml.

In some embodiments, said L-histidine hydrochloride may be present at a concentration of 2.8 to 3.3 mg/ml.

In some embodiments, said L-histidine hydrochloride may be present at a concentration of 2.96 mg/ml.

In some embodiments of the invention, the composition may have a pH of from 5.5 to 6.5.

In some embodiments of the invention, the composition may have a pH of from 5.5 to 6.0.

In some embodiments, the composition may have a pH of 5.5.

The aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab of the present invention may further contain a suitable solubilizer.

In some embodiments, said solubilizer may be poloxamer 188.

In some embodiments, said poloxamer 188 may be present in an amount that is greater than 0 mg/ml but equal to or less than 1 mg/ml.

In some embodiments, said poloxamer 188 may be present in an amount of 0 mg/ml, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 04 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 20 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 100 mg/ml;

(c) sodium acetate trihydrate at a concentration of 0.2 to 2.5 mg/ml and (d) acetic acid to a pH of 4.5 to 5.5.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 20 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 100 mg/ml;

(c) sodium acetate trihydrate at a concentration of 1.7 to 1.8 mg/ml and (d) acetic acid to pH 5.0.

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In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 20 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 100 mg/ml;

(c) sodium acetate trihydrate at a concentration of 1.742 mg/ml and (d) acetic acid to pH 5.0.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 100 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 80 mg/ml;

(c) sodium acetate trihydrate at a concentration of 0.2 to 2.5 mg/ml and (d) acetic acid to a pH of 4.5 to 5.5.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 100 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 80 mg/ml;

(c) sodium acetate trihydrate at a concentration of 1.7 to 1.8 mg/ml and (d) acetic acid to a pH of 5.0 to 5.5.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 100 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 80 mg/ml;

(c) sodium acetate trihydrate at a concentration of 1.742 mg/ml and (d) acetic acid to a pH of 5.0 to 5.5.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 100 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 80 mg/ml;

(c) sodium acetate trihydrate at a concentration of 1.742 mg/ml and (d) acetic acid to pH 5.0.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 20 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 100 mg/ml;

(c) L-histidine at a concentration of 0.2 to 2.5 mg/ml (d) L-histidine hydrochloride at a concentration of 0.2 to 3.5 mg/ml;

(e) wherein said composition has a pH of from 5.5 to 6.5.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 20 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 100 mg/ml;

(c) L-histidine at a concentration of 0.92 mg/ml and (d) L-histidine hydrochloride at a concentration of 2.96 mg/ml;

(e) wherein said composition has a pH of 5.5.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 100 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 80 mg/ml;

(c) L-histidine at a concentration of 0.2 to 2.5 mg/ml and (d) L-histidine hydrochloride at a concentration of 0.2 to 3.5 mg/ml;

(e) wherein said composition has a pH of from 5.5 to 6.5.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 100 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 80 mg/ml;

(c) L-histidine at a concentration of 0.92 mg/ml and (d) L-histidine hydrochloride at a concentration of 2.96 mg/ml;

(e) wherein said composition has a pH of 5.5 to 6.0.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 100 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 80 mg/ml;

- 8 046787 (c) L-histidine at a concentration of 0.92 mg/ml and (d) L-histidine hydrochloride at a concentration of 2.96 mg/ml;

(e) wherein said composition has a pH of 5.5.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

I) composition per 1 ml:

Prolgolimab 20.0 mg Sodium acetate trihydrate 1.742 mg Trehalose dihydrate 100 mg Acetic acid ice. up to pH 5.0 Water for injections up to 1 ml.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

II) composition per 1 ml:

Prolgolimab 20.0 mg Trehalose dihydrate 100 mg L-histidine 0.92 mg L-histidine hydrochloride 2.96 mg Water for injections up to 1 ml.

The aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab of the present invention may further contain a suitable solubilizer.

In some embodiments, said solubilizer may be poloxamer 188.

In some embodiments, said poloxamer 188 may be present in an amount that is greater than 0 mg/ml but equal to or less than 1 mg/ml.

In some embodiments, said poloxamer 188 may be present in an amount of 0 mg/ml, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 20 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 100 mg/ml;

(c) sodium acetate trihydrate at a concentration of 0.2 to 2.5 mg/ml and (d) acetic acid to a pH of 4.5 to 5.5;

(e) poloxamer 188 at a concentration greater than 0 mg/ml but equal to or less than 1 mg/ml.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 20 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 100 mg/ml;

(c) sodium acetate trihydrate at a concentration of 1.7 to 1.8 mg/ml and (d) acetic acid to pH 5.0;

(e) poloxamer 188 at a concentration greater than 0 mg/ml but equal to or less than 1 mg/ml.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 20 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 100 mg/ml;

(c) sodium acetate trihydrate at a concentration of 1.742 mg/ml and (d) acetic acid to pH 5.0;

(e) poloxamer 188 at a concentration greater than 0 mg/ml but equal to or less than 1 mg/ml.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 100 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 80 mg/ml;

(c) sodium acetate trihydrate at a concentration of 0.2 to 2.5 mg/ml and (d) acetic acid to a pH of 4.5 to 5.5;

(e) poloxamer 188 at a concentration greater than 0 mg/ml but equal to or less than 1 mg/ml.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 100 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 80 mg/ml;

- 9 046787 (c) sodium acetate trihydrate at a concentration of 1.7 to 1.8 mg/ml and (d) acetic acid to a pH of 5.0 to 5.5;

(e) poloxamer 188 at a concentration greater than 0 mg/ml but equal to or less than 1 mg/ml.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 100 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 80 mg/ml;

(c) sodium acetate trihydrate at a concentration of 1.742 mg/ml and (d) acetic acid to a pH of 5.0 to 5.5;

(e) poloxamer 188 at a concentration greater than 0 mg/ml but equal to or less than 1 mg/ml.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 100 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 80 mg/ml;

(c) sodium acetate trihydrate at a concentration of 1.742 mg/ml and (d) acetic acid to pH 5.0;

(e) poloxamer 188 at a concentration greater than 0 mg/ml but equal to or less than 1 mg/ml.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 20 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 100 mg/ml;

(c) L-histidine at a concentration of 0.2 to 2.5 mg/ml and (d) L-histidine hydrochloride at a concentration of 0.2 to 3.5 mg/ml;

(e) wherein said composition has a pH of 5.5 to 6.5;

(f) poloxamer 188 at a concentration greater than 0 mg/ml but equal to or less than 1 mg/ml.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 20 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 100 mg/ml;

(c) L-histidine at a concentration of 0.92 mg/ml and (d) L-histidine hydrochloride at a concentration of 2.96 mg/ml;

(e) wherein said composition has a pH of 5.5;

(f) poloxamer 188 at a concentration greater than 0 mg/ml but equal to or less than 1 mg/ml.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 100 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 80 mg/ml;

(c) L-histidine at a concentration of 0.2 to 2.5 mg/ml and (d) L-histidine hydrochloride at a concentration of 0.2 to 3.5 mg/ml;

(e) wherein said composition has a pH of 5.5 to 6.5;

(f) poloxamer 188 at a concentration greater than 0 mg/ml but equal to or less than 1 mg/ml.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 100 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 80 mg/ml;

(c) L-histidine at a concentration of 0.92 mg/ml and (d) L-histidine hydrochloride at a concentration of 2.96 mg/ml;

(e) wherein said composition has a pH of from 5.5 to 6.0;

(f) poloxamer 188 at a concentration greater than 0 mg/ml but equal to or less than 1 mg/ml.

In one embodiment, the present invention provides an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 100 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 80 mg/ml;

(c) L-histidine at a concentration of 0.92 mg/ml and (d) L-histidine hydrochloride at a concentration of 2.96 mg/ml;

(e) wherein said composition has a pH of 5.5;

(f) poloxamer 188 at a concentration greater than 0 mg/ml but equal to or less than 1 mg/ml.

In some embodiments of the present invention, said poloxamer 188 may be present in an amount of 0 mg/ml, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0 .6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml.

In some embodiments, the aqueous pharmaceutical composition of the anti-PD-1 antibody of the present invention can be administered parenterally.

- 10 046787

In some embodiments, the aqueous pharmaceutical composition of the anti-PD-l antibody of the present invention can be administered intramuscularly.

In some embodiments, the aqueous pharmaceutical composition of the anti-PD-1 antibody of the present invention can be administered subcutaneously.

In some embodiments, the aqueous pharmaceutical composition of the anti-PD-1 antibody of the present invention can be administered intravenously.

In some embodiments, the aqueous pharmaceutical composition of the anti-PD-1 antibody of the present invention can be administered intravenously as an infusion.

In some embodiments, the aqueous pharmaceutical composition of the anti-PD-1 antibody of the present invention can be administered intravenously as an infusion over a period of 60 minutes; if well tolerated, the infusion duration can be reduced to 30 minutes.

In one embodiment, the aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab of the present invention may be contained in a vial.

In some embodiments, said vial may be a glass vial.

In some embodiments, said vial may have a volume of from 1 ml to 50 ml.

In some embodiments, said vial may have a volume of from 1 ml to 20 ml.

In some embodiments, said vial may have a volume of 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, 20 ml, 25 ml, 30 ml , 35 ml, 40 ml, 45 ml or 50 ml.

In one embodiment, the aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab of the present invention may be contained in a syringe.

In some embodiments, said syringe may have a capacity of 1 ml.

In some embodiments, said syringe may have a capacity of 2 ml.

In one embodiment, the aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab of the present invention may be contained in a pre-filled syringe.

In some embodiments, said prefilled syringe may have a capacity of 1 ml.

In some embodiments, said prefilled syringe may have a capacity of 2 ml.

In accordance with another broad aspect of the present invention, there is provided a method for preparing an aqueous pharmaceutical composition suitable for administration to a subject for inhibiting the activity of the PD-1 protein. The method includes combining a pharmaceutically effective amount of the anti-PD-1 antibody prolgolimab with an acetate-based buffering agent; and an effective amount of trehalose. The method includes combining a pharmaceutically effective amount of the anti-PD-1 antibody prolgolimab with a histidine-based buffering agent; and an effective amount of trehalose.

In some embodiments, poloxamer 188 may be added as a solubilizer.

In accordance with another broad aspect, the use of an aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab, as defined herein, is provided for the treatment of cancer.

In some embodiments, the use of an aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab for the treatment of malignancy, which may be selected from the group consisting of melanoma, including unresectable or metastatic melanoma, early stages of melanoma before and after radical treatment; lung cancer, non-small cell lung cancer (NSCLC), including unresectable or metastatic non-small cell lung cancer; non-squamous non-small cell lung cancer, squamous cell lung cancer; small cell lung cancer, including unresectable or metastatic small cell lung cancer; early stage lung cancer before and after radical treatment; cervical cancer, including metastatic cervical cancer, early stages of cervical cancer before and after radical treatment; head and neck tumors, including squamous cell carcinoma of the head and neck; Hodgkin's lymphoma; tumors of the stomach and intestines, metastatic squamous cell carcinoma of the esophagus; bladder cancer, including metastatic urothelial carcinoma, kidney cancer; endometrial cancer, including metastatic endometrial cancer, early stages of endometrial cancer before and after radical treatment; breast cancer, including metastatic breast cancer, early stages of endometrial cancer before and after radical treatment; liver cancer, including metastatic or inoperable liver cancer, early stages of liver cancer before and after radical treatment; unresectable or metastatic solid tumor, including unresectable or metastatic solid tumor with signs of microsatellite instability.

In another broad aspect, the use of an aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab for the treatment of cancer is provided, comprising administering a pharmaceutically effective amount of the aqueous pharmaceutical composition as defined herein.

In accordance with another broad aspect, the use of an aqueous pharmaceutical

- 11 046787 anti-PD-1 antibody composition containing:

(a) prolgolimab at a concentration of 15 to 40 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 80 to 110 mg/ml;

(c) sodium acetate trihydrate at a concentration of 0.2 to 2.5 mg/ml and (d) acetic acid to pH 4.5-5.5, for the treatment of malignancy in a subject in need thereof.

In some embodiments, said prolgolimab may be at a concentration of 15 to 25 mg/ml.

In some embodiments, said prolgolimab may be at a concentration of 20 mg/ml.

In some embodiments, said trehalose dihydrate may be present at a concentration of 95 to 105 mg/ml.

In some embodiments, said trehalose dihydrate may be present at a concentration of 100 mg/ml.

In some embodiments, said sodium acetate trihydrate may be present at a concentration of 1.6 to 1.9 mg/ml.

In some embodiments, said sodium acetate trihydrate may be present at a concentration of 1.7 to 1.8 mg/ml.

In some embodiments, said sodium acetate trihydrate may be present at a concentration of 1.742 mg/ml.

In some embodiments, said acetic acid may be added to pH 5.0.

In some embodiments, said acetic acid may be present at a concentration of from 0.04 to 0.77 mg/ml.

In some embodiments, said acetic acid may be present at a concentration of 0.40 to 0.50 mg/ml.

In some embodiments, said acetic acid may be present at a concentration of 0.43 mg/ml.

According to another broad aspect, the use of an aqueous pharmaceutical composition of an anti-PD-1 antibody is provided, comprising:

(a) prolgolimab at a concentration of 5 to 40 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 70 to 110 mg/ml;

(c) L-histidine at a concentration of 0.2 to 2.5 mg/ml and (d) L-histidine hydrochloride at a concentration of 0.2 to 3.5 mg/ml;

for the treatment of malignant neoplasm in a subject in need of it.

In some embodiments, said prolgolimab may be at a concentration of 15 to 25 mg/ml.

In some embodiments, said prolgolimab may be at a concentration of 20 mg/ml.

In some embodiments, said trehalose dihydrate may be present at a concentration of 95 to 105 mg/ml.

In some embodiments, said trehalose dihydrate may be present at a concentration of 100 mg/ml.

In some embodiments, said L-histidine may be present at a concentration of 0.7 to 1.0 mg/ml.

In some embodiments, said L-histidine may be present at a concentration of 0.92 mg/ml.

In some embodiments, said L-histidine hydrochloride may be present at a concentration of 2.8 to 3.3 mg/ml.

In some embodiments, said L-histidine hydrochloride may be present at a concentration of 2.96 mg/ml.

In some embodiments of the invention, the composition may have a pH of from 5.5 to 6.5.

In some embodiments, the composition may have a pH of 5.5.

One embodiment of the invention provides the use of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 20 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 100 mg/ml;

(c) sodium acetate trihydrate at a concentration of 0.2 to 2.5 mg/ml and (d) acetic acid to a pH of 4.5 to 5.5;

for the treatment of malignant neoplasm in a subject in need of it.

One embodiment of the invention provides the use of an aqueous pharmaceutical

- 12 046787 anti-PD-1 antibody composition containing:

(a) prolgolimab at a concentration of 20 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 100 mg/ml;

(c) sodium acetate trihydrate at a concentration of 1.7 to 1.8 mg/ml and (d) acetic acid to pH 5.0;

for the treatment of malignant neoplasm in a subject in need of it.

One embodiment of the invention provides the use of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 20 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 100 mg/ml;

(c) sodium acetate trihydrate at a concentration of 1.742 mg/ml and (d) acetic acid to pH 5.0;

for the treatment of malignant neoplasm in a subject in need of it.

One embodiment of the invention provides the use of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 20 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 100 mg/ml;

(c) L-histidine at a concentration of 0.2 to 2.5 mg/ml and (d) L-histidine hydrochloride at a concentration of 0.2 to 3.5 mg/ml;

(e) wherein said composition has a pH of 5.5 to 6.5;

for the treatment of malignant neoplasm in a subject in need of it.

One embodiment of the invention provides the use of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 20 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 100 mg/ml;

(c) L-histidine at a concentration of 0.92 mg/ml and (d) L-histidine hydrochloride at a concentration of 2.96 mg/ml;

(e) wherein said composition has a pH of 5.5;

for the treatment of malignant neoplasm in a subject in need of it.

One embodiment of the invention provides the use of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

I) composition per 1 ml:

Prolgolimab 20.0 mg Sodium acetate trihydrate 1.742 mg Trehalose dihydrate 100 mg Acetic acid ice. up to pH 5.0 Water for injections up to 1 ml;

for the treatment of malignant neoplasm in a subject in need of it.

One embodiment of the invention provides the use of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

II) composition per 1 ml:

Prolgolimab 20.0 mg Trehalose dihydrate 100 mg L-histidine 0.92 mg L-histidine hydrochloride 2.96 mg Water for injections up to 1 ml:

for the treatment of malignant neoplasm in a subject in need of it.

The aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab of the present invention may further contain a suitable solubilizer.

In some embodiments, said solubilizer may be poloxamer 188.

In some embodiments, said poloxamer 188 may be present in an amount that is greater than 0 mg/ml but equal to or less than 1 mg/ml.

In some embodiments, said poloxamer 188 may be present in an amount of 0 mg/ml, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 04 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml.

In some embodiments, use of said aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab may comprise administering the composition at a dose of the anti-PD-1 antibody prolgolimab of 1 mg/kg body weight.

In some embodiments of the invention, the use of said aqueous pharmaceutical

- 13 046787 A composition of the anti-PD-1 antibody prolgolimab may comprise administration of the composition at a dose of the anti-PD-1 antibody prolgolimab of 3 mg/kg body weight.

In some embodiments, use of said aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab may include administering the composition every 2 weeks.

In some embodiments, use of said aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab may include administering the composition once every 2 weeks.

In some embodiments, use of said aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab may include administering the composition every 3 weeks.

In some embodiments, use of said aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab may include administering the composition once every 3 weeks.

In some embodiments, use of said aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab may include administering the composition at a dose of the anti-PD-1 antibody prolgolimab of 1 mg/kg body weight every 2 weeks.

In some embodiments, use of said aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab may include administering the composition at a dose of the anti-PD-1 antibody prolgolimab of 1 mg/kg body weight once every 2 weeks.

In some embodiments, use of said aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab may include administering the composition at a dose of 3 mg/kg body weight of the anti-PD-1 antibody prolgolimab every 3 weeks.

In some embodiments, use of said aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab may include administering the composition at a dose of 3 mg/kg body weight of the anti-PD-1 antibody prolgolimab once every 3 weeks.

In some embodiments, use of said aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab may include administering the composition parenterally.

In some embodiments of the invention, said parenteral administration may be intravenous, subcutaneous or intramuscular administration.

In some embodiments, use of said aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab may include administering the composition intravenously as an infusion.

In some embodiments, the aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab of the present invention may be administered intravenously as a 60-minute infusion, which may be reduced to 30 minutes if tolerated.

In some embodiments, the malignancy is melanoma, including unresectable or metastatic melanoma, early stages of melanoma before and after curative treatment; lung cancer, non-small cell lung cancer (NSCLC), including unresectable or metastatic non-small cell lung cancer.

In one embodiment, the invention provides a method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 15 to 40 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 80 to 110 mg/ml;

(c) sodium acetate trihydrate at a concentration of 0.2 to 2.5 mg/ml and (d) acetic acid to pH 4.5-5.5.

In some embodiments, said prolgolimab may be at a concentration of 15 to 25 mg/ml.

In some embodiments, said prolgolimab may be at a concentration of 20 mg/ml.

In some embodiments, said trehalose dihydrate may be present at a concentration of 95 to 105 mg/ml.

In some embodiments, said trehalose dihydrate may be present at a concentration of 100 mg/ml.

In some embodiments, said sodium acetate trihydrate may be present at a concentration of 1.6 to 1.9 mg/ml.

In some embodiments, said sodium acetate trihydrate may be present at a concentration of 1.7 to 1.8 mg/ml.

In some embodiments, said sodium acetate trihydrate may be present at a concentration of 1.742 mg/ml.

- 14 046787

In some embodiments, said acetic acid may be added to pH 5.0.

In some embodiments, said acetic acid may be present at a concentration of from 0.04 to 0.77 mg/ml.

In some embodiments, said acetic acid may be present at a concentration of 0.40 to 0.50 mg/ml.

In some embodiments, said acetic acid may be present at a concentration of 0.43 mg/ml.

In one embodiment, the invention provides a method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 5 to 40 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 70 to 110 mg/ml;

(c) L-histidine at a concentration of 0.2 to 2.5 mg/ml; and (d) L-histidine hydrochloride at a concentration of 0.2 to 3.5 mg/ml.

In some embodiments, said prolgolimab may be at a concentration of 15 to 25 mg/ml.

In some embodiments, said prolgolimab may be at a concentration of 20 mg/ml.

In some embodiments, said trehalose dihydrate may be present at a concentration of 95 to 105 mg/ml.

In some embodiments, said trehalose dihydrate may be present at a concentration of 100 mg/ml.

In some embodiments, said L-histidine may be present at a concentration of 0.7 to 1.0 mg/ml.

In some embodiments, said L-histidine may be present at a concentration of 0.92 mg/ml.

In some embodiments, said L-histidine hydrochloride may be present at a concentration of 2.8 to 3.3 mg/ml.

In some embodiments, said L-histidine hydrochloride may be present at a concentration of 2.96 mg/ml.

In some embodiments of the invention, the composition may have a pH of from 5.5 to 6.5.

In some embodiments, the composition may have a pH of 5.5. In one embodiment, the invention provides a method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 20 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 100 mg/ml;

(c) sodium acetate trihydrate at a concentration of 0.2 to 2.5 mg/ml and (d) acetic acid to a pH of 4.5 to 5.5.

In one embodiment, the invention provides a method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 20 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 100 mg/ml;

(c) sodium acetate trihydrate at a concentration of 1.7 to 1.8 mg/ml and (d) acetic acid to pH 5.0.

In one embodiment, the invention provides a method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 20 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 100 mg/ml;

(c) sodium acetate trihydrate at a concentration of 1.742 mg/ml and (d) acetic acid to pH 5.0.

In one embodiment, the invention provides a method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 20 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 100 mg/ml;

(c) L-histidine at a concentration of 0.2 to 2.5 mg/ml and (d) L-histidine hydrochloride at a concentration of 0.2 to 3.5 mg/ml;

- 15 046787 (e) where the specified composition has a pH from 5.5 to 6.5.

In one embodiment, the invention provides a method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

(a) prolgolimab at a concentration of 20 mg/ml as an antibody;

(b) trehalose dihydrate at a concentration of 100 mg/ml;

(c) L-histidine at a concentration of 0.92 mg/ml and (d) L-histidine hydrochloride at a concentration of 2.96 mg/ml;

(e) wherein said composition has a pH of 5.5.

In one embodiment, the invention provides a method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

I) composition per 1 ml:

Prolgolimab 20.0 mg Sodium acetate trihydrate 1.742 mg Trehalose dihydrate 100 mg Acetic acid ice. up to pH 5.0 Water for injections up to 1 ml.

In one embodiment, the invention provides a method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising: II) a 1 ml formulation of:

Prolgolimab 20.0 mg Trehalose dihydrate 100 mg L-histidine 0.92 mg L-histidine hydrochloride 2.96 mg Water for injections up to 1 ml.

The aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab of the present invention may further contain a suitable solubilizer.

In some embodiments, said solubilizer may be poloxamer 188.

In some embodiments, said poloxamer 188 may be present in an amount that is greater than 0 mg/ml but equal to or less than 1 mg/ml.

In some embodiments, said poloxamer 188 may be present in an amount of 0 mg/ml, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of said aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab at a dose of the anti-PD-1 antibody prolgolimab of 1 mg/kg body weight.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of said aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab at a dose of the anti-PD-1 antibody prolgolimab of 3 mg/kg body weight.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of said aqueous pharmaceutical composition of the anti-PD1 antibody prolgolimab every 2 weeks.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of said aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab once every 2 weeks.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of said aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab every 3 weeks.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of said aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab once every 3 weeks.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of said aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab at a dose of the anti-PD-1 antibody prolgolimab of 1 mg/kg body weight every 2 weeks.

- 16 046787

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of said aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab at a dose of the anti-PD-1 antibody prolgolimab of 1 mg/kg body weight once every 2 weeks.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of said aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab at a dose of the anti-PD-1 antibody prolgolimab of 3 mg/kg body weight every 3 weeks.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of said aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab at a dose of the anti-PD-1 antibody prolgolimab of 3 mg/kg body weight once every 3 weeks.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of said aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab parenterally.

In some embodiments of the invention, said parenteral administration may be intravenous, subcutaneous or intramuscular administration.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of said aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab intravenously as an infusion.

In some embodiments, the aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab of the present invention may be administered intravenously as a 60-minute infusion, which may be reduced to 30 minutes if tolerated.

In some embodiments, the malignancy is melanoma, including unresectable or metastatic melanoma, early stages of melanoma before and after curative treatment; lung cancer, non-small cell lung cancer (NSCLC), including unresectable or metastatic non-small cell lung cancer.

In one embodiment, the invention provides a method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of prolgolimab.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of prolgolimab at a dose of 1 mg/kg.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of prolgolimab at a dose of 3 mg/kg.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of prolgolimab every 2 weeks.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of prolgolimab once every 2 weeks.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of prolgolimab every 3 weeks.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of prolgolimab once every 3 weeks.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of prolgolimab at a dose of 1 mg/kg every 2 weeks.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of prolgolimab at a dose of 1 mg/kg once every 2 weeks.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of prolgolimab at a dose of 3 mg/kg every 3 weeks.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of prolgolimab at a dose of 3 mg/kg once every 3 weeks.

In some embodiments, a method of treating malignant neoplasms

- 17 046787 administration to a subject in need thereof may involve administering a therapeutically effective amount of prolgolimab parenterally.

In some embodiments of the invention, said parenteral administration may be intravenous, subcutaneous or intramuscular administration.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of prolgolimab intravenously as an infusion.

In some embodiments, prolgolimab may be administered intravenously as a 60-minute infusion; if well tolerated, the infusion duration may be reduced to 30 minutes.

In some embodiments, the malignancy is melanoma, including unresectable or metastatic melanoma, early stages of melanoma before and after curative treatment; lung cancer, non-small cell lung cancer (NSCLC), including unresectable or metastatic non-small cell lung cancer.

In one embodiment, the invention provides an aqueous pharmaceutical composition suitable for administration to a subject to inhibit the activity of the PD-1 protein, wherein the pharmaceutical composition contains for each 1 ml of the pharmaceutical composition:

Prolgolimab 20.0 mg Sodium acetate trihydrate 1.742 mg Trehalose dihydrate 100 mg Acetic acid ice. up to pH 5.0 Water for injections up to 1 ml.

In one embodiment, the invention provides an aqueous pharmaceutical composition suitable for administration to a subject to inhibit the activity of the PD-1 protein, wherein the pharmaceutical composition contains for each 1 ml of the pharmaceutical composition:

Prolgolimab 20.0 mg Trehalose dihydrate 100 mg L-histidine 0.92 mg L-histidine hydrochloride 2.96 mg Water for injections up to 1 ml.

In some embodiments, said aqueous pharmaceutical composition suitable for administration to a subject to inhibit PD-1 protein activity may be administered at a dose of the anti-PD-1 antibody prolgolimab of 1 mg/kg body weight.

In some embodiments, said aqueous pharmaceutical composition suitable for administration to a subject to inhibit PD-1 protein activity may be administered at a dose of the anti-PD-1 antibody prolgolimab of 3 mg/kg body weight.

In some embodiments, said aqueous pharmaceutical composition suitable for administration to a subject to inhibit PD-1 protein activity may be administered every 2 weeks.

In some embodiments, said aqueous pharmaceutical composition suitable for administration to a subject to inhibit PD-1 protein activity may be administered once every 2 weeks.

In some embodiments, said aqueous pharmaceutical composition suitable for administration to a subject to inhibit PD-1 protein activity may be administered once every 3 weeks.

In some embodiments, the aqueous pharmaceutical composition suitable for administration to a subject to inhibit PD-1 protein activity may be administered at a dose of the anti-PD-1 antibody prolgolimab of 1 mg/kg body weight every 2 weeks.

In some embodiments, the aqueous pharmaceutical composition suitable for administration to a subject to inhibit PD-1 protein activity may be administered at a dose of the anti-PD-1 antibody prolgolimab of 1 mg/kg body weight once every 2 weeks.

In some embodiments, said aqueous pharmaceutical composition suitable for administration to a subject to inhibit PD-1 protein activity may be administered at a dose of the anti-PD-1 antibody prolgolimab of 3 mg/kg body weight every 3 weeks.

In some embodiments, said aqueous pharmaceutical composition suitable for administration to a subject to inhibit PD-1 protein activity may be administered at a dose of the anti-PD-1 antibody prolgolimab of 3 mg/kg body weight once every 3 weeks.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of prolgolimab parenterally.

- 18 046787

In some embodiments of the invention, said parenteral administration may be intravenous, subcutaneous or intramuscular administration.

In some embodiments, a method of treating cancer in a subject in need thereof may comprise administering a therapeutically effective amount of prolgolimab intravenously as an infusion.

In some embodiments, prolgolimab may be administered intravenously as a 60-minute infusion; if well tolerated, the infusion duration may be reduced to 30 minutes.

In some embodiments, the malignancy is melanoma, including unresectable or metastatic melanoma, early stages of melanoma before and after curative treatment; lung cancer, non-small cell lung cancer (NSCLC), including unresectable or metastatic non-small cell lung cancer.

Exemplary Embodiments

The present invention relates to suitable aqueous pharmaceutical compositions of the anti-PD-1 antibody prolgolimab. In one embodiment of the invention, the aqueous pharmaceutical composition of prolgolimab may contain an acetate-based buffer and trehalose. Poloxamer 188 can be added as a solubilizer. In yet another embodiment, the aqueous pharmaceutical composition of prolgolimab may contain a histidine-based buffer and trehalose. Poloxamer 188 can be added as a solubilizer.

A histidine-based buffer can be formed by combining L-histidine with histidine hydrochloride or, optionally, with hydrochloric acid or other acids. It should be understood that while histidine hydrochloride can be used as the salt for the histidine buffer, any other histidine salt can be used for the histidine buffer without departing from the teachings of the present invention.

An acetate buffer can be formed by combining acetic acid with sodium acetate trihydrate. It should be understood that while sodium acetate trihydrate can be used as the salt for the acetate buffer, any other acetate salt, such as potassium acetate, can be used for the acetate buffer without departing from the teachings of the present invention.

In addition, the composition of the present invention may further include one or more other suitable excipients that are well known to those skilled in the art.

In some embodiments, the liquid pharmaceutical composition is formulated such that it is shelf stable in the sense that no further protein aggregation or modification occurs beyond the stability at zero time point.

In one embodiment, the inventors have unexpectedly produced highly concentrated aqueous pharmaceutical compositions of the anti-PD-1 antibody prolgolimab, where prolgolimab can be at a concentration of 90 to 150 mg/ml. In some embodiments of the present invention, the specified prolgolimab may be at a concentration of 90 mg/ml, 95 mg/ml, 100 mg/ml, 105 mg/ml, 110 mg/ml, 115 mg/ml, 120 mg/ml, 125 mg/ml ml, 130 mg/ml, 135 mg/ml, 140 mg/ml, 145 mg/ml, 150 mg/ml.

Such highly concentrated aqueous pharmaceutical compositions of the anti-PD-1 antibody prolgolimab of the present invention showed colloidal stability under vigorous stirring (800 rpm) for 120 hours and high thermal stability when heated at 50 and 37°C, and also acceptable for parenteral administration viscosity less than 50 cP.

The above compositions are suitable for parenteral administration, such as intravenous, subcutaneous, intradermal, intraarterial, intrathecal, intraperitoneal, intraarticular and/or intramuscular administration.

The provided pharmaceutical compositions can be administered to an individual in need of treatment by systemic injection, for example, by intravenous or subcutaneous or intramuscular injection; or by injection or application to the appropriate site, for example, by direct injection or direct application to the site when the site is accessible by surgery; or through topical application.

The above compositions can be administered to an individual in need of treatment intravenously as an infusion.

In some embodiments, the aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab of the present invention may be administered intravenously as a 60-minute infusion, which may be reduced to 30 minutes if tolerated.

The aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab of the present invention can be used after dilution. To do this, the required amount of the composition from the bottle is transferred into an infusion container containing a sterile 0.9% sodium chloride solution or a sterile 5% dextrose solution. The concentration of this composition in the prepared solution can range from 0.5 to 10 mg/ml. The prepared solution is mixed by carefully turning

- 19 046787 lining the infusion container to avoid foaming.

Methods of treatment and use of an aqueous composition.

In another embodiment, the present invention provides a method of treating a mammal, comprising administering to the mammal a therapeutically effective amount of pharmaceutical compositions wherein the concentration of the anti-PD-1 antibody prolgolimab is from 15 to 40 mg/ml, according to the present invention, wherein the mammal may have a disease or disorder , which can be effectively treated with the anti-PD-1 antibody prolgolimab of the present invention.

In another embodiment, the present invention provides a method of treating a mammal, comprising administering to the mammal a therapeutically effective amount of pharmaceutical compositions wherein the concentration of the anti-PD-1 antibody prolgolimab is 20 mg/ml, according to the present invention, wherein the mammal may have a disease or disorder that can be effectively treated with the anti-PD-1 antibody prolgolimab of the present invention.

In a preferred embodiment, the mammal is a human.

In another embodiment, the present invention provides a method of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of pharmaceutical compositions wherein the concentration of the anti-PD-1 antibody prolgolimab is from 15 to 40 mg/ml, according to the present invention, wherein the subject may have a disease or disorder that can be effectively treated with the anti-PD-1 antibody prolgolimab of the present invention.

In another embodiment, the present invention provides a method of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of pharmaceutical compositions wherein the concentration of the anti-PD-1 antibody prolgolimab is 20 mg/ml of the present invention, wherein the subject may have a disease or disorder , which can be effectively treated with the anti-PD-1 antibody prolgolimab of the present invention.

In a preferred embodiment, the subject is a human.

In one embodiment, the invention relates to a method of treating melanoma, comprising administering to a subject in need thereof a therapeutically effective amount of one of the present compositions, wherein the concentration of the anti-PD-1 antibody prolgolimab is from 15 to 40 mg/ml, according to this invention.

In one embodiment, the invention relates to a method of treating melanoma, comprising administering to a subject in need thereof a therapeutically effective amount of one of the following compositions, wherein the concentration of the anti-PD-1 antibody prolgolimab is 20 mg/ml, according to this invention.

In one embodiment, the invention relates to a method of treating unresectable melanoma, comprising administering to a subject in need thereof a therapeutically effective amount of one of the following compositions of the anti-PD-1 antibody prolgolimab, wherein the concentration of the anti-PD-1 antibody prolgolimab is from 15 to 40 mg/ml, according to this invention.

In one embodiment, the invention relates to a method of treating inoperable melanoma, comprising administering to a subject in need thereof a therapeutically effective amount of one of the following compositions of the anti-PD-1 antibody prolgolimab, wherein the concentration of the anti-PD-1 antibody prolgolimab is 20 mg/ml, according to the present invention .

In one embodiment, the invention relates to a method of treating metastatic melanoma, comprising administering to a subject in need thereof a therapeutically effective amount of one of the provided compositions of the anti-PD-1 antibody prolgolimab, wherein the concentration of the anti-PD-1 antibody prolgolimab is from 15 to 40 mg/ml, according to this invention.

In one embodiment, the invention relates to a method of treating metastatic melanoma, comprising administering to a subject in need thereof a therapeutically effective amount of one of the provided compositions of the anti-PD-1 antibody prolgolimab, wherein the concentration of the anti-PD-1 antibody prolgolimab is 20 mg/ml, according to the present invention .

In one embodiment, the invention relates to a method of treating early stages of melanoma before and after radical treatment, comprising administering to a subject in need thereof a therapeutically effective amount of one of the following compositions of the anti-PD-1 antibody prolgolimab, wherein the concentration of the anti-PD-1 antibody prolgolimab is between 15 up to 40 mg/ml, according to this invention.

In one embodiment, the invention relates to a method of treating early stages of melanoma before and after curative treatment, comprising administering to a subject in need thereof a therapeutically effective amount of one of the provided compositions of the anti-PD-1 antibody prolgolimab, wherein the concentration of the anti-PD-1 antibody prolgolimab is 20 mg /ml, according to this invention.

In one embodiment, the invention relates to a method of treating lung cancer, comprising administering to a subject in need thereof a therapeutically effective amount of one of the provided compositions of the anti-PD-1 antibody prolgolimab, wherein the concentration of the anti-PD-1 antibody prolgolimab is from 15 to 40 mg/ml, according to this invention.

In one embodiment, the invention relates to a method of treating lung cancer, comprising administering to a subject in need thereof a therapeutically effective amount of

- 20 046787 one of the above compositions of the anti-PD-1 antibody prolgolimab, where the concentration of the anti-PD-1 antibody prolgolimab is 20 mg/ml, according to this invention.

In one embodiment, the invention provides a method of treating non-small cell lung cancer (NSCLC), comprising administering to a subject in need thereof a therapeutically effective amount of one of the following compositions of the anti-PD-1 antibody prolgolimab, wherein the concentration of the anti-PD-1 antibody prolgolimab is from 15 to 40 mg/ml, according to this invention.

In one embodiment, the invention provides a method of treating non-small cell lung cancer (NSCLC), comprising administering to a subject in need thereof a therapeutically effective amount of one of the following compositions of the anti-PD-1 antibody prolgolimab, wherein the concentration of the anti-PD-1 antibody prolgolimab is 20 mg/ml , according to this invention.

In one embodiment, the invention provides a method of treating unresectable or metastatic non-small cell lung cancer, comprising administering to a subject in need thereof a therapeutically effective amount of one of the following compositions of the anti-PD-1 antibody prolgolimab, wherein the concentration of the anti-PD-1 antibody prolgolimab is from 15 to 40 mg/ml, according to this invention.

In one embodiment, the invention relates to a method of treating unresectable or metastatic non-small cell lung cancer, comprising administering to a subject in need thereof a therapeutically effective amount of one of the following compositions of the anti-PD-1 antibody prolgolimab, wherein the concentration of the anti-PD-1 antibody prolgolimab is 20 mg/ml , according to this invention.

In one embodiment, the invention provides a method of treating melanoma in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

I) composition per 1 ml:

Prolgolimab 20.0 mg Sodium acetate trihydrate 1.742 mg Trehalose dihydrate 100 mg Acetic acid ice. up to pH 5.0 Water for injections up to 1 ml.

In one embodiment, the invention provides a method of treating melanoma in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

II) composition per 1 ml:

Prolgolimab 20.0 mg Trehalose dihydrate 100 mg L-histidine 0.92 mg L-histidine hydrochloride 2.96 mg Water for injections up to 1 ml.

In one embodiment, the invention provides a method of treating unresectable melanoma in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

I) composition per 1 ml:

Prolgolimab 20.0 mg Sodium acetate trihydrate 1.742 mg Trehalose dihydrate 100 mg Acetic acid ice. up to pH 5.0 Water for injections up to 1 ml.

In one embodiment, the invention provides a method of treating unresectable melanoma in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

II) composition per 1 ml:

Prolgolimab 20.0 mg Trehalose dihydrate 100 mg L-histidine 0.92 mg L-histidine hydrochloride 2.96 mg Water for injections up to 1 ml.

In one embodiment, the invention provides a method of treating metastatic melanoma in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

I) composition per 1 ml:

Prolgolimab 20.0 mg Sodium acetate trihydrate 1.742 mg Trehalose dihydrate 100 mg Acetic acid ice. up to pH 5.0 Water for injections up to 1 ml.

In one embodiment, the invention provides a method of treating metastatic melanoma in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

II) composition per 1 ml:

Prolgolimab 20.0 mg Trehalose dihydrate 100 mg L-histidine 0.92 mg L-histidine hydrochloride 2.96 mg Water for injections up to 1 ml.

In one embodiment, the invention provides a method of treating early stages of melanoma before and after radical treatment in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

I) composition per 1 ml:

Prolgolimab 20.0 mg

Sodium acetate trihydrate 1.742 mg

Trehalose dihydrate 100 mg

Acetic acid ice. up to pH 5.0

Water for injections up to 1 ml.

In one embodiment, the invention provides a method of treating early stages of melanoma before and after radical treatment in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

II) composition per 1 ml:

Prolgolimab 20.0 mg Trehalose dihydrate 100 mg L-histidine 0.92 mg L-histidine hydrochloride 2.96 mg Water for injections up to 1 ml.

In one embodiment, the invention provides a method of treating lung cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

I) composition per 1 ml:

Prolgolimab 20.0 mg Sodium acetate trihydrate 1.742 mg Trehalose dihydrate 100 mg Acetic acid ice. up to pH 5.0 Water for injections up to 1 ml.

In one embodiment, the invention provides a method of treating lung cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

II) composition per 1 ml:

Prolgolimab 20.0 mg Trehalose dihydrate 100 mg L-histidine 0.92 mg L-histidine hydrochloride 2.96 mg Water for injections up to 1 ml.

In one embodiment, the invention provides a method of treating non-small cell lung cancer (NSCLC) in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

- 22 046787

I) composition per 1 ml:

Prolgolimab 20.0 mg Sodium acetate trihydrate 1.742 mg Trehalose dihydrate 100 mg Acetic acid ice. up to pH 5.0 Water for injections up to 1 ml.

In one embodiment, the invention provides a method of treating non-small cell lung cancer (NSCLC) in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

II) composition per 1 ml:

Prolgolimab 20.0 mg Trehalose dihydrate 100 mg L-histidine 0.92 mg L-histidine hydrochloride 2.96 mg Water for injections up to 1 ml.

In one embodiment, the invention provides a method of treating unresectable or metastatic non-small cell lung cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

I) composition per 1 ml:

Prolgolimab 20.0 mg Sodium acetate trihydrate 1.742 mg Trehalose dihydrate 100 mg Acetic acid ice. up to pH 5.0 Water for injections up to 1 ml.

In one embodiment, the invention provides a method of treating unresectable or metastatic non-small cell lung cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition of an anti-PD-1 antibody comprising:

II) composition per 1 ml:

Prolgolimab 20.0 mg Trehalose dihydrate 100 mg L-histidine 0.92 mg L-histidine hydrochloride 2.96 mg Water for injections up to 1 ml

In one embodiment, the invention provides a method of treating melanoma, comprising administering to a subject in need thereof a therapeutically effective amount of the anti-PD-1 antibody prolgolimab.

In one embodiment, the invention provides a method of treating unresectable melanoma, comprising administering to a subject in need thereof a therapeutically effective amount of the anti-PD-1 antibody prolgolimab.

In one embodiment, the invention provides a method of treating metastatic melanoma, comprising administering to a subject in need thereof a therapeutically effective amount of the anti-PD-1 antibody prolgolimab.

In one embodiment, the invention provides a method of treating early stages of melanoma before and after radical treatment, comprising administering to a subject in need thereof a therapeutically effective amount of the anti-PD-1 antibody prolgolimab.

In one embodiment, the invention provides a method of treating lung cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the anti-PD-1 antibody prolgolimab.

In one embodiment, the invention provides a method of treating non-small cell lung cancer (NSCLC), comprising administering to a subject in need thereof a therapeutically effective amount of the anti-PD-1 antibody prolgolimab.

In one embodiment, the invention provides a method for treating unresectable or metastatic non-small cell lung cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the anti-PD-1 antibody prolgolimab.

A therapeutically effective amount of the anti-PD-1 antibody prolgolimab of the present invention

- 23 046787 nia, and aqueous compositions comprising the anti-PD-1 antibody prolgolimab of the present invention, in the proposed compositions depends on the condition being treated, the severity of the condition, previous therapy and medical history of the patient, and response to the therapeutic agent. The appropriate dose can be adjusted according to the judgment of the attending physician so that it can be administered to the patient once or through multiple administrations.

In one embodiment, the effective amount of anti-PD-1 antibody per dose to a patient is about 0.01 to 10 mg per kilogram of body weight, or about 1 to 10 mg per kilogram of body weight, or about 0.05 mg per kilogram body weight, or approximately 0.25 mg per kilogram of body weight, or approximately 0.5 mg per kilogram of body weight, or approximately 1 mg per kilogram of body weight, or approximately 2 mg per kilogram of body weight, or approximately 3 mg per kilogram of body weight body weight, or approximately 4 mg per kilogram of body weight, or approximately 5 mg per kilogram of body weight, or approximately 6 mg per kilogram of body weight, or approximately 7 mg per kilogram of body weight, or approximately 8 mg per kilogram of body weight, or approximately 9 mg per kilogram of body weight, or approximately 10 mg per kilogram of body weight.

The dosing frequency may typically be about once per week, or about once every 2 weeks, or about once every 3 weeks.

In another embodiment, a suitable dose for administration by infusion may comprise 5-450 mg/dose, or may contain 40 mg, or 50 mg, or 60 mg per dose; or may contain 70 mg, or 80 mg, or 90 mg, or 100 mg per dose; or may contain 110 mg, or 120 mg, or 130 mg, or 140 mg per dose; or may contain 150 mg, or 160 mg, or 170 mg, or 180 mg per dose; or may contain 190 mg, or 200 mg, or 210 mg, or 220 mg per dose; or may contain 230 mg, or 240 mg, or 250 mg, or 260 mg per dose; or may contain 270 mg, or 280 mg, or 290 mg per dose, or may contain 300 mg, or 310 mg, or 320 mg, or 330 mg, or 340 mg, or 350 mg per dose; or may contain 360 mg, or 370 mg, or 380 mg, or 390 mg, or 400 mg, or 410 mg, or 420 mg, or 430 mg, or 440 mg, or 450 mg per dose.

In one embodiment of the invention, the dose may be delivered through one or more infusions. The dose may be delivered through one, two or three infusions. In some embodiments, the duration of therapy may be one or more infusions. In some embodiments, improvement in the patient's condition can be achieved through treatment over an extended period of time. In some embodiments, the duration of therapy may be until disease progression or lifelong.

In another embodiment, the pharmaceutical compositions of the present invention may be prepared in bulk form and, as such, the components of the pharmaceutical composition are present in amounts greater than may be required for administration and are suitably diluted prior to administration.

Alternatively, the pharmaceutical composition may be frozen, spray dried, or lyophilized and reconstituted in a suitable sterile carrier before use. Lyophilization can be performed using methods known in the art, which include various steps such as freezing, annealing, primary and secondary drying.

The pharmaceutical compositions can be administered as a single therapeutic agent or in combination with additional therapeutic agents as needed. Thus, in one embodiment, the present methods of treatment and/or prophylaxis are used in combination with the administration of a therapeutically effective amount of another active agent. The other active agent may be administered before, during, or after administration of the pharmaceutical compositions of the present invention. The other active agent may be administered as part of the present composition or, alternatively, as a separate formulation.

Administration of the proposed pharmaceutical compositions can be carried out in a variety of ways, including parenteral, oral, buccal, nasal, rectal and topical administration. Parenteral administration may include, without limitation, transdermal, subcutaneous, intravenous, intraarterial, intraperitoneal, intradermal, intracardiac, intraventricular, intracranial, intratracheal, intrathecal, intramuscular injection, intravitreal injection.

The pharmaceutical compositions of the present invention are particularly suitable for parenteral administration, i.e. subcutaneously, intramuscularly, intravenously, intraperitoneally, into the spinal cord, into the joints, intrasynovially and/or intrathecally. Parenteral administration can be done by bolus injection or continuous infusion. Pharmaceutical compositions for injection may be in unit dosage form, such as, but not limited to, ampoules, vials, prefilled syringes, or multi-dose containers with an added preservative. In addition, a number of recent approaches to drug delivery have been developed, and the pharmaceutical compositions of the present invention are suitable for administration by these new routes, for example, BD Physioject™, Inject-ease®, Genject®, injector pens such as GenPen®, and needleless device

- 24 046787 va, such as MediJector®40 and BioJector®. The pharmaceutical composition of the present invention can also be adapted for as yet undiscovered routes of administration.

Also see Langer, 1990, Science, 249: 1527-1533.

The proposed pharmaceutical compositions can also be formulated as a depot drug. Such long-acting formulations can be administered by implantation (eg, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the formulations can be modified using suitable polymeric or hydrophobic materials (eg, as an emulsion in a suitable oil), or ion exchange resins, or as sparingly soluble derivatives, eg, as a sparingly soluble salt.

Pharmaceutical compositions may optionally be provided in a vial, package, or dispenser, which may contain one or more unit dosage forms containing the active ingredient. In one embodiment, the dispenser device may comprise a syringe containing a single dose of liquid formulation ready for injection. The syringe may be accompanied by instructions for administration.

In another embodiment, the present invention relates to a kit or container containing an aqueous pharmaceutical composition according to the invention. The concentration of the antibody in the aqueous pharmaceutical composition can vary over a wide range, but is typically within the range of about 1 to about 200 mg/ml. The kit may also come with instructions for use.

The method for obtaining the above compositions involves adding acetate buffering agents to the aqueous phase, followed by adding, in any order, the following components: trehalose, prolgolimab, and/or a solubilizer selected from the group: polysorbate 20, polysorbate 80, poloxamer 188 or a combination thereof.

The method for obtaining the above compositions involves adding histidine buffer agents to the aqueous phase, followed by adding, in any order, the following components: trehalose, prolgolimab, and/or a solubilizer selected from the group: polysorbate 20, polysorbate 80, poloxamer 188 or a combination thereof.

Case study

Below are examples of a study to determine the reagents and their concentrations for the preparation of an aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab of the present invention.

The studies and examples presented herein are for illustrative purposes only, which demonstrate the suitability of certain components used in aqueous pharmaceutical compositions of the anti-PD-1 antibody prolgolimab. It is understood that other methods may be used by one of ordinary skill in the art without departing from the teachings of the present invention.

The suitability of the aqueous compositions of the present invention was tested through the illustrative methods described herein.

Example 1: Preparation of stable formulations of the anti-PD-1 antibody prolgolimab.

Antibody samples with a concentration of 5 mg/ml were obtained in Stirred Cell concentration cells (Millipore) under pressure. To do this, the antibody in its original composition was placed in a cell, with continuous stirring, the protein was concentrated under a stream of compressed air to a concentration of 10 mg/ml, after which at least a 10-fold volume of an aqueous solution with the target composition, including buffers, osmotic agents, and, if necessary, additional water-soluble stabilizers. Upon completion of the diafiltration process, the antibody was concentrated to a concentration of about 10 mg/ml, unloaded from the cell, and the exact protein concentration was determined by UV spectrophotometry. The appropriate solution of excipients was then added to the sample to obtain a solution with a target protein concentration of 5 ± 0.2 mg/mL.

Protein samples with a concentration of more than 50 mg/ml were obtained in Pellicon cassettes (Millipore) in tangential flow mode. To do this, the antibody in the original composition was placed in a container for diafiltration, the protein was concentrated to a concentration of 50-100 mg/ml, after which the system was connected to a supply of at least 10 times the volume of a solution with the target composition containing buffers, osmotic agents and, if necessary, additional water-soluble stabilizers. It is allowed to add a concentrate of osmotic agents and water-soluble stabilizers after completion of diafiltration. Upon completion of the diafiltration process, the antibody was concentrated to a concentration above the target, discharged from the system, and the exact protein concentration was determined. The appropriate solution of excipients was then added to the sample to obtain a solution with the target protein concentration.

When preparing formulations containing solubilizers, surfactant concentrates were added to the antibody after completion of diafiltration and concentration during the final dilution of the antibody with a solution of excipients to the target concentration.

- 25 046787

Before aseptic filling into the final container (eg, glass or polymer vessel, vial or syringe), the antibody solution was filtered through a 0.22 μm pore size membrane.

Example 2. Determination of protein concentration in the test samples.

Protein concentration was determined using UV spectrophotometry at 280 nm in UV transparent plates.

Each sample was diluted with an appropriate solution of excipients to a concentration of ~0.5 mg/ml. 150 μl of the diluted sample was placed into the well of the UV spectrophotometry plate. The optical density of the solutions placed in the plate was measured using a plate spectrophotometer at a wavelength of 280 nm. The corresponding solution of excipients was used as a reference solution.

Protein concentration (C) in mg/ml was calculated using the formula

I _ A(280)*b ε*1 ' where A ₂₈₀ is the optical density value at a wavelength of 280 nm; ε is the extinction coefficient of the protein under study;

b is the total dilution factor of the sample;

l is the thickness of the layer in the well of the tablet; for 150 µl 1 = 0.42 cm.

Example 3. PEG aggregation study.

A solution of PEG 6000 was prepared with a mass concentration of 20-25% in the studied composition of excipients. The resulting solutions were filtered through a 0.45 μm Durapore filter.

The calculated amount of sample, solution of excipients and 20-25% PEG 6000 solution was transferred into 96-well plates for UV spectrophotometry so that in a number of wells there was a PEG6000 concentration from 0 to 18%, and the protein concentration in each well was 1 mg/ ml. All solutions obtained in the wells were mixed well by pipetting.

After this, the degree of turbidity of the solutions was assessed visually, and the optical density of the solutions was measured at a wavelength of 400 nm.

Protein precipitation in the presence of PEG is due to a volume displacement effect, that is, the protein is sterically displaced from solvent regions by the polymer. This causes the protein to concentrate until its solubility is exceeded and a precipitate forms. The less stable the sample, the lower the concentration of PEG 6000 it will form visible aggregates (opalescence).

Example 4. Determination of colloidal stability using the shake test method.

The test samples were divided into 2 parts of 200 μl and placed in glass vials, 1 vial for each composition was placed in a refrigerator for storage at 2 -8 ° C, the rest were placed in a shaker and shaken at a speed of 800 rpm at a temperature of 2 - 8°C for the specified time. After the end of the stress, the tubes were shaken on a vortex and transferred for analysis.

Example 5. Determination of colloidal stability by cryoconcentration method.

The test samples were divided into 2 parts and placed in polymer tubes: 1 tube for each composition was placed in the refrigerator for storage at 2-8°C, the rest were placed in the freezer and stored at a temperature of minus 16-20°C for the specified time. After the end of the stress, the tubes were removed from the freezer, kept at room temperature until the contents were completely thawed, the solutions were mixed using vortex and transferred for analysis.

Example 6. Determination of thermal stability using the thermostress method.

The test samples were divided into 2 parts and placed in separate glass vials: 1 vial for each composition was placed in the refrigerator for storage at 2-8°C, the rest were placed in a thermostat and incubated at the required temperature for the specified time. After heating was completed, the vials were removed from the thermostat, kept at room temperature for about 15 minutes, and submitted for analysis.

Example 7. Determination of sample homogeneity by size exclusion (E) high-performance liquid chromatography (HPLC).

Column Tosoh TSK-GelG3000SW _X L 7.8 mm IDx30 cm, cat. No. 08541.

Column temperature: 25°C.

Mobile phase flow rate: 0.7 ml/min.

Injection volume: 10 µl.

Sample concentration: 5 mg/ml.

Detector wavelength: 220 nm.

Elution duration: 25 min.

Mobile phase: disodium hydrogen phosphate b/v 7.1 mg/ml.

Sodium chloride 17.54 mg/ml.

The pH of the mobile phase was adjusted to 7.0 with orthophosphoric acid.

The change in purity after stress was calculated using the formula:

Δ = (proportion of the main peak after stress - fraction of the main peak before stress).

- 26 046787

Example 8. Determination of charge heterogeneity (profile of charged forms) of samples on a Caliper Labchip GXII device.

Preparation of analyzed samples.

Samples were diluted to a concentration of 1 mg/ml. To 200 μl of the resulting solution, 2 μl of a solution of carboxypeptidase B (CpB) with a concentration of 5 mg/ml was added, mixed and incubated for 2 hours at a temperature of 37°C. The test samples were dialyzed against three changes of water. To carry out dialysis, the test solutions were placed in Amicon Ultra centrifuge tubes with a volume of 0.5 ml and centrifuged for 10 minutes at a rotor speed of 10,000 rpm in an Eppendorf Centrifuge 5417R centrifuge. The absorption intensity of the solutions was measured using a Cary 50 Bio spectrophotometer relative to water. Samples of the studied series were prepared with a concentration of 2 mg/ml. A 96-well Bio-Rad plate was filled with 3 µl of Labelling Buffer (from the HT Protein Charge Variant Labeling Kit), 15 µl of test solutions and 3 µl of Dye Mixture solution (from the HT Protein Charge Variant Labeling Kit). The plate was placed in a dark place for 10 minutes, after which 36 μl of water was added to each well and mixed by pipetting. Centrifuge for 1 min at 1000 rpm in an Eppendorf Centrifuge 5417R centrifuge.

Preparation of working solutions and filling the chip.

The preparation of working solutions and preparation of the chip were carried out in accordance with the manufacturer’s method using the HT Protein Charge Variant Labeling Kit. Running an analysis is standard procedure. Analysis method HT Protein Charge Variant 90s.

Example 9. Determination of sample purity using a Caliper Labchip GXII instrument under reducing and non-reducing conditions.

Preparation of analyzed samples.

To prepare denaturing and reducing solutions, 700 μl of NT Protein Express Sample Buffer was used. To restore the samples, 24.5 μl of 1 M dithiothreitol (DTT) was added. An alkylating agent, 24.5 μl of 1 M iodoacetamide (IAM), was added to the buffer for non-reducible samples.

For each sample, two microtubes were prepared: 35 μl of denaturing buffer was added to one, and 35 μl of reducing buffer was added to the other. Samples were diluted to a concentration of 2 mg/ml. 5 μl of sample was added to each pair of tubes. The samples were denatured at 100°C for 5 min. The tubes were mixed by vortex, after which 70 μl of water was added to each tube and mixed by vortex. Transfer 44 μl of each sample into the wells of a 96-well plate.

Preparation of working solutions and filling the chip.

Working solutions and chip preparation were carried out in accordance with the manufacturer’s method using the HT Protein Express Reagent Kit. Running an analysis is standard operation. HT analysis method Protein Express 200.

Example 10. Determination of the profile of charged forms of samples by ion exchange (IO) high-performance liquid chromatography (HPLC).

Column: ProPac WCX-10 Analytical, 4x250 mm.

Pre-column: Pro Race WCX-10G, 4x50 mm.

Column temperature: 30°C.

Mobile phase flow rate: 0.7 ml/min.

Injection volume: 50 µl.

Sample concentration: 1 mg/ml.

Detector wavelength: 220 nm.

Elution duration: 60 min.

Mobile phase:

Eluent A: 0.03 M 2-monfolinoethanesulfonic acid (MES), pH 6.0.

Eluent B: 0.03 M MES, 0.5 M NaCl, pH 6.0.

Eluent gradient A: 86% - 0% - 86%.

Before analysis, the test sample was treated with a solution of carboxypeptidase B at a temperature of +37 ± 1°C for 2 hours.

The absolute change in the profile of charged forms after stress was calculated using the formula:

Δ = |content of acidic fractions before stress - content of acidic fractions after stress| + |content of the dominant fraction before stress - content of the dominant fraction after stress| + |content of alkaline fractions before stress - content of alkaline fractions after stress|.

Example 11. Determination of low molecular weight impurities by vertical gel electrophoresis (VEP) in polyacrylamide gel (PAGE) under reducing and non-reducing conditions.

PAGE was prepared in glass plates in the presence of sodium dodecyl sulfate, consisting of a concentrating layer - 4% PAGE and a separating layer: under reducing conditions - 12.5% PAGE, under non-reducing conditions - 8% PAGE.

Assembled and installed the electrophoresis chamber in accordance with the operating instructions

- 27 046787 device for vertical electrophoresis. Samples were prepared by diluting samples with purified water to a final concentration of 1 mg/mL. A volume equivalent to 40 μg was taken and the prepared samples of the test sample were mixed in a ratio of 3:1 (v/v) with a 4-fold sample loading buffer solution containing 2-mercaptoethanol (reducing conditions) and not containing 2-mercaptoethanol (non-reducing conditions) , mixed. The resulting solutions were incubated at a temperature of (99 ± 1)°C for 3 min (samples containing 2-mercaptoethanol) and at a temperature of (99 ± 1)°C for 1 min (samples not containing 2-mercaptoethanol). The solutions were cooled to room temperature, mixed and applied to PAGE wells under a layer of electrode buffer solution.

Electrophoresis was carried out in constant current mode using a water cooling system. The operating parameters of the power source were set: when the dye front passed through the concentrating gel, the current voltage was 110 V. After the dye front entered the lower separating gel by 5 - 7 mm, the current voltage was increased to 180 V. The power source was turned off when the dye front reached the lower boundary of the gel .

After completion of electrophoresis, the gels were separated from the glasses and the proteins were fixed in a fixing solution for 16-18 hours at room temperature. Then the gels were stained (in acid blue 83 solution) and washed until the bands were clearly visualized. The gels were scanned. The purity and impurities of the test samples were assessed using GelPro software.

Example 12 Determination of relative specific activity.

The relative specific activity of anti-PD-1 monoclonal antibody samples was assessed by the ability to specifically bind to the PD-1 protein on the membrane surface of Jurkat PD-1 NFAT cells. The day before the assay, PDL-1 was immobilized in the wells of culture plates. The next day, the plates were washed and 50 μl/well of phytohemagglutinin-P solution (PanEco, Russia, cat. no. M021) was added. Using the Freedom Evo robotic station, serial dilutions of the standard and test samples were prepared and added to culture plates at 10 μl/well. 40 μl/well of Jurkat PD-1 NFAT cell suspension was added to culture plates. The plates were incubated for 4-6 hours at 37°C, 5% CO2. All procedures described above were carried out under aseptic conditions.

Upon completion of incubation, 100 μl/well of BioGlo solution (Promega, USA, cat. no. G7941) was added and the luminescence level was determined.

Using Magellan software, four-parameter curves of the dependence of the average luminescence value on protein concentration were constructed for solutions of the standard sample and the test sample located on the same plate.

The relative specific activity of the test sample in percent (RP) was calculated using the formula

ED. _rst

RP =---—-100%,

EDi.. test, where ED ₅₀ st is the value of the half effective dose of the standard sample, ng/ml;

ED ₅₀ test - the value of the half effective dose of the test sample, ng/ml.

The final result was taken as the average value of relative specific activity, calculated from three independent determinations (determined on 3 different culture plates).

Example 13. Determination of viscosity.

The dynamic viscosity of the solutions under study was determined by rotational viscometry on a Brookfield CAP2000+L viscometer.

Example 14. Investigation of the nature of the buffer solution.

Compositions under study.

Four buffer solutions were selected for this study. When choosing the molarity and pH of the buffer, restrictions on possible subcutaneous administration were taken into account, as well as the pI of the antibody (the pH of the buffer solutions under study was selected at the minimum possible physiological value).

- 28 046787

Composition (per 1 ml):

Acetate, pH 5.0 Prolgolimab 5 mg Sodium acetate trihydrate (t/g) 0.436 mg Glacial acetic acid (ice) up to pH 5.0 Citrate, pH 5.0 Prolgolimab 5 mg Sodium citrate dihydrate (d/g) 0.932 mg Anhydrous citric acid (w/w) 0.35.2 mg Histidine, pH 5.5 Prolgolimab 5 mg L-histidine 0.23 mg L-histidine hydrochloride monohydrate (m/g)0.74 mg Phosphate, pH 6.0 Prolgolimab 5 mg Sodium dihydrogen phosphate monohydrate (m/g) 1.21 mg Sodium hydrogen phosphate anhydrous (w/w) 0.17 mg

Determination of colloidal stability by PEG aggregation method.

The study was carried out in triplicate for each image. The results are presented in table. 1 and Fig. 1.

Table 1

Average optical density of solutions immediately after preparation at a wavelength of 400 nm

PEG 6000, Ϊ 0 2 4 6 8 10 12 14 16 18 Acetate,pfl 5.0 0L5 9 0.061 0.063 0.063 0.062 0.064 0.066 0.063 0.069 0.070 _Citrate pH 5.0 0.05 6 0.050 0.053 VNya Histidine, pH 5.5 0.05 8 0.062 0.062 0.063 0.064 0.066 Phosphate,pH 6.0 0.05 3 0.061 0.061

As a result of the study, the samples in histidine and acetate buffer solutions showed the highest colloidal stability in the presence of PEG. Citrate and phosphate formulations were excluded from further study because aggregation at 6% PEG is an indication of poor colloidal stability. Based on the results obtained, acetate and histidine buffer solutions were selected for further selection of pH and molarity of the buffer solution.

Example 15. Selection of pH and buffer capacity of the solution.

____________________Study compositions (per 1 ml)

Designation pH 5.5 pH 6.0 pH 6.5 5 ntv Histidine buffer solution Prolgolimab 5 mg L-histidine 0.23 mg L-hissidine g/x m/g 0.74 mg Prolgolimab 5 mg L-histidine 0.45 mg L-histidine g/x m/g 0.45 mg Prolgolimab 5 mg L-histidine 0.58 mg L-histidine g/x m/g 0.27 mg 10 Histidine buffer solution Prolgolimab 5 mg L-histidine 0.46 mg L-histidine g/x m/g 1.48 mg Prolgolimab 5 mg L-tistidine 0.9 mg L-histidine g/x m/g 0.9 mg Prolgolimab 5 mg L-histidine 1.16 mg L-histidine g/x m/g 0.54 mg 20 shM Histidine buffer solution Prolgolimab 5 mg L-histidine 0.92 mg L-histidine g/x m/g 2.96 mg Prolgolimab 5 mg L-tistidine 1.8 mg L-histidine g/x m/g 1.8 mg Prolgolimab 5 mg L-histidine 2.32 mg L-histidine g/x m/g 1.08 mg

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5 pC Acetate buffer solution Prolgolimab 5 mg Sodium acetate t/t 0.24 mg Acetic acid up to pH 4.5 Prolgolimab 5 mg Sodium acetate t/g 0.44 mg Acetic acid to pH up to 5.0 Prolgolimab 5 mg Sodium acetate t/g 0.58 mg Acetic acid up to pH 5.5 10 iM Acetate buffer solution Prolgolimab 5 mg Sodium acetate t/g 0.49 mg Acetic acid up to pH 4.5 Prolgolimab 5 mg Natrm acetate t/g 0.87 mg Acetic acid to pH up to 5.0 Prolgolimab 5 mg Sodium acetate t/g 1.16 mg Acetic acid up to pH 5.5 20 iM Acetate buffer solution Prolgolimab 5 mg Sodium acetate t/g 0.98 mg Acetic acid up to pH 4.5 Prolgolimab 5 mg Sodium acetate t/g ig 1.74 Acetic acid to pH up to 5.0 Prolgolimab 5 mg Sodium acetate t/g 2.31 mg Acetic acid up to pH 5.5

The study was carried out using the thermostress method at 50°C for 72 hours. The results are presented in table. 2. The homogeneity of the samples was analyzed by E-HPLC and electrophoresis on a Labchip device. The profile of the charged forms of the samples was analyzed on a Labchip instrument.

table 2

Summary results of quality indicators before and after thermal stress______________

5 shI Histidine pH 5.5 10 shI Histidine pH Ξ.ϊ - , 7........................................................ ...... 5 inM Histidine pH 6.0 10 isH Histidine pH 6.3 2? rii Histidine pH 6.0 5 ml-1 Histidine pH 6.5 10 ibM Histidine pH c.r. 2 0 rll Histiline pH6. Ξ 5 iM Acetate pH 4.5 10 rll Acetate pH 4 . 5 20 nM Acetate pH i. 5 5 ml-I Acetate pH 5.0 theory"., 5.5 a. οι A.51 5.3 ..ate'.' tW™ -it . : NIMI .No.. GDP m. .№ kt/йй ^{ω ,ffc3} lb | -cc. Yu ω НйгШ ⁿ mН ¹ ΕΒΒΙ № Ή ·' ^,g kYYM ^{,n L} ' KЗжьь! ^{113 1} 'la: ₽H pract. noc.te 50 °C 72h 5 . 72 = . With: 5.63 6.12 6.20 6.17 6.67 6. ZE 6.34 5.03 4.27 4.66 5.43 Βχοι 3: HPLC,' Seder .t.aniye monom era, % 93.46 93.55 S3.53 93.4 0 92.57 93.61 93.70 93.59 93.74 93.79 93.53 93.59 53.55 ΓΪΞΖ ΚΞ2- Sode monomial Lafc EF ed. uel. , =5 9 2.70 95.4 9 99.05 92. 97 92. 94 95.95 92.94 9 9.02 9 9.07 92. 93 9 2.22 9 5.26 99.20 troll Zhanie era, EF Chip EF non-ed. us l. ,% = 4.50 95.05 94.12 93.99 94.12 93.99 94.09 93.75 93.52 90.95 90.29 92.25 93.31 e/HPLC', Δ content monomer, % 1¾^¾¾¾ kyvvyZ ozhv yZhiyoSHYY I _ -7.-13 SHI — 7.42 -7.4 6 -0.41 -9.42 Change D kiol. fractions, % nivni -24.2-7 -25.7- NO NO· BAT -+2^.2 and vnv··· + 25.69 + 26.46 Thermal stress containment LaoChio* d dsminj. fractions, % -15.-75 -15.21 MIV -16.16 INN ZHYYY -15.34 -1 ^t.41 -14.06 -15.61 -16.64 c- 72 h fractions, D slot fractions, % IMI -5.77 NININ yyyya - - = . 61 them -:. s about nnnnn -7.64 '. Izme-ι seder ks iske ZhaY EF p=d. USL.g ΐ ΗΒΗ 0.:5 research institute NIN PIM IM NINI HHI -1.09 -0.62 : leniya ra _g EF Γ.1Ζ EF non-ed. _:.P. , % -2.34 -1.11 - 2 . £ £ - 2 . Ξ = - 2. 43 pii ipm - 1 . 35 him 10 Eli Acetate pH 5.0 5.33 5.31 93.65 99.23 92.75 -2 E.ΞΞ -15.71 -9:5 -0.63 5.20 93. €С 9:.:5 94.67 nnnnnnn -15.75 -9.54 him iin 5 eM Acetate pH 5.5 5.5 6.11 j^MI 93.55 99.17 92.43 -15.15 -9.2: NO NO iin 10 eM Acetate pH 5.5 5.71 5.72 93.43 99.37 92.50 +25.40 -15.40 TIN them 20 eM Acetate pH 5.5 5.66 53.67 9:.95 93.94 +26.42 -16.6S -9.7 4 -1.97

* The change was calculated using the formula Δ = fraction content after stress - fraction content before stress

- best result

worst result

- input control or average result

The study showed that the presence of a monoclonal antibody against PD-1 (prolgolimab) in an aqueous solution leads to an increase in pH levels. The greatest pH stabilization is provided by 20 mM buffer solutions.

All studied samples demonstrated high aggregation stability on E-HPLC, since the increase in impurities during thermal stress was no more than 0.5% for all samples. The best

- 30 046787 stability was shown by samples based on histidine with pH 5.5-6.0, as well as on acetate with pH 5.0-5.5.

All compositions demonstrated similar stability of the profile of charged forms; the quantitative difference in the change in the content of fractions between compositions was within the error limits of the method.

According to the results of gel electrophoresis under reducing conditions, all histidine-based formulations demonstrated higher stability, while under non-reducing conditions, better performance was obtained for acetate buffer solutions.

Based on the obtained stability data in terms of pH, purity and profile of the charged forms of the monoclonal antibody against PD-1, the following compositions of excipients are recommended for further work.

20 mM Acet 5.0 /20 mM Acetate buffer solution pH 5.0 Sodium acetate t/g Acetic acid, ice. 1.74 mg/ml up to pH 5.0 20 mM His 5.5 /20 mM Histidine buffer solution pH 5.5 L-histidine L-histidine hydrochloride m/g 0.92 mg/ml 2.9 6mg/mp

Example 16. Selection of pharmaceutical composition based on histidine.

Compositions under study.

To screen a stable pharmaceutical composition based on a 20 mM histidine buffer solution with pH 5.5, the following excipients were taken: mannitol, trehalose dihydrate, sucrose (osmotic agents). All tested solutions are isotonic.

- 31 046787

Prolgslimab Stabilizer! Solubilizers 2o; mH His pH _: 5.5 Osmotic Οι gates Mannktol Trehalose d/g Sucrose 1-Prolia Glinin IN A oh oh Oh cha To ABOUT •To; Poloxamer 188 1 5 g J up to/1 ml 2 5 5 3 WITH . 2 up to 1 ml 3 5 50 5 . 5 up to/ 1 ml 4 5 50 5 . 3 de 1 ml 5 -5 50 C 2 up to 1 ml ;·5 50 WITH . 5 up to 1 ml 7/ ·5 50 WITH . IN de 1 ml 8 ; /5; 5 2: . : up to 1 ml 9 ;;5 5 23.5 bi. de 1 ml 10 ; ;5 5 23.5 5 . 5 de 1 ml eleven 5 5 2: . : WITH . 3 de 1 ml 12 5' s; 23.5 WITH . 2 up to 1 ml 13 ;;5; 5 2: . ζ WITH . 5 up to'/ 1 ml 14 /5 5 2 5.3 G. 8 before; 1 ml •Ιΐτ /5 33;. 5 / . 0 before; 1 ml 16; '·5· 33. 5 7. 5 WITH . 2 up to 1 ml 17 5 33. 5 ~ . 5 WITH . 5 up to/ 1 ml 18 '·;5· zz. h; T . 5 =:. ? DO/ 1 ml 19 ;·5· 33. 5 “. 5 WITH . 2 up to 1 ml 20; /5 Zz, 5 . 5 5 . 5 before; 1 ml ;ζΐ; ;;5; 3 3 . 5 “. 5 WITH . 8 up to/1 ml ;2£ 5 2: ·: ' to/ 1 md 23 5 2: : up to/1 ml; ; 24 _... :. h before;;; 1 ml 25 5 2:: :. h before; 1 ml 26 5 2: s WITH . 2 up to 1 ml 27 5 2:: WITH . 5 up to/ 1 mp 2Ε 5 222 S. I up to/1 ml 29 5 2 5.3 up to ;1 ml

- 32 046787

Study formulations (mg to 1 ml)

2: h-> 1C 23.8 C 2 up to 1 ml _·. B 1C 23.5 S, 5 -With 1 ml 32 G- 1C 1 . i C « 8 ds 1 ml 3 1C 23.5 WITH . ζ up to 1 ml 34 Σ' 1C 2 z. B ,- ζ ds 1 ml 33 - 1C YU . 3 t o up to-<1 ml ? : G 67 7 - DO '·1·ml G 67 7 C L - έ up to 71 ml - = = 67 . G WITH . 5 ds 1 ml -J 5 67 '. G P I up to 1 ml L with B 67 7" And . Z ds 1 ml 4 1 - 67 7~ WITH . 5 up to 1 yal / " ¢7 .· . W 0.8 DS 1 ML 4 3 □ - ^! J up to>--1 ml 44 _ ^: JL· And . ζ up to 1 ml - - _ · C , ό ds_ml - : - 207 S, U TO <:1 ML / T G 2 27 L·. ώ ds 1 ml ί 2 - jus g Ξ before)! ml 4 3 G 107 p I up to -71 ml 5 C - 1C ίί.: up to 1 ml from 1 - - - YU up to 1 ml L - 1 s £— Z . Z G up to 1 mp 53 5 1C ιί. z. : S: I up to 1 ml 54 1C i ζ . With L·. Ζ up to 71 ml Ts K. G 1C Ξ. Ξ g Ξ up to 1 ml 56 5 1C ώ ί . :ζ ds 1 ml 57 5 67 .· . G up to 71 ml 58 5 67 .· . ABOUT L. Ζ DO<1 ml 59G 5 67 -7 - G up to 1 ml 60 <5; ·: ~ '. G 7^0 up to 1 ml '61- 5 67 7 C b' - z. up to -1 ml 62 5 >2 / 7 ζ — ds 7 ml 63· 5 67 ί. □ f”i·Q DO/1 ml

The stability of the samples was studied using the thermostress method at 50°C for 72 hours, the shake test for 120 hours at a speed of 800 rpm, as well as single freezing at a temperature of minus 16-20°C and thawing at a temperature of +25 ± 1° WITH. The turbidity of solutions was assessed by spectrophotometry at 400 nm. The homogeneity of the samples was analyzed by E-HPLC and electrophoresis on a Labchip device. The profile of the charged forms of the samples was analyzed on a Labchip instrument.

- 33 046787

Table 3

Summary results of quality indicators before and after stress exposure, obtained as a result of analysis by gel filtration and UV spectrophotometry of samples

No. _O1 Forgive me Composition of excipients , mg/ml Containing nieirnshera- 3 Ε3ΖΧ, %·.·. ' 97.54 Change in E HPLC purity, % Optical density, 400: nm Selection for isoform profile analysis IN K k i< And yo S Ϊ b h e ft in Sucrose L-Proline Glycine Η β ®: ft ΰ 11 ο c « 0 I: Poloxamer -8 8 Shaketest: 120 h -5.30 Zamsrszka -15 ³ C, razmsszka +25 ^and C -1.05 Terktstrezs, +53 ^a N 96 h -1.37 Shake- test: 120 h 0.1232 Freezing -13°C, defrosting -25°C 0.0-00 2 5 5: 0.2 97.52 -2.26 -1.33 -1.77 9.1795 0.0735 8 5 5: 0.5 97.54 FDI -1.72 -1.54 0.1703 VINNIE 4 5 50 ύ.3 97.4Ε nnnnn -1.90 -28.94 0.1622 nnnnn 5 5 50 2.2 97.14 0.12 -1.15 0.1852 0.07.52 b 5 50 0.5 97.53 -2.32 -3.22 -1.29 0.1740 0.0723 7 5 50 0.8 97.48 -0.72 -4.45 -1.24 0.2017 IND·· 8 5 5 2-.. z 97.5Οι -2.22 -1.11 -1.50 0.0747 9 5 . 5 - ^; 2-. . 8 0.2 8 8 . 8 5 -0.71 -2.16 -2.75 0.1541 0.0753 10 58 /5: 23.8 0.5 98.12 NIV -2.40 - 3.0 7 0.14 5 6 NNNNN eleven 58 _: D 23.8 0.8 98.23 -2.67 0.1477 And··· 12 D 5 28.8 3.2 97.93 -WITH. 58 -1.30 -2.33 0.1557 0.0797 13 5 _; 5 28. Ξ 0.5 97.90 -0.31 -1. 98 -2.04 0.1545 0.0759 14 5 ;5^ 28.8 ύ. 8 97.95 -2.6£ -2.12 -2.05 0.1440 0.0781 15 5 33. 5 . 5 93.00 IC -1.2-7 -1.39 NNNN nnnny 16 5 33. 5 7.5 0.2 97.94 NNNN -1.42 •ABOUT. 1100 nnnny 17 5 33. 5 7.5 0. 5 97.84 neither -1. 85 0.1035 PINNIE 18 5 33. ·:5: 7.5 0. 8 97.3 -2.08 -2.90 0.1094 19 5 33 5 . 5 8.2 97.3-0 IN· -1.75 -1.22 research institute neither 20 5 33. • ,5: . 8 0.5 97.32 nnnnn -2.44 -1.35 NNNN pnnny 21 5 33. 5: . 5 0.8 97.93 NNNN -2.87 -1.37 0.1115 nnnny 22 5 100 97. 33 -0.69 -0.58 0.С849 0.0716 V 23 5 100 And . ζ 97.90 -0.64 -0.58 -0.32 :.1_85 0.074 /y 24 5 130 0. :· 97.8-3 SBBS NNNNN NNNNN 0.1030 nnnnn V 25 5 1:: η. 97.88 NNNN nnnnn NNNNN 0.1110 NCII V 28 5 100 8.2 97.90 -2.66 -:. 85 -0.32 0.1096 0.0730 V 27 5 1:: 0.5 97.92 -7.5s -0.82 -0.80 0.1124 0.0741 ^; V 23 I yuo 0.8 97.82 -0. 53 -:.=5 -0.5 3 0.1147 0.0740 : ^V 29 5: O: 28. : 97.74 -:..4 6 -0.91 -1.31 /ZhOV® 5.0725 thirty 10 28.8. 0.2- 97.86 -:.5e -1.02 -1.84 0.1399 S.077' 31 • L :1α 28.8 0.5 97.73 NNV· -1.13 -1.72 0.1476 NININ |NIdRR| madmm 32 5 20 28.8 0.8 37.85 IN· -1.17 -1.90 0.1375 PNIN 33 ⁵ < ι: 28. 8 8 . 2 97.80 . <9 -1.14 -1.35 0.1499 S. 07 5Ξ 34 5 ID 28.8 0.5 97.77 -5.58 -1.05 -1.53 0.1450 0.076С 35 A ι: 2 8 . Ε 0.8 97.73 . 52 -1.03 -1.41 0.1409 0.0751 38 5 •G 7.5 97.89 -0.61 -S..5-2 -0.33 0.0725 iv^grmd^^a1 : V 37 '5' S' 7.5 0.2 9 ¹ . 8 8 -0. 86 -· -0.39 0.1006 0.0743 V 33 5 67 7.5 0.5 97.84 Research Institute nnnnn nnnnn -1.22 0.1209 NININ : V 33 ;5 Έ7 7.5 0.3 97.94 NNNN 1- -1.95 0.1057 U ... . 40 5 67 7.5 8.2 97.35 -:.72 -0.73 -0.50 . - 0.0739 V 41 5 57 7.5 0.5 97.81 -:. 51 -3.57 -0.30 0.0369 0.0744 G V 42 5 57 7.5 0.8 93.27 -1.14 -1.04 -0.72 0.0967 UZh-MZhd : V 43 5 ΙΰΟ 97.30 -0 . about -P.31 -ύ.50 ,. 7° 58 '· .-LO- :V 44 :· 100 0.2 97.-3 . 29 -:·.3ί -1.50 0.12.: 0.0792 ² V

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45 5 199 ϋ. 5· 97.7? Research Institute| MINN -- - 3.1327 NO NO· Cv 4 E 5 199 ϋ. 3 97.35 iiiiii opinions BAT 3·. 1233· ninnn V 47 0 199 3.2 97.3ϋ -3.41 -9.44 9.122 ^p 0.9-73 V 4 □ 5 Ιύϋ j, 5 97.34 “E; -<··. 46 -9.42 3'. 1 Li with 1 9.97.92 V 4 9 5 Ιϋύ WITH'. 3 97.3:1 -Z.ZE 3.11 ^g 1 ININ· V 5 3 5 12 13 . Ξ 97 > E ϋ III|I| - C, 9 5 -1,. 4 3 3.3е£5 3.0741 V 51 5 13 23.3 3ι. 2 97.72 -3.54 - ? , 9 Ό -1, 33 0.1364 2.9773 V 52 5 eleven 23. Ξ ύ. 5· 97.7th -1.33 - - 0.1691 INNI| 53 1 = 23.3 ϋ. 3 97.34 n·· -1.97 9.1454 V 54 O 1.3 23.3 3.2 3 3.24 -3.39 -1.35 -1.73 9.1391 3.9763 V 55 O 1 - 23.3 .j. 5 97.33 -3.32 -1.33 £.7 0.1321 9.0-32 V q ς ζ> 12 2Ξ. WITH |J „ 5 97.52 -WITH. 73 -1.27 S i.1302 s.o-eg 7 ^R 7 ; 67 7.5 97.37 -3.61 -S--.4& -0.15 9.9733 V 5 3 5 67 7 5 ΰ. 2 97.37 -3.34 -3.44 -9.39 3.1199 9.9773 v 59 5 67 7.5 Ci. 5- 97. from 5 iinm NNIN| 9. 1129 V 3 3 5 7.5 ϋ.3 97.7 4 nnny nnnny NI|M| 9.1121 pninn· 61 5 67 7.5 C.2 E7.63 I" dmmea 7.16 V 62 5 67 7 5 ϋ. 5 ch7 2 innn at £3 5 67 7.5 and.. 8 97.75 . UJ1 ... ~:- ^L -. G . 7... . -.- .. : about 37 V

best result

- worst result

- input score or average result

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Table 4

Summary results of the study of the acid-base profile of samples before and after stress exposure

» Composition of excipients in 20 mM tistkudshova ••buffer solution with: pH· 5.5, mg/ml Total change: weight of otao-alkaline profile BY MODULE**, % Selection.' . on . lio—· . fil- ny ./drying·. Er olg zlimab Ulnisol Tregalove: D/g Sakharov TO o d, c 1st GlkMCN Polkssrbat thirty Loloxamer 1&S Shaketest 120: h /Daissance -13 ^a C, desensitization: +25° C: Thermostress:: -Ξ1 22: = 122 5 . '24 S.6B ;.·: V 23 5 12 : 2.2 8.81 6.43 36.39 24 : 12 : 2.7 And···· 1.2'2 34.67 25 G 122 2.2 2.22 4.92 3 2.32 26 =· 122 :.O q* $5 . . 7.82 06 61 27 7 12 : Oh 5 4. S3 O1WS6 2'_ V 2 V 5 i; : 2.2 2.22 With. 94 2 3.1- 36: 5 2.5 2 1.32 32.62 y·· 37 5 " .5 2.2 5.34 1.61 32 6 5 38 5 -.5 2.7 ^- . 12 2.22 36.07 39 5 G ~.7 2.2 9. OS 1.90 ::36.83 40: = r :.: 2.2 1.15 41 = r ~.= 2.2 2 V'· 42: 5 ~.7 :. h 11.61 2.95 35.86 43 5 i:? 2. 0.40 36.38 T ₄₄ = 2.2 10.22 2.67 41.24 45 5 i:.' 2.7 : .54 1.72 41.14 46 5 i: / 2.2 6.3 2. 47 5 1'2 :. 2 9.20 2.24 3.24 48: 5 12 2 With eleven 3.43 34. EE 49 5 12 2 2. 2 2. 2 2 3.42 :36.52 50 5 12 1? 2 2.22 1.2 3 :35.62 57 5 ^_ .5 0.98 V· 58 _: 5 ΐ~· ~.7 2.2 10.78 1.68 34.48 59:· 5 , : 2.7 2. 2 2 0.72 60: 5 '.5 2.2 11.31 1.2 3 33.52 61 5 ^_ , Ξ 2.2 12.0 6 1.5“ :34.34 62 5 έ ^_ .7 2.5 2.2 2 0.95 T ^: 63/ 5 6" 2.2 12.06 3.24 34.92

The change was calculated using the formula Δ = (fraction content after stress - fraction content before stress) * Absolute change was calculated using the formula Δ = Kpsl content. fractions before stress - acid content, fractions after stress - alkaline content. fractions before stress - content of cracks, fractions after stress ¹ + 'content of dominant, fractions before stress - content of dominant, fractions after stress^

- best result

- worst result

- iodine value or average result

In table Figure 3 shows the negative effect of mannitol on the thermal and colloidal stability of the monoclonal antibody against PD-1 in a 20 mM histidine buffer solution with pH 5.5: during thermal stress for 96 hours, the increase in impurities on E-HPLC ranged from 1.24 to 3.18 % during a shake test for 120 hours, the solutions acquired visible aggregation. Compositions with mannitol also showed negative stability results during cryoconcentration: after one freeze-thaw cycle, the increase in impurities on E-HPLC ranged from 1.05 to 4.45%, which is much higher than this figure for other compositions.

Formulations containing L-proline also demonstrated low colloidal stability due to the formation of visible aggregation during the shake test. After freezing-thawing and thermal stress tests, the increase in impurities of formulations with L-proline averaged more than 1% on E-HPLC.

The experiment revealed high thermal and colloidal stability of prolgolimab (no significant changes in quality indicators under all stress influences) in formulations with trehalose dihydrate without the presence of solubilizers, such as polysorbate 80 and poloxamer 188.

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The greatest stabilizing effect on the monoclonal antibody against PD-1 in a 20 mM histidine buffer solution is exerted by trehalose dihydrate, sucrose, and their combinations with glycine. These formulations can be used for a lyophilic dosage form of a monoclonal antibody.

Evaluation of the stability of promising histidine-based formulations during freeze drying.

The samples that demonstrated the best stability at the previous screening stage were examined for the possibility of obtaining a lyophilisate for the preparation of a solution for infusion.

For this purpose, solutions containing 20 mg/ml or 100 mg/ml of the monoclonal antibody to PD-1 prolgolimab were poured into hydrolytic class I glass vials; the vials were loosely sealed with a rubber stopper with a slot. Vials with the solution were placed in a freeze-drying chamber. Freeze drying was carried out in automatic mode. The freezing stage was carried out at a temperature of minus 40°C, primary drying was carried out at a pressure of (0.10 ± 0.03) mbar, during secondary drying the temperature was increased, the pressure was (0.05 ± 0.02) mbar. After the drying process was completed, the pressure was reduced to the required vacuum value of 0.76 ± 0.03 mbar, the vials were sealed with rubber stoppers, and then the vacuum was released to atmospheric pressure. The sealed vials were transferred for further research. Lyophilized samples of the anti-PD-1 monoclonal antibody prolgolimab were stored at 2-8°C.

To confirm protein stability, samples were reconstituted after lyophilization. The monomer content was assessed by E-HPLC, and the profile of charged forms was analyzed on a Labchip instrument. The pH value was also determined before and after lyophilization. The results are presented in table. 5.

During freeze-drying of selected histidine-based compositions and after reconstitution of the lyophilisates, a slight pH shift was observed, which was within the error limits of the method. All formulations demonstrated high stability in terms of E-HPLC purity and charged species profile after reduction. However, a noticeable difference in the purity of the samples was demonstrated by gel electrophoresis in the LabChip Caliper system. The most stable samples in terms of this indicator are compositions Nos. 22, 27, 36, containing trehalose dihydrate. Formulations in L-proline also showed a significant change in the profile of charged forms and are therefore not recommended for use. The possibility of lyophilization of formulations No. 22 and 27 with a protein concentration of 100 mg/ml was demonstrated.

Table 5

Summary table of the results of quality indicators of formulations based on a 20-mM histidine buffer solution with pH 5.5 before and after lyophilization

No. Composition of excipients, ig/mp: Yes lif. After lyof. &, (absolute change) wear lyophilization Psolgslkmab Mannitsl Tregalsea d/g and a 1 07 L-Proline Glycine O'Sh; V J U o ϋ To with h—I.. to her- S o i k pH: E HPLC pH: E HPLC pH E BELC Changing fraction content, LabChip Clean _f % Purity, : %; ACID, fractions * _R % faction dominant ★, % hel. factions* Total change in acid:-x-alkali in psophila modulo” * J. . . % Protein concentration in solution 20<mg/ml 22 20 1С0 5 JSC 98 87 5.66 99.00 GB And- +0.24 -0.23? -0.03 Μ··· 27 20 ICO 0.5 5.42 98.86 5.64 98.97 · WITH.' mmi +2.55 -0.47 :-2.07 pl h; 20 67 7.5 5.60 98.89 5.65 98.96 <: NIN + 1 32 -1.19 -0.14 IMNNN 20 67 7.5 0.5 5.45 98.89 5.64: 98.98 0.19 + 1 2S +0.30: -0.85 2-1-' - 20 100 5.38 98.87 5.65 99.02 0.2? nnn· +2 03 -1.71 -0.31 4.06 57 20 67 7.5 5.42 98.88 5.62 99.02 -4.11 +3.06 +1.05 8.22 62 20 67 7.5 0.5 5.40 98.87 5.73, 98.99 0.33 +2 39 :-2.38: 0 8 20 /5· 28.S 5.71 98 8ΐ 5.70 98.48 /Safe -0.39 +5.12 -3.88 -1.23 10 23 29' 20: 10 28.8 5.43: 98.88 5.90 98.95 0 47 and··· -6 67 ,-2.75: -3.92 13 34 507 20 10 28.8 5.50 98 89 5.76 98.94 0.26 ININ + 1 74 +0tZZ: :-2.12 4.24 Concentration of oelok in solution 100 mg 'ml 22 100 100 5.44 99 13 : 5.61 98.81 0.17 -0.22,· +1.46 :-0.45: -0.03 1.94 27: 100 100 0.5 5.44 99.05: 5.75 98.76 0.31 -0.16 +1.47 -0.80 -0.68 2.95

* The change was calculated using the formula Δ = (the content of the fraction after stress - the content of the fraction before stress) * * The absolute change was calculated using the formula Δ = (the content of acidic fractions before stress - the content of acidic fractions after stress] + [the content of alkalis, fractions before stress — content of al. fractions after stress] + | content of dominant fraction before stress — content of dominant fraction after stress] — best result — worst result — input control/average result |

Obtaining a highly concentrated form and confirming stability during accelerated storage.

Based on the results of screening in liquid and lyophilic dosage forms, the following formulations were selected for accelerated stability studies:

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Prolgolimab 20 - 150 mg/ml Prolgolimab 20 - 150 mg/ml P-histidine 0.92 mg/ml P-histidine 0.92 mg/ml L-histidine h/x m/g 2.96 mg/ml L-histidine h/x m/g 2.96 mg/ml Trehalose dihydrate 100 mg/ml Trehalose dihydrate 80 mg/ml Water for injections up to 1 ml Water for injections up to 1 ml

The content of trehalose dihydrate was reduced to equalize the osmolality of solutions with increased concentrations of monoclonal antibody against PD-1 to physiological levels (about 300 mOsm/kg), as well as to reduce the viscosity of solutions to less than 100 cP. The stability of the samples was confirmed by accelerated storage at +37°C, the analysis was carried out by E HPLC, IO HPLC and VEF, and the relative specific activity of prolgolimab was determined. The results are presented in table. 6 and 7.

Table 6

Results of the stability study ri 37°C Compound Protein concentration Index Input control 2. not l ;4;;n.ed; Prollozhab 20 - 150 mg/ml; ;L-histidine·· 0.92 mg/m* 'E-histijyau g/x m/g 2.96 mg/mi 1 rehalose citytigrat 100 mg/mp 'SO-'-'W/mk' ' Sbci protein _f mg/ml 19.9 20,.-1 20.0 100 mg/ml;; 101.X 100.6 ;101.9; 1'50 mg/ml; 14918 /151.3 150.4 2 0: mg/ml ' : pH; 5.5 5.5 5.5 100 mg/ml 5.6 /5.6; 5.6 1'50 mg/ml;; 5.6 5.6 --5/.7 '20;;mg/ml· VISCOSITY:, cF IN·· - - 100 mg/ml 40 - - 150 mg/ml: '122';· - - Purity S E3ZX 20 mg/ml; Aggregate content,% 0.40 0.67 0.21 Monomer content, % '99 _: .'.ts 92.65 92.32 100 mg/ml:; ; Content;-/a.-1’p=egatoB;,;;-% 0.57 0.97 I. 23 Monomer content, % 99.22 95.39 97.21 15C mg/ml Aggregate content, % 0.51 :1.11/ 1.75 Monomer content, % 99.19 97.92 97.09 20 mg/ml ;VEF; Harm·. Terms ; Monomer content, % 95.33/ 94.27 94.06 '100 mg/ml' . 95.27 94.79 /93:.:93-- 150 mg/ml 95.40 94.61 93.90 20 mg/?sh ZEF b before, conditions Monomer content, %; 93.36 91.96 ^/91:.14/, ......100 mg'/ml; '93.28/' 91.79 90.89 150 mg/ml: 93.21., 91.64 90.52 Profile of charged forms ,ΗΟ Ξ3ΙΧ) ΙΒ'/μγ/μπ·' Acid fraction, % 16.72 25.22 31.03 1 Domshir... ;;|ra:ktsi i ,/.;%' ' 50.27 54.07 56.11 Alkaline fraction, % 32.41 20.71 12.26 100 mg/ml Acid fraction, % 17.00 25.95 32.04 Dominant ·..fraction, % 50.66 /53.91/ 55.26 Alkaline fraction, % 32.34 20.14 12.10 150 mg/ml·. Acid fraction, % 16.95 26.09 ;32.19; . Compound' Protein concentration Index : Input.; control; ;2'';ed; 4 weeks Dominant Thrace,% 49;.-87' 54.36 56.11 Alkaline fraction, % .33.18;' /19.55/ c.<70; 2 C mg/ml Relative/specific activity, % 105 97 11'0·; 100 mg/ml;. 101 104' 97' ' ' 150 mg/ml .' ::97- 100 105

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Table 7

Results of stability study at 37°C

Compound Protein concentration Index Incoming control 2 no 4weeks Prolgolimab 20 150 mg/ml L-histidine 0.92 i/ml L-histidine: g/x m/g 2.96 mg/ml Trehalose ditidate Ξ0 mg/ml 2 0 mg/ml White life span: mg/ml: 20.4 20.2 2D.S 100 mg/ml 100.3 102.8 101.2 150 mg/ml /152.2 1507 153.0 20 mg/ml pH :5.4 <55 5.6- 100 mg/ml 5.5: 55 5.6/ 150 mg/ml 5.6 55 5.7 20 mg/ml Viscosity, cP -5 ..... - - 100 mg/ml 2 in - - 150 mg/ml 96/ - - Purity E VYAH 20 mg/ml Contents of aggregates, 1 0.41 0.46 073 Monomer content, 1 99.09 98.72 98.30 100 mg/ml Aggregate content, % 0.55 0.98 1.10 • Content/!®measure, A 99.26 98.60 9'79 150 mg/ml Aggregate content, :;%< 0.60 0.92 1.26 Monomer content, 1 99.27 98.16 97.55 20 mg/ml VEF under ed. conditions Monomer content, i 95.45 9479 94.15 100 mg/ml 95.59 94.52 93.SS 150 mg/ml 95.26 94.40 93.76 20 mg/ml VE4 in edition conditions Monomer content, % 93.18 91.82 91.10 -100 mg/ml 93.04 91.62 9S77 150 mg/ml 93.00 9L25 9С.28 Profile zvdyazhen1M/;fprm/ ^: (ZHVEZH) 20 mg/ml Acid fraction, 1 15.3 V 24.11 30.66 Domgair. fraction, Ϊ 51.81 55.09 56.31 Alkaline fraction, Ϊ 32.81 2C.S0 13.03 100 mg/ml .whole faction, * 16.66 24.91 31.64 Dozhnir. fraction, % 50.65 53.05 :56.17- Alkaline fraction, % ........... :32.-69 22.04 12.19 150 mg/ml fox faction, * :17-.17: 25.36 3252: Dominant fraction, Ϊ 50.34 54.06 57.81 Alkaline fraction, ί 32.49 2C.5S 9.6 20 mg/ml Relative specific activity: % 100 '103 94 100 mg/ml 89..... 94 105 150 mg/ml 91 99; 108

Example 17. Selection of pharmaceutical composition based on acetate.

Compositions under study.

To screen a stable pharmaceutical composition based on a 20 mM acetate buffer solution with pH 5.0, the following excipients were taken: mannitol, trehalose dihydrate, sucrose (osmotic agents), L-proline (osmotic agent and stabilizer), glycine (osmotic agent and stabilizer ), polysorbate 80 and poloxamer P188 (solubilizers). All tested solutions are isotonic.

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Compositions studied (per 1 ml)

V w R O h R O $ Stabilizers 20 mM.- YceC pH 5.5 Osmotic agents / Solubilizers Mannits-l Trehalose d/g "in p D&Y K at 70 L-Prodin 1 Polysorbate 80 Poloxamer 188 1 5 50 Before 1: south 2 5 50 0.5 Before 1 No.1 3 5 50 0.5 Before 1 ml: 4 5 5 28.8 before 1 mp:· 5/ 5 5 28.8 0.5 ...... before 1: ML: b 5 5 28.8 0.5 Before 1 ml: 7' 5 33.5 7.5 Before 1: ml 8 5 33.5 7.5 0.5 before: 1: mp/ 9 5 33.5 7.5 0.5 BEFORE 1: mp/ 10 5 100 BEFORE 1 ml AND 5 100 0.5 before: 1 ml/ ..... 12 5 100 0.5 BEFORE: 1 ml 13 5 1C 28.8 BEFORE· 1: mp: 14 5 10 28.8 0.5 :BEFORE: 1 MP: 15 5 1G 28.8 0.5 before 1 ml: 16 5 67 7.5 Before 1 MP: 17 5 5 7.5 0.5 before 1: ml 18 5 67 7.5 0.5 Before 1 ml 19 5 100 /Before 1 ml/ 20 5 100 0.5 Before 1 ml ' 21 5 100 0.5 BEFORE 1' ILO:/ 22 5 57 7.5 Before 1: ml 23 5 With ^- 7.5 0.5 BEFORE 1 ^: ml 24 5 67 7.5 0.5 BEFORE 1 ML::

The stability of the samples was studied using the thermostress method at 50°C for 72 hours, the shake test for 120 hours at a speed of 800 rpm, as well as single freezing at minus 16-20°C and thawing at a temperature of +25 ± 1°C. The turbidity of solutions was assessed by spectrophotometry at 400 nm. The homogeneity of the samples was analyzed by E-HPLC and electrophoresis on a Labchip device. The profile of the charged forms of the samples was analyzed on a Labchip instrument.

- 40 046787 gel" in

Table 8

» Composition of excipients, MG/ML< Monomer content E VEEX, % Change in HPLC E purity, % Sltich. density, 4СО nm Selection for acid-base profile analysis Prolgolimab Mannitol Trehalose d/g Sucrose L-PRELII | Glycine | OU: ΟΪ X in < w from (F (F S'·R O HEDGEHOG Kolliphor isа Paketest; 123 h Freezing -13 ^s C, defrosting +2 = ^s C Therms-stress, +50 ³ S 96 h Eyktest/ 12 3 hours Freezing -13 ^s C, defrosting +25 ^s C 1 5 50 97.71 -1.63 -2.48 0.0’49 O.C''S 2 5 50 0.5 97.66 -P.35 -3.19 -1.66 S.0749 0.0’51 3 5 50 0.5 97.54 -S.S7 -9.12 -1.25 C.C6SS 0.0’21 4 5 5 28.8 97.56 -N.NW -2.19 0.0809 0.0’97 5 5 5 2S.S 0.5 97.74 -P.36 -1.15 -2.59 S.O32O 0.0804 b 5 5 2S.S 0.5 97.66 -P.28 -1.Ό ' ' 0.0773 ' 0.0'58 AND 5 33.5 5 97.58 -С-.С5 -1.15 -1.54 0.0745 0.0777 8 5 33.5 0.5 97.52 -0.21 -2.84 -2.08 0.0’96 O.S^'b 9 5 33.5 ' 5 0.5 / 97.58 -S.S9 -9.44 -1.59 0.0’33 0.0831 10 5 ICO ^ς?63 -S.;9. .. . .. -2.17 . .. 0.0744.. . .0.076’ .. and AND W luj 0.5 97.60 -0.19 -0.48 -?zs 0.0778 0.0801 AND 12 5 ICO 0.5 97.52 -0.10 -0.22 -3.10 0.0’25 0.0'33· .......V· 13 5 10 2S..S 97.48 -P.17 -0.38 -1.54 0.C7S1 0.0802 14 5 10 28.8 0.5 | 97.55 . -P.12 . gO., -1 . . -1.93 . 0.0797. . 0.0319. 15 AND 10 28.8 1°' ⁵ 97.64 -P.26 -0.98 -1.91 0.0756 0.0^2 16 5 6' AND 1 97.71 -0.36 -0.45 -P.32 0.0754 0.0765 V G 5 6' 7.5 0.5 ..... 97.57 -0.31 -0.45 -0.53 0.3'76 0.SZh5 .......V...... 1S 5 6' '5 0.5 97.68 -0.36 -338 0.0’25 0.0’32 1 19 5 ICO 97.56 -P.26 -0.34 -0.31 S.S’92 0.0819 V 20 ⁵ 1 ICO 0.5 97.53 -032 -3.05 0.0313 0.0821 V: 21 5 ICO °' ⁵ 97.50 -P.29 -038 -3.34 C.C7S3 0.0767 V 22 5 b 5 97.50 -P.27 -035 -0.0s 0.0’37 0.08С’ .V 23 5 b ^- .5 0.5 97.43 -O.-SS -0:22- 0.0312 V 24 5 63 7.5 0.5 97.50 AI -0Zh -0.01 0.0751 0.0’61 V

- best result

- worst result

- exemplary control or average result

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Table 9

Results of acid-base profile of samples ._________

and Composition of excipients, ig/ni'·· Total change in acid-base profile to module**, % Selection for lyophile N’/K drying Prolgolimab Mannitol Trehalose d/g ίη o < a "· X Y ABOUT' F X a·k Glycine 0 0 hefassitod 83T hschitttoya Shaketest'· 120 h Zayorizka -19°С _G sizes. +2 5 °C Sermostress, +50 °C •96 F ι: 5 100 5.16 1.4S 29 28 V And 5 100 C.5 5.53 1.52. 33.76 G 12 5 100 0 5 IIIMsh 1.91 33.89' V 15 5 G 7.5 b 59 1.09 33.47 1' 5 5' '.2 0.5 5.S1 O.3 34.7? 18 2 5' 7.2 ?: - ·.: 34.95 ι; 2 100 5.33 1.DS 33.48 V •20 180 0.5 6.92 0.93 34.2'8 21 •5· 100 0.2 b.Z 1.02 34.06 V- 222·· /5 6' 5.94 2.46 34.38 23 5· -.5 0.5 ddd I. ^- , 33.63 /24·· ζ" 7.5 0.2 5+6 1.37 33.60 V

* The change was calculated using the formula Δ= (the content of the fraction after stress - the content of the fraction before stress) ** The absolute change was calculated using the formula D = | acid content fractions before stress - acid content. fractions after stress] + [content of cracks, fractions before stress - content of cracks. fractions of stress] + |contents dzminir. fractions before stress - content dominant. fractions after stress]

- best result

- worst result

- early control or average result

As a result of the work, the negative effect of mannitol on the thermal and colloidal stability of the monoclonal antibody against PD-1 in a 20 mM acetate buffer solution with pH 5.0 was shown: during thermal stress for 96 hours, the increase in impurities on E HPLC ranged from 0.48 to 2, 59%; during a shake test for 120 hours, all tested solutions did not acquire visible aggregation (see UV spectrophotometry results). Compositions with mannitol also showed negative stability results during cryoconcentration: after one freeze-thaw cycle, the increase in impurities on E-HPLC ranged from 0.69 to 9.44%, which is much higher than this figure for other compositions.

Compositions containing L-proline also demonstrated low thermal stability in terms of purity when studied by E-HPLC: the increase in impurities after 96 hours of stress was 1.64-1.93%. After freezing-thawing, the increase in impurities in formulations with L-proline did not exceed the average value of the most stable formulations in the group. The experiment did not reveal the effect of solubilizers on the thermal or colloidal stability of the protein. Prolgolimab in formulations with trehalose dihydrate showed high stability under all stress conditions in the absence of surfactants such as polysorbate 80 and poloxamer 188.

The greatest stabilizing effect on the monoclonal antibody against PD-1 in a 20 mM acetate buffer solution is exerted by trehalose dihydrate, sucrose, and their combinations with glycine. These compositions can be used to prepare a lyophilic dosage form of a monoclonal antibody.

Determination of the stability of promising acetate-based compositions during freeze drying.

The samples that demonstrated the best stability at the previous screening stage were examined for the possibility of obtaining a lyophilisate for the preparation of a solution for infusion.

For this purpose, solutions containing 20 mg/ml or 100 mg/ml of monoclonal antibody against PD-1 were poured into glass vials of hydrolytic class I; the vials were loosely sealed with a rubber stopper with a slot. Vials with the solution were placed in a freeze-drying chamber. Freeze drying was carried out in automatic mode. The freezing stage was carried out at a temperature of minus 40°C, primary drying was carried out at a pressure of (0.10 ± 0.03) mbar, during secondary drying the temperature was increased, the pressure was (0.05 ± 0.02) mbar. After the drying process was completed, the pressure was reduced to the required vacuum value of 0.76 ± 0.03 mbar, the vials were sealed with rubber stoppers, and then the vacuum was released to atmospheric pressure. The sealed vials were transferred for further research. Lyophilized anti-PD-1 monoclonal antibody samples were stored at 2-8°C.

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To confirm protein stability, samples were reconstituted after lyophilization. The monomer content was assessed by E-HPLC, and the profile of charged forms was analyzed on a Labchip instrument. The pH value was also determined before and after lyophilization. The results are presented in table. 8.

During freeze-drying of selected acetate-based formulations and after reconstitution of the lyophilisates, a significant pH shift was observed in the range of 0.26-0.45. This shift may be due to the loss of acetate ions during the drying process. Thus, acetate-based compositions can be recommended for freeze-drying, provided that this phenomenon is taken into account.

All formulations demonstrated high stability in terms of E-HPLC purity and charged species profile after reduction. During the work, the possibility of lyophilization of compositions No. 10 and 12 with a protein concentration of 100 mg/ml was demonstrated.

Table 10

Results of the stability of formulations based on a 20-mM acetate buffer solution with pH 5.0 after lyophilization

» Composition of excipients, mg/ml Before liof. After lyof. Δ, [absolute change) after lyophilization Proltolimab S' in 1 x * Trehalose d/g Sucrose si 3 Polysorbate Sj Polzisaier 1== pH E HPLC pH E HPLC pH E HPLC Changing fraction content, LabChip Cleanliness, ΐ Purity, % ?fi; clean, 1 L У7.2~. factions *, % . dstnir Δ gap = FRANCE *, % Total change in acid-base profile by modulus**, ?%g £shares *, %;) Protein concentration in solution 2C mg/ml 10 2C '00 4.99 98.91 5.44 99.05 1.66; 0.90; :-2.56 10' 20 100 ° ² 5.02 98.91 5.38 98/97 2.07 -159 -048 eleven? 20 ::: 0.5 5.00 98.91 5.35 98.97 w -2.68 -0.35 1(G 20 ΐϋΟ ¹ 0.8 5.02 98.91 5.39 98.92 1-91? ;-1.73? -0.18 .......o ................Ί 12 ^: 20 '.TO· 0.2 5.05 98.93 5.34 98.95 <29| 1.44? )-0.42) -1.01 12 20 ΐϋο 0.5 5.02 99.01 5.28 98.94 -0.26 J.Q. 1.80? -0.86; -0.94 12 ^,: 20 iOO 0.8 5.01 99.19 5.43 98.96 1.91; -1.0.2) :-0.88 19 20; 5.00 99.19 5.37 98.9S 2.37) -1.90 7-047 21? 20 ::: 0.5 5.01 VD? 5.33 98.95 2.45 -1.56 -0.89 24 ^; 20 67 K.5 0.5 5.04 99/22? 5 35· 9902 3:09 -2.14 -0.95 618 Conc protein concentration in solution 100 mg/ml 10? 100 100; 5.10 99.10 5.55 98.75 -0.45 -0.34 2.17; )-1.44/ ? -0/73/ 4.34 12) 100 100 0.2 5.06 99.05 5.41 98.76; -0.35 -0.35 1.04 -1.68 -1.36 4 08

“The change was calculated using the formula Δ= (content of the fraction after stress - content of the fraction before stress) **. The absolute change was calculated using the formula D = (content of acidic fractions before stress - content of sol:sl. fractions after stress] t [alkaline content. fractions before stress - content of fractions after stress] + [content of lominir, fractions before stress - content of fraction after stress]

- best result·?·

- worst result]

- input control or average result

Obtaining a highly concentrated form and confirming stability during accelerated storage.

Based on the results of screening in liquid and lyophilic dosage forms, the following compositions were selected for accelerated stability studies:___________________________

About l golim b 20 - 150 w/nl Preloslicab 20 - 150 mg/ml Sodium acetate t/g 1.712 mg/ml Sodium acetate t/g 1.71.2 mg/ml Acetic acid, ice. up to pH 5.0 Acetic acid, ice. up to pH 5.0 Trehalose dahydrate ICC mg/ml Trehalose dihydrate ^0 mg/ml Water for injection up to 1 ml Water for injections up to 1 ml

The content of trehalose dihydrate was reduced to equalize the osmolality of solutions with increased concentrations of monoclonal antibody against PD-1 to physiological levels (about 300 mOsm/kg), as well as to reduce the viscosity of solutions to less than 100 cP. The samples were stored at +37°C and analyzed by E HPLC, IO HPLC and VEF methods, and the specific activity of the protein was determined. The results are presented in table. 9 and 10.

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Table 11

Results of stability study at 37°C

Compound Protein concentration: Index Incoming control 2<week 4 weeks · Prolgolimab· /20/ - <150 mg/ml Sodium acetate t/t 1.7 42 mt/ml Acetic acid, ice. up to pH 5.0 Trehalose dihydrate 100 mg/ml 20 mg/mp Total protein, mg/ml 21.5 /20.85 20.89/ 100 mg/mp 102.2 102.9 10 0.3 150 mg/m 152.1 150.0 151.6 <2 0 <f/mp< pH 5.2 /5-.1 5.2 10 0 mg/mp 5.3 <5<.<3 5.3 . 150 mg/mp 5.4 5.4 5.4 2 0 mg/mp· Viscosity, - сР< 6 - - 100 mg/mp 33 - < 150 mg/mp 110 - - Purity E HPLC 20 mg/mp Content • units, % 0.34 -0:.41: 0.68 Monomer content, :% 99.17 98.85 98.39 100 kg/ml Content of aggregates, :%< 0.57 0.99 1.03 Content- Monomer, Chz< 99.25 98.64 <97.8 8 15·0· mg/mp Content units, %:< 0.64 0.99 1.35 Content:: ...... Monomer, %<< 99.20 93.08 97.64 <20- ^: mg/mp· VEF <g<< ed. conditions Monomer content, %< <95.45 94.23 94.01 100 yg/ml /95129 94.60 94.12 150 mg/ml /95.60 94.15 93.79 2 0 mg/mp VEF · in< < int. conditions 93.05 91.40 91.14 100 mg/ml: <93.22 /91.05 90.95/ Compound Protein concentration Index Input control·. 2 < week <4 weeks/ 150 mg/mp •Monomer content, % /93.79 90.87/ 90.22 Charged Species Profile (HPLC) 2 0 mg/mp Acid: fraction, % <15.63 /24.15 /29.32 Dominant. fraction, % 50.81 55.96 58.03 Alkaline fraction: % 33.56 19.89 12.65 100 mg/ml Acid fraction, <%' /16.02 23.76 /30.44 Dominant. fraction,:%' 51.01 55.33 <57.57 Alkaline fraction, % 32.97 <20.91/ 11.99 150 mg/mp Acid fraction, Ϊ 16.31 24.4 8 33.23 Dominir. fraction ,<% 53.99 54.07 57.51 Alkaline fraction, % <32.7 0 21.45 <9.<26 2 0 mg/mp Relative specific activity, % 8 9 93/ •99 100 mg/ml 107 <112 <97< 150 mt/my: ..... 92 106 103

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Table 12

Results of stability study at 37°C

Compound Protein concentration Index Input control 2 weeks 4 weeks Prolgolimab 20 - 150 mg/ml Sodium acetate t/g 1.742 mg/ml Acetic acid ice. up to pH 5.0 Trehalose dihydrate 80 mg/ml 20 mg/ml Total protein, .mg/ml 1-9.9 20.4 20.5 100 mg/ml 103..5 102.1 102.0 150 mg/ml 150..2 153.8 153.0 20 mg/ml pH 5.1 5.2 5.2 100 mg/ml 5.3 5.2 5.3 150 mg/ml 5.4 5.4 5.4 20 mg/ml Viscosity, cP 5 100 mg/ml- 26 - - 150 mg/ml 93 - - Purity E HPLC 20 mg/ml Aggregate content, % 0.49 0.79 0.98 Monomer content, % 99.22 98.85 98.47 100 mg/ml Aggregate content, % 0.52 1.09 1.23 Content .monomer, % 99.04 98.36 97.72 150 mg/ml Aggregate content, % 0.64 1.15 1.55

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Content msnszhra, ·ΐ /99.00 97.80 97. 14 20 mg/ml BEF and ed. conditions Monoger content, % 95.47 94 South Ossetia 94. 16 103 mg/ml 95.47 94.81 93.91 151 mg/ml 95.47 94. 63 93.94 20 mg/ml VEF in: vered, conditions'; Monomer content, % 93.20 91.09 91.14 103 mg/ml 93.20/ 91.35/ 90.16 150 mg/ml 93.20 91.3 0 90.97 She asked to dress up: -: яь:х germ ΪΖ053ί·.Χ; 20 mg/ml Sour fraction, % 100 23.75 28.29 Dominant fraction,% 49.37 54.86 5У32 Alkaline fraction, 9 32.43 21.39 14.40 101 μγ/ι-l Acid fraction, % 16.66 2-.. 91 29.30 Dominant fraction,% 50.65 53.15 57.31 Alkaline fraction, % 32.69 22.04 13.01 150 mg/ml Acidic: fraction, ϊ· ' 16.81 24.11 31.52 Dominant fraction,% 5 0.34 54. 39 57.31 Alkaline fraction, '%- 32.3 5 21.8· 10.67 20 mg/ml Relative specific active with a * 130 133 94 101 mg/ml 89 94 105 153 mg/ml 91 99 108

While the invention has been described with reference to preferred embodiments, it should be understood that changes and modifications may be made as will be apparent to those skilled in the art. Such changes and modifications should be considered within the scope and scope of the present invention.

Illustrative, non-limiting examples of implementation of the present invention have been described in detail above. This detailed description is provided merely to provide one skilled in the art with additional details for the practice of preferred aspects of the present teachings, and is not intended to limit the scope of the present invention. In addition, each of the additional features and ideas disclosed above and below may be used individually or in combination with other features or ideas.

Moreover, the combinations of features and steps disclosed in the detailed description below, as well as the experimental examples, may not be necessary for the practice of the invention in the broadest sense, and are provided only to describe in detail specific examples for the present invention. Moreover, the features of the specific examples described above and the independent and dependent claims set forth herein may be combined in ways not specifically and directly indicated to realize additional useful embodiments of the present invention.

Additional case studies have been conducted using various aqueous pharmaceutical compositions containing the anti-PD-1 antibody prolgolimab for the treatment of malignancies such as melanoma, including unresectable or metastatic melanoma, early stage melanoma before and after radical treatment, non-small cell lung cancer lung cancer (NSCLC), including unresectable or metastatic non-small cell lung cancer. Typical pharmaceutical compositions used for these studies are characterized by the following compositions below (Table 12.1).

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Table 12.1

I. Composition per 1 ml: Prolgolimab

Sodium acetate trihydrate Trehalose dihydrate Acetic acid ice. Water for injections

20.0 mg

1.742 mg

100 mg to pH 5.0 to 1 ml.

II. Composition per 1 ml: Prolgolimab Trehalose dihydrate L-histidine

L-histidine hydrochloride Water for injection

20.0 mg

100 mg

0.92 mg

2.96 mg to 1 ml.

Basic information about the clinical development of the investigational drug.

The pharmacokinetics, pharmacodynamics, safety and immunogenicity of BCD-100 (prolgolimab) as monotherapy when administered intravenously were studied in a phase I dose-escalation clinical trial. This study demonstrated the safety and benefits of the study product in patients with advanced forms of malignant neoplasms of various locations (melanoma, NSCLC) and, based on the results of the study, two modes of administration of BCD-100 were selected for further clinical development, 3 mg/kg IV once a day 3 weeks and 1 mg/kg IV once every 2 weeks.

The objective of Study No. BCD-100-2/MIRACULUM of BCD-100 in patients with unresectable or metastatic melanoma was to evaluate the pharmacokinetics, efficacy, safety and immunogenicity of monotherapy in two administration regimens: 3 mg/kg IV once every 3 weeks and 1 mg/kg IV once every 2 weeks. The primary endpoint (ORR) was assessed at the interim analysis at 6 months. BCD-100 demonstrated a favorable safety profile across all dose regimens. Both dose regimens demonstrated efficacy of approximately 60% CR and 30% ORR in patients with unresectable or metastatic melanoma.

Example 18. Use of prolgolimab (BCD-100) for the treatment of patients with advanced forms of malignant neoplasms of various locations, phase I study, BCD-100-1.

Study design.

Study No. BCD-100-1 examined the pharmacokinetics and tolerability of BCD100 (prolgolimab). Study No. BCD-100-1 (NCT03050047) was a multicenter, open-label, phase I study in patients with solid tumors conducted in the Russian Federation. The primary objective of the study was to evaluate the pharmacokinetics and clinical pharmacological parameters of BCD-100. The main characteristics of the BCD-100-1 study are shown in Table. 1.

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Table 13

Characteristics, research BCD-100-1

Multicenter, open, non-comparative/multi-cohort study of pharmacokinetic and pharmacodynamic properties, safe-: titanic and immunogenic: properties of the drug BCD-too (ZAO BIOCAD, Russia):(!/ •phase): in patients with recurrent diseases forms of ^: malignant neoplasms of various types: localizations: BCD-100-1 Phase I (Russian •Federation) PK, PD, tolerability, safety: in, -pilot: effectiveness— ESSENCE 1' immunogenicity Study of pharmacokinetic, pharmacodan: mic properties, ^safety but the axis and immunogenicity of the drug: BCD-100: (prolgeitima b) with intravenous administration at an increasing dose in ionotherapy: 15 partners personal and types of unstable people :malignant neoplasms: /b Patients in: cohort 1: bw/kg, 6 patients in the 3 mg/kg cohort, and: 3 patients in: 10 mg/kg cohort Multicentre: /three open-label multicohort study with escalation: doses: (phase I) /3 months · BCD-100 at a dose of 0.3 mg/kg IV every /2: weeks, Erez: F: weeks (two: administrations)/ escala gdaya: up to up to 1 mg/kg BCD-100 at a dose of 1: mg/kg IV every: 2: weeks (Q2W) /in/ for 85 days (main period: studies, 6 injections)/ BCD-100 3 mg/kg IV every 2 weeks (Q2W) for: 85 days Pharmacoische tika: AP^ i o-CMj ,: AUCro—·. , Sshkh, _Tsh , Tch, Kei, 01, Vd after a single use of the study drug; /Ciin. during: •b use of the investigational drug Farmaxdin mika: % -employment : receptors. PD-1 : with BCD-103 after 4 hours -/after /first/ administration, 336 hours after /first: introductions (but until next dose} , and: before: 6th administration^ Inmuogenic with ^b: ^CAT detection rate, NAG/

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• (OSIOENO “period.: research, 6 introductions BCD-100 at a dose of 10 mt/kg IV: every 2 weeks -(121/- for B5 days (-OCH0EH0·· th study period·: ania, € -administrations': J..... Safety b: Frequency of occurrence and severity associated. techniques :resulting from the :drug.:: adverse events (AEs) based on the evaluation of the 4 Data and Safety Monitoring Commission Effective: : Pilot evaluation of antitumor activity of BCD-100 based on CT results (RECIST 1.1 and irRC criteria)

The first patient was prescribed a starting dose of BCD-100 - 0.3 mg/kg every two weeks. In the absence of dose-limiting toxicity within 4 weeks, the patient's dose was increased to 1 mg/kg every two weeks. After the first patient, the study continued according to the classical design with dose escalation 3+3, i.e. in the absence of EBRT, a cohort of 3 patients was sequentially enrolled into the study every 4 weeks at the next dose level. At each dose level, BCD-100 was administered for 85 days (about 3 months) or until signs of DLT or disease progression appeared.

If DLT events at the current dose level were observed in one patient, the next cohort of three patients received the drug at the same level, that is, a total of six patients began receiving a given dose of BCD-100. If DLT events were observed in two or more patients at the same dose level, dose titration and administration of the drug to new patients were stopped.

The duration of follow-up for patients after therapy with BCD-100 was 126 days. During this period, monitoring of adverse events, medical examinations, and blood tests were carried out (once every 2 weeks - the first 28 days, then once every 28 days).

Research results.

Summary of patient population characteristics.

The study included 15 patients with common forms of malignant solid neoplasms of various locations (melanoma, including choroidal melanoma, NSCLC, renal cell cancer, mesothelioma), aged 18 years and older, of both sexes. To be included in the study, patients had to have an ECOG score of 0-2 and at least one measurable target lesion according to RECIST 1.1 (not including bone metastases). A description of the disease characteristics of the patients and the distribution of patients is presented in Table. 14, 15.

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Table 14

Disease Characteristics of the Patient Population (BCD-100-1 Study)

Index All patients BCD-100 1 mg/kg BCD-100 3 mg/kg BCD-100 10 mg/kg Meaning P (n = 15) (n=6) (n=6) (n=3) P % n 6 π % n ZZiagnsv Melanoma 9 60.00 '4/ 66.67 3 5C 2 66, 67 NSCLC 4· 26, 67 /;i; 16, 67 2 33.33 L;; S3.33 ;;1: Renal cell carcinoma 1 6, 67 0 0 1; 16.67 07 0.03 71; Yez o te:pi oka 1 6.67 Έ /16.-6 7 0 : 3 :.73 /1; •Stage III 1 2 8, 6 72; S3, 33 1 16, 87 1 33.33 ;/i; IV eleven "3.33 /4/ 68.67 : 8 3.33 ^? 2·· 66.67 ;;;1; .......... ..previous treatment X^zurria- 10 66.67 74; 66/67 /4/ 66.67 2 66.67 71; Radiation therapy 2 13.33 ;;1; 16, 67 /1/ 16.67 0 3.7 3 ;/i; Other therapy ² ΐ 4 8, 67 ;;3·; EC, 3-C /2/ 33.33 ^; 27 86, 87 3.6-4 Chemotherapy for inoperable conditions 10 88, 6 5 83.33 /3- SC, 7-3 2 66, 67 3,775 One line 6 4C, jC /2/ S3, S3 2/ 33.33 2 66.67 3, 6 4 C Pae or more •LINES 4 2 6, 67 73; EC, 3-C 71·; 16, 67 0 0 3, 44C Duration of illness । [bear] ....... Fresher meaning 26 _r 31 28, 13 2C, 83 38.38 1.683 Median 17.07 11, E.C. 8.2 3 2C, 13 Minimum -, ? 2 _f j7 , 6 7 17.07 Maximum 31.2C "2.77 91.2 s ^7.93 NW 31.31 2 3.28 85.21 34.2 3 Note: ....... - comparison of military cohorts was carried out based on the two-sided exact Feeder criterion ² sex “other therapy” implies the use of such agents as interferon, targeted drugs, anticancer vaccines

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Table 15

Patient distribution (BCD-100-1 study)

Patient Cohort Dose of BCD-100 (mg/kg) Number of injections Further participation eleven 0.3-1 6 According to the protocol 08-02 1 1 6 In accordance with the protocol 10-03 1 1 5 Dropped out due to disease progression 08-04 1 1 6 According to the protocol 12-05 1 1 5 Dropped out due to disease progression 12-06 1 1 6 In accordance with the protocol 12-07 2 3 6 According to the protocol 12-08 2 3 6 According to the protocol 13-09 2 3 6 According to the protocol 13-10 2 3 3 Dropped out due to SAE 13-11 2 3 6 According to the protocol 01-12 2 3 € According to the protocol 13-13 3 10 6 According to the protocol 13-14 3 10 6 According to the protocol 08-15 3 10 5 Dropped out due to disease progression

The final analysis included data from 15 patients (6 patients from the BCD-100 1 mg/kg cohort, including 1 patient after dose escalation, 6 patients from the BCD-100 3 mg/kg cohort, and 3 patients from the BCD-100 10 mg/kg cohort kg). All patients received therapy during the main phase of the study (85 days).

All patient cohorts were balanced for basic demographic characteristics. The distribution of all patients by gender was even: women - 53.33% (8 patients), men - 46.67% (7 patients). The median age of patients was 56 years (minimum 35 years, maximum 77 years).

According to the characteristics of the underlying disease, patients with melanoma predominated in the population (9/15 patients). The population also included 4 patients with NSCLC, 1 patient with pleural mesothelioma, and 1 patient with renal cell carcinoma. The duration of the disease at the time of screening in the entire population was (median) 17.07 months, minimum - 0.6 months, maximum - 91.2 months.

Summary of clinical safety information obtained during the phase I study.

Safety was examined in a population that included all patients who received at least one dose of study drug (n=15). Each of the 15 patients had AEs and/or SAEs, the total number of events was 247. Statistical analysis did not reveal differences between the cohorts in the number of AEs (p = 0.567, Kruskal-Wallis test). Summary table 16 with data on the safety of the drug is given below.

AEs corresponding to grade 3 severity according to the CTCAE scale of 4.03 were noted in 40.00% (6 of 15) of patients - two patients in each cohort. In 4 of 6 of these patients, AEs grade 3 or higher were considered treatment related by the investigator: in a patient receiving BCD-100 at a dosage of 1 mg/kg, in a patient receiving

BCD-100 at a dosage of 3 mg/kg, and in two patients receiving BCD-100 at a dosage of 10 mg/kg. Many of these adverse events were hematologic in nature.

SAEs were reported in three patients receiving 1 mg/kg BCD-100 and one patient receiving 3 mg/kg BCD-100.

The study identified one case of dose-limiting toxicity (DLT). Patient 13-09, who received two doses of BCD-100 at a dosage of 3 mg/kg, had hormonal imbalance (TSH decreased) and developed autoimmune thyroiditis (immune-mediated adverse event of grade 2 according to CTCAE 4.03 criteria). The Data and Safety Monitoring Board considered this case to be a DLT event. Patient 13-09 continued to receive study therapy, and the course of his disease was characterized as stable according to RECIST 1.1 and irRC criteria.

In addition to autoimmune thyroiditis, immune-mediated AEs were noted in 33.33% (5 of 15) of patients: 33.33% (2 of 6) of patients receiving 1 mg/kg BCD-100, 50.00% (3 of 6 ) patients receiving 3 mg/kg BCD-100, but none of the patients receiving 10 mg/kg BCD-100. All of these AEs were grade 1 events according to the CTCAE 4.03 scale, and were not considered to be DLT events.

AEs/SAEs resulted in temporary discontinuation of study therapy in three patients: 16.67% (1 of 6) of patients receiving 1 mg/kg BCD-100 and 33.33% (2 of 6) of patients receiving 3 mg/kg BCD-100. kg BCD100. All AEs due to which therapy was temporarily discontinued were of an immune nature (hyperthyroidism

- 51 046787 oz of the 1st degree of severity in patient 10-03, who received 1 mg/kg BCD-100; grade 1 autoimmune thyroiditis in patient 13-10, and grade 2 autoimmune thyroiditis in patient 13-09, both patients received 3 mg/kg BCD-100). Patient 10-03's investigational therapy was suspended. Patients 13-09 and 13-10 were prescribed glucocorticoids for the disease, after which study therapy was resumed. None of the breaks in therapy exceeded the norms (2-4 weeks) established in the study protocol.

In three patients, the study drug was discontinued due to disease progression (two of them after 5 injections at a dosage of 1 mg/kg and one after 5 injections at a dosage of 10 mg/kg). The reason for discontinuation in all cases was disease progression and not BCD-100 use.

The SAE led to discontinuation of therapy and then to the death of one of the subjects.

Patient 13-10, who underwent three injections of the drug at a dosage of 3 mg/kg, experienced a grade 5 hemorrhage in the right hemisphere. The investigator considered that this AE was likely related to the study treatment.

There were no differences between the patient groups in terms of changes in laboratory and physiological parameters over time. Although analysis of these parameters revealed isolated examples of statistically significant differences, these differences were considered to reflect natural variability within the normal range. Abnormalities detected by the results of a biochemical blood test and a blood coagulation test (increased levels of transaminases, bilirubin, abnormalities in blood clotting parameters, etc.) in most cases were typical for the study population (patients with inoperable neoplasms), expected, and transient ; normalization of parameters passed without consequences; no additional therapy was required.

The described results demonstrate a favorable safety profile of BCD-100 at any of the studied dosages when administered intravenously.

Table 16

Safety data (study BCD-100-1)

Proportion of patients All patients (a = 15) Cohorts BCD-100, 1 mg/kg (n = 6) BCD-100, 3 mg/kg (n = 6) BCD-100, 10 mg/kg (n = 3) Any AE 15 (100%) 6 (100%) 6 (100%) 3 (100%) - therapy-related 11 (73%) 4 (671) 5 (83%) 2 (671) AE grade 3-5 6 (401) 2 (33%) 2 (33%) 2 (671) - therapy-related 4 (271) 1 (17%) 1 (17%) 2 (67%) sleeping 3 (201) 2 (33%) 1 (17%) 0 (0%) - therapy-related 1 (7%) 0 (0%) 1 (171) 0 (0%) Dose-limiting toxicity 1 (7%) 0 (01) 1 (17%) 0 (0%) Immune-mediated AEs 5 (33%) 2 (33%) 3 (50%) 0 (0%) Early elimination due to AND 1(7%) 0 (0%) 1 (17%) 0 (0%) Treatment cycle delayed due to AEs 3 (251) 1 (171) 2 (33%) 0 (0%) Immunogenicity (appearance of antibodies to the drug) 0 (0%) 0 (0%) 0 (0%) 0 (0%)

Immunogenicity.

Immunogenicity assessments were performed at screening, on day 28 of study therapy, at the end of the study period (on day 85), and among patients continuing to receive BCD-100 therapy, also every 42 days thereafter. Immunogenicity assessment was carried out in two stages: the first stage was screening for the content of drug binding antibodies (CAT) in patient serum samples, and the second stage was screening of samples with a positive CAT titer for the content of neutralizing antibodies (HAT).

All patients whose serum samples were available for analysis at the time of screening and on subsequent testing days were included in the immunogenicity analysis (n=15). No samples were found to contain CAT. Due to the lack of samples with CAT, HAT analysis was not performed.

Summary of pharmacokinetics and metabolism information obtained during the phase I study.

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The pharmacokinetics (PK) analysis included data from patients who did not have more than 3 blood serum samples missed/lost/spoilt to determine the concentration of BCD-100 after the first administration of the drug (n=15).

The study of pharmacokinetics (both after the first administration of the drug and throughout all administrations) was carried out in the following groups: in the first patient (BCD-100 dose 0.3 mg/kg) (n=1); in the first cohort of patients (BCD-100 dose 1 mg/kg) (n=5); in the second cohort of patients (BCD-100 dose 3 mg/kg) (n=6); in the third cohort of patients (BCD-100 dose 10 mg/kg) (n=3).

The pharmacokinetic analysis included assessment of standard PK characteristics of distribution and elimination of the study drug after repeated intravenous administration. The results of the analysis are presented in table. 17.

Blood samples for pharmacokinetic analysis (serum BCD-100 levels) were collected from all patients enrolled in the study at each dose level. The following time points for sampling were used: before the first administration of the drug; after 30 minutes, 2 hours (±15 minutes), 4 hours (±15 minutes), 6 hours (±15 minutes), 24 hours (±1 hour), 48 hours (±2 hours), 192 hours (±8 hours ), and 336 hours (±8 hours) after the first administration of the drug (before the second administration), and before each subsequent administration of the drug once every two weeks. Blood samples were collected for analysis of serum BCD-100 concentrations at the same time as samples were taken for immunogenicity analysis (to assess the possible effect of antibodies to the drug on the pharmacokinetic parameters of BCD-100). The concentration of BCD-100 in blood serum was determined using a validated ELISA method.

Table 17

Pharmacokinetic parameters (BCD-100-1 study)

Mean (median [lower quartile; upper quartile]) 1 mg/kg (n=5) 3 mg/kg (n=6) 1C mg/kg (n=3) p value ¹ S-ah (MCG ML; 17.36 ¢13.9 [13.1; 15.4]) 50.65 (49.4 [17.4; 74.7]) 110,467 (93.100 [85.80; 152.50]) 0.0103 Tezkh (h) 8.1(6 [4; b]) 12.333 (6[4:b]) 0.5 AKS(O-336 h) C(µg/ml)>'h) 1934.815 (2201.575 [1576.75; 2360.925]) 8972.413 ¢8482.938 [3397.3; 14254.5]) 12927.308 (13441.075 [7382.850; 17958,000]) 0.0088 AUC(0-=) ((µg/ml) *h) 3664.334 ¢3491.898 [2348.363; 3510.258]) 18071.061 ¢15978.296 [14630.459; 21616.064]) 27943.841 (27373.114 [11989.993; 44468.417]) 0.0123 T1 2(h) 305.511 (288.811 [248.440; 319.423]) 470.936 ¢447.192 [305.351; 597.541]) 315.015 (336.479 [247.553; 361,014]) 1 sph (µg/ml) 3,260 (2,600 [1,800; 3,600]) 15,817 ¢14,100 [10,300; 21,800]) 30,833 ¢28,700 [12,900; 50,900]) ¹ Kruskal-Wallis criterion

All parameters were measured after a single administration of the drug, with the exception of the C _min indicator, which was calculated for 6 administrations of the drug (12 weeks).

Statistically significant differences between the three dose cohorts were noted for AUC _{(0-336 hours)} (p=0.0088, Kruskal-Wallis test); AUMC( _{0-336 h} )) (p=0.0088, Kruskal-Wallis test); AUC _(0l-/ ., (p=0.0123, Kruskal-Wallis test); _Cmax (p=0.0103, Kruskal-Wallis test). At doses of 3 mg/kg and 10 mg/kg, more favorable pharmacokinetic profiles compared with a dose of 1 mg/kg; however, PK, efficacy and safety indicators were not associated with dosage.

BCD-100 drug concentration profiles from the first dose to week 10 in all dose cohorts (1 mg/kg cohort [n=5], 3 mg/kg cohort [n=6], and 10 mg/kg cohort [n =3]) are shown in Fig. 1 and 2.

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Calculation of PK parameters showed that the concentration of BCD-100 varies in direct proportion to the administered dose, reaching a peak between 30 minutes and 6 hours after administration and gradually decreasing thereafter. There was no relationship between the half-life and the amount of drug administered. The T1 _/2 value turned out to be typical for monoclonal antibodies (12-18 days).

Summary of pharmacodynamic information obtained during the phase I study.

All patients whose serum samples were available for analysis at the time of screening and on subsequent testing days were included in the pharmacodynamic analysis (n=15).

The pharmacodynamics study was carried out in the following groups:

in the first patient (BCD-100 dose 0.3 mg/kg) (n=1);

in the first cohort of patients (BCD-100 dose 1 mg/kg) (n=5);

in the second cohort of patients (BCD-100 dose 3 mg/kg) (n=6);

in the third cohort of patients (BCD-100 dose 10 mg/kg) (n=3).

BCD-100 saturation of the PD-1 receptor helps assess the interaction of the study drug with its target and is the most important pharmacodynamic marker of the activity of a PD-1 monoclonal antibody, since blockade of the PD-1 receptor initiates an antitumor immune response. Blood sampling to assess pharmacodynamics (% saturation of the PD-1 receptor with BCD-100) was performed in all patients. Samples for PD assessment were taken before drug administration, 4 hours and 336 hours after administration (but before the second administration), and before the sixth drug administration.

The results demonstrate high saturation of the PD-1 receptor by BCD-100 (95 to 100%) at all dose levels and in all cell populations (Table 18).

Table 18

PD-1 Receptor Saturation (BCD-100-1 Study)

Cohort Index CD4- CD8+ ABOUT CD4+ HLA-DR- CDS-HLA-DR- R ^: Visit 1 Visit 5 Visit 1.0 Visit 1 Visit 5 Visit 10 Visit I11||| Visit 5 Visit 10 Visit 1 Visit Visit 10 Average 95.60 95.64 91.65 96.42 95.55 94,.65 97.83 98.17 96.86 97.19 97.43 97.01 Median 95.21 97.72 95.44 96.62 98.35 97.08 98.45 99.10 99.15 97.17 98.81 97.89 Kogor- Minimum Maximum 89.89 86.24 83.41 89.48 100.0 83.09 100.0 88.13 94.00 100.0 94.87 100.0 91.42 93.70 100.0 90.71 100.0 93.15 111 BCD10U 1 mg/kg (n = 5) N^vartile In quartile 99.36 9-.32 99.03 97.01 96.10 83.41 |E,71 65 0 96.23 0 97.81 98.75 88.13 0.71 65 0 97.83 0 97.58 100.00 91.42 0.71 65 0 95.24 0 98.14 100.00 93.15 0.71 65 99.21 98.20 96.10 99.75 98.50 98.75 98.86 99.31 100.00 99.85 99.51 100.00 CO 3.92 531 7.14 4.25 7.01 5.71 2.28 2.05 4.73 2.78 3.82 3.51 sq.% -and about 5 I 5 7.80 4.41 7.34 6.03 2.33 2.09 4.88 2.86 3.93 3.62 Average 98.14 96.24 99.19 99.48 95=72 99.58 97.46 99.04 99.24 98.75 97.88 99.05 Median 99.94 95.84 100.00 100.0 0 96.27 100.00 100.0 0 99.53 99.54 100.0 0 98.63 99.90 Kogor- Minimum 93.84 92.83 96.29 97.39 90.80 98.08 87.30 97.26 98.00 95.31 91.75 95.95 that Maxim 100.0 100.0 100.0 100.0 100.0 100.0 BCD- m 0 99.99 100.00 0.06 0 98.26 100.00 0.01 0 0 99.95 0.51 0 0 100.00 0.93 100 Nquartile 95 100.0 47 100.0 34 55 3 mg/kg 96.93 93.85 99.65 0 94.52 99.83 0 97.93 98.88 98.43 98.25 99.40 (11=6) Vquartile 100.0 100.0 1000 100.0 100.0 100.0 99.08 100.00 98.18 100.00 0 99.84 0 100.00 CO 2D4 2.95 1.63 1.17 2.81 0.84 5.68 1.16 0.81 2.04 3.10 1.75 sq.% 2.80 3.07 1.64 1.17 2.94 0.85 5.83 1=17 0.82 2.06 3.17 1.77 Average 97.00 98.61 98.71 96.86 100.0 0 97.82 98.89 98.72 100.00 99.37 100.0 0 100.00 Cohort BCD100 10 mg.'kg (n = 3) Median 97.45 99.25 98.71 98.06 100.0 0 97.82 98.82 99.02 100.00 100.0 0 100.0 0 100.00 Minimum 93.66 96.58 97.41 92.58 100.0 0 95.64 98.13 97.13 100.00 98.10 100.0 0 100.00 Maxim m 99 ₌ S9 100.0 0 100.00 0.60 65 99.94 100.0 0 100.00 0.15 61 99.66 100.0 0 100.00 0.36 79 100.0 0 100.0 0 100.00 - N. quartile 93.66 96.58 97.41 92.58 100.0 0 95.64 98.18 97.13 100.00 98.10 100.0 0 100.00 IN. 100.0 100.0 100.0 100.0 100.0 quartile 99.89 0 100.00 99.94 0 100.00 99.66 0 100.00 0 0 100.00 CO 3.14 1=80 1.83 3.82 0.00 3=08 0.74 1=46 0.00 1.10 0.00 0.00 3.24 1.82 1.86 3.95 0.00 3=15 0.75 1.48 0.00 1.10 0.00 0.00 p-value ¹ 0.323 6 0.416 0.047 0.108 3 0.032 0.1589 0.318 5 0.654 7 0.1804 0.264 8 0.140 7 0.3018

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Summary of clinical efficacy information obtained during the phase I study.

Fourteen of 15 patients receiving BCD-100 were included in the efficacy population. One patient had treatment discontinued due to a SAE (death); At the time of the patient's death, tumor response to therapy had not been assessed using CT.

Overall response rate (partial response rate + complete response rate) and disease control rate (stable disease + partial response + complete response) were determined after 85 days of BCD-100 therapy. The efficacy assessment after 85 days of treatment with BCD-100 was based on analysis of CT scan results. Tumor response was assessed using RECIST 1.1 and Immune-Related RECIST (irRECIST) criteria.

According to the irRECIST criteria, which were developed taking into account immunotherapy, disease control was achieved in 28.57% (4 of 14) of patients. The overall response rate was 7.14% (1 of 14). There were no significant differences in response rates between the different patient cohorts (Table 19).

Table 19

Tumor response (BCD-100-1 study)

Patient Dose BCD100 b£g/kg Diagnosis Mutations Reply by RECIST 1.1 □tweg by irRECIST BCD therapy 103 10-01 o ₅ z^1 Metastatic melanoma BRAF V600E- o^et Partial answer (pChO) Continued 9 months (confirmed PR after b and: 9 months) 08-02 1 Locally spread injured NSCLC Not testvrova. leia: on EGFR.H ALK Disease progression Stabilization of zabschievash (1gSZ) Lasted 8 months (confirmed by SZ after 6 months) 10-Yu 1 Mezhzhsh vyshassha BRAF V600E- Progression get sick" Progression of saftiesm: (YPZ) Discontinued 08-04 1 Metastatic mesothelioma shiri Progression diseases Stabilization of the disease (DSD) Discontinued (no-sa clinical symptoms) 12-05 1 Nerezeiab^etia and melanoma BRAF V600E- Disease progression Disease progression (PDP) Discontinued 12-06 1 yizhitiksh MSHYAI1 BRAF V600E+ Progression diseases Disease progression (DP) Discontinued 12-07 3 Metastatic NSCLC EGFR+ Disease progression Progression of scoring (IP) Discontinued

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f-08 3 Metastatic NSCLC EGFR+, ALK test Not see me off! Xia Disease progression Disease progression (DPD) Discontinued 13-09 3 Metastatic melanoma IMF- Stabilization of the disease Disease stabilization (DSD) Lasted 2 months (lymphadenectomy) 13-11 3 Nerezeyaabemna. I have melanoma IMF- Disease progression Disease progression (1gPD) Discontinued 01-12 3 Metastatic renal- cell carcinoma Progression we get sick Disease progression (DS) Discontinued 13-13 10 Metastatic NSCLC EGFR.-, ALK- Stable disease Stabilization of the disease (DSD) Continued 1 month 13-14 10 Metastatic melanoma IMF- Disease progression Disease progression (iPD) Discontinued 08-15 10 Metastatic melanoma IMF- Disease progression Disease progression (1mPD) Discontinued

Conclusion.

The analysis of the data obtained clearly demonstrates the favorable safety profile of the drug BCD-100 when used in a wide range of doses from 0.03 to 10.0 mg/kg. For further clinical study, it seems optimal to choose 2 modes of administration of the drug BCD-100: 1 mg/kg once every 2 weeks and 3 mg/kg once every 3 weeks.

Example 19 Prolgolimab (BCD-100) for the treatment of patients with unresectable/metastatic melanoma, phase II study, BCD-100-2/MIRACULUM.

Study design.

Study No. BCD-100-2/MIRACULUM (MIRACULUM, NCT03269565) is a multicenter, open-label, randomized study of the pharmacokinetics, efficacy, safety and immunogenicity of BCD-100 (prolgolimab) as monotherapy in patients with unresectable/metastatic melanoma, previously untreated or treated. . Comparisons were made between BCD-100 administered intravenously at a dose of 3 mg/kg every three weeks and BCD-100 administered intravenously at a dose of 1 mg/kg every two weeks. The study is currently ongoing in the Russian Federation and Belarus. Data for 1 year of therapy were obtained and analyzed.

Each efficacy parameter was assessed separately for each group. The primary efficacy endpoint was overall response rate (partial response + complete response rate) by irRECIST in study participants receiving BCD-100. The best response to therapy over the above period was taken into account when calculating ORR (overall response rate) and disease control. Secondary endpoints for assessing effectiveness were progression-free and overall survival of patients after 12 months from the start of therapy, rate of achieving disease control (rate of achieving stabilization + partial response + complete response), time to achieve response to therapy, duration of response to therapy (Fig. 3 , table 20).

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Table 20

Characteristics of the BCD-100-2/MIRACULUM study (NCT03269565)

Name Study code: (ots>ana) Objectives of the study Patients Design/duration Therapy Endpoints International FC, security, 126 Randomized BCD-100, Primary multicenter efficiency and patient open inside- (FC): open immunogenicity comrade s international venno _g AUCo-t, randomized melano multicenter 1 mg/kg Cliaxj Tia*, research Research my clinical once every T TZ J-Ng LyHg efficiency, efficiency efficiency, (n=63 research from two Cl, etc. pharmacokinetics pharmacokinetics, IN subjects with weeks after KIC properties, safety and each unresectable/meta many times security and immunogenicity from static melanoma BCD-100, leg immunogenicity two doses groups) inside- intravenous drug BCD- modes Main period: 52 indeed, leg 100 (JSC BCD applications weeks; subsequent 3 mg/kg introduction from "BIOCAD", 100 (JSC observation: 12 weeks once every ispalzova Russia) in ion therapy in patients with unresectable or metastatic melanoma BCD-1002/HIRACULUM (НСТ03269565) Phase III (Russian Federation, Republic of Belarus] "BZhKAD" _T Russia) as monotherapy in patients with unresectable/metastatic melanoma. (total 64 weeks) three weeks using two dose regimens Immunogenicity: Frequency of detection of SAG, ΗΆΤ after repeated intravenous administration of the drug with used two dose regimens

Research results.

Summary of patient population characteristics.

The study included 131 patients over the age of 18 years. The patient population included both men and women with advanced melanoma, including choroidal melanoma. This population included patients who took the place of those who dropped out before the first administration of BCD100 or due to serious deviations from the Protocol. The resulting modified ITT population, consisting of patients who received at least one dose of BCD-100, consisted of 126 patients. To be included in the study, patients had to meet an ECOG score of 0-1 and have at least one measurable target lesion according to RECIST 1.1 (excluding bone metastases) (Fig. 4, Table 21).

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Table 21

Disease characteristics in the patient population (BCD-100-2/MIRACULUM study)

Index BCD-ICO. 1 mg/kg BCD-1C0. 3kg/kg ρ value η = 63 η = 63 ^r 1 '" Dkagshkz 1 at the time of screening) Unresectable melanoma stage IZ 4 635 1 1.59 0.365 ² Unresectable melanoma stage IZI 0 07’0 1 1.59 IjCOij- Metastatic melanoma GS stage 59 93.65 61 96-83 0.6S0 ^: Gizmizhnpy™ virii Mixed cell melanoma 3 4.76 2 3.17 LCOO: Dpitetptpi_tap-zhetyatp'»tnyaya ^: mvlyaigmya 23 36.51 26 41.27 D715- Spindle line and tire 5 7.94 5 “ _; 94 LCO'O ² Nevus cell melanoma 1 179 0 070 LCDO ² N.A. 31 4Y21 thirty 47.62 1 _G SEA- Tumors of atypical localizations Melanoma of atypical location 4 6J5 5 7.94 LC00 ² Melanoma chorisidea 3 4.76- 5 794 071S ² Mitted mucous membrane 1 1.59 0 070 LOGO ² intestine Steppe"-ietaizhgiyesvgo defeat AJCC classification Mn 0 th S-0 0 070 LOGO ² M1a 6 9.52 8 1270 G M1Ъ 13 20.54 8 1270 0.339 ¹ M1s 44 59.S4 47 “4.50 0.691 ¹ AJCC Classification 8 mm | 17 1 26.98 I 9 1 1<29 I | 0.123 ¹ Metastatic lesion of the central nervous system Available 17 26.98 9 14.29 0.123 ¹ Absent 46 73.02 54 85.71 0.123 ¹

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-M>-S status. ^: ' Pszitlv ntk /33<</ 52D8 7:31/: <49.21 0.359- 'Negative /12/7. 19.05 14// 22.22 ClS26 ^: No traces of guilt were investigated /18/:, 25.57 18 23.5' 1,000 ¹ BRAF status 3RAF mmtaikya sonar’zhena 21 33.33 24 :38.1/ ..... 0710 ³ BRAF no entry detected /25// 39.68 22 34.92 0713- No study conducted /1’7/ 26.95 17/ 26.95 1,000- Median BTS (tumor volume at screening) Median BTS | 71mm/ | 1 9’ mm 1 0.0-f LDH level at screening Norm /-42/// 66.67 741/ 65.05 1,000 ¹ Above the nose·.:ye 21 33.33 --:22:7 34.92 1,000 ¹ Previous therapy Early stage therapy Neoadjuvant therapy 0 0.00 0 0.30 1,000 ² Surgery 59 93.65 -9 “7.73 0.32 ² Adjuvant therapy <23::// 36.51/ :/-:247 /:/38,:1-- 1,000 ¹ Late stage therapy Chemotherapy for common disease Do not proceed (the drug BCD-lOO is used on the 1st line! /46/ ~z _; To 47 “4.60 1,000 ¹ Conducted 7177 26.95 16 25.40 1,000 ³ 1 line of precursor teralin (BCD-:OS drug is used in 2 /15 23.81 8 K7 0.156 ³ .2 backstay ' (the drug BCD-US is used e 3 „nwHWHji · · : 2: 3.17 8 127 0.0 95- Other (immuiot^hapia, /vazhdinmerapia) 6 9.52 <5 7>4 1,000 ² /Radiation therapy / 4 635 7 7 11.11 0.5 Z ² Duration of disease (months) Average 3373 31.32 0.1" 4- 2. 1 unit 19.55 / 15,447 Mmniuyag' 0.53 ..... '0/13 Mzsimum/...... ..... ..........: 315.83 ......./227.09 CO -6.12 45.86/ Note: -Pearson's test with Koethe's correction, -two-sided Fisher's exact test, -Mann-Whitney test. HA - no data

The safety analysis included all patients who received at least one dose of study drug (modified intent-to-treat (mITT) population, n=126).

The effectiveness analysis was carried out in two patient populations:

all patients who received at least one administration of study drug (mITT population, n=126);

Patients who received at least one dose of study drug and had at least one routine follow-up CT scan to assess response (per protocol (PP) population, n=114).

The pharmacokinetic analysis included data from patients who received at least one administration of BCD-100 and did not have missing/lost/spoilt required samples according to the current version of the protocol at the time of determination (n=125).

Summary of clinical efficacy information from Study No. BCD-1002/MIRACULUM.

The results of the primary endpoint efficacy assessment (ORR) indicate sufficient effectiveness of BCD-100 in both dose regimens in all patient populations studied. Thus, ORR and disease control in the RR population were 40.68 and 67.80% in group 1 and 32.73 and 52.73% in group 2 of therapy. Similar efficacy rates were observed in the mITT population. Thus, the expected target response rate (28%) and the critical r value (11 responses) were achieved in both treatment groups.

Subgroup analysis by line of therapy demonstrated the high efficacy of BCD-100 at a minimum dose of 1 mg/kg in a population of previously untreated patients (ORR was 50.00%).

The results of the analysis of effectiveness for secondary endpoints confirmed the conclusion about

- 59 046787 sufficient effectiveness of the drug BCD-100 in both doses. The DFS, OS, and duration of response rates were comparable to those of the best-in-class PD-1 inhibitors.

The 12-month DFS was 41.27% for patients in the BCD-100 1 mg/kg group and 34.92% for patients in the BCD-100 3 mg/kg group. Median PFS was 5.78 months (95% CI 3.52 - -) for patients in the BCD-100, 1 mg/kg group and 2.33 months (95% CI 2.07-10.25) for patients in the BCD group -100.3 mg/kg (p=0.400, log-rank test). A detailed assessment of progression-free survival in each group, according to lines of therapy (according to irRECIST criteria, in the mITT population), did not reveal statistically significant differences. BCD-100 was equally effective when used in treatment-naive patients and when used in previously treated patients.

At a median follow-up of 13.8 months (95% CI 13.2-14.7), the median overall survival for patients in the BCD-100 1 mg/kg group was not reached (95% CI - ¹ - -) (1 in this case and further in the text means not achieved). The 12-month OS was 74.60% for patients in the BCD-100 1 mg/kg group. The median OS for patients in the BCD-100 3 mg/kg group was 15 months (95% CI 9.99 - -) with a median follow-up of 14.5 months (95% CI 13.9-15.2). The 12-month OS was 53.97% for patients in the BCD-100 3 mg/kg group. A detailed assessment of overall survival in each group, according to lines of therapy, did not reveal statistically significant differences. BCD-100 was equally effective when used in treatment-naive patients and when used in previously treated patients. Median OS was not achieved in any of the subgroups in patients receiving BCD-100 at a dose of 1 mg/kg (p=0.800; log-rank test). Median OS was 16.8 months (95% CI 9.33 - -) for previously untreated patients and 15 months (95% CI 7.46 - -) for previously treated patients in the drug group

BCD-100, 3 mg/kg (p=0.900; log-rank test) (Table 22, Table 23, Table 24, Table 25, Fig. 5, Fig. 6, Fig. 7, Fig. 8 ).

Table 22

Primary endpoint reflecting the effectiveness of the drug in the PP population (population of patients treated in accordance with the protocol) according to the results of the BCD-100-2/MIRACULUM study

Performance parameters irRECIST BCD-100, 1 mg/kg (n=59) BCD-100, 3 mg/kg (n=55) p value G. % iligjjgi % Partial answer (40) 19 32.20 16 29.09 0,: Full answer (PO) 5 8.47 2 3.64 0.440 ² Stabilization (ST) 16 27.12 eleven 20.00 0.501 ¹ Progression (PR) ² 19 32.20 26 47.27 0.146 ¹ Disease control (DC) 40 67.80 29 52.73 0.146 ¹ Obrev response rate (ORR) 24 40.68 18 32.73 0.493 ¹

Note: Pearson's test with Yates' correction, ² two-sided Fisher's exact test ( ² Confirmed by data from 1 routine CT evaluation at visit S).

Summary of clinical safety information from Study No. BCD-1002/MIRACULUM.

BCD-100 demonstrated a favorable safety profile at both dose regimens (Table 23).

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Table 23

Safety profile of the drug BCD-100 based on the results of the BCD-100-2/MIRACULUM study

Proportion of patients with (with); BCD-130 _g 1 mg/kg p=63 η (?) ECE-10C, 3 mg/kg p=b3 p (%) Meaning R AE 54 (85.71%) 54 (35.71%) 1,030 ¹ AEs associated, according to the researchers, with the therapy 35 (55,561) 34 (53.97%) 1,000 ¹ AEs of 3 or more severity degrees according to CTSAE 4.03 IB (28.57%) 21 (33, 33%) 0.699 ¹ AEs of 3 or more severity according to C'TSAE 4.03 associated, in the opinion of the researchers, with the therapy performed 0 (12.70%) 2 (3.17%) 0.095 ³ SAE 5 (7.94%) 13 (15.s7%) 0.271 ² SM associated, according to researchers, with the therapy 2 (3.17%) 0 (0.00%) 0.496 ³ ion 23 (36.51%) 22 (34.92%) 1,000 ¹ ionE of 3 or more degrees severity according to CTSAE 4.03 5 (7.94%) 1 (1.59%) 0.207 ² Cancellation of therapy due to the development of AEs 3 (4.76%) 2 (3.17%) 1,000 ³ Cancellation of therapy due to the development of ionAEs 1 (1.59%) 1 (1.59%) 1,000 ² Note: Pearson's ^J2 test with Yates's test, two-tailed Fisher's exact test

Based on the above data, we can conclude that the results obtained in this study do not contradict the known data on the safety profile of monoclonal antibodies to the PD-1 receptor. There were no statistically significant differences in the safety profile of the drug at the studied doses, with the exception of the number of SAEs followed by death (a significantly higher number was noted among patients in the BCD-100 group, 3 mg/kg).

Immunogenicity.

All patients whose serum samples were available for analysis at the time of screening and on subsequent testing days were included in the immunogenicity analysis (n=121). None of the patients had CAT to BCD-100.

Summary of pharmacokinetics and metabolism information from Study No. BCD-100-2/MIRACULUM.

The analysis showed that a single intravenous administration of the drug BCD-100 at a dose of 1 to 3 mg/kg is characterized by a linear increase in the concentration of the test substance in the blood plasma, followed by a uniform decrease, while the half-life of the drug, regardless of the dose administered to the body, is characterized by the usual duration for immunoglobulins of the IgG class is from 11.5 to 17 days (Fig. 9, Fig. 10).

The trend towards a dose-dependent increase in the concentration of the test substance in the blood plasma (according to the parameters C _max and AUC _0-t , _SS ) also persists after repeated administration of the drug BCD-100, which corresponds to the literature data on the PK profile of other anti-PD-1 antibody drugs.

The persistent preservation of C _min at a sufficient level during the course of treatment demonstrated in the clinical study No. BCD-100-2/MIRACULUM indicates that therapeutic concentrations of the drug are maintained as when using the drug BCD-100 at a dose of 1 mg/kg once a day 2 weeks, and when using the drug at a dose of 3 mg/kg once every 3 weeks. Thus, the use of BCD-100 in both dose regimens is justified from the standpoint of pharmacokinetic properties.

Assessment of the T1 _/2 parameter, including a comparative assessment between treatment groups, was difficult due to the absence of a typical terminal period of the drug elimination phase in a number of patients after a single administration.

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Claims (68)

In general, the drug BCD-100, both at a dose of 1 mg/kg once every 2 weeks and at a dose of 3 mg/kg once every 3 weeks, was characterized by pharmacokinetic indicators that ensured a long stay of the drug in the body sufficient to maintain a stable therapeutic concentration against the background repeated administration. No dose dependence was observed regarding the efficacy and safety of the drug. Summary of pharmacodynamic information obtained during the phase I study. The percentage of PD-1 receptor saturation with BCD-100 in all leukocyte subpopulations studied did not differ between treatment groups. PD-1 saturation of >99% in the population of activated T helper cells and cytotoxic leukocytes was recorded in 33.33% of patients included in the analysis of this indicator (14 of 42). However, there was no statistically significant difference between the treatment groups: 8 patients received BCD-100 at a dose of 1 mg/kg, 6 patients at a dose of 3 mg/kg. Ki-67 is a universal marker of cell proliferation, which is due to its presence in the cell only during the division process and destruction within 1.5-2 hours after the end of mitosis. The Ki-67 marker is detected in the telomere region, centromeres and nucleoli. The percentage of Ki-67 positive cytotoxic T lymphocytes increased during therapy in both groups, but no significant differences were found. Analysis of the proportion of Ki-67 positive cytotoxic T lymphocytes also did not reveal statistically significant differences between the groups. The percentage of Th9 in the total population of T-helper cells during therapy increased mainly in group 2 (BCD-100, 3 mg/kg Q3W), but no significant differences were found. There were no significant differences in intergroup comparisons. According to the available literature, an early increase in Th9 levels is associated with a better response to nivolumab therapy in melanoma ³ ( ³ Nonomura Y., Otsuka A., Nakashima S., Seidel JA, Kitoh A., Dainichi T., Nakajima S., Sawada Y ., Matsushita S., Aoki M., Takenouchi T., Fujimura T., Hatta N., Koreeda S., Fukushima S., Honda T., Kabashima K. Peripheral blood Th9 cells are a passible pharmacodynamic biomarker of nivolumab treatment efficacy in metastatic melanoma patients. Qncoimmunology. 2016 Oct 18; 5(12):e1248327. It should be noted that these results were obtained on a limited sample of patients (n=42), heterogeneous in gender and characteristics of the underlying disease. A comparative analysis of the proportion of Th9 in the total population of T-helper cells in subgroups with different types of response to therapy with BCD-100 did not reveal statistically significant differences in both dose cohorts. However, the presented graphs show a tendency towards a better response to therapy in patients with initially elevated Th9 levels (Fig. 11, Fig. 12). Conclusion. The obtained data on the effectiveness, safety and pharmacokinetic properties of BCD-100 are sufficient to justify the use of the drug both in a dose regimen of 1 mg/kg once every 2 weeks, and in a dose regimen of 3 mg/kg once every 3 weeks. The international, multicenter, randomized, open-label, phase II study BCD-100-2/MIRACULUM in a population of patients with unresectable and metastatic melanoma demonstrated significant therapeutic benefits over the known efficacy of chemotherapy, and results comparable to the best existing treatments. Taking into account the efficacy indicators obtained in the study with a favorable safety profile, the use of BCD-100 in routine clinical practice meets the balance of risk and benefit in this patient population. CLAIM 1. An aqueous pharmaceutical composition of an anti-PD-1 antibody containing: (a) prolgolimab at a concentration of 15 to 40 mg/ml as an antibody; (b) trehalose dihydrate at a concentration of 80 to 110 mg/ml; (c) sodium acetate trihydrate at a concentration of 1.6 to 1.9 mg/ml and (d) acetic acid to pH 4.5-5.5. 2. An aqueous pharmaceutical composition according to claim 1, where said prolgolimab is in a concentration of 15 to 25 mg/ml. 3. An aqueous pharmaceutical composition according to claim 2, where said prolgolimab is at a concentration of 20 mg/ml. 4. An aqueous pharmaceutical composition according to any one of claims 1 to 3, where said trehalose dihydrate is in a concentration of 95 to 105 mg/ml. 5. An aqueous pharmaceutical composition according to claim 3, where said trehalose dihydrate is at a concentration of 100 mg/ml. 6. An aqueous pharmaceutical composition according to claim 1, where said sodium acetate trihydrate is in a concentration of 1.7 to 1.8 mg/ml. - 62 046787 7. An aqueous pharmaceutical composition according to claim 6, where said sodium acetate trihydrate is at a concentration of 1.742 mg/ml. 8. An aqueous pharmaceutical composition according to any one of claims 1 to 3, wherein said acetic acid is added to a pH of 5.0. 9. An aqueous pharmaceutical composition of anti-PD-1 antibody containing: (a) prolgolimab at a concentration of 90 to 150 mg/ml as an antibody; (b) trehalose dihydrate at a concentration of 50 to 110 mg/ml; (c) sodium acetate trihydrate at a concentration of 1.6 to 1.9 mg/ml and (d) acetic acid to a pH of 4.5 to 5.5. 10. An aqueous pharmaceutical composition according to claim 9, where said prolgolimab is in a concentration of 90 to 110 mg/ml. 11. An aqueous pharmaceutical composition according to any one of claims 9, 10, where said prolgolimab is at a concentration of 100 mg/ml. 12. An aqueous pharmaceutical composition according to claim 9, where said trehalose dihydrate is in a concentration of 75 to 85 mg/ml. 13. An aqueous pharmaceutical composition according to claim 12, where said trehalose dihydrate is at a concentration of 80 mg/ml. 14. An aqueous pharmaceutical composition according to claim 9, where said sodium acetate trihydrate is in a concentration of 1.7 to 1.8 mg/ml. 15. An aqueous pharmaceutical composition according to claim 14, where said sodium acetate trihydrate is at a concentration of 1.742 mg/ml. 16. An aqueous pharmaceutical composition according to claim 9, wherein said acetic acid is added to a pH of 5.0 to 5.5. 17. An aqueous pharmaceutical composition of anti-PD-1 antibody containing: (a) prolgolimab at a concentration of 5 to 150 mg/ml as an antibody; (b) trehalose dihydrate at a concentration of 70 to 110 mg/ml; (c) L-histidine at a concentration of 0.7 to 1.0 mg/ml; and (d) L-histidine hydrochloride at a concentration of 2.8 to 3.3 mg/ml. 18. An aqueous pharmaceutical composition according to claim 17, where said prolgolimab is in a concentration of 15 to 40 mg/ml. 19. An aqueous pharmaceutical composition according to claim 18, where said prolgolimab is in a concentration of 15 to 25 mg/ml. 20. An aqueous pharmaceutical composition according to claim 19, where said prolgolimab is at a concentration of 20 mg/ml. 21. An aqueous pharmaceutical composition according to any one of claims 17 to 20, where said trehalose dihydrate is in a concentration of 95 to 105 mg/ml. 22. An aqueous pharmaceutical composition according to claim 21, where said trehalose dihydrate is at a concentration of 100 mg/ml. 23. An aqueous pharmaceutical composition according to claim 17, where said prolgolimab is in a concentration of 90 to 110 mg/ml. 24. An aqueous pharmaceutical composition according to claim 23, where said prolgolimab is at a concentration of 100 mg/ml. 25. An aqueous pharmaceutical composition according to claims 17, 23, 24, where said trehalose dihydrate is in a concentration of 75 to 85 mg/ml. 26. An aqueous pharmaceutical composition according to claim 25, where said trehalose dihydrate is at a concentration of 80 mg/ml. 27. An aqueous pharmaceutical composition according to claim 17, where said L-histidine is at a concentration of 0.92 mg/ml. 28. An aqueous pharmaceutical composition according to claim 17, where said L-histidine hydrochloride is at a concentration of 2.96 mg/ml. 29. An aqueous pharmaceutical composition according to claim 17, wherein said composition has a pH of 5.5 to 6.5. 30. An aqueous pharmaceutical composition according to claim 29, wherein said composition has a pH of 5.5 to 6.0. 31. An aqueous pharmaceutical composition according to any one of claims 1, 17, additionally containing a suitable solubilizer. 32. An aqueous pharmaceutical composition according to claim 31, wherein said solubilizer is poloxamer 188. 33. The aqueous pharmaceutical composition according to claim 32, wherein said poloxamer 188 is present in an amount that is greater than 0 mg/ml but equal to or less than 1 mg/ml. 34. An aqueous pharmaceutical composition of an anti-PD-1 antibody containing: (a) prolgolimab at a concentration of 20 mg/ml as an antibody; (b) trehalose dihydrate at a concentration of 100 mg/ml; (c) sodium acetate trihydrate at a concentration of 0.2 to 2.5 mg/ml and (d) acetic acid to a pH of 4.5 to 5.5. - 63 046787 35. An aqueous pharmaceutical composition of anti-PD-l antibody according to claim 34, containing: (a) prolgolimab at a concentration of 20 mg/ml as an antibody; (b) trehalose dihydrate at a concentration of 100 mg/ml; (c) sodium acetate trihydrate at a concentration of 1.7 to 1.8 mg/ml and (d) acetic acid to pH 5.0. 36. An aqueous pharmaceutical composition of anti-PD-1 antibody according to claim 35, containing: (a) prolgolimab at a concentration of 20 mg/ml as an antibody; (b) trehalose dihydrate at a concentration of 100 mg/ml; (c) sodium acetate trihydrate at a concentration of 1.742 mg/ml and (d) acetic acid to pH 5.0. 37. An aqueous pharmaceutical composition of an anti-PD-1 antibody containing: (a) prolgolimab at a concentration of 100 mg/ml as an antibody; (b) trehalose dihydrate at a concentration of 80 mg/ml; (c) sodium acetate trihydrate at a concentration of 0.2 to 2.5 mg/ml and (d) acetic acid to a pH of 4.5 to 5.5. 38. An aqueous pharmaceutical composition of anti-PD-1 antibody according to claim 37, containing: (a) prolgolimab at a concentration of 100 mg/ml as an antibody; (b) trehalose dihydrate at a concentration of 80 mg/ml; (c) sodium acetate trihydrate at a concentration of 1.7 to 1.8 mg/ml and (d) acetic acid to a pH of 5.0 to 5.5. 39. An aqueous pharmaceutical composition of anti-PD-1 antibody according to claim 38, containing: (a) prolgolimab at a concentration of 100 mg/ml as an antibody; (b) trehalose dihydrate at a concentration of 80 mg/ml; (c) sodium acetate trihydrate at a concentration of 1.742 mg/ml and (d) acetic acid to a pH of 5.0 to 5.5. 40. An aqueous pharmaceutical composition of an anti-PD-1 antibody containing: (a) prolgolimab at a concentration of 20 mg/ml as an antibody; (b) trehalose dihydrate at a concentration of 100 mg/ml; (c) L-histidine at a concentration of 0.2 to 2.5 mg/ml and (d) L-histidine hydrochloride at a concentration of 0.2 to 3.5 mg/ml; (e) wherein said composition has a pH of from 5.5 to 6.5. 41. An aqueous pharmaceutical composition of anti-PD-1 antibody according to claim 40, containing: (a) prolgolimab at a concentration of 20 mg/ml as an antibody; (b) trehalose dihydrate at a concentration of 100 mg/ml; (c) L-histidine at a concentration of 0.92 mg/ml and (d) L-histidine hydrochloride at a concentration of 2.96 mg/ml; (e) wherein said composition has a pH of 5.5. 42. An aqueous pharmaceutical composition of an anti-PD-1 antibody containing: (a) prolgolimab at a concentration of 100 mg/ml as an antibody; (b) trehalose dihydrate at a concentration of 80 mg/ml; (c) L-histidine at a concentration of 0.2 to 2.5 mg/ml and (d) L-histidine hydrochloride at a concentration of 0.2 to 3.5 mg/ml; (e) wherein said composition has a pH of from 5.5 to 6.5. 43. An aqueous pharmaceutical composition of anti-PD-1 antibody according to claim 42, containing: (a) prolgolimab at a concentration of 100 mg/ml as an antibody; (b) trehalose dihydrate at a concentration of 80 mg/ml; (c) L-histidine at a concentration of 0.92 mg/ml and (d) L-histidine hydrochloride at a concentration of 2.96 mg/ml; (e) wherein said composition has a pH of 5.5 to 6.0. 44. An aqueous pharmaceutical composition of an anti-PD-1 antibody containing: (a) prolgolimab at a concentration of 20 mg/ml as an antibody; (b) trehalose dihydrate at a concentration of 100 mg/ml; (c) sodium acetate trihydrate at a concentration of 1.742 mg/ml; (d) acetic acid to pH 5.0. 45. An aqueous pharmaceutical composition of an anti-PD-1 antibody containing: (a) prolgolimab at a concentration of 20 mg as an antibody; (b) trehalose dihydrate at a concentration of 100 mg; (c) sodium acetate trihydrate at a concentration of 1.742 mg; (d) acetic acid to pH 5.0, and (e) water for injection to 1 ml. 46. An aqueous pharmaceutical composition according to any one of claims 34-45, further containing a suitable solubilizer. - 64 046787 47. An aqueous pharmaceutical composition according to claim 46, wherein said solubilizer is poloxamer 188. 48. The aqueous pharmaceutical composition according to claim 47, wherein said poloxamer 188 is present in an amount that is greater than 0 mg/ml but equal to or less than 1 mg/ml. 49. Use of an aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab according to any one of claims 1 to 48 for the treatment of cancer in a subject in need thereof. 50. Use according to claim 49, where the malignant neoplasm is selected from the group containing melanoma, including inoperable or metastatic melanoma, early stages of melanoma before and after radical treatment; lung cancer, non-small cell lung cancer (NSCLC), including unresectable or metastatic non-small cell lung cancer; non-squamous non-small cell lung cancer, squamous cell lung cancer; small cell lung cancer, including unresectable or metastatic small cell lung cancer; early stage lung cancer before and after radical treatment; cervical cancer, including metastatic cervical cancer, early stages of cervical cancer before and after radical treatment; head and neck tumors, including squamous cell carcinoma of the head and neck; Hodgkin's lymphoma; tumors of the stomach and intestines, metastatic squamous cell carcinoma of the esophagus; bladder cancer, including metastatic urothelial carcinoma, kidney cancer; endometrial cancer, including metastatic endometrial cancer, early stages of endometrial cancer before and after radical treatment; breast cancer, including metastatic breast cancer, early stages of endometrial cancer before and after radical treatment; liver cancer, including metastatic or inoperable liver cancer, early stages of liver cancer before and after radical treatment; unresectable or metastatic solid tumor, including unresectable or metastatic solid tumor with signs of microsatellite instability. 51. Use of an aqueous pharmaceutical composition of the anti-PD-1 antibody prolgolimab according to any one of claims 1-8, 17-21, 34-36, 40, 41 for the treatment of malignancy in a subject in need thereof. 52. Use according to claim 51, where the specified aqueous pharmaceutical composition is administered at a dose of the anti-PD-1 antibody prolgolimab 1 mg/kg body weight. 53. Use according to claim 51, where said aqueous pharmaceutical composition is administered at a dose of the anti-PD-1 antibody prolgolimab of 3 mg/kg body weight. 54. Use according to claim 51, wherein said aqueous pharmaceutical composition is administered every 2 weeks. 55. Use according to claim 51, wherein said aqueous pharmaceutical composition is administered every 3 weeks. 56. Use according to claim 51, where the specified aqueous pharmaceutical composition is administered at a dose of the anti-PD-1 antibody prolgolimab 1 mg/kg body weight every 2 weeks. 57. Use according to claim 51, wherein said aqueous pharmaceutical composition is administered at a dose of the anti-PD-1 antibody prolgolimab of 3 mg/kg body weight every 3 weeks. 58. Use according to claim 51, wherein said aqueous pharmaceutical composition is administered parenterally. 59. Use according to claim 58, wherein said parenteral administration is intravenous, subcutaneous or intramuscular administration. 60. Use according to claim 58, wherein said aqueous pharmaceutical composition is administered intravenously as an infusion. 61. Use according to claim 51, where the malignant neoplasm is melanoma, including inoperable or metastatic melanoma, early stages of melanoma before and after radical treatment; lung cancer, non-small cell lung cancer (NSCLC), including unresectable or metastatic non-small cell lung cancer. 62. A method of treating a malignant neoplasm in a subject in need thereof, comprising administering a therapeutically effective amount of an aqueous pharmaceutical composition according to any one of claims 1-8, 17-21, 34-36, 40, 41. 63. The method according to claim 62, where the specified aqueous pharmaceutical composition is administered at a dose of the anti-PD-1 antibody prolgolimab of 1 mg/kg body weight. 64. The method according to claim 62, where the specified aqueous pharmaceutical composition is administered at a dose of the anti-PD-1 antibody prolgolimab of 3 mg/kg body weight. 65. The method according to claim 62, wherein said aqueous pharmaceutical composition is administered every 2 weeks. 66. The method according to claim 62, wherein said aqueous pharmaceutical composition is administered every 3 weeks. 67. The method according to claim 62, where the specified aqueous pharmaceutical composition is administered at a dose of the anti-PD-1 antibody prolgolimab 1 mg/kg body weight every 2 weeks. 68. The method according to claim 62, where the specified aqueous pharmaceutical composition is administered at a dose of the anti-PD-1 antibody prolgolimab 3 mg/kg body weight every 3 weeks. -